CN110082332B - Method for detecting alkaline phosphatase by manganese dioxide modified up-conversion nano material - Google Patents
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- MIJPAVRNWPDMOR-ZAFYKAAXSA-N L-ascorbic acid 2-phosphate Chemical compound OC[C@H](O)[C@H]1OC(=O)C(OP(O)(O)=O)=C1O MIJPAVRNWPDMOR-ZAFYKAAXSA-N 0.000 description 3
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- SIWVEOZUMHYXCS-UHFFFAOYSA-N oxo(oxoyttriooxy)yttrium Chemical compound O=[Y]O[Y]=O SIWVEOZUMHYXCS-UHFFFAOYSA-N 0.000 description 1
- UZLYXNNZYFBAQO-UHFFFAOYSA-N oxygen(2-);ytterbium(3+) Chemical compound [O-2].[O-2].[O-2].[Yb+3].[Yb+3] UZLYXNNZYFBAQO-UHFFFAOYSA-N 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/38—Diluting, dispersing or mixing samples
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- 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
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
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- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
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- Pathology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
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Abstract
The invention discloses a method for detecting alkaline phosphatase by using manganese dioxide modified up-conversion nanoparticles. Yb and Tm doped yttrium tetrafluorideSodium (NaYF)4Yb, Tm) is about 2 nm, and the emission spectrum is 471 nm. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) up-conversion nanoparticles and manganese dioxide modified up-conversion nanoparticles, has simple synthesis method, simple and convenient operation for detecting alkaline phosphatase, no need of expensive instruments, rapidness and sensitivity, can avoid interference of autofluorescence in biological sample detection, and has low toxicity. The method has the linear range of 0.1U/L-100U/L for alkaline phosphatase and the detection limit of 0.41U/L, and has excellent application prospect in the analysis and detection of actual biological samples and biological imaging.
Description
The application is supported by the project on the national science fund (21375095) and the youth project of the science fund (No.17JCQNJC05800) in Tianjin.
Technical Field
The invention belongs to the technical field of biological analysis and detection, and relates to an application of a lamellar amorphous manganese dioxide modified up-conversion nano material in detection of cancer marker alkaline phosphatase.
Background
Up-converting nanomaterials can typically convert two or more photons of low energy into photon emissions of high energy, also known as anti-stokes luminescence, since the luminescence of the up-converting material is absorption of low energy light of long wavelength (typically 980 nm near infrared light) emitting high energy light of short wavelength. The up-conversion process of the rare earth doping mainly depends on the stair-step energy level of the rare earth ions, and the electrons of the inner layer 4f of the rare earth ions hardly interact with the matrix, so that the absorption and emission spectra of the doped rare earth ions are similar to those of free ions, extremely sharp peaks are shown, meanwhile, the anti-Stokes displacement is large, the interference of autofluorescence is avoided, the toxicity is low, the tissue penetration depth is high, and excellent optical properties are shown, so that the up-conversion nano material is widely concerned by researchers in biosensing and imaging.
Alkaline phosphatase, a group of isozymes, is widely present in human bones, liver, intestine and kidney, can remove small biological molecules, and phosphate groups carrying free hydroxyl groups on proteins and nucleic acids, and the process is called dephosphorylation, which plays an important role in biological transduction and regulation of intracellular protein activity. Normal alkaline phosphatase levels in humans ranged from 46-190U/L. The abnormal level of alkaline phosphatase is related to various diseases (bone diseases such as rickets, osteomalacia, malignant bone tumor, malignant tumor bone metastasis, and diseases of liver and gallbladder such as extrahepatic biliary tract obstruction, liver cancer, liver cirrhosis and capillary bile duct hepatitis) in human bodies, and has great significance for early diagnosis and screening of cancers. The traditional method for detecting alkaline phosphatase comprises an isotope labeling method, a mass spectrometry method, an electrochemical method, a colorimetric method, surface enhanced resonance Raman scattering and the like. The methods have the defects of large instrument consumption, low detection speed, high cost, complex sample processing process and the like. Fluorescence has received attention from researchers because of its rapid, simple and low cost advantages. The method utilizes the unique optical property of the manganese dioxide modified up-conversion nano material and the hydrolysis of alkaline phosphatase and 2-phosphoric acid-L-ascorbic acid to realize the rapid and sensitive detection of the alkaline phosphatase through the linear change value of fluorescence.
The methods for detecting cancer markers reported at present mainly include colorimetric methods, fluorescence methods, chromatography methods and electrochemical methods, and these methods have the disadvantages of poor selectivity, low sensitivity, long time consumption, high cost, poor biocompatibility, unavoidable interference of autofluorescence in actual sample detection, and the like, so that it is necessary to develop a method for detecting alkaline phosphatase by using manganese dioxide modified up-conversion nanomaterial with simple operation, rapid reaction and high sensitivity.
Disclosure of Invention
The invention aims to provide a method for detecting a cancer marker by using manganese dioxide modified up-conversion nano material, which is simple to operate, quick to react and high in sensitivity.
In order to achieve the aim, the invention discloses a method for detecting alkaline phosphatase by using manganese dioxide modified up-conversion nano-materials, which is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a pipette, uniformly mixing, and reacting at 37 ℃ for 15-120 min;
moving 2 mL of manganese dioxide modified up-conversion solution, adding 600 mu L of high-purity water, adding 400 mu L of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 10-60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer.
The invention also discloses a synthesis method of the manganese dioxide modified up-conversion nano material, which is characterized by comprising the following steps:
1. ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nano particles are synthesized by a hydrothermal method, polyethyleneimine is used as a coating agent, and the molar ratio of Y, Yb and Tm is 75: 25: 0.3, and the method is characterized by comprising the following steps:
(1) pre-treatment nitration: respectively weighing Y2O3, Yb2O3, Tm2O3Adding three rare earth oxides with a molar ratio of 75: 25: 0.3 into a beaker A, sequentially adding 2 mL of concentrated nitric acid and 2 mL of high-purity water, and placing a small beakerHeating in an electrothermal sleeve, continuously adding 5 mL of high-purity water after the oxide is completely nitrified to form nitrate, continuously adding 5 mL of high-purity water when the oxide is to be evaporated to dryness, measuring the pH value of the solution, repeatedly adding the high-purity water in the way until the pH value of the solution is about 7, and adding 5 mL of high-purity water after the solution is evaporated to dryness for later use.
(2) Synthesizing: sodium hydroxide and ammonium fluoride were weighed 10 mmol each into beaker B and 0.15g of different molecular weight Polyethylenimine (PEI) (Mw =10000, Mw =1800, Mw = 600) were weighed in an additional step into beaker a. Then, 4.5 mL of ethylene glycol, 3 mL of highly purified water, was added to the A beaker, and 4.5 mL of highly purified water, 3 mL of ethylene glycol, was added to the B beaker. The A, B beakers were sonicated for 5-10 minutes, followed by magnetic stirring for 5-10 minutes, with the sonication being alternated until the contents of the beakers were dissolved for about 50-60 minutes. And transferring the transparent solution containing the rare earth ions and the Polyethyleneimine (PEI) in the beaker A into a beaker B, stopping stirring after the transfer is completed, standing, transferring into a polytetrafluoroethylene reaction kettle, heating to 200 ℃, reacting for 12 hours, and cooling to room temperature.
(3) Washing: and (3) uniformly stirring the reacted solution by using a glass rod, washing the solution by using ethanol and the reaction solution according to the volume ratio of 3:1, centrifuging the solution to remove supernatant, washing the solution for three times by using high-purity water, centrifuging the solution for three times, and drying the obtained up-conversion nanoparticles in a vacuum drying oven for 12 hours.
2. The synthesis of up-conversion nano particles modified by manganese dioxide is characterized by comprising the following steps:
(1) 0.1 mol/L, preparation of 25 ℃ MES buffer:
weighing 2.012 g2- (N-morpholino) ethanesulfonic acid, dissolving in 100 mL water, adjusting pH of the solution with (0.5 mol/L) sodium hydroxide =6, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
（2）KMnO4preparing a solution:
taking 8 mM KMnO4Solutions, diluted to different concentrations respectively, are prepared into solutions of 4 mM, 3 mM, 2mM, 1 mM, 500 μ M, 400 μ M, 300 μ M, 200 μ M, 100 μ M;
(3) preparation of an upconversion solution:
0.0200 g of the dried up-conversion nano material is weighed and dissolved in 8 mL of high-purity water to be uniformly mixed by ultrasound.
(4) Taking 500. mu.L of the upconversion solution, 250. mu.L of MES solution and 250. mu.L of KMnO with different concentrations4Putting the solution into a 1.5 mL centrifuge tube, uniformly mixing, performing ultrasonic treatment for 15-30 min, centrifuging the obtained solution, removing supernatant, adding 1 mL high-purity water, performing centrifugal washing, repeating for three times, and adding water to dilute the obtained solution to 4 mL.
The invention has the advantages and positive effects that:
(1) the manganese dioxide modified up-conversion nano material disclosed by the invention has the advantages of good biocompatibility, low toxicity, large tissue penetration depth and small damage to organisms.
(2) The invention adopts a fluorescence analysis method, is simple and quick, has low cost, does not need expensive instruments, and can avoid the interference of autofluorescence when the material is excited by near infrared light, thereby further improving the detection sensitivity.
(3) Compared with other detection methods, the method has higher detection selectivity because the alkaline phosphatase specifically hydrolyzes the 2-phospho-L-ascorbic acid.
(4) The linear range of the alkaline phosphatase detection is wide and is 0.1-100U/L, and the detection limit is low and is 0.41U/L;
the comparison of this method with other methods is shown in table 1:
drawings
FIG. 1 upconversion nanomaterial (NaYF)4Yb, Tm);
FIG. 2 Transmission Electron Microscopy (TEM) of manganese dioxide modified upconverting nanomaterials;
FIG. 3 upconversion nanomaterials (NaYF)4Yb, Tm) indicating that Polyethyleneimine (PEI) is successfully coated on the surface of the upconversion material;
FIG. 4 is a fluorescence spectrum of polyethyleneimine coated upconversion nanomaterials with different relative molecular masses;
FIG. 5 is a graph of optimization of reaction time for alkaline phosphatase and 2-phospho-L-ascorbic acid;
FIG. 6 is a graph showing the optimization of detection time in the reaction system;
FIG. 7 is a linear range diagram of detection of alkaline phosphatase by manganese dioxide modified upconverting nanoparticles.
Detailed Description
The foregoing features and advantages will become more apparent and be readily understood from the following further description of the present invention, taken in conjunction with the accompanying specific embodiments. The chemical reagents used in the following examples were analytically pure, high purity water was purchased from Hangzhou Waha purified water, alkaline phosphatase (ALP) and 2-phospho-L-Ascorbic Acid (AAP) were purchased from Shanghai-derived Biotech Co., Ltd, 2- (N-morpholino) ethanesulfonic acid was provided by Beijing Solebao Tech Co., Ltd, ytterbium oxide, yttrium oxide, thulium oxide, ammonium fluoride, sodium hydroxide, ethanol and ethylene glycol were purchased from shinko refinish chemical research institute, and polyethyleneimine (Mw =10000, Mw =1800, Mw = 600) was purchased from Affa Angsa (chemical Co., Ltd.).
Example 1
Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticles:
(1) pre-treatment nitration: respectively weighing Y2O3, Yb2O3, Tm2O3Adding three rare earth oxides with a molar ratio of 75: 25: 0.3 into a beaker A, sequentially adding 2 mL of concentrated nitric acid and 2 mL of high-purity water, placing a small beaker into an electric heating sleeve for heating, continuing to add 5 mL of high-purity water after the oxides are completely nitrified and become nitrates, continuing to add 5 mL of high-purity water when the oxides are about to be evaporated to dryness, measuring the pH value of the solution, repeatedly adding the high-purity water until the pH value of the solution is about 7, and adding 5 mL of high-purity water for later use after the oxides are evaporated to dryness.
(2) Synthesizing: sodium hydroxide and ammonium fluoride were weighed into beaker B at 10 mmol each and 0.15g of Polyethyleneimine (PEI) of different molecular weight (Mw = 600) was weighed into beaker a. Subsequently, 4.5 mL of ethylene glycol, 3 mL of highly purified water, was added to beaker A, and 4.5 mL of highly purified water, 3 mL of ethylene glycol, was added to beaker B. The A, B beakers were sonicated for 5-10 minutes, followed by magnetic stirring for 5-10 minutes, with the sonication being alternated until the contents of the beakers were dissolved for about 50-60 minutes. And transferring the transparent solution containing the rare earth ions and the Polyethyleneimine (PEI) in the beaker A into a beaker B, stopping stirring after the transfer is completed, standing, transferring into a polytetrafluoroethylene reaction kettle, heating to 200 ℃, reacting for 12 hours, and cooling to room temperature.
(3) Washing: and (3) uniformly stirring the reacted solution by using a glass rod, washing the solution by using ethanol and the reaction solution according to the volume ratio of 3:1, centrifuging the solution to remove supernatant, washing the solution for three times by using high-purity water, centrifuging the solution for three times, and drying the obtained up-conversion nanoparticles in a vacuum drying oven for 12 hours.
Example 2
Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticles:
(1) pre-treatment nitration: respectively weighing Y2O3, Yb2O3, Tm2O3Adding three rare earth oxides with a molar ratio of 75: 25: 0.3 into a beaker A, sequentially adding 2 mL of concentrated nitric acid and 2 mL of high-purity water, putting the small beaker into an electric heating jacket for heating, continuously adding 5 mL of high-purity water after the oxides are completely nitrified into nitrates, continuously adding 5 mL of high-purity water when the solution is about to be evaporated to dryness, measuring the pH value of the solution, repeatedly adding the high-purity water until the pH value of the solution is about 7, and adding 5 mL of high-purity water for later use after the solution is evaporated to dryness.
(2) Synthesizing: sodium hydroxide and ammonium fluoride were weighed into beaker B at 10 mmol each and 0.15g of Polyethyleneimine (PEI) of different molecular weight (Mw = 1800) was weighed into beaker a. Then, 4.5 mL of ethylene glycol, 3 mL of highly purified water, was added to the A beaker, and 4.5 mL of highly purified water, 3 mL of ethylene glycol, was added to the B beaker. The A, B beakers were sonicated for 5-10 minutes, followed by magnetic stirring for 5-10 minutes, with the sonication being alternated until the contents of the beakers were dissolved for about 50-60 minutes. And transferring the transparent solution containing the rare earth ions and the Polyethyleneimine (PEI) in the beaker A into a beaker B, stopping stirring after the transfer is completed, standing, transferring into a polytetrafluoroethylene reaction kettle, heating to 200 ℃, reacting for 12 hours, and cooling to room temperature.
(3) Washing: and (3) uniformly stirring the reacted solution by using a glass rod, washing the solution by using ethanol and the reaction solution according to the volume ratio of 3:1, centrifuging the solution to remove supernatant, washing the solution for three times by using high-purity water, centrifuging the solution for three times, and drying the obtained up-conversion nanoparticles in a vacuum drying oven for 12 hours.
Example 3
Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticles:
(1) pre-treatment nitration: respectively weighing Y2O3, Yb2O3, Tm2O3Adding three rare earth oxides with a molar ratio of 75: 25: 0.3 into a beaker A, sequentially adding 2 mL of concentrated nitric acid and 2 mL of high-purity water, placing a small beaker into an electric heating sleeve for heating, continuing to add 5 mL of high-purity water after the oxides are completely nitrified and become nitrates, continuing to add 5 mL of high-purity water when the oxides are about to be evaporated to dryness, measuring the pH value of the solution, repeatedly adding the high-purity water until the pH value of the solution is about 7, and adding 5 mL of high-purity water for later use after the oxides are evaporated to dryness.
(2) Synthesizing: sodium hydroxide and ammonium fluoride were weighed into beaker B at 10 mmol each and 0.15g of Polyethyleneimine (PEI) of different molecular weight (Mw = 10000) was weighed into beaker a. Then, 4.5 mL of ethylene glycol, 3 mL of highly purified water, was added to the A beaker, and 4.5 mL of highly purified water, 3 mL of ethylene glycol, was added to the B beaker. The A, B beakers were sonicated for 5-10 minutes, followed by magnetic stirring for 5-10 minutes, with the sonication being alternated until the contents of the beakers were dissolved for about 50-60 minutes. And transferring the transparent solution containing the rare earth ions and the Polyethyleneimine (PEI) in the beaker A into a beaker B, stopping stirring after the transfer is completed, standing, transferring into a polytetrafluoroethylene reaction kettle, heating to 200 ℃, reacting for 12 hours, and cooling to room temperature.
(3) Washing: and (3) uniformly stirring the reacted solution by using a glass rod, washing the solution by using ethanol and the reaction solution according to the volume ratio of 3:1, centrifuging the solution to remove supernatant, washing the solution for three times by using high-purity water, centrifuging the solution for three times, and drying the obtained up-conversion nanoparticles in a vacuum drying oven for 12 hours.
Example 4
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis of up-conversion nano particles modified by manganese dioxide is characterized by comprising the following steps:
(1) 0.1 mol/L, preparation of 25 ℃ MES buffer:
weighing 2.012 g of 2- (N-morpholino) ethanesulfonic acid, dissolving in 100 mL of water, adjusting pH of the solution with (0.5 mol/L) sodium hydroxide =6, fixing volume with high-purity water in a 100 mL volumetric flask, and storing at low temperature;
（2）KMnO4preparing a solution:
taking 8 mM KMnO4Solutions, diluted to different concentrations respectively, are prepared into solutions of 2.5mM, 2mM, 1.5mM, 1 mM, 750 μm, 500 μm, 250 μm, 200 μm, 100 μm, 50 μm;
(3) preparation of an upconversion solution:
weighing 0.0200 g of the dried up-conversion nano material, dissolving in 8 mL of high-purity water, and ultrasonically mixing;
(4) 500. mu.L of the upconverting solution, 250. mu.L of MES solution, 250. mu.L of KMnO of different concentrations4Putting the solution into a 1.5 mL centrifuge tube, uniformly mixing, performing ultrasonic treatment for 20-30 min, centrifuging the obtained solution, removing supernatant, adding 1 mL high-purity water, performing centrifugal washing, repeating for three times, and adding water to dilute the obtained solution to 4 mL.
Example 5
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of the manganese dioxide modified upconversion nanoparticles is as shown in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 30 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer, wherein the details are shown in FIG. 5.
Example 6
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 60 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer, wherein the details are shown in FIG. 5.
Example 7
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 90 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer, wherein the details are shown in FIG. 5.
Example 8
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of the manganese dioxide modified upconversion nanoparticles is as shown in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 120 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer, wherein the details are shown in FIG. 5.
Example 9
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 70 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 10 min, and detecting the change value of fluorescence intensity by using a fluorescence spectrophotometer, which is detailed in figure 6.
Example 10
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high-purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 70 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 30min, and detecting the change value of fluorescence intensity by using a fluorescence spectrophotometer, which is detailed in figure 6.
Example 11
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 70 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 50 min, and detecting the change value of fluorescence intensity by using a fluorescence spectrophotometer, which is detailed in figure 6.
Example 12
1. Ytterbium and thulium doped sodium yttrium tetrafluoride (NaYF)4Yb, Tm) upconversion nanoparticle synthesis method reference example 3;
2. the synthesis method of manganese dioxide modified up-conversion nanoparticles is as described in example 4;
3. the detection of alkaline phosphatase by using the manganese dioxide modified up-conversion nano material is characterized by comprising the following steps of:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g (Mw = 157.6) Tris-HCl and dissolving in 100 mL high purity water; adjusting the pH of the solution to =7.4 with (0.5 mol/L) sodium hydroxide, adding high-purity water to a constant volume in a 100 mL volumetric flask, and storing at low temperature;
(2) preparation of 2-phosphoric acid-L-Ascorbic Acid (AAP) solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 70 min;
transferring 2 mL of manganese dioxide modified up-conversion solution, adding 600 muL of high-purity water, adding 400 muL of Tris-HCl (pH = 7.4) buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 60 min, and detecting the change value of fluorescence intensity by using a fluorescence spectrophotometer, which is detailed in figure 6.
Claims (2)
1. A method for detecting alkaline phosphatase by using manganese dioxide modified up-conversion nano-materials is characterized by comprising the following steps:
(1) 0.1 mol/L, preparation of Tris-HCl buffer solution at 25 ℃:
weighing 1.576 g, Mw =157.6Tris-HCl, and dissolving in 100 mL of high-purity water; adjusting the pH of the solution to be 7.4 by using 0.5 mol/L sodium hydroxide, fixing the volume of the solution in a 100 mL volumetric flask by using high-purity water, and storing the solution at low temperature;
(2) preparation of 2-phosphoric acid-L-ascorbic acid AAP solution:
weighing 0.2577 g AAP, dissolving in 8 mL high-purity water, and diluting to 4 mM solution;
(3) preparation of alkaline phosphatase solution:
respectively diluting alkaline phosphatase with alkaline phosphatase buffer solution to different concentrations to prepare solutions of 250U/L, 100U/L, 50U/L, 25U/L, 10U/L, 5U/L, 1U/L, 0.5U/L and 0.1U/L;
(4) detection of alkaline phosphatase by manganese dioxide modified up-conversion nanoparticles:
taking 500 muL 2-phosphoric acid-L-Ascorbic Acid (AAP) solution and 500 muL alkaline phosphatase solution with different concentrations by using a liquid transfer gun, uniformly mixing, and reacting at 37 ℃ for 15-120 min;
adding 600 mu L of high-purity water into 2 mL of manganese dioxide modified up-conversion solution, adding 400 mu L of LTris-HCl and pH =7.4 buffer solution, adding 1 mL of 2-phosphoric acid-L-Ascorbic Acid (AAP) and alkaline phosphatase reaction products with different concentrations, uniformly mixing, reacting for 10-60 min, and detecting a fluorescence intensity change value by using a fluorescence spectrophotometer;
1. ytterbium and thulium doped sodium yttrium tetrafluoride NaYF4The Yb and Tm upconversion nano particle is synthesized by a hydrothermal method, polyethyleneimine is used as a coating agent, the molar ratio of Y to Yb to Tm is 75: 25: 0.3, and the method comprises the following steps:
(1) pre-treatment nitration: respectively weighing Y2O3, Yb2O3, Tm2O3Adding three rare earth oxides with a molar ratio of 75: 25: 0.3 into a beaker A, sequentially adding 2 mL of concentrated nitric acid and 2 mL of high-purity water, placing a small beaker into an electric heating sleeve for heating, continuing to add 5 mL of high-purity water after the oxides are completely nitrified and become nitrates, continuing to add 5 mL of high-purity water when the oxides are about to be evaporated to dryness, measuring the pH value of the solution, repeatedly adding the high-purity water until the pH value of the solution is about 7, and adding 5 mL of high-purity water for later use after the oxides are evaporated to dryness;
(2) synthesizing: weighing 10 mmol each of sodium hydroxide and ammonium fluoride into beaker B, continuing to weigh 0.15g of polyethyleneimine of different molecular weights, Mw =10000, Mw =1800, Mw =600 into beaker a, followed by adding 4.5 mL of ethylene glycol 3 mL of high purity water into beaker a and 4.5 mL of high purity water 3 mL of ethylene glycol into beaker B;
respectively carrying out ultrasonic treatment on A, B two beakers for 5-10 minutes, then carrying out magnetic stirring for 5-10 minutes, alternately carrying out ultrasonic stirring until substances in the beakers are dissolved for about 50-60 minutes, transferring a transparent solution containing rare earth ions and Polyethyleneimine (PEI) in the beaker A into the beaker B, stopping stirring after the transfer is completed, standing, transferring the transparent solution into a polytetrafluoroethylene reaction kettle, heating to 200 ℃, reacting for 12 hours, and cooling to room temperature;
(3) washing: uniformly stirring the reacted solution by using a glass rod, washing the solution by using ethanol and the reaction solution according to the volume ratio of 3:1, centrifuging the solution to remove supernatant, washing the solution for three times by using high-purity water, centrifuging the solution for three times, and drying the obtained up-conversion nanoparticles in a vacuum drying oven for 12 hours;
2. synthesis of manganese dioxide modified up-conversion nanoparticles:
(1) 0.1 mol/L, preparation of 25 ℃ MES buffer:
weighing 2.012 g of 2- (N-morpholino) ethanesulfonic acid, dissolving in 100 mL of water, adjusting the pH of the solution to =6 with 0.5 mol/L sodium hydroxide, fixing the volume with high-purity water in a 100 mL volumetric flask, and storing at low temperature;
（2）KMnO4preparing a solution:
taking 8 mM KMnO4The solutions were diluted to different concentrations to prepare 4 mM, 3 mM, 2mM, and 1 m solutionsM, 500 μ M, 400 μ M, 300 μ M, 200 μ M, 100 μ M solution;
(3) preparation of an upconversion solution:
weighing 0.0200 g of the dried up-conversion nano material, dissolving in 8 mL of high-purity water, and ultrasonically mixing;
(4) 500. mu.L of the upconverting solution, 250. mu.L of MES solution, 250. mu.L of KMnO of different concentrations4Putting the solution into a 1.5 mL centrifuge tube, uniformly mixing, performing ultrasonic treatment for 15-30 min, centrifuging the obtained solution, removing supernatant, adding 1 mL high-purity water, performing centrifugal washing, repeating for three times, and adding water to dilute the obtained solution to 4 mL.
2. The method for detecting alkaline phosphatase using manganese dioxide modified upconversion nanomaterial as claimed in claim 1, wherein the alkaline phosphatase sample is dissolved in an alkaline phosphatase buffer solution according to the following formula: 0.1M Tris-HCl, 0.1M sodium chloride, 2mM magnesium chloride, autoclaved.
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