CN112414977A - Alkaline phosphatase detection reagent, preparation method thereof and alkaline phosphatase detection method - Google Patents
Alkaline phosphatase detection reagent, preparation method thereof and alkaline phosphatase detection method Download PDFInfo
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- CN112414977A CN112414977A CN201910773827.9A CN201910773827A CN112414977A CN 112414977 A CN112414977 A CN 112414977A CN 201910773827 A CN201910773827 A CN 201910773827A CN 112414977 A CN112414977 A CN 112414977A
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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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
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention belongs to the technical field of inorganic semiconductor materials, and particularly relates to an alkaline phosphatase detection reagent, a preparation method thereof and an alkaline phosphatase detection method. The alkaline phosphatase detection reagent provided by the invention comprises: the composite material comprises a transition metal disulfide quantum dot/iron ion composite, an ascorbate substance and a solvent, wherein the ascorbate substance is dissolved in the solvent, the transition metal disulfide quantum dot/iron ion composite is dispersed in the solvent, and iron ions in the transition metal disulfide quantum dot/iron ion composite are adsorbed on the surface of the transition metal disulfide quantum dot. The alkaline phosphatase detection reagent provided by the invention can realize rapid detection of alkaline phosphatase, is simple and convenient to operate and has high sensitivity.
Description
Technical Field
The invention belongs to the technical field of inorganic semiconductor materials, and particularly relates to an alkaline phosphatase detection reagent, a preparation method thereof and an alkaline phosphatase detection method.
Background
Alkaline phosphatase is an enzyme which is very important in physiological processes, catalyzes hydrolysis and dephosphorylation of various substrates including proteins, nucleic acids, carbohydrates, etc., and plays an important role in cellular metabolism and nerve signal transduction, etc. It has been found that the activity of alkaline phosphatase is closely related to various diseases, such as the activity of alkaline phosphatase in serum of patients with breast cancer, prostate cancer and liver dysfunction is higher than normal level, while the activity of alkaline phosphatase in serum of patients with hypophosphatemia is relatively low. Therefore, establishing a rapid and sensitive detection method for alkaline phosphatase activity is of great significance for clinical diagnosis and biomedical research.
At present, the detection method of alkaline phosphatase mainly comprises the following steps: the method comprises the following steps of (1) chromatography, electrochemiluminescence, electrochemistry, colorimetry, fluorescence and the like, wherein the chromatography has better sensitivity and quantitative accuracy and is the most common method for detecting the content of alkaline phosphatase. However, the above methods basically require expensive equipment, time-consuming and complicated pretreatment procedures, and some organic solvents used in the detection process are not environmentally friendly.
Disclosure of Invention
The invention mainly aims to provide an alkaline phosphatase detection reagent and a preparation method thereof, and another aim of the invention is to provide a detection method of alkaline phosphatase, aiming at solving the technical problems that the existing detection method of alkaline phosphatase needs expensive instruments and equipment, the pretreatment procedure of a sample is complex and time-consuming, and the like.
In order to achieve the purpose of the invention, the invention provides the following specific technical scheme:
an alkaline phosphatase detection reagent comprising: the composite material comprises a transition metal disulfide quantum dot/iron ion composite, an ascorbate substance and a solvent, wherein the ascorbate substance is dissolved in the solvent, the transition metal disulfide quantum dot/iron ion composite is dispersed in the solvent, and iron ions in the transition metal disulfide quantum dot/iron ion composite are adsorbed on the surface of the transition metal disulfide quantum dot.
The alkaline phosphatase detection reagent provided by the invention comprises: transition metal disulfide quantum dot/iron ion complex, ascorbate class material and solvent, and the iron ion in transition metal disulfide quantum dot/iron ion complex adsorbs the surface of transition metal disulfide quantum dot. Under the excitation of visible light, iron ions are adsorbed on the surface of the transition metal disulfide quantum dots, valence band electrons excited in a conduction band in the transition metal disulfide quantum dots are transferred to a d-orbit of the iron ions, so that the fluorescence of the transition metal disulfide quantum dots is quenched, and when alkaline phosphatase exists in a detection system, an ascorbate substance is decomposed under the catalytic action of the alkaline phosphatase to form ascorbic acid, and the ascorbic acid can reduce the iron ions into ferrous ions and destroy the compounding of the transition metal disulfide quantum dots and the iron ions, so that the fluorescence of the transition metal disulfide quantum dots is recovered. Therefore, in the alkaline phosphatase detection reagent provided by the invention, the transition metal disulfide quantum dots are used as fluorescent labels, the iron ions adsorbed on the surfaces of the transition metal disulfide quantum dots are used as detection switches, the ascorbate substances are used as synthesis precursors of ascorbic acid, when no alkaline phosphatase exists in a detection system, the transition metal disulfide quantum dots and the iron ions are kept in a composite state, and the fluorescence emitted by the transition metal disulfide quantum dots in a visible light range is quenched; when alkaline phosphatase exists in the detection system, the iron ions are reduced into ferrous ions, the compounding of the transition metal disulfide quantum dots and the iron ions is damaged, the fluorescence emitted by the transition metal disulfide quantum dots in the visible light range is recovered, the rapid detection of the alkaline phosphatase can be realized according to the fluorescence emission signals, the operation is simple and convenient, and the sensitivity is high.
Accordingly, a method for preparing an alkaline phosphatase detection reagent, comprising:
providing transition metal disulfide quantum dots, iron salt and an ascorbate substance, mixing the transition metal disulfide quantum dots, the iron salt and the ascorbate substance in a solvent, so that iron ions of the iron salt are adsorbed on the surfaces of the transition metal disulfide quantum dots, and the ascorbate substance is dissolved in the solvent to obtain the alkaline phosphatase detection reagent.
According to the preparation method of the alkaline phosphatase detection reagent, transition metal disulfide quantum dots, iron salt and ascorbate are mixed in the solvent, so that iron ions of the iron salt are adsorbed on the surfaces of the transition metal disulfide quantum dots, and the ascorbate is dissolved in the solvent, so that the preparation method is simple and convenient to operate and easy for mass production.
Accordingly, a method for detecting alkaline phosphatase, comprising the steps of:
providing a sample to be detected and the alkaline phosphatase detection reagent or the alkaline phosphatase detection reagent prepared by the preparation method;
mixing and incubating the sample to be detected and the alkaline phosphatase detection reagent to obtain a mixed solution;
and irradiating the mixed solution by adopting visible light, and determining whether alkaline phosphatase exists in the sample to be detected according to the fluorescence emission signal of the mixed solution.
The method for detecting the alkaline phosphatase comprises the steps of mixing and incubating a sample to be detected and an alkaline phosphatase detection reagent, irradiating the mixed solution by adopting visible light, and determining whether the alkaline phosphatase exists in the sample to be detected according to a fluorescence emission signal of the mixed solution; when alkaline phosphatase exists in the sample to be detected, the mixed solution generates a fluorescence emission signal. The method is simple, short in detection time, high in sensitivity and wide in application range.
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FIG. 1 is a flow chart of a method for detecting alkaline phosphatase according to a preferred embodiment of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In order to solve the technical problems that the existing alkaline phosphatase detection method needs expensive instruments and equipment, the sample pretreatment process is complex and time-consuming, and the like, the embodiment of the invention provides the following specific technical scheme:
an alkaline phosphatase detection reagent comprising: the composite material comprises a transition metal disulfide quantum dot/iron ion composite, an ascorbate substance and a solvent, wherein the ascorbate substance is dissolved in the solvent, the transition metal disulfide quantum dot/iron ion composite is dispersed in the solvent, and iron ions in the transition metal disulfide quantum dot/iron ion composite are adsorbed on the surface of the transition metal disulfide quantum dot.
The alkaline phosphatase detection reagent provided by the embodiment of the invention comprises: transition metal disulfide quantum dot/iron ion complex, ascorbate class material and solvent, and the iron ion in transition metal disulfide quantum dot/iron ion complex adsorbs the surface of transition metal disulfide quantum dot. Under the excitation of visible light, iron ions are adsorbed on the surface of the transition metal disulfide quantum dots, valence band electrons excited in a conduction band in the transition metal disulfide quantum dots are transferred to a d-orbit of the iron ions, so that the fluorescence of the transition metal disulfide quantum dots is quenched, and when alkaline phosphatase exists in a detection system, an ascorbate substance is decomposed under the catalytic action of the alkaline phosphatase to form ascorbic acid, and the ascorbic acid can reduce the iron ions into ferrous ions and destroy the compounding of the transition metal disulfide quantum dots and the iron ions, so that the fluorescence of the transition metal disulfide quantum dots is recovered. Therefore, in the alkaline phosphatase detection reagent provided by the invention, the transition metal disulfide quantum dots are used as fluorescent labels, the iron ions adsorbed on the surfaces of the transition metal disulfide quantum dots are used as detection switches, the ascorbate substances are used as synthesis precursors of ascorbic acid, when no alkaline phosphatase exists in a detection system, the transition metal disulfide quantum dots and the iron ions are kept in a composite state, and the fluorescence emitted by the transition metal disulfide quantum dots in a visible light range is quenched; when alkaline phosphatase exists in the detection system, the iron ions are reduced into ferrous ions, the compounding of the transition metal disulfide quantum dots and the iron ions is damaged, the fluorescence emitted by the transition metal disulfide quantum dots in the visible light range is recovered, the rapid detection of the alkaline phosphatase can be realized according to the fluorescence emission signals, the operation is simple and convenient, and the sensitivity is high.
Specifically, the transition metal disulfide quantum dot is a semiconductor material with photoelectric properties, and can emit fluorescence under the excitation of visible light. In an embodiment of the present invention, the transition metal disulfide quantum dot/iron ion complex is dispersed in the solvent, and iron ions in the transition metal disulfide quantum dot/iron ion complex are adsorbed on the surface of the transition metal disulfide quantum dot. The valence state of the iron ions is +3, and the iron ions are adsorbed to the surface of the transition metal disulfide quantum dots through electrostatic interaction to form a transition metal disulfide quantum dot/iron ion complex. Under the excitation of visible light, valence band electrons in the transition metal disulfide quantum dots are excited into a conduction band and then transferred to a d-orbital of iron ions, resulting in quenching of fluorescence of the transition metal disulfide quantum dots. In the embodiment of the invention, the transition metal disulfide quantum dot is used as a fluorescent label, the iron ions adsorbed on the surface of the transition metal disulfide quantum dot are used as a detection switch, and whether alkaline phosphatase exists in a sample to be detected can be indicated by detecting a fluorescent signal emitted in a visible light range. When the complex state of the transition metal disulfide quantum dots and iron ions is destroyed, the fluorescence of the transition metal disulfide quantum dots is restored. For example, when the iron ions are reduced to ferrous ions, the ferrous ions are dissociated from the transition metal disulfide quantum dots, and fluorescence emitted from the transition metal disulfide quantum dots in the visible light range is recovered.
In the embodiment of the invention, the basic chemical formula of the transition metal disulfide quantum dot is preferably MS2Wherein M is selected from Mo, W, V, Nb, Ta, Ti, Zr, Hf or Re, and the transition metal disulfide quantum dot has a graphene-like two-dimensional structure and excellent photoelectric properties. Preferably, the transition metal disulfide quantum dots are two-dimensional transition metal disulfides and have a single-layer structure, the two-dimensional transition metal disulfides are direct band gap materials, high-intensity fluorescence can be obtained by carrying out light irradiation on a small amount of materials, the light emitting effect is good, and the detection sensitivity of alkaline phosphatase can be effectively improved. Further, the transition metal disulfide quantum dot is a transition metal disulfide quantum dot with a water-soluble ligand modified on the surface, and hasThe composite material has good water solubility, is easy to disperse in a water environment, promotes the transition metal disulfide quantum dots to be fully mixed with ferric salt and ascorbyl ester substances, and is beneficial to adsorbing iron ions on the surfaces of the transition metal disulfide quantum dots to form transition metal disulfide quantum dot/iron ion composites so as to further improve the detection sensitivity of alkaline phosphatase. In some embodiments, the water-soluble ligand comprises at least one of a carboxylate ion, a nitrate ion, a sulfate ion, an ammonium ion, a nitrite ion, a sulfonate ion, and a sulfinate ion. Furthermore, the particle size of the transition metal disulfide quantum dots is 2-5nm, the transition metal disulfide quantum dots in the particle size range emit light with single wavelength, and the light color is pure; when the particle size is larger than 5nm, the light color is not pure, which may cause a decrease in detection sensitivity.
As a preferred embodiment, the preparation of the transition metal disulfide quantum dots comprises the steps of:
a. providing a transition metal salt, a sulfur source and a nonpolar solvent, wherein the mass ratio of the transition metal salt to the sulfur source is (30-80): 1;
b. mixing the transition metal salt, the sulfur source and the nonpolar solvent, and carrying out heating reaction.
More specifically, in step a, the transition metal salt refers to an organic salt or an inorganic salt for providing a transition metal atom, including but not limited to ammonium metavanadate, molybdenum nitrate, tungsten nitrate, niobium nitrate, hafnium sulfate, and the like. The sulfur source refers to an organic or inorganic substance for providing a sulfur atom, including, but not limited to, elemental sulfur, octanethiol, thiophenol, diethyldisulfide, N-acetyl-L-cysteine, borane dimethylsulfide, and the like. The nonpolar solvent is used as a medium for the reaction of the transition metal salt and the sulfur source, and is preferably a water-insoluble organic solvent which is miscible with water and can cause delamination or turbidity, including but not limited to benzene, carbon tetrachloride, dichloroethane, chloroform, tetrachloromethane, and the like. In the embodiment of the invention, the mass ratio of the transition metal salt to the sulfur source is preferably (30-80):1, so as to ensure that the prepared product is a two-dimensional transition metal disulfide with uniform morphology. In some preferred embodiments, the transition metal salt and the sulfur source are present in a mass ratio of 30:1, 34:1, 36:1, 39:1, 43:1, 48:1, 50:1, 53:1, 56:1, 60:1, 63:1, 65:1, 68:1, 71:1, 75:1, 76:1, 80: 1.
In step b, the transition metal salt, the sulfur source and the nonpolar solvent are mixed, and the transition metal disulfide quantum dots are synthesized through a solvothermal reaction. The step of mixing the transition metal salt, the sulfur source and the nonpolar solvent may refer to the conventional operations in the art, and the transition metal salt and the sulfur source may be respectively dissolved in the nonpolar solvent and then mixed, or may be simultaneously added to the nonpolar solvent and uniformly mixed. Preferably, in the step of mixing the transition metal salt, the sulfur source and the nonpolar solvent, 1 to 10g of the transition metal salt is mixed per 100 to 1000mL of the nonpolar solvent to ensure complete dissolution of the transition metal salt and promote the reaction.
Further, in the step of heating reaction, the reaction temperature is 200-500 ℃, and the reaction time is 1-24 hours, so that the particle size of the prepared transition metal disulfide quantum dots is ensured to be 2-5nm, and the detection sensitivity of alkaline phosphatase is ensured. In some embodiments, the step of performing a heating reaction is performed in a high temperature reaction vessel; in other embodiments, the pressure of the autoclave is set to 1-10MPa, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10MPa, to accelerate the reaction rate and control the particle size of the transition metal disulfide produced.
Further, after the step of performing the heating reaction, the method further comprises: and mixing a water-soluble ligand with the reaction liquid containing the transition metal disulfide quantum dots, and carrying out ultrasonic reaction to modify the water-soluble ligand on the surfaces of the transition metal disulfide quantum dots. In some embodiments, the reaction solution is cooled to room temperature, a water-soluble ligand is added, and then ultrasound is performed at 1-10 ℃ to modify the water-soluble ligand on the surface of the transition metal disulfide quantum dots. In other embodiments, the mass ratio of the transition metal disulfide quantum dots to the water-soluble ligand in the reaction solution is (50-100):1, preferably 50:1, 53:1, 56:1, 60:1, 63:1, 65:1, 68:1, 71:1, 75:1, 76:1, 80:1, 81:1, 85:1, 87:1, 90:1, 93:1, 95:1, 98:1, 100: 1. In still other embodiments, the water-soluble ligand is selected from organic compounds containing at least one of carboxylate, nitrate, sulfate, ammonium, nitrite, sulfonate, and sulfinate ions, such as methacrylic acid, ethacrylic acid, dilute sulfuric acid, dilute nitric acid, and the like.
In an embodiment of the invention, the iron ions in the transition metal disulfide quantum dot/iron ion complex are derived from an iron salt dissolved in the solvent, the iron salt being used to provide the iron ions. Preferably, the mass ratio of the iron salt to the transition metal disulfide quantum dots is (1-100) to (1-50). In some embodiments, the mass ratio of the iron salt to the transition metal disulfide quantum dot is 1:50, 1:40, 1:30, 1:20, 1:10, 1:7, 1:5, 1:3, 1:1, 2:1, 4:1, 5:1, 7:1, 10:1, 15:1, 20:1, 24:1, 29:1, 33:1, 37:1, 41:1, 49:1, 52:1, 60:1, 66:1, 70:1, 76:1, 82:1, 90:1, 95:1 or 100:1, so as to ensure that when the iron ion is complexed with the transition metal disulfide quantum dot, the fluorescence emitted by the transition metal disulfide quantum dot in the visible light range can be completely quenched, ensure accuracy and improve detection sensitivity for alkaline phosphatase. Further, the iron salt includes at least one of ferric nitrate, ferric sulfate, and ferric chloride.
Specifically, the ascorbyl ester substances refer to organic matters containing ascorbic acid groups and can be catalyzed and decomposed by alkaline phosphatase to generate ascorbic acid. In the embodiment of the invention, the ascorbyl ester substance is used as a synthetic precursor of ascorbic acid, and is decomposed to form ascorbic acid under the catalytic action of alkaline phosphatase, the ascorbic acid can reduce iron ions into ferrous ions, and the transition metal disulfide quantum dots are damaged to be compounded with the iron ions, so that the fluorescence emitted by the transition metal disulfide quantum dots in a visible light range is recovered. Preferably, the ascorbyl ester comprises at least one of ascorbyl phosphate and/or a salt thereof, ascorbyl palmitate and/or a salt thereof, ascorbyl laurate and/or a salt thereof, ascorbyl stearate and/or a salt thereof, ascorbyl oleate and/or a salt thereof, ascorbyl linoleate and/or a salt thereof, and ascorbyl benzoate and/or a salt thereof. In a preferred embodiment, the ascorbate is selected from ascorbic acid-2-phosphate or trisodium ascorbate-2-phosphate, and in one embodiment, the ascorbate is trisodium ascorbate-2-phosphate, and the trisodium L-ascorbate-2-phosphate is decomposed to form L-ascorbic acid under the catalytic action of alkaline phosphatase.
In the embodiment of the invention, the mass ratio of the transition metal disulfide quantum dots to the ascorbyl ester substances is 1 (3-10), and more preferably 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9 or 1:10, so that the detection sensitivity to alkaline phosphatase is effectively improved.
As a preferred embodiment, the iron ion in the transition metal disulfide quantum dot/iron ion complex is derived from an iron salt dissolved in the solvent, the mass ratio of the iron salt to the transition metal disulfide quantum dot is (1-100): (1-50); and the mass ratio of the transition metal disulfide quantum dots to the ascorbyl ester substances is 1 (3-10).
In the embodiment of the present invention, the solvent is preferably at least one of water, buffer solution and alcohol, such as physiological saline, buffer solution, alcohol aqueous solution, etc., for dissolving the ascorbate-based substance and dispersing the transition metal disulfide quantum dot/iron ion complex. Further, the alkaline phosphatase detection reagent may be in the form of a kit, in some embodiments, a transition metal disulfide quantum dot/iron ion complex is stored in the solvent and loaded in a container, such as a bottle, tube, or cup, labeled reagent a; the ascorbyl ester substance is stored in the solvent and loaded in a container such as a bottle, a tube or a cup and marked as a reagent B, and when in use, the reagent A and the reagent B are fully mixed; in other embodiments, the transition metal disulfide quantum dot/iron ion complex, the ascorbate species, and the solvent are simultaneously stored in the solvent and loaded into a container such as a bottle, tube, or cup.
The following is a method for preparing the alkaline phosphatase detecting reagent according to the embodiment of the present invention.
Accordingly, a method for preparing an alkaline phosphatase detection reagent, comprising:
s01, providing transition metal disulfide quantum dots, iron salt and ascorbyl ester substances;
s02, mixing the transition metal disulfide quantum dots, the iron salt and the ascorbate in a solvent to enable iron ions of the iron salt to be adsorbed on the surfaces of the transition metal disulfide quantum dots, and dissolving the ascorbate in the solvent to obtain the alkaline phosphatase detection reagent.
According to the preparation method of the alkaline phosphatase detection reagent provided by the embodiment of the invention, the transition metal disulfide quantum dots, the iron salt and the ascorbyl ester substance are mixed in the solvent, so that iron ions of the iron salt are adsorbed on the surfaces of the transition metal disulfide quantum dots, and the ascorbyl ester substance is dissolved in the solvent, so that the preparation method is simple and convenient to operate and easy for mass production.
Specifically, in step S01, the transition metal disulfide quantum dots, the iron salt, and the ascorbate are substantially the same as those of the above-mentioned alkaline phosphatase detection reagent, and are not repeated herein for brevity.
In step S02, the transition metal disulfide quantum dots, the iron salt, and the ascorbate are mixed in a solvent, such that iron ions of the iron salt are adsorbed onto the surfaces of the transition metal disulfide quantum dots during the mixing process, forming transition metal disulfide quantum dot/iron ion complexes, and at the same time, the ascorbate is dissolved in the solvent. In the embodiment of the present invention, the step of mixing the transition metal disulfide quantum dots, the iron salt, and the ascorbate in the second solvent may refer to a conventional operation in the art, such as mixing at room temperature. Further, the solvent is substantially the same as that of the above-mentioned alkaline phosphatase detection reagent, and in the embodiment of the present invention, the solvent is mainly used for uniformly dispersing the transition metal disulfide quantum dots, the iron salt and the ascorbate, dissolving the ascorbate, and promoting iron ions to be complexed with the transition metal disulfide quantum dots during the mixing process, and preferably, the polarity of the second solvent is greater than 6, such as an aqueous medium, including but not limited to physiological saline, buffer, alcohol aqueous solution, and the like.
In the embodiment of the invention, the adding sequence of the transition metal disulfide quantum dots, the iron salt and the ascorbate can be adjusted and changed according to different experimental environments. In some embodiments, the transition metal disulfide quantum dots are uniformly dispersed in a solvent, the iron salt is slowly added until the fluorescence emission signal of the mixed solution in the visible range is completely quenched, and then the ascorbate species is added. In other embodiments, the transition metal disulfide quantum dots and the ascorbate are mixed uniformly in a solvent, and then the iron salt is added slowly until the fluorescence emission signal of the mixed solution in the visible light range is completely quenched. Therefore, the excessive iron ions in the detection system can be avoided, and the detection sensitivity is improved.
Preferably, the mass ratio of the iron salt to the transition metal disulfide quantum dots is (1-100): (1-50), and the mass ratio of the transition metal disulfide quantum dots to the ascorbate is preferably 1 (3-10), so that on one hand, a fluorescence emission signal of the transition metal disulfide quantum dots in a visible light range is completely quenched, and on the other hand, ascorbic acid generated by catalysis of alkaline phosphatase is ensured to be capable of destroying a transition metal disulfide quantum dot/iron ion complex in a first detection system, so that fluorescence emitted by the transition metal disulfide quantum dots in the visible light range is recovered, and the detection reagent is ensured to have higher sensitivity. Furthermore, the mass ratio of the ascorbyl esters to the iron salt is (1-10): (1-5), preferably 1:5, 1:4, 1:3, 1:2, 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1, so as to ensure that the ascorbic acid generated by the decomposition of the ascorbyl esters can effectively destroy the combination of iron ions and transition metal disulfide quantum dots, ensure the accuracy and sensitivity of the alkaline phosphatase detection reagent of the embodiment of the invention even if the generated ascorbic acid is in trace amount, and avoid false negative results.
Accordingly, referring to FIG. 1, a method for detecting alkaline phosphatase comprises the following steps:
s03, providing a sample to be detected and the alkaline phosphatase detection reagent or the alkaline phosphatase detection reagent prepared by the preparation method;
s04, mixing and incubating the sample to be detected and the alkaline phosphatase detection reagent to obtain a mixed solution;
s05, irradiating the mixed solution by adopting visible light, and determining whether alkaline phosphatase exists in the sample to be detected according to the fluorescence emission signal of the mixed solution.
According to the detection method of the alkaline phosphatase provided by the embodiment of the invention, a sample to be detected and an alkaline phosphatase detection reagent are mixed and incubated, then the mixed solution is irradiated by visible light, and whether the alkaline phosphatase exists in the sample to be detected is determined according to a fluorescence emission signal of the mixed solution; when alkaline phosphatase exists in the sample to be detected, the mixed solution generates a fluorescence emission signal. The method is simple, short in detection time, high in sensitivity and wide in application range.
Specifically, in step S04, the sample to be tested is mixed and incubated with the alkaline phosphatase detection reagent, wherein the alkaline phosphatase detection reagent comprises: when the sample to be detected contains alkaline phosphatase, the ascorbate substances can be catalytically decomposed to form ascorbic acid, and then the ascorbic acid reduces the iron ions compounded with the transition metal disulfide quantum dots to form ferrous ions, so that the compounding between the transition metal disulfide quantum dots and the iron ions is destroyed, and the fluorescence emitted by the transition metal disulfide quantum dots in a visible light range is recovered. The sample to be tested includes, but is not limited to, blood sample, food, medicine, etc. The step of adding the sample to be tested to the alkaline phosphatase detection reagent may be performed by a conventional method in the art. Preferably, in the step of mixing and incubating the sample to be tested and the alkaline phosphatase detection reagent, the incubation temperature is 10-30 ℃ and the incubation time is 1-24 hours. In some embodiments, the incubation temperature is 10, 13, 15, 17, 20, 22, 25, 28, 30 ℃. In other embodiments, the incubation time is 1, 5, 7, 10, 13, 15, 17, 19, 21, 24 hours.
Specifically, in step S05, the mixed solution is irradiated with visible light, and whether alkaline phosphatase is present in the sample to be tested is determined according to the fluorescence emission signal of the mixed solution. If the fluorescence emission signal exists in the mixed solution, the alkaline phosphatase exists in the sample to be detected; and if the fluorescence emission signal is not detected in the mixed solution, the alkaline phosphatase does not exist in the sample to be detected. In some preferred embodiments, the fluorescence emission signal of the mixed solution is detected by scanning with a spectrophotometer, so that the operation is simple, convenient, rapid and sensitive.
In order to make the above-mentioned details and operations of the present invention clearly understood by those skilled in the art, and to make the progress of the alkaline phosphatase detection reagent and the alkaline phosphatase detection method obvious, the practice of the present invention will be illustrated by examples.
Example 1
The embodiment provides a method for detecting alkaline phosphatase, which comprises the following specific process flows:
1. providing an alkaline phosphatase detection reagent
1) Preparation of transition metal disulfide Quantum dots
Weighing 3.5g of ammonium metavanadate and 0.1g N-acetyl-L-cysteine, and respectively dissolving the ammonium metavanadate and the N-acetyl-L-cysteine in 150mL of dichloroethane to obtain a transition metal salt solution and a sulfur source;
ultrasonically mixing a transition metal salt solution and a sulfur source, adding the mixture into a high-pressure reaction kettle, heating the mixture for 2 hours at 250 ℃, cooling the mixture to room temperature, and purifying the supernatant by silica gel column chromatography to obtain two-dimensional VS2Quantum dots;
weighing 3g two-dimensional VS2Quantum dots and 0.05g of methacrylic acid are uniformly scattered in water, and then ultrasonic treatment is carried out for 3 hours at the temperature of 2 ℃ to obtain two-dimensional VS with carboxylate radical modified on surface2And (4) quantum dots.
2) Selecting iron salt as ferric nitrate;
3) the ascorbyl ester is L-ascorbic acid-2-trisodium phosphate.
2. Detection of alkaline phosphatase in a test sample
1) Weighing 3g of two-dimensional VS with carboxylate radical modified on surface2Quantum dots and 0.8g of L-ascorbic acid-2-trisodium phosphate were mixed in physiological saline;
2) weighing 20.2g of ferric nitrate, and dissolving the ferric nitrate in physiological saline to prepare ferric salt solution; then, slowly adding an iron salt solution into the mixed solution obtained in the step 1) until the fluorescence emitted by the mixture in the visible light range is completely quenched, and constructing a detection system;
3) adding a sample to be detected into the detection system constructed in the step 2), incubating for 3 hours at room temperature, and scanning and detecting whether a fluorescence emission signal in a visible light range exists in the mixed solution by adopting a spectrophotometer.
Example 2
The embodiment provides a method for detecting alkaline phosphatase, which comprises the following specific process flows:
1. providing an alkaline phosphatase detection reagent
1) Preparation of transition metal disulfide Quantum dots
Weighing 7.8g of niobium nitrate and 0.15g of borane dimethylsulfide, and respectively dissolving the niobium nitrate and the borane dimethylsulfide in 900mL of benzene to obtain a transition metal salt solution and a sulfur source;
ultrasonically mixing a transition metal salt solution and a sulfur source, adding the mixture into a high-pressure reaction kettle, heating the mixture for 20 hours at 450 ℃, cooling the mixture to room temperature, and purifying the supernatant by silica gel column chromatography to obtain two-dimensional NbS2Quantum dots;
weighing 7g of two-dimensional NbS2Quantum dots and 0.065g of water-soluble ligand containing nitrate ions, uniformly dispersed in benzene, and thenPerforming ultrasonic treatment at 8 ℃ for 10 hours to obtain two-dimensional NbS with the surface modified with nitrate radical2And (4) quantum dots.
2) Selecting ferric salt as ferric sulfate;
3) the ascorbyl ester is L-ascorbic acid-2-trisodium phosphate.
2. Detection of alkaline phosphatase in a test sample
1) 6.5g of two-dimensional NbS with a surface modified by nitrate radical is weighed2Quantum dots and 0.6g of L-ascorbic acid-2-trisodium phosphate are uniformly dispersed in benzene;
2) weighing 2.5g of ferric sulfate, and uniformly dispersing in benzene to prepare a ferric salt solution; then, slowly adding an iron salt solution into the mixed solution obtained in the step 1) until the fluorescence emitted by the mixture in the visible light range is completely quenched, and constructing a detection system;
3) adding a sample to be detected into the detection system constructed in the step 2), incubating for 10 hours at room temperature, and scanning and detecting whether a fluorescence emission signal in a visible light range exists in the mixed solution by adopting a spectrophotometer.
Example 3
The embodiment provides a method for detecting alkaline phosphatase, which comprises the following specific process flows:
1. providing an alkaline phosphatase detection reagent
1) Preparation of transition metal disulfide Quantum dots
Weighing 5.5g of hafnium sulfate and 0.25g of octanethiol, and respectively dissolving the hafnium sulfate and the octanethiol in 500mL of benzene to obtain a transition metal salt solution and a sulfur source;
ultrasonically mixing a transition metal salt solution and a sulfur source, adding the mixture into a high-pressure reaction kettle, heating the mixture for 10 hours at 350 ℃, cooling the mixture to room temperature, and purifying the supernatant by silica gel column chromatography to obtain two-dimensional HfS2Quantum dots;
weighing 5g of two-dimensional HfS2Quantum dots and 0.09g of water-soluble ligand containing sulfonate ions are uniformly scattered in benzene, and then ultrasonic treatment is carried out for 6 hours at 4 ℃ to obtain two-dimensional HfS with the surface modified with nitrate ions2And (4) quantum dots.
2) Selecting ferric salt as ferric chloride;
3) the ascorbyl ester is L-ascorbic acid-2-trisodium phosphate.
2. Detection of alkaline phosphatase in a test sample
1) Weighing 5.5g of two-dimensional HfS with sulfonate group modified on surface2Quantum dots and 0.78g of L-ascorbic acid-2-trisodium phosphate are uniformly dispersed in ethanol;
2) weighing 10.6g of ferric chloride, and uniformly dispersing in ethanol to prepare ferric salt solution; then, slowly adding an iron salt solution into the mixed solution obtained in the step 1) until the fluorescence emitted by the mixture in the visible light range is completely quenched, and constructing a detection system;
3) adding a sample to be detected into the detection system constructed in the step 2), incubating for 20 hours at room temperature, and scanning and detecting whether a fluorescence emission signal in a visible light range exists in the mixed solution by adopting a spectrophotometer.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (13)
1. An alkaline phosphatase detection reagent comprising: the composite material comprises a transition metal disulfide quantum dot/iron ion composite, an ascorbate substance and a solvent, wherein the ascorbate substance is dissolved in the solvent, the transition metal disulfide quantum dot/iron ion composite is dispersed in the solvent, and iron ions in the transition metal disulfide quantum dot/iron ion composite are adsorbed on the surface of the transition metal disulfide quantum dot.
2. The alkaline phosphatase detection reagent according to claim 1, wherein the iron ions in the transition metal disulfide quantum dot/iron ion complex are derived from an iron salt dissolved in the solvent, and the mass ratio of the iron salt to the transition metal disulfide quantum dot is (1-100): 1-50; and/or
The mass ratio of the transition metal disulfide quantum dots to the ascorbyl ester substances is 1 (3-10).
3. The alkaline phosphatase detection reagent according to claim 1, wherein the transition metal disulfide quantum dot is a two-dimensional transition metal disulfide having a monolayer structure.
4. The alkaline phosphatase detection reagent according to claim 1, wherein the transition metal disulfide quantum dot is a transition metal disulfide quantum dot with a water-soluble ligand modified on the surface.
5. The alkaline phosphatase detection reagent according to claim 1, wherein the transition metal disulfide quantum dot has the basic chemical formula of MS2Wherein M is selected from Mo, W, V, Nb, Ta, Ti, Zr, Hf or Re.
6. The alkaline phosphatase detection reagent according to claim 1, wherein the ascorbyl ester compound comprises at least one of ascorbyl phosphate and/or a salt thereof, ascorbyl palmitate and/or a salt thereof, ascorbyl laurate and/or a salt thereof, ascorbyl stearate and/or a salt thereof, ascorbyl oleate and/or a salt thereof, ascorbyl linoleate and/or a salt thereof, and ascorbyl benzoate and/or a salt thereof.
7. A method for preparing an alkaline phosphatase detection reagent, comprising:
providing transition metal disulfide quantum dots, iron salt and an ascorbate substance, mixing the transition metal disulfide quantum dots, the iron salt and the ascorbate substance in a solvent, so that iron ions of the iron salt are adsorbed on the surfaces of the transition metal disulfide quantum dots, and the ascorbate substance is dissolved in the solvent to obtain the alkaline phosphatase detection reagent.
8. The method of claim 7, wherein the mass ratio of the iron salt to the transition metal disulfide quantum dots is (1-100): 1-50; and/or
The mass ratio of the transition metal disulfide quantum dots to the ascorbyl ester substances is 1 (3-10).
9. The production method according to claim 7, wherein the transition metal disulfide quantum dot is a two-dimensional transition metal disulfide having a monolayer structure; and/or
The transition metal disulfide quantum dots are transition metal disulfide quantum dots with surfaces modified with water-soluble ligands; and/or
The basic chemical formula of the transition metal disulfide quantum dot is MS2Wherein M is selected from Mo, W, V, Nb, Ta, Ti, Zr, Hf or Re.
10. The preparation method according to claim 7, wherein the preparation of the transition metal disulfide quantum dots comprises the following steps:
providing a transition metal salt, a sulfur source and a nonpolar solvent, wherein the mass ratio of the transition metal salt to the sulfur source is (30-80): 1;
mixing the transition metal salt, the sulfur source and the nonpolar solvent, and carrying out heating reaction.
11. The method according to claim 10, wherein the heating reaction is carried out at a reaction temperature of 200 to 500 ℃ for 1 to 24 hours.
12. A method for detecting alkaline phosphatase, comprising the steps of:
providing a sample to be tested, and the alkaline phosphatase detecting reagent according to any one of claims 1 to 6 or the alkaline phosphatase detecting reagent prepared by the preparation method according to any one of claims 7 to 11;
mixing and incubating the sample to be detected and the alkaline phosphatase detection reagent to obtain a mixed solution;
and irradiating the mixed solution by adopting visible light, and determining whether alkaline phosphatase exists in the sample to be detected according to the fluorescence emission signal of the mixed solution.
13. The method according to claim 12, wherein the incubation of the sample with the alkaline phosphatase detecting reagent is carried out at 10-30 ℃ for 1-24 hours.
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