CN116675666A - Preparation method and application of pH fluorescent probe based on rhodamine near-infrared dye - Google Patents

Abstract

The application discloses preparation and application of a pH fluorescent probe based on rhodamine near infrared dye, wherein the structural formula of the fluorescent probe is as follows:the preparation method of the fluorescent probe is simple, the product yield is high, and the fluorescent probe is suitable for large-scale popularization and application. When the probe QL-2 is excited at 580nm, the fluorescence is strongest under the acidic condition, and the fluorescence intensity gradually decreases along with the enhancement of the alkalinity of the solution; when excited at 700nm, the fluorescent intensity is strongest under alkaline conditions, and gradually decreases as the acidity of the solution increases. Both probes can detect pH 2-10, and the probesQL-2 has good linear ratio relation, can effectively remove the influence of the background, and improves the sensitivity and accuracy of detection.

Description

Preparation method and application of pH fluorescent probe based on rhodamine near-infrared dye

technical field

The invention belongs to the technical field of fluorescent probes, and in particular relates to a preparation method and application of a rhodamine near-infrared dye-based pH fluorescent probe.

Background technique

Intracellular pH plays an indispensable role as an important physiological parameter that plays an important regulatory role in various events in the cell, including ion transport, enzyme activity, cell growth, calcium regulation, endocytosis, cell adhesion etc. As one of the key factors in cell generation and metabolism, pH value plays a vital role in maintaining cell growth, proliferation and apoptosis. In addition, the pH in different subcellular organelles in the cell is different, and the pH value is also different due to the specific function of each organelle. For example, the pH of lysosomes and endosomes is about 4-6; the pH of cytoplasm and nucleus is about 7; the pH of mitochondria and nuclear regions is about 8.0.

The methods for detecting pH value mainly include colorimetric analysis, electrochemical method and other methods. Compared with these methods, fluorescence microscopy has shown superior performance, and with the appropriate selection of fluorescent probes, intracellular pH can be labeled at the micron scale. Dynamic pH changes in living cells can be visualized in situ and in real time under a fluorescence microscope. In order to achieve intracellular pH fluorescence imaging, a variety of fluorescent probes have been constructed, which have different response sites and fluorescence mechanisms.

In recent years, in order to study more accurate detection techniques, fluorescent probes have been widely used in the detection of various substances and in biological neighborhoods because of their simple operation, high selectivity, and good temporal and spatial resolution. Fluorescent probes detect substances that rely on increases or decreases in fluorescence intensity, and ratiometric pH-responsive fluorescent probes with dual absorption or emission wavelengths allow self-calibration of the two bands and exclude most environmental variables such as probe concentration, instrumentation parameters, photobleaching, etc., which can provide quantitative analysis of pH.

Fluorescent probes are one of the effective means to detect pH, and are widely used in the fields of industry, environment and medical inspection. Compared with the electrochemical method, this method can better eliminate background interference, and the sensitivity is also greatly improved, but it is applied in the field of life, and the detection range of the fluorescent probe that has been reported to detect pH is relatively low. Most of them have a good linear relationship in a weak acid environment, but for a strong acid environment such as gastric acid, ribozyme, and lysosome, the sensitivity is not enough, so there is a defect that it cannot be applied. Moreover, the background interference of these fluorescent probes in the detection of organisms is relatively large, which greatly limits their applications. Therefore, it is very important to develop fluorescent probes that can detect pH.

Contents of the invention

Based on the technical problems existing in the background technology, the present invention proposes the preparation method and application thereof of pH fluorescent probes based on rhodamine near-infrared dyes. Simple, high yield, suitable for large-scale popularization and application.

The present invention is realized through the following technical solutions:

The pH fluorescent probe based on rhodamine near-infrared dye, the molecular formula of the probe is C ₅₆ H ₅₄ N ₂ O ₇ ²⁺ , and the structural formula QL-2 is as follows:

.

In the present invention, the preparation method of the fluorescent probe based on the pH of rhodamine near-infrared dye may further comprise the steps:

(1) Add cyclohexanone dropwise to 20ml of concentrated sulfuric acid under ice-bath conditions, then add 2-(4-diethylamino-hydroxybenzoyl)benzoic acid, after the addition is complete, under nitrogen protection, 90°C Heat and stir for 3 h. After the reaction was completed, it was cooled to room temperature, poured into ice, and HClO ₄ was added. Filtered, washed 3 times with cold distilled water, and dried at room temperature to obtain compound 1 as a red solid;

(2) Put compound 1, 1,4-hydroxyisophthalaldehyde and 0.1 g sodium ethoxide in a round bottom flask, add ethanol to the flask, and heat the reaction under nitrogen protection. The obtained crude product was purified by column chromatography to obtain fluorescent probe QL-2.

The synthetic route of the pH fluorescent probe is as follows:

.

Preferably, the molar ratio of compound 1 and 4-hydroxyisophthalaldehyde in step (2) is 1:2.

Preferably, the heating reaction condition in step (2) is 80° C. for 3 hours.

Preferably, the column chromatography purification method described in step (2) is: extract the aqueous phase with dichloromethane, remove the solvent by rotary distillation, dissolve the solid with a small amount of dichloromethane, and use dichloromethane and dichloromethane with a volume ratio of 10:1 Methanol mixed solvent column chromatography separation.

In the present invention, the application of the rhodamine near-infrared dye-based pH fluorescent probe is used to detect the pH of different systems.

Preferably, the system includes an aqueous system, an organic system or a biological system.

The pH fluorescent probe QL-2 prepared by the method proposed in the present invention.

Preferably, the method steps of detection are as follows:

S1: Solution preparation: 1mmol/ml probe solution was prepared with dimethyl sulfoxide solution as solvent, different pH solutions, all pH solutions were prepared with hydrochloric acid solution and PBS.

S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer (Hitachi Group), add 2ml of solvent and 20 μl of probe solution into the quartz dish, mix well to keep the concentration at 10 μmol/ml, the probe QL- 2 Scan at 520 nm and 740 nm as excitation wavelengths.

Beneficial effect

(1) The pH fluorescent probe based on rhodamine near-infrared dye in the present invention uses rhodamine dye to synthesize a pH-responsive fluorescent probe, which has near-infrared emission.

(2) The preparation method of the rhodamine near-infrared dye-based pH fluorescent probe of the present invention is simple, the yield of the prepared product is high, and it is suitable for large-scale popularization and application.

(3) The rhodamine near-infrared dye-based pH fluorescent probe of the present invention has good selectivity and stability for pH detection. The probe QL-2 can effectively eliminate the background effect, and when the pH value is 2-10, the probe has a good linear ratio relationship, which improves the accuracy and sensitivity of detection.

Description of drawings

Fig. 1 is the synthetic route diagram of the pH fluorescent probe based on the near-infrared dye rhodamine proposed by the present invention;

Fig. 2 is the UV absorption figure of pH fluorescent probe QL-2 based on rhodamine near-infrared dye proposed by the present invention under different pH conditions;

Fig. 3 is the fluorescence spectrogram of fluorescent probe QL-2 proposed by the present invention in different pH solutions;

Fig. 4 is the ratio pH response linear fitting under the excitation of fluorescent probe QL-2580 nm and 700 nm in different pH solutions proposed by the present invention;

Fig. 5 is the fluorescence spectrum of the probe when different ions exist in the water phase proposed by the present invention (pH=2, pH=7.4, pH=10);

Fig. 6 is the mass spectrum of the rhodamine near-infrared dye-based pH fluorescent probe QL-2 proposed by the present invention.

Detailed ways

In order to enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention is clearly and completely described below. Other similar embodiments obtained below shall all belong to the protection scope of the present application. The present invention will be further explained below in conjunction with specific implementation examples.

Implementation Case 1

The preparation method steps of the rhodamine near-infrared dye synthetic pH fluorescent probe proposed by the present invention are as follows:

(1) Add 2.0 ml of cyclohexanone dropwise to 20 ml of concentrated sulfuric acid in an ice bath, and then add 1.5 g of 2-(4-diethylamino-hydroxybenzoyl)benzoic acid. Heat and stir at 90°C for 3 h. After the reaction was completed, it was cooled to room temperature, poured into 150 g of ice, and then added 2.0 ml of 70% HClO ₄ . Filtered, washed 3 times with cold distilled water, and dried at room temperature to obtain compound 1;

(2) Put compound 1 (2.0 g, 1.33 mmol), 4-hydroxyisophthalaldehyde (0.5, 0.67 mmol), 0.5 g of sodium ethoxide in a round bottom flask, add 20 ml of ethanol to the flask, and place under nitrogen protection Reflux at 80°C for 3 h. After the reaction, it was cooled to room temperature, extracted with 50 ml of dichloromethane, washed with distilled water three times, and the solvent was removed under reduced pressure. The resulting crude product was purified on silica gel (eluted with dichloromethane:methanol=10:1 v/v). The product QL-2 is a purple-black solid with a yield of 77%. ¹ HNMR (400 MHz, DMSO) δ 10.99 (s, 1H), 9.87 (d, J = 35.5 Hz, 2H), 8.11 – 7.66(m, 15H), 7.13 (s, 12H), 7.14 – 6.89 (m, 6H), 6.63 – 6.19 (m, 11H), 6.20 (s,13H), 3.22 (d, J = 6.8 Hz, 6H), 2.18 (d, J = 113.5 Hz, 11H), 1.57 (s, 12H), 1.13 (dd, J = 36.2, 29.2 Hz, 12H), 0.92 (dt, J = 20.1, 7.3 Hz, 13H). ¹³ C NMR(101 MHz, DMSO) δ 169.53, 168.21, 163.49, 162.01, 159.47, 15 1.31, 148.54, 137.18, 137.14, 133.73, 130.37, 128.77, 122.11, 121.10, 97.38, 70.23, 59.05, 57.48, 55.86, 44.24, 44.07, 35.57, 31. 73, 29.47, 29.02, 27.15, 27.00, 20.40, 12.92, 12.86, 12.82.

ESI-MS calculated for C ₅₆ H ₅₄ N ₂ O ₇ ²⁺ [M+H] ⁺ 867.3920, found 867.4001.

The synthetic route is as follows:

Implementation Case 2

1 Fluorescence sensing of pH with fluorescent probes

(1) Fluorescence spectrum test

The probe and its fluorescence properties to pH were investigated by fluorescence spectrometer. The specific test steps are as follows:

S1: Solution preparation: 1mmol/ml probe solution was prepared with dimethyl sulfoxide solution as solvent, and different pH solutions were prepared with hydrochloric acid solution and PBS for all pH solutions. Add a fluorescent probe with an initial concentration of 1 mM, so that the concentration of the fluorescent probe in the solution is 10 μM, and the pH gradient of the solution is 2, 3, 4, 5, 6, 7, 8, 9, 10, and stand for 0.5 h Make the pH solutions of different gradients fully react with the fluorescent probe.

S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer, add 2 ml of solvent and 20 μl of probe solution into the quartz dish, mix well to keep the concentration constant at 10 μmol/ml, and probe QL-2 at 580 nm and 700 nm were used as excitation wavelengths to scan, and the emission peaks were obtained at 675 nm and 750 nm.

(2) Determination of the molar concentration of the probe solution

QL-2 is a small molecule probe with a specific relative molecular mass, which can well determine the molar concentration of the probe. Therefore, we directly weighed 1mmol of QL-2 and directly dissolved it in 1ml of dimethyl sulfoxide solution.

S1: Using F-7100 fluorescence spectrophotometer (Hitachi Group), add 2 ml of solvent and 20 μl of probe solution into the quartz dish, mix well to keep the concentration at 10 μmol/ml, and probe QL-2 at 580 nm and 700 nm were used as excitation wavelengths to scan, and the emission peaks were obtained at 675 nm and 750 nm.

The ultraviolet absorption spectrum in the range of 400-900 nm was tested with an ultraviolet spectrophotometer. The results are shown in Figure 2. The absorption wavelengths of QL-2 are at 570 nm and 750 nm, respectively, which provides the preliminary experimental basis for the fluorescence spectral titration experiment.

(3) Fluorescence sensing of pH by the probe

Probe QL-2 was scanned with excitation wavelengths of 580 nm and 700 nm, and the emission peaks were obtained at 675 nm and 750 nm. As we changed the pH in the aqueous phase, we found that the fluorescence intensity at 675 nm gradually decreased with increasing alkalinity, but the fluorescence intensity at 750 nm gradually increased (Fig. 3a, 3b). pH was linearly correlated with F ₆₇₅ /F ₇₅₀ (Fig. 4), and the correlation coefficient was 0.99163.

Probe QL-2 is sensitive to other ions and amino acids (K ⁺ , Ca ²⁺ , Zn ^{2 +} , Na ⁺ , Fe ³⁺ , Fe ²⁺ , Cu ⁺ , S ₂ O ₃ ²⁻ , S ²⁻ , SO ₃ ^{2 −} ,SO ₄ ²⁻ , HSO ₃ ²⁻ ,NO ₂ ⁻ ,Cr ₂ O ₇ ^2- ,SO ₄ ^2- , H ₂ O ₂ , Trp(Tryptophan),Cys(Cysteine,Leu(Leucine),Thr(Threonine )) Weak or almost no response (Fig. 5).

Claims (8)

1. A pH fluorescent probe based on rhodamine near-infrared dye, characterized in that the molecular formula of the probe is C ₅₆ H ₅₄ N ₂ O ₇ ²⁺ , and the structural formula QL-2 is as follows: 2. the preparation method of the fluorescent probe based on the pH of rhodamine near-infrared dye according to claim 1, is characterized in that, comprises the following steps: (1) Add cyclohexanone dropwise to 20ml of concentrated sulfuric acid under ice-bath conditions, then add 2-(4-diethylamino-hydroxybenzoyl)benzoic acid, after the addition is complete, under nitrogen protection, 90°C Heated and stirred for 3 h; after the reaction was completed, cooled to room temperature, poured into 150 g of ice, and then added 2.0 ml, 70% HClO ₄ ; filtered, washed 3 times with cold distilled water, and dried at room temperature to obtain compound 1 as a red solid; (2) Put compound 1, 1,4-hydroxyisophthalaldehyde and 0.1 g sodium ethoxide in a round-bottomed flask, add ethanol to the flask, and heat the reaction under the protection of nitrogen; the obtained crude product is purified by column chromatography to obtain fluorescence Probe QL–2; The synthetic route of pH fluorescent probe QL-2 is as follows: ; . 3. The preparation method according to claim 2, characterized in that the molar ratio of compound 1 and 4-hydroxyisophthalaldehyde in step (2) is 1:2. 4. The preparation method according to claim 2, characterized in that the heating reaction condition in step (2) is 80°C for 3 hours. 5. The preparation method according to claim 2, characterized in that, the column chromatography purification method described in step (2) is: remove the water phase by extraction with dichloromethane, remove the solvent by rotary distillation, and dissolve a small amount of dichloromethane The solid was separated by column chromatography with a mixed solvent of dichloromethane and methanol at a volume ratio of 10:1. 6. A fluorescent probe according to claim 1 is used in the preparation and detection of pH reagents in different systems. 7. The application according to claim 6, characterized in that said system comprises water system, organic system or biological system. 8. application according to claim 7, is characterized in that, the method step of detection is as follows: S1: Preparation of solution: prepare 1mmol/ml probe solution with dimethyl sulfoxide solution as solvent, different pH solutions, all pH solutions are prepared with hydrochloric acid solution and PBS; S2: Fluorescence spectrum test: Using F-7100 fluorescence spectrophotometer, add 2 ml of solvent and 20 μl of probe solution into the quartz dish, mix well to keep the concentration at 10 μmol/ml, probe QL-2 at 520 nm and 740 nm were scanned as excitation wavelengths.