A dual-ratio optical detection probe, its preparation method and application

Abstract

This invention discloses a dual-ratio optical detection probe, its preparation method, and its application, comprising the following steps: dispersing 16,17-dihydroxyanthrone violet and anhydrous potassium carbonate in N,N-dimethylformamide, stirring, then rapidly adding 4-bromomethylphenylboronic acid pinacol ester, and reacting at 120°C for 12 hours; slowly adding the reaction solution dropwise into methanol, allowing it to settle for 24 hours, then filtering, washing, and extracting three times, collecting the organic layer by rotary evaporation and drying, and purifying by silica gel column chromatography to obtain the anthrone violet derivative VTP; mixing a tetrahydrofuran solution of the anthrone violet derivative VTP at a predetermined concentration with a tetrahydrofuran solution of an amphiphilic surfactant, and injecting the mixture into a predetermined volume of water, purifying to obtain a water-soluble optical probe. This invention solves the problems of low stability, poor specificity, and high cost of most near-infrared fluorescent or photoacoustic organic probes, exhibiting excellent water solubility and biocompatibility, and can eliminate interference from other reactive oxygen free radicals under normal physiological conditions.

Description

Technical Field

[0001] This invention relates to the field of organic light-emitting materials technology, specifically to a dual-ratio optical detection probe, its preparation method, and its application. Background Technology

[0002] Near-infrared optical probes, with their advantages of high sensitivity, low interference, good penetration depth, and resolution, can accurately and comprehensively analyze various physiological and pathological processes when combined with spectrophotometers, fluorescence spectrometers, confocal laser scanning microscopes, and animal fluorescence / photoacoustic imaging systems. They have become one of the important tools for detecting various indicators and biomolecules in biological samples. - ONOO is widely present in many diseased organs, such as liver damage and diabetes. - While changes can be observed, most near-infrared fluorescence / photoacoustic probes cannot yet detect peroxynitroso (ONOO) with high specificity. - ).

[0003] In addition, the existing detection of ONOO - Near-infrared optical probes mainly consist of cyanine and boron dipyrrole (BODIPY) dyes, which may be affected by H2O2, ·OH, and ClO during detection. - , 1 Interference from reactive oxygen species such as O2. Furthermore, these dyes may suffer from low stability, poor specificity, and high cost, hindering their widespread application. Summary of the Invention

[0004] The purpose of this invention is to overcome or at least partially solve the above-mentioned problems by proposing a dual-ratio optical detection probe, its preparation method, and its application.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a method for preparing a dual-ratio optical detection probe, comprising the following steps:

[0006] S1. Disperse 16,17-dihydroxyanthrone purple and anhydrous potassium carbonate in N,N-dimethylformamide, stir at 20-80℃ for 0.25-1h, then rapidly add 4-bromomethylphenylboronic acid pinacol ester, and heat to 120℃ to react for 12h.

[0007] S2. After the reaction is complete, the reaction solution of S1 is slowly added dropwise to methanol. After settling for 24 hours, the solution is filtered, washed with methanol and water, and extracted three times with dichloromethane. The organic layer is collected, evaporated and dried, and purified by silica gel column chromatography to obtain the anthrone purple derivative VTP.

[0008] S3. Mix the tetrahydrofuran solution of anthrone violet derivative VTP at a preset concentration and the tetrahydrofuran solution of the amphiphilic surfactant evenly.

[0009] S4. Under ultrasonic conditions, rapidly inject the mixed solution from S3 into a predetermined volume of water and continue ultrasonic treatment until the nanoparticles are fully formed.

[0010] S5. Under heating conditions, tetrahydrofuran in the aqueous solution is blown away with nitrogen gas, and after ultrafiltration purification, a water-soluble probe with near-infrared fluorescence and photoacoustic dual ratio detection capabilities is obtained for ONOO. - Specific detection.

[0011] In a preferred embodiment, in S1, the concentration of 16,17-dihydroxyanthrone purple in N,N-dimethylformamide is 100 mM, and the molar ratio of 16,17-dihydroxyanthrone purple, anhydrous potassium carbonate, and pinacol ester of 4-bromomethylphenylboronic acid is 1:2 to 8:2 to 6.

[0012] In a preferred embodiment, in S2, the volume of methanol is 20 times the volume of the reaction solution.

[0013] In a preferred embodiment, in step S3, the concentrations of the anthrone violet derivative VTP and the amphiphilic surfactant are 1 mg / mL and 20 mg / mL, respectively, with a volume ratio of 2:5. The amphiphilic surfactant includes, but is not limited to, nonionic, cationic, anionic, and zwitterionic surfactants, such as F127, DSPE-PEG, etc.

[0014] In a preferred embodiment, in step S4, the preset volume of water is 9 mL, the ultrasonic time is 2-3 min, and the volume of the mixed solution is 1.4 mL.

[0015] In a preferred embodiment, the heating temperature in step S5 is 50°C.

[0016] A dual-ratio optical detection probe is prepared according to the method described above. The probe comprises anthrone violet derivative VTP and an amphiphilic surfactant. The fluorescence intensity change of the probe at two excitation wavelengths of 660 nm and 808 nm constitutes a fluorescence-based excitation ratio probe. The probe utilizes the change in absorption spectrum before and after the response to achieve ratiometric photoacoustic detection.

[0017] An application of a dual-ratio optical detection probe: The probe prepared according to the method described above is used in the field of optical detection, particularly for detecting peroxynitrite (ONOO-) in biological samples; the probe can specifically detect ONOO-. -Excluding H2O2, ·OH, and ClO - , 1 Interference from reactive oxygen species (O2); the probe does not show a significant response to H2O2 in aqueous solution, but only responds under alkaline and heated conditions;

[0018] The probe can also be used to detect ONOO- in diseased organs such as liver damage and diabetes, achieving high sensitivity and high specificity through fluorescence and photoacoustic dual ratio imaging technology.

[0019] Compared with existing technologies, the present invention has the following advantages: The present invention solves the problems of low stability, poor specificity, and high cost of most near-infrared fluorescent or photoacoustic organic probes, possesses excellent water solubility and biocompatibility, and can eliminate interference from other reactive oxygen species under normal physiological conditions, specifically detecting ONOO. - . Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the synthesis reaction of the dual-ratio optical detection probe of the present invention;

[0021] Figure 2 The absorption spectra of the molecules and raw materials obtained in Example 1 are shown.

[0022] Figure 3 The ratio of the material absorption at 750 nm to 640 nm of the molecule obtained in Example 1 in tetrahydrofuran as a function of ONOO - Concentration change curve;

[0023] Figure 4 The tetrahydrofuran solution of the molecule obtained in Example 1 was prepared by adding ONOO. - Then, the changes in fluorescence intensity under 660nm excitation (detection wavelength >900nm) and under 808nm excitation are shown in the graph;

[0024] Figure 5 The ratio of the photoacoustic intensity at 750 nm to the photoacoustic intensity at 660 nm of the probe obtained in Example 1 varies with ONOO. - A graph showing the concentration change;

[0025] Figure 6 The ratio of fluorescence intensity excited at 808 nm and 660 nm by the probe obtained in Example 1 as a function of ONOO - Concentration change graph;

[0026] Figure 7 The probe obtained in Example 1 was used for photoacoustic detection of liver injury. The comparison of photoacoustic intensity of the liver at 660nm and 750nm between the control group and the liver injury group after probe injection is shown.

[0027] Figure 8 The fluorescence intensity ratio of the control group and the liver injury group when excited at 808 nm and 660 nm after probe injection is shown for fluorescence detection of liver injury using the probe obtained in Example 1. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example 1

[0030] Please see Figures 1-8 As shown, the present invention provides a technical solution: a method for preparing a dual-ratio optical detection probe, comprising the following steps:

[0031] Step 1: Prepare 16,17-dihydroxyanthrone purple (2.44 g, 5 mmol), anhydrous potassium carbonate (2.76 g, 20 mmol) and N,N-dimethylformamide (20 mL), mix and stir at 80 °C for 0.5 h, then rapidly add 4-bromomethylphenylboronic acid pinacol ester (4.46 g, 15 mmol) and heat to 120 °C for 12 h.

[0032] Step 2: After the reaction is complete, the S1 reaction solution is slowly added dropwise to methanol. After settling for about 24 hours, it is washed with methanol and water, filtered, and the liquid phase is collected and extracted three times with dichloromethane. The organic layer is collected and evaporated by rotary evaporation and dried.

[0033] Step 3: Add 400 μL of VTP tetrahydrofuran solution (1 mg / mL) to F127 tetrahydrofuran solution (1 mL, 1 mg / mL) and mix well.

[0034] Step 4: Under ultrasonic conditions, rapidly inject the above solution into 9 mL of water and maintain ultrasonication for 3 minutes;

[0035] Step 5: Under conditions of 50℃ and continuous stirring, the tetrahydrofuran in the aqueous solution is blown away with nitrogen gas, and after ultrafiltration purification, the product with specific detection capability for ONOO is obtained. - A dual-ratio optical detection probe.

[0036] Experimental results show that the yield of VTP is 55%; Figure 2 As shown, the absorption peak of VTP is around 640 nm, while the absorption peak of the raw material is around 800 nm, proving the successful synthesis of the product; Figure 3As shown, the ratio of the absorbance of the tetrahydrofuran solution of dye VTP at 750 nm and 660 nm varies with ONOO. - The concentration increases and the level rises; for example... Figure 4 As shown, the tetrahydrofuran solution of VTP was reacted with the addition of ONOO. - Subsequently, the fluorescence intensity decreased significantly under 660 nm excitation (detection wavelength > 900 nm), but increased significantly under 808 nm excitation; the tetrahydrofuran solution of dye VTP also exhibited a similar effect to hydrogen peroxide. Figure 3 and Figure 4 The response.

[0037] After water-soluble modification using F127, such as Figure 5 As shown, the ratio of the photoacoustic intensity of the obtained probe at 750 nm and 660 nm varies with ONOO - The concentration increases with increasing concentration, showing a linear relationship in the range of 0–50 μM, and can be used for ratiometric photoacoustic detection.

[0038] like Figure 6 As shown, the ratio of fluorescence intensity excited by the obtained probe at 808 nm and 660 nm varies with ONOO - The concentration increases with increasing fluorescence intensity, exhibiting a linear relationship within the range of 0–50 μM, and can be used for ratiometric fluorescence detection. The obtained probe does not show a significant response to H₂O₂ in aqueous solution, but only responds under alkaline and heated conditions, and is resistant to other common interfering substances such as ·OH and ClO₂. - , 1 There was no response from O2 or other substances, indicating that the probe was not responding to ONOO. - It has high specificity.

[0039] Using acetaminophen (APAP) as a drug-induced acute liver injury model in mice, after intravenous injection of the probe, such as Figure 7 As shown, the photoacoustic signal of the liver at 660 nm is significantly lower than that of normal mice, while the photoacoustic signal at 750 nm is significantly higher than that of normal mice. Therefore, it can be used for ratiometric photoacoustic imaging detection in a mouse model of liver injury. Figure 8 As shown, the ratio of fluorescence intensity in the liver region of liver-injured mice under excitation at 808 nm and 660 nm was significantly higher than that in normal mice, which can be used for ratiometric fluorescence imaging detection in mouse liver injury models.

[0040] Example 2

[0041] The concentration of 4-bromomethylphenylboronic acid pinacol ester in Example 1 was adjusted to 10 mmol, and the other steps were the same as in Example 1. The amount of 4-bromomethylphenylboronic acid pinacol ester was reduced so that the yield of VTP was 38%. The optical properties of the obtained product were similar to those in Example 1, and it was able to respond to ONOO-.

[0042] Example 3

[0043] The concentration of 4-bromomethylphenylboronic acid pinacol ester in Example 1 was adjusted to 20 mmol, and the other steps were the same as in Example 1. The amount of 4-bromomethylphenylboronic acid pinacol ester was further increased so that the yield of VTP was 58%. The optical properties of the obtained product were similar to those in Example 1, and it was able to respond to ONOO-.

[0044] Example 4

[0045] In Example 1, the amphiphilic surfactant was replaced by DSPE-PEG, and the other steps were the same as in Example 1. The resulting optical probe had similar performance to that of Example 1 and was able to produce a specific response to ONOO-.

[0046] In summary, this invention designs a novel stimulus-responsive material based on anthrone violet derivatives and constructs a dual-ratio optical detection probe that combines excellent near-infrared optical properties with specific response capability, and can be used for the specific detection of ONOO-.

[0047] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for preparing a dual-ratio optical detection probe, characterized in that, Includes the following steps: S1. Disperse 16,17-dihydroxyanthrone purple and anhydrous potassium carbonate in N,N-dimethylformamide and stir at 20-80℃ for 0.25-1h. Then, rapidly add 4-bromomethylphenylboronic acid pinacol ester and heat to 120℃ to react for 12h. S2. After the reaction is complete, the reaction solution of S1 is slowly added dropwise to methanol. After settling for 24 hours, the solution is filtered, washed with methanol and water, and extracted three times with dichloromethane. The organic layer is collected, evaporated and dried, and purified by silica gel column chromatography to obtain the anthrone purple derivative VTP. S3. Mix the tetrahydrofuran solution of anthrone violet derivative VTP at a preset concentration and the tetrahydrofuran solution of an amphiphilic surfactant evenly. The amphiphilic surfactant is polyoxyethylene-polyoxypropylene-polyoxyethylene triblock copolymer F127 or distearate phosphatidylethanolamine-polyethylene glycol DSPE-PEG. S4. Under ultrasonic conditions, rapidly inject the mixed solution from S3 into a predetermined volume of water and maintain ultrasonic treatment. S5. Under heating conditions, tetrahydrofuran in the aqueous solution is blown away with nitrogen gas, and after ultrafiltration purification, a water-soluble probe with near-infrared fluorescence and photoacoustic dual ratio detection capabilities is obtained for ONOO. - Specific detection.

2. The method for preparing a dual-ratio optical detection probe according to claim 1, characterized in that: In S1, the concentration of 16,17-dihydroxyanthrone purple in N,N-dimethylformamide is 100 mM, and the molar ratio of 16,17-dihydroxyanthrone purple, anhydrous potassium carbonate, and pinacol ester of 4-bromomethylphenylboronic acid is 1:2~8:2~6.

3. The method for preparing a dual-ratio optical detection probe according to claim 2, characterized in that: In step S2, the reaction solution from step S1 is slowly added dropwise to methanol, wherein the volume of methanol is 20 times the volume of the reaction solution.

4. The method for preparing a dual-ratio optical detection probe according to claim 3, characterized in that: In S3, the concentrations of the anthrone violet derivative VTP and the amphiphilic surfactant are 1 mg / mL and 20 mg / mL, respectively, with a volume ratio of 2:

5.

5. The method for preparing a dual-ratio optical detection probe according to claim 4, characterized in that: In step S4, the preset volume of water is 9 mL, the ultrasonic time is 2-3 min, and the volume of the mixed solution is 1.4 mL.

6. The method for preparing a dual-ratio optical detection probe according to claim 5, characterized in that: In step S5, the heating temperature is 50°C.

7. A dual-ratio optical detection probe, characterized in that: The dual-ratio optical detection probe is prepared according to any one of claims 1 to 6. The probe is composed of anthrone violet derivative VTP and an amphiphilic surfactant. The fluorescence intensity change of the probe at two excitation wavelengths of 660 nm and 808 nm constitutes a fluorescence-based excitation ratio probe. The probe realizes ratiometric photoacoustic detection by utilizing the change in absorption spectrum before and after the response.

8. The application of the probe prepared by the method of any one of claims 1 to 6 in the preparation of a peroxynitrosamine reagent for detecting oxygen in biological samples, wherein the probe is capable of specifically detecting ONOO. - Exclude H2O2, ·OH, and ClO − , 1 Interference from reactive oxygen species in O2.

9. The application of the probe prepared by the method of any one of claims 1 to 6 in the preparation of ONOO- reagent for detecting liver injury in diseased organs.

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