AIE ratio-dependent fluorescent probe and synthesis and application thereof
By designing the AIE ratio fluorescent probe TPNP and using the 545 nm and 420 nm fluorescence intensity ratios for self-calibration, the problems of ACQ effect and environmental interference in sulfur dioxide detection in plants were solved, and high selectivity and high sensitivity sulfur dioxide detection was achieved.
Patent Information
- Application Number
- CN202510437125.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-09
- Publication Date
- 2025-07-11
AI Technical Summary
When existing fluorescent probes detect sulfur dioxide derivatives in plants, there is an aggregation-induced quenching effect (ACQ), which leads to weakening of fluorescence, making it difficult to meet the needs of high concentration detection. A single wavelength signal is susceptible to probe concentration and environmental interference, and high-precision detection cannot be achieved.
A ratio-type fluorescence probe TPNP is designed, using D-π-A structure, and self-calibrating using the fluorescence intensity ratio at 545 nm and 420 nm, identify sulfur dioxide and destroy the conjugated system through Michael addition reaction to achieve ratio-type detection.
It realizes high selectivity and high sensitivity sulfur dioxide detection in plants, has fast response time, is suitable for high concentration and aggregated environments, reduces background interference, and improves detection reliability and accuracy.
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Figure CN120289518A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fluorescent probes, and particularly relates to an AIE ratiometric fluorescent probe for detecting sulfur dioxide in plants, and its synthesis and application. Background Art
[0002] Sulfur dioxide (SO2), as one of the main components of air pollution, can enter tissues through plant stomata and be converted into sulfite (SO3²⁻) and bisulfite (HSO3⁻), interfering with the redox balance of plant cells, inhibiting the function of chloroplasts, and causing typical stress symptoms such as photosynthesis inhibition and leaf chlorosis. Accurately monitoring the spatio-temporal distribution dynamics of SO2 derivatives in plants has important scientific value for evaluating the degree of environmental pollution stress and analyzing the plant stress response mechanism.
[0003] Traditional detection methods such as ion chromatography and electrochemical analysis rely on tissue disruption and extraction, with the inherent defects of destroying sample integrity and being unable to achieve in-situ real-time monitoring. Fluorescent probe technology has become an important tool for in-vivo detection due to its high sensitivity, spatio-temporal resolution ability, and non-invasive characteristics. However, conventional fluorescent probes generally face the aggregation-caused quenching (ACQ) effect, where fluorescence decreases sharply at high concentrations or in the solid state, severely restricting their application reliability in complex biological systems.
[0004] The breakthrough development of aggregation-induced emission (AIE) materials provides a new idea for solving the ACQ bottleneck. AIE chromophores have weak fluorescence in the dispersed state, while the luminescence intensity is significantly enhanced in the aggregated state due to the restriction of intramolecular motion (RIM) effect, which is particularly suitable for high-concentration detection environments and solid-state imaging scenarios. However, most existing AIE-based SO2 probes adopt a single-wavelength intensity signal output mode, which is easily affected by probe concentration distribution, instrument parameter fluctuations, and environmental interference factors, resulting in an increase in quantitative analysis errors and making it difficult to meet the high-precision detection requirements of the complex microenvironment in plants.
[0005] Ratiometric fluorescent probes construct a dual-emission channel and use the ratio of fluorescence intensities at two characteristic wavelengths for self-calibration, which can effectively eliminate background interference and significantly improve the detection reliability. At present, the research on ratiometric AIE probes for SO2 derivatives is still in its infancy, and most existing designs focus on animal cell systems, failing to fully consider the unique cell wall barrier effect, vacuolar compartmentalized storage, and chloroplast targeting requirements of plant cells, resulting in low probe penetration efficiency and poor subcellular localization specificity, severely restricting their application potential in plant in-vivo detection. Therefore, it is of great significance to develop an AIE ratiometric fluorescent probe for highly selective and sensitive detection of SO2 in plants. Summary of the Invention
[0006] The object of the present invention is to overcome the deficiencies of the prior art and provide an AIE ratiometric fluorescent probe for detecting SO2 in plants, its synthesis method and application.
[0007] The object of the present invention can be achieved by the following technical solutions: An AIE ratiometric fluorescent probe TPNP, whose structural formula is shown as follows: 。
[0008] This fluorescent probe uses triphenylamine as the AIE fluorophore and energy donor (D), diethyl cyanomethylphosphonate as the energy acceptor (A), and introduces a C=C double bond as the SO2 recognition site, and designs and synthesizes an AIE luminophore TPNP with a typical D-π-A structure. The probe TPNP has a large conjugated system and has a strong fluorescence emission signal at a long wavelength (545 nm). As Figure 1 shown, when the probe responds to SO2, SO2 reduces the C=C double bond of the probe through a Michael addition reaction to a C-C single bond, destroying the conjugated system of the probe molecule, resulting in a rapid decrease in fluorescence at 545 nm, and at the same time inducing a significant increase in fluorescence at 420 nm, realizing the ratiometric detection of SO2.
[0009] The synthesis route of the above AIE ratiometric fluorescent probe TPNP is as Figure 2 shown.
[0010] The synthesis method of the AIE ratiometric fluorescent probe TPNP includes: Dissolve 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde and diethyl cyanomethylphosphonate in an organic solvent, add a catalytic amount of piperidine, and heat under reflux for reaction; After the reaction is completed, cool the reaction solution, filter it under reduced pressure, wash and dry the obtained solid, and purify it to obtain an orange-yellow solid, which is the fluorescent probe TPNP.
[0011] In some embodiments, the organic solvent is preferably anhydrous ethanol.
[0012] In some embodiments, the molar ratio of 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde to diethyl cyanomethylphosphonate is 1:(1.2 - 2.0).
[0013] In some embodiments, the molar ratio of 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde to piperidine is 100:(3 - 5).
[0014] In some embodiments, the reaction temperature is 70 - 90 °C.
[0015] In some embodiments, the reaction time is 6 - 12 hours.
[0016] In some embodiments, the purification is column chromatography separation, and the column chromatography conditions are as follows: ethyl acetate and petroleum ether are used as eluents, and the volume ratio is 1:(2 - 10); the silica gel is 200 - 300 mesh.
[0017] The present invention also provides the application of the above AIE ratiometric fluorescent probe in detecting SO2.
[0018] The present invention also provides the application of the above AIE ratiometric fluorescent probe in detecting SO2 in plants.
[0019] Advantages of the present invention: 1. The fluorescent probe of the present invention realizes ratiometric monitoring and imaging of SO2 in plants through fluorescence at 545 nm and 420 nm, and has the advantages of low background interference and small light damage to biological samples.
[0020] 2. The fluorescent probe of the present invention has enhanced fluorescence in the aggregated state or at high concentration, has typical AIE luminescence properties, overcomes the ACQ effect, and is suitable for the high-viscosity microenvironment of plant tissues.
[0021] 3. The fluorescent probe of the present invention has high selectivity, specifically recognizes SO2, and does not respond to other active substances in plants.
[0022] 4. The fluorescent probe of the present invention has a fast response time (1 min) and high sensitivity for recognizing SO2.
[0023] 5. The synthesis steps of the probe of the present invention are simple, the raw materials are easily available, the reaction conditions are mild, and the yield is high. Description of the Drawings
[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.
[0025] Figure 1 Shows the reaction principle of the fluorescent probe TPNP of the present invention for detecting SO2; Figure 2 Shows the synthesis route of the fluorescent probe TPNP of the present invention; Figure 3 Is the 1H NMR spectrum of the fluorescent probe TPNP of the present invention (the solvent is CDCl3); Figure 4 Is the 13C NMR spectrum of the fluorescent probe TPNP of the present invention (the solvent is CDCl3); Figure 5 1H NMR spectrum of the fluorescent probe TPNP of the present invention (solvent: CDCl3); Figure 6 High-resolution mass spectrum of the fluorescent probe TPNP of the present invention; Figure 7 UV and fluorescence titration diagrams of the fluorescent probe TPNP of the present invention for the recognition of SO2; Figure 8 Lowest detection limit diagram of the fluorescent probe TPNP of the present invention for the recognition of SO2; Figure 9 Kinetic curve diagram of the fluorescent probe TPNP of the present invention for the recognition of SO2; Figure 10 UV and fluorescence selectivity diagrams of the fluorescent probe TPNP of the present invention for the recognition of SO2; Figure 11 AIE performance test of the fluorescent probe TPNP of the present invention; Figure 12 Fluorescence imaging diagram of the fluorescent probe TPNP of the present invention for the recognition of SO2 in tobacco root tips.
[0026] Detailed Description of the Invention The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts fall within the scope of protection of the present invention.
[0027] Example 1 Synthesis of AIE ratiometric fluorescent probe: In a 50 mL round-bottom flask, accurately weigh 4'-(diphenylamine)-[1,1'-biphenyl]-4-carbaldehyde (Compound T1, 349.4 mg, 1 mmol) and diethyl cyanomethylphosphonate (Compound T2, 265.7 mg, 1.5 mmol) and dissolve them in 10 mL of anhydrous ethanol solvent. Then, add piperidine (2.6 mg, 0.03 mmol) dropwise to the flask. The above mixed system is heated and stirred at 80 °C for 10 hours.
[0028] After the reaction, filter and collect the precipitate, wash it three times, and the crude product is purified by silica gel column chromatography. The eluent is ethyl acetate: petroleum ether = 1:3 to obtain the target orange-yellow probe compound with a yield of 80%.
[0029] 1H NMR measurement: 1 1H NMR (400 MHz, Chloroform- d) δ 7.99 - 7.89 (m, 3H), 7.61 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.7 Hz, 2H), 7.24 - 7.17 (m, 4H), 7.10 - 6.96 (m, 8H), 4.16 (dq, J = 13.8, 7.1 Hz, 4H), 1.34 (t, J = 7.0 Hz, 6H). 13C NMR measurement: 13 13C NMR (101 MHz, Chloroform- d ) δ 158.42 (d, J = 7.2 Hz), 148.51, 147.30, 145.26, 132.43, 131.27, 130.77 (d, J = 18.0 Hz), 129.43, 127.87, 126.92, 124.95, 123.55, 123.03, 115.82 (d, J = 10.3 Hz), 97.56, 63.59 (d, J = 5.8 Hz), 16.29 (d, J = 6.4 Hz). 31P NMR measurement: 31 31P NMR (162 MHz, Chloroform- d ) δ 11.66. The 1H NMR spectrum is as shown in Figure 3 , the 13C NMR spectrum is as shown in Figure 4 , and the 31P NMR spectrum is as shown in Figure 5 .
[0030] High resolution mass spectrometry measurement: HR-MS (m / z) calcd for chemical formula C 31 H 29 N2NaO3P + [M] + : 531.1813, Found: 531.1808. The high resolution mass spectrum is as shown in Figure 6 .
[0031] Example 2 Dissolve the fluorescent probe TPNP synthesized in Example 1 in DMSO solution to prepare a fluorescent probe stock solution with a concentration of 1.0 mM for standby.
[0032] Prepare a stock solution of NaHSO3 (SO2 derivative) using ultrapure water. Then, test the spectral response of the probe to the SO2 derivative in a phosphate buffer solution (PBS, pH 7.4, 10 mM, containing 10 mM CTAB).
[0033] As Figure 7 shown in Fig. a, the individual probe TPNP (10 μM) has a strong ultraviolet absorption peak at 420 nm in the solution. When the SO2 derivative (0 - 100 μM) is added, its conjugated structure is disrupted, the absorption at 420 nm gradually disappears, and a new absorption peak appears at 340 nm.
[0034] From the fluorescence emission spectra ( Figure 7 Figs. b and 7c), when excited at a wavelength of 420 nm, the probe TPNP (10 μM) has obvious fluorescence emission at 545 nm. When excited at a wavelength of 350 nm, the probe TPNP has almost no fluorescence emission.
[0035] After the probe responds to the SO2 derivative (0 - 100 μM), its fluorescence at 545 nm decreases rapidly, and the fluorescence at 420 nm increases significantly. The above experiments show that the probe can achieve ratio detection of the SO2 derivative at two wavelengths of 545 nm and 420 nm.
[0036] As shown in the appendix Figure 8 Figs., by linearly fitting the fluorescence intensities of the probe TPNP at 545 nm and 420 nm with the concentration of the added SO2 derivative, a high linear correlation is presented. According to the lowest detection limit equation LOD = 3σ / k, the detection limit is calculated to be 20.1 nM. This indicates that the probe TPNP has high sensitivity for the detection of SO2.
[0037] Example 3 This example evaluates the response kinetics of the probe TPNP to SO2.
[0038] Add the SO2 derivative (0, 20 μM, 50 μM, 100 μM) to the solution containing the probe TPNP (10 μM), and record the change in the fluorescence ratio of the probe I 420nm / 545nm .
[0039] As shown in the appendix Figure 9 Figs., when no SO2 derivative is added, the fluorescence ratio of the probe TPNP hardly changes within 0 to 1000 seconds. 420nm / 545nm However, after the SO2 derivative is added, its fluorescence ratio of I
[0040] 420nm / 545nm 420nm / 545nm increases significantly within 100 seconds.
[0041] The experimental results show that the probe has a fast response time for the detection of SO2.
[0042] Example 4 There is a complex physiological environment with multiple coexisting components in plants, including various endogenous active molecules (such as reactive oxygen species, glutathione, etc.) and metal ions. Therefore, the probe needs to have a highly specific recognition ability to exclude the interference of non-target substances, which is the key prerequisite for its reliable application in in vivo plant detection.
[0043] In a buffer solution containing 10 μM of the probe TPNP, and then various other potential competitive analytes (TBHP, H2O2, GSH, Hcy, Cys, Lys, NO, Ac - , Br - , I - , ClO-, CO3 2- , HCO3 2- , H2PO4 - , HPO4 2- , HS - , S2O3 2- , NO2 - , NO3 - ) and SO2 derivatives (NaHSO3, 100 μM) were added to the system in sequence. The fluorescence emission spectra were measured using a fluorescence emission spectrometer, and the fluorescence emission curves were recorded.
[0044] As shown in FIGS. Figure 10 a and 10b, the probe TPNP exhibits good photophysical properties in the PBS buffer system: when the excitation wavelength is 420 nm, it shows a significant fluorescence emission peak at 554 nm; while at an excitation wavelength of 350 nm, no obvious fluorescence signal is observed at 420 nm. It is worth noting that even when 10-fold concentrations of potential interfering substances (TBHP, H2O2, GSH, Hcy, Cys, Lys, NO, Ac - , Br - , I - , ClO-, CO3 2- , HCO3 2- , H2PO4 - , HPO4 2- , HS - , S2O3 2- , NO2 - , NO3 - ) are added to the system, the fluorescence emission spectrum of the probe TPNP remains stable, without peak position shift or significant intensity change.
[0045] Only when SO2 (100 μM) was introduced into the system, the fluorescence intensity at 554 nm was quenched, while the intensity of the newly emerged emission peak at 420 nm was significantly enhanced.
[0046] The above experimental results indicate that the probe has a high selective recognition ability for SO2 and its derivatives, and can effectively overcome the influence of complex matrices in plants.
[0047] Example 5 To further study the luminescence behavior of the probe TPNP, the changes in the emission spectra of this example were tested in DMSO solutions with different water contents.
[0048] In 3 mL of mixed solutions of DMSO and water with different ratios (the ratio of water is 0% - 100%), the probe TPNP solution (10 μM) was added, and then on a fluorescence spectrometer, using 420 nm as the excitation wavelength, the fluorescence emission of the probe was tested.
[0049] As Figure 11 shown, with the gradual increase in the water content in the solution, the fluorescence intensity of the probe TPNP generally showed an increasing trend. When the water content reached 99%, the fluorescence intensity of the probe TPNP increased by 20 times respectively. The experiment shows that when the probe TPNP is dispersed in the solution, there is almost no fluorescence emission, while it aggregates in aqueous solution, generating an obvious fluorescence signal, and has typical AIE luminescence properties.
[0050] Example 6 In this example, the application potential of the fluorescent probe TPNP in the detection and imaging of SO2 derivatives in tobacco root tips was explored.
[0051] Four-leaf stage seedlings of tobacco seedlings with uniform growth were selected and hydroponically cultured with Hoagland's nutrient solution. The tobacco seedlings were incubated with the probe TPNP (10 μM) at room temperature for 30 min, washed with distilled water and then imaged by a laser scanning confocal microscope. Subsequently, NaHSO3 (100 μM) was added and incubated for 60 min. After the treatment was completed, the tobacco seedlings were washed with distilled water and then imaged.
[0052] As Figure 12 shown, when imaging and observing after incubating tobacco seedlings with the TPNP solution (10 μM) for 30 minutes, the results showed that significant fluorescence signals were observed in the green fluorescence channel ( Figure 12 A2), while weak fluorescence signals were presented in the tobacco root tips under the blue fluorescence channel ( Figure 12 A3).
[0053] Subsequently, under the same experimental conditions, a solution of NaHSO3 (100 μM) was additionally added to the culture solution of tobacco seedlings that had been incubated with the probe TPNP, and the incubation was continued for 60 minutes. The subsequent imaging ( Figure 12 B2-12B3) revealed a significant decrease in the intensity of green fluorescence, while the blue fluorescence signal was significantly enhanced.
[0054] Figure 12 A1 and 12B1 are photos of the tobacco root tips under visible light. The photos show that the root tip tissues are intact, indicating that the probe causes less damage to the tissues. The experimental results of this series of tobacco seedling root tips strongly prove that the probe TPNP of the present invention has the ability to effectively achieve fluorescence ratio imaging and detection of SO2 in living plants.
[0055] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.
Claims
1. An AIE ratiometric fluorescent probe, the structural formula of which is as follows: 。 2. The synthesis method of the AIE ratiometric fluorescent probe according to claim 1, comprising: Dissolve 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde and diethyl cyanomethylphosphonate in an organic solvent, add a catalytic amount of piperidine, and heat under reflux for reaction; After the reaction is completed, cool the reaction solution, filter it under reduced pressure, wash and dry the obtained solid, and purify it to obtain an orange-yellow solid, namely the fluorescent probe TPNP.
3. The synthesis method according to claim 2, characterized in that, The organic solvent is anhydrous ethanol.
4. The synthesis method according to claim 2, characterized in that, The molar ratio of 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde to diethyl cyanomethylphosphonate is 1:(1.2 - 2.0).
5. The synthesis method according to claim 2, characterized in that, The molar ratio of 4'-(diphenylamino)-[1,1'-biphenyl]-4-carbaldehyde to piperidine is 100:(3 - 5).
6. The synthesis method according to claim 2, characterized in that, The reaction temperature is 70 - 90 °C.
7. The synthesis method according to claim 2, characterized in that, The reaction time is 6 - 12 hours.
8. The synthesis method according to claim 2, wherein The purification is column chromatography separation, and the column chromatography conditions are as follows: ethyl acetate and petroleum ether are used as eluents, and the volume ratio is 1:(2 - 10); the silica gel is 200 - 300 mesh.
9. The application of the AIE ratiometric fluorescent probe according to claim 1 or the AIE ratiometric fluorescent probe obtained by the synthesis method according to claims 2 - 8 in the detection of SO2.
10. The application of the AIE ratiometric fluorescent probe according to claim 1 or the AIE ratiometric fluorescent probe obtained by the synthesis method according to any one of claims 2 - 8 in the detection of SO2 in plants.