CN112869707A

CN112869707A - Double-channel ratio fluorescence sensor for detecting pH through near-infrared second window and preparation method and application thereof

Info

Publication number: CN112869707A
Application number: CN202110044323.0A
Authority: CN
Inventors: 张凡; 赵梦瑶; 汪剑波
Original assignee: Fudan University
Current assignee: Fudan University
Priority date: 2021-01-14
Filing date: 2021-01-14
Publication date: 2021-06-01
Also published as: CN114271792B; CN114271792A

Abstract

The invention belongs to the technical field of biological fluorescence sensing, and particularly relates to a near-infrared second window pH detection dual-channel ratio fluorescence sensor and a preparation method and application thereof. The fluorescent sensor of the present invention relates to a dye comprising: a nitrogen-doped boron dipyrrole near-infrared fluorescent dye NAB with near-infrared second window emission and a pH response near-infrared rhodamine fluorescent dye NRh; the two dyes are wrapped by methoxypolyethylene glycol phospholipid in different molar ratios to construct a two-channel ratio fluorescence sensor pTAS for detecting pH by a near-infrared second window capable of adjusting a detection transition point; the sensor can be used for visually detecting the change of the pH value in the body of the mouse.

Description

Double-channel ratio fluorescence sensor for detecting pH through near-infrared second window and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biological materials, and particularly relates to a dual-channel ratio fluorescence sensor for detecting pH by a near-infrared second window, and a preparation method and application thereof.

Background

Currently, molecular imaging techniques such as tomography (X-CT), Ultrasound (UI), Positron Emission Tomography (PET) and Magnetic Resonance Imaging (MRI) are widely used for medical diagnosis of diseases and the like. However, these current imaging techniques have their own drawbacks, such as radiation, poor spatial resolution, and inability to achieve dynamic real-time monitoring. Fluorescence imaging is widely used in the field of biotechnology, especially in fluorescence imaging of biological tissues, due to its advantages of real-time, non-invasive, high resolution, small amount of sample required, etc. Compared with the near-infrared first window (700 nm-900 nm) fluorescence imaging, the near-infrared second window (900 nm-1700 nm) fluorescence imaging is limited by the fact that biological tissues have strong absorption and scattering in the wave band range, so that the signal-to-noise ratio and the tissue penetration depth are low, and due to the fact that the biological tissues in the wave band have weak absorption and scattering, the imaging quality is high, the penetration depth is deep, and the near-infrared second window becomes one of the research hotspots in the field of recent fluorescence imaging. At present, some inorganic materials such as carbon nanotubes, rare earth down-conversion nanoparticles and quantum dots realize the emission of a near infrared second window region, but heavy metal elements contained in the inorganic materials have potential biological toxicity, slowly metabolize after entering a living body and have poor solubility in water, so that the application value of the inorganic materials is greatly limited. Compared with inorganic materials, the organic dye has smaller relative molecular weight, is easy to metabolize, can realize the emission of a near-infrared second window region, and is beneficial to clinical transformation. The organic fluorescent dye in the near infrared second window region has attracted much attention.

Recently, many groups of subjects have developed organic small molecule fluorescent dyes with near-infrared second window emission and applied to living organism imaging. Most of the dyes belong to a fluorescence always-on state, and after the dyes are intravenously fed into an organism, a fluorescence signal can be continuously emitted no matter whether the dyes reach a target tissue, so that the background signal is very high, the ratio of the target to the background signal is small, and the sensitivity and specificity of fluorescence detection are further limited; in addition, there are some reports of single-channel imaging fluorescent probes based on target-targeted activation, and such probes based on single-channel fluorescence enhancement mode imaging are often limited by the excitation light source and the biological tissue environment, thereby affecting the accuracy of fluorescence detection. Therefore, the development of a dual-channel ratio fluorescence sensor for near-infrared second window detection realizes the sensitivity, specificity and precision of imaging detection diagnosis, and becomes a problem to be solved for promoting the near-infrared second window technology diagnosis detection and clinical application.

Disclosure of Invention

The invention aims to provide a dual-channel ratio fluorescence sensor for detecting pH by a near-infrared second window with an adjustable detection conversion point, which has the advantages of simple preparation process, good biocompatibility, high detection sensitivity and high accuracy, and a preparation method and application thereof.

The invention provides a dual-channel ratio fluorescence sensor for detecting pH by a near-infrared second window, which is characterized in that aza-BODIPY dye (marked as NAB) and pH-responsive near-infrared rhodamine fluorescent dye (marked as NRh) are adopted, and the two dyes are wrapped by methoxy polyethylene glycol phospholipid to construct a fluorescence sensor, which is marked as pTAS; the method realizes double-channel ratio fluorescence detection through energy resonance transfer (FRET) between dyes, and realizes adjustment of detection point conversion through simple regulation and control of the molar ratio of NAB to NRh so as to meet the imaging requirements of different subintervals of a detection object.

Firstly, the invention mainly designs and prepares new aza-BODIPY and pH response rhodamine fluorescent dye emitted by a near-infrared second window on the basis of previous work, so as to construct a dual-channel ratio fluorescent sensor, and realizes the adjustment of the conversion of detection points by simply regulating and controlling the molar ratio of the two dyes, and the design idea and the compound structure are not reported in documents.

Secondly, aiming at the problems of poor imaging detection sensitivity, specificity, accuracy and the like caused by the fact that the existing near-infrared second window imaging dye and single-channel fluorescence activated probe are always turned on, the invention synthesizes and constructs the two-channel ratio fluorescence sensor for detecting the pH value by the near-infrared second window. The sensor realizes accurate visual monitoring of the change process of acidity and alkalinity of microenvironment of mouse lesions.

The invention firstly provides an organic micromolecular fluorescent dye NAB and a pH response dye NRh emitted by a near-infrared second window, and the structural formulas of the compounds are as follows:

the invention also provides a preparation method of the two compounds, wherein the synthetic route of the organic small-molecule fluorescent dye NAB is as follows:

the synthesis of the organic micromolecule fluorescent dye NAB comprises the following specific steps:

(1) synthesis of intermediate 2

Dissolving a compound 1(5- (N, N-diphenyl) -2-thiopheneacetone) and 3,4, 5-trimethoxybenzaldehyde in ethanol, adding 40% of potassium hydroxide or sodium hydroxide at room temperature, and stirring for 10-12 hours; after the reaction is finished, precipitating a solid, filtering, washing with cold ethanol, and drying to obtain an intermediate red solid compound 2; wherein the feeding molar ratio of the compound 1 to the 3,4, 5-trimethoxybenzaldehyde and potassium hydroxide is 1 (1.0-1.3) to 2-2.5;

(2) synthesis of intermediate 3

Placing the intermediate 2, nitromethane and potassium carbonate in methanol, and stirring and refluxing for 6-8 hours at 90-100 ℃; after the reaction is cooled, performing rotary evaporation on a solvent methanol, and adding ethyl acetate and water for liquid separation and extraction; drying the organic phase by anhydrous sodium sulfate, filtering, performing rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain an intermediate 3; wherein the feeding molar ratio of the intermediate 2 to the nitromethane and the potassium carbonate is 1 (10-20) to (10-20);

(3) synthesis of intermediate 4

Dissolving the intermediate 3 and ammonium acetate in ethanol, and stirring and refluxing for 24 hours at 100 ℃; after the reaction is cooled, removing half solvent ethanol by rotary evaporation, and filtering to obtain a blue-black intermediate 4; wherein the feeding molar ratio of the intermediate 3 to the ammonium acetate is 1: 20-1: 25;

(4) synthesis of the dye NAB

Mixing the intermediate 4 in anhydrous dichloromethane, sequentially adding diisopropylethylamine and boron trifluoride diethyl etherate under the protection of nitrogen, reacting and stirring for 1-2 hours, adding dichloromethane, washing with water, separating liquid, drying, performing rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain a dye NAB; wherein the feeding molar ratio of the intermediate 4 to the diisopropylethylamine to the boron trifluoride diethyl etherate is 1 (5: 7) to 10-13.

The synthesis route of the pH response dye NRh is as follows:

the specific steps for the synthesis of the pH-responsive dye NRh are as follows.

(1) Synthesis of intermediate 7

Mixing a compound 5 (9-o-benzoic acid-6-diethylamino-1, 2,3, 4-tetrahydroxanthene salt), a compound 6 (4-phenyliminopropene-6-diethylamino-1, 2, 3-trihydroxanthene) and sodium acetate in acetic anhydride, and stirring at room temperature for 3-6 hours under the protection of nitrogen; after the reaction is finished, removing the solvent by rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain an intermediate 7; wherein the feeding molar ratio of the compound 5 to the compound 6 to the sodium acetate is 1 (1.2-1.3) to 1-2.

(2) Synthesis of the dye NRh

Dissolving the intermediate 7 and 2, 6-dimethylaniline in N, N-dimethylformamide, adding a PyBOP condensing agent and diisopropylethylamine under the protection of nitrogen, and stirring at room temperature for 10-12 hours; after the reaction is finished, adding ethyl acetate and water for liquid separation and extraction; drying the organic phase by using anhydrous sodium sulfate, filtering, performing rotary evaporation to obtain an oily substance, and finally performing column chromatography to obtain a dye NRh; wherein the feeding molar ratio of the intermediate 7 to the 2, 6-dimethylaniline and the PyBOP condensing agent/diisopropylethylamine is 1 (1.0-1.5) to 1.5-2.

The dye NAB and the pH-responsive NRh prepared by the invention can be used for preparing a dual-channel ratio fluorescence sensor for detecting pH in a near-infrared second window, and the preparation method comprises the following specific steps.

Dissolving near-infrared fluorescent dyes NAB and NRh and methoxyphospholipid polyethylene glycol in chloroform, stirring for 0.4-1 h, performing rotary evaporation to remove a solvent, performing vacuum drying, heating to 60 ℃, adding deionized water at 40-60 ℃ for dissolution, performing ultrasonic treatment, cooling to room temperature, and performing ultrafiltration concentration through an ultrafiltration tube with the temperature of 30KD or 10KD to obtain a final two-channel ratio fluorescence sensor, which is marked as pTAS; the mass percentage of the near-infrared dye NAB/NRh and the methoxyphospholipid polyethylene glycol is 1 (100-24), the molar ratio of the near-infrared dye NAB to the NRh is (1: 1-15) (wherein the molar ratio of the NAB to the NRh is 1:1 and is marked as pTAS-1, the molar ratio of the NAB to the NRh is 1:5 and is marked as pTAS-2, the molar ratio of the NAB to the NRh is 1:10 and is marked as pTAS-3, the molar ratio of the NAB to the NRh is 1:15 and is marked as pTAS-4), and the concentration of the final pH fluorescence sensor is 0.005-0.02 mM (calculated by the dye NAB).

The dual-channel ratiometric fluorescence sensor pTAS prepared by the invention is excited by a laser with 808nm in a neutral or alkaline environment, and the sensor only emits fluorescence of aza-BODIPY NAB at 940nm because NRh does not generate open loop response and does not generate absorption of a receptor; under the acidic environment, the dye NRh in the sensor is subjected to open loop response by acid, and the NAB energy donor and NRhH⁺The energy acceptor generates FRET, when the 808nm laser is excited, the sensor generates NRhH⁺1026nm fluorescence of the energy acceptor, thus enabling near-infrared second window to pH two-channel ratio fluorescence detection (fig. 1).

The dual-channel ratio fluorescence sensor pTAS prepared by the invention can be used for accurately and visually monitoring the acidic change and alkaline change process of the microenvironment pH of the mouse focus.

In the invention, a near-infrared second window fluorescence emission spectrogram (figure 2) capable of being observed is obtained by exciting a dual-channel ratio fluorescence sensor pTAS-1-4 constructed by four dyes NAB/NRh with different molar ratios by a laser with a wavelength of 808nm in disodium hydrogen phosphate citric acid buffer solutions with different pH values, wherein the sensor emits fluorescence of an NAB donor at 940nm in neutral and alkaline environments, and the fluorescence of the NAB donor at 940nm is gradually weakened and NRhH is obtained as the pH value is reduced to 4.88 in an acidic environment⁺The 1026nm fluorescence of the energy acceptor is gradually increased.

In the invention, a dual-channel fluorescence sensor pTAS-1-3 obtains a ratio fluorescence change (F) after being excited by a laser with a wavelength of 808nm in disodium hydrogen phosphate citric acid buffer solutions with different pH values_940nm/F_1026nm) According to the spectrogram changing along with the pH, pH detection conversion points of the sensors pTAS-1-3 are respectively 6.49,6.76and 6.92 according to a Boltzmann formula, andand the corresponding detection intervals are respectively 6.11-6.88,6.43-7.09 and 6.63-7.22 (figure 3).

In the invention, the dual-channel fluorescence sensors pTAS-2 and pTAS-3 carry out accurate visual monitoring on the change process of acidity and alkalinity of mouse tumor microenvironment pH (figure 4).

Drawings

FIG. 1 shows the principle of the dye contained in the dual channel ratiometric fluorescence sensor for detecting pH in the near infrared second window and adjusting the pH detection point of the sensor according to the present invention. Wherein, a is the structural change and energy transfer (FRET) generation of the dye NAB and the pH-responsive dye NRh under different pH conditions, and b is the principle of adjusting the pH detection point of the sensor.

FIG. 2 is a diagram of a near-infrared fluorescence emission spectrum of a second window, which is observed by a dual-channel ratiometric fluorescence sensor for detecting pH in a near-infrared second window, in a disodium hydrogen phosphate citric acid buffer solution with different pH values and by excitation with a laser of 808 nm. Wherein a is pTAS-1, b is pTAS-2, c is pTAS-3 and d is pTAS-4.

FIG. 3 shows the ratio fluorescence change (F) obtained by exciting the dual-channel fluorescence sensors pTAS-1-3 of the invention with a laser of 808nm in disodium hydrogen phosphate citric acid buffer solutions with different pH values_940nm/F_1026nm) Spectrum as a function of pH.

FIG. 4 is a diagram of two-channel fluorescence sensors pTAS-2 and pTAS-3 for visually monitoring the change process of acidity and alkalinity of mouse tumor microenvironment pH. The kit comprises a micro-needle pH electrode, a tumor micro-tumor blood vessel clamping device, a micro-needle pH electrode, a ratiometric fluorescence imaging device and a micro-needle pH electrode detection device, wherein a is a ratiometric fluorescence imaging result in a microenvironment pH reduction process caused by mouse tumor blood vessel clamping, b is a ratiometric fluorescence imaging result in a microenvironment pH increase process caused by mouse tumor intratumoral injection of sodium bicarbonate, c is a comparison between a ratiometric fluorescence quantitative pH value and a micro-needle pH electrode detection result in a tumor microenvironment pH reduction process, and d is a comparison between the ratiometric fluorescence quantitative pH.

Detailed Description

The invention is further illustrated by the following specific examples.

Example 1:

the preparation of near-infrared two-window NAB dye has the following structural formula:

the method comprises the following specific steps.

(1) Synthesis of intermediate 2

Compound 1(1.17g,4mmol) and 3,4, 5-trimethoxybenzaldehyde (0.78g,4mmol) were weighed out and dissolved in 20mL of absolute ethanol, 40% potassium hydroxide (1.5mL) was added under ice water bath, stirring was continued for 12 hours at room temperature, yellow solid precipitated, filtered and washed with cold ethanol to obtain compound 1(1.2g, 63% yield).¹H NMR(400MHz,CDCl₃)δ:7.67(d,J＝15.2Hz,1H),7.63(d,J＝4.4Hz,1H),7.38–7.34(m,4H),7.30–7.28(m,4H),7.23–7.19(m,3H),6.83(s,2H),6.41(d,J＝4.0Hz,1H),3.91(s,6H),3.89(s,3H)；¹³C NMR(100MHz,CDCl₃)δ:180.7,163.1,153.6,146.5,142.2,133.3,130.9,129.9,125.9,125.5,120.7,113.4,105.7,61.2,56.5.HR–MS(ESI):calculated for C₂₈H₂₅N₂O₄S[M+Na]⁺:494.1397,found 494.1388；

(2) Synthesis of intermediate 3

Weighing intermediate 2(0.94g,2mmol) and nitromethane (1.5mL) to dissolve in 20mL of methanol solution, adding potassium carbonate (0.56g,4mmol), and stirring and refluxing at 100 ℃ for 6 hours; after the reaction is cooled, the solvent methanol is evaporated in a rotary manner, and ethyl acetate (40mL) and water (30mL) are added for liquid separation and extraction; the organic phase was dried over anhydrous sodium sulfate, filtered, rotary evaporated to an oil and separated by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate) to give intermediate 3(0.55g, 52% yield).¹H NMR(400MHz,CDCl₃)δ:7.38(d,J＝4.4Hz,1H),7.37–7.33(m,4H),7.26–7.24(m,4H),7.22–7.19(m,2H),6.44(s,2H),6.30(d,J＝4.4Hz,1H),4.84–4.79(m,1H),4.71–4.66(m,1H),4.08–4.06(m,1H),3.83(s,6H),3.81(s,3H),3.22–3.08(m,2H)；¹³C NMR(100MHz,CDCl₃)δ:188.3,163.7,153.7,146.3,137.7,135.0,134.0,130.4,130.0,126.2,125.6,112.8,104.7,79.6,61.0,56.4,41.2,40.7.HR–MS(ESI):calculated for C₂₉H₂₈N₂O₆S[M+H]⁺:533.1741,found 533.1747；

(3) Synthesis of the dye NAB

Weighing the intermediate 3(1.50g,4.3mmol) and ammonium acetate (172mg,2.2mmol), mixing in 30mL ethanol, heating at 100 ℃ for reflux reaction for 24h, removing half solvent ethanol by rotary evaporation after the reaction is cooled, and filtering to obtain a bluish black intermediate 4 which is directly used in the next step; dissolving the black solid in 20mL of dry dichloromethane, adding diisopropylethylamine (0.8mL) and boron trifluoride diethyl etherate (1mL) in sequence under the protection of nitrogen, stirring for 1-2 hours, adding dichloromethane, washing with water, separating, drying, performing rotary evaporation to obtain an oil, and separating by silica gel column chromatography (eluent: dichloromethane) to obtain the dye NAB (0.12g, yield 12%).¹H NMR(400MHz,CDCl₃)δ:8.21(d,J＝4.4Hz,2H),7.39–7.35(m,8H),7.32–7.29(m,8H),7.22–7.19(m,4H),6.83(s,4H),6.59(d,J＝4.8Hz,2H),3.89(s,6H),3.73(s,12H)；¹³C NMR(100MHz,CDCl₃)δ:159.5,153.3,146.7,146.4,145.5,140.2,138.9,134.1,130.0,128.7,125.8,125.1,123.8,118.1,117.7,109.9,106.5,61.2,56.2.HR–MS(ESI):calculated for C₅₈H₄₈BF₂N₅O₆S₂[M+H]⁺:1024.3189,found1024.3188；

Example 2:

the preparation method of the near-infrared two-window pH fluorescence response rhodamine structural dye NRh has the following structural formula:

the method comprises the following specific steps.

(1) Synthesis of intermediate 7

Dissolving compound 5(0.94g,2mmol) and compound 6(0.77g,2mmol) in 20mL of acetic anhydride, adding sodium acetate (0.33g,4mmol) under nitrogen, and stirring at room temperature for 3 hours; after completion of the reaction, the solvent was removed by rotary evaporation under reduced pressure in vacuo to give an oil, which was then subjected to silica gel column chromatography (eluent: dichloromethane/methanol) to give intermediate 7(0.32g, yield 21%);

(2) synthesis of the dye NRh

Dissolving intermediate 7(200mg,0.26mmol) and 2, 6-dimethylaniline (50mg,0.4mmol) in N, N-dimethylformamide, adding PyBOP condensing agent (200mg,0.26mmol) and diisopropylethylamine (70mg) successively under nitrogen protection, and stirring at room temperature for 12 hours; after the reaction, ethyl acetate (100mL) was added and washed with two batches of water (30 mL); the organic phase was dried over anhydrous sodium sulfate, filtered, rotary evaporated to an oil and finally separated by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate) to give the dye NRh (80mg, 40% yield).¹H NMR(400MHz,CDCl₃)δ:8.04(d,J＝7.6Hz,1H),7.70–7.63(m,1H),7.61–7.57(m,1H),7.39(d,J＝4.4Hz,1H),7.29–7.27(m,1H),7.08–7.05(m,1H),7.00(d,J＝7.2Hz,1H),6.83–6.80(m,3H),6.50(d,J＝8.4Hz,1H),6.38(s,2H),6.31–6.28(m,3H),6.12(s,1H),3.41–3.31(m,8H),2.44(m,4H),2.22(s,3H),1.99–1.94(m,2H),1.76–1.68(m,6H),1.38(s,3H),1.22(t,J＝6.8Hz,6H),1.15(t,J＝6.8Hz,6H)；¹³C NMR(100MHz,CDCl₃)δ:166.7,156.2,154.8,151.2,149.0,148.5,148.1,147.7,138.8,138.4,131.6,129.2,128.5,127.9,126.2,125.5,124.9,124.2,120.5,120.0,111.1,110.2,107.7,107.6,107.5,106.0,98.3,97.8,70.9,44.5,44.4,29.9,26.0,25.3,24.5,21.8,21.3,19.3,18.7,12.7,12.5.HR–MS(ESI):calculated for C₅₂H₂₅₅N₃O₃[M+H]⁺:770.4316,found770.4310.。

Example 3:

the preparation of the near-infrared second window pH two-channel ratio fluorescence sensor pTAS takes pTAS-3 with the mole ratio of the near-infrared dye NAB to the NRh of 1:10 as an example, and comprises the following specific steps:

respectively taking a near-infrared fluorescent dye NAB chloroform solution (20 mu L,1.25mM) and an NRh chloroform solution (100 mu L,2.5mM) to dissolve in 5mL chloroform, then weighing a methoxy DSPE-2000 chloroform solution, stirring for 0.5 hour at room temperature according to the mass ratio of the total weight of the dye to the methoxy DSPE-2000 being 1:24, removing the solvent by rotary evaporation, drying in vacuum, heating to 60 ℃, adding deionized water at 40-60 ℃ to dissolve, performing ultrasonic treatment, cooling to room temperature, and performing ultrafiltration concentration to 5mL through an ultrafiltration tube with 30KD or 10KD to obtain the final pH fluorescent sensor pTAS-3, wherein the NAB/NRh concentration is 5 mu M:50 mu M.

Example 4:

fluorescence emission spectrograms of the near-infrared second window pH dual-channel ratio fluorescence sensor pTAS-1-4 at different pH values are as follows: taking pTAS-3 as an example, the sensor was added to disodium hydrogenphosphate citric acid buffers of different pH values to prepare a solution with a concentration of 1. mu.M/10. mu.M of the dye NAB/NRh, after equilibration, the fluorescence emission spectrum was measured after excitation with a laser of 808nm, and the result is shown in FIG. 2, where the fluorescence intensity ratio F is_940nm/F_1026nmThe variation is as in figure 3.

Example 5:

the two-channel fluorescent sensors pTAS-2 and pTAS-3 image the change of the mouse tumor microenvironment pH. The method comprises the following specific steps:

injecting 150 muL pTAS-2 probe (NAB/NRh concentration is 10 muM/50 muM) into tail vein of anesthetized tumor-bearing mouse, clamping tumor blood supply vessel by minimally invasive surgery, irradiating whole body of mouse with 808nm external laser with power density of 30mW/cm²Fluorescence imaging images of channel 1(900-1700nm) and channel 2(1000-1700nm) were collected (see FIG. 4).

Injecting 150 μ L pTAS-3 probe (NAB/NRh concentration is 10 μ M/100 μ M) into tail vein of anesthetized tumor-bearing mouse, clamping tumor blood supply vessel by minimally invasive surgery, irradiating whole body of mouse with 808nm external laser, and making laser workThe specific density is 30mW/cm²Fluorescence imaging images of channel 1(900-1700nm) and channel 2(1000-1700nm) were collected (see FIG. 4).

Claims

1. A double-channel ratio fluorescence sensor for detecting pH through a near-infrared second window is characterized in that nitrogen-doped boron dipyrromethene dye is adopted and is marked as NAB, and a pH-responsive near-infrared rhodamine fluorescent dye is marked as NRh; the two dyes are wrapped by methoxy polyethylene glycol phospholipid to construct a fluorescent sensor which is marked as pTAS; the method realizes double-channel ratio fluorescence detection through energy resonance transfer between dyes, and realizes adjustment of detection point conversion through molar ratio regulation and control of NAB and NRh so as to meet imaging requirements of different subintervals of a detection object; wherein the mass percentage of NAB to NRh and the methoxyphospholipid polyethylene glycol is 1 (100-24), and the regulation range of the molar ratio of NAB to NRh is 1 (1-15); the structural formula of the NAB and NRh compound is as follows:

。

2. the preparation method of the dual-channel ratiometric fluorescence sensor for detecting pH in the near-infrared second window according to claim 1, which comprises the following steps:

dissolving near-infrared fluorescent dyes NAB and NRh and methoxyphospholipid polyethylene glycol in chloroform, stirring for 0.4-1 h, performing rotary evaporation to remove a solvent, performing vacuum drying, heating to 60 ℃, adding deionized water at 40-60 ℃ for dissolution, performing ultrasonic treatment, cooling to room temperature, and performing ultrafiltration concentration through an ultrafiltration tube with the temperature of 30KD or 10KD to obtain a final two-channel ratio fluorescence sensor, which is marked as pTAS; the mass percentage of the near-infrared dye NAB to NRh and the methoxyphospholipid polyethylene glycol is 1 (100-24), and the molar ratio of the near-infrared dye NAB to NRh is 1 (1-15).

3. The preparation method according to claim 2, wherein the near-infrared fluorescent dye NAB is synthesized by the following steps:

the specific steps of NAB synthesis are as follows:

(1) synthesis of intermediate 2

Dissolving 5- (N, N-diphenyl) -2-thiopheneacetone compound 1 and 3,4, 5-trimethoxybenzaldehyde in ethanol, adding 40% potassium hydroxide or sodium hydroxide at room temperature, and stirring for 10-12 hours; after the reaction is finished, precipitating a solid, filtering, washing with cold ethanol, and drying to obtain an intermediate red solid compound 2; wherein the feeding molar ratio of the compound 1 to the 3,4, 5-trimethoxybenzaldehyde and potassium hydroxide is 1 (1.0-1.3) to 2-2.5;

(2) synthesis of intermediate 3

Placing the intermediate 2, nitromethane and potassium carbonate in methanol, and stirring and refluxing for 6-8 hours at 90-100 ℃; after the reaction is cooled, performing rotary evaporation on a solvent methanol, and adding ethyl acetate and water for liquid separation and extraction; drying the organic phase by anhydrous sodium sulfate, filtering, performing rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain an intermediate 3; wherein the feeding molar ratio of the intermediate 2 to the nitromethane and the potassium carbonate is as follows: 1 (10-20) and (10-20);

(3) synthesis of intermediate 4

Dissolving the intermediate 3 and ammonium acetate in ethanol, and stirring and refluxing for 24 hours at 100 ℃; after the reaction is cooled, removing half solvent ethanol by rotary evaporation, and filtering to obtain a blue-black intermediate 4; wherein the feeding molar ratio of the intermediate 3 to the ammonium acetate is 1 (20-25);

(4) synthesis of the dye NAB

Mixing the intermediate 4 in anhydrous dichloromethane, sequentially adding diisopropylethylamine and boron trifluoride diethyl etherate under the protection of nitrogen, reacting and stirring for 1-2 hours, adding dichloromethane, washing with water, separating liquid, drying, performing rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain a dye NAB; wherein the feeding molar ratio of the intermediate 4 to diisopropylethylamine and boron trifluoride diethyl etherate is as follows: 1 (5-7) and (10-13).

4. The preparation method according to claim 2, wherein the near-infrared fluorescent dye NRh is synthesized by the following steps:

the NRh synthesis comprises the following specific steps:

(1) synthesis of intermediate 7

Mixing 9-o-benzoic acid-6-diethylamino-1, 2,3, 4-tetrahydroxanthene salt compound 5, 4-phenyliminopropene-6-diethylamino-1, 2, 3-trihydroxanthene compound 6and sodium acetate in acetic anhydride, and stirring for 3-6 hours at room temperature under the protection of nitrogen; after the reaction is finished, removing the solvent by rotary evaporation to obtain an oily substance, and separating by using column chromatography to obtain an intermediate 7; wherein the feeding molar ratio of the compound 5 to the compound 6 to the sodium acetate is as follows: 1 (1.2-1.3) and (1-2);

(2) synthesis of the dye NRh

Dissolving the intermediate 7 and 2, 6-dimethylaniline in N, N-dimethylformamide, adding a PyBOP condensing agent and diisopropylethylamine under the protection of nitrogen, and stirring at room temperature for 10-12 hours; after the reaction is finished, adding ethyl acetate and water for liquid separation and extraction; drying the organic phase by using anhydrous sodium sulfate, filtering, performing rotary evaporation to obtain an oily substance, and finally performing column chromatography to obtain a dye NRh; wherein the feeding molar ratio of the intermediate 7 to the 2, 6-dimethylaniline and the PyBOP condensing agent/diisopropylethylamine is as follows: 1 (1.0-1.5) and (1.5-2).

5. The application of the near-infrared second window pH detection dual-channel ratiometric fluorescence sensor of claim 1 in precise visual monitoring of the change of acidity and the change of alkalinity of microenvironment of mouse lesions.

6. The fluorescent dye for emission of the near-infrared second window is characterized by being a nitrogen-doped boron dipyrrole fluorescent dye and a rhodamine dye, wherein the nitrogen-doped boron dipyrrole fluorescent dye is marked as NAB dye, and the rhodamine dye is marked as NRh dye; the structures of the compounds are respectively as follows:

。