阅读说明：本技术 光控螺吡喃整体柱的制备及在细胞代谢物检测中的应用 (Preparation of light-controlled spiropyran monolithic column and application of light-controlled spiropyran monolithic column in cell metabolite detection ) 是由 汪夏燕 裴彤 刘珍 邵云龙 李利杰 郭广生 于 2020-12-28 设计创作，主要内容包括：光控螺吡喃整体柱的制备及在细胞代谢物检测中的应用,属于分析化学与生物化学领域。该系统包括：光控螺吡喃整体柱的制备、光控单元的搭建及高效液相色谱与质谱联用系统。将丙烯酰基螺吡喃、二甲基丙烯酸乙二醇酯(EDMA)、致孔剂按比例配成有机预聚合溶液,然后向活化及烷基化后的毛细管内注入螺吡喃预聚合溶液。在一定水浴温度下引发热聚合制备光控螺吡喃整体柱,该整体柱具有光控极性转换特性；通过将整体柱接入液质联用系统中可实现在可见光与紫外光先后照射下整体柱极性转变进而对A549细胞代谢物分离与检测,最终在可见光条件下实现对A549细胞中能量分子如ATP、ADP的检测,紫外光下三羧酸循环中间体、戊糖磷酸循环中间体的选择性检测。(The preparation of light-operated spiropyran monolithic column and its application in cell metabolite detection belong to the field of analytical chemistry and biochemistry. The system comprises: the preparation of the light-operated spiropyran monolithic column, the construction of a light-operated unit and a high performance liquid chromatography and mass spectrometry combined system. Proportionally preparing acryloyl spiropyran, ethylene glycol dimethacrylate (EDMA) and a pore-foaming agent into an organic prepolymerization solution, and then injecting the spiropyran prepolymerization solution into the activated and alkylated capillary. Initiating thermal polymerization at a certain water bath temperature to prepare a light-controlled spiropyran monolithic column, wherein the monolithic column has a light-controlled polarity conversion characteristic; the polarity of the whole column is changed under the sequential irradiation of visible light and ultraviolet light by connecting the whole column into a liquid chromatography-mass spectrometry system, so that the metabolite of the A549 cell is separated and detected, and finally, the detection of energy molecules such as ATP and ADP in the A549 cell and the selective detection of tricarboxylic acid cycle intermediates and pentose phosphate cycle intermediates under the ultraviolet light are realized under the condition of visible light.)

1. A preparation method of a light-operated spiropyran monolithic column is characterized by comprising the following steps:

1, 4-butanediol, isopropanol and chloroform are used as a pore-forming agent, ethylene glycol dimethacrylate (EDMA) and acryloyl spiropyran are used as monomers, the pore-forming agent and the monomers are added with an initiator, and are injected into a capillary tube with an alkylated inner surface, and thermal polymerization is initiated in water bath at 50-70 ℃ to prepare the light-controlled acryloyl spiropyran monolithic column.

2. A process for preparing a light-operated spiropyran monolithic column according to claim 1, characterized in that,

the synthesis procedure of the acryloyl spiropyran monomer used was as follows: adding 1.0eq of 2, 3, 3-trimethylindole and 1.2eq of 2-bromoethanol into a three-neck flask containing anhydrous acetonitrile, refluxing for 24 hours under the protection of nitrogen, cooling to room temperature, spin-drying the residual solvent, and adding the crude product in diethyl ether: the volume ratio of trichloromethane is 1:1 to obtain a pink solid product 1; dissolving 11.0 eq of the product in a round-bottom flask containing ultrapure water, adding 1.6eq of KOH under the ice bath condition until the solution turns yellow, stirring at room temperature for 30min, extracting by using ethyl acetate, drying by using anhydrous sodium sulfate, and concentrating an organic phase to obtain a yellow oily product 2; ③ taking 21.0 eq of the product to be put into a three-neck flask containing 1.3eq of 5-nitro salicylaldehyde dissolved in absolute ethyl alcohol, refluxing for 4h under the protection of nitrogen, cooling to room temperature, filtering, washing by using ethyl alcohol, and drying to obtain a mauve solid product 3; and fourthly, putting 31.0 eq of the product into a three-neck flask containing dichloromethane, dropwise adding 1.3eq of acryloyl chloride dissolved in the dichloromethane, stirring at room temperature for 6 hours, washing with water after the reaction is finished, collecting a dichloromethane layer, drying with anhydrous sodium sulfate, and adding ethyl acetate: n-hexane volume ratio of 1: 5 was passed through the column as a mobile phase and the fraction having an Rf value of 0.7 was collected.

3. The method for preparing a light-controlled spiropyran monolithic column according to claim 1, wherein the mass ratio of the acryloyl spiropyran monomer to the pore-forming agent is 1: 2; the mass ratio of EDMA to acryloyl spiropyran is 1:1, wherein the mass ratio of the pore-forming agent 1, 4-butanediol, isopropanol and chloroform is 5: 5: 10, the initiator is azobisisobutyronitrile, and the polymerization time is 5-7 h.

4. The preparation method of the light-controlled spiropyran monolithic column as claimed in claim 1, wherein the capillary used has a specification of 50-100 μm in inner diameter and 160-360 μm in outer diameter, and the outer surface is coated with polyethylene.

5.A light-controlled spiropyran monolithic column produced according to the method of any one of claims 1 to 3.

6. Use of a light-operated spiropyran monolithic column prepared by the method of any one of claims 1-3 for the detection of cell metabolites.

7. Use according to claim 6, characterized in that it comprises the following steps:

step one, preparation of a cell metabolite sample: placing a 6-hole plate for culturing A549 cells on ice, adding an extraction solvent, transferring to a refrigerator, standing at-20 ℃ for 5min, extracting metabolites in the cells, scraping the cells from the wall by using a cell scraper, transferring a cell suspension into a centrifuge tube, centrifuging, and collecting a supernatant in the centrifuge tube for later use;

step two, visible light separation and detection of cell metabolites: constructing a high performance liquid chromatography mass spectrometry system for the prepared light-controlled spiropyran monolithic column, and separating and detecting A549 cell metabolites under visible light after the light-controlled spiropyran monolithic column is irradiated for 300 s;

step three, ultraviolet light separation and detection of cell metabolites: and (3) carrying out ultraviolet illumination on the light-controlled spiropyran monolithic column subjected to the second step for 240s, and then separating and detecting the A549 cell metabolites.

8. The use according to claim 6, characterized in that in step one, the A549 cells are seeded at a cell density of 3X 10 in 6-well plates⁵A hole;

the cells inoculated in the first step are at 37 ℃ and CO₂Culturing in 5% incubator for 48 hr.

When the A549 cells are extracted in the first step, the extraction solvent is isopropanol.

9. Use according to claim 6, characterized in that in step two and in step three, a monolithic column of optically controlled spiropyran is used in combination with a LC-MS: comprises a sample introduction system, a pressure system, a separation system and a detection system 4; placing a sample introduction bottle containing metabolites on a sample table at 4 ℃, wherein the sample introduction volume is 1 mu L, introducing the samples into a light-controlled spiropyran monolithic column under the drive of a mobile phase for separating the metabolites, connecting an elastic quartz capillary tube equal-inner-diameter spray needle at the outlet end of the monolithic column, realizing the electrospray ionization of the nanoliter flow rate samples, and detecting in a mass spectrum positive ion mode;

the mobile phase used was a 1:1 by volume methanol/water solution, and the flow rate used was 0.15. mu.L/min.

10. Use according to claim 6, characterized in that the visible light in step two is an incandescent lamp, and the ultraviolet light in step three is a 365nm ultraviolet lamp; the design of double light source opposite irradiation is adopted, so that the whole column can be guaranteed to be uniformly illuminated in all directions;

under visible light, the separation and detection of energy molecules such as ATP, ADP and the like in A549 cells are realized, and under ultraviolet light, the selective separation and detection of tricarboxylic acid cycle intermediates and pentose phosphate cycle intermediates are realized.

Technical Field

The invention relates to a preparation method of a light-operated spiropyran monolithic column and application of the light-operated spiropyran monolithic column in cell metabolite detection, and belongs to the fields of analytical chemistry and biochemistry.

Background

Metabonomics is an emerging field following genomics, transcriptomics and proteomics, and realizes monitoring of dynamic level in organisms mainly through qualitative and quantitative analysis of metabolites. Chromatographic techniques are widely used for the separation of metabolites due to their strong separation capacity. Depending on the type of metabolite, different separation methods may be selected. In general, reverse phase liquid chromatography is suitable for separating non-polar and weakly polar metabolites, and hydrophilic interaction chromatography is suitable for separating more polar metabolites. However, to date, no single chromatographic column has been able to achieve separation of all polar metabolites from a sample. Therefore, the ion suppression effect due to insufficient separation inevitably affects the detection of a trace amount of metabolites.

The spiropyran compound means a compound having an SP³Aromatic rings bound by carbon atoms, by SP³The generic name of a class of organic compounds with photochromic properties in which the carbon atom is linked to another pyran ring. Under the irradiation of ultraviolet light, the spiropyran molecules are subjected to reversible conversion between a closed ring body and an open ring body, and the polarity, the color and the fluorescence property of the compound are changed. By utilizing the property of the spiropyran compound and combining with instruments such as a fluorescence microscope, an ultraviolet spectrophotometer and the like, the high-sensitivity and high-selectivity detection of metabolites with strong nucleophilicity such as S, F and the like can be realized. However, a technology for separating and detecting cell metabolites with different polarities in one sample injection by using a spiropyran molecule to prepare a capillary monolithic column and quickly changing the polarity of the monolithic column through light control has not been reported.

Disclosure of Invention

The light-operated spiropyran monolithic column is prepared and used for detecting A549 cell metabolites. Based on the fact that the molecule of the spiropyran compound is subjected to reversible conversion of a closed ring body and an open ring body under the condition of different wavelengths of light, the selective adsorption and separation of metabolite molecules with different polarities are carried out. The system is applied to realize the separation and detection of energy molecules such as ATP, ADP and the like in A549 cells under visible light, and the selective separation and detection of tricarboxylic acid cycle intermediates and pentose phosphate cycle intermediates under ultraviolet light.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a preparation method of a light-operated spiropyran monolithic column is characterized by comprising the following steps:

1, 4-butanediol, isopropanol and chloroform are used as a pore-forming agent, ethylene glycol dimethacrylate (EDMA) and acryloyl spiropyran are used as monomers, the pore-forming agent and the monomers are added with an initiator, and the mixture is injected into an alkylated capillary tube and is subjected to thermal polymerization in water bath at 50-70 ℃ to prepare a light-controlled acryloyl spiropyran monolithic column;

the synthesis procedure of the acryloyl spiropyran monomer used was as follows: adding 2, 3, 3-trimethylindole (1.0eq) and 2-bromoethanol (1.2eq) into a three-neck flask containing anhydrous acetonitrile, refluxing for 24 hours under the protection of nitrogen, cooling to room temperature, spin-drying the residual solvent, and reacting the crude product in diethyl ether: the volume ratio of trichloromethane is 1:1 to obtain a pink solid product 1; dissolving the product 1(1.0eq) in a round-bottom flask containing ultrapure water, adding KOH (1.6eq) under the ice bath condition until the solution turns yellow, stirring at room temperature for 30min, extracting by using ethyl acetate, drying by using anhydrous sodium sulfate, and concentrating an organic phase to obtain a yellow oily product 2; ③ taking the product 2(1.0eq) to a three-neck flask containing 5-nitro salicylaldehyde (1.3eq) dissolved in absolute ethyl alcohol, refluxing for 4 hours under the protection of nitrogen, cooling to room temperature, filtering, washing with ethyl alcohol, and drying to obtain a mauve solid product 3; putting the product 3(1.0eq) into a three-neck flask containing dichloromethane, dropwise adding acryloyl chloride (1.3eq) dissolved in dichloromethane, stirring at room temperature for 6 hours, washing with water after the reaction is finished, collecting a dichloromethane layer, drying with anhydrous sodium sulfate, and adding ethyl acetate: n-hexane volume ratio of 1: 5 was passed through the column as a mobile phase and the fraction having an Rf value of 0.7 was collected.

When the monolithic column is prepared, the mass ratio of the monomer to the pore-forming agent is 1: 2; the mass ratio of the monomer EDMA to the acryloyl spiropyran is 1:1, preparing a pore-forming agent 1, 4-butanediol, isopropanol and chloroform in a mass ratio of 5: 5: 10, the initiator is azobisisobutyronitrile, and the polymerization time is 5-7 h.

The specification of the capillary tube is 50-100 mu m in inner diameter and 160-360 mu m in outer diameter, and the outer surface of the capillary tube is provided with a polyethylene coating.

The application of the prepared optically-controlled spiropyran monolithic column in cell metabolite detection is characterized by comprising the following steps:

step one, preparation of a cell metabolite sample: placing a 6-well plate for culturing A549 cells on ice, adding an extraction solvent, transferring to a refrigerator at-20 ℃, standing for 5min to extract metabolites in the cells, scraping the cells from the wall by using a cell scraper, transferring a cell suspension into a centrifuge tube, centrifuging (12000rpm, 4 ℃, 10min), and collecting a supernatant in the centrifuge tube for later use.

Step two, visible light separation and detection of cell metabolites: constructing a high performance liquid chromatography mass spectrometry system for the prepared light-controlled spiropyran monolithic column, and separating and detecting A549 cell metabolites under visible light after the light-controlled spiropyran monolithic column is irradiated for 300 s;

step three, ultraviolet light separation and detection of cell metabolites: and (3) irradiating the optically controlled spiropyran monolithic column subjected to the second step with ultraviolet light for 240s (wherein lambda is 365nm, and 2x 6W), and then separating and detecting the A549 cell metabolites.

When the A549 cells are inoculated in the 6-well plate in the step one, the cell density is 3 multiplied by 10⁵A hole.

The cells inoculated in the first step are at 37 ℃ and CO₂Culturing in 5% incubator for 48 hr.

When the A549 cells are extracted in the first step, the extraction solvent is isopropanol.

In the second step and the third step, the used light-controlled spiropyran monolithic column is combined with a liquid chromatography-mass spectrometry system: comprises 4 parts of a sample introduction system, a pressure system, a separation system, a detection system and the like; placing a sample introduction bottle containing metabolites on a sample table at 4 ℃, wherein the sample introduction volume is 1 mu L, introducing the samples into a light-controlled spiropyran monolithic column under the drive of a mobile phase to separate the metabolites, connecting an elastic quartz capillary tube equal-inner-diameter spray needle at the outlet end of the monolithic column, realizing the electrospray ionization of the nanoliter flow rate samples, and detecting in a mass spectrum positive ion mode.

In the second step and the third step, the mobile phase is methanol/water solution with the volume ratio of 1: 1.

The flow rate used in step two and step three was 0.15. mu.L/min.

The visible light in the second step is incandescent lamp (2x 6W), and the ultraviolet light in the third step is 365nm ultraviolet lamp (2x 6W).

And the whole column is ensured to uniformly receive light all around by adopting a double-light-source opposite irradiation design.

The invention can obtain the following effects:

the light-operated spiropyran monolithic column is prepared and used for separating and detecting A549 cell metabolites. In the system, the method is adopted to realize the detection of energy molecules in A549 cells under visible light, and tricarboxylic acid cycle intermediates and pentose phosphate cycle intermediates can be selectively detected under ultraviolet light. Different kinds of metabolites are separated, the influence of the ion suppression effect on the detection of trace metabolites is effectively reduced, and the method is a good method for separating and detecting trace substances in the metabolites. Compared with the traditional liquid chromatography-mass spectrometry method, the detection of more metabolites is realized by switching the polarity of the monolithic column while realizing small-volume sample injection.

According to the invention, under ultraviolet light, the acryloyl spiropyran monolithic column is used, under the action of C-O bond heterolysis, isomerization or rearrangement of electronic configuration is carried out on the spiropyran compound from a closed ring body to form a large conjugated plane, so that the polarity is changed, and the selective and specific adsorption of amino acid, lipid and nucleic acid in cells is realized, and the purpose of separation and detection is achieved.

The light-controlled spiropyran monolithic column-liquid chromatography-mass spectrometry combined system is used for separating and detecting metabolites of A549 cells, and analyzing and detecting data to obtain the detection data, the system is used for separating and detecting energy molecules in the A549 cells according to different colors under visible light, and selective separation and detection of tricarboxylic acid cycle intermediates and pentose phosphate cycle intermediates are realized according to different colors under ultraviolet light.

Drawings

FIG. 1 is a schematic diagram of a light-controlled spiropyran monolithic column-LC-MS system.

FIG. 2 is a graph showing the change in contact angle of acryloyl spiropyrans upon irradiation with visible light and ultraviolet light.

Fig. 3 is an isopropyl alcohol: 1, 4-butanediol: chloroform was added at a ratio of 5: 5: scanning electron micrographs of the end faces of monolithic capillary columns polymerized for different times at a ratio of 10.

FIG. 4 shows that under different conditions, the light-controlled spiropyran monolithic column-LC-MS system detects the types of metabolites.

1. High performance liquid chromatography, 2 light control units, 3 elastic quartz capillary equal inner diameter spray needles, 4 mass spectra, 5 and 8.365nm ultraviolet lamps (2x 6W), 6 and 9 incandescent lamps (2x 6W) and 7 light control spiropyran monolithic columns.

Detailed Description

In order to make the technical solutions of the present invention better understood, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides preparation of a light-operated spiropyran monolithic column and application thereof in cell metabolite detection, and the specific implementation steps are as follows:

s1, the synthesis process of the acryloyl spiropyran used for preparing the monolithic column is as follows: adding 2, 3, 3-trimethylindole (1.0eq) and 2-bromoethanol (1.2eq) into a three-neck flask containing 10mL of anhydrous acetonitrile, refluxing for 24h under the protection of nitrogen, cooling to room temperature, spin-drying the residual solvent, and dissolving the crude product in 5mL of diethyl ether: 1, trichloromethane (v: v) ═ 1:1 to obtain a pink solid product 1; ② adding the product 1(1.0eq) into a round-bottom flask containing a proper amount of ultrapure water, adding KOH (1.6eq) under the ice bath condition, stirring for 30min at room temperature when the solution becomes yellow, extracting by using ethyl acetate, drying by using anhydrous sodium sulfate, and concentrating the organic phase to obtain a yellow oily product 2; ③ taking the product 2(1.0eq) to a three-neck flask containing 5-nitro salicylaldehyde (1.3eq) dissolved in absolute ethyl alcohol, refluxing for 4 hours under the protection of nitrogen, cooling to room temperature, filtering, washing with ethyl alcohol, and drying to obtain a mauve solid product 3; putting the product 3(1.0eq) into a three-neck flask containing 5mL of dichloromethane, slowly dropwise adding acryloyl chloride (1.3eq) dissolved in dichloromethane into the three-neck flask by using a constant-pressure dropping funnel, stirring the mixture at room temperature for 6 hours, washing the mixture by using water after the reaction is finished, collecting a dichloromethane layer, drying the dichloromethane layer by using anhydrous sodium sulfate, and adding ethyl acetate: n-hexane ═ 1: 5(v: v) was passed through the column as a mobile phase, and the fraction having an Rf value of 0.7 was collected.

S2, modification of the capillary: taking a fused quartz capillary tube with an inner diameter of 75 μm, an outer diameter of 360 μm and a length of 15cm, washing the inner wall of the fused quartz capillary tube for 1h by using 1.0mol/L NaOH respectively, washing the fused quartz capillary tube by using ultrapure water until the pH of the liquid flowing out from the tail end is close to 7, and then using CH₃The capillary was rinsed with OH for 30min and blown dry with nitrogen. Capillary injection after treatment γ -MAPS: CH (CH)₃The mixed solution of OH (v/v ═ 3: 7) was reacted at 40 ℃ for 14 h. After the reaction, the residual gamma-MAPS in the capillary was washed with methanol and N was used₂And drying the capillary tube to obtain the modified capillary tube.

S3, preparing the light-operated spiropyran monolithic column by a thermal polymerization method

Accurately weighing 0.0302g of 1, 4-butanediol, 0.0298g of isopropanol and 0.0603g of trichloromethane to prepare a ternary pore-foaming agent solution, wherein the mass ratio is 1:1:2, adding 0.0453g of acryloyl spiropyran monomer, 0.0451g of EDMA and 0.0013g of azobisisobutyronitrile to fully vibrate and ultrasonically degas an organic prepolymerization solution, injecting the organic prepolymerization solution into an alkylated capillary, and plugging two ends by using silica gel; the capillary tube filled with the organic prepolymerization solution was placed in a water bath and heated to polymerize by heating azodiisobutyronitrile (2,2' -Azobis (2-methyl propionitril), AIBN) by free radical reaction for 7 h. After the thermal polymerization reaction was completed, the reaction was terminated by flushing the capillary with methanol and removing the unreacted solution. The optically controlled spiropyran monolith was activated by washing with methanol before each use. Isopropanol of the light-controlled acryloyl spiropyran monolith prepared in this example: 1, 4-butanediol: the mass ratio of chloroform is 5: 5: a scanning electron micrograph of the monolithic column end face of the capillary at 10 scale is shown in FIG. 3

S4. extraction experiment of metabolite of A549 cells:

taking A549 cells in exponential growth phase at 1 × 10⁵One/hole, 3X 10⁵One/hole, 5X 10⁵The cells were seeded at a density of cells per well in 6-well plates, and 2mL of the cell suspension and 0.5mL of the cell culture medium were added to each well and cultured for 48 h. The growth state of the cells after 48h of culture was characterized using a biomicroscope. When the cell density is 1X 10⁵Each hole is formed, after 48 hours of culture, cells in a 6-hole plate are sparse, and the anchorage rate is only 60%; when the cell density is 3X 10⁵The cells per well are cultured for 48 hours, the growth state of the cells in the 6-well plate is good, and the anchorage rate is higher and can reach 90%; when the cell density is 5X 10⁵After culturing for 48h, the adherence rate of the cells in the 6-well plate is higher, the phenomenon of double layers occurs, and the cell morphology is poorer, so the cell inoculation density on the 6-well plate is determined to be 3 multiplied by 10⁵Per well. Count using PVC cytometry tubes when seeding density is 3X 10⁵After 48h of culture per well, the cell volume was 8. mu.L, and therefore, 130. mu.L of isopropanol was added to extract metabolites in A549 cells.

Inoculating cells on a 6-well plate, culturing for 24h, placing the 6-well plate on ice, washing the cells for 3 times by using PBS, adding 130 mu L of isopropanol to extract metabolites in the cells, scraping the cells from the 6-well plate by using a scraper, transferring the cells into a centrifuge tube, centrifuging for 15min at 4 ℃, and taking supernatant to perform light-controlled spiropyran monolithic column-liquid chromatography-mass spectrometry separation detection.

S5.A549 cell metabolite visible light separation detection experiment

The sample vial containing the metabolite was placed on a 4 ℃ sample stage, and 1 μ L of the sample was aspirated through the sample needle using methanol: the intracellular metabolites obtained by extraction were separated with water (v/v) ═ 50:50 as a mobile phase, and after irradiation with visible light for 300 seconds, the whole column was colorless, and metabolite detection was performed in the ESI positive ion mode, and 57 metabolites were detected in the visible light state.

S6.A549 cell metabolite ultraviolet light separation detection experiment

The same conditions as above were applied: methanol: separating the intracellular metabolites obtained by extraction with water (v/v) ═ 50:50 as a mobile phase and at a flow rate of 0.15 muL/min, and separating and detecting the A549 cell metabolites after ultraviolet irradiation of 240s (lambda ═ 365nm,2x 6W) on the monolithic column to make the monolithic column in a purple state; the number of metabolites detected in the UV state is up to 83.

The constructed light-controlled spiropyran monolithic column-liquid chromatography-mass spectrometry combined system is used for detecting A549 cell metabolites, the types of the metabolites detected in an ultraviolet state are 83 at most, 33 metabolites are detected only under the irradiation of ultraviolet light, the types of the metabolites detected in a visible light state are 57, 8 compounds are detected only in the visible light state, and the types of the metabolites detected by direct sample injection are 46 at least; (2) most amino acids can be detected in three states, and the types of the amino acids detected by using the visible light to irradiate the integral column are the most; (3) separating and detecting amino acid derivatives, amines, alkalis, organic acids and sugar metabolites under ultraviolet irradiation; (4) tricarboxylic acid cycle intermediates, pentose phosphate pathway intermediates, glucose related metabolites, and heterocyclic metabolites are detectable only under ultraviolet irradiation conditions; (5) energy molecules such as ATP, ADP, etc. can only be detected under visible light conditions. The result shows that the spiropyran monolithic column selectively acts with different metabolites under the irradiation of ultraviolet light and visible light respectively, and further realizes the separation and detection of different metabolites under the irradiation of ultraviolet light and visible light.

The preparation of the optically controlled spiropyran monolithic column provided by the embodiment of the invention and the application thereof in cell metabolite detection are described in detail above, the embodiment of the invention and the application of the obtained product are explained in the text by using specific examples, and the description of the above examples is only used for helping to understand the method and the core idea of the invention; meanwhile, for a person skilled in the art, according to the idea of the present invention, the specific implementation manner and the application range may be changed, and the changes and the modifications belong to the protection scope of the present invention; in view of the above, the present disclosure should not be construed as limiting the invention.