CN109959640B - Method for detecting content of formaldehyde in cells by using water-soluble polymer through Hangqi reaction - Google Patents
Method for detecting content of formaldehyde in cells by using water-soluble polymer through Hangqi reaction Download PDFInfo
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- CN109959640B CN109959640B CN201711338115.1A CN201711338115A CN109959640B CN 109959640 B CN109959640 B CN 109959640B CN 201711338115 A CN201711338115 A CN 201711338115A CN 109959640 B CN109959640 B CN 109959640B
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- 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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Abstract
The invention provides a method for detecting the content of formaldehyde in cells, which uses a water-soluble polymer with an β -diketone structure as a raw material and detects the content of formaldehyde in cells through Hangqi reaction.
Description
Technical Field
The invention belongs to the field of materials, and particularly relates to a method for detecting the content of formaldehyde in cells by using water-soluble polymers through a Hangqi reaction.
Background
Formaldehyde is an aldehyde with the simplest structure, and is a substance which brings toxicity and pollution to human bodies and the environment. Some endogenous formaldehyde is also produced in human body by the action of some demethylase and oxidase, and the content of formaldehyde in human body cells is generally 400 mu mol/L. There are many reports that excessive formaldehyde in the body can cause some diseases, such as Alzheimer's disease, and some cancers. Therefore, it is necessary and urgent to develop a method for detecting the formaldehyde content in cells. The traditional method for detecting formaldehyde in cells mainly designs some fluorescent probes, and has the defects of complex structure, low sensitivity, low detection limit and low real-time property. The traditional method also comprises the steps of taking acetylacetone and ammonium salt as raw materials, and detecting the content of formaldehyde in cells through a Hanzi reaction. But acetylacetone has higher toxicity, weak fluorescence signal, difficult storage and certain defects in the aspect of use and popularization.
Disclosure of Invention
The invention aims to provide a method for detecting the content of formaldehyde in cells, which uses a water-soluble polymer with an β -diketone structure as a raw material and detects the content of formaldehyde in cells through Hangqi reaction.
The method for detecting the content of formaldehyde in cells provided by the invention comprises the following steps:
1) preparing a detection solution for detecting formaldehyde in cells;
2) contacting the detection liquid for detecting formaldehyde in the cells with the cells, removing the detection liquid after the cells swallow, adding a formaldehyde solution with a known concentration, detecting the fluorescence intensity on the cells, and repeating the operation to detect the fluorescence intensity on the cells after a series of formaldehyde solutions with known concentrations are added, so as to obtain the relation between the fluorescence intensity on the cells and the formaldehyde concentration;
3) contacting the detection liquid for detecting the formaldehyde in the cell with the formaldehyde content to be detected, removing the detection liquid after the cell swallows, detecting the fluorescence intensity on the cell, and obtaining the formaldehyde content in the cell according to the relation between the fluorescence intensity and the formaldehyde concentration obtained in the step 2).
In the step 1) of the method, the detection solution for detecting intracellular formaldehyde is a solution of a water-soluble polymer with an β -diketone structure and an ammonium salt.
The solvent used may be any buffer suitable for cell detection, such as PBS buffer, as known to those skilled in the art.
The water-soluble polymer with β -diketone structure can be specifically a polymer shown in formula I:
in the formula I, x, y and n all represent polymerization degrees, wherein x is 20-50, y is 20-50, and n is 10-30.
The ammonium salt may be selected from: ammonium chloride, ammonium sulfate, ammonium acetate, etc., and specifically ammonium acetate may be mentioned.
The molar ratio of the water-soluble polymer having an β -diketone structure (calculated on the basis of the amount of diketone substance) to the ammonium salt can be 1:1 to 1: 10.
The water-soluble polymer with β -diketone structure shown in the formula I is prepared by copolymerizing polyethylene glycol methyl methacrylate shown in the formula II and acetoacetic acid ethylene glycol methacrylate shown in the formula III:
in formula II, n represents the degree of polymerization, and n is 10 to 30.
In step 2) of the above method, the concentration of the series of formaldehyde solutions with known concentrations is a concentration value in the range of 0 to 100. mu. mol/L.
The detection solution for detecting intracellular formaldehyde used in the above method also belongs to the protection scope of the present invention.
The invention aims to provide a method for detecting formaldehyde in cells by a Hanzi reaction, and the used water-soluble polymer with β -diketone structure comprises a copolymer of polyethylene glycol methyl methacrylate and acetoacetic acid ethylene glycol methacrylate, but is not limited to the copolymer.
The invention further improves the traditional Hangqi reaction for detecting formaldehyde, and the traditional method uses acetylacetone as the source of β -diketone, compared with the prior method, the acetylacetone has lower safety and the fluorescence intensity of the product is weak, so the polymer with diketone structure is adopted to improve the safety and enhance the fluorescence intensity.
The invention provides a method for detecting formaldehyde in cells by using a controllable polymer structure. One is that the molecular weight of polyethylene glycol methyl methacrylate which is one of the raw materials of the copolymer can be regulated, and the other is that the proportion of the two raw materials can be regulated, thereby achieving the purposes of regulating and controlling the water solubility, the detection limit and the like of the polymer.
The invention provides a method idea for post-modification of an intracellular polymer, wherein the post-modification of the polymer is a common method for modifying the polymer, and the method idea is provided for possible macromolecule bio-orthogonal reaction in the future by changing a reaction system into a cell.
The polymer with β -diketone structure provided by the invention has very good water solubility, and is easy to be used in biological systems.
The invention provides a method for preparing formaldehyde detection solution from polymer, which is convenient for long-term storage and transportation due to stable polymer structure and lays a foundation for possible commercialization.
The invention detects the content of formaldehyde in cells by the fluorescence intensity obtained by Hangqi reaction.
The invention discloses a method for detecting the content of formaldehyde in cells according to the fluorescence intensity of a product generated by a Hangqi reaction by synthesizing a water-soluble polymer with a diketone structure and forming formaldehyde detection liquid with ammonium salt. The polymer and ammonium salt can be dissolved in PBS buffer solution, and can be stored in the form of solution, thus being convenient for long-term storage and transportation.
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FIG. 1 shows the equation for the copolymerization of polyethylene glycol methyl methacrylate and acetoacetic acid ethylene glycol methacrylate.
FIG. 2 shows the chemical reaction formula of the principle of formaldehyde detection in the copolymer-based detection solution.
FIG. 3 is a comparison of fluorescence spectra of formaldehyde detection for copolymer and acetylacetone based detection solutions, respectively. (wherein a is the result of acetylacetone test, and b is the result of copolymer test)
FIG. 4 is a comparison of fluorescence intensity of formaldehyde for detection with time for copolymer and acetylacetone based detection solutions, respectively. (wherein a is the result of acetylacetone test, and b is the result of copolymer test)
FIG. 5 shows the measurement of the detection limit for formaldehyde of the copolymer and acetylacetone as the respective basic detection solutions. (wherein a is the result of acetylacetone test, and b is the result of copolymer test)
FIG. 6 shows the specificity of detection of formaldehyde for copolymer and acetylacetone based detection solutions. (wherein a is the result of acetylacetone test, and b is the result of copolymer test)
FIG. 7 is a confocal laser scanning microscope photograph of formaldehyde detection in the copolymer and acetylacetone-based detection solutions. (wherein the a/a 'diagram shows the result of acetylacetone test; the b/b' diagram shows the result of copolymer test; the c/c 'diagram shows a blank control without any detection solution; the exciter of the a/b/c diagram is 408 nm; the exciter of the a'/b '/c' diagram is 488 nm; and the a "/b"/c "diagrams show the fusion of the former two, respectively).
FIG. 8 is a comparison of the cell safety of the copolymer and acetylacetone. (wherein a is the result of acetylacetone test, and b is the result of copolymer test)
Detailed Description
The present invention will be described below with reference to specific examples, but the present invention is not limited thereto.
The experimental methods used in the following examples are all conventional methods unless otherwise specified; reagents, materials and the like used in the following examples are commercially available unless otherwise specified.
Examples 1,
Preparation of mono-and water-soluble diketone structure polymer
According to the preparation route of FIG. 1, polyethylene glycol methyl methacrylate (molecular weight 950) and ethylene glycol acetoacetate methacrylate were mixed in a ratio of 1:1 in terms of the amounts of the substances.
Adding the mixed polyethylene glycol methyl methacrylate and acetoacetic acid ethylene glycol methacrylate into a polymerization tube, adding an initiator azobisisoheptonitrile, adding a solvent dimethylformamide, and sealing by using a rubber plug.
Nitrogen was introduced for 30 minutes to evacuate the air inside the polymerization tube. The mixture was heated in an oil bath at 70 ℃ for 8 hours with stirring.
The polymerization solution was precipitated by adding ether and centrifuged (10000 rpm, 10 minutes) to obtain a precipitated solid.
And cooling the obtained polymer solid by using liquid nitrogen, and cooling and draining to obtain the water-soluble diketone structure polymer.
Secondly, preparing formaldehyde detection liquid
And (3) dissolving the prepared polymer with a certain mass and ammonium salt with a certain mass in a PBS buffer solution to obtain a formaldehyde detection solution, and storing at room temperature. Wherein the ratio of the amounts of polymer (calculated on the basis of the amount of diketone) and ammonium salt is 1:2.5, and ammonium acetate is used as ammonium salt.
Third, formaldehyde detection
Adding the prepared formaldehyde detection solution into an L929 cell culture dish. After the cells are swallowed for 30 minutes, the detection solution is removed, and after 3 times of washing with PBS buffer solution, a formaldehyde solution with a trace concentration (100 mu mol/L) is added for simulation detection. And (3) observing cell fluorescence: all the liquid was removed from the cell culture dish and the fluorescence on the cells was observed using a confocal laser microscope. As shown in FIG. 7 b/b'.
Example 2 fluorescence Spectroscopy test experiment
1) After the polymer was obtained according to the preparation method of example 1, a formaldehyde detection solution was prepared from the polymer and ammonium salt at a certain concentration. Acetylacetone and ammonium salt with certain mass are prepared into the traditional formaldehyde detection solution. The ratio of the amount of acetylacetone to the amount of ammonium salt is 1:2.5, and the ammonium salt is ammonium acetate.
2) And adding a trace amount of formaldehyde solution (the concentration is 100 mu mol/L), taking 360 nanometers as an excitation wavelength, and detecting the sensitivity and the timeliness of the formaldehyde solution and the formaldehyde solution through a fluorescence spectrum test. As shown in fig. 3 and 4.
As can be seen from fig. 3 and 4: in comparison, the detection sensitivity of the polymer-based detection solution is higher than that of the traditional acetylacetone, and the polymer-based detection solution has stronger fluorescence intensity.
Example 3 detection Limit test experiment
The water-soluble polymer prepared in example 1 and ammonium acetate were taken to prepare a formaldehyde detection solution (polymer concentration 20 mg/mL; ammonium acetate concentration 2mg/mL), which was compared with conventional acetylacetone and ammonium acetate detection solutions (polymer concentration 1 mg/mL; ammonium acetate concentration 2mg/mL), and reacted with formaldehyde solutions of different concentrations, respectively, to detect the fluorescence intensity values measured with a multi-well microplate reader, and the detection limit was determined based on the linear relationship between the fluorescence intensity values and formaldehyde concentrations. The results are shown in FIG. 5. From fig. 5a, b, it can be seen that: in comparison, the formaldehyde detection effect of the detection liquid based on the macromolecule can achieve the effect similar to the traditional acetylacetone formaldehyde detection effect.
Example 4 specificity test experiment
The water-soluble polymer prepared in example 1 and ammonium acetate are prepared into formaldehyde detection solution, compared with the traditional acetylacetone and ammonium acetate detection solution, the formaldehyde detection solution reacts with different substance solutions (with the same concentration) respectively, the fluorescence luminosity value measured by a porous microplate reader is detected, and the specificity of detecting formaldehyde is measured according to the fluorescence luminosity value. The results are shown in FIG. 6.
From fig. 6a, b, it can be seen that: the formaldehyde detection solution based on the macromolecule can achieve the specific detection of formaldehyde by the traditional acetylacetone detection solution, and even has better effect.
Example 5 cell safety test experiment
The cell viability of the water-soluble polymer and acetylacetone of example 1 was tested by L929 cells. The measurement is carried out by adopting a CCK-8 calibration method and testing the absorbance value by utilizing a microplate reader.
The results of comparing the cell safety of high and small molecule acetylacetone were calculated with the cell standard of 100% in normal medium and 0% in normal medium without cell standard, and are shown in fig. 8.
From fig. 8a, b, it can be seen that: in comparison, the safety of the high molecular polymer for detecting the substance is much better than that of the traditional acetylacetone. It is almost non-toxic even in high concentration, so it has very good biocompatibility.
Claims (8)
1. A detection solution for detecting formaldehyde in cells is a solution of water-soluble polymer with β -diketone structure and ammonium salt;
the water-soluble polymer with the β -diketone structure is a polymer shown in a formula I:
in the formula I, x, y and n all represent polymerization degrees, wherein x is 20-50, y is 20-50, and n is 10-30.
2. The detection solution for detecting intracellular formaldehyde according to claim 1, characterized in that:
the ammonium salt is selected from: ammonium chloride, ammonium sulfate, ammonium acetate;
the molar ratio of the water-soluble polymer with β -diketone structure to the ammonium salt is 1:1-1: 10.
3. The polymer of formula I in claim 1.
4. A method of preparing a polymer of formula I in claim 1, comprising: copolymerizing polyethylene glycol methyl methacrylate represented by formula II with acetoacetic acid ethylene glycol methacrylate represented by formula III:
in formula II, n represents the degree of polymerization, and n is 10 to 30.
5. Use of the detection solution for detecting intracellular formaldehyde according to claim 1 or 2 for the detection of intracellular formaldehyde content.
6. Use according to claim 5, characterized in that: the detection of the intracellular formaldehyde content is realized by a Hangqi reaction.
7. A method for detecting the content of formaldehyde in cells comprises the following steps:
1) preparing a detection solution for detecting intracellular formaldehyde according to claim 1 or 2;
2) contacting a detection liquid for detecting formaldehyde in cells with the cells, removing the detection liquid after the cells swallow the detection liquid, adding a formaldehyde solution with a known concentration, detecting the fluorescence intensity on the cells, and repeating the operation to detect the fluorescence intensity on the cells after a series of formaldehyde solutions with known concentrations are added, so as to obtain the relation between the fluorescence intensity on the cells and the formaldehyde concentration;
3) contacting a detection liquid for detecting formaldehyde in the cell with the formaldehyde content to be detected, removing the detection liquid after the cell swallows, detecting the fluorescence intensity on the cell, and obtaining the formaldehyde content in the cell according to the relation between the fluorescence intensity and the formaldehyde concentration obtained in the step 2).
8. The method of claim 7, wherein: in step 2), the concentration of the series of formaldehyde solutions with known concentrations is a concentration value in the range of 0-100 [ mu ] mol/L.
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