CN113461609A - Sulfatase-responsive AIE nano probe and preparation method and application thereof - Google Patents
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Abstract
The invention discloses an AIE nano probe responding to sulfatase, a preparation method and application thereof. The probe is a nano probe with an AIE signal amplification function for monitoring sulfatase, and takes a hydrophobic quinoline-malononitrile (DQM) derivative as a core and a hydrophilic sulfate bond as a response group of the sulfatase. The probe provided by the invention is simple and easy to prepare and convenient to use, and can be used for quickly detecting the sulfatase from the fluorescence intensity change before and after response, so that the aim of quick detection is fulfilled. The probe is characterized by no fluorescence, but can generate obvious fluorescence signal enhancement after fast reaction with the sulfatase, thereby realizing the selective and fast detection of the sulfatase. Therefore, the probe of the present invention can be used as an effective tool for examinationThe method for detecting the sulfatase in the tumor has the potential of being used as an inhalation contrast agent for tumor diagnosis.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to an AIE nano probe responding to sulfatase, a preparation method and application thereof.
Background
Sulfatases are a highly conserved family of proteins with high homology in structure and function. They can catalyze the hydrolysis of sulfate ester bonds from a variety of substrates, including glycosaminoglycans, sulfatides, and steroid sulfates. Sulfatase is associated with a variety of pathophysiological conditions, such as hormone-dependent cancers, lysosomal storage diseases, dysplasias and bacterial pathogenesis. The vast majority of breast tumors overexpress the enzyme, and there is evidence that sulfatase plays a role in prostate cancer. Therefore, detection of sulfatase is beneficial for diagnosing cancers with high expression of sulfatase and understanding the pathological activity of sulfatase.
In 2001, the Tang-loyalty team proposed the concept of Aggregation-induced emission (AIE) which describes an abnormal phenomenon opposite to the ACQ effect, showing weak or no fluorescence in solution, but emitting intense fluorescence once aggregated. The main factors of solid state light emission of AIE molecules are intramolecular motion limitations, including intramolecular rotation and vibration limitations. When the molecules are aggregated, the intramolecular movement, which originally consumes the energy of the excited state of the molecules, is suppressed, so that the non-radiative transition, which quenches the fluorescence of the molecules, is greatly suppressed, and the molecules emit light mainly by releasing energy through the radiative transition. Meanwhile, AIE molecules generally have a distorted molecular structure and are difficult to form pi-pi stacking during aggregation, thereby also reducing the attenuation of excited state energy through non-radiative channels. The methods for limiting intramolecular movement are mainly electrostatic attraction, hydrogen bonding, hydrophobic interaction and solubility change. Among these, hydrophobic interactions are the main driving force for protein folding. Proteins are very stable in water when hydrophobic side chains in the protein aggregate inside the protein, rather than being solvated by water. Thus, in aqueous media, the amphiphilic organic light emitting agent is driven into the hydrophobic domain or pores in the protein fold structure by hydrophobic interactions in the aqueous media. Due to the limited volume of the pores, intramolecular movement is restricted so that the luminescent agent forms aggregates. The mechanism of the AIE restricted intramolecular movement makes this approach suitable for the design of AIE biological probes. Based on the excellent properties of AIE photoluminescent agents, specific enzyme bioprobes have been designed by integration with recognition units, showing many advantages, including low background interference, high signal-to-noise ratio and excellent photostability.
Currently, the fluorescent probe has the advantages of high sensitivity, good selectivity, no wound and the like, and is a reliable method for detecting sulfatase. However, few fluorescent probes have been developed for detecting sulfatase activity in vivo due to their short emission wavelength, small Stokes shift, or limitations of quenching effects caused by aggregation. Thus, there is an urgent need for deep tissue penetrating fluorescent probes for imaging of sulfatase in vivo.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the problems in the prior art, the invention provides a sulfatase-responsive AIE nano probe, which can be specifically hydrolyzed by sulfatase to release a fluorophore and realize secondary enhancement of a fluorescence signal, is used for detecting the sulfatase and effectively solves the problem of the limitation of quenching effect caused by short emission wavelength, small Stokes displacement or aggregation of a fluorescent probe.
The invention also provides a preparation method and application of the sulfatase-responsive AIE nanoprobe.
The technical scheme is as follows: in order to achieve the above object, the AIE nanoprobe responding to sulfatase, which is abbreviated as DQM-SULF, is composed of a fluorophore and a sulfate, and has a structural formula shown in the following formula I:
the preparation method of the sulfatase responding AIE nano probe comprises the following steps:
(1) dissolving 2-methylquinoline and iodoethane in anhydrous acetonitrile, stirring and reacting under the protection of inert gas, cooling a reaction mixture to room temperature, and filtering, washing and drying a precipitate in vacuum to obtain a compound 2;
(2) dissolving the compound 2 in ethanol, then adding malononitrile and sodium ethoxide for reaction, filtering precipitates after the reaction is finished, and washing to obtain a compound 3;
(3) dissolving the compound 3 and 4-hydroxybenzaldehyde in anhydrous acetonitrile containing piperidine, reacting under the protection of inert gas, cooling the reaction mixture to room temperature, evaporating the solvent under reduced pressure, and purifying the residue to obtain a compound DQM-OH;
(4) dropwise adding sodium tert-butoxide dissolved in a tetrahydrofuran solution into the tetrahydrofuran solution dissolved with DQM-OH; adding trimethyl ammonium sulfur trioxide copolymer for reaction; the solvent was evaporated from the reaction mixture and the residue was purified to give the compound probe DQM-SULF.
The reaction route is as follows:
preferably, in the step (1), 2-methylquinoline and iodoethane are dissolved in anhydrous acetonitrile and stirred for 20-24h at 85-90 ℃ under the protection of nitrogen inert gas. After the reaction is finished, cooling the reaction mixture to room temperature, and performing vacuum filtration by using a Buchner funnel, wherein a large amount of precipitate is at the bottom of the reaction mixture; the solid crude product was washed with cold acetonitrile without further purification and dried overnight to give compound 2.
Preferably, in the step (2), the compound 2 is dissolved in dry ethanol, then malononitrile and sodium ethoxide are added, stirring is carried out for 0.5-1h at 0-4 ℃, then stirring is carried out for 2-3h at room temperature, and after the reaction is finished, the precipitate is filtered and washed with cold ethanol for multiple times to obtain the compound 3.
Preferably, in step (3), compound 3 and 4-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile containing piperidine, the mixture is stirred overnight at 85-90 ℃ under nitrogen protection, after the reaction mixture is cooled to room temperature, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel chromatography to obtain compound DQM-OH.
Preferably, step (4) is carried out by adding dropwise sodium tert-butoxide dissolved in tetrahydrofuran solution at 20 to 22 ℃ to the tetrahydrofuran solution in which DQM-OH is dissolved, after 15 to 20min, adding trimethylammonium sulfur trioxide copolymer in solid form, after 30 to 40min, evaporating the solvent from the reaction mixture and purifying the residue by means of a silica gel chromatography column to give the probe DQM-SULF.
The invention relates to application of an AIE nano probe responding to sulfatase in response detection of the sulfatase.
The probe of the invention is applied to the preparation of a tool for detecting the responsiveness of sulfatase.
Preferably, the process of the responsiveness detection is as follows: adding sulfatase solutions with different concentrations into a reaction system containing the probe, quickly mixing the reaction solution, incubating, transferring the incubated reaction solution into an English cuvette, and measuring the ultraviolet absorption and fluorescence emission spectra.
The invention relates to application of an AIE nano probe responding to sulfatase in imaging of cell endogenous sulfatase.
Preferably, the imaging process is as follows: digesting and centrifuging 4T1 cells in logarithmic growth phase to prepare cell suspension, adding the cell suspension into a laser confocal culture dish, respectively using probes with different concentrations for co-incubation, and finally using a laser confocal microscope for cell imaging.
The probe of the invention is applied to the preparation of an imaging tool for detecting sulfatase in tumor cells.
The AIE probe structure provided by the invention can hydrolyze the AIE probe into a fluorophore with strong fluorescence in the presence of sulfatase, then combine with a hydrophobic domain of the sulfatase, and further amplify an AIE fluorescence signal through hydrophobic interaction. The catalytic domain and the hydrophobic domain of the sulfatase can turn on and enhance AIE fluorescence, thereby detecting the sulfatase with high sensitivity and signal-to-noise ratio.
The probe is a nano probe with an AIE signal amplification function for monitoring sulfatase, a hydrophobic quinoline-malononitrile (DQM) derivative is used as a core, a hydrophilic sulfate bond is used as a response group of the sulfatase, a novel nano probe for detecting the sulfatase is synthesized, the AIE probe is hydrolyzed by the sulfatase, a fluorophore is released, secondary enhancement of a fluorescence signal is realized, and the endogenous sulfatase of cells and the sulfatase overexpressed in tumors can be imaged. The probe is used as an effective tool for detecting sulfatase in tumors and has the potential of being used as an inhalation contrast agent for tumor diagnosis.
The probe prepared by the invention is hydrolyzed by sulfatase to become a fluorophore, and then the fluorophore is combined into a hydrophobic cavity of the sulfatase to realize secondary enhancement of a fluorescence signal, so that the limitation of quenching effect caused by short emission wavelength, small Stokes shift or aggregation of the fluorescence probe can be effectively overcome. The probe provided by the invention has a hydrophilic group and a hydrophobic group, and the amphipathic structure enables the probe to be self-assembled into a loose nano probe in an aqueous solution.
Has the advantages that: compared with the prior art, the invention has the following advantages:
(1) the probe is simple to synthesize and convenient to use, and the mechanism of response to sulfatase is based on that sulfatase catalyzes hydrolysis of a sulfate ester bond to release a fluorophore DQM-OH.
(2) The sulfatase can be rapidly detected from the fluorescence intensity change before and after response, and the purpose of rapid detection is realized.
(3) The probe is characterized by no fluorescence, but can generate obvious fluorescence signal enhancement after fast reaction with the sulfatase, thereby realizing the selective and fast detection of the sulfatase. Therefore, the probe can be used as an effective tool for detecting sulfatase in tumors, and has the potential of being used as an inhalation contrast agent for tumor diagnosis.
Drawings
FIG. 1 is a high resolution mass spectrum of probe DQM-SULF;
FIG. 2 is the trend of fluorescence intensity of probe DQM-SULF (5 μ M) in PBS buffer as a function of sulfatase (0-50U/mL) concentration and the linear relationship;
FIG. 3 shows fluorescence emission spectra of the fluorophore DQM-OH before and after reaction with sulfatase (50U/mL), wherein the upper curve is DQM-OH + SULF;
FIG. 4 is an image of probe DQM-SULF at different concentrations in 4T1 cells.
Detailed Description
The invention is further illustrated by the following figures and examples.
The experimental methods used in the present invention are all conventional methods unless otherwise specified. Materials, reagents and the like used in the experiments can be obtained from commercial sources unless otherwise specified. All reagents used in the examples below were either commercially available, analytically pure or chemically pure.
Wherein the sulfatase (SULF for short) is sulfate from Roman snail and purchased from sigma-aldrich, and the enzyme activity can hydrolyze 1.0 micromole of p-nitrophthalic acid sulfate per hour under the conditions of 37 ℃ and pH value of 5.0.
Trimethylammonium sulfur trioxide copolymer was purchased from Meclin reagent Inc.
Example 1
The preparation method of the sulfatase responding AIE nano probe comprises the following steps:
(1) synthesis of fluorophore: 2-methylquinoline (2.86g,20mmol) and iodoethane (3.90g,25mmol) were weighed out and dissolved in 25mL of anhydrous acetonitrile and stirred at 85 ℃ for 24h under nitrogen. After the reaction was completed, the reaction mixture was cooled to room temperature, and then a large amount of precipitate was formed at the bottom of the flask, which was then vacuum-filtered using a Buchner funnel. The crude product was washed with cold acetonitrile without further purification. After drying overnight, compound 2 was obtained.
(2) Compound 2(1.72g,10mmol) was weighed out and dissolved in 10mL of dry ethanol, followed by the addition of malononitrile (1g,15mmol) and sodium ethoxide (1.02g,15 mmol). The reaction was stirred at 0 ℃ for 0.5h and then at room temperature for a further 3 h. After completion of the reaction, the precipitate was filtered and washed three times with cold ethanol to give compound 3.
(3) Compound 3(470mg,2mmol) and 4-hydroxybenzaldehyde (244mg,2mmol) were weighed out and dissolved in 20mL of anhydrous acetonitrile containing piperidine (1mL) and stirred under nitrogen overnight at 85 ℃. After the reaction mixture was cooled to room temperature, the solvent was evaporated under reduced pressure, and the residue was purified by a silica gel chromatography column (dichloromethane: methanol ═ 10:1) to obtain a compound DQM-OH.
(4) Probe DQM-SULF: to a continuously stirred solution (5mL) of tetrahydrofuran dissolved with DQM-OH (509mg,1.5mmol) at 22 ℃ was added dropwise a solution of sodium tert-butoxide (144mg,1.5mmol) dissolved in tetrahydrofuran solution (3 mL). After 15min, trimethylammonium sulfur trioxide copolymer (278mg,2mmol) was added in solid form. After further reaction for 30min, the solvent was evaporated from the reaction mixture under reduced pressure. The residue was purified by silica gel chromatography (dichloromethane: methanol ═ 10:1) to give probe DQM-SULF.
1H NMR(300MHz,DMSO-d6)δ8.90(d,J＝8.3Hz,1H),8.07(d,J＝8.8Hz,1H),7.89-7.93(m,1H),7.67(d,J＝8.5Hz,2H),7.57-7.62(m,1H),7.26-7.38(m,2H),6.99(s,1H),6.85(d,J＝8.5Hz,2H),4.55(q,J＝6.9Hz,2H),1.40(t,J＝7.0Hz,3H).13C NMR(75MHz,DMSO-d6)δ159.8,155.6,152.7,149.9,140.4,139.9,138.3,134.2,130.5,129.4,125.6,121.1,120.7,119.5,118.6,117.4,116.2,107.2,47.3,44.7,14.1.HRMS:m/z calcd for C22H16N3O4S[M-H]-:418.0862,found:418.0861.
FIG. 1 is a high-resolution mass spectrum of probe DQM-SULF, which shows the success of synthesizing probe DQM-SULF.
Example 2
Weighing 4.2mg of the probe DQM-SULF prepared in the embodiment, dissolving in 10mL of DMSO solution, and preparing into standard solution with the concentration of 1 mM; similarly, 3.4mg of the fluorescence DQM-OH prepared in the example was dissolved in 10mL DMSO solution to prepare a standard solution with a concentration of 1 mM; the sulfatase was prepared into a standard solution of 100U/mL with PBS buffer (10Mm, pH 7.4). To a reaction system PBS buffer (10mM, pH 7.4, 1% DMSO) containing the probe DQM-SULF (5 μ M), a sulfatase solution (0-50U/mL) was added at various final concentrations, taking 4mL EP tubes. Incubated in a constant temperature shaker at 37 ℃ for 30 min. Under the excitation of the wavelength of 440nm, the slit width is set to 10.0/10.0nm, and the fluorescence emission spectrum of the solution in the wavelength band of 450-800nm is collected. As shown in FIG. 2, the fluorescence intensity curve of the probe DQM-SULF increases with increasing concentration of sulfatase. In addition, the detection limit is 2.1U/L and the detection range is 0-50U/mL, which indicates that the probe has high sensitivity and can be used for detecting endogenous sulfatase in a biological system.
Example 3
The preparation steps of the probe DQM-SULF solution and the sulfatase solution related to the embodiment are the same as the embodiment 2, and other specific steps are as follows: to a reaction system PBS buffer (10mM, pH 7.4, 1% DMSO) containing a probe DQM-SULF (5. mu.M), a sulfatase solution was added at a final concentration of 50U/mL, and incubated at 37 ℃ for 30min in a constant temperature shaker. Under the excitation of the wavelength of 440nm, the slit width is set to 10.0/10.0nm, and the fluorescence emission spectrum of the solution in the wavelength band of 450-800nm is collected. As shown in fig. 3, the AIE fluorescence signal is amplified for the fluorophore and enzyme response compared to the fluorophore alone. The probe prepared by the invention can release fluorophore, realize secondary enhancement of fluorescence signals and effectively solve the problem of the limitation of quenching effect caused by short emission wavelength, small Stokes shift or aggregation of the fluorescence probe. In addition, the enzyme responds to other compounds with AIE properties such as tetraphenylethylene, 1- (4-hydroxyphenyl) -1,2, 2-triphenylethylene, 1,1,2,3,4, 5-hexaphenylsilole and the like by the above method, and secondary enhancement of the fluorescence signal is not achieved.
Example 4
The preparation steps of the probe DQM-SULF solution related to the embodiment are the same as those of the embodiment 2, and other specific steps are as follows: 4T1 cells in logarithmic growth phase were digested, centrifuged, and prepared in 10% FBS-containing DMEM medium at 5X 104cell/mL, the cell suspension was added to a laser confocal culture dish, and 100. mu.L of cell suspension was placed in 5% CO per dish2Adherent monolayers were formed by overnight incubation in an incubator at 37 ℃ and when the cell density reached 60-70%, co-incubation in the incubator for 2h with different final concentrations of the probe DQM-SULF (0-30. mu.M) solution, respectively, followed by removal of the mixture, washing three times with PBS and fixation with 4% paraformaldehyde. And finally, performing cell imaging by using an FV-1000 laser confocal microscope. As shown in FIG. 4, as the concentration of the probe increases, the fluorescence in the cell increases. Therefore, the probe DQM-SULF can be effectively activated by endogenous sulfatase in living cells, and has good application prospect in the fields of biology and medicine.
Example 5
Example 5 was prepared identically to example 1, except that: stirring the mixture in the step (1) for 20 hours at 90 ℃ under the protection of nitrogen inert gas. Step (2) compound 2 was dissolved in dry ethanol, and then malononitrile and sodium ethoxide were added, stirred at 4 ℃ for 1h, and then stirred at room temperature for another 2 h. And (3) stirring overnight at 90 ℃ under the protection of nitrogen. And (4) dropwise adding sodium tert-butoxide dissolved in the tetrahydrofuran solution into the tetrahydrofuran solution dissolved with DQM-OH at the temperature of 20 ℃, adding a trimethyl ammonium sulfur trioxide copolymer in a solid form after 20min, evaporating the solvent from the reaction mixture after 40min, and purifying the residue by a silica gel chromatographic column to obtain the probe DQM-SULF.
Claims (10)
1. An AIE nanoprobe responding to sulfatase, which is characterized in that the structural formula is shown as the following formula I:
2. a preparation method of an AIE nano probe responding to sulfatase is characterized by comprising the following steps:
(1) dissolving 2-methylquinoline and iodoethane in anhydrous acetonitrile, stirring and reacting under the protection of inert gas, cooling the reaction mixture to room temperature, and filtering, washing and drying the precipitate in vacuum to obtain a compound 2;
(2) dissolving the compound 2 in ethanol, adding malononitrile and sodium ethoxide for reaction, filtering precipitates after the reaction is finished, and washing to obtain a compound 3;
(3) dissolving the compound 3 and 4-hydroxybenzaldehyde in anhydrous acetonitrile containing piperidine, reacting under the protection of inert gas, cooling the reaction mixture to room temperature, evaporating the solvent under reduced pressure, and purifying the residue to obtain a compound DQM-OH;
(4) dropwise adding sodium tert-butoxide dissolved in a tetrahydrofuran solution into the tetrahydrofuran solution dissolved with DQM-OH; adding trimethyl ammonium sulfur trioxide copolymer for reaction; the solvent was evaporated from the reaction mixture and the residue was purified to give the compound probe DQM-SULF;
the reaction route is as follows:
3. the preparation method according to claim 2, wherein the 2-methylquinoline and the iodoethane are preferably dissolved in anhydrous acetonitrile in the step (1), and stirred for 20-24h at 85-90 ℃ under the protection of nitrogen inert gas. After the reaction is finished, cooling the reaction mixture to room temperature, and performing vacuum filtration by using a Buchner funnel, wherein a large amount of precipitate is at the bottom of the reaction mixture; the solid crude product was washed with acetonitrile without further purification and dried overnight to give compound 2.
4. The preparation method according to claim 2, wherein the compound 2 is dissolved in dry ethanol in the step (2), then malononitrile and sodium ethoxide are added, stirring is carried out at 0-4 ℃ for 0.5-1h, then stirring is carried out at room temperature for 2-3h, and after the reaction is completed, the precipitate is filtered and washed with ethanol to obtain the compound 3.
5. The preparation method according to claim 2, characterized in that in step (3), the compound 3 and 4-hydroxybenzaldehyde are dissolved in anhydrous acetonitrile containing piperidine, the mixture is stirred overnight at 85-90 ℃ under nitrogen protection, after cooling the reaction mixture to room temperature, the solvent is evaporated under reduced pressure, and the residue is purified by silica gel chromatography to obtain the compound DQM-OH.
6. The preparation method according to claim 2, characterized in that the step (4) of adding sodium tert-butoxide dissolved in tetrahydrofuran solution dropwise at 20-22 ℃ to the tetrahydrofuran solution in which DQM-OH is dissolved, after 15-20min, trimethylammonium sulfur trioxide copolymer in solid form is added, after 30-40min, the solvent is evaporated from the reaction mixture, and the residue is purified by silica gel chromatography to give the probe DQM-SULF.
7. Use of the sulfatase-responsive AIE nanoprobe of claim 1 in the detection of sulfatase responsiveness.
8. The application of claim 7, wherein the responsiveness detection procedure is: adding sulfatase solutions with different concentrations into a reaction system containing the probe, quickly mixing the reaction solution, incubating, transferring the incubated reaction solution into an English cuvette, and measuring the ultraviolet absorption and fluorescence emission spectra.
9. Use of the sulfatase-responsive AIE nanoprobe of claim 1 in the imaging of cellular endogenous sulfatase.
10. The use according to claim 9, wherein the imaging process is: digesting and centrifuging 4T1 cells in logarithmic growth phase to prepare cell suspension, adding the cell suspension into a laser confocal culture dish, respectively using probes with different concentrations for co-incubation, and finally using a laser confocal microscope for cell imaging.
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| CN114436892A (en) * | 2022-01-27 | 2022-05-06 | 中国药科大学 | Abeta-responsive light-activated fluorescent probe and preparation method and application thereof |
| CN114634497A (en) * | 2022-02-25 | 2022-06-17 | 东南大学 | Cysteine/homocysteine response AIE fluorescent probe and preparation method and application thereof |
| CN116514787A (en) * | 2023-04-23 | 2023-08-01 | 曲阜师范大学 | Activated near infrared fluorescence/photoacoustic dual-mode living body imaging probe for detecting sulfatase and preparation method and application thereof |
| CN114736153B (en) * | 2022-05-16 | 2023-08-25 | 扬州工业职业技术学院 | AIE type azo enzyme fluorescent small molecular probe and preparation method thereof |
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| CN106478576A (en) * | 2016-09-29 | 2017-03-08 | 华南理工大学 | A kind of for detecting fluorescence probe of carboxy-lesterase and preparation method and application |
| CN109608474A (en) * | 2018-12-13 | 2019-04-12 | 湖北大学 | A kind of compound and its preparation method and application detecting tyrosinase |
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| CN106478576A (en) * | 2016-09-29 | 2017-03-08 | 华南理工大学 | A kind of for detecting fluorescence probe of carboxy-lesterase and preparation method and application |
| CN109608474A (en) * | 2018-12-13 | 2019-04-12 | 湖北大学 | A kind of compound and its preparation method and application detecting tyrosinase |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114436892A (en) * | 2022-01-27 | 2022-05-06 | 中国药科大学 | Abeta-responsive light-activated fluorescent probe and preparation method and application thereof |
| CN114634497A (en) * | 2022-02-25 | 2022-06-17 | 东南大学 | Cysteine/homocysteine response AIE fluorescent probe and preparation method and application thereof |
| CN114634497B (en) * | 2022-02-25 | 2023-12-22 | 东南大学 | Cysteine/homocysteine response AIE fluorescent probe and preparation method and application thereof |
| CN114736153B (en) * | 2022-05-16 | 2023-08-25 | 扬州工业职业技术学院 | AIE type azo enzyme fluorescent small molecular probe and preparation method thereof |
| CN116514787A (en) * | 2023-04-23 | 2023-08-01 | 曲阜师范大学 | Activated near infrared fluorescence/photoacoustic dual-mode living body imaging probe for detecting sulfatase and preparation method and application thereof |
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| CN113461609B (en) | 2022-05-17 |
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