Ratio fluorescent probe responding to NADH as well as preparation method and application of ratiometric fluorescent probe

Abstract

The invention discloses a ratiometric fluorescent probe responding to NADH (Nicotinamide Adenine Dinucleotide) as well as a preparation method and application of the ratiometric fluorescent probe. The structure of the ratiometric fluorescent probe responding to the NADH is as follows: # imgabs0 #. The ratio fluorescent probe responding to the NADH shows specific ratio fluorescent response to the NADH at 460 nm and 590 nm, and the detection limit of the ratio fluorescent probe is as low as 42 nM. Cytotoxicity tests show that the fluorescent probe is low in biotoxicity, confocal fluorescence microscopic imaging experiments show that the fluorescent probe is good in light stability in HeLa cells, NADH level changes can be sensitively detected through ratio fluorescence imaging, cancer cells and normal cells are effectively distinguished, the fluorescent probe is suitable for ratio fluorescence imaging and detection of the NADH in the cells, and the fluorescent probe has good application prospects. Tumor distribution can be quickly and accurately visualized in an in-situ spraying mode, and the method is used for fluorescent surgical navigation.

Description

Technical Field

[0001] The present invention relates to a ratiometric fluorescence probe responsive to NADH, a preparation method and uses thereof, so as to realize ratiometric fluorescence imaging for detecting NADH in cells and rapidly and accurately visualize tumors by means of in-situ spraying, which has the advantages of high selectivity, high sensitivity and low biotoxicity. Background Art

[0002] Cancer is one of the main causes of human death and an important obstacle to extending human lifespan. With the emergence of laparoscopic and robotic technologies, minimally invasive liver surgery, especially hepatectomy, has made remarkable progress. However, the minimally invasive resection of abdominal tumors is still limited by the insufficient ability of tumor-specific visualization, making it difficult to achieve precise separation of tumors. Fluorescence imaging technology can provide real-time visualization of tumors, helping to accurately identify tumors, and thus is widely used in the medical field. For example, the developed fluorescence probe such as indocyanine green (ICG) can achieve tumor visualization by illuminating tumors, but its clinical application is severely limited due to its non-specific imaging of normal tissues. In addition, most fluorescence probes for tumor imaging need to be intravenously injected to identify tumors, which requires a long waiting time and their effects are affected by the distribution of blood vessels in tissues. Therefore, it is very necessary to develop an ideal fluorescence probe that can quickly outline the tumor boundary by means of in-situ spraying.

[0003] With the continuous development of fluorescence probe technology, fluorescence probes have been successfully applied to distinguish tumor cells from normal cells. They usually utilize the differences in biomarker expression or microenvironment characteristics between cancer cells and normal cells to achieve specific labeling. Among them, different from normal cells, tumor cells support the rapid proliferation and anabolism of tumor cells by enhancing the glycolysis process, and more reduced nicotinamide adenine dinucleotide (NADH) is produced in this process. Therefore, developing a fluorescence probe capable of sensitively detecting NADH is expected to be applied to the identification of tumors and normal tissues. Although there are reports on fluorescence probes for specifically detecting NADH in the prior art, fluorescence probes that can achieve highly accurate detection of NADH through ratiometric fluorescence signals are still scarce. And ratiometric fluorescence probes can improve the signal-to-noise ratio by monitoring the changes in two emission peaks. Therefore, it is very urgent and important to develop a ratiometric fluorescence probe responsive to NADH. Summary of the Invention

[0004] The present invention aims to provide a ratiometric fluorescence probe responsive to NADH, a preparation method and uses thereof. The technical problem to be solved is to obtain, through molecular design, a probe that can detect NADH through ratiometric fluorescence signals, so as to realize real-time monitoring of NADH levels in living cells and tissues by ratiometric fluorescence imaging, which has the advantages of high selectivity, high sensitivity, good photostability, etc. Cytotoxicity tests show that the ratiometric fluorescence probe of the present invention has good cell compatibility.

[0005] The ratio fluorescence probe of the present invention that responds to NADH, briefly denoted as XND, has the following structural formula:

[0006] .

[0007] The preparation method of the ratio fluorescence probe of the present invention that responds to NADH includes the following steps:

[0008] Step 1: Add compound 3 (1.023 g, 3.0 mmol), 4-methylumbelliferone (0.528 g, 3.0 mmol), and cesium carbonate (1.467 g, 4.5 mmol) to the reaction system in sequence, and use 50 mL of acetone as the reaction solvent. The solution is refluxed for 8 hours under nitrogen protection and then cooled to room temperature. The mixture is washed with saturated brine, extracted with dichloromethane, the organic phases are combined and the solvent is removed by rotary evaporation under reduced pressure. The crude product is purified by column chromatography to finally obtain 0.88 g of compound 4 with a yield of 67.0%.

[0009] Step 2: Mix compound 4 (0.437 g, 1 mmol), 3-quinolinecarboxaldehyde (0.157 g, 1 mmol), and two drops of piperidine with 10 mL of ethanol. The solution is refluxed for 12 hours under nitrogen protection, and after the reaction is completed, it is cooled to room temperature. The mixture is washed with saturated brine, extracted with dichloromethane, the organic phases are combined, and then the solvent is evaporated under reduced pressure. The crude product is purified by column chromatography to finally obtain 0.46 g of compound 5 with a yield of 79.3%.

[0010] Step 3: Dissolve compound 5 (0.200 g, 0.347 mmol) in 10 mL of acetonitrile, add 0.5 mL of methyl iodide, and reflux for 24 hours. After the reaction is completed, add 30 mL of ethyl acetate to the system, stir well at 0 °C, filter under reduced pressure, wash with ethyl acetate, and collect the filter cake. Finally, 0.16 g of the target product XND is obtained with a yield of 65.0%.

[0011] In Step 1, the eluent for purifying the crude product by column chromatography is petroleum ether:ethyl acetate = 15:1, v / v.

[0012] In Step 2, the eluent for purifying the crude product by column chromatography is petroleum ether:ethyl acetate = 10:1, v / v.

[0013] Among them, Compound 2 and Compound 3 can be synthesized by the methods reported in the literature (Silswal A., Koner A. L. Tracking endoplasmic reticulum viscosity during ferroptosis and autophagy using a molecular rotor probe [J]. Chemical Communications, 2023, 59(13): 1769-1772.).

[0014] The synthesis process of the ratio fluorescence probe responsive to NADH of the present invention is as follows:

[0015]

[0016] The application of the ratio fluorescence probe of the present invention in the preparation of NADH detection reagents.

[0017] The ratio fluorescence probe XND of the present invention can be used to prepare NADH detection reagents to detect the NADH level in living cells; in addition, it can also rapidly and accurately visualize tumor distribution and perform fluorescence surgical navigation by means of in-situ spraying.

[0018] Furthermore, when detected by fluorescence emission spectroscopy, the fluorescence intensity ratio I 590 nm / I 460 nm has a linear relationship with the NADH concentration.

[0019] Furthermore, the detection reagent can distinguish tumor cell tissues and normal cell tissues by fluorescence imaging.

[0020] Specifically, the detection method includes the following steps:

[0021] Dissolve XND of the present invention in DMSO (5 mL) to prepare a stock solution of 2 mM. Take 15 μL of the XND stock solution and add it to 3 mL of PBS solvent (30% acetonitrile is added as a co-solvent) to obtain a test solution with a final concentration of 10 μM. The spectral properties of XND are detected by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. In the absence of NADH, XND shows only one absorption peak at about 440 nm, while in the presence of NADH, a new absorption peak appears at 537 nm, and its absorption value shows a gradient upward trend with the increase of NADH concentration (0-35 μM), while the absorption value at 440 nm remains stable throughout the process without obvious change ( Figure 1a). Subsequently, its fluorescence emission properties were further investigated. The test found that the probe XND itself had a broad peak around 460 nm and showed a weak fluorescence emission peak at 590 nm ( Figure 1 b). After adding NADH, the fluorescence peak intensity at 590 nm increased significantly with the increase of NADH concentration (0 - 35 μM), and the enhancement degree showed a good dose-dependence with the NADH concentration. It is worth noting that during this process, the fluorescence peak intensity and peak shape at 460 nm remained stable without obvious changes. This property enables it to be used as an ideal internal reference signal for NADH detection, thus realizing ratiometric fluorescence detection. To further verify the quantitative detection ability of the probe, the quantitative relationship between the fluorescence intensity ratio (I 590 nm / I 460 nm ) of the probe XND and the NADH concentration (0 - 35 μM) was analyzed ( Figure 1 c). The results showed that there was a good linear relationship between the two, and the correlation coefficient R 2 reached 0.9911. The detection limit (LOD) was calculated to be as low as 42 nM by LOD = 3δ / k.

[0022] Subsequently, the reaction kinetics of the probe XND to NADH was further evaluated. As Figure 1 shown in d, the addition of NADH caused a sharp increase in the fluorescence intensity of the probe XND solution at 590 nm, while the fluorescence intensity at 460 nm remained basically unchanged. By real-time monitoring the change of fluorescence intensity, it was found that the reaction system reached kinetic equilibrium within 36 minutes, and then entered a plateau period and remained stable. The fluorescence emission spectral characteristics of the probe XND in the presence of potential interferents and NADH were further investigated. The experimental results showed that the I 590 nm / I 460 nm value of the probe XND increased significantly after adding NADH, while it remained stable when other analytes (including GSH, Cys, Hcy, H2O2, etc.) were introduced ( Figure 1 e), showing good selective recognition ability. As Figure 1 shown in f, the fluorescence intensity ratio (I 590 nm / I 460 nm ) of the probe XND remained stable in the pH range of 4 - 9. After adding NADH, the fluorescence intensity ratio (I 590 nm / I 460 nmIt shows a significant enhancement and remains stable in the pH range of 4 - 9, indicating that XND has a wide pH application range in NADH recognition and can adapt to complex physiological environments.

[0023] Existing NADH fluorescent probes based on organic small molecules cannot be detected by ratio fluorescence signals. The probe molecule of the present invention can achieve ratio fluorescence detection. Through the internal calibration mechanism of dual emission wavelengths, it effectively eliminates environmental interference, thereby providing more reliable detection results.

[0024] Traditional tumor imaging fluorescent probes mostly rely on the intravenous injection route and need to go through processes such as blood circulation distribution and tumor enrichment. The imaging cycle is relatively long, and the imaging effect is affected by the density and permeability of tumor blood vessels. However, the probe molecule of the present invention can quickly and accurately visualize tumor tissues by spraying.

[0025] The ratio fluorescence probe of the present invention that responds to NADH shows specific ratio fluorescence responses to NADH at 460 and 590 nm, and its detection limit is as low as 42 nM. It has good response ability to NADH in solutions, cells, and tissues. Cytotoxicity tests show the relatively low biotoxicity of this fluorescent probe. Confocal fluorescence microscopy imaging experiments show that this fluorescent probe has good photostability in HeLa cells, can sensitively detect changes in NADH levels through ratio fluorescence imaging, effectively distinguish cancer cells from normal cells, is suitable for intracellular NADH ratio fluorescence imaging and detection, and can quickly and accurately visualize tumor distribution by in-situ spraying and be used for fluorescence surgical navigation. Brief Description of the Drawings

[0026] Figure 1 Shows the changes of (a) absorption spectrum and (b) fluorescence spectrum of XND (10 μM) with the increase of NADH concentration (0 - 35 μM); (c) linear relationship between the fluorescence intensity ratio (I 590 nm / I 460 nm ) and NADH concentration; (d) time-dependent changes of the fluorescence intensity of XND before and after adding 35 μM NADH; (e) fluorescence intensity ratio (I 590 nm / I 460 nm ) of the probe XND after adding potential interferents, 1 - 20: Blank, Na + , K + , Ca 2+ , Mg 2+ , Fe 2+ , Cu 2+ , HCO3 - , Cl - , ONOO- ,ClO - ,H2O2,·OH,Cys,GSH,Hcy,serine,S 2- ,glycine,NADH; (f) The fluorescence intensity ratio (I 590 nm / I 460 nm ) of probe XND in the presence and absence of 35 μM NADH at different pH values.

[0027] Figure 2 are the LC-MS spectra of XND before and after the addition of NADH.

[0028] Figure 3 is the cytotoxicity after treating HeLa cells with different concentrations (0, 10, 20, 30, 40 μM) of XND for 24 hours.

[0029] Figure 4 In (a), confocal images of XND (10 μM) in HeLa cells with and without NADH (30 μM) treatment, scale bar: 20 μm; (b) are the fluorescence intensities of the green and red channels in Fig. (a); (c) is the fluorescence intensity ratio (I red / I green ) in Fig. (a).

[0030] Figure 5 are the confocal images of three cancer cells (HeLa, HepG2, Hepa1-6) and three normal cells (AML-12, NIH / 3T3, MH-S) after treatment with XND (10 μM) for 30 minutes, scale bar: 20 μm.

[0031] Figure 6 is (a) a schematic diagram of fluorescence surgical navigation; (b) fluorescence imaging of tumor-bearing mice and image-guided surgical resection after spraying 100 μM XND (the fluorescent imaging area in the abdominal cavity is marked by the white circle); 1: tumor, 2: liver, 3: kidney, 4: spleen, 5: lung, 6: heart.

[0032] Figure 7 is the H&E staining of tumor tissues and adjacent tissues.

[0033] Figure 8 is the ex vivo organ fluorescence imaging 30 minutes after spraying 100 μM of probe XND and 100 μM of ICG into the abdominal cavity. Detailed implementation manners

[0034] The technical solution of the present invention will be further described below through embodiments.

[0035] Example 1: Synthesis of XND

[0036] Compound 4 (0.200 g, 0.347 mmol) was dissolved in 10 mL of acetonitrile, 0.5 mL of methyl iodide was added, and the mixture was refluxed for 24 hours. After the reaction was completed, 30 mL of ethyl acetate was added to the system, and the mixture was stirred thoroughly at low temperature, filtered under reduced pressure, washed with ethyl acetate, and the filter cake was collected. Finally, 0.16 g of XND was obtained with a yield of 65.0%.

[0037] 1 H NMR (400 MHz, DMSO-d6, ppm) δ 9.97 (s, 1H), 9.51 (s, 1H), 8.48 (d, J = 8.9 Hz, 1H), 8.37 (d, J = 8.2 Hz, 1H), 8.30 (s, 1H), 8.28 – 8.22 (m, 1H), 8.03 (t, J = 7.6 Hz, 1H), 7.96 (d, J = 15.7 Hz, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.59 (t, J = 9.3 Hz, 2H), 7.51 (d, J = 15.7 Hz, 1H), 7.32 (t, J = 7.7 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 6.87 (d, J = 7.9 Hz, 2H), 6.13 (s, 1H), 4.60 (s, 3H), 4.44 (t, J = 7.0 Hz, 2H), 4.09 (t, J = 6.3 Hz, 2H), 2.32 (s, 3H), 2.02 (p, J = 7.2 Hz, 2H), 1.76 (p, J = 6.5 Hz, 2H). 1313C NMR (101 MHz, DMSO-d6, ppm) δ 163.12, 162.10, 160.65, 155.21, 153.91, 150.70, 145.55, 141.00, 138.40, 137.48, 136.77, 136.44, 131.49, 131.10, 129.99, 129.33, 129.25, 126.94, 126.08, 123.87, 122.34, 121.65, 119.85, 115.91, 115.10, 113.58, 112.94, 112.17, 111.65, 109.11, 101.72, 76.34, 68.32, 46.63, 46.19, 26.54, 26.10, 18.67. ESI-MS m / z: [XND - I - + C 38 H 31 N4O3 + calcd, 591.2391; found, 591.2379.

[0038] Example 2: Spectral Test of XND

[0039] The XND of the present invention was dissolved in DMSO (5 mL) to prepare a 2 mM mother liquor. 15 μL of the XND mother liquor was added to 3 mL of PBS solvent (30% acetonitrile was added as a co-solvent) to obtain a test solution with a final concentration of 10 μM for spectral testing. The spectral properties of XND were detected by ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy. In the absence of NADH, XND showed only one absorption peak at about 440 nm, while in the presence of NADH, a new absorption peak appeared at 537 nm, and its absorption value showed a gradient upward trend with the increase of NADH concentration (0 - 35 μM), while the absorption value at 440 nm remained stable throughout the process without obvious change ( Figure 1 a). Subsequently, its fluorescence emission characteristics were further studied. The test found that the probe XND itself had a broad peak at about 460 nm and showed a weak fluorescence emission peak at 590 nm ( Figure 1 ​b). After adding NADH, the fluorescence peak intensity at 590 nm increased significantly with the increase of NADH concentration (0 - 35 μM), and the degree of its enhancement showed a good dose - dependence with the NADH concentration. It is worth noting that during this process, the fluorescence peak intensity and peak shape at 460 nm remained stable without obvious changes. This characteristic enables it to be used as an ideal internal reference signal for NADH detection, thus realizing ratio - type fluorescence detection. To further verify the quantitative detection ability of the probe, the fluorescence intensity ratio (I 590 nm / I 460 nm ) of probe XND and the quantitative relationship with NADH concentration (0 - 35 μM) were analyzed ( Figure 1 c). The results showed that there was a good linear relationship between the two, and the correlation coefficient R² reached 0.9911. The detection limit (LOD) was calculated to be as low as 42 nM by LOD = 3δ / k.

[0040] Subsequently, the reaction kinetics of probe XND with NADH was further evaluated. As Figure 1 shown in d, the addition of NADH caused the fluorescence intensity of probe XND solution at 590 nm to increase sharply, while the fluorescence intensity at 460 nm remained basically unchanged. By real - time monitoring the change of fluorescence intensity, it was found that the reaction system reached kinetic equilibrium within 36 minutes, and then entered a plateau and remained stable. The fluorescence emission spectral characteristics of the probe XND in the presence of potential interferents and NADH were further investigated. The experimental results showed that the I 590 nm / I 460 nm value of probe XND increased significantly after adding NADH, while remained stable when introducing other analytes (including GSH, Cys, Hcy, H2O2, etc.) ( Figure 1 e), showing good selective recognition ability. As Figure 1 shown in f, the fluorescence intensity ratio (I 590 nm / I 460 nm ) of probe XND remained stable in the pH range of 4 - 9. The fluorescence intensity ratio (I 590 nm / I 460 nm ) of probe XND after adding NADH showed significant enhancement and remained stable in the pH range of 4 - 9, indicating that XND has a wide pH application range in NADH recognition and can adapt to complex physiological environments.

[0041] Example 3: Study on the response mechanism of probe XND to NADH

[0042] To deeply explore this reaction process, two samples were prepared for testing: one sample was the probe XND, and the other sample was the mixed solution after the reaction of XND and NADH, which had been subjected to extraction and concentration. In the total ion chromatogram (TIC), a chromatographic peak of the probe XND appeared at about 1.43 minutes, and the corresponding mass spectrometry peak was 591.2360 ( Figure 2 ). This result was basically consistent with the theoretical molecular weight of the probe XND ([XND - I - + C 38 H 31 N4O3 + calcd, 591.2391). For the extraction and concentration solution after the reaction of XND and NADH, the test results revealed a more complex reaction process. In the total ion chromatogram, the peak that appeared at about 1.37 minutes should be attributed to the residual probe XND that had not reacted completely. This phenomenon indicated that in the actual reaction process, there might be some probe XND that did not react fully with NADH. It should be noted that a new peak was observed at 8.94 minutes ( Figure 2 ). The mass spectrometry results showed that the molecular mass corresponding to this peak was 593.2535, which corresponded to the molecular mass of the reaction product XNDH calculated according to the chemical reaction ([XNDH + H] + C 38 H 33 N4O3 + calcd, 593.2547). This key finding confirmed that after the reaction of the probe XND and NADH, the expected product XNDH was generated.

[0043] Example 4: Cytotoxicity Test

[0044] Before applying the probe XND for cell imaging, the toxicity of the probe needs to be tested, and the MTT method was used for the experiment. HeLa cells were cultured for 24 h by adding 0 μM, 10 μM, 20 μM, 30 μM, and 40 μM of the probe XND respectively, and it was found that the cell survival rate always remained above 90% ( Figure 3 ). Therefore, the probe XND has low toxicity to HeLa cells and can be used for biological applications.

[0045] Example 5: Exogenous Imaging of NADH by XND in HeLa Cells

[0046] In HeLa cells, after staining with 10 μM of the probe XND, it was observed through a fluorescence microscope that bright fluorescence signals were shown in both the green channel and the red channel ( Figure 4 ​a). Among them, the fluorescence signal of the red channel mainly comes from endogenous NADH in cells, indicating that probe XND can react with NADH in cells and produce a fluorescence response. Next, exogenous NADH (30 μM) was added to the stained HeLa cells. The results showed that the red fluorescence signal increased significantly by 1.49 times, while the green fluorescence signal remained basically stable without obvious changes, reflecting the increase in the intracellular NADH level ( Figure 4 b). The change in the fluorescence ratio image further confirmed this result, and its color gradually changed from the initial yellow-green to blue-green ( Figure 4 a). These results indicate that probe XND can sensitively respond to the changes in the NADH level in HeLa cells.

[0047] Example 6: Probe XND is used to distinguish normal cells and cancer cells

[0048] To verify the discrimination ability of probe XND, four typical cancer cell lines (HeLa cells, 4T1 cells, HepG2 cells, and Hepa1-6 cells) and three normal cell lines (AML-12 cells, NIH / 3T3 cells, and MH-S cells) were selected for experiments. The imaging results showed that in cancer cells, the fluorescence signal of the red channel was significantly higher than that in normal cells ( Figure 5 ). Further analysis of the fluorescence ratio images found that the fluorescence ratio images of normal cells mainly showed orange-red, while those of cancer cells mainly showed yellow-green. These results indicate that probe XND can effectively distinguish cancer cells and normal cells by imaging NADH.

[0049] Example 7: Probe XND is used for in vivo fluorescence imaging and surgical navigation of tumor-bearing mice

[0050] The ability of probe XND to rapidly image tumor tissues in vivo by spraying and its application potential in surgical navigation were evaluated in an H22 orthotopic tumor-bearing mouse model. To simulate the actual tumor resection process during surgery, after opening the mouse abdomen, 100 μM XND was evenly sprayed on the exposed abdominal cavity area ( Figure 6 a). As Figure 6 shown in b, within 2 minutes after in situ spraying, the red fluorescence signal from probe XND could be detected, and this fluorescence basically stabilized after 10 minutes and maintained a high-intensity fluorescence signal for at least 20 minutes, showing the excellent performance of probe XND for rapid response and stable imaging in vivo. The imaging results showed bright fluorescence signals emitted by the tumor tissues, forming a sharp contrast with the adjacent normal tissues ( Figure 6b). This significant difference in fluorescence signals provides an intuitive visual basis for the precise identification of tumor boundaries during surgery. Subsequently, the tumor was completely resected based on the fluorescence signals emitted by the tumor tissue. To further verify the specificity and accuracy of the probe, tissues near the surgical incision were collected for H&E staining analysis ( Figure 7 ). The results showed that the tissues fluorescently labeled with probe XND exhibited typical features of hepatocellular carcinoma, including nuclear atypia and increased nuclear-cytoplasmic ratio. No obvious lesions were observed in the tissue sections with weaker fluorescence on the other side. These results indicate that probe XND can rapidly illuminate tumor tissues in mice by in-situ spraying and achieve precise surgical navigation through significant fluorescence signal changes, and has the ability to serve as a visualization tool for tumor resection guidance.

[0051] Indocyanine green (ICG) is a near-infrared fluorescent dye widely used in the medical field, especially playing an important role in tumor identification and surgical navigation. Then, the capabilities of probe XND and indocyanine green (ICG) in fluorescence imaging of tumors were compared. A mixed solution of 100 μM probe XND and 100 μM ICG was evenly sprayed into the abdominal cavity of mice, and after waiting for 30 minutes, ex vivo imaging of each organ was performed. The imaging results showed that probe XND could well distinguish tumor tissues from normal tissues ( Figure 8 ). However, obvious fluorescence also existed in the normal liver tissue in the ICG fluorescence channel, indicating that it could not well achieve tumor identification by spraying within a short time.

Claims

1. A ratiometric fluorescence probe responsive to NADH, abbreviated as XND, characterized in that Its structure is as follows: 。 2. The preparation method of the ratiometric fluorescence probe according to claim 1, characterized in that It includes the following steps: Step 1: Compounds 3, 4-methylumbelliferone and cesium carbonate are successively added to the reaction system. Using acetone as the reaction solvent, reflux the reaction for 8 hours under nitrogen protection. After the reaction is completed, cool it to room temperature, and obtain Compound 4 after separation and purification; Step 2: Mix Compound 4, 3-quinolinecarboxaldehyde and piperidine with ethanol, reflux for 12 hours under nitrogen protection. After the reaction is completed, cool it to room temperature, and obtain Compound 5 after separation and purification; Step 3: Dissolve Compound 5 in acetonitrile, add methyl iodide, reflux the reaction for 24 hours, and obtain the target product XND after the reaction is completed; The synthetic route is as follows: 。 3. Use of the ratio fluorescence probe according to claim 1 in the preparation of a NADH detection reagent.

4. According to the use described in claim 3, wherein: When detected by fluorescence emission spectroscopy, the fluorescence intensity ratio I 590nm / I 460nm has a linear relationship with the NADH concentration.

5. According to the use described in claim 3, wherein: The detection reagent can distinguish tumor cell tissues and normal cell tissues through fluorescence imaging.

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