Method for detecting isoprene in plants by utilizing deferritin encapsulated coumarin derivative fluorescent probe

Abstract

The invention relates to a method for detecting isoprene in plants, in particular to a method for detecting isoprene in plants based on a coumarin derivative fluorescent probe packaged by deferritin. The invention aims to solve the problems of difficulty in long-time storage of a sample, weak detection interference resistance, response lag and the like in an isoprene detection process. The preparation method comprises the following steps: 1, preparing 3-amino-7-bromocoumarin; (2) preparing 3-amino-7-pyrrole coumarin; (3) preparing 3-phenylmaleimide-7-pyrrole coumarin, and preparing the 3-phenylmaleimide-7-pyrrole And 4, preparing the deferritin encapsulated coumarin derivative fluorescent probe. The use method comprises the following steps: sealing and storing isoprene gas in an extracted plant sample in a gas bag, and carrying out constant-temperature reaction on the isoprene gas and the packaged coumarin derivative fluorescent probe for preparing the deferritin by utilizing a gas circulating device, so as to carry out fluorescence detection on a solution. The method has the advantages of simple operation, high sensitivity and strong anti-interference capability, and realizes the detection of isoprene in plants in the aqueous solvent.

Description

Technical Field

[0001] The present invention relates to an analytical method for detecting isoprene in plants by utilizing a coumarin derivative fluorescent probe encapsulated by apoferritin. Background Art

[0002] Isoprene (C₅H₄) is an important volatile organic compound released by plants and is widely involved in plant physiological metabolism, stress response, and atmospheric chemical processes. As a plant secondary metabolite, the release dynamics of isoprene can reflect plant photosynthesis efficiency, stress response level, and developmental stage characteristics, playing an important role in agricultural ecological monitoring, environmental quality assessment, and plant physiological research. Currently, plant isoprene detection mainly relies on technologies such as gas chromatography-mass spectrometry, proton transfer reaction mass spectrometry, and Raman spectroscopy. However, these methods generally suffer from problems such as bulky equipment, high operational specialization, and limited real-time monitoring capabilities. In particular, in complex matrix environments, they are susceptible to cross-interference from other volatile substances such as aldehydes and ketones, resulting in insufficient detection specificity. For example, the published patent "A method for detecting isoprene by surface-enhanced Raman spectroscopy" (Patent No.: CN116148241A) requires complex oxidation pretreatment of isoprene. Compared with this published patent, the present invention is simple to operate, has excellent anti-interference performance, high selectivity, and rapid detection, and has great application potential.

[0003] In response to the above technical bottlenecks, the present invention innovatively proposes a coumarin derivative fluorescent probe system based on apoferritin encapsulation, which utilizes the rigid hydrophobic cavity of apoferritin to efficiently load a coumarin derivative fluorescent probe with isoprene-specific recognition function, and realizes signal amplification and specific activation through the intramolecular charge transfer mechanism. Compared with traditional carriers (such as bovine serum albumin), the iron core cavity structure of apoferritin significantly improves the probe loading density and environmental stability. At the same time, the organic fluorescent probe is given the characteristic of water solubility, making it better adapted to the plant cell environment. The present invention overcomes the selectivity and anti-interference problems of in situ detection of isoprene in complex matrices through the "reaction-signal amplification" dual-function design.

[0004] In view of the shortcomings of isoprene analysis technology, such as high environmental sensitivity, lengthy detection cycle and insufficient specificity, the present invention provides a method for detecting isoprene in plants using a coumarin derivative fluorescent probe encapsulated by apoferritin.

[0005] A method for preparing a coumarin derivative fluorescent probe encapsulated with apoferritin is specifically carried out according to the following steps:

[0006] 1. Preparation of 3-amino-7-bromocoumarin: Glycine methyl ester hydrochloride and 5-bromosalicylaldehyde were added to high-purity water at room temperature and premixed at a rotation speed of 200 rpm to 300 rpm. Triethylamine was added to adjust the pH of the system to 9; the entire reaction system was refluxed at 70° C. to 90° C. for 30 minutes until the formation of an orange solid suspension was observed; the reaction mixture was cooled to room temperature and continued to stir to promote the precipitation of a precipitate; after precipitation was complete, the product was collected by suction filtration and washed with water; the crude product was recrystallized from ethanol to obtain an orange solid, i.e., 3-amino-7-bromocoumarin; the molar ratio of 5-bromosalicylaldehyde to glycine methyl ester hydrochloride in step 1 was 1:(1.5-2.5);

[0007] 2. Preparation of 3-amino-7-pyrrolecoumarin: palladium acetate, tri-tert-butylphosphine, sodium tert-butoxide and tetrahydropyrrole were added to benzene at room temperature, pre-mixed at 200 rpm to 300 rpm for 30 min to 40 min, and then 3-amino-7-bromocoumarin was added and refluxed at 90°C to 100°C for 8h to 10h; then the desired product was extracted with ethyl acetate, and the crude product was purified by silica gel column chromatography and eluted with petroleum ether and ethyl acetate, then rotary evaporated at 40°C to 50°C, and vacuum dried at 50°C to 70°C for 24h to 36h to obtain a light yellow product. a color solid, namely 3-amino-7-pyrrolcoumarin; the molar ratio of 3-amino-7-bromocoumarin to palladium acetate in step 2 is 1:(10-15); the molar ratio of 3-amino-7-bromocoumarin to tri-tert-butylphosphine in step 2 is 1:(6-9); the molar ratio of 3-amino-7-bromocoumarin to sodium tert-butoxide in step 2 is 1:(2-3); the molar ratio of 3-amino-7-bromocoumarin to tetrahydropyrrole in step 2 is 1:(1.2-1.5); the molar ratio of 3-amino-7-bromocoumarin to benzene in step 2 is 1:(8-10);

[0008] 3. Preparation of 3-phenylmaleimide-7-pyrrolecoumarin: palladium acetate, tri-tert-butylphosphine, sodium tert-butoxide and N-(4-bromophenyl)maleimide were added to benzene at room temperature, pre-mixed at 200 rpm to 300 rpm for 30 min to 40 min, and then 3-amino-7-pyrrolecoumarin was added and refluxed at 120° C. to 130° C. for 8 h to 10 h; the desired product was extracted with ethyl acetate, and the crude product was purified by silica gel column chromatography and eluted with petroleum ether, ethyl acetate and dichloromethane, and then the solvent was removed by rotary evaporation at 40° C. to 50° C., and then vacuum dried at 50° C. to 70° C. for 24 h to 36 h to obtain a yellow solid, i.e., 3-phenylmaleimide-7-pyrrolecoumarin. imide-7-pyrrolocoumarin; the molar ratio of 3-phenylmaleimide-7-pyrrolocoumarin to palladium acetate in step three is 1:(10-15); the molar ratio of 3-phenylmaleimide-7-pyrrolocoumarin to tri-tert-butylphosphine in step three is 1:(6-9); the molar ratio of 3-phenylmaleimide-7-pyrrolocoumarin to sodium tert-butoxide in step three is 1:(2-4); the molar ratio of 3-phenylmaleimide-7-pyrrolocoumarin to N-(4-bromophenyl)maleimide in step three is 1:(1.4-1.6); the molar ratio of 3-phenylmaleimide-7-pyrrolocoumarin to benzene in step three is 1:(10-15);

[0009] 4. Preparation of apoferritin-encapsulated coumarin derivative fluorescent probe: 50 mM PBS buffer solution, pH 7.4 and 100 μM apoferritin aqueous solution were heated to 50°C to 60°C and maintained for 20 min to 30 min, 1 mM coumarin derivative dimethyl sulfoxide solution was added, and premixed at a speed of 200 rpm to 300 rpm for 30 min to 40 min. The temperature was reduced to 20°C to 30°C, and the mixture was transferred to a dialysis bag and dialyzed for 2 h to 5 h to obtain apoferritin-encapsulated coumarin derivative fluorescent probe; the volume ratio of the 100 μM apoferritin aqueous solution to the PBS buffer solution in step 4 was 1: (9 to 11); the volume ratio of the 100 μM apoferritin aqueous solution to the 1 mM coumarin derivative dimethyl sulfoxide solution in step 4 was 1: (15 to 20).

[0010] An application of a coumarin derivative fluorescent probe encapsulated by apoferritin specifically comprises the following steps:

[0011] I. Determination of Isoprene Standard Concentration: Place a solution of a coumarin derivative fluorescent probe encapsulated with apoferritin and high-purity water in a sample vial. Add 5-10 mL of isoprene gas of varying concentrations to a gas bag. Circulate the gas using a gas circulation device at room temperature for 5-10 minutes. Record the fluorescence spectra of isoprene at varying concentrations at an excitation wavelength of 397 nm and the fluorescence intensity at 486 nm. Analyze the isoprene content based on the fluorescence enhancement of the fluorescent probe.

[0012] II. Detection of isoprene concentration in eucalyptus samples: Cover the eucalyptus branches with a 1 L air bag. Extract 5 mL to 10 mL of isoprene gas from the bag every 3 hours for 4 to 18 hours. Detect the isoprene gas according to step 1 above. Analyze the isoprene content based on the fluorescence enhancement of the coumarin derivative fluorescent probe.

[0013] Advantages of the present invention: (1) The probe is encapsulated with apoferritin, making it water-soluble and overcoming the pollution of traditional organic solvents, meeting the requirements of environmentally friendly technology. (2) The coumarin derivative probe encapsulated with apoferritin inhibits the aggregation and inactivation of the probe molecule in an aqueous environment through the cavity confinement effect, and enhances its photostability, overcoming the problem of easy photobleaching of traditional organic fluorescent probes. (3) Based on the intramolecular charge transfer effect of the coumarin probe and the isoprene-specific cycloaddition reaction, the anti-interference ability, response rate and recognition accuracy of the detection system are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is a high-resolution mass spectrum of a coumarin derivative fluorescent probe encapsulated by apoferritin in Example 1; HRMS (M+H) + 402.14499; by Figure 1 It can be seen that the apoferritin-encapsulated coumarin derivative fluorescent probe was successfully synthesized;

[0015] Figure 2 1 is a graph showing the fluorescence response of isoprene to the apoferritin-encapsulated coumarin derivative fluorescent probe in Example 1. The isoprene concentration in the standard sample of Example 1 ranges from 2 ppm to 620 ppm. The fluorescence intensity of the coumarin derivative fluorescent probe increases with increasing isoprene concentration.

[0016] Figure 3 : is a linear relationship graph of the isoprene concentration and the change in fluorescence intensity F / F0 of the apoferritin-encapsulated coumarin derivative fluorescent probe in Example 1; F is the fluorescence intensity of the fluorescent probe after adding isoprene, F0 is the fluorescence intensity of the fluorescent probe when no isoprene is added, and X is the isoprene concentration; the graph is plotted with the change in fluorescence intensity F / F0 of the coumarin derivative fluorescent probe as the ordinate and the isoprene concentration X as the abscissa. The results are shown in FIG. Figure 3As shown in the figure, the isoprene concentration showed a linear relationship with the change in the fluorescence intensity of the fluorescent probe F / F0. The linear regression equation for isoprene concentration in the range of 2 ppm to 620 ppm was F / F0 = 1.0267 + 0.0073X (R2 = 0.9974). The detection limit of this method for isoprene was 1.2 ppm.

[0017] Figure 4 The effect of reaction time on the isoprene and apoferritin encapsulated coumarin derivative fluorescent probe in Example 1; after adding isoprene, the fluorescence intensity of the apoferritin encapsulated coumarin derivative fluorescent probe increased rapidly. Figure 4 It can be seen that the detection of isoprene can be completed in about 5 min;

[0018] Figure 5 The effect of photobleaching on isoprene and apoferritin-encapsulated coumarin derivative fluorescent probe in Example 1; under photobleaching, the apoferritin-encapsulated coumarin derivative fluorescent probe still has the ability to recognize isoprene; Figure 5 It can be seen that 70% of the fluorescence intensity is still maintained after 10 h of photobleaching;

[0019] Figure 6 is a graph showing the change in isoprene release over time in the actual sample eucalyptus in Example 2; Figure 6 It can be seen that isoprene release exhibits an M-shaped distribution, primarily driven by temperature. Isoprene synthase activity shows a significant positive correlation with temperature. Within the 25-35°C range, enzyme activity increases with increasing temperature, promoting the synthesis and release of isoprene. When the temperature exceeds 40°C, enzyme activity decreases, likely due to protein denaturation. DETAILED DESCRIPTION

[0020] Example 1:

[0021] A method for preparing a coumarin derivative fluorescent probe encapsulated with apoferritin is specifically completed by the following steps:

[0022] 1. Preparation of 3-amino-7-bromocoumarin: Add 1.2 g of glycine methyl ester hydrochloride and 0.2 g of 5-bromosalicylaldehyde to high-purity water at room temperature, pre-mix at 250 rpm, and add triethylamine to adjust the pH of the system to 9. The entire reaction system was refluxed at 90 ° C for 30 minutes, and the reaction progress was monitored by thin layer chromatography until the formation of an orange solid suspension was observed. Subsequently, the reaction mixture was cooled to ambient temperature and continued to stir to promote the precipitation of the precipitate. After complete precipitation, the product was collected by suction filtration and washed with water. Finally, the crude product was recrystallized to obtain an orange-yellow solid, namely 3-amino-7-bromocoumarin;

[0023] 2. Preparation of 3-amino-7-pyrrolecoumarin: Add 0.112g of palladium acetate, 0.161g of tri-tert-butylphosphine, 0.23g of sodium tert-butoxide, and 0.54g of tetrahydropyrrole to benzene at room temperature. Premix at 250rpm for 30min, then add 0.87g of 3-amino-7-bromocoumarin and reflux at 100°C for 10h. The desired product is then extracted with ethyl acetate, and the crude product is purified by silica gel column chromatography and eluted with petroleum ether and ethyl acetate. The solvent is then removed by rotary evaporation at 45°C, and vacuum dried at 60°C for 36h to obtain a light yellow solid, i.e., 3-amino-7-pyrrolecoumarin.

[0024] 3. Preparation of 3-phenylmaleimide-7-pyrrolecoumarin: 0.112g of palladium acetate, 0.161g of tri-tert-butylphosphine, 0.23g of sodium tert-butoxide, and 1.9g of N-(4-bromophenyl)maleimide were added to benzene at room temperature and premixed at 250rpm for 30min. 1.45g of 3-amino-7-pyrrolecoumarin was then added. The mixture was refluxed at 130°C for 10h and then cooled to room temperature. The desired product was then extracted with ethyl acetate. The crude product was purified by silica gel column chromatography and eluted with petroleum ether, ethyl acetate, and dichloromethane. The solvent was then removed by rotary evaporation at 45°C and vacuum dried at 60°C for 46h to obtain a yellow solid, namely 3-phenylmaleimide-7-pyrrolecoumarin.

[0025] 4. Preparation of apoferritin-encapsulated coumarin derivative fluorescent probe: 50 mM PBS buffer solution, pH 7.4, and 100 μM apoferritin aqueous solution were heated at 60°C for 30 min, 1 mM coumarin derivative dimethyl sulfoxide solution was added, and premixed at 250 rpm for 30 min. The temperature was lowered to 25°C, and the mixture was transferred to a dialysis bag for dialyzation to obtain apoferritin-encapsulated coumarin derivative fluorescent probe;

[0026] Example 2:

[0027] A method for detecting isoprene in plants using an apoferritin-encapsulated coumarin derivative fluorescent probe is specifically accomplished by the following steps:

[0028] I. Determination of isoprene standard concentration: Place 200 μL of apoferritin-encapsulated coumarin derivative fluorescent probe solution and 1800 μL of high-purity water in a sample vial. Add 5 mL of isoprene gas of varying concentrations to the gas bag and circulate the gas at room temperature for 5 minutes using a gas circulation device. Record the fluorescence spectra of isoprene at varying concentrations at an excitation wavelength of 397 nm and the fluorescence intensity at 486 nm. Analyze the isoprene content based on the fluorescence enhancement of the fluorescent probe.

[0029] II. Detection of isoprene concentration in eucalyptus samples: Cover the eucalyptus branches with a 1 L air bag. Extract 5 mL of isoprene gas from the bag every 3 hours for 4 to 18 hours. Detect the isoprene gas according to step 1 above. Analyze the isoprene content based on the fluorescence enhancement of the coumarin derivative fluorescent probe.

Claims

1. A method for preparing a coumarin derivative fluorescent probe encapsulated by apoferritin, characterized in that Prepared according to the following steps:

1. Preparation of 3-amino-7-bromocoumarin: Glycine methyl ester hydrochloride and 5-bromosalicylaldehyde are added to high-purity water at room temperature and pre-mixed at a rotation speed of 200 rpm to 300 rpm. Triethylamine is added to adjust the pH of the system to 7 to 9; the entire reaction system is refluxed at 70° C. to 90° C. for 30 minutes, and the reaction progress is monitored by thin-layer chromatography until the formation of an orange solid suspension is observed; the reaction mixture is cooled to ambient temperature and continued to be stirred to promote the precipitation of a precipitate. After precipitation is complete, the product is collected by suction filtration and washed with water; the crude product is recrystallized from ethanol or methanol to obtain an orange solid, i.e., 3-amino-7-bromocoumarin; the molar ratio of 5-bromosalicylaldehyde to glycine methyl ester hydrochloride in step 1 is 1:(1.5 to 2.5); 2. Preparation of 3-amino-7-pyrrolecoumarin: palladium acetate, tri-tert-butylphosphine, sodium tert-butoxide and tetrahydropyrrole were added to benzene at room temperature; premixed at 200 rpm to 300 rpm for 30 min to 40 min, 3-amino-7-bromocoumarin was added and refluxed at 90°C to 100°C for 8h to 10h; the desired product was extracted with ethyl acetate, and the crude product was purified by silica gel column chromatography and eluted with eluents of petroleum ether and ethyl acetate, and then the solvent was removed by rotary evaporation at 40°C to 50°C, and vacuum dried at 50°C to 70°C for 24h to 36h to obtain a light Yellow solid, i.e. 3-amino-7-pyrrolecoumarin; the molar ratio of 3-amino-7-bromocoumarin to palladium acetate in step 2 is 1:(10-15); the molar ratio of 3-amino-7-bromocoumarin to tri-tert-butylphosphine in step 2 is 1:(6-9); the molar ratio of 3-amino-7-bromocoumarin to sodium tert-butoxide in step 2 is 1:(2-3); the molar ratio of 3-amino-7-bromocoumarin to tetrahydropyrrole in step 2 is 1:(1.2-1.5); the molar ratio of 3-amino-7-bromocoumarin to benzene in step 2 is 1:(8-10); 3. Preparation of 3-phenylmaleimide-7-pyrrolocoumarin: N-(4-bromophenyl)maleimide and 3-amino-7-pyrrolocoumarin were added to benzene at room temperature; premixed at 200 rpm to 300 rpm for 30 to 40 minutes, refluxed at 120° C. to 130° C. for 8 to 10 hours, and then cooled to room temperature; the desired product was extracted with ethyl acetate, and the crude product was purified by silica gel column chromatography and eluted with petroleum ether, ethyl acetate, and dichloromethane. The solvent was then removed by rotary evaporation at 40° C. to 50° C., and vacuum dried at 50° C. to 70° C. for 24 to 36 hours to obtain a yellow solid, namely 3-phenylmaleimide- 7-pyrrocoumarin; the molar ratio of 3-phenylmaleimide-7-pyrrocoumarin to palladium acetate in step three is 1:(10-15); the molar ratio of 3-phenylmaleimide-7-pyrrocoumarin to tri-tert-butylphosphine in step three is 1:(6-9); the molar ratio of 3-phenylmaleimide-7-pyrrocoumarin to sodium tert-butoxide in step three is 1:(2-4); the molar ratio of 3-phenylmaleimide-7-pyrrocoumarin to N-(4-bromophenyl)maleimide in step three is 1:(1.4-1.6); the molar ratio of 3-phenylmaleimide-7-pyrrocoumarin to benzene in step three is 1:(10-15); 4. Preparation of apoferritin-encapsulated coumarin derivative fluorescent probe: 50 mM PBS buffer solution, pH 7.4, and 100 μM apoferritin aqueous solution were heated at 50°C to 60°C for 20 min to 30 min, and 1 mM coumarin derivative dimethyl sulfoxide solution was added; premixed at a speed of 200 rpm to 300 rpm for 30 min to 40 min, the temperature was reduced to 20°C to 30°C, and the mixture was transferred to a dialysis bag and dialyzed for 2 h to 5 h to obtain apoferritin-encapsulated coumarin derivative fluorescent probe; the volume ratio of the 100 μM apoferritin aqueous solution to the PBS buffer solution in step 4 was 1: (9 to 11); the volume ratio of the 100 μM apoferritin aqueous solution to the 1 mM coumarin derivative dimethyl sulfoxide solution in step 4 was 1: (1 to 1.5).

2. A method for detecting isoprene in plants using a coumarin derivative fluorescent probe encapsulated by apoferritin, characterized in that This is done by: I. Determination of isoprene standard concentration: Place a solution of a coumarin derivative fluorescent probe encapsulated with apoferritin and high-purity water in a sample vial. Add 5-10 mL of isoprene gas of varying concentrations to a gas bag. Circulate the gas using a gas circulation device at room temperature for 5-10 minutes. Record the fluorescence spectra of isoprene at varying concentrations at an excitation wavelength of 397 nm and the fluorescence intensity at 486 nm. Analyze the isoprene content based on the fluorescence enhancement of the fluorescent probe. II. Detection of isoprene concentration in eucalyptus samples: Cover a eucalyptus branch with a 1 L air bag. Extract 5 mL to 10 mL of isoprene gas from the bag every 3 hours between 7:00 and 22:

00. Detect the isoprene gas according to step 1 above. Analyze the isoprene content based on the fluorescence enhancement of the coumarin derivative fluorescent probe.

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