AIE-based fluorescent probe, preparation method thereof and application of AIE-based fluorescent probe in preparation of COX-2 inhibitor screening kit

Abstract

The invention provides an AIE-based fluorescent probe, a preparation method thereof and application of the AIE-based fluorescent probe in preparation of a COX-2 inhibitor screening kit. The probe disclosed by the invention can be used for determining the activity of COX-2 in vitro, the probe has a good response relationship and selectivity on COX-2, the detection limit is as low as 0.007 mu g / mL, the probe has relatively high sensitivity, and the fluorescent probe can be used for screening anti-inflammatory active components from lotus seed pots.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to an AIE-based fluorescent probe, its preparation method, and its application in the preparation of a COX-2 inhibitor screening kit. Background Technology

[0002] Inflammation is a defensive response of the body to harmful stimuli; however, excessive inflammation can cause serious damage to the body, even leading to loss of bodily function and death. Inflammation is closely related to many common diseases, including arthritis, colitis, diabetic nephropathy, and cardiovascular diseases. Cyclooxygenase-2 (COX-2) is rapidly expressed under the stimulation of a series of pro-inflammatory cytokines and regulates local and systemic inflammatory responses by catalyzing the conversion of arachidonic acid into prostaglandins, and has been considered one of the important targets for anti-inflammatory and analgesic effects. Currently, various small-molecule fluorescent probes have been developed to achieve the fluorescence detection of COX-2. However, traditional fluorophores may encounter aggregation quenching (ACQ) problems at high concentrations or in the solid state, which will reduce detection sensitivity and limit the application of probes. In recent years, fluorescent probes based on aggregation-induced emission (AIE) have gradually emerged. These AIE materials have the advantages of avoiding fluorescence attenuation in the aggregated state, excellent photostability, and high sensitivity, and have been widely used in fields such as biosensing, bioimaging, and photodynamic therapy.

[0003] Traditional Chinese medicine (TCM) theory holds that the core pathogenesis of inflammation is often the accumulation of pathogenic factors and masses, primarily due to heat toxins and blood stasis. Lotus seedpods, a traditional Chinese medicine, are used to treat metrorrhagia, carbuncles, lochia retention, and postpartum blood stasis due to their properties of resolving blood stasis, stopping bleeding, clearing heat, and detoxifying. They also provide good relief for pain and swelling caused by blood stasis. Modern pharmacological studies have shown that lotus seedpod extract can reduce the production of pro-inflammatory cytokines TNF-α and interleukin-6 in LPS-stimulated HepG2 cells, inhibit the expression levels of inflammatory mediators COX-2 and iNOS proteins, and improve liver toxicity induced by excessive acetaminophen. Summary of the Invention

[0004] In view of this, the present invention aims to overcome the defects in the prior art and proposes an AIE-based fluorescent probe, its preparation method and its application in the preparation of a COX-2 inhibitor screening kit for specific detection of COX-2 activity, so as to solve the problems of insufficient sensitivity and poor specificity of existing COX-2 activity detection tools.

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a fluorescent probe based on AIE, the structural formula of which is shown in Formula III: Formula III.

[0006] Secondly, the present invention also provides a method for preparing the above-mentioned AIE-based fluorescent probe. The preparation of the AIE-based fluorescent probe of the present invention requires first synthesizing a fluorescent group and a linker group, and then obtaining the final probe through a coupling reaction.

[0007] Includes the following steps: Step 1: 2,3-bis(4-bromophenyl)-2-butenidonitrile is coupled with triphenylamine 4-borate to generate the compound shown in Formula I; the reaction formula is shown below:

[0008] Formula I Step 2: The compound shown in Formula I undergoes a substitution reaction with 6-amino-1-hexanol to generate the compound shown in Formula II; the reaction formula is shown below:

[0009] Formula I Formula II Step 3: The compound shown in Formula II undergoes an esterification reaction with indomethacin to generate the compound shown in Formula III, which is the AIE-based fluorescent probe; the reaction formula is shown below:

[0010] Formula II and Formula III.

[0011] Preferably, the substitution reaction in step one further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, and an organic solvent, wherein the base is selected from one or more of potassium carbonate, potassium phosphate, cesium carbonate, and sodium carbonate; the palladium catalyst is selected from one or more of tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, dichloro(triphenylphosphine)palladium, and 1,3-bis(2,6-diisopropylphenyl)imidazolium-2-ylpyridinium dichloride; and the organic solvent is selected from one or more of tetrahydrofuran, toluene, and 1,4-dioxane.

[0012] Preferably, the molar ratio of bis(4-bromophenyl)-2-butenidonitrile, triphenylamine 4-boronic acid, base, and palladium catalyst is 100:(50~60):(500~800):(3~5).

[0013] Preferably, the reaction conditions for the substitution reaction in step one are: a temperature of 60~80℃ and a reaction time of 12~16h.

[0014] Preferably, the coupling reaction in step two further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, a ligand, and an organic solvent. The base is selected from one or more of cesium carbonate, potassium phosphate, sodium tert-butoxide, and potassium tert-butoxide; the palladium catalyst is selected from one or more of tris(dibenzylacetone)palladium, tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, and palladium acetate; the ligand is selected from one or more of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphine-2'-methylbiphenyl, and 1,1'-bis(diphenylphosphine)ferrocene; and the organic solvent is selected from one or more of toluene, 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide.

[0015] Preferably, the molar ratio of the compound represented by Formula I, 6-amino-1-hexanol, palladium catalyst, ligand, and base is 365:(400~480):(15~22):(30~40):(650~800).

[0016] Preferably, the reaction conditions for the coupling reaction in step two are: a temperature of 80~120℃ and a reaction time of 8~16h.

[0017] Preferably, the esterification reaction in step three further includes the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine, and an organic solvent in an inert atmosphere, wherein the organic solvent is selected from one or more of dichloromethane, anhydrous diethyl ether, tetrahydrofuran, and N,N-dimethylformamide.

[0018] Preferably, the molar ratio of the compound represented by Formula II, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 165:(220~280):(400~550):(25~42).

[0019] Preferably, the reaction conditions for the esterification reaction in step three are: room temperature and reaction time of 8-14 hours.

[0020] Thirdly, the present invention also provides the application of the above-mentioned AIE-based fluorescent probe in COX-2 activity detection or COX-2 inhibitor screening.

[0021] Fourthly, the present invention also provides the application of the above-mentioned AIE-based fluorescent probe in the preparation of COX-2 activity detection kits or COX-2 inhibitor screening kits.

[0022] Fifthly, the present invention provides a method for detecting COX-2 activity using an AIE-based fluorescent probe, the detection method comprising the following steps: (1) Dilute the COX-2 stock solution to obtain COX-2 detection solutions of different concentrations; weigh the probe solutions prepared by the above AIE-based fluorescent probe powder, dispense them and store them in a -80℃ refrigerator for later use; (2) Add probe solution, COX-2 solution of different concentrations and PBS buffer to a 96-well plate in sequence to obtain a mixed solution. Shake the mixed solution and place it in an incubator for incubation. (3) Measure the emission spectrum of the incubated mixture at an excitation wavelength of 460 nm and record the fluorescence intensity at 600 nm to obtain the COX-2 concentration data of the mixture, thus realizing the COX-2 activity detection; The detection method for COX-2 has a detection range of 0~0.5 μg / mL, and the linear equation is F-F0=769.26C+9.5256(R). 2 =0.9972), where F is the fluorescence intensity of the COX-2+ probe; F0 is the fluorescence intensity without COX-2, and the detection limit is 0.007 μg / mL.

[0023] Preferably, in step (2), the final concentration of the probe solution in the mixed solution is 5 μM, the PBS buffer is a PBS buffer with pH=6.8, the incubation temperature is 37°C, and the incubation time is 20 minutes.

[0024] Preferably, in step (1), the AIE-based fluorescent probe powder is dissolved in PBS buffer with 10% THF.

[0025] Preferably, in step (2), 20 μL of 50 μM probe solution, 20 μL of COX-2 solution of different concentrations and 160 μL of PBS buffer are added sequentially to the 96-well plate.

[0026] Sixthly, the present invention also provides a method for testing COX-2 activity inhibition using an AIE-based fluorescent probe, the detection method comprising the following steps: (1) COX-2 was diluted with PBS buffer to obtain COX-2 detection solutions of different concentrations; TPADFN-IMC powder based on AIE was weighed and prepared into probe solutions, which were then aliquoted and stored at -80℃ for later use. (2) First, add the sample solution to be tested, PBS, and COX-2 solution to the 96-well plate, incubate, then add the probe solution, incubate again, and measure the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The inhibition rate I was calculated using the formula: I(%)=(F-Fs) / (F-F0)×100%, where F is the fluorescence intensity of COX-2 and + probes; Fs is the fluorescence intensity of +, COX-2 and + probes in the sample to be tested; and F0 is the fluorescence intensity without COX-2.

[0027] Preferably, in step (2), 20 μL of the sample solution to be tested, 40 μL of PBS, and 20 μL of 0.4 μg / mL COX-2 solution are added to the 96-well plate, and after incubation, 20 μL of 50 μM probe solution is added.

[0028] Compared with the prior art, the present invention has the following advantages: This invention constructs a fluorescent probe based on the aggregation-induced emission (AIE) effect for detecting COX-2 and screening natural COX-2 inhibitors. This probe offers advantages such as simple operation, low cost, and high sensitivity, and is composed of the FRET effect between R-CDs and CoOOHNSs. Verification has shown that this probe can measure COX-2 activity in vitro, exhibiting a good response relationship and selectivity to COX-2, with a detection limit as low as 0.007 μg / mL and high sensitivity. This method can also be used to screen anti-inflammatory active ingredients from lotus receptacles. Attached Figure Description

[0029] Figure 1 The image shows the FT-IR spectrum of the AIE-based fluorescent probe TPADFN-IMC prepared in Example 1. Figure 2 This is a schematic diagram showing the effect of the AIE-based fluorescent probe TPADFN-IMC prepared in Example 1; probe concentration: 5 μM; where (a) fluorescence emission spectrum of TPADFN-IMC in different solvents; (b) fluorescence spectrum of TPADFN-IMC in different solvents; (c) fluorescence of TPADFN-IMC in mixed solvents with different water contents; detection solvent: THF / PBS buffer; (d) change of fluorescence spectrum of TPADFN-IMC with different water contents; detection solvent: THF / PBS buffer; (e) change of fluorescence intensity of TPADFN-IMC with different water contents; detection solvent: THF / PBS buffer; (f) fluorescence lifetime decay curve of TPADFN-IMC; Figure 3 Figure 1 shows the stability verification of the probe; detection solvent: 10% THF, probe concentration: 5 μM; Figure 2 shows the effect of UV irradiation time on the fluorescence intensity of TPADFN-IMC; (b) the effect of storage time on the fluorescence intensity of TPADFN-IMC; (c) optimization of probe concentration conditions in the detection system; (d) optimization of pH conditions of the buffer solution in the detection system; (e) optimization of incubation temperature conditions in the detection system; (f) optimization of incubation time conditions in the detection system. Figure 4A graph showing the activity of AIE probes in detecting COX-2 (10% THF, probe concentration: 5 μM); where (a) the change in fluorescence intensity of AIE probes with COX-2 concentration; (b) the response relationship of AIE probes to COX-2; and (c) and (d) the anti-interference test of AIE probes. Figure 5 The diagram shows the fluorescence enhancement mechanism of the TPADFN-IMC probe; (a) the particle size of the AIE probe before binding with COX-2; (b) the particle size of the AIE probe after binding with COX-2; (c) visualization of the docking of the AIE probe with COX-2 molecules; and (d) the interacting residues of the AIE probe with COX-2. Figure 6 The inhibition rate of COX-2 by different concentrations of celecoxib; Figure 7 The figures show the liquid chromatogram of the lotus seedpod, the preparation of the components, and the evaluation of the COX-2 inhibitory activity of the components; where (a) is the liquid chromatogram of the lotus seedpod and the preparation of the components; and (b) is the evaluation of the COX-2 inhibitory activity of the components. Figure 8 These are potential anti-inflammatory active ingredients in lotus seedpods; Figure (a) shows the chemical structure of the anti-inflammatory active ingredient, and (b) shows seven ICs containing COX-2 inhibitory active ingredients. 50 value; Figure 9 A schematic diagram showing the molecular docking of the seven active ingredients with COX-2; Figure 10 The COX-2 enzyme inhibition rate of the combination of lotus seedpod extract and active ingredients. Detailed Implementation

[0030] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention.

[0031] In this document, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0032] In this document, when values ​​are described as ranges, it should be understood that such disclosure includes disclosure of all possible subranges within that range, as well as the specific numerical values ​​falling within that range, regardless of whether the specific numerical value or specific subrange is explicitly specified.

[0033] In this article, the terms "multiple" or "more than" are used unless otherwise specified, referring to a quantity greater than or equal to 2. For example, "one or more" means one or more types.

[0034] In this document, the terms "preferred" and "more preferred" are used only to describe implementation methods or embodiments with better effects, and should be understood as not constituting a limitation on the scope of protection of this invention.

[0035] In this document, terms such as "further" are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of protection of this invention.

[0036] In this article, the term "and / or" describes an association between objects, indicating that three relationships can exist. For example, A and / or B means: A or B, or A and B.

[0037] In this document, the term "about" means a specified value of + / - 10%, preferably + / - 5%, and more preferably + / - 1%.

[0038] In this article, the terms “include,” “including,” “have,” “contain,” etc., are all open-ended terms, meaning that they include but are not limited to.

[0039] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar to or equivalent to those described herein may be used in the implementation or testing of this invention.

[0040] The following is the synthesis route of the AIE fluorescent probe in Example 1:

[0041] The present invention will be described in detail below with reference to embodiments.

[0042] Example 1: Preparation of AIE-based fluorescent probe TPADFN-IMC Preparation of the fluorescent group: Under nitrogen protection, 3.1 g of 2,3-bis(4-bromophenyl)-2-butenidonitrile, 1.1 g of triphenylamine 4-borate, 5.5 g of anhydrous potassium carbonate, and 0.28 g of tetra(triphenylphosphine)palladium were added to a 250 mL reaction flask. Then, 120 mL of tetrahydrofuran and 20 mL of water were added. The reaction flask was placed on a magnetic stirrer with constant temperature heating, and the temperature was raised to 60 °C and stirred for 15 h. After the reaction was completed, tetrahydrofuran was removed by vacuum evaporation (EYELA-1300 rotary evaporator, Tokyo Rika Co., Ltd., Japan). The remaining system was extracted three times with water / dichloromethane (volume ratio 1:1). The organic phase was collected and filtered through a Buchner funnel. After the filtrate was evaporated under reduced pressure, the crude product was separated by silica gel column chromatography with petroleum ether / dichloromethane (V:V=4:1) as the eluent. The corresponding eluent was collected and evaporated to give compound 1 (1.36 g, yield 63%).

[0043] Preparation of the coupling product of the linking group: Under nitrogen protection, 810 mg of compound 1, 206 mg of 6-amino-1-hexanol, 66.8 mg of tris(dibenzylacetone)palladium, 90 mg of 1,1'-binaphthyl-2,2'-bis(diphenylphosphine) and 950 mg of cesium carbonate were added to a 100 mL reaction flask. After dissolving in 30 mL of toluene, the mixture was stirred and heated to 100 °C for 12 h. After the reaction was completed, the mixture was extracted three times with water / dichloromethane (v / v ratio 1:1), and the organic phase was collected and filtered. The filtrate was dried under reduced pressure, and the crude product was separated by silica gel column chromatography with petroleum ether / ethyl acetate (v:v = 8:1) as the eluent. The eluent was collected and evaporated to dryness to give compound 2 (240.3 mg, yield 28%).

[0044] Final preparation of AIE-based fluorescent probe TPADFN-IMC: Under nitrogen protection, compound 2200 mg, indomethacin 121 mg, 189 mg of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide and 8.1 mg of 4-dimethylaminopyridine were added to a 100 mL reaction flask and dissolved in 40 mL of dry dichloromethane. The mixture was stirred at room temperature for 10 h. After the reaction, the mixture was extracted three times with water / dichloromethane (volume ratio 1:1). The organic phase was collected, dried over anhydrous magnesium sulfate, filtered, and the filtrate was dried under reduced pressure. The crude product was separated by silica gel column chromatography using petroleum ether / dichloromethane (V:V=10:1) as the eluent. The corresponding eluent was collected and evaporated to dryness to obtain AIE-based fluorescent probe TPADFN-IMC (205.1 mg, yield 67%), abbreviated as TPADFN-IMC, which was stored at 4 °C.

[0045] Structural characterization of intermediates and probes: The structures of compounds 1-3 were identified using a nuclear magnetic resonance (NMR) spectrometer (Avance Ⅲ 400, Burker, Germany) and a mass spectrometer (Q-TOF 6250, Agilent Technologies, USA); the samples were dissolved in deuterated chloroform, and the proton NMR spectra were measured. 1 HNMR and carbon spectroscopy (HNMR) 13 CNMR): Compound 1 1 HNMR (400M HZ, Chloroform-d) δ7.92 (2H, d, J=8.5 HZ), 7.72 (6H, m), 7.52 (2H, d, J=8.7 HZ), 7.30 (4H, dd, J=8.5, 7.3 HZ), 7.5 (m, 6H), 7.08 (2H, t, J=7.3 HZ), 13CNMR (101 MHz, Chloroform-d) δ 147.43, 144.41, 132.71, 132.47, 131.24, 130.33, 129.87, 129.55, 129.36, 127.98, 127.19, 126.45, 125.87, 125.04, 123.63, 123.21, 122.82, 116.83; Mass spectrometry (m / z) [M+H] + =553.1072 (molecular formula C) 34 H 22 BrN3); Compound 2 1 HNMR (400M HZ, Chloroform-d) δ7.84 (3H, m), 7.69 (1H, m), 7.54 (1H, d, J=8.8 HZ), 7.32 (5H, d, J=8.0 HZ), 7.17 (7H, d, J=8.4 HZ), 7.08 (2H, t, J=7.7 HZ), 6.66 (1H, d, J=9.3 HZ), 3.72 (1H, m), 3.24 (1H, dd, J=14.0, 6.9 HZ), 1.72 (6H, d, J=7.7 HZ), 1.47 (4H, m), 13 CNMR (101 MHz, Chloroform-d) δ 147.45, 142.90, 133.05, 132.35, 131.30, 130.67, 130.25, 129.40, 129.20, 127.82, 126.93, 124.91, 124.79, 123.34, 120.21, 118.30, 117.36, 112.17, 62.81, 43.26, 32.62, 29.25, 26.87, 25.57; Mass spectrometry (m / z) [M+H] + =589.2956 (molecular formula C) 40 H 36 N4O); Compound 3 (TPADFN-IMC) 1HNMR (600M HZ, Chloroform-d) δ7.81 (4H, phenyl, dd, J=19.3,8.7 HZ), 7.67 (4H, phenyl, dd, J=16.1,8.5 HZ), 7.51 (2H, phenyl, d, J=8.6 HZ), 7.46 (2H, phenyl, d, J=8.5 HZ), 7.29 (4H, phenyl, m), 7.15 (6H, phenyl, m), 7.06 (2H, phenyl, t, J=7.4 HZ), 6.98 (1H, phenyl, d, J=2.5 HZ), 6.87 (1H, phenyl, d, J=9.0 HZ), 6.68 (1H, phenyl, dd, J=9.0, 2.5 HZ), 6.63 (2H, phenyl, d, J=8.9 HZ), 4.12 (2H, -CH2-O-, t, J=6.5HZ), 3.84 (3H, -OCH3, s), 3.67 (2H, -CH2-C=O, s), 2.40 (3H, -CH3, s), 1.62 (5H, m), 1.32 (5H, m), 13 CNMR (101 MHz, Chloroform-d) δ 170.96, 168.35, 156.01, 147.45, 135.93, 133.85, 132.34, 131.20, 130.67, 130.24, 129.39, 129.17, 127.81, 126.93, 124.78, 123.32, 120.25, 114.96, 112.72, 112.15, 111.58, 101.48, 64.83, 55.76, 43.13, 30.51, 28.54, 26.54, 25.65; Mass spectrometry (m / z) [M+H] + =929.3581 (molecular formula C) 59 H 50 ClN5O4) confirmed the successful synthesis of the probe.

[0046] Furthermore, Fourier transform infrared spectroscopy was used to further confirm the characteristic functional groups of the TPADFN-IMC molecule. (3391.85 cm⁻¹) -1 The broad absorption peaks at 2922.32 and 2856.17 cm⁻¹ are attributed to the stretching vibrations of NH₃. -1 This is the CH stretching vibration of the methyl and methylene groups on the linking group. 2204.96 cm⁻¹ -1 The absorption peaks are due to the stretching vibration of the cyano group. (1088.48, 1069.16 cm⁻¹) -1The absorption peaks are for the C-Cl rocking vibration of the aromatic ring, at 1605.97, 1518.15, and 1488.93 cm⁻¹. -1 Multiple absorption peaks appear nearby, belonging to C=C stretching vibrations, reflecting the conjugated structure of the benzene ring. 1324.70 cm⁻¹ -1 The absorption peak is due to the stretching vibration of CN in the -CON- group. These results indicate that TPADFN-IMC is formed by linking indomethacin to a fluorescent molecule via a coupling group. Figure 1 ).

[0047] Optical property characterization of the probe: After obtaining TPADFN-IMC, its photophysical properties were investigated using solvents of different polarities, including dimethyl sulfoxide (DMSO), acetonitrile (CH3CN), N,N-dimethylformamide (DMF), methanol (MeOH), ethanol (EtOH), tetrahydrofuran (THF), and dichloromethane (CH2Cl2). The UV-Vis absorption spectra of the probe in different solvents were measured using a UV-Vis spectrophotometer (Carry90, Agilent Technologies, USA). The results showed that the maximum absorption wavelength of TPADFN-IMC in CH2Cl2 and acetonitrile was 450 nm. However, the maximum absorption wavelength of TPADFN-IMC in DMF, MeOH, EtOH, and THF red-shifted to 460 nm, and the maximum absorption peak in DMSO red-shifted to 475 nm. This may be due to solvent effects (…). Figure 2 a) Fluorescence spectra were measured using a fluorescence spectrophotometer (model FL7000, Hitachi, Japan). The emission spectrum of TPADFN-IMC changed with increasing solvent polarity. In EtOH, the emission wavelength was 560 nm; in DMSO, the emission wavelength red-shifted to 580 nm, and the fluorescence intensity decreased. Furthermore, in 10% THF PBS buffer, the probe fluorescence intensity increased significantly due to the AIE effect. Figure 2 b). The aggregation-induced emission properties of TPADFN-IMC were further investigated. THF and PBS buffers were selected as benign and non-benign solvents for the probe, respectively. The state of TPADFN-IMC in the mixed solvent system was controlled by adjusting the proportion of non-benign solvent in the mixed solvent system. Figure 2 As shown in c, the probe showed almost no fluorescence in the THF solution. However, as the water content of the THF solution increased from 10% to 90%, the fluorescence of TPADFN-IMC under a 360 nm UV lamp gradually brightened. Figure 2 d represents the fluorescence emission spectrum of TPADFN-IMC at water contents ranging from 10% to 90%. The fluorescence intensity of TPADFN-IMC continuously increases with increasing undesirable solvent content. For example... Figure 2As shown in Figure e, the fluorescence emission intensity of TPADFN-IMC significantly increased when the water content exceeded 70%, and approached its highest level (λex = 460 nm) in a 90% aqueous solution. These results indicate that TPADFN-IMC exists in a dispersed form in mixed solvent systems with high water content, and increasing the water content can cause the fluorescent probe molecules to gradually aggregate, leading to enhanced fluorescence. Therefore, a 10% THF PBS buffer was selected as the probe dissolution environment in the following study. The fluorescence lifetime decay fitting is shown below. Figure 2 As shown in f, the average lifetime is 4.95 ns, indicating that the probe molecule has a short residence time in the excited state energy level and has good stability.

[0048] Example 2: Fluorescence detection of COX-2 activity Pre-detection solution preparation: Accurately weigh an appropriate amount of COX-2 and prepare a 0.5 μg / mL stock solution with PBS buffer (0.1 M, pH=6.8). Dilute the stock solution to obtain a COX-2 detection solution of 0.001~0.8 μg / mL according to experimental requirements. Weigh TPADFN-IMC powder and prepare a 50 μM probe solution with THF / PBS buffer (10:90 v / v, pH=6.8). Aliquot the solution and store it at -80℃ for later use.

[0049] COX-2 activity assay procedure: Add 20 μL LPADFN-IMC probe solution (50 μM), 20 μL COX-2 solution of different concentrations and 160 μL PBS buffer (pH=6.8) to a 96-well plate in sequence. Shake for 2 minutes and then incubate at 37°C for 20 minutes. Use a multi-mode microplate reader to measure the fluorescence intensity of the mixture at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The excitation and emission slit widths are both set to 5 nm.

[0050] COX-2 inhibition activity test: First, add 20 μL of the test sample solution, 40 μL of PBS buffer (pH=6.8) and 20 μL of COX-2 solution to a 96-well plate and incubate at 37℃ for 10 minutes; then add 20 μL of LTPADFN-IMC probe solution and continue incubation at 37℃ for 20 minutes. Measure the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The inhibition rate is calculated using the following formula: Inhibition rate (%) = (F-Fs) / (F-F0)×100%, where F is the fluorescence intensity containing COX-2 and the probe, Fs is the fluorescence intensity containing the test sample, COX-2 and the probe, and F0 is the fluorescence intensity of the probe without COX-2. Celecoxib was selected as the positive control drug, and 0~0.5 μM celecoxib solutions were prepared for parallel experiments.

[0051] Example 3 (1) Sample preparation Preparation of lotus seedpod extract: Weigh 1 g of lotus seedpod powder and place it in a 50 mL stoppered conical flask. Add 20 mL of 80% methanol, seal tightly, and weigh. Place the conical flask in an ultrasonic cleaner and extract ultrasonically for 30 minutes at an ultrasonic power of 400 W. After standing and cooling, make up the lost mass. Centrifuge the resulting solution at 13400 rpm for 6 minutes. Filter the supernatant through a 0.22 μm filter membrane and store the filtrate at 4℃ for later use.

[0052] Preparation of reference solution: Accurately weigh (+)-catechin, piperidin, proanthocyanidin B1, and myricetin-3- O Appropriate amounts of glucoside, hyperoside, isoquercitrin, and quercetin reference standard powders were prepared into reference standard stock solutions with methanol concentrations of 2.0 mg / mL or 1.0 mg / mL, respectively. These stock solutions were stored in a refrigerator at 4°C and diluted with methanol to the appropriate concentration as needed before use.

[0053] Chromatographic conditions for fraction collection Lotus seedpod fractions were prepared using a high-performance liquid chromatography (HPLC) system with an automated fraction collector. The chromatographic column was an ACQUITYUPLCBEHC18 (50 mm × 2.1 mm, 1.7 μm). The mobile phase consisted of 0.1% formic acid aqueous solution (phase A) and methanol (phase B). The gradient elution program was as follows: 0-28 min: phase B content 5% → 27%; 28-35 min: phase B content 27% → 33%; 35-53 min: phase B content maintained at 33%; 53-80 min: phase B content 33% → 70%; 80-81 min: phase B content maintained at 70%. The flow rate was 1.0 mL / min, the column temperature was 35℃, the detection wavelength was 254 nm (photodiode array detector), and the injection volume was 10 μL. Fractions were collected in time mode, with a total of 17 fractions collected at the following times: Fr.1 (5.2-5.9 min), Fr.2 (6.0-6.6 min), and Fr.3 (6.0-6.6 min). min), Fr.3 (9.3-9.9 min), Fr.4 (11.8-12.6 min), Fr.5 (15-15.7 min), Fr.6 (18.5-19.5 min), Fr.7 (21.6-22.4 min), Fr.8 (25.1-25.9 min), Fr.9 (42.5-43.3 min), Fr.10 (43.4-44.5 min), Fr.11 (44.6-45.7 min), Fr.12 (52.5-54.2 min), Fr.13 (54.3-56.6 min), Fr.14 (62-63 min), Fr.15 (64.2-65.1 min), Fr.16 (65.9-66.8 min), Fr.17 (69.7-70.7 (min); after four enrichment cycles, the collected fractions were dried with nitrogen and redissolved in methanol to a suitable concentration for subsequent activity testing.

[0054] (2) Characterization of lotus seedpod extract Qualitative analysis of the active components of lotus seedpods was performed using ultra-high performance liquid chromatography-tandem time-of-flight mass spectrometry. The analytical column was a Phenomenex Kinetex C18 (100 mm × 2.1 mm, 1.7 μm), and the guard column was an Agilent EclipseXDB-C18 (1.8 μm, 2.1 × 5 mm). The mobile phase was 0.1% formic acid aqueous solution (phase A) and acetonitrile (phase B). The gradient elution program was as follows: phase B content remained at 5% for 0–2 min; phase B content increased from 5% to 30% for 2–15 min; phase B content increased from 30% to 64% for 15–26 min; and phase B content increased from 64% to 90% for 26–35 min. The flow rate was 0.3 mL / min, the column temperature was 30 °C, and the injection volume was 2 μL.

[0055] Mass spectrometry parameter settings: Ion source was electrospray ionization (ESI), recording both positive and negative ion modes simultaneously; drying gas flow rate 9.0 L / min, drying gas temperature 350℃, nebulizer gas pressure 40 psig, fragmentation voltage 120 V, capillary voltage 3500 V, quadrupole RF 750 V, skimmer voltage 65 V; collision energies were set to 10 V and 30 V for MS / MS analysis, with a mass range of 50-1000; MS and MS data were processed using SCIEXOS software. 2 Ion fragment information was used to determine the molecular formula, relative molecular mass error, and characteristic ion fragments of the compound. By comparing with published literature and the PubMed, PubChem, and ChemSpider databases, the chemical components in the lotus seedpod extract were determined.

[0056] (3) Molecular docking The crystal structure of COX-2 (PDB ID: 3NL1) was downloaded from the Protein Database (PDB) for molecular docking experiments. AutodockVina was used as the docking engine, and Gasteigercharge was employed to calculate electrostatic interactions. The docking parameters were set as follows: gridbox center X=44.7, Y=-35.9, Z=25.3, and dimensions X=107, Y=91, Z=115. The three-dimensional structures of the screened active compounds were downloaded from the PubChem database and optimized for minimum energy in Chem3D software. AutoDockTool was used to add hydrogen and charge to the ligands and adjust chemical bonds. All ligands were docked 100 times to ensure sufficient sampling. The optimal conformation was determined by calculating the binding energy, and the binding sites and hydrogen bond interactions between the active compounds and the COX-2 active pocket were analyzed.

[0057] Example 4: Stability of TPADFN-IMC To investigate the photostability of TPADFN-IMC, the effects of UV irradiation time and storage time on its fluorescence intensity were examined. After the probe was irradiated with a 365 nm UV lamp for a period of time, its fluorescence intensity was recorded at the optimal excitation wavelength. Figure 3 a and Figure 3 As shown in b, TPADFN-IMC exhibits good stability within 10 hours of irradiation. Even after 16 hours of irradiation, no significant change in the probe's fluorescence intensity was observed, indicating that ultraviolet light has minimal impact on the probe's fluorescence intensity, and the synthesized probe possesses good resistance to photobleaching. Furthermore, the fluorescence intensity of the TPADFN-IMC solution remained stable after being stored at 4°C for 8 days, further demonstrating the probe's good stability.

[0058] Example 5: Optimization of conditions for COX-2 detection by TPADFN-IMC Probe concentration optimization: With the COX-2 concentration fixed at 0.4 μg / mL, the TPADFN-IMC concentration was varied (1~10 μM), and the fluorescence intensity difference (F-F0, where F is the fluorescence intensity after adding COX-2 and F0 is the fluorescence intensity without adding COX-2) was measured. The results showed that F-F0 was maximized when the probe concentration was 5 μM; therefore, the optimal probe concentration was determined to be 5 μM. Figure 3 c).

[0059] Buffer pH optimization: Considering the cyano functional group structure of the TPADFN-IMC molecule, the pH of the buffer used in the detection system may affect the fluorescence emission of the probe itself and the enzyme activity of COX-2. The effect of buffer pH in the range of 5.5–8.0 on fluorescence intensity was investigated. At pH=6.8, F-F0 was the highest, and COX-2 enzyme activity remained at a high level. Therefore, PBS buffer with pH=6.8 was selected as the detection system. Figure 3 d).

[0060] Optimization of incubation conditions: Incubation temperature has a significant impact on the activity of COX-2, therefore, the temperature during the detection process was optimized. The effects of incubation temperature (25~45℃) and time (5~30 minutes) on fluorescence intensity were investigated. The results showed that the fluorescence intensity reached its stable peak at 37℃ for 20 minutes. Therefore, the optimal incubation temperature was determined to be 37℃ and the optimal incubation time was 20 minutes. Figure 3 e and Figure 3 f).

[0061] Example 6: Quantitative Detection of COX-2 Fluorescence Under optimal detection conditions, the relationship between TPADFN-IMC fluorescence intensity and COX-2 activity was investigated using different concentrations of COX-2. Figure 4 As shown in Figure a, as the concentration of COX-2 increases, the fluorescence intensity of TPADFN-IMC gradually increases with the increase of COX-2 concentration, and the emission wavelength does not shift significantly. Figure 4 b shows the relationship between the fluorescence intensity change at 600 nm of the probe and the COX-2 concentration. Within the range of 0–0.5 μg / mL, a good linear relationship exists between the fluorescence intensity change and the COX-2 concentration, with the linear equation being F - F0 = 769.26C + 9.5256 (R²). 2 =0.9972). Here, F represents the fluorescence intensity of the probe at 600 nm in the presence of COX-2, F0 represents the fluorescence intensity of the probe at 600 nm in the absence of COX-2, C is the concentration of COX-2 in μg / mL, and its detection limit is 0.007 μg / mL. In summary, this probe exhibits excellent sensitivity for COX-2 detection.

[0062] Specificity Test: Specific recognition of the analyte is one of the important characteristics of a fluorescent probe. To evaluate the selectivity of TPADFN-IMC, various potential interferences, including metal cations, anions, small biomolecules, and proteases, were introduced in the presence and absence of COX-2. The results showed that the fluorescence intensity of the system did not change significantly in the presence of these interfering substances, and only COX-2 significantly enhanced the probe fluorescence intensity, indicating that the probe has good specificity for COX-2. (Figure) Figure 4 c and Figure 4 d).

[0063] Example 7: Investigation into the fluorescence enhancement mechanism of TPADFN-IMC Given that the fluorescence intensity increased upon the addition of COX-2 to the TPADFN-IMC solution, and this increase continued with increasing COX-2 concentration, it was hypothesized that the fluorescence enhancement process involved the binding of the probe to the active pocket of COX-2, forming aggregates that restricted intramolecular movement, leading to the enhanced fluorescence intensity. To elucidate the mechanism of COX-2-induced fluorescence enhancement in TPADFN-IMC, the self-assembly behavior of TPADFN-IMC in the presence and absence of COX-2 was first investigated using DLS. The results showed that the average diameter of the TPADFN-IMC (5 μM) aggregates was 18.2 nm (see...). Figure 5 a). When exposed to COX-2 (0.4 μg / mL), the average diameter of TPADFN-IMC changed to 168.3 nm ( Figure 5 b). DLS results provided evidence for the self-assembly of TPADFN-IMC and COX-2 enhancing the AIE process. Molecular docking was used to investigate the spatial conformation and binding mode of the probe TPADFN-IMC into the active cavity of COX-2. COX-2 (PDB: 3NL1) was selected as the research model. Figure 5 As shown in c, the docking results indicate that the recognition group indomethacin in TPADFN-IMC tends to insert into the hydrophobic cavity inside COX-2. Furthermore, the methoxy, carbonyl, and cyano groups on TPADFN-IMC can form non-covalent bonds with amino acid residues in the hydrophobic pocket of COX-2, such as CYS-21, TYR-116, GLN-42, CYS-26, and ABG-22, enabling TPADFN-IMC to be firmly anchored in specific cavities within COX-2. Figure 5 d), thereby restricting the intramolecular movement of the probe and producing the AIE effect, which enhances fluorescence emission.

[0064] Example 8 Evaluation of COX-2 inhibitory activity To examine the applicability of the established AIE fluorescence detection system for evaluating the inhibitory activity of COX-2 in samples, celecoxib, a classic COX-2 inhibitor, was used as the positive control. During the interaction between the probe and COX-2, when an inhibitor is introduced into the system, it competes with TPADFN-IMC for the hydrophobic active pocket within the COX-2 sample. The inhibitor occupies the active site, resulting in no binding site for the probe TPADFN-IMC to COX-2, thus exhibiting fluorescence intensity inhibition. The results are as follows: Figure 6 As shown, COX-2 activity was inhibited with increasing celecoxib concentration, which limited the enzyme-probe binding process, resulting in weaker fluorescence enhancement compared to when no corresponding inhibitor was added. The calculated IC50 of celecoxib was 0.12 μM, consistent with previously reported results. These results demonstrate that the method established in this study is sensitive and reliable, and can be used to evaluate the COX-2 inhibitory activity of samples.

[0065] Example 9: Screening and Identification of Anti-inflammatory Active Ingredients from Lotus Receptacle Screening of active components: The 17 lotus seedpod components collected by HPLC were subjected to COX-2 inhibitory activity tests. The inhibition rate of each component was calculated, and active components with inhibition rates greater than 60% were screened out, including Fr.4, Fr.6, Fr.7, Fr.10, Fr.12, Fr.13, Fr.15, and Fr.17. Figure 7 a). This indicates that these components contain potential COX-2 inhibitors, which may be the material basis for the anti-inflammatory activity of lotus seedpods.

[0066] Identification of active ingredients: UPLC-Q-TOF-MS / MS was used to identify the above active components. Combined with comparison with reference standards, secondary fragment information, and literature data, a total of 19 chemical components were identified. The compound identification process is illustrated using component 12 as an example. Figure 7 As shown in b, a quasi-molecular ion peak with m / z 609.1389 [MH] was detected at 13.264 min in negative ion mode. - Based on secondary mass spectrometry fragments, it is inferred that rhamnose (C6H) has been removed. 12 O6) produces fragment ions with m / z 463.0873, which further lose glucose to generate (C) with m / z 300.0247. 15 H8O7 - The compound was compared with rutin standard, and the molecular ion peak, chromatographic retention time, and secondary mass spectrometry fragments were consistent, confirming that the compound was rutin. A quasi-molecular ion peak (m / z 463.0851 [MH]) was detected at both 14.731 min and 15.895 min. -The two are isomers. Based on secondary mass spectrometry fragmentation, it is inferred that the sugar group was removed to generate a fragment at m / z 300.0253. After comparison with the standard, the compounds were identified as hyperoside and isoquercitrin, respectively.

[0067] Selection and Validation of Active Compounds: Based on literature reports, availability of standards, and sample measurability, (+)-catechins, piperidin, proanthocyanidin B1, and myricetin-3- were ultimately selected. O - Seven potential active ingredients, including glucoside, hyperoside, isoquercitrin, and quercetin, with structures as follows: Figure 8 As shown in a. The COX-2 inhibitory activity of the seven active ingredients was verified, and the results showed that all had varying degrees of inhibitory effect, among which (+)-catechin, taxanein, and quercetin had stronger inhibitory effects. Figure 8 b); Molecular docking was used to analyze the interaction between seven active compounds screened from lotus seedpods and COX-2, and the lowest binding energy conformations of each chemical component were visualized. Figure 9 The binding energies of all seven components to COX-2 are less than -5.0 kcal / mol, with quercetin having the lowest binding energy (-9.7 kcal / mol). Quercetin can form seven hydrogen bonds with the GLY-121, ALA-142, GLU-451, GLY-30, and CYS-32 residues of COX-2 through its phenolic hydroxyl groups.

[0068] Quantitative analysis: The results showed that the contents of (+)-catechin ranged from 0.910 to 2.469 mg / g, hyperoside from 0.817 to 2.275 mg / g, and isoquercitrin from 0.942 to 2.688 mg / g. The total contribution of the seven components to the COX-2 inhibitory activity of lotus seedpod extract was 54%.

[0069] To determine the contribution rate of the screened anti-inflammatory active ingredients in lotus seedpods, the total inhibition rate of the mixed standard of active compounds was calculated, such as... Figure 10 As shown, calculated based on the area under the curve, the total contribution of the screened active compounds to the COX-2 inhibitory activity of lotus seedpod extract was 54%. Both showed the same trend with increasing dilution factor, further indicating that the seven screened compounds are the main material basis for the anti-inflammatory activity of lotus seedpod.

[0070] This invention establishes a fluorescence detection strategy based on aggregation-induced emission (AIE) effect. A highly sensitive and specific detection of COX-2 activity is achieved by designing and synthesizing the TPADFN-IMC probe. This probe is then combined with chromatographic separation and mass spectrometry identification techniques to screen anti-inflammatory active components in lotus seedpods (a traditional Chinese medicine). This probe solves the aggregation quenching (ACQ) problem of traditional fluorescent probes, exhibits good photostability and anti-interference ability, and has a detection limit as low as 0.007 μg / mL. Furthermore, the seven screened active components clarify the material basis of the anti-inflammatory properties of lotus seedpods, providing a powerful tool for the development of COX-2 inhibitors and the study of the anti-inflammatory mechanisms of traditional Chinese medicine.

[0071] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A fluorescent probe based on AIE, characterized in that: Its structural formula is shown in Formula III: Formula III.

2. The method for preparing the AIE-based fluorescent probe according to claim 1, characterized in that: Includes the following steps: Step 1: 2,3-bis(4-bromophenyl)-2-butenidonitrile is coupled with triphenylamine 4-borate to generate the compound shown in Formula I; the reaction formula is shown below: , Formula I; Step 2: The compound shown in Formula I undergoes a substitution reaction with 6-amino-1-hexanol to generate the compound shown in Formula II; the reaction formula is shown below: , Formula I; Formula II; Step 3: The compound shown in Formula II undergoes an esterification reaction with indomethacin to generate the compound shown in Formula III, which is the AIE-based fluorescent probe; the reaction formula is shown below: , Formula II and Formula III.

3. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: The substitution reaction in step one further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, and an organic solvent. The base is selected from one or more of potassium carbonate, potassium phosphate, cesium carbonate, and sodium carbonate. The palladium catalyst is selected from one or more of tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, dichloro(triphenylphosphine)palladium, and 1,3-bis(2,6-diisopropylphenyl)imidazolium-2-ylpyridinium dichloride. The organic solvent is selected from one or more of tetrahydrofuran, toluene, and 1,4-dioxane. The molar ratio of bis(4-bromophenyl)-2-butenidonitrile, triphenylamine 4-boronic acid, base, and palladium catalyst is 100:(50~60):(500~800):(3~5); The reaction conditions for the substitution reaction in step one are: temperature of 60~80℃ and reaction time of 12~16h.

4. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: The coupling reaction in step two further includes being carried out under an inert atmosphere in the presence of a base, a palladium catalyst, a ligand, and an organic solvent. The base is selected from one or more of cesium carbonate, potassium phosphate, sodium tert-butoxide, and potassium tert-butoxide; the palladium catalyst is selected from one or more of tris(dibenzylacetone)palladium, tetra(triphenylphosphine)palladium, dichloro[1,1'-bis(diphenylphosphine)ferrocene]palladium, and palladium acetate; the ligand is selected from one or more of 1,1'-binaphthyl-2,2'-bisdiphenylphosphine, 2-dicyclohexylphosphine-2',6'-dimethoxybiphenyl, 2-dicyclohexylphosphine-2'-methylbiphenyl, and 1,1'-bis(diphenylphosphine)ferrocene; and the organic solvent is selected from one or more of toluene, 1,4-dioxane, tetrahydrofuran, and N,N-dimethylformamide. The molar ratio of the compound represented by Formula I, 6-amino-1-hexanol, palladium catalyst, ligand, and base is 365:(400~480):(15~22):(30~40):(650~800). The reaction conditions for the coupling reaction in step two are: temperature of 80~120℃ and reaction time of 8~16h.

5. The method for preparing an AIE-based fluorescent probe according to claim 2, characterized in that: Step three of the esterification reaction also includes the presence of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, 4-dimethylaminopyridine and an organic solvent in an inert atmosphere, wherein the organic solvent is selected from one or more of dichloromethane, anhydrous diethyl ether, tetrahydrofuran and N,N-dimethylformamide. The molar ratio of the compound represented by Formula II, indomethacin, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 165:(220~280):(400~550):(25~42). The reaction conditions for the esterification reaction in step three are: room temperature and reaction time of 8-14 hours.

6. The application of the AIE-based fluorescent probe as described in claim 1 in COX-2 activity detection or COX-2 inhibitor screening.

7. The use of the AIE-based fluorescent probe as described in claim 1 in the preparation of a COX-2 activity detection kit or a COX-2 inhibitor screening kit.

8. A method for detecting COX-2 activity using the AIE-based fluorescent probe as described in claim 1, characterized in that: The detection method includes the following steps: (1) Dilute the COX-2 stock solution to obtain COX-2 detection solutions of different concentrations; weigh the probe solution prepared by the AIE-based fluorescent probe powder as described in claim 1, dispense it and store it in a -80℃ refrigerator for later use; (2) Add probe solution, COX-2 solution of different concentrations and PBS buffer to a 96-well plate in sequence to obtain a mixed solution. Shake the mixed solution and place it in an incubator for incubation. (3) Measure the emission spectrum of the incubated mixture at an excitation wavelength of 460 nm and record the fluorescence intensity at 600 nm to obtain the COX-2 concentration data of the mixture, thus realizing the COX-2 activity detection; The detection method described above has a detection range of 0–0.5 μg / mL for COX-2, and the linear equation is F - F0 = 769.26C + 9.5256 (R0). 2 =0.9972), where F is the fluorescence intensity of the COX-2+ probe; F0 is the fluorescence intensity without COX-2; C is the concentration of COX-2 in μg / mL; and the detection limit is 0.007 μg / mL.

9. The detection method according to claim 8, characterized in that: In step (2), the final concentration of the probe solution in the mixed solution is 5 μM, the PBS buffer is pH 6.8, the incubation temperature is 37°C, and the incubation time is 20 minutes; in step (1), the AIE-based fluorescent probe powder is dissolved in 10% THF PBS buffer.

10. A method for COX-2 activity inhibition testing using the AIE-based fluorescent probe of claim 1, characterized in that: The testing method includes the following steps: (1) COX-2 was diluted with PBS buffer to obtain COX-2 detection solutions of different concentrations; AIE fluorescent probe TPADFN-IMC powder was weighed and prepared into probe solutions, which were then aliquoted and stored at -80℃ for later use. (2) First, add the sample solution to be tested, PBS, and COX-2 solution to the 96-well plate, incubate, then add the probe solution, incubate again, and measure the fluorescence intensity at an excitation wavelength of 460 nm and an emission wavelength of 600 nm. The inhibition rate I was calculated using the formula: I(%)=(F-Fs) / (F-F0)×100%, where F is the fluorescence intensity of COX-2+ and the probe; Fs is the fluorescence intensity of the sample, COX-2+ and the probe; and F0 is the fluorescence intensity without COX-2.

Images