Hemicyanine-based red / near-infrared two-region dual-response fluorescent probe and application thereof in detection of NAD (P) H and viscosity

Abstract

The invention discloses a red / near-infrared two-region dual-response fluorescent probe based on a hemicyanine skeleton and application of the fluorescent probe in detection of NAD (P) H and viscosity. The probe is designed based on an extended pi conjugated skeleton and an A-pi-A structure, and dual-channel detection of NAD (P) H and viscosity is realized through a unique response mechanism. 3-methylquinoline is used as a response site of NAD (P) H, active hydrogen in the NAD (P) H attacks an aromatic pi bond of a quinoline ring, structural rearrangement is initiated, the quinoline ring is converted into Cy-NH with a D-pi-A structure, and fluorescence enhancement of an NIR-II region is triggered through an enhanced intramolecular charge transfer (ICT) effect. The response to viscosity depends on a benzo [c, d] indole molecular rotor, in a high-viscosity environment, intramolecular rotation is limited, a distorted intramolecular charge transfer (TICT) non-radiative decay pathway is inhibited, a system is converted from a TICT state to an ICT state, and fluorescence is remarkably recovered. According to the mechanism, the Cy-N can realize high-selectivity and high-sensitivity detection on two target objects under the condition of no spectral crosstalk.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of chemical analysis and detection, and particularly relates to a red / near-infrared two-region dual-response fluorescent probe based on a hemicyanine and application thereof in detection of NAD(P)H and viscosity. BACKGROUND

[0002] Fluorescence imaging technology has been widely used in biomedical detection, cell imaging and disease diagnosis due to its high sensitivity, high spatial and temporal resolution, and real-time non-invasive monitoring. However, traditional fluorescent probes generally have inherent defects such as short emission wavelength (mostly in the visible light region) and small Stokes shift, which limits their application in deep tissue imaging.

[0003] In recent years, near-infrared two-region (NIR-II, 900-1700 nm) fluorescence imaging technology provides a new solution to the above problems. Compared with traditional visible light and near-infrared one-region (NIR-I, 650-900 nm) imaging, NIR-II imaging has deeper tissue penetration, higher spatial resolution and lower tissue scattering and autofluorescence background, showing great application prospects. In the development of NIR-II fluorescent probes, small organic molecule dyes have become the focus of current research due to their strong chemical modification, relatively low synthesis cost, good biocompatibility and easy metabolism.

[0004] Hemicyanine dyes are an important class of near-infrared organic fluorophores with a tunable push-pull electron system (A-π-A). This class of dyes usually has high molar extinction coefficient, tunable fluorescence quantum yield, easy synthesis and functional modification. By extending the π conjugated system or enhancing the intramolecular charge transfer (ICT) effect, the emission spectrum can be red-shifted and covered in the NIR-II region, significantly improving the penetration depth and signal-to-noise ratio of in vivo imaging, and having great potential in deep tumor imaging, fine visualization of blood vessels and detection of small lesions.

[0005] From the perspective of detection objects, nicotinamide adenine dinucleotide (phosphate) [NAD(P)H] is a key coenzyme in cells, and its concentration level directly reflects the energy metabolism state and redox balance of cells, which is a key biomarker for studying metabolic abnormal diseases such as cancer and neurodegenerative diseases. On the other hand, microenvironment viscosity is a key physical parameter that affects the transport and signal transduction of intracellular substances, and its abnormal increase is closely related to mitochondrial dysfunction, malignant tumors and atherosclerosis. Therefore, accurate and real-time detection of NAD(P)H and viscosity is crucial for revealing the mechanism of life activities and early diagnosis of diseases.

[0006] Currently, there are many fluorescent probes for single detection of NAD(P)H or viscosity. However, NIR-II fluorescent probes capable of simultaneously and independently detecting these two key parameters without crosstalk and high sensitivity using a single probe are still rarely reported. Developing such multifunctional integrated probes is of great significance for a comprehensive understanding of complex cell physiology and pathological processes, and realizing multi-parameter correlation analysis and high-precision disease diagnosis. It is also a technical difficulty that needs to be broken through in the current molecular imaging field. SUMMARY

[0007] In order to solve the deficiency of one-sided detection of existing fluorescent probes, the purpose of the present application is to provide a red / near-infrared two-region fluorescent probe with dual-channel response of NAD(P)H and viscosity, and a preparation method and detection application thereof. The probe provided by the present application can not only realize independent detection of two channels, but also realize high sensitivity, high selectivity detection, strong specificity and excellent anti-interference ability, and can realize precise and real-time monitoring of the changes of two key parameters, solving the deficiency of existing tools in specificity and synchronous detection.

[0008] To achieve the above purpose, the technical scheme adopted by the present application is as follows: In a first aspect, the structure formula of the fluorescent probe according to the present application is as follows: The fluorescent probe prepared by the present application is referred to as Cy-N, and Cy-N hereinafter refers to the fluorescent probe of the above structure formula.

[0009] In a second aspect, the preparation method of the fluorescent probe according to the present application is as follows: 1) The synthesis of probe Cy-N can be carried out in two steps, in which compounds 1 and 2 have been reported in the literature; Compound 1: Xu, W., Liu, S., Chen, Z., Wu, F., Cao, W., Tian, Y., et al., Bichromatic imaging with hemicyanine fluorophores enables simultaneous visualization of non-alcoholic fatty liver disease and metastatic intestinal cancer [J]. Analytical Chemistry. 2022, 94 (39): 13556-13565. Compound 2: Wei, H., Yu, Y., Wu, G., Wang, Y., Duan, S., Han, J., et al., Dual-responsive fluorescent probe for imaging NAD(P)H and mitochondrial viscosity and its application in cancer cell ferroptosis [J]. Sensors and Actuators B: Chemical. 2022, 350: 130862. 2) Compound 1 and Compound 2 were dissolved in an organic solvent at a molar ratio of 1.0: (0.8-1.4), and the solution was heated to 80-120 ℃ and refluxed for 10-18 h. The color of the solution changed from yellow to brown red. The reaction was monitored by TCL plate, and after the reaction was completed, the solution was naturally cooled to room temperature. The solvent was removed under reduced pressure, and the obtained solid was separated and purified by silica gel column chromatography (400 mesh), with dichloromethane and methanol (volume ratio V 二氯甲烷 :V 甲醇 =20:1) as eluent to obtain Compound 3; 3) Compound 3 and methyl triflate were dissolved in a dry organic solvent at a molar ratio of 1.0: (0.8-2.5) and stirred at room temperature for 24 h. The reaction was monitored by TCL, and after the reaction was completed, the crude product was obtained by removing the solvent under reduced pressure, and was separated and purified by silica gel column chromatography, with silica gel particle size of 400 mesh and eluent ratio of V 二氯甲烷 :V 甲醇 =10:1 to obtain the target compound Cy-N.

[0010] The present application provides a single-molecule fluorescent probe Cy-N based on an extended π-conjugated skeleton, which has an A-π-A structure and a large Stokes shift, and can realize double-channel detection without spectral interference. The probe uses benzo[c,d]indole as an electron acceptor and a molecular rotor, and extends the conjugated system through a thiophene bridge. Its detection principle is as shown in Figure 15 : In a high viscosity environment, the intramolecular rotation is limited, and the fluorescence is restored; in the presence of NAD(P)H, the recognition site 3-methylquinoline is reduced to an electron-rich amine, forming Cy-NH with a D-π-A structure, triggering NIR-II fluorescence enhancement, thereby realizing high selectivity and high sensitivity double response for viscosity and NAD(P)H.

[0011] In a third aspect, the application provides the use of the fluorescent probe in the detection of NAD(P)H and viscosity, comprising the following steps: 1) Preparation of PBS-EtOH buffer solution: 0.36 g of disodium hydrogen phosphate and 0.11 g of sodium dihydrogen phosphate were weighed respectively and dissolved in 100 mL of double distilled water, a molar concentration of 0.5 M sodium hydroxide solution and hydrochloric acid solution were used to adjust the pH value to 7.4, and then transferred into a 250 mL volumetric flask, and then diluted to the mark to obtain a PBS buffer solution (pH = 7.4) with a molar concentration of 10 mM. The PBS solution and ethanol were mixed in a volume ratio of 1:1, and subsequent property tests were carried out in the PBS-EtOH (v / v = 1 / 1, 10 mM, pH 7.4) buffer system.

[0012] 2) Probe stock solution: The probe Cy-N was dissolved in dimethyl sulfoxide to obtain a 5 mM Cy-N stock solution, which was stored in a 4 °C refrigerator in the dark for standby.

[0013] 3) System solution preparation for detecting NAD(P)H: NADH (as a representative of NAD(P)H) was dissolved in deionized water to prepare a 10 mM NADH stock solution for standby. The probe Cy-N (10 μM) and different concentrations of NADH stock solution were mixed in the PBS-EtOH (v / v = 1:1, 10 mM, pH 7.4) buffer system. The spectrum test was carried out at room temperature using a 1 cm standard cuvette.

[0014] 4) System solution preparation for detecting viscosity: Different viscosity systems were prepared using different volume ratios of water and glycerol. The final concentration of the probe was 10 μM. The spectrum test was carried out at room temperature using a 1 cm standard cuvette.

[0015] The beneficial effects of the detection probe of the present application are: 1) In the preparation process of the fluorescent probe of the present application, the response group can be modified according to the specific target, and the preparation method has strong expandability; 2) The detection probe belongs to an activated probe, and the emission wavelength is in the near-infrared two region, so that deep penetration and high-fidelity imaging can be realized on biological imaging; 3) It has no crosstalk dual-channel imaging capability; 4) The recognition sensitivity of NAD(P)H and viscosity is high, and the specificity is strong. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 It is the nuclear magnetic resonance hydrogen spectrum of the fluorescent probe Cy-N of the present application (the solvent is TFA).

[0017] Figure 2 It is the nuclear magnetic resonance carbon spectrum of the fluorescent probe Cy-N of the present application (the solvent is TFA).

[0018] Figure 3Mass spectrum of the fluorescent probe Cy-N of the present application.

[0019] Figure 4 High resolution mass spectrum of the fluorescent probe Cy-N of the present application after response to NAD(P)H.

[0020] Figure 5 UV absorption spectrum and fluorescence spectrum of the probe Cy-N before and after response to NAD(P)H.

[0021] Figure 6 UV titration graph (a) of the probe Cy-N after response to different concentrations of NAD(P)H (0-50 μM), and linear fitting graph (b) of absorbance at 808 nm and NAD(P)H concentration.

[0022] Figure 7 Fluorescence titration graph (a) of the probe Cy-N after response to different concentrations of NAD(P)H (0-50 μM), and linear fitting graph (b) of fluorescence intensity at 944 nm and NAD(P)H concentration.

[0023] Figure 8 Time dependence (a) and pH stability graph (b) of the probe Cy-N in response to NAD(P)H.

[0024] Figure 9 Selectivity of the probe Cy-N to NAD(P)H.

[0025] Figure 10 Fluorescence quantum yield calculation of the probe Cy-N after response to NAD(P)H. (a) UV-visible absorption spectrum of different concentrations of ICG (0.02-0.1 μM); (b) fluorescence emission spectrum of the corresponding concentration of ICG; (c) linear fitting curve of fluorescence integral area of ICG under 808 nm excitation and the corresponding absorbance; (d) UV-visible absorption spectrum of different concentrations of Cy-N (1-5 μM); (e) fluorescence emission spectrum of the corresponding concentration of Cy-N; (f) linear fitting curve of fluorescence integral area of Cy-NH under 808 nm excitation and the corresponding absorbance.

[0026] Figure 11 UV absorption spectrum and fluorescence spectrum of the probe Cy-N before and after response to viscosity.

[0027] Figure 12 Fluorescence emission graph (a) of the probe Cy-N after response to different viscosity ratios, and linear fitting graph (b) of fluorescence intensity at 633 nm and viscosity.

[0028] Figure 13 pH stability graph of the probe Cy-N in response to viscosity.

[0029] Figure 14 This demonstrates the viscosity selectivity of the probe Cy-N.

[0030] Figure 15 This is a schematic diagram of the detection mechanism of the probe Cy-N. Detailed Implementation

[0031] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0032] This invention provides a red / near-infrared II (NIR-II) dual-response fluorescent probe (code-named Cy-N) based on a hemicyanine skeleton, which can simultaneously detect NAD(P)H and viscosity with high selectivity and high sensitivity. The synthesis method and detection applications are described in detail below through specific examples.

[0033] The probe synthesis route is as follows: first step: Step Two: Step 3: .

[0034] I. Examples of Synthesis of Probe Cy-N Example 1: Synthesis of Compound 3 (Toluene / n-Butanol System) Synthesis of Compound 3: Compound 1 (1 mmol, 0.323 g) and Compound 2 (1 mmol, 0.239 g) were dissolved in a mixed solvent of toluene / n-butanol (V / V = 5 mL / 5 mL) under a N2 atmosphere and refluxed at 100 °C for 12 h. The reaction was monitored by a TCL plate. After the reaction was complete, the mixture was allowed to cool naturally to room temperature, and the solvent was removed under reduced pressure. The resulting solid was purified by silica gel column chromatography (400 mesh) using dichloromethane and methanol (V / V = 5 mL / 5 mL). 二氯甲烷 :V 甲醇 (20:1), to obtain compound 3.

[0035] 1 H NMR (400 MHz, DMSO- d6) δ 9.39 (d, J=2.3 Hz, 1H), 9.33 (d, J=7.4 Hz,1H), 9.08 (d, J=15.7 Hz, 1H), 8.81 (s,1H), 8.72 (d, J=8.1 Hz, 1H), 8.38 (d, J=7.4 Hz, 1H), 8.34 (d, J=8.2 Hz, 1H), 8.30 (d, J=4.0 Hz, 1H), 8.19 (d, J=7.8Hz, 1H), 8.15 (d, J=4.0 Hz, 1H), 8.10–8.05 (m, 2H), 7.96 (t, J=7.8 Hz, 1H),7.83 (t, J=7.8 Hz,1H), 7.73–7.68 (m, 2H), 4.86 (q, J=7.3 Hz, 2H), 1.55 (t, J=7.2 Hz, 3H). 13 C NMR (101 MHz, DMSO- d 6) δ 160.09, 149.38, 147.65, 147.20, 144.38,141.09, 138.46, 134.62, 132.12, 130.80, 129.86, 128.60, 127.70, 127.17,125.61, 122.90, 118.89, 112.85, 41.23, 15.52. ESI-MS: m / z calcd. for C 28 H 21 IN2S + , [M-I] + , 417.1420; found, 417.2121. Example 2: Synthesis of compound 3 (ethanol system) Compound 1 (1 mmol, 0.323 g) and compound 2 (1 mmol, 0.239 g) were dissolved in super dry ethanol (20 mL) under N2 atmosphere, 200 μL piperidine was added, and refluxed at 80 °C for 12 h. The reaction was monitored by TLC plate, after the reaction was completed, it was naturally cooled to room temperature, diluted with 20 mL of diethyl ether, and the crude product was crystallized out. The obtained solid was separated and purified by silica gel column chromatography (400 mesh), eluent was dichloromethane and methanol (volume ratio V 二氯甲烷 ︰V 甲醇 =20︰1), to obtain compound 3.

[0036] Example 3: Synthesis of compound 3 (acetic anhydride system) Synthesis of compound 3: Compound 1 (1 mmol, 0.323 g) and compound 2 (1 mmol, 0.239 g) were dissolved in a solution of anhydrous acetic anhydride (20 mL) under N2 atmosphere, and sodium acetate (2 mmol, 0.164 g) was added to the mixed solution, which was refluxed at 80 °C for 10 h. The reaction was monitored by TLC plate, and after the reaction was completed, it was naturally cooled to room temperature, and the solvent was removed under reduced pressure. The obtained solid was separated and purified by silica gel column chromatography (200-300 mesh), with dichloromethane and methanol (volume ratio V 二氯甲烷 ︰V 甲醇 =40︰1) as eluent to obtain compound 3.

[0037] Example 4: Synthesis of probe Cy-N Compound 3 (1 mmol, 0.509 g) and methyl trifluoromethanesulfonate (2 mmol, 210 μL) were dissolved in dichloromethane (10 mL) and stirred at room temperature for 24 h. The reaction was monitored by TLC, and after the reaction was completed, the crude product was obtained after the solvent was removed under reduced pressure, and was separated and purified by silica gel column chromatography, with silica gel particle size of 400 mesh and eluent ratio of V 二氯甲烷 ︰V 甲醇 =10︰1 to obtain the target compound Cy-N.

[0038] 1 H NMR (400 MHz, TFA- d ) δ 10.12 (s, 1H), 9.83 (s, 1H), 9.45 (d, J=7.4Hz, 1H), 9.31 (d, J=15.6 Hz, 1H), 9.13 (d, J=8.1Hz, 1H), 8.95 (d, J=8.1 Hz,1H), 8.91 (d, J=9.1 Hz, 1H), 8.87–8.78 (m, 2H), 8.70–8.63 (m, 2H), 8.61 (d, J=7.3 Hz, 1H), 8.48 (d, J=7.9 Hz, 1H), 8.44 (d, J=8.5 Hz,2H), 8.12 (d, J=15.7Hz, 1H), 5.37 (s, 3H), 5.31 (dd, J=14.1, 7.2 Hz, 2H), 2.30 (t, J=7.1 Hz, 3H). 13 C NMR (101 MHz, TFA- d) δ 148.91, 146.12, 145.52, 145.01, 144.93, 140.88, 140.57, 140.51, 139.95, 139.47, 136.62, 133.97, 133.88, 133.35, 133.31, 132.48, 132.28, 132.26, 131.53, 130.52, 129.81, 126.08, 121.29, 119.92, 56.65, 47.78, 44.09, 16.52, 16.43. ESI-MS: m / z calcd. for Cy-N(C 30 H 24 F3IN2O3S2 + , [M-I - -OTf] 2+ / 2), 216.0825; found, 216.0424. Example II. Application of probe Cy-N in NAD(P)H and viscosity detection The following experiments are verified for the response to NAD(P)H using NADH as an example, NAD(P)H: is a collective term, indicating "NADH or NADPH", often used to refer to this kind of reducing power coenzyme.

[0039] Example 5: NAD(P)H response performance test The probe Cy-N was added to the PBS-EtOH (v / v = 1:1, 10 mM, pH 7.4) buffer solution to make its final concentration 10 μM, and the test solution was prepared. Then NADH (50 μM) was added to the system, and incubated at 37 ℃ for 30 min, and its ultraviolet absorption spectrum and fluorescence spectrum were measured, respectively. As shown in Figure 5 the results, Cy-N has a maximum absorption peak at 532 nm, and after the addition of NADH, the absorption at this position is weakened, and a new absorption peak at 810 nm appears and is significantly enhanced. Correspondingly, in terms of fluorescence performance, the probe itself has weak fluorescence in the near-infrared region, while after reaction with NADH, a strong fluorescence opening signal can be observed at 944 nm. The significant changes in the above absorption and fluorescence signals confirm the high sensitivity and high selectivity of Cy-N in response to NAD(P)H.

[0040] Example 6: NAD(P)H concentration titration and linear relationship establishment The probe Cy-N (10 μM) was mixed with a series of different concentrations of NADH (0-50 μM) in PBS-EtOH buffer solution and incubated at 37 ℃ for 30 min. Subsequently, the UV-Vis absorption spectrum and fluorescence emission spectrum of each reaction system were measured, respectively. The results are shown in Figures 6-7 By analyzing the linear relationship between absorbance or fluorescence intensity at a specific wavelength and NADH concentration, a working curve for quantitative detection of NADH was established. The curve shows that the fluorescence intensity of Cy-N at 944 nm has a good linear relationship with the concentration of NADH, proving that the probe can be used for the detection of NADH.

[0041] Example 7: Response kinetics and pH stability test First, Cy-N (10 μM) was mixed with NADH (50 μM) in PBS-EtOH buffer (pH 7.4) and reacted at 37 ℃, and the fluorescence spectrum change was monitored at different time points to obtain the reaction time course curve, by which the optimal reaction time required to reach fluorescence stability was determined. Subsequently, Cy-N (10 μM) was mixed with NADH (50 μM) in PBS-EtOH buffer systems with different pH values, and the fluorescence spectrum was measured after incubation at 37 ℃ for 30 min to systematically investigate the effect of pH on the recognition performance of the probe. The results are shown in Figure 8 Under physiological pH conditions, the response of the probe to NADH did not show significant influence.

[0042] Example 8: Selectivity test To systematically evaluate the selective recognition ability of the probe Cy-N to NAD(P)H, the effect of various potential interfering substances on its fluorescence response was investigated. In the PBS-EtOH buffer system containing Cy-N (10 μM), different types of metal ions (such as Na⁺, K⁺, Ca²⁺, Mg²⁺, Zn²⁺, Fe²⁺, Fe³⁺, etc.) and biologically active molecules (including glutathione, cysteine, glucose, ascorbic acid, etc.) were introduced, and the mixed system was incubated at 37 ℃ for 30 min, and then the fluorescence spectrum was measured. The results are shown in Figure 9 By comparing the fluorescence signal difference between adding each interferent and adding only NAD(P)H, it is proved that Cy-N has a high specific response to NAD(P)H.

[0043] NaCl, KCl, MgCl2 6H2O, ZnCl2, FeCl2 4H2O, FeCl3 6H2O, Na2CO3, NaNO3, Na2SO4 were dissolved in double-distilled water, respectively, to prepare different mother liquor of analyte, and the molar concentration of all mother liquor was 10 mM; various biomolecules (GSH, Gly, Ser, Ala, Hcy, Cys, Glu, Vitamin, NADP + , NAD + , Glycerol) were dissolved in double-distilled water, respectively, and the concentration of mother liquor was 100 mM.

[0044] Example 9: Fluorescence quantum yield determination The fluorescence quantum yield of Cy-N was determined with ICG (Ф f = 13% in DMSO at 25 °C) as reference. ICG and Cy-NH (the product of Cy-N reacting with NADH) were diluted with DMSO and PBS / EtOH, respectively, to obtain five different concentrations, and the absorbance value at 808 nm of each concentration was less than 0.1. Under 808 nm excitation, the fluorescence emission spectrum of 900 ~ 1400 nm region was collected. The obtained emission integral was linearly related to the absorbance at 808 nm. The quantum yield calculation formula was as follows: Ф f,sam = Ф f,ref ×(n sam / n ref ) 2 ( k sam / k ref ) In the formula, sam represents Cy-NH, ref represents ICG, Ф f，sam is the quantum yield of Cy-NH, Ф f，ref is the quantum yield of ICG, and n is the refractive index of the measurement solvent. k , and ICG is the linear fitting slope of the emission integral to the 808 nm absorbance.

[0045] The fluorescence quantum yield of Cy-N was 3.5% obtained by the quantum yield calculation formula. The results are shown in Figure 10 .

[0046] Example 10: Viscosity response performance test Cy-N was dissolved in pure water, water / glycerol mixed solution (V / V = 4:6) and pure glycerol, respectively, and the final concentration of the probe was controlled to be 10 μM. By changing the ratio of water to glycerol, a system environment with different viscosity ratios was constructed. Subsequently, the ultraviolet absorption spectrum and fluorescence emission spectrum of Cy-N in each system were determined, respectively. The results are shown inFigure 11 As shown in Figure 7, Cy-N exhibited a weak absorption band at 532 nm and negligible fluorescence intensity at 633 nm in low viscosity solvent medium. With the increase of glycerol ratio, both the absorption intensity at 532 nm and the fluorescence intensity at 633 nm increased significantly. The significant changes of the above absorption and fluorescence signals confirmed the high sensitivity and highly selective response of Cy-N to viscosity.

[0047] Example 11: Viscosity titration and linear relationship establishment The viscosity response behavior of probe Cy-N (10 μM) was systematically studied in water-glycerol mixed system. By adjusting the ratio of water to glycerol (0-100%, v / v), a series of environments with different viscosities were constructed, and the fluorescence emission spectra of Cy-N in each system were measured. Taking the maximum emission peak intensity as the index, the response relationship of its change with the glycerol volume fraction (i.e. the environmental viscosity) was analyzed, and the standard working curve was drawn accordingly. The results are shown in Figure 8. Figure 12 As shown in Figure 8, the fluorescence intensity of Cy-N at 633 nm showed good linear correlation with the medium viscosity, confirming that the probe could be used for quantitative detection of microenvironment viscosity changes.

[0048] Example 12: pH stability of viscosity response The probe Cy-N (10 μM) was dissolved in a series of PBS-EtOH buffer solutions with different pH values, and then 60% glycerol was added to each system to construct a high-viscosity microenvironment, and the fluorescence emission intensity was measured at 633 nm wavelength. This experiment aimed to systematically evaluate the effect of pH value on the fluorescence behavior of Cy-N under high viscosity conditions. The results are shown in Figure 9. Figure 13 As shown in Figure 9, under physiological pH conditions, the response of the probe to viscosity did not show significant influence.

[0049] Example 13: Viscosity selectivity test After Cy-N (10 μM) was dissolved in PBS-EtOH buffer, different kinds of metal ions, active biomolecules (including glutathione, cysteine, ascorbic acid, etc.) and 95% glycerol solution were introduced into the system, respectively. After mixing evenly, the fluorescence emission spectra of each system were measured. The results are shown in Figure 10. Figure 14 As shown in Figure 10, it was proved that Cy-N had a high specificity response to viscosity, and the results showed that it had good selective recognition potential in complex biological systems.

Claims

1. A red / near-infrared dual-response fluorescent probe based on hemicyanine, characterized in that, The probe is named Cy-N, and its structure is as follows: 。 2. A method for preparing the fluorescent probe as described in claim 1, characterized in that, Includes the following steps: (1) Compound 1 and compound 2 were co-dissolved in an organic solvent and reacted at 80~100℃ for 10~18h. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography to obtain intermediate compound 3. (2) Compound 3 was reacted with methyl trifluoromethanesulfonate in an inert organic solvent at 25-40°C for 20-26 h. After the reaction was completed, the mixture was cooled to room temperature and the solvent was removed under reduced pressure. The product was purified by silica gel column chromatography to obtain the target fluorescent probe Cy-N.

3. The preparation method according to claim 2, characterized in that: In step (1), the structural formula of compound 1 is as follows: The structural formula of compound 2 is The molar ratio of compound 1 to compound 2 is 1.0: (0.8~1.4). The organic solvent is selected from one of the following systems: a) A mixed solvent of toluene and n-butanol, in which no additional catalyst is required for the reaction; b) Ethanol, and piperidine is added to the system as a catalyst; the amount of piperidine added is 0.1~0.3 mL per millimol of compound 1; c) Anhydrous acetic anhydride, and sodium acetate is added to the system as a catalyst; the molar ratio of sodium acetate to compound 1 is (1.5~2.5):

1.

4. The preparation method according to claim 2, characterized in that: In step (2), the structural formula of compound 3 is as follows: The molar ratio of compound 3 to methyl trifluoromethanesulfonate is 1.0: (0.8~2.5); the inert organic solvent is ultra-dry dichloromethane.

5. The application of the fluorescent probe as described in claim 1 in the detection of NAD(P)H and viscosity, characterized in that: The fluorescent probe can simultaneously detect NAD(P)H and viscosity in two independent optical channels, and the spectral signals of the two channels are free from crosstalk.

6. The application according to claim 5, characterized in that: When detecting NAD(P)H, an excitation light of 808 nm was used to monitor the near-infrared II fluorescence enhancement signal at 944 nm. When measuring viscosity, a wavelength of 532 nm was used as the excitation light, and the red fluorescence enhancement signal at 633 nm was monitored.

7. The application according to claim 5 or 6, characterized in that: The detection can be performed in an aqueous solution, an organic phase, or a PBS-ethanol mixed buffer system, wherein the pH of the PBS-ethanol mixed buffer system is 7.4, the volume ratio of PBS solution to ethanol is 1:1, and the concentration of PBS solution is 10 mM.

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