Visual enzyme activated fluorescent probe, synthetic method, biological application and method for detecting beta-Gal activity

Abstract

The invention relates to a visual enzyme activated fluorescent probe, a synthesis method, biological application and a method for detecting beta-Gal activity. The chemical structure of the fluorescent probe comprises D-galactose residues and a cyanine dye matrix, and the cyanine dye can be released under the action of beta-Gal to generate a fluorescence enhancement effect. The fluorescent probe provided by the invention can realize high-sensitivity, high-selectivity and real-time in-situ detection of beta-Gal activity, is suitable for imaging analysis of living cell and living animal levels, and provides a powerful tool for pathological research and clinical diagnosis of beta-Gal related diseases.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological detection, and particularly relates to a visual enzyme-activated fluorescent probe, a synthesis method, biological application and a method for detecting β-Gal activity. BACKGROUND

[0002] Colorectal cancer (CRC) is the third most commonly diagnosed cancer worldwide and the second leading cause of cancer-related deaths. β-Galactosidase (β-Gal) is an exoglycosidase that can specifically hydrolyze glycosidic bonds, and is a classic biomarker of cell aging, and is also closely related to a variety of physiological and pathological processes. Therefore, it is of great clinical significance to develop a specific and efficient quantitative detection method for β-Gal activity.

[0003] Existing techniques for detecting β-Gal activity include positron emission tomography, bioluminescence and activity detection kits, but these methods have defects such as complex operation, high detection limit, and inability to achieve real-time in situ visualization detection in vivo. Fluorescence imaging detection technology has become a research hotspot due to its low detection limit, imaging capability and quasi-in situ monitoring performance. However, the reported β-Gal fluorescent probes based on traditional chromophores have problems such as poor biocompatibility and small Stokes shift (<100 nm). Derivatives of cyanine dyes have great potential in biomolecular detection due to their strong intramolecular charge transfer (ICT) effect and "push-pull" substituent pairs, and have advantages such as structural stability and excellent fluorescence properties. However, there are still few β-Gal fluorescent probes based on cyanine dyes, so it is of great value to develop a high-performance cyanine dye-based β-Gal fluorescent probe. SUMMARY

[0004] The present application aims to solve the problems of existing β-Gal detection methods, such as complex operation, high detection limit, poor biocompatibility, and inability to achieve real-time in situ visualization detection, and provides a visual enzyme-activated fluorescent probe, a synthesis method, biological application and a method for detecting β-Gal activity. The fluorescent probe can achieve high sensitivity, high selectivity, real-time in situ detection of β-Gal activity, and is suitable for imaging analysis at the level of living cells and living animals.

[0005] The first object of the present application is to provide a visual enzyme-activated fluorescent probe, which is achieved by the following technical solution: A visual enzyme-activated fluorescent probe, characterized in that the chemical structure of the fluorescent probe contains a D-galactose residue and a cyanine dye parent body, and can release a cyanine dye under the action of β-Gal, producing a fluorescence enhancement effect.

[0006] As a further improvement of the fluorescent probe, the structural formula of the fluorescent probe is: As a further improvement of the fluorescent probe, the fluorescent probe has weak self-fluorescence in an aqueous solution, and generates strong fluorescence at 576 nm after reacting with β-Gal, with an excitation wavelength of 496 nm.

[0007] A second object of the present application is to provide a synthesis method of a visualized enzyme-activated fluorescent probe, which is achieved by the following technical scheme: A synthesis method of a visualized enzyme-activated fluorescent probe, characterized by comprising the following steps: Step 1: weigh o-hydroxybenzaldehyde and 2-benzoylpyridine, add appropriate amount of acetic acid and ammonium acetate, continuously introduce high-purity nitrogen (purity ≥ 99.99%) into the reaction system to exclude air, maintain a nitrogen atmosphere, control the nitrogen flow rate to be 10-15 mL / min, heat to 120 ℃, and heat under magnetic stirring for 12-16 h to obtain an intermediate product I; the structural formula of the intermediate product I is:

[0008] Step 2: weigh the intermediate product I and α-acetobromogalactose, add appropriate amount of cesium carbonate, sodium sulfate and acetonitrile, react at room temperature for 3 hours, after the reaction is completed, the filtrate is concentrated by rotary evaporation to obtain a crude product, which is then purified by column chromatography to obtain an intermediate product II, and the structural formula of the intermediate product II is:

[0009] Step 3: add appropriate amount of potassium carbonate (K2CO3) and methanol (CH3OH) to the intermediate product II, and react at room temperature for 30 min to obtain the target fluorescent probe.

[0010] As a further improvement of the synthesis method, in step 1, the molar ratio of o-hydroxybenzaldehyde and 2-benzoylpyridine is 1: (1-1.2), the amount of acetic acid is 3-5 times the equivalent of o-hydroxybenzaldehyde, and the amount of ammonium acetate is 0.5-1 times the equivalent of o-hydroxybenzaldehyde.

[0011] As a further improvement of the synthesis method, in step 2, the molar ratio of intermediate product I and α-acetobromogalactose is 1: (1.1-1.2), the amount of cesium carbonate is 5-10 times the equivalent of intermediate product I, the amount of sodium sulfate is 5-10 times the equivalent of intermediate product I, and the amount of acetonitrile is 5-10 times the equivalent of intermediate product I.

[0012] As a further improvement of the synthesis method, in step 3, the amount of potassium carbonate is 3-5 times the equivalent amount of intermediate II, and the amount of methanol is 5-10 times the equivalent amount of intermediate II.

[0013] A third object of the present application is to provide an application of the visualized enzyme-activated fluorescent probe in detecting β-Gal activity.

[0014] As a further improvement of the application, the detection includes quantitative detection of β-Gal in vitro solution, and visualized detection of endogenous and exogenous β-Gal in living cells.

[0015] As a further improvement of the application, the detection further includes real-time in situ imaging detection of β-Gal activity in BALB / c nude mice.

[0016] A fourth object of the present application is to provide a method for detecting β-Gal activity by visualizing enzyme-activated fluorescent probes, which is achieved by the following technical solutions: A method for detecting β-Gal activity, characterized by comprising the following steps: (1) mixing the fluorescent probe with the sample to be detected, incubating for 25 minutes to obtain a reaction product; (2) detecting the fluorescence intensity of the reaction product at 576 nm under excitation light, and determining the activity of the sample to be detected according to the standard curve, wherein the excitation wavelength of the excitation light is 496 nm.

[0017] As a further improvement of the method for detecting β-Gal activity, the sample to be detected includes biological fluids, living cells or living tissues.

[0018] Compared with the prior art, the present application has the following advantages: 1) High sensitivity: the fluorescent probe of the present application has a detection limit of 0.024 U / mL for β-Gal, and the fluorescence enhancement factor can reach 11.3 times, which can realize accurate detection of low concentration β-Gal.

[0019] 2) High selectivity: the fluorescent probe of the present application only produces specific fluorescence response to β-Gal in the presence of various biological interference substances, which can effectively avoid false positive results.

[0020] 3) Excellent stability: the fluorescent probe of the present application has good light stability and pH stability, and is suitable for detection under physiological conditions.

[0021] 4) Good biocompatibility: the fluorescent probe of the present application has low cytotoxicity and can be used for living cell detection.

[0022] 5), Real-time in situ detection: the fluorescent probe of the application can quickly respond to beta-Gal (reaching the fluorescence peak within 25 minutes), and realize real-time and long-term visual monitoring of beta-Gal activity in living cells and living animals, thereby providing a powerful tool for pathological research and clinical diagnosis of beta-Gal related diseases. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The synthetic route map of the fluorescent probe (CS-betaGal) prepared in the application.

[0024] Figure 2 The working principle of CS-betaGal for detecting beta-Gal and the electron microscope photo comparison.

[0025] Figure 3A The fluorescence spectrum change curve graph of CS-betaGal reacting with different concentrations of beta-Gal; wherein, 1: 9 U / mL; 2: 8 U / mL; 3: 7 U / mL; 4: 6 U / mL; 5: 5 U / mL; 6: 4 U / mL; 7: 3.5 U / mL; 8: 3 U / mL; 9: 2 U / mL; 10: 1 U / mL; 11: 0 U / mL.

[0026] Figure 3B The standard curve graph of the fluorescence intensity of CS-betaGal reacting with different concentrations of beta-Gal at 576 nm and the concentration of beta-Gal.

[0027] Figure 4A The fluorescence spectrum change graph of CS-betaGal reacting with beta-Gal at different time points; wherein, 1: 30 minutes; 2: 25 minutes; 3: 20 minutes; 4: 15 minutes; 5: 10 minutes; 6: 5 minutes; 7: 0 minutes.

[0028] Figure 4B The curve graph of the fluorescence intensity of CS-betaGal reacting with beta-Gal at 576 nm and the time.

[0029] Figure 5 The selective response histogram of CS-betaGal to various substances; wherein, 1: CS-betaGal alone; 2: AlCl3; 3: CaCl2; 4: NO3⁻; 5: ONOO⁻; 6: ClO⁻; 7: NaHS; 8: L-cysteine; 9: glutathione; 10: beta-glucuronidase; 11: lysozyme; 12: esterase; 13: human serum albumin; 14: cytochrome C; 15: immunoglobulin; 16: myoglobin; 17: beta-Gal.

[0030] Figure 6 The light stability comparison curve of CS-betaGal and fluorescein.

[0031] Figure 7 The fluorescence intensity curve of CS-βGal with or without β-Gal at different pH.

[0032] Figure 8 The toxicity histogram of CS-βGal to LoVo and HeLa cells.

[0033] Figure 9 The imaging graph of CS-βGal in different cells; wherein, a-c: HeLa cells; d-f: LoVo cells; g-i: LoVo+exogenous β-Gal; j-l: LoVo+inhibitor.

[0034] Figure 10 The real-time imaging graph of CS-βGal in nude mice; from left to right, from top to bottom, 0, 15 minutes, 30 minutes, 2 hours, 12 hours, 24 hours. DETAILED DESCRIPTION

[0035] The application will be further described in conjunction with specific drawings and examples, but the application is not limited to the following examples. The methods described below are all conventional methods unless otherwise specified, and the raw materials described are all available from public commercial channels unless otherwise specified.

[0036] Example 1: Preparation of visual enzyme-activated fluorescent probe (CS-βGal) Step 1: Take o-hydroxybenzaldehyde (C7H6O2) and 2-benzoylpyridine (C12H9NO) in a molar ratio of 1: (1~1.2), add acetic acid (Acetic acid) and ammonium acetate (ammonium acetate), wherein the amount of acetic acid is 3~5 times the equivalent of o-hydroxybenzaldehyde, and the amount of ammonium acetate is 0.5~1 times the equivalent of o-hydroxybenzaldehyde; continuously introduce high-purity nitrogen (purity ≥ 99.99%) into the reaction system to exclude air, maintain a nitrogen atmosphere, control the nitrogen flow rate to be 10-15 mL / min, heat to 120 ℃, and heat under magnetic stirring for 12~16h to obtain intermediate product I; Step 2: Take the intermediate product I and α-acetobromogalactose (α-Acetobromogalactose) in a molar ratio of 1: (1.1~1.2), add cesium carbonate (CsCO3), sodium sulfate (Na2SO4) and acetonitrile (H3CN), wherein the amount of cesium carbonate is 5~10 times the equivalent of intermediate product I, the amount of sodium sulfate is 5~10 times the equivalent of intermediate product I, acetonitrile is the solvent, and the amount of acetonitrile is 5~10 times the equivalent of intermediate product I; react at room temperature for 3 hours, after the reaction is completed, the crude product is obtained by suction filtration and rotary evaporation of the filtrate, and then purified by column chromatography to obtain intermediate product II; Step 3: Add potassium carbonate (K2CO3) and methanol (CH3OH) to the intermediate product II, wherein the amount of potassium carbonate is 3-5 times the equivalent of the intermediate product II, methanol is the solvent, and the amount of methanol is 5-10 times the equivalent of the intermediate product II; react at room temperature for 30 min to obtain the target fluorescent probe, visual enzyme-activated fluorescent probe (CS-βGal), and then verify its structure by nuclear magnetic resonance hydrogen spectrum, mass spectrum and other means.

[0037] The chemical structure of the fluorescent probe synthesized in the above steps contains a D-galactose residue and a cyanine dye parent. In an aqueous solution, the CS-βGal itself has weak fluorescence

see Figure 2 Photo A in the figure

see Figure 2 Photo B in the figure

[0038] 2. Instruments: Fluorescence spectrophotometer, fluorescence microscope, in vivo imaging system, enzyme label instrument, etc.

[0039] (II) Performance test steps 1. Spectral property test: Dissolve CS-βGal in anhydrous DMSO to prepare a 1×10⁻²M stock solution; dilute the stock solution to 10 μM with PBS buffer, mix with different concentrations of β-Gal (0-12 U / mL), incubate at 37°C for 25 min, and then detect the fluorescence spectrum (excitation wavelength 496 nm) on the fluorescence spectrophotometer, the detection results are shown in Figure 3A . Record the change of fluorescence intensity at 576 nm, draw the standard curve, and calculate the detection limit and linear range of the probe (3Sb / m, where Sb is the standard deviation of the blank signal, and m is the slope of the standard curve)

see Figure 3B

[0040] 2. Response time test: Mix 10 μΜ CS-βGal with 9 U / mL β-Gal in PBS buffer, incubate at 37℃, detect fluorescence spectrum (excitation wavelength 496 nm) at 0, 5, 10, 15, 20, 25, 30 minutes respectively

see Figure 4A

see Figure 4B

[0041] 3. Selective test: Mix 10 μΜ CS-βGal with the following substances respectively in PBS buffer (incubate at 37℃ for 25 minutes): AlCl3, CaCl2, NO3⁻, ONOO⁻, ClO⁻, NaHS, L-cysteine, glutathione, β-glucuronidase, lysozyme, esterase, human serum albumin, cytochrome C, immunoglobulin, myoglobin, β-Gal, detect the fluorescence intensity at 576 nm

see Figure 5

[0042] 4. Stability test: 4.1) Light stability: 10 μΜ CS-βGal and fluorescein were placed under the same light conditions respectively, and the fluorescence intensity was detected every 20 minutes

see Figure 6

[0043] 4.2) pH stability: in PBS buffer at pH 4-10, the fluorescence intensity of 10 μΜ CS-βGal with or without 9 U / mL β-Gal was detected respectively

see Figure 7

see Figure 8

[0044] 2. Cell imaging: CS-βGal was co-incubated with HeLa cells (low expression of β-Gal), LoVo cells (high expression of β-Gal), and LoVo cells pretreated with exogenous β-Gal or β-Gal inhibitor (D-galactose), and the changes in intracellular fluorescence signals were observed by fluorescence microscopy. The specific steps are as follows: HeLa cells and LoVo cells were seeded in confocal culture dishes, and after 24 hours of culture, 10 μM CS-βGal was added and incubated at 37°C for 30 minutes. For the pretreatment group, LoVo cells were first incubated with exogenous β-Gal for 0.5 hours or with 50 μM D-galactose for 1 hour, and then CS-βGal was added for incubation. The excitation wavelength was 488 nm under fluorescence microscopy, and the cell fluorescence images were observed and recorded

see Figure 9

[0045] 3. In vivo imaging: CS-βGal (100 μM) was injected intraperitoneally into BALB / c nude mice, and fluorescence imaging was performed at different time points to detect changes in intraperitoneal fluorescence signals. The specific steps are as follows: BALB / c nude mice were injected intraperitoneally with 100 μM CS-βGal, and imaging was performed at 15 minutes and 30 minutes after injection using an in vivo imaging system (excitation 488 nm, emission 550-650 nm)

see Figure 10

[0046] The above description is only the preferred embodiments of the present application, and the above specific embodiments are not a limitation on the present application. Various modifications and changes can occur within the scope of the technical idea of the present application, and any modification, change or equivalent replacement made by those skilled in the art based on the above description belongs to the scope of protection of the present application.

Claims

1. A visualizing enzyme-activatable fluorescent probe, characterized in that: The chemical structure of the fluorescent probe comprises a D-galactose residue and a cyanine dye parent body, and the cyanine dye can be released under the action of β-Gal, thereby producing a fluorescence enhancement effect.

2. The visualizable enzyme-activated fluorescent probe according to claim 1, characterized in that: The structural formula of the fluorescent probe is:

3. The visualizable enzyme-activated fluorescent probe of claim 1, wherein: The fluorescent probe has weak self-fluorescence in an aqueous solution, and produces strong fluorescence at 576 nm after reacting with β-Gal, with an excitation wavelength of 496 nm.

4. A method of visualizing the synthesis of enzyme-activated fluorescent probes, characterized by The method comprises the following steps: Step 1: A certain amount of o-hydroxybenzaldehyde and 2-benzoylpyridine are weighed, and a certain amount of acetic acid and ammonium acetate is added, high-purity nitrogen is continuously introduced into the reaction system to exclude air, a nitrogen atmosphere is maintained, the nitrogen flow is controlled to be 10-15 mL / min, and the temperature is raised to 120 DEG C, and the reaction is heated under magnetic stirring for 12-16 hours to obtain an intermediate product I; the structural formula of the intermediate product I is: Step 2: A certain amount of cesium carbonate, sodium sulfate and acetonitrile is added to the intermediate product I and α-acetyl bromide galactose, and the reaction is carried out at room temperature for 3 hours. After the reaction is completed, the crude product is obtained by filtration and rotary evaporation concentration of the filtrate, and then purified by column chromatography to obtain an intermediate product II, and the structural formula of the intermediate product II is: Step 3: A certain amount of potassium carbonate and methanol is added to the intermediate product II, and the reaction is carried out at room temperature for 30 min to obtain the target fluorescent probe.

5. The method of visualizing synthesis of enzyme-activated fluorescent probes according to claim 4, wherein: In step 1, the molar ratio of o-hydroxybenzaldehyde and 2-benzoylpyridine is 1:(1-1.2), the amount of acetic acid is 3-5 times the amount of o-hydroxybenzaldehyde, and the amount of ammonium acetate is 0.5-1 times the amount of o-hydroxybenzaldehyde; in step 2, the molar ratio of intermediate product I and α-acetyl bromide galactose is 1:(1.1-1.2), the amount of cesium carbonate is 5-10 times the amount of intermediate product I, the amount of sodium sulfate is 5-10 times the amount of intermediate product I, and the amount of acetonitrile is 5-10 times the amount of intermediate product I; in step 3, the amount of potassium carbonate is 3-5 times the amount of intermediate product II, and the amount of methanol is 5-10 times the amount of intermediate product II.

6. The application of a visual enzyme-activated fluorescent probe in detecting β-Gal activity.

7. Use of the visualizable enzyme-activated fluorescent probe according to claim 6 for detecting β-Gal activity, characterized in that: The detection includes quantitative detection of β-Gal in an in-vitro solution, and visual detection of endogenous and exogenous β-Gal in living cells.

8. Use of the visualized enzyme-activated fluorescent probe according to claim 6 for detecting β-Gal activity, characterized in that: The detection also includes real-time in-situ imaging detection of β-Gal activity in a BALB / c nude mouse.

9. A method of detecting β-Gal activity, characterized by The method comprises the following steps: (1) The fluorescent probe in claim 1 is mixed with a sample to be detected, and incubated for 25 minutes to obtain a reaction product; (2) The fluorescence intensity of the reaction product at 576 nm under excitation light is detected, and the activity of the sample to be detected is determined according to a standard curve, and the excitation wavelength of the excitation light is 496 nm.

10. The method for detecting β-Gal activity according to claim 9, characterized by: The sample to be detected includes a biological fluid, living cells or living tissues.

Images