阅读说明：本技术 pH比率型荧光探针SP-DCCH的制备方法及生物成像应用 (preparation method of pH ratio type fluorescent probe SP-DCCH and biological imaging application ) 是由 何晓俊 沈建良 于 2020-04-27 设计创作，主要内容包括：本发明公开了一种pH比率型荧光探针SP-DCCH的制备方法及生物成像应用,用于酸性以及弱碱条件下与生物成像,通过检测该探针在不同pH缓冲液(1.68～7.21)中的荧光光谱,可以看出随着pH值的降低,pH比率型荧光探针在λ＝525nm处的荧光强度逐渐降低,在λ＝629nm处的荧光强度逐渐升高,说明pH比率型荧光探针对pH非常敏感,因此在pH检测方面具有良好的应用前景。(The invention discloses a preparation method of a pH ratio type fluorescent probe SP-DCCH and a biological imaging application, which are used for biological imaging under acidic and weak alkali conditions, and the fluorescence spectra of the probe in different pH buffer solutions (1.68-7.21) are detected, so that the fluorescence intensity of the pH ratio type fluorescent probe at the position of lambda 525nm is gradually reduced along with the reduction of the pH value, and the fluorescence intensity at the position of lambda 629nm is gradually increased, which shows that the pH ratio type fluorescent probe is very sensitive to pH, and therefore, the pH ratio type fluorescent probe has a good application prospect in the aspect of pH detection.)

1. A pH ratio type fluorescent probe SP-DCCH, characterized in that: the molecular formula of the molecular probe is C₃₈H₄₂N₄O₄The structural formula is as follows:

2. the method for preparing a pH ratio type fluorescent probe SP-DCCH as claimed in claim 1, characterized by comprising the following steps:

the volume molar concentration of a mixed solution prepared from an anhydrous ethanol solution containing 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-formaldehyde is 0.02-0.1mol/L, 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is added, the mixed solution is mixed for reaction, and after filtration, the mixed solution is washed by a washing solution to obtain a product, wherein the molar ratio of the 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-formaldehyde to the 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is as follows: 1: 1-2.

3. The method of claim 2, wherein the washing solution is cooled ethanol or diethyl ether or a mixture thereof.

4. The method for preparing pH ratio type fluorescent probe SP-DCCH as claimed in claim 2, wherein the molar ratio of 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-carbaldehyde and 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is 1: 1.

5. Use of the pH ratio type fluorescent probe SP-DCCH according to any one of claims 1 to 4 for detecting, identifying pH value in environment or in biological sample.

6. Use of the pH ratio fluorescent probe SP-DCCH for detecting, identifying pH values in the environment or in biological samples according to claim 5, characterized in that the pH ratio fluorescent probe is used for detecting pH values in normal cells and in vivo using fluorescence imaging.

7. Use of the pH ratio type fluorescent probe SP-DCCH according to claim 5 or 6 for detecting, identifying pH value in acidic or weakly alkaline environment or in biological sample.

8. The use of the pH ratio type fluorescent probe SP-DCCH of claim 7 for detecting, identifying pH in an acidic or slightly alkaline environment or in a biological sample with a precise detection range of pH 4.0-6.0.

9. The method for detecting pH value of SP-DCCH as claimed in any of claims 1 to 4, wherein the pH value is determined by measuring fluorescence intensity of pH value at wavelength of 525nm, 629nm with 456nm as excitation wavelength by fluorescence spectrophotometry and calculating fluorescence emission intensity ratio of F525/F629.

10. The method for detecting the pH value of the pH ratio type fluorescent probe SP-DCCH according to claim 9, wherein the curve relationship between the fluorescence emission intensity ratio of F525/F629 and the pH value detection result is as follows: y is 3.39 x-13.1.

Technical Field

The invention relates to the field of fluorescent imaging molecular probes, in particular to a preparation method and biological imaging application of a pH ratio type fluorescent probe SP-DCCH.

Background

The pH in the intracellular environment plays an important role in cellular activity. It is closely related to cell proliferation, apoptosis, ion transport, etc. Intracellular pH abnormalities may cause a series of adverse effects, even induce cell mutations and the like. Recent studies have shown that changes in pH may be indistinguishable from certain diseases, such as neurodegenerative diseases, cardiovascular diseases and cancer.

Therefore, it is important to accurately detect the change of intracellular pH, which can provide useful information for our study of cellular physiological and pathological processes. The traditional methods for detecting pH include electrochemical analysis, nuclear magnetic resonance, ultraviolet absorption spectroscopy, electrochemical methods and the like, and although the methods also have good accuracy and sensitivity, the methods have the defects of complex sample processing technology, high instrument cost, low sensitivity and the like. Compared with the analysis methods, the fluorescence analysis method has the remarkable advantages of simple operation, high sensitivity, short time consumption, specificity selection, in-situ imaging, noninvasive detection and the like. Therefore, the fluorescence analysis method is widely applied to the fields of environmental science, military, biological medicine, gene detection and the like.

Fluorescent probes of various pH have been known. However, these probes all show more or less different disadvantages in complex biological systems, such as photobleaching, lower loading rates and short action times. Therefore, it is necessary to design a pH fluorescent probe with good light stability, high selectivity and stable action time. Due to the complexity of the cells themselves and the changing internal environment, the use of ratiometric fluorescence analysis has become an effective detection method that can counteract most of the effects of probe concentration, external environmental changes and photobleaching. Thus, ratiometric fluorescence analysis may provide more reliable detection under complex conditions.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a pH fluorescent probe with good stability and high selectivity.

In order to achieve the aim, the invention provides a pH ratio type fluorescent probe SP-DCCH, wherein the molecular probe molecular formula is C₃₈H₄₂N₄O₄The structural formula is as follows:

the invention also provides a preparation method of the pH ratio type fluorescent probe, which comprises the following steps:

the volume molar concentration of a mixed solution prepared from an anhydrous ethanol solution containing 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-formaldehyde is 0.02-0.1mol/L, 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is added, the mixed solution is mixed for reaction, and after filtration, the mixed solution is washed by a washing solution to obtain a product, wherein the molar ratio of the 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-formaldehyde to the 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is as follows: 1: 1-2.

As a further improvement, the washing solution is cooled ethanol or diethyl ether or a mixed solution of the cooled ethanol and the diethyl ether.

As a further improvement, the molar ratio of 6-tert-butyl-1 ', 3', 3 '-trimethylspiro [2, 2' -indoline ] -8-carbaldehyde to 7- (diethylamino) -2-oxo-2H-methylene-3-carbohydrazide is 1: 1.

The invention also provides application of the pH ratio type fluorescent probe in detecting and identifying the pH value in the environment or in a biological sample.

As an application range of the present invention, the application of the pH ratio type fluorescent probe for detecting, discriminating pH values in the environment or in a biological sample and the application of the pH ratio type fluorescent probe for detecting pH values in normal cells and in vivo using fluorescence imaging are described.

As an application range of the invention, the application of the pH ratio type fluorescent probe in detecting and identifying the pH value in an acidic or weakly alkaline environment or in a biological sample.

As an application mode of the present invention, the pH value is measured by fluorescence spectrophotometry at an excitation wavelength of 456nm and at wavelengths of 525nm and 629nm, and the pH value is measured by calculating the fluorescence emission intensity ratio of F525/F629.

Further, the curve relationship between the fluorescence emission intensity ratio of F525/F629 and the pH detection result is as follows: y is 3.39 x-13.1.

Further, the application of the pH ratio type fluorescent probe in detecting and identifying the pH value in an acidic or weakly alkaline environment or in a biological sample has the accurate detection range of the pH value between 4.0 and 6.0.

The invention has the following advantages: the invention designs a novel pH ratio type fluorescent probe and uses the novel pH ratio type fluorescent probe for biological imaging under acidic and weak base conditions. By detectingThe fluorescence spectra of the probe in different pH buffers (1.68-7.21) show that the fluorescence intensity of the pH ratio type fluorescent probe at the position of lambda 525nm is gradually reduced along with the reduction of the pH value, and the fluorescence intensity at the position of lambda 629nm is gradually increased, which indicates that the pH ratio type fluorescent probe is very sensitive to pH, and the probe molecules are gradually changed into an open-loop structure from a closed-loop structure along with the reduction of the pH value and can be kept stable for a long time. Through repeated experiments, the probe has high reversibility and strong stability in buffer solutions with pH values of 1.68 and 7.21. And the ratio of fluorescence intensities (F) obtained by mixing different cations and anions₅₂₅/F₆₂₉) The detection result shows that the ion is not easily influenced by other ions. Subsequent confocal fluorescence imaging of the pH ratio type fluorescent probe in MRC-5 cells at different pH indicated that the probe had good cell membrane permeability, and fluorescence imaging of MRC-5 cells at different time points at pH 1.68 indicated that the probe was stable and long lasting. In biological imaging, the zebra fish experiment also proves a conjecture. Therefore, the pH ratio type fluorescent probe designed by the invention can be used for detecting the pH in a solution, living cells and zebra fish, and has the advantages of simple synthesis method, convenient operation and no need of harsh conditions, thereby having good application prospect in the aspect of pH detection.

Drawings

FIG. 1: and (3) a synthetic route of a probe SP-DCCH.

FIG. 2: (a) absorption spectra of SP-DCCH (20 μ M) in EtOH-PBS (1/100, V/V) at various pH values (7.21 to 1.68); (b) fluorescence spectra of SP-DCCH (10 μ M) in EtOH/PBS (1/100, V/V) at various pH values (7.30 to 1.68,. lambda.ex 456nm, Ex/Em 2/2 nm); (c) fluorescence intensities at 525nm and 629nm at different pH values; (d) fluorescence intensity ratio of SP-DCCH (10. mu.M) in EtOH-PBS buffer at different pH values (F525/F629). Illustration is shown: linear relationship between F525/F629 and pH (4.20-7.21).

FIG. 3: (a) the fluorescence intensity ratio of SP-DCCH (10. mu.M) to EtOH-PBS solutions at various pH values (F525/F629) as a function of time; (b) reversible fluorescence intensity ratio (F525/F629) of SP-DCCH (10. mu.M) between pH 7.35 and 1.10; (c) SP-DCCH (10. mu.M) fluorescence and color photographed at various pH valuesColor; (d) in buffer solutions at pH7.21 and 3.02 with different cations (1-12: 1: SP-DCCH, 2: Zn, respectively)²⁺，3：Ni²⁺，4：Cd²⁺，5：Al³⁺，6：Fe³⁺，7：pb²⁺，8：Co²⁺，9：Fe²⁺，10：Cr³⁺，11：Mn²⁺，12：Cu²⁺) Fluorescence intensity ratio (F525/F629) of mixed SP-DCCH (10. mu.M); (e) in buffer solutions at pH7.21 and 3.02 with different anions (A-L: A: SP-DCCH, B: SO, respectively)₄ ^2-，C：CIO^-，D：HS^-，E：PO₄ ^3-，F：NO₃ ^-，G：Cl^-，H：CO₃ ^2-，I：S₂O₃ ^2-，J：SO₃ ^2-K: br-, L: SCN-) fluorescence intensity ratio of mixed SP-DCCH (10 μ M) (F525/F629).

FIG. 4: SP-DCCH and SP-DCCH + H⁺Theoretical calculation of molecular orbital

FIG. 5: confocal fluorescence imaging of SP-DCCH (10. mu.M) in MRC-5 cells at different pH conditions.

FIG. 6: confocal fluorescence imaging of SP-DCCH (10. mu.M) in P.aeruginosa at different pH conditions.

FIG. 7: confocal fluorescence imaging of SP-DCCH (10. mu.M) in zebrafish under different pH conditions.

Detailed Description

The present invention will be further described in detail with reference to examples and effect examples, but the scope of the present invention is not limited thereto.