A near-infrared fluorescent probe based on xanthene and its preparation method and application

Abstract

The present invention relates to the field of uranyl ion detection technology, and in particular to a near-infrared fluorescent probe based on xanthene and its preparation method and application. The present invention provides a novel near-infrared fluorescent probe based on xanthene. The probe is combined with UO2 2+ The fluorescence is enhanced after binding and can be successfully applied to UO2 in living mice 2+ Biological imaging and understanding UO2 2+ distribution and toxicity in biological systems.

Description

Technical Field

[0001] The present invention relates to the technical field of uranyl ion detection, and in particular to a xanthene-based near-infrared fluorescent probe and a preparation method and application thereof. Background Art

[0002] Uranium is a key element in the nuclear fuel cycle. Due to the environmental risks posed by its radioactive waste, uranium dioxide (UO2), it has attracted widespread attention. Uranium dioxide is often expressed in the form of uranyl ions (Uranyl ions, UO2 2 + Uranium dioxide has radiotoxicity and chemical toxicity, posing a major threat to ecosystems and human health. Excessive UO2 in the body 2+ Accumulation can lead to serious health consequences, including kidney, liver, and nervous system damage. In addition, UO2 2+ It can also cause genetic damage, which can increase the risk of cancer and other life-threatening diseases.

[0003] Traditional UO2 2+ Detection methods such as atomic absorption spectroscopy and electrochemical analysis are known for their high sensitivity. However, these techniques have significant disadvantages, including high equipment cost, complex operating procedures, and unsuitability for real-time monitoring. Recently, fluorescent probes have become popular for UO2 detection due to their fast response speed and ease of operation. 2+ Despite this, conventional fluorescent probes primarily emit in the visible spectrum, which limits their ability to penetrate biological tissues. To overcome these limitations, near-infrared (NIR) fluorescence imaging has emerged as a powerful alternative, offering deeper tissue penetration, higher spatial resolution, and reduced background noise, making it more suitable for in vivo monitoring of physiological and pathological processes.

[0004] Therefore, the near-infrared fluorescence technology was used to develop UO2 2+ Specific probes have important research value. Summary of the Invention

[0005] The present invention provides a xanthene-based near-infrared fluorescent probe, its preparation method, and its application. This invention achieves uranyl ion imaging in living mice for the first time, breaking through the previous limitation of cell imaging. By monitoring fluorescence in the mouse eyeball, the dynamic changes in uranyl ion concentration in the blood can be tracked. Furthermore, the present invention uses a near-infrared fluorescent probe made from a specific rhodamine derivative and 3-hydroxy-2-naphthaldehyde, enabling selective detection of uranyl ions with high sensitivity and rapid response.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a near-infrared fluorescent probe based on xanthene, the structural formula of which is shown in the following formula (1):

[0008]

[0009] This new xanthene-based near-infrared fluorescent probe is doped with a UO2 2+ (Hydroxynaphthalen-2-yl)methyleneamino recognition ligand that forms a stable complex. This ligand reacts with UO2 2+ After binding, the rhodamine moiety undergoes ring opening, triggering fluorescence and achieving UO2 2+ The novel xanthene-based near-infrared fluorescent probe was also modified with a rigid diamine donor group to enhance its electron-donating properties. This modification resulted in a red-shift of the emission spectrum to the near-infrared region, thereby improving tissue penetration and reducing autofluorescence in biological tissues.

[0010] Furthermore, the applicant tested the prepared novel near-infrared fluorescent probe based on xanthene to verify its performance. The relevant results showed that: UO2 2+ When the concentration increased from 0mM to 30mM, the fluorescence signal and UO2 2+ A clear linear relationship was established between the concentrations of 2% and 1%, proving that the probe is suitable for quantitative detection. The new near-infrared fluorescent probe based on xanthene showed high selectivity, high sensitivity and 2+ The rapid response makes it suitable for long-term fluorescence imaging and detection.

[0011] The present invention provides a method for preparing a near-infrared fluorescent probe based on xanthene, comprising: dissolving a rhodamine derivative and 3-hydroxy-2-naphthaldehyde in an organic solvent, and stirring the mixture to react to obtain a near-infrared fluorescent probe based on xanthene; wherein the structural formula of the rhodamine derivative is shown in the following formula (2):

[0012]

[0013] The method provided by the present invention prepares a novel xanthene-based near-infrared fluorescent probe (RU), which is obtained by reacting a specific rhodamine derivative (compound 1) and 3-hydroxy-2-naphthaldehyde as synthetic raw materials. The reaction formula is shown in the following formula (3):

[0014]

[0015] Preferably, the molar ratio of the rhodamine derivative to the 3-hydroxy-2-naphthaldehyde is 1:(1.5-3).

[0016] More preferably, the molar ratio of the rhodamine derivative to the 3-hydroxy-2-naphthaldehyde is 1:(1.5-2).

[0017] More preferably, the molar ratio of the rhodamine derivative to the 3-hydroxy-2-naphthaldehyde is 1:(2-3).

[0018] More preferably, the molar ratio of the rhodamine derivative to the 3-hydroxy-2-naphthaldehyde is 1:2.

[0019] Preferably, the organic solvent is anhydrous ethanol; and the ratio of the sum of the masses of the rhodamine derivative and 3-hydroxy-2-naphthaldehyde to the volume of the anhydrous ethanol is (2-5) mg / mL.

[0020] Further preferably, the ratio of the sum of the masses of the rhodamine derivative and 3-hydroxy-2-naphthaldehyde to the volume of the anhydrous ethanol is (4-5) mg / mL.

[0021] Preferably, the stirring reaction temperature is 80-100° C., and the stirring reaction time is 12-24 h.

[0022] Preferably, after the stirring reaction is completed, the reaction mixture is concentrated under reduced pressure and then washed with deionized water to obtain a crude product; the crude product is loaded on a silica gel column, and dichloromethane and methanol are used as eluents, and gradient elution is performed in a ratio of 100:1 to 10:1, and chromatography purification is performed to obtain a white solid, i.e., a near-infrared fluorescent probe based on xanthene.

[0023] The present invention also provides the use of the above-mentioned xanthene-based near-infrared fluorescent probe or the xanthene-based near-infrared fluorescent probe prepared by the above-mentioned method in the detection and / or imaging of uranyl ions in living mammals.

[0024] The xanthene-based near-infrared fluorescent probe provided by the present invention can be used not only for detection and imaging in living cells but also in living animals, a feat not previously achieved in the prior art. This present invention provides, for the first time, a near-infrared probe that can be used for in vivo detection / imaging.

[0025] Preferably, the detection is quantitative detection with a detection range of 0 to 30 mM and a detection limit of 11 nM.

[0026] Preferably, the application tracks the dynamic changes of uranyl ion concentration by tracking the changes of fluorescence intensity in blood.

[0027] The present invention also provides the use of the above-mentioned xanthene-based near-infrared fluorescent probe or the xanthene-based near-infrared fluorescent probe prepared by the above-mentioned method in preparing a uranyl ion detection kit for living mammals and / or preparing a uranyl ion imaging reagent for living mammals.

[0028] Therefore, the present invention has the following beneficial effects:

[0029] (1) The present invention provides a novel near-infrared fluorescent probe based on xanthene. The probe is combined with UO2 2+ The fluorescence is enhanced after binding and can be successfully applied to UO2 in living mice 2+ Biological imaging and understanding UO2 2+ distribution and toxicity in biological systems.

[0030] (2) The present invention provides a preparation method, which utilizes a special rhodamine derivative to react with 3-hydroxy-2-naphthaldehyde to generate a near-infrared fluorescent probe with a new structure.

[0031] (3) The novel xanthene-based near-infrared fluorescent probe provided by the present invention can track UO2 in the blood by monitoring the fluorescence in the mouse eyeball. 2+ Dynamic changes in concentration.

[0032] (4) The novel near-infrared fluorescent probe based on xanthene provided by the present invention exhibits high selectivity, high sensitivity and 2+ The rapid response makes it suitable for long-term fluorescence imaging and detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 RU probe in CDCl3 1 HNMR spectrum.

[0034] Figure 2 RU probe in CDCl3 13 CNMR spectroscopy.

[0035] Figure 3 is the HRMS spectrum of the RU probe.

[0036] Figure 4 For RU probe and UO2 2+ Performance results diagram, where: (a) 10μM RU and 100μM UO2 2+ Absorbance response of RU. The inset in a is the color change of RU; (b) 10 μM RU and 100 μM UO2 2+ (c) Fluorescence response of 10 μM RU at different UO2 concentrations from 0 to 30 μM 2+ Fluorescence intensity response at 665 nm under different concentrations; (d) Fluorescence intensity and UO2 2+ Linear relationship between concentrations; (e) Fluorescence response of 10 μM RU to 100 μM of different analytes at 665 nm, numbers 1 to 14 represent Na + 、UO2 2+ 、Ag + 、Zn 2 + 、Al3+ 、Ni 2+ 、Y 3+ 、Ti 3+ 、Nd 3+ 、Ba 2+ 、Pr 3+ 、Sm 3+ 、Mn 2+ and La 3+ ; (d) Bar graph of 10 μM RU and 100 μM of different analytes; All assays were performed in PBS (pH 7.4) containing 5% DMSO, λex = 580 nm.

[0037] Figure 5 Under the excitation of 150W xenon lamp, 100μM UO2 2+ Photostability graph in the presence of 10 μM RU.

[0038] Figure 6 For 100μM UO2 2+ Fluorescence intensity response diagram of 10μM RU at 665nm in the presence of and other analytes, where numbers 1 to 14 represent: no, Na + 、Ag + 、Zn 2+ 、Al 3+ 、Ni 2+ 、Y 3+ 、Ti 3+ 、Nd 3+ 、Ba 2+ 、Pr 3+ 、Sm 3+ 、Mn 2+ and La 3+ .

[0039] Figure 7 For UO2 2+ Time scans were performed in the presence of 10 μM RU.

[0040] Figure 8 100 μM UO in different pH buffers + 2+ Fluorescence intensity graph in the presence of 10 μM RU.

[0041] Figure 9 0.5mM RU probe with 1mM UO2 2+ HPLC-MS analysis of the reactions.

[0042] Figure 10 UO2 2+ ([AIU]+[UO2 2+ ]=10μM) and the binding response relationship between the RU probe.

[0043] Figure 11 RU to UO2 2+ Stern-Volmer plot of fluorescence quenching, where F min and F are respectively in the absence and presence of UO2 2+ The fluorescence intensity recorded under the condition of low concentration of UO2 is shown in the inset. 2+ The linear Stern-Volmer plot of fluorescence quenching was obtained below.

[0044] Figure 12 RU to UO2 2+ DFT analysis diagram (B3LYP / 6-31G(d,p) method, H2O solvent).

[0045] Figure 13 The graph shows the MTT assay results at different RU concentrations.

[0046] Figure 14 Different concentrations of RU and 10 μM exogenous UO2 2+ Figure 2: HeLa cell imaging; (i) cells were incubated with 10 μM RU, (ii) cells were pretreated with 10 μM RU and then incubated with 5 μM UO2 2+ (iii) cells were pretreated with 10 μM RU and then incubated with 10 μM UO2 2+ (iv) cells were pretreated with 10 μM RU and then incubated with 20 μM UO2 2+ Incubate together.

[0047] Figure 15 The left picture is the imaging result of RU. The right picture is the treatment result corresponding to the left picture. In the left picture, the four groups of treatments from left to right are: 1. Mouse intravenous injection of 100μL PBS, 2. Mouse intravenous injection of 100μL RU (50μM), 3. Mouse intravenous injection of 100μL RU (50μM) followed by injection of 100μL UO2 2+ (50μM), 4 mice were intravenously injected with 100μL RU (50μM) and then 100μL UO2 2+ (100 μM), groups 1, 2, 3, and 4 correspond to the data of 1, 2, 3, and 4 in the right figure, respectively.

[0048] Figure 16 The following is the mouse imaging diagram of RU changing with time; A is the RU to UO2 using the mouse hind limbs 2+In vivo test results: Group a in Figure A was injected subcutaneously with 25 μL of Probe (50 μM) and then injected with 25 μL of PBS 20 minutes later; Group b in Figure A was injected subcutaneously with 25 μL of Probe (50 μM) and then injected with 25 μL of UO2 20 minutes later. 2+ (100 μM); B is the quantitative fluorescence intensity measured at different time points after subcutaneous injection of ions into mice.

[0049] Figure 17 The results of the blood fluorescence experiment in mice were shown in Figure 2. The three treatment groups were: mice were intravenously injected with 100 μL RU (100 μM), mice were intravenously injected with 200 μL UO2 2+ (50μM), mice were intravenously injected with 200μL UO2 2+ (100 μM); 10 mM EDTA was used as anticoagulant. DETAILED DESCRIPTION

[0050] The present invention will be further described below with reference to specific embodiments. Those skilled in the art will be able to implement the present invention based on these descriptions. Furthermore, the embodiments of the present invention described below generally represent only a portion of the present invention, rather than all of the embodiments. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention.

[0051] Instruments used in this section: NMR and HPLC-MS analyses were performed on Brucker 400 and Waters instruments, respectively. UV-visible absorption and fluorescence spectroscopy were performed using Hitachi U 3010 and F2500, respectively.

[0052] The preparation of rhodamine derivatives (compound 1) in this section refers to the literature: Zhang Y, Wang S, Sun Y, Xu H, Xu Z, Liang X, et al. Evaluation of a biomarker (NO) dynamics in inflammatory zebrafish and periodontitis saliva samples via a fast-response and sensitive fluorescent probe. Bioorganic Chemistry. 2024; 143: 107014. 3-Hydroxy-2-naphthaldehyde was purchased from Bidex Pharmaceuticals.

[0053] Example 1

[0054] A rhodamine derivative (Compound 1, 200 mg, 0.4 mmol) and 3-hydroxy-2-naphthaldehyde (138 mg, 0.8 mmol) were accurately weighed and added to 100 mL of anhydrous ethanol. After complete dissolution, the mixture was stirred at 80°C overnight (approximately 12 h) to complete the reaction according to formula (1). After the reaction was complete, the reaction mixture was concentrated under reduced pressure and then washed with deionized water to obtain a crude product. The crude product was loaded onto a silica gel column and eluted with dichloromethane and methanol in a gradient ratio of 100:1 to 10:1. Chromatographic purification was performed to obtain a white solid, which was a near-infrared fluorescent probe based on xanthene, denoted as RU probe.

[0055]

[0056] The obtained RU probe was subjected to NMR and MS tests, and the results were as follows: Figures 1 to 3 shown. 1 HNMR(400MHz, CDCl3)10.83(d,J=18.9Hz,1H),9.17(d,J=11.4Hz,1H), δ8.00(d,J=5.7Hz,1H),7.66(d,J= 8.2Hz,1H),7.62-7.55(m,2H),7.51(d,J=7.0,2H),7.35(d,J=8.3,Hz,1H),7.24-7.12(m,3H),6.49(d,J= 8.9Hz,2H),6.37-6.13(m,2H),5.67(s,1H),3.61(t,J=10.2,1H),3.47-3.35(m,1H),3.31(q,J=7.3Hz,4H ), 3.20 (t, J = 4.1Hz, 1H), 2.50 (d, J = 9.0Hz, 4H), 2.10-1.92 (m, 3H), 1.42-1.30 (m, 1H), 1.12-1.14 (m, 6H).

[0057] 13 CNMR (101MHz, CDCl3) δ164.56,155.27,153.61,151.07,148.99,146.36,137. 44,135.19,132.21,129.32,128.01,127.45,126.20,124.24,123.24,121.14 ,111.15,107.14,105.01,103.90,97.87,96.64,77.05,67.03,60.41,56.55,54.39,53.45,47.34,44.33,39.77,30.21,29.73,23.58,21.07,14.22,12.64.

[0058] HR-MS (ESI), C 41 H 40 N5O3[M+H] + Calculated value: 650.3131; Found value: 650.3134. The results show that a near-infrared fluorescent probe based on xanthene was synthesized.

[0059] Application Example 1

[0060] This application case studies UO2 2+ Response relationship between ions and RU probe

[0061] Assay method: Prepare a 1M RU stock solution in DMSO and store at -4°C. Dilute the 1M RU to 10 μM in PBS (pH 7.4, 5% DMSO) and perform fluorescence spectroscopy. Measure emission at 580 nm within the 600-800 nm range using a 10 nm slit width.

[0062] Specific results such as Figure 4 As shown, Figure 4 a shows that when UO2 is introduced 2+ Previously, 10 μM RU had no absorption at 560 nm. However, after adding 100 μM UO2 2+ After adding 10μM RU, a clear absorption peak appeared and the color changed from colorless to blue. This colorimetric change highlights the role of RU in UO2 2+ potential in visual inspection. Figure 4 In b, the fluorescence intensity of the RU probe increased by 76 times at 665 nm, and the fluorescence quantum yield increased from 0.3% to 31.8%, which was a significant improvement. Figure 4 c shows that when UO2 2+ When the concentration increases from 0 to 30 mM, the fluorescence signal is related to UO2 2+ A clear linear relationship was established between the concentrations, demonstrating that the probe is suitable for quantitative detection. Figure 4 d shows that the detection limit of RU was determined as low as 11 nM using the formula 3σ / k.

[0063] In addition, the RU probe was irradiated with a xenon lamp, and the results were as follows: Figure 5 As shown. It was observed that RU and UO2 2+ The recognition product exhibited excellent resistance to photobleaching, with fluorescence intensity decreasing by only 4% after one hour of continuous excitation. This stability further demonstrates the applicability of the RU probe for long-term fluorescence imaging and detection.

[0064] Application Example 2

[0065] This application case studies the interference effects of other metal ions on the RU probe

[0066] Assay method: A 1 M RU stock solution in DMSO was prepared and stored at -4°C. The 1 M RU was diluted to 10 μM in PBS (pH 7.4, 5% DMSO) and then analyzed by fluorescence spectroscopy. Emission at 580 nm was measured over a range of 600-800 nm with a slit width of 10 nm. Stock solutions (1 mM) of the corresponding metal species were prepared using AgNO₃, NaCl, Al(NO₃)₃·9H₂O, BaCl₂, ZnCl₂, MnCl₂, NiCl₂, Y₂(NO₃)₆·6H₂O, Pr(NO₃)₃·6H₂O, Sm(NO₃)₃·6H₂O, CoCl₂·6H₂O, La(NO₃)₃·6H₂O, and NdCl₃·6H₂O.

[0067] Specific results such as Figure 4 e: In order to study the effect of RU on UO2 2+ The selectivity of the RU probe was evaluated for the fluorescence response to other metal ions, including Na + 、Ag + 、Al 3+ 、Ba 2+ 、Zn 2+ 、Mn 2+ 、Ni 2+ 、Y 3+ 、Ti 3+ 、Nd 3+ 、Pr 3+ 、Sm 3+ and La 3+ The results showed that RU exhibited excellent selectivity only in UO2 2+ The fluorescence was significantly enhanced in the presence of Figure 6 The interference test confirmed the resistance of RU probe to interference from competing metal ions, indicating the effectiveness of RU probe in complex environments. Figure 7 By analyzing the different concentrations of UO2 2+ The response time of RU was evaluated by measuring the fluorescence response of 10μM, 20μM and 30μM. 2+ After that, the probe reached fluorescence saturation within 28 seconds and 30 μM UO2 was added. 2+ The fluorescence saturation was achieved within 18 seconds, proving that the RU probe can realize real-time monitoring of UO2 2+ Quick response.

[0068] Application Example 3

[0069] This application example studies the effect of pH on RU probe interference.

[0070] Figure 4f is the pH value response to RU probe: in the absence of UO2 2+ In the case of , the fluorescence increases slightly under acidic conditions (pH < 5.2) due to ring opening. Figure 8 It can be seen that even in a weakly acidic environment, the introduction of UO2 2+ It also leads to a significant increase in fluorescence. It is worth noting that RU can effectively detect UO2 in the pH range of 5 to 9. 2+ , indicating that it is suitable for detecting UO2 under different pH conditions 2+ These findings highlight the excellent selectivity, high reactivity, and rapid response of RU, and highlight the sensitive and reliable detection of UO2 in complex environmental matrices. 2+ potential.

[0071] Application Example 4

[0072] This application case studies the RU probe and UO2 2+ Mechanism of interaction

[0073] In order to clarify the relationship between RU probe and UO2 2+ The mechanism of interaction was analyzed by HPLC-MS. Figure 9 It shows that a clear peak corresponding to the RU molecular weight (650.71) is observed at 7.46 minutes. 2+ After elution, the peak disappeared and a new signal peak appeared at 5.63 minutes with a molecular weight of 920.39, which was consistent with the predicted product mass.

[0074] Figure 10 Analyze and study RU and UO2 2+ The binding stoichiometry between UO2 2+ When the molar fraction of RU-UO2 is 0.5, the fluorescence intensity reaches the highest, which strongly indicates that the binding stoichiometry is 1:1. 2+ Complexation involves a single binding site. Figure 11 RU-UO2 was calculated using the Benesi-Hildebrand equation 2+ The binding constant (Ka) of the complex is 2.88×10 5 M -1 , which confirms the effect of RU on UO2 2+ Strong chelating ability.

[0075] In addition, the applicant used density functional theory to calculate the 2+ The molecular orbitals of the complex products were analyzed in detail and the results are reported in Figure 12The calculation results show that the LUMO energy level of the product is significantly lower than that of RU, and the HOMO-LUMO energy gap significantly promotes the excitation of photogenerated excitons in the entire molecule, thereby promoting the ICT process and ultimately leading to fluorescence enhancement.

[0076] Application Example 5

[0077] This application example investigates the live cell imaging capabilities of the RU probe.

[0078] Assay method: HeLa cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FBS and 100 U / mL penicillin-streptomycin. 4 The cells were seeded at a density of 100 cells / well in 15 mm glass bottom culture dishes and incubated for 24 hours. 2+ The cells were treated with 5 μM (0 μM, 5 μM, 10 μM and 20 μM) for 1 hour and then washed three times with PBS. The cells were then incubated with 10 μM probe in DMEM for 30 minutes. After further washing, the cells were imaged using a Leica Stellaris 5 confocal microscope. The cytotoxicity of RU was assessed using a standard MTT assay.

[0079] Based on RU UO2 2+ The applicant evaluated its bioimaging potential in living cells based on its powerful recognition ability. The cytotoxicity of RU was evaluated using the MTT assay on HeLa cells. Figure 13 As shown, after 12 h of incubation with 10 μM RU probe, cell viability remained above 90%. Notably, even at a higher concentration of 50 μM, cell viability exceeded 80%. These findings indicate that the RU probe exhibits relatively low cytotoxicity, making it suitable for bioimaging.

[0080] On the basis of the above, different concentrations of UO2 2+ (5 μM, 10 μM and 20 μM) were treated with the cells and then incubated with 10 μM RU. Figure 14 Observation Figure 14 It can be seen that cells treated with RU alone did not show any fluorescent signal. 2+ The treated cells showed strong red near-infrared fluorescence signals, the intensity of which increased with the increase of UO2 2+ Importantly, the fluorescence observed in the nuclei of cells treated with Hoechst remained stable, indicating that cells were 2 + These results indicate that RU can effectively detect exogenous UO2 in living cells.2 + , highlighting its potential for bioimaging applications.

[0081] Application Example 6

[0082] This application example investigates the in vivo imaging capabilities of the RU probe in mice.

[0083] Measurement method:

[0084] ① Mouse imaging

[0085] SPF-grade Balb / c mice (22–25 g) were purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. and housed in the SPF-grade laboratory animal room of Jianghan University. All animal experiments were approved by the Animal Ethics Committee. In vivo imaging was performed using the IMAGING 200pro system with excitation / emission wavelengths of 600–650 nm / 650–700 nm. Mice were intravenously injected with PBS, RU probe, or UO2. 2+ Groups 1 and 2 were injected with 100 μL PBS, and groups 3 and 4 were injected with 50 μM and 100 μM UO2, respectively. 2+ 100 μL each. Subsequently, 100 μL RU probe (50 μM) was injected into all groups except Group 1. Mice were anesthetized with isoflurane before imaging.

[0086] ② Spectral analysis of mouse blood

[0087] SPF Balb / c mice (22-25 g) were intravenously injected with 200 μL PBS (control) or 200 μL UO2 2+ (50 μM or 100 μM). 20 minutes later, blood was collected from the retroorbital plexus and mixed with 50 μL of ethylenediaminetetraacetic acid (EDTA, 10 mM) as an anticoagulant. A 300 μL aliquot of the blood was mixed with 500 μL of PBS and then with 100 μL of RU (100 μM). Fluorescence was measured at 580 nm.

[0088] Mice were intravenously injected with 100 μL PBS or 100 μL UO2 at different concentrations (50 μM or 100 μM). 2+ , then inject 100 μL RU (50 μM). Figure 15 As shown, mice injected with PBS and RU showed minimal fluorescence. In contrast, mice injected with UO2 2+ Mice treated with UO2 and RU showed a significant increase in red fluorescence intensity within 30 minutes, mainly concentrated in the abdominal area due to the high concentration of abdominal organs and blood vessels. 2+ The concentration of UO2 increased, indicating that the probe has different 2+ Concentration is sensitive.

[0089] Further research on UO2 2+ In vivo imaging studies in mice at different times were performed, e.g. Figure 16 Mice were subcutaneously injected with 25 μL RU (50 μM), and then injected with 25 μL PBS or UO2 20 minutes later. 2+ (100μM), no fluorescence signal was observed in the control group injected with PBS, while the fluorescence signal was observed in the control group injected with UO2 2+ The fluorescence signal of the experimental group increased rapidly, reached the maximum value within 5 minutes, and showed no obvious attenuation after 45 minutes, indicating that the RU probe can quickly and sensitively detect UO2 in the body for a long time. 2+ .

[0090] Blood samples were collected from the eyes of mice to verify the clinical applicability of RU. 200 μL PBS (as a control) or 200 μL UO2 was injected intravenously into the living mice. 2+ (50μM or 100μM). After 20 minutes, blood was collected from the retroorbital venous plexus of the mouse, and then the blood was measured with 100μL RU (100μM) probe. The results were as follows Figure 17 As shown in the figure, no fluorescence was observed in the eye blood of mice injected with PBS alone. However, fluorescence was detected in the blood of mice injected with UO2 2+ The red fluorescence intensity of the group was significantly enhanced, and the fluorescence intensity was similar to that of UO2 2+ There is a clear correlation between the concentrations. This is UO2 2+ The first successful fluorescence detection in mouse ocular blood. These results demonstrate the utility of the RU probe for in vivo detection of UO2 in mammals. 2+ potential.

[0091] In summary, the present invention has achieved the first UO2 2+ Imaging breaks through the previous limitation of only being able to image cells; by monitoring fluorescence in mouse eyes, it is possible to track the dynamic changes in uranyl ion concentration in the blood. Furthermore, the present invention uses a near-infrared fluorescent probe made from a specific rhodamine derivative and 3-hydroxy-2-naphthaldehyde, enabling selective detection of uranyl ions with high sensitivity and rapid response.

Claims

1. A near-infrared fluorescent probe based on xanthene, characterized in that: Its structural formula is shown in the following formula (1):

2. The method for preparing a near-infrared fluorescent probe based on xanthene according to claim 1, wherein: include: After a rhodamine derivative and 3-hydroxy-2-naphthaldehyde are dissolved in an organic solvent, the mixture is stirred and reacted to obtain a near-infrared fluorescent probe based on xanthene; wherein the structural formula of the rhodamine derivative is shown in the following formula (2):

3. The preparation method according to claim 2, wherein The molar ratio of the rhodamine derivative to the 3-hydroxy-2-naphthaldehyde is 1:(1.5-3).

4. The preparation method according to claim 2, wherein The organic solvent is anhydrous ethanol; the ratio of the sum of the masses of the rhodamine derivative and 3-hydroxy-2-naphthaldehyde to the volume of the anhydrous ethanol is (2-5) mg / mL.

5. The preparation method according to claim 2, wherein The temperature of the stirring reaction is 80-100° C., and the time of the stirring reaction is 12-24 hours.

6. The preparation method according to claim 2 or 5, characterized in that After the stirring reaction is completed, the reaction mixture is concentrated under reduced pressure and then washed with deionized water to obtain a crude product; the crude product is loaded on a silica gel column, and dichloromethane and methanol are used as eluents, and gradient elution is performed in a ratio of 100:1 to 10:

1. Chromatographic purification is performed to obtain a white solid, which is a near-infrared fluorescent probe based on xanthene.

7. Use of the xanthene-based near-infrared fluorescent probe according to claim 1 or the xanthene-based near-infrared fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 in the detection and / or imaging of uranyl ions in living mammals.

8. The use according to claim 7, characterized in that The detection is a quantitative detection with a detection range of 0 to 30 mM and a detection limit of 11 nM.

9. The use according to claim 7, characterized in that The application tracks the dynamic changes of uranyl ion concentration by tracking the changes of fluorescence intensity in blood.

10. Use of the xanthene-based near-infrared fluorescent probe according to claim 1 or the xanthene-based near-infrared fluorescent probe prepared by the preparation method according to any one of claims 2 to 6 in preparing a uranyl ion detection kit for use in living mammals and / or preparing a uranyl ion imaging reagent for use in living mammals.

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