A hypochlorous acid responsive fluorescent compound, a ratio type fluorescent probe and application thereof

Abstract

The application belongs to the technical field of nanobiomaterials, and particularly relates to a hypochlorous acid responsive fluorescent compound, a ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid, and further discloses a preparation method and application thereof. The ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid is mainly prepared by a nanocomprecipitation method through energy donors CNPPV and energy receptors MeOTPATBT fluorescent compounds. In the fluorescent probe, the presence of hypochlorous acid can oxidize the sulfur atom of MeOTPATBT, and then block the energy transmission from CNPPV to MeOTPATBT, so that the fluorescent spectrum of the Pdots probe changes in ratio, the ratio detection of hypochlorous acid is realized, and the fluorescent probe has the advantages of high specificity, anti-interference and high stability.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of nanobiomaterials, and particularly relates to a hypochlorous acid responsive fluorescent compound, a ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid, and further discloses a preparation method and application thereof. BACKGROUND

[0002] Hypochlorous acid / hypochlorite (HClO / ClO - ) is one of the important reactive oxygen species (ROS) in the body, which is produced by hydrogen peroxide and chloride ions under the catalysis of peroxidase, and is considered to be the number one killer of invading pathogens, and plays an important role in many pathological and physiological processes. In the body, hypochlorous acid / hypochlorite (HClO / ClO - ) mainly exists in various organs, tissues and cells, and plays an important role in maintaining the redox balance in cells, especially in the immune defense of the body against invading bacteria and other pathogens. Although the presence of endogenous hypochlorite can destroy invading bacteria and pathogens to protect human health, however, excessive content of HClO may lead to various diseases, such as arthritis, lung injury, Alzheimer's disease, diabetes, neurodegenerative diseases, cardiovascular diseases and even cancer. Therefore, it is of great significance to establish a reliable, rapid and accurate detection and analysis method for HClO / ClO - , and to realize real-time visual detection of hypochlorous acid for monitoring and diagnosis of such diseases.

[0003] At present, the methods for detecting HClO / ClO - mainly include reduction titration, electrochemical test, chromatographic analysis, colorimetric analysis and fluorescent probe technology. The traditional detection methods mostly have the disadvantages of high analysis cost, need for expensive instruments and complex methods, while the fluorescent probe technology has the characteristics of rapid response, high sensitivity and specificity to the analyte, and can be used as an effective biological analysis means for detecting HClO. At present, the fluorescent probes for detecting HClO have been deeply studied in the aspects of design, synthesis and biological application, and various types of hypochlorous acid probes such as switch type probes and ratio type probes have been developed. The design of many hypochlorous acid fluorescent probes starts from the reaction site protection and deprotection and selective oxidation strategy, that is, these reaction sites have reaction specificity with HClO, and HClO changes the fluorescence spectrum by blocking the PET process or changing the ICT, FRET, ESIPT and other processes. The ratio type fluorescent probe can better eliminate the interference of background signal, overcome the shortcomings of single wavelength change probe, such as the influence of instrument parameter fluctuation and uneven distribution of probe, and can be self-calibrated through two wavelengths, which is more conducive to obtaining accurate fluorescence detection signal, and has become a research hotspot in the field of hypochlorous acid detection probes. SUMMARY

[0004] To this end, the technical problem to be solved by the present application is to provide a hypochlorous acid-responsive fluorescent compound MeOTPATBT.

[0005] The second technical problem to be solved by the present application is to provide a ratio-type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid, which is a MeO-CNPPV Pdots fluorescent probe designed and synthesized based on the principle of fluorescence resonance energy transfer (FRET), has good specificity and good photochemical stability, and can detect hypochlorous acid in a ratio.

[0006] The third technical problem to be solved by the present application is to provide a preparation method and application of the above-mentioned hypochlorous acid-responsive compound and ratio-type semiconductor polymer quantum dot fluorescent probe.

[0007] To solve the above technical problems, the present application provides a hypochlorous acid-responsive fluorescent compound, which is denoted as MeOTPATBT and has the structure shown in the following formula (I), and the English name of the compound is 4,4'-(benzo[c][1,2,5]thiadiazole-4,7-diylbis(thiophene-5,2-diyl))bis(N,N-bis(4-methoxyphenyl)aniline):

[0008]

[0009] The present application also discloses a method for preparing the hypochlorous acid-responsive fluorescent compound, which comprises the step of performing Suzuki coupling reaction of 4-boronic acid ester-4',4'-dimethoxytriphenylamine shown in formula (1) with 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole shown in formula (2).

[0010]

[0011] Specifically, the Suzuki coupling reaction comprises the step of performing reaction in the presence of potassium carbonate, tetrabutylammonium bromide and tetrakis(triphenylphosphine)palladium;

[0012] Preferably, the amount of potassium carbonate is 6-10 equivalents, the amount of tetrabutylammonium bromide is 0.1-0.15 equivalents, and the amount of tetrakis(triphenylphosphine)palladium is 0.1-0.15 equivalents, based on 1 equivalent of the compound shown in formula (2);

[0013] More preferably, the amount of potassium carbonate is 8 equivalents, the amount of tetrabutylammonium bromide is 0.125 equivalents, and the amount of tetrakis(triphenylphosphine)palladium is 0.125 equivalents, based on 1 equivalent of the compound shown in formula (2).

[0014] The present application also discloses a ratio type Pdots fluorescent probe for detecting hypochlorous acid, which comprises a fluorescent semiconductor polymer as an energy donor, the fluorescent compound MeOTPATBT as claimed in claim 1 as an energy acceptor, and an amphiphilic polymer as a wrapping agent.

[0015] Preferably, the fluorescent semiconductor polymer comprises CNPPV, i.e. poly(5-(2-ethylhexyloxy)-2-methoxy-cyanophthalocyanine).

[0016] Preferably, the amphiphilic polymer comprises PS-PEG-COOH (polystyrene-polyethylene glycol-carboxyl) and / or PSMA.

[0017] Specifically, in the fluorescent probe, the mass ratio of the MeOTPATBT to the CNPPV is = 1:180-220, and the mass ratio of the total amount of the MeOTPATBT and the CNPPV to the amphiphilic polymer is = 1:0.8-1.2.

[0018] Preferably, in the fluorescent probe, the mass ratio of the MeOTPATBT to the CNPPV is = 1:200, and the mass ratio of the total amount of the MeOTPATBT and the CNPPV to the amphiphilic polymer is = 1:1.

[0019] Specifically, the fluorescent probe further comprises 3-aminopropyl triethoxysilane (APTES).

[0020] Preferably, the mass ratio of the APTES to the MeOTPATBT is 1:180-220, preferably 1:200.

[0021] The present application also discloses a method for preparing the ratio type fluorescent probe for detecting hypochlorous acid, which comprises the step of performing nano-precipitation reaction on selected amounts of the MeOTPATBT, the fluorescent semiconductor polymer and the amphiphilic polymer.

[0022] Preferably, the method further comprises the step of adding 3-aminopropyl triethoxysilane (APTES) to perform nano-precipitation reaction.

[0023] Specifically, the nano-precipitation reaction comprises the step of performing ultrasonic treatment; specifically, the power of the ultrasonic treatment step is 20-60 KH Z , and the treatment time is 3-8 minutes.

[0024] Preferably, the solvent system of the nano-precipitation reaction comprises tetrahydrofuran (THF).

[0025] The application also discloses use of the fluorescent compound for preparing the ratio type fluorescent probe for detecting hypochlorous acid.

[0026] The application also discloses the ratio type fluorescent probe for detecting hypochlorous acid in the HClO / ClO - detection field.

[0027] Preferably, the HClO / ClO - detection includes endogenous, exogenous or water environment ClO - detection.

[0028] The hypochlorous acid responsive fluorescent compound in the application is a coupling compound with a thiophene structure, the fluorescent compound can be used as an energy acceptor to have sensitive response to hypochlorous acid, the sulfur atom of the thiophene can be oxidized by the hypochlorous acid, and then the FRET process inside the probe is affected, so that the fluorescent emission spectrum is obviously changed, and the ratio type nanometer fluorescent probe for detecting hypochlorous acid can be prepared.

[0029] The ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid in the application is an A-D (D: energy donor, A: energy acceptor) ratio type hypochlorous acid fluorescent probe designed and synthesized based on the principle of fluorescence resonance energy transfer, the probe includes an energy donor and an energy acceptor and meets the energy transfer condition, that is, the emission spectrum of the energy donor and the absorption spectrum of the energy acceptor have great overlap, and the acceptor emission spectrum is sensitive to hypochlorous acid, so that the ratio detection of hypochlorous acid can be realized. Compared with the probe with single wavelength change, the ratio type fluorescent probe can better eliminate the interference of background signals, and through self-calibration of two wavelengths, accurate detection signals can be obtained.

[0030] The ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid in the application is mainly made of an energy donor CNPPV and an energy acceptor MeOTPATBT fluorescent compound through a nanometer coprecipitation method. In the fluorescent probe, the sulfur atom on the thiophene ring in the MeOTPATBT can be oxidized by the hypochlorous acid, and then the energy transfer from the CNPPV to the MeOTPATBT is blocked, so that the fluorescent spectrum of the Pdot probe changes in ratio. Experiments show that the fluorescent intensity of the probe at a red light region of 680 nm is obviously weakened, and the fluorescent intensity at 600 nm is enhanced, and the fluorescent change (I 600nm / I 680nmThe ratio detection of hypochlorous acid can be realized. The ratio type semiconductor polymer quantum dot fluorescent probe for detecting hypochlorous acid has small interference from the outside (ions, active oxygen, active nitrogen), high specificity, anti-interference and high stability. Compared with the traditional 'on-off' type fluorescent probe, the ratio type fluorescent probe has the advantages of low background fluorescence and strong anti-interference ability for the analysis and detection of hypochlorous acid, and can realize the detection of active oxygen in RAW 264.7 cells and mice.

[0031] The introduction of 3-aminopropyl triethoxysilane (APTES) helps to improve the stability of the probe, so that the stability of the probe is greatly enhanced. The interference experiment also proves that the probe with APTES has better selectivity and stronger anti-interference ability. BRIEF DESCRIPTION OF DRAWINGS

[0032] In order to make the content of the present application more easily understood, the present application will be further described in detail below according to the specific embodiments of the present application and in combination with the drawings, in which,

[0033] Figure 1 A is the synthesis process of the fluorescent compound MeOTPATBT; B is the principle of the response of the fluorescent compound MeOTPATBT to hypochlorous acid;

[0034] Figure 2 It is a synthesis process diagram of the ratio type fluorescent probe MeO-CNPPV Pdots in the embodiments of the present application;

[0035] Figure 3 It is the normalized absorption and emission spectrum of each single component of the Pdots probe in Example 1 in the experimental examples of the present application (the left curve in the same component is the absorption spectrum, and the right curve is the emission spectrum);

[0036] Figure 4 It is the response speed result of the Pdots probe to hypochlorous acid in Example 1 in the experimental examples of the present application;

[0037] Figure 5 It is the cell fluorescence microscope image (A) and the quantitative red channel fluorescence signal columnar statistical chart (B) of the RAW 264.7 cells after being treated by different schemes in the experimental examples of the present application and then dyed by the Pdots probe in Example 1;

[0038] Figure 6Fig. 1 shows the in vivo fluorescence imaging (A) and the fluorescence signal columnar statistics (B) of mice treated with PBS, LPS, NAC, LPS+NAC and NAC+LPS, and the in vivo fluorescence imaging (C) and the fluorescence signal columnar statistics (D) of mice treated with PBS, LPS, ABAH, LPS+ABAH and ABAH+LPS, for the responsive results of endogenous hypochlorous acid in cells in the experimental examples of the present application.

[0039] Figure 7 Fig. 2 shows the in vivo fluorescence imaging (A) and the fluorescence signal columnar statistics (B) of the knee joint site of mice injected with LPS. DETAILED DESCRIPTION

[0040] The following examples further illustrate the present application, but should not be construed as limiting the application. Modifications and variations of the methods, steps and conditions of the application are possible without departing from the spirit of the application.

[0041] In the following example embodiments of the present application, the structures of raw materials and the corresponding information are as follows:

[0042] Fluorescent semiconductor polymer CNPPV: i.e. poly(5-(2-ethylhexyloxy)-2-methoxy-cyanophthalaldehyde), abbreviated as CNPPV, CAS No. 194292-84-7;

[0043] Amphiphilic polymer PS-PEG-COOH: i.e. polystyrene-polyethylene glycol-carboxyl;

[0044] Amphiphilic PSMA, i.e. polystyrene maleic anhydride copolymer, CAS No. 9011-13-6;

[0045] 3-aminopropyltriethoxysilane (APTES): CAS No. 919-30-2.

[0046] Preparation Example 1 Synthesis of fluorescent compound MeOTPATBT

[0047]

[0048] As Figure 1The synthesis process shown in Figure A involves coupling the above-mentioned compound 4-boronate-4',4'-dimethoxytriphenylamine (2.5 mmol, 2.5 eq.) with the compound 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (1 mmol, 1 eq.) using a Suzuki coupling reaction under nitrogen protection.

[0049] The reaction conditions were as follows: a toluene (3 mL) solution containing potassium carbonate (2 mol / L, 2 mL), tetrabutylammonium bromide (0.025 mmol, 0.125 eq.), and tetra(triphenylphosphine)palladium (0.125 mmol, 0.125 eq.) was reacted at 85 °C for 24 h. The organic phase was then extracted with dichloromethane and water to separate it. The mixture was concentrated by rotary evaporation using a circulating water vacuum pump, and the concentrate was obtained by mixing petroleum ether and dichloromethane (volume ratio V). 石油醚 :V 二氯甲烷 The mixture was purified and separated by silica gel column chromatography at a ratio of 2:1 to obtain the purple solid compound MeOTPATBT.

[0050] like Figure 1 As shown in Figure B, the fluorescent compound MeOTPATBT is sensitive to hypochlorous acid. The sulfur atom of thiophene can be oxidized by hypochlorous acid, causing its absorption peak to change. This, in turn, affects the FRET process of CNPPV to MeOTPATBT inside the Pdots probe, resulting in a significant change in the fluorescence emission spectrum.

[0051] Preparation Example 2: Synthesis of the fluorescent compound MeOTPATBT

[0052] like Figure 1 The synthesis process shown in Figure A involves coupling the above-mentioned compound 4-boronate-4',4'-dimethoxytriphenylamine (2.5 mmol, 2.5 eq.) with the compound 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (1 mmol, 1 eq.) using a Suzuki coupling reaction under nitrogen protection.

[0053] The reaction conditions were as follows: a toluene (3 mL) solution containing potassium carbonate (2 mol / L, 2 mL), tetrabutylammonium bromide (0.02 mmol, 0.1 eq.), and tetra(triphenylphosphine)palladium (0.15 mmol, 0.15 eq.) was reacted at 85 °C for 24 h. The organic phase was then extracted with dichloromethane and water, separated, concentrated by rotary evaporation using a circulating water vacuum pump, and distilled with petroleum ether and dichloromethane (volume ratio V). 石油醚 :V 二氯甲烷 The mixture was purified and separated by silica gel column chromatography at a ratio of 2:1 to obtain the purple solid compound MeOTPATBT.

[0054] Preparation Example 3: Synthesis of the fluorescent compound MeOTPATBT

[0055] As Figure 1 shown in the synthetic process of A, the compound 4-boronic acid ester-4',4'-dimethoxytriphenylamine (2.5 mmol, 2.5 eq.) is coupled with the compound 4,7-bis(5-bromo-2-thienyl)-2,1,3-benzothiadiazole (1 mmol, 1 eq.) by Suzuki coupling reaction under nitrogen protection.

[0056] The reaction conditions are as follows: potassium carbonate (2 mol / L, 2 mL), tetrabutylammonium bromide (0.03 mmol, 0.15 eq.), a solution of tetrakis(triphenylphosphine)palladium (0.1 mmol, 0.1 eq.) in toluene (3 mL), reaction at 85°C for 24 h, extraction with dichloromethane and water, separation of the organic phase, rotary evaporation concentration with a circulating water vacuum pump, and purification and separation by silica gel column chromatography with petroleum ether and dichloromethane (volume ratio V 石油醚 :V 二氯甲烷 = 2:1) to obtain the compound MeOTPATBT in purple solid state.

[0057] Synthesis of MeO-CNPPV Pdots probe

[0058] The MeO-CNPPV Pdots probe described in this example is prepared by the nanometer precipitation method, and the specific synthesis process is as shown in Figure 2 .

[0059] The fluorescent compound MeOTPATBT prepared in Preparation Example 1 is dissolved in THF to obtain a stock solution of 2 mg / mL for standby; another fluorescent semiconductor polymer CNPPV is dissolved in THF to obtain a stock solution of 2 mg / mL for standby; and then the polymer PS-PEG-COOH is dissolved in THF to obtain a stock solution of 5 mg / mL for standby; and then the APTES stock solution is diluted 100 times with THF for standby.

[0060] The above stock solutions are used to prepare 0.4 mL of a mixture THF solution, which contains MeOTPATBT:CNPPV = 1:200, MeOTPATBT+CNPPV (i.e. fluorescent dye):PS-PEG-COOH = 1:1, and 10 μL of APTES dilution liquid. Z Under the action of strong ultrasonic waves (40 kHz, 5 minutes), the above 0.4 mL of mixture THF solution is quickly injected into 2 mL of ultrapure water for mixing, and the mixture is treated by ultrasonic waves in an ice water bath to form a uniform and clear solution.

[0061] The reaction solution was removed by rotary evaporation under reduced pressure, and filtered using a 0.2 μm cellulose membrane filter to obtain a MeO-CNPPV Pdots probe stock solution with a final concentration of 200 μg / mL.

[0062] Example 2 Synthesis of MeO-CNPPV Pdots probe

[0063] The MeO-CNPPV Pdots probe described in this example was prepared by the nanoprecipitation method, and the specific synthesis process is as shown in Figure 2 .

[0064] The fluorescent compound MeOTPATBT prepared in Preparation Example 1 was dissolved in THF to obtain a stock solution of 2 mg / mL, which was prepared for use; another fluorescent semiconductor polymer CNPPV was dissolved in THF to obtain a stock solution of 2 mg / mL, which was prepared for use; and the polymer PS-PEG-COOH was dissolved in THF to obtain a stock solution of 5 mg / mL, which was prepared for use.

[0065] Using the above stock solutions, 0.4 mL of a mixture THF solution was prepared, which contained MeOTPATBT: CNPPV = 1:200, MeOTPATBT + CNPPV (i.e. fluorescent dye): PS-PEG-COOH = 1:1. Under the action of strong ultrasonic waves (40 kHz, 5 minutes), the above 0.4 mL of mixture THF solution was rapidly injected into 2 mL of ultrapure water to mix, and the mixture was treated by ultrasonic waves in an ice water bath to form a uniform and clear solution. Z

[0066] The reaction solution was removed by rotary evaporation under reduced pressure, and filtered using a 0.2 μm cellulose membrane filter to obtain a MeO-CNPPV Pdots probe stock solution with a final concentration of 200 μg / mL.

[0067] Example 3

[0068] The MeO-CNPPV Pdots probe described in this example was prepared by the nanoprecipitation method, and the specific synthesis process is as shown in Figure 2 .

[0069] The fluorescent compound MeOTPATBT prepared in Preparation Example 1 was dissolved in THF to obtain a stock solution of 2 mg / mL, which was prepared for use; another fluorescent semiconductor polymer CNPPV was dissolved in THF to obtain a stock solution of 2 mg / mL, which was prepared for use; and the polymer PS-PEG-COOH was dissolved in THF to obtain a stock solution of 5 mg / mL, which was prepared for use; and the APTES stock solution was diluted 100 times with THF, which was prepared for use.

[0070] ​The above stock solutions were used to prepare 0.4 mL mixture THF solutions, which contained MeOTPATBT: CNPPV = 1 : 180, MeOTPATBT + CNPPV (i.e. fluorescent dye): PS-PEG-COOH = 1 : 1.2, and 10 μL APTES diluent. The 0.4 mL mixture THF solutions were rapidly injected into 2 mL ultrapure water under the action of strong ultrasound (40 KH Z zation (40 KH

[0071] The reaction solution was filtered using a 0.2 μm cellulose membrane filter to obtain a MeO-CNPPV Pdots probe stock solution with a final concentration of 200 μg / mL.

[0072] Example 4: Synthesis of MeO-CNPPV Pdots probe

[0073] The MeO-CNPPV Pdots probe described in this example was prepared by a nanometer precipitation method, and the specific synthesis process is shown in Figure 2

[0074] The fluorescent compound MeOTPATBT prepared in Preparation Example 1 was dissolved in THF to obtain a stock solution of 2 mg / mL; another fluorescent semiconductor polymer CNPPV was dissolved in THF to obtain a stock solution of 2 mg / mL; and the polymer PS-PEG-COOH was dissolved in THF to obtain a stock solution of 5 mg / mL.

[0075] The above stock solutions were used to prepare 0.4 mL mixture THF solutions, which contained MeOTPATBT: CNPPV = 1 : 180, MeOTPATBT + CNPPV (i.e. fluorescent dye): PS-PEG-COOH = 1 : 1.2. The 0.4 mL mixture THF solutions were rapidly injected into 2 mL ultrapure water under the action of strong ultrasound (40 KH Z zation (40 KH

[0076] The reaction solution was filtered using a 0.2 μm cellulose membrane filter to obtain a MeO-CNPPV Pdots probe stock solution with a final concentration of 200 μg / mL.

[0077] Example 5

[0078] The MeO-CNPPV Pdots probe described in this example was prepared by a nanometer precipitation method.

[0079] ​The fluorescent compound MeOTPATBT prepared in Preparation Example 1 was dissolved in THF to obtain a stock solution of 2 mg / mL, which was used as prepared; the fluorescent semiconductor polymer CNPPV was also dissolved in THF to obtain a stock solution of 2 mg / mL, which was used as prepared; and PSMA was dissolved in THF to obtain a stock solution of 5 mg / mL, which was used as prepared; and a stock solution of APTES was diluted 100 times with THF, which was used as prepared.

[0080] Using the above stock solutions, respectively, 0.4 mL of a mixture THF solution was prepared, which contained MeOTPATBT: CNPPV = 1:220, MeOTPATBT + CNPPV (i.e. fluorescent dye): PSMA = 1:0.8, and 10 μL of the APTES dilution was added. Under the action of strong ultrasonic waves (40 KH Z z, 5 minutes), the above 0.4 mL of the mixture THF solution was rapidly injected into 2 mL of ultrapure water for mixing, and the mixture was treated with ultrasonic waves in an ice water bath to form a uniform and clear solution.

[0081] The reaction solution was removed of THF by rotary evaporation under reduced pressure, and filtered using a 0.2 μm cellulose membrane filter to obtain a final MeO-CNPPV Pdots probe stock solution with a concentration of 200 μg / mL.

[0082] Example 6 Synthesis of MeO-CNPPV Pdots probe

[0083] The MeO-CNPPV Pdots probe described in this example was prepared by a nanometer precipitation method.

[0084] The fluorescent compound MeOTPATBT prepared in Preparation Example 1 was dissolved in THF to obtain a stock solution of 2 mg / mL, which was used as prepared; the fluorescent semiconductor polymer CNPPV was also dissolved in THF to obtain a stock solution of 2 mg / mL, which was used as prepared; and PSMA was dissolved in THF to obtain a stock solution of 5 mg / mL, which was used as prepared.

[0085] Using the above stock solutions, respectively, 0.4 mL of a mixture THF solution was prepared, which contained MeOTPATBT: CNPPV = 1:220, MeOTPATBT + CNPPV (i.e. fluorescent dye): PSMA = 1:0.8. Under the action of strong ultrasonic waves (40 KH Z z, 5 minutes), the above 0.4 mL of the mixture THF solution was rapidly injected into 2 mL of ultrapure water for mixing, and the mixture was treated with ultrasonic waves in an ice water bath to form a uniform and clear solution.

[0086] The reaction solution was removed of THF by rotary evaporation under reduced pressure, and filtered using a 0.2 μm cellulose membrane filter to obtain a final MeO-CNPPV Pdots probe stock solution with a concentration of 200 μg / mL.

[0087] Experimental Example

[0088] 1. Photophysical properties test of the MeO-CNPPV Pdots fluorescent probe

[0089] The probe described in Example 1 was subjected to probe spectroscopy in phosphate buffer (PB). To eliminate errors during UV absorption spectroscopy, a baseline scan was performed first, and the sample was tested after stabilization. The results are shown in the attached figure. Figure 3 As shown.

[0090] like Figure 3 The results shown indicate that the emission spectrum of CNPPV overlaps with the absorption spectrum of MeOTPATBT. Since the hypochlorous acid response affects the energy transfer between the two substances, thus causing a change in the spectrum, this probe can detect hypochlorous acid.

[0091] 2. Hypochlorous acid response test of the MeO-CNPPV Pdots fluorescent probe

[0092] This embodiment uses the probe described in Example 1 for testing. The fluorescence spectrum was tested using 440 nm as the excitation wavelength of the sample, and the test temperature was 25 °C. The results are shown in [Figure Number]. Figure 4 As shown.

[0093] like Figure 4 The results shown indicate that the probe reacts completely with hypochlorous acid after 5 minutes of addition. Therefore, 5 minutes is chosen as the reaction time between the probe and hypochlorous acid.

[0094] 3. The response of the MeO-CNPPV Pdots fluorescent probe to different concentrations of exogenous hypochlorous acid in RAW 264.7 cells.

[0095] RAW 264.7 cells were seeded in cell culture dishes and incubated overnight. After cell attachment, they were incubated for 24 hours in DMEM medium containing MeO-CNPPV Pdots at a concentration of 10 μg / mL. Then, after washing three times with PBS buffer, the cells were incubated for 30 minutes with different concentration gradients of hypochlorous acid solutions (0, 50 μmol / mL, 100 μmol / mL, and 150 μmol / mL). Figure 5 The results shown indicate that the fluorescence intensity at 600 nm increases with increasing hypochlorous acid concentration, demonstrating that the MeO-CNPPV Pdots fluorescent probe responds accurately and effectively to different concentrations of exogenous hypochlorous acid within cells.

[0096] 4. The response of the MeO-CNPPV Pdots fluorescent probe to endogenous hypochlorous acid in RAW 264.7 cells.

[0097] This example tests the responsiveness of the probe described in Example 1 to endogenous hypochlorous acid in RAW 264.7 cells using LPS as the endogenous hypochlorous acid inducer and NAC and ABAH as the hypochlorous acid inhibitors.

[0098] RAW 264.7 cells were seeded in cell culture dishes and incubated overnight. After the cells adhered, the cells were treated in different ways (i.e. PBS, LPS, NAC, LPS + NAC / ABAH and NAC / ABAH + LPS) and then further incubated with the probe described in Example 1 in the incubator for 24 hours. After the probe completely entered the cells, the fluorescence of the cells was imaged and the fluorescence intensity at 600 nm was calculated. As shown in the results in Figure 6 As shown in the results in Figure 6, the red fluorescence of the cells pretreated with LPS was the strongest. After NAC / ABAH treatment, the fluorescence intensity of the cells decreased significantly, while the fluorescence intensity of the cells pretreated with NAC / ABAH increased significantly after LPS stimulation, which indicated that the MeO-CNPPV Pdots fluorescent probe could effectively respond to endogenous probes in cells.

[0099] 5. Monitoring of mouse arthritis and treatment process by the MeO-CNPPV Pdots fluorescent probe

[0100] BALB / c mice were randomly divided into three groups. The first group of mice was injected with 25 μL of normal saline in the right knee as a negative control group. The second group of mice was injected with an equal amount of LPS with a concentration of 10 mg / mL in the right knee for 3 consecutive days to induce arthritis as a positive control. The third group of mice was first injected with an equal amount of LPS with a concentration of 5 mg / mL in the right knee for 3 consecutive days to induce arthritis, and then injected with an equal amount of NAC with a concentration of 60 mg / mL for 3 consecutive days for treatment. On the seventh day, 25 μL of 8 mg / mL MeO-CNPPV Pdots fluorescent probe was injected into the joint of the mice, and then in vivo fluorescence imaging was performed and the fluorescence intensity at 600 nm was calculated. As shown in the results in Figure 7 As shown in the results in Figure 7, the knee joint of the mice injected with LPS showed stronger fluorescence, and the fluorescence of the knee joint of the mice decreased significantly after NAC treatment, which indicated that the MeO-CNPPV Pdots had the potential to visualize ClO - mediated arthritis in vivo.

[0101] In summary, the MeO-CNPPV Pdots fluorescent probe prepared in the present application can be used for the detection of hypochlorous acid and has the advantages of high selectivity and good detection stability.

[0102] Obviously, the above embodiments are merely example for clearly illustrating but not limitation to the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments need not and can not be enumerated. The obvious changes or variations derived from the above description are still within the protection scope of the present application.

Claims

1. A ratiometric fluorescent probe for detecting hypochlorous acid, characterized in that, Prepared by nanocomprecipitation from the following components: Energy donor: CNPPV; Energy acceptor: fluorescent compound MeOTPATBT, having the structure shown below: ； Polymer for encapsulation: PS-PEG-COOH or PSMA; Optionally containing: 3-aminopropyl triethoxysilane APTES.

2. The fluorescent probe according to claim 1, wherein In the fluorescent probe, the mass ratio of MeOTPATBT to CNPPV is = 1:180-220, the mass ratio of the total amount of MeOTPATBT and CNPPV to the amphiphilic polymer is = 1:0.8-1.2, and if APTES is contained, the mass ratio of APTES to MeOTPATBT is 1:180-220.

3. A method for preparing the ratiometric fluorescent probe for detecting hypochlorous acid according to claim 1 or 2, characterized in that, The method comprises the steps of: adding a selected amount of MeOTPATBT, CNPPV and amphiphilic polymer to carry out nanocomprecipitation; and adding 3-aminopropyl triethoxysilane to carry out nanocomprecipitation.

4. Use of the fluorescent probe of claim 1 or 2 in non-diagnostic purposes, including detection of exogenous hypochlorous acid in environmental water bodies or extracellularly. detection.

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