Double-ratio type optical detection probe as well as preparation method and application thereof

Abstract

The invention discloses a double-ratio type optical detection probe and a preparation method and application thereof, and the preparation method comprises the following steps: dispersing 16, 17-dihydroxy anthrone violet and anhydrous potassium carbonate in N, N-dimethylformamide, stirring, rapidly adding 4-bromomethyl phenylboronic acid pinacol ester, heating to 120 DEG C, and reacting for 12 hours; slowly dropwise adding the reaction solution into methanol, settling for 24 hours, carrying out suction filtration, washing and extraction for three times, collecting an organic layer, carrying out rotary evaporation and drying, and purifying through silica gel column chromatography to obtain the anthrone violet derivative VTP; the preparation method comprises the following steps: uniformly mixing a tetrahydrofuran solution of an anthrone violet derivative VTP with a preset concentration and a tetrahydrofuran solution of an amphiphilic surfactant, injecting into water with a preset volume, and purifying to obtain the water-soluble optical probe. The invention solves the problems of low stability, poor specificity and high cost of most near-infrared fluorescent or photoacoustic organic probes, has good water solubility and biocompatibility, and can eliminate interference of other reactive oxygen free radicals in a normal physiological environment.

Description

Technical Field

[0001] The present invention relates to the technical field of organic luminescent materials, in particular to a dual ratio optical detection probe and a preparation method and application thereof. Background Art

[0002] Near-infrared optical probes have the advantages of high sensitivity, low interference, good penetration depth and resolution. By combining with spectrophotometers, fluorescence spectrometers, confocal laser scanning microscopes and animal fluorescence / photoacoustic imaging systems, they can accurately and comprehensively analyze various physiological and pathological processes and 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. - However, most near-infrared fluorescence / photoacoustic probes cannot detect peroxynitrite (ONOO - ).

[0003] In addition, currently available detection of ONOO - The near-infrared optical probes are mainly cyanine and boron dipyrrolidone (BODIPY) dyes, which may be affected by H2O2, ·OH, ClO - , 1 In addition, these dyes may have problems such as low stability, poor specificity and high cost, which are not conducive to large-scale promotion and application. Summary of the invention

[0004] The purpose of the present invention is to overcome the above problems or at least partially solve the above problems, and to propose a dual ratio optical detection probe and a preparation method and application thereof.

[0005] To achieve the above object, 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-dihydroxyanthraquinone violet and anhydrous potassium carbonate in N,N-dimethylformamide, stir at 20-80°C for 0.25-1h, quickly add 4-bromomethylphenylboronic acid pinacol ester, heat to 120°C and react for 12h;

[0007] S2. After the reaction is completed, the reaction solution of S1 is slowly dripped into methanol, filtered after settling for 24 hours, washed with methanol and water, and extracted three times with dichloromethane, the organic layer is collected and rotary evaporated, dried, and purified by silica gel column chromatography to obtain the anthrone violet derivative VTP;

[0008] S3, uniformly mixing a tetrahydrofuran solution of anthrone violet derivative VTP and a tetrahydrofuran solution of an amphiphilic surfactant of preset concentrations;

[0009] S4, under ultrasonic conditions, quickly inject the mixed solution of S3 into a preset volume of water, and maintain ultrasonic treatment until the nanoparticles are completely formed;

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

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

[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 S3, the concentrations of the anthrone violet derivative VTP and the amphiphilic surfactant are 1 mg / mL and 20 mg / mL, respectively, and the volume ratio of the two is 2:5; the amphiphilic surfactant includes but is not limited to nonionic, cationic, anionic and zwitterionic surfactants, such as F127, DSPE-PEG,

[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 amount of the mixed solution is 1.4 mL.

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

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

[0017] An application of a dual ratio optical detection probe. The probe prepared according to the preparation method of the dual ratio optical detection probe is applied in the field of optical detection, especially for detecting peroxynitrite (ONOO-) in biological samples; the probe can specifically detect ONOO -, excluding H2O2, ·OH, ClO - , 1 The probe has no obvious response to H2O2 in aqueous solution and can only respond under alkaline and heated conditions;

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

[0019] Compared with the prior art, the present invention has the following beneficial effects: the present invention solves the problems of low stability, poor specificity and high cost of most near-infrared fluorescent or photoacoustic organic probes, has good water solubility and biocompatibility, and can eliminate the interference of other active oxygen free radicals under normal physiological conditions, and specifically detect ONOO - . BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the synthesis reaction of the dual ratiometric 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 absorption ratio of the molecule obtained in Example 1 at 750nm and 640nm in tetrahydrofuran varies with the absorption of ONOO - Concentration change curve graph;

[0023] Figure 4 The tetrahydrofuran solution of the molecule obtained in Example 1 was added with ONOO - After that, the change of fluorescence intensity under 660nm excitation (detection wavelength>900nm) and under 808nm excitation;

[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 changes with the ONOO - A graph showing changes in concentration;

[0025] Figure 6 The ratio of the fluorescence intensity of the probe obtained in Example 1 at 808 nm and 660 nm is proportional to the fluorescence intensity of ONOO - Concentration variation graph;

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

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

[0028] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] See also Figure 1-Figure 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-dihydroxyanthraquinone violet (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, quickly add 4-bromomethylphenylboronic acid pinacol ester (4.46 g, 15 mmol), heat to 120 ° C and react for 12 h;

[0032] Step 2: After the reaction is completed, the S1 reaction solution is slowly dripped into methanol, and after settling for about 24 hours, it is rinsed with methanol and water, filtered, and the liquid phase is collected and extracted three times with dichloromethane, and the organic layer is collected and rotary evaporated and dried;

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

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

[0035] Step 5: At 50°C and under constant stirring, the tetrahydrofuran in the aqueous solution was blown away with nitrogen, and the ONOO with specific detection was obtained after ultrafiltration purification. - Dual ratiometric optical detection probe.

[0036] The experimental results show that the yield of VTP is 55%; Figure 2 As shown, the absorption peak of VTP is around 640nm, while the absorption peak of the raw material is around 800nm, which proves the successful synthesis of the product; Figure 3As shown in Figure 2, the absorbance ratio of the dye VTP tetrahydrofuran solution at 750nm and 660nm changes with the concentration of ONOO - Increases with increasing concentration; Figure 4 As shown, the tetrahydrofuran solution of VTP is added with ONOO - After that, the fluorescence intensity under 660nm excitation (detection wavelength>900nm) decreased significantly, and the fluorescence intensity under 808nm excitation increased significantly; the tetrahydrofuran solution of the dye VTP can also produce a similar reaction to hydrogen peroxide. Figure 3 and Figure 4 Response.

[0037] After water-soluble modification with F127, Figure 5 As shown in Figure 2, the ratio of the photoacoustic intensity of the probe at 750nm and 660nm increases with the increase of ONOO - It increases with the increase of concentration and shows a linear relationship in the range of 0 to 50uM, and can be used for ratiometric photoacoustic detection.

[0038] like Figure 6 As shown in Figure 2, the ratio of the fluorescence intensity of the obtained probe excited at 808 nm and 660 nm increases with the concentration of ONOO - The probe has no obvious response to H2O2 in aqueous solution, but can respond only under alkaline and heated conditions, and has no significant effect on other common interferences, such as OH, ClO - , 1 O2 and others did not respond, indicating that the probe was sensitive to ONOO - Has higher specificity.

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

[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, and 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 could 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 could respond to ONOO-.

[0044] Example 4

[0045] In Example 1, the amphiphilic surfactant was replaced by DSPE-PEG from F127, and the other steps were the same as those in Example 1. The performance of the obtained optical probe was close to that in Example 1, and it was able to produce a specific response to ONOO-.

[0046] In summary, the present invention designs a new stimulus-responsive material based on anthrone violet derivatives and constructs a dual-ratio optical detection probe, which has excellent near-infrared optical properties and specific response capabilities and can be used for the specific detection of ONOO-.

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

Claims

1. A method for preparing a dual ratiometric optical detection probe, characterized in that: The following steps are involved: S1. Disperse 16,17-dihydroxyanthraquinone violet and anhydrous potassium carbonate in N,N-dimethylformamide, stir at 20-80°C for 0.25-1h, then quickly add 4-bromomethylphenylboronic acid pinacol ester, heat to 120°C and react for 12h; S2. After the reaction is completed, the reaction solution of S1 is slowly dripped into methanol, filtered after settling for 24 hours, washed with methanol and water, and extracted three times with dichloromethane, the organic layer is collected and rotary evaporated, dried, and purified by silica gel column chromatography to obtain the anthrone violet derivative VTP; S3, uniformly mixing a tetrahydrofuran solution of anthrone violet derivative VTP and a tetrahydrofuran solution of an amphiphilic surfactant of preset concentrations; S4, under ultrasonic conditions, quickly inject the mixed solution of S3 into a preset volume of water, and maintain ultrasonic treatment; S5. Under heating conditions, tetrahydrofuran in the aqueous solution was blown off with nitrogen, and a water-soluble probe with near-infrared fluorescence and photoacoustic dual ratio detection capability was obtained after ultrafiltration purification for ONOO - Specific detection.

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

3. The method for preparing a dual ratiometric optical detection probe according to claim 2, characterized in that: In S2, the volume of methanol is 20 times the volume of the reaction solution.

4. The method for preparing a dual ratiometric 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, and the volume ratio between the two is 2:5; the amphiphilic surfactant includes but is not limited to nonionic, cationic, anionic and zwitterionic surfactants.

5. The method for preparing a dual ratiometric 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 amount of the mixed solution is 1.4 mL.

6. The method for preparing a dual ratiometric optical detection probe according to claim 5, characterized in that: In the 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 the preparation method of any one of claims 1 to 6, wherein the probe includes an anthrone violet derivative VTP and an amphiphilic surfactant, and the change in fluorescence intensity of the probe at two excitation wavelengths of 660nm and 808nm constructs a fluorescence-based excitation ratio probe; the probe utilizes the change in absorption spectrum before and after response to realize ratiometric photoacoustic detection.

8. An application of a dual ratio optical detection probe, characterized in that: The probe prepared by the preparation method of the dual ratio optical detection probe according to any one of claims 1 to 6 is applied to the field of optical detection, in particular, to the detection of peroxynitrite (ONOO-) in biological samples; the probe can specifically detect ONOO - , excluding H2O2, ·OH, ClO - ,1O2 active oxygen interference; the probe has no obvious response to H2O2 in aqueous solution, and can only respond under alkaline and heated conditions; The probe can also be used to detect ONOO- in pathological organs such as liver damage and diabetes, and achieve high-sensitivity and high-specificity detection through fluorescence and photoacoustic dual ratio imaging technology.

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