CN114507191A - Synthesis of pH fluorescent probe and method for applying pH fluorescent probe in cell imaging - Google Patents

Abstract

The invention belongs to the technical field of analytical chemistry, and particularly relates to a synthesis method of a pH fluorescent probe and a method for applying the pH fluorescent probe in cell imaging. The method comprises the following steps: step 1: mixing acetonitrile and water in a volume ratio of 4:1 to obtain a solvent A, and dissolving 0.50g of 2-cyano-6-hydroxybenzothiazole and 0.43g of 2-aminobenzenethiol in the solvent A to obtain a mixture B; step 2: mixture B is stirred at about 1500 rpm and 0.30g (2.84mmol) of K are added₂CO₃Continuously stirring for 30min at room temperature; and step 3: extracting the product obtained in the step 2 twice by using dichloromethane, taking the aqueous phase, adjusting the pH of the aqueous phase to 1 by using HCl, monitoring the pH change by using pH test paper, and generating light yellow precipitate; and 4, step 4: will cutQualitative filter paper, put in buchner funnel, pumped with water to reduce pressure and filtered, washed twice, and the filter cake in buchner funnel was taken to obtain 0.31g of probe HP, which was applied to cells to image pH.

Description

Synthesis of pH fluorescent probe and method for applying pH fluorescent probe in cell imaging

Technical Field

The invention belongs to the technical field of analytical chemistry, and particularly relates to a synthesis method of a pH fluorescent probe and a method for applying the pH fluorescent probe in cell imaging.

Background

The acid-base balance in the organism plays an important role in the normal operation of the organism. Under normal physiological conditions, the cytosolic pH is typically maintained at around 7.2, and intracellular ion-proton exchange and metabolism regulate the cytoplasmic pH. Since intracellular pH abnormality induces the generation of free radicals, destroys contractility of cells, causes necrosis or apoptosis of cells, thereby causing various diseases such as sunstroke, cancer, Alzheimer's disease, etc., the monitoring of intracellular pH is of great significance.

Fluorescence has been widely used in the detection of disease-related molecules in organisms as a simple, effective, non-invasive and real-time detection means, and thus fluorescence has been selected as our detection means for real-time monitoring of pH changes in cells and tissues.

The existing inorganic nano material, organic fluorescent molecule and organic-inorganic hybrid nano material are applied to pH detection, wherein the organic fluorescent material has various types, has conjugated heterocycles and various chromophores, and can adjust the fluorescence emission wavelength by modifying and introducing unsaturated groups and chromophores, so the organic molecular fluorescent probe has wide application.

The pH detection fluorescent probe is mainly based on two types of design principles: 1. acid-base neutralization based on phenols, amino groups and N-heterocyclic compounds; 2. based on the nucleophilic addition reaction of hydroxide ions to cyanine pigments. The acid-base neutralization reaction rate based on the phenol and N heteroatoms is high, and the probe is a quick-response fluorescent probe.

The benzothiazole compound has a rigid planar structure, has the properties of higher fluorescence quantum yield, low toxicity and the like, and has important application in the aspects of photoelectric materials and fluorescent probes. Because the N atom of the benzothiazole ring and the hydroxyl on the aromatic ring connected with the N atom can generate an intramolecular excited state proton transfer process, the fluorescence enhancement and the red shift of the emission wavelength are obvious, and the detection method has important application in the aspect of detection of bio-related small molecules. Because the molecular fluorescence wavelength is generally short blue-green light, the penetration depth of the molecular fluorescence is short, and the application in the aspect of biology has certain limitation, the synthesis of the probe with the longer fluorescence emission wavelength has important significance.

Disclosure of Invention

Aiming at the technical problems, the invention provides a method for synthesizing a pH fluorescent probe and applying the pH fluorescent probe in cell imaging, and synthesizes a biphenyl benzothiazole compound HP, wherein the biphenyl benzothiazole compound has a larger conjugated system, the fluorescence emission is red-shifted to yellow light, and the change of the pH has obvious influence on the luminous state of molecules. As the solution pH gradually increased (5.6< pH <8.0), the fluorescence of the solution at both 500nm and 568nm gradually increased; as the pH continued to increase (pH >8.0), fluorescence increased dramatically at 568nm, with fluorescence at 500nm being covered and indistinguishable. The probe has high selectivity, and the pH detection process is not influenced by other ions. Because the long distance between the two fluorescence emission wavelengths of 500nm and 568nm is short, and the two fluorescence emission wavelengths are difficult to distinguish in laser confocal imaging, the fluorescence at 568nm is taken as the detection wavelength, and the HP can be used as an off-on type fluorescent probe for monitoring the intracellular pH in real time. The fluorescence intensity gradually increased with increasing intracellular pH (pH range of 4.0-10.0), and the increase was more pronounced at pH 8.0-10.0.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

a synthetic method of a pH fluorescent probe comprises the following steps:

step 1: mixing acetonitrile and water to obtain a solvent A, and dissolving 2-cyano-6-hydroxybenzothiazole and 2-aminobenzenethiol in the solvent A to obtain a mixture B;

step 2: stirring the mixture B and adding K₂CO₃Continuously stirring for 30min at room temperature;

and step 3: extracting the product obtained in the step 2 with dichloromethane twice, taking the aqueous phase and adjusting the aqueous phase to pH 1 with HCl, and generating light yellow precipitate;

and 4, step 4: and shearing qualitative filter paper, placing the qualitative filter paper in a Buchner funnel, pumping air by using a water pump, carrying out reduced pressure filtration, washing twice, taking a filter cake in the Buchner funnel, and synthesizing to prepare the probe HP.

Preferably, the volume ratio of acetonitrile to water of step 1 is 4: 1.

Preferably, the ratio of the substances of the step 12-cyano-6-hydroxybenzothiazole and the 2-aminobenzenethiol is 1: 1.

The application of the pH fluorescent probe in cell imaging comprises the following steps:

step 1: cells were incubated in a buffer solution of PBS with a probe concentration of 10. mu.M for 20 minutes at 37 ℃;

step 2: cells were imaged by confocal laser microscopy after incubation in buffer solutions containing 10 μ M nigericin at different pH's for 30 minutes at 37 ℃ when the intracellular pH and the extracellular buffer solution pH were the same.

Compared with the prior art, the invention has the beneficial effects that:

(1) the fluorescent probe HP synthesized by the method has better selectivity, and common ions or molecules in a biological system have no obvious influence on the fluorescent probe.

(2) The fluorescent probe HP of the invention presents a sensitive change trend to the pH change in the solution, and the change is obvious by imaging the pH in the cell through laser confocal.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention.

In the drawings:

FIG. 1 is a flow chart of a method of the present invention;

FIG. 2 shows UV-VIS absorption spectra (a) and fluorescence spectra (b) of probe HP in different solvents with excitation wavelength (Ex) of 400 nm;

fig. 3(a) uv-vis absorption and fluorescence spectra of compound HP at pH 5.6 and 10.1; (b) fluorescence of compound HP in buffer solutions of different pH; (c) the change trend of the fluorescence at 568nm and the fitting effect of the change trend; (d) linear fitting of fluorescence at 568nm at pH greater than 9.4;

FIG. 4 shows two forms of the probe HP in different pH solvents;

FIG. 5 shows the HOMO (a, c), LOMO (b, d) orbital electron cloud profiles of two forms of the compound H-1(a, b) and H-2(c, d);

FIG. 6 shows d of Probe HP₆Gradually adding NaOH to DMSO solution D₂The change trend of nuclear magnetic resonance hydrogen spectrum when in O solution;

FIG. 7 is a selective spectrum of compound HP for other ions;

FIG. 8 is a bar graph showing the selectivity of compound HP (1-23 are each Na)⁺，K⁺，NH₄ ⁺，Ag⁺，Mg²⁺，Ca²⁺，Ba²⁺，Co²⁺，Ni^{2 +}，Cu²⁺，Zn²⁺，Cd²⁺，Hg²⁺，Sn²⁺，Pb²⁺，Al³⁺，F^-，Cl^-，Br^-，NO₃ ^-，SO₄ ^2-，CH₃COO^-，OH^-)；

FIG. 9 MTT assay for Probe HP: hela cells were incubated for 24 hours in culture with various concentrations (0.4. mu.M, 0.8. mu.M, 1.6. mu.M, 3.2. mu.M, 6.3. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M) of Compound HP;

FIG. 10 confocal laser mapping of Hela cells (orange fluorescence channel: a-e; bright field channel: f-j; superimposed field: k-o) incubated with HP (10 μ M) at 37 ℃ for 20min followed by incubation with buffer solutions of different pH containing 10 μ M nigericin (no compound HP control: a, f, k; pH 4.0: b, g, l; pH 6.0: c, h, M; pH 8.0: d, i, n; pH 10.0: e, j, o) for 30min, orange fluorescence channel: 565 + -25 nm, lambda_ex＝405nm,scale bar＝10μm；

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are some, but not all, embodiments of the present invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example (b):

a synthetic method of a pH fluorescent probe comprises the following steps:

step 1: mixing acetonitrile and water in a volume ratio of 4:1 to obtain a solvent A, and dissolving 0.50g of 2-cyano-6-hydroxybenzothiazole and 0.43g of 2-aminobenzenethiol in the solvent A to obtain a mixture B;

step 2: mixture B is stirred at about 1500 rpm and 0.30g (2.84mmol) of K are added₂CO₃Continuously stirring for 30min at room temperature;

and 3, step 3: extracting the product obtained in the step 2 twice by using dichloromethane, taking the aqueous phase, adjusting the pH of the aqueous phase to 1 by using HCl, monitoring the pH change by using pH test paper, and generating light yellow precipitate;

and 4, step 4: shearing qualitative filter paper, placing the qualitative filter paper in a Buchner funnel, pumping air by a water pump for reduced pressure filtration, washing with water twice, and taking a filter cake in the Buchner funnel, wherein the filter cake is the target compound probe HP, and 0.31g of the probe HP is obtained.

The application of the prepared pH fluorescent probe in cell imaging comprises the following steps:

step 1: cells were incubated in a buffer solution of PBS with a probe concentration of 10. mu.M for 20 minutes at 37 ℃;

step 2: cells were imaged by confocal laser microscopy after incubation in buffer solutions containing 10 μ M nigericin at different pH's for 30 minutes at 37 ℃ when the intracellular pH and the extracellular buffer solution pH were the same.

The experimental process comprises the following steps:

1. spectral properties in different solvents

Dissolving the prepared probe HP in different organic solvents, diluting to 10 mu mol/L, and respectively testing an ultraviolet-visible absorption spectrum and a fluorescence spectrum, wherein as shown in figure 2, the absorption and fluorescence spectrum of a compound are greatly influenced by the solvent effect, as shown in table 1, in an aqueous solution, the maximum absorption wavelength of the probe HP is obviously red-shifted; the wavelength of the maximum emission peak of the fluorescence spectrum in different solvents is gradually red-shifted along with the increase of the polarity of the solvents.

TABLE 1 maximum absorption wavelength (. lamda.max), molar absorptivity (. epsilon.) and maximum emission wavelength (. Em.) of Probe HP in common solvents

Solvent	λmax a/nm	εb/mol-1·L·cm-1	Em/nm
				Water	376	1.43×104	Very weak
DMF	366	2.27×104	452
				DMSO	367	2.21×104	454
CH3OH	360	2.63×104	451
				CH3CH2OH	362	2.41×104	449
Ethyl acetate	360	2.42×104	435
				Acetonitrile	358	2.70×104	448
THF	363	2.53×104	440
				DCM	365	3.19×104	441

2. UV-visible absorption and fluorescence spectra as a function of pH

The probe HP was characterized by UV-visible absorption and fluorescence spectroscopy as a function of the pH of the solution. As the pH of the buffer solution increased (pH 5.6 and pH 10.1), there was some increase in the absorption of the solution (see fig. 3 a). In a buffered solution at pH 5.6, the solution is almost non-fluorescent due to quenching of fluorescence by the presence of protons in the solution; the fluorescence of the solution at 500nm gradually increased with increasing pH (5.6< pH <8.0) (FIG. 3b) (0.20% absolute quantum yield of fluorescence peak at 500 nm); when the pH is increased continuously (the pH is more than 8.0), the fluorescence enhancement at 568nm is more obvious (the absolute quantum yield is 4.50 percent), because the compound HP exists in the solution in different forms under acidic and alkaline conditions (as shown in figure 4, H-1 and H-2 exist in two forms), the fluorescence at 500nm belongs to the fluorescence of a compound molecule H-1 type, and under the strong acidic condition, the proton quenching effect is realized, and the fluorescence is extremely weak; as the pH of the solution increases to neutrality, the proton quenching effect is weakened, and the fluorescence of the H-1 form gradually appears; when the pH value of the solution is increased to be alkaline, H-1 molecules lose protons and exist in the solution as negative ions H-2 type (shown in figure 2), an H-2 type conjugated system and a push-pull electronic structure are changed, obvious intramolecular electron transfer (ICT) is presented, fluorescence red shift is presented, and obvious fluorescence is presented at 568 nm.

By calculation of the fluorescence emission of the compounds, as shown in Table 2, the fluorescence wavelengths of the two existing forms (H-1 and H-2) were calculated to be 476nm and 558nm, respectively, in accordance with the experimental data of 500nm and 568 nm; the HOMO-LOMO energy level difference of two existing forms is obtained through calculation, and it can be seen that the HOMO-LOMO energy level of the existing form H-2 is small, which indicates that electrons are easily excited and obvious Intramolecular Charge Transfer (ICT) exists, as shown in fig. 5: HOMO and LOMO orbital electron cloud profiles), the emission wavelength is longer, consistent with the red-shift of the emission wavelength in the experiment.

TABLE 2 theoretical calculation and experimental fluorescence emission wavelength data for two existing forms

Formation	Calculated Em Wavelength/nm	Em Wavelength/nm	HOMO-LUMO energy level
				H-1	476	500	4.04eV
H-2	558	568	3.20eV

Fitting the 568nm fluorescence intensity (FIG. 3c) yields the pK of compound HP_a9.91 (dissociation constant K)_a＝1.23×10^-10) And pK of phenol_a9.89, it is said that the compound HP is slightly less acidic than phenol because N and S atoms on the thiazole ring attached to the benzene ring have electron donating ability, so that the benzene ring is rich in electrons, and thus the ability to dissociate protons is reduced and the acidity is reduced.

At pH > 9.4, the fluorescence intensity of the solution changes almost linearly (FIG. 3d), and a linear fit to this segment is performed, with good linearity, R²0.9618, indicating that the fluorescence of the oxyanion form H-2 does not exhibit concentration quenching effects in this range, and that the pH of the solution can be quantitatively analyzed according to this standard curve.

3. Nuclear magnetic titration

The change condition of H atoms in the compound can be tracked through nuclear magnetic resonance hydrogen spectroscopy, and the change of the probe HP in DMSO solutions with different pH values is explored; d of Compound HP₆In the NMR spectrum of DMSO solution (FIG. 6a), we can see that the phenolic hydroxyl active hydrogen is displayed at the chemical shift delta of 10.23ppm, D of NaOH is gradually added₂The proton peak of the hydrogen spectrum shows change when in O solution: first, when a small amount of solution is added, the phenolic hydroxyl group is activated by D₂Substitution of OGradually disappear; continued addition of NaOH D₂In O solution, all active hydrogen of phenolic hydroxyl is neutralized to form an oxygen anion type H-2, and hydrogen (3,5,6) on a benzene ring B connected with the oxygen anion obviously moves to a high field due to the electron-rich shielding effect of the oxygen anion (as shown in figure 6 e); the hydrogen atoms (1,2,4) on the benzene ring a are less affected and thus have less tendency to move to high fields.

4. Ion selectivity data

To exclude the effect of other ions on the fluorescence properties of compound HP, we performed ion-selective experiments. To a solution of compound HP in PBS (pH 5.6) was added the other common cations and anions (1-23 Na, respectively)⁺，K⁺，NH₄ ⁺，Ag⁺，Mg²⁺，Ca²⁺，Ba²⁺，Co²⁺，Ni²⁺，Cu²⁺，Zn²⁺，Cd²⁺，Hg²⁺，Sn²⁺，Pb²⁺，Al³⁺，F^-，Cl^-，Br^-，NO₃ ^-，SO₄ ^2-，CH₃COO^-，OH^-) There was no significant change in fluorescence (see FIGS. 7 and 8). The experimental result proves that common anions and cations have no obvious influence on the fluorescence property of the compound HP, and are only influenced by the pH value of the solution, so that the ion selectivity is good.

5. Cytotoxicity and imaging

The cytotoxicity of probe HP was tested by means of a cell viability test (MTT), and the cell viability of Hela cells was still greater than 90% after incubation of Hela cells in culture media containing different concentrations (0.4. mu.M, 0.8. mu.M, 1.6. mu.M, 3.2. mu.M, 6.3. mu.M, 12.5. mu.M, 25. mu.M, 50. mu.M, 75. mu.M, 100. mu.M) of probe for 24 hours (see FIG. 9).

The probe is applied to Hela cells to monitor and image the intracellular pH. The cells were incubated at 37 ℃ for 20 minutes in a buffer solution of PBS (pH 7.4) with a probe concentration of 10 μ M and then at 37 ℃ for 30 minutes in a buffer solution of nigericin at various pH (pH 4.0,6.0,8.0,10.0) containing 10 μ M (nigericin)Rilimin is a polycyclic ether carboxylic acid compound prepared by reacting Rilimin with a compound of formula (I)⁺/K⁺Exchange to balance intracellular and extracellular pH), cells were imaged by confocal laser microscopy (see fig. 10). The results show that when the cytoplasmic solution is acidic, the probe is less fluorescent; the fluorescence of the probe HP gradually increased with increasing pH, and the fluorescence increased more significantly at pH 8.0-10.0. The probe can be used for detecting the change of the intracellular pH value in real time.

The invention synthesizes a pH-responsive fluorescence probe HP, the fluorescence is enhanced at 568nm along with the increase of the pH of the solution (5.6< pH <8.0), and the fluorescence change is more obvious within the range of pH 8.0-10.0. The probe has high selectivity, and when the probe is applied to the real-time monitoring of intracellular pH, the result shows that: when the intracellular solution is acidic, the fluorescence of the probe is weak; the fluorescence of probe HP gradually increased with increasing pH. The probe HP can be used as an off-on type fluorescent probe for monitoring the change of the intracellular pH value in real time.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims (4)

1. A synthetic method of a pH fluorescent probe is characterized in that: the method comprises the following steps:

step 1: mixing acetonitrile and water to obtain a solvent A, and dissolving 2-cyano-6-hydroxybenzothiazole and 2-aminobenzenethiol in the solvent A to obtain a mixture B;

step 2: stirring mixture B and adding K₂CO₃Continuously stirring for 30min at room temperature;

and step 3: extracting the product obtained in the step 2 with dichloromethane twice, taking the aqueous phase and adjusting the aqueous phase to pH 1 with HCl, and generating light yellow precipitate;

and 4, step 4: and shearing qualitative filter paper, placing the qualitative filter paper in a Buchner funnel, pumping air by using a water pump, carrying out reduced pressure filtration, washing twice, taking a filter cake in the Buchner funnel, and synthesizing to prepare the probe HP.

2. The method for synthesizing a pH fluorescent probe according to claim 1, wherein: the volume ratio of acetonitrile to water in step 1 is 4: 1.

3. The method for synthesizing a pH fluorescent probe according to claim 2, wherein: the mass ratio of the 12-cyano-6-hydroxybenzothiazole to the 2-aminobenzenethiol in the step is 1: 1.

4. Use of a pH fluorescent probe prepared according to the method of any one of claims 1 to 3 for cellular imaging, characterized in that: the method comprises the following steps:

step 1: cells were incubated for 20min at 37 ℃ in a buffered solution of PBS with probe HP concentration of 10. mu.M;

step 2: cells were imaged by confocal laser microscopy after incubation in buffer solutions containing 10 μ M nigericin at different pH's for 30 minutes at 37 ℃ when the intracellular pH and the extracellular buffer solution pH were the same.

Images