ATP (adenosine triphosphate)-targeted hydrogen sulfide near-infrared nano fluorescent probe as well as preparation method and application thereof
By targeting ATP with a hydrogen sulfide near-infrared nanofluorescent probe and using ZIF-90 to encapsulate H2S-responsive compounds, the problems of false positives and complex synthesis of existing fluorescent probes were solved, achieving highly specific and sensitive imaging of acute liver injury.
Patent Information
- Application Number
- CN202510891092.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-30
- Publication Date
- 2025-09-19
AI Technical Summary
Existing fluorescent probes are prone to produce false positive signals in the diagnosis of acute liver injury. In addition, there are few probes that respond to both ATP and H2S simultaneously, and the synthesis process is complicated, which limits their application.
A hydrogen sulfide near-infrared nanofluorescent probe targeting ATP was used, and the H2S response compound was encapsulated by the nanomaterial ZIF-90. The H2S response compound was released by the coordination effect of ATP and reacted with H2S to generate a near-infrared fluorescence signal.
It achieves high-specificity imaging, reduces false-positive interference, is suitable for deep tissue imaging, simplifies the preparation process, and improves detection accuracy and sensitivity.
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Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of fluorescent probes, and in particular relates to a hydrogen sulfide near-infrared nano fluorescent probe targeting ATP, and a preparation method and application thereof. Background Art
[0002] With the development of society and the continuous improvement of people's living standards and quality of life, the incidence of acute liver injury has also shown an increasing trend year by year, and has now become the second largest cause of liver injury. Early and accurate diagnosis and imaging identification of acute liver injury is one of the keys to improving the cure rate of acute liver injury. Fluorescence imaging technology has become one of the most promising methods for early and accurate diagnosis of acute liver injury due to its advantages such as convenience, real-time imaging, good spatial resolution imaging rate and high sensitivity. However, the fluorescent probes currently developed usually only respond to a single microenvironmental feature in acute liver injury, which is prone to interference from "false positive" signals due to insufficient specificity. By combining the detection of two important biomarkers in the occurrence and development of acute liver injury, the false positive signal problem existing in most current fluorescent probes will be effectively improved.
[0003] H2S, an important gaseous signaling molecule, is primarily produced in the liver through the metabolism of methionine and cysteine. Endogenous H2S levels can influence human health. Small amounts of H2S can promote cell growth and protect cells from oxidative damage, while excessive amounts can exacerbate the development and progression of liver diseases. H2S also plays a crucial role in regulating liver function, participating in multiple metabolic pathways. The liver is not only the primary organ producing H2S but also a key site for regulating H2S metabolism and function. Furthermore, abnormal H2S levels are closely associated with various liver diseases. Therefore, H2S is a key microenvironmental characteristic of acute liver injury. ATP is another key microenvironmental characteristic of acute liver injury and serves as the primary source of cellular energy. More importantly, ATP and H2S are closely linked in the development of acute liver injury. Mitochondrial dysfunction is closely linked to the pathophysiology of acute liver injury and is associated with abnormal changes in ATP and H2S concentrations. H2S regulates mitochondrial ATP synthesis through S-sulfhydrylation of ATP synthase on the inner mitochondrial membrane. The development of near-infrared fluorescent probes activated by ATP and H2S, which are interrelated in acute liver injury, holds promise for highly specific imaging of acute liver injury, addressing the need for early, precise diagnosis and treatment of acute liver injury. However, currently, there are very few fluorescent probes that can simultaneously respond to both ATP and H2S microenvironments, and the complex synthesis, purification, and isolation processes of such probes often limit their widespread application. Summary of the Invention
[0004] In response to the above technical problems, the present invention provides a hydrogen sulfide near-infrared nanofluorescent probe targeting ATP, as well as a preparation method and application thereof.
[0005] The present invention adopts the following technical solutions: The present invention targets ATP-targeting hydrogen sulfide near-infrared nanofluorescent probe, which is composed of a nanomaterial ZIF-90 and an H2S-responsive compound encapsulated within the ZIF-90. The chemical structure of the H2S-responsive compound is as follows: The structure of the ZIF-90 is as follows: The present invention's hydrogen sulfide near-infrared nanofluorescent probe targeting ATP encapsulates an H2S-responsive compound within ZIF-90 through nanomaterial self-assembly, thereby forming a stable nanofluorescent probe. In the presence of ATP, the coordination of Zn²⁺ with ATP destroys the structure of ZIF-90, thereby releasing an H2S-responsive compound that responds to H2S. Simultaneously, H2S reacts with the H2S-responsive compound, converting its ether group into a hydroxyl group, thereby simultaneously targeting ATP and responding to H2S, generating a strong near-infrared fluorescence signal for analysis and detection of acute liver injury (e.g., Figure 1 shown).
[0006] The preparation method of the hydrogen sulfide near-infrared nanofluorescent probe targeting ATP of the present invention comprises the following steps: (1) Mixing zinc acetate dihydrate, 2-methylimidazole, and H2S-responsive compound in N,N-dimethylformamide solution; (2) Stir at room temperature for 4-6 minutes and add N,N-dimethylformamide to stabilize the structure; (3) The nanoparticles were purified by centrifugation, washed and freeze-dried to obtain the near-infrared nanofluorescent probe CySO3NO2@ZIF-90.
[0007] The molar ratio of zinc acetate dihydrate to 2-methylimidazole was 1:2; the concentration of the H2S-responsive compound in the reaction system was 1-2 mg / mL; the centrifugation was performed at 10,000 rpm for 5 minutes, and the freeze-drying time was 3 hours.
[0008] The present invention provides a simple preparation method for a near-infrared nano-fluorescent probe with a fast synthesis speed. The target product can be obtained by simply adding zinc acetate dihydrate, 2-methylimidazole and an H2S-responsive fluorescent probe into an N,N-dimethylformamide solution in steps and stirring for 5 minutes.
[0009] The present invention also provides the use of a hydrogen sulfide near-infrared nano-fluorescent probe targeting ATP in the preparation of a fluorescent imaging detection reagent for acute liver injury.
[0010] The nanofluorescent probe can highly selectively target ATP and respond to two microenvironmental characteristics of H2S, and can only light up the near-infrared fluorescence signal when stimulated by ATP and H2S.
[0011] The fluorescence imaging detection reagent is used for co-activation detection of ATP and H2S in acute liver injury cells, and the near-infrared fluorescence signal emission wavelength is 650-710 nm.
[0012] The H2S-responsive near-infrared nanofluorescent probe targeting ATP of the present invention can respond to the H2S microenvironmental characteristics while targeting ATP, generating a strong near-infrared fluorescence signal for analysis and detection, thereby reducing background signal interference and achieving highly specific imaging of acute liver injury. It has good application prospects in the early and accurate diagnosis and imaging of acute liver injury.
[0013] Compared with the prior art, the beneficial effects of this application are: 1. Dual-marker collaborative detection to improve specificity By simultaneously targeting ATP and H2S response, two key biomarkers of acute liver injury, the "false positive" interference of traditional single-marker probes is significantly reduced, achieving highly specific imaging.
[0014] Pathological correlation between ATP and H2S: The probe utilizes the synergistic changes of the two in liver damage (such as abnormal ATP synthesis and imbalance of H2S metabolism caused by mitochondrial dysfunction) to enhance detection accuracy.
[0015] 2. Advantages of near-infrared fluorescence signals Low background interference: The near-infrared band (emission wavelength 705 nm) has strong penetrability, which can reduce the interference of spontaneous fluorescence of biological tissues and improve the signal-to-noise ratio.
[0016] Deep tissue imaging: Suitable for real-time monitoring of deep tissues such as the living liver, providing a visualization tool for early diagnosis of acute liver injury.
[0017] 3. Structural design and ease of preparation No need for complex modification: The H2S-responsive compound CySO3NO2 is encapsulated by ZIF-90 nanomaterials, avoiding the tedious steps of introducing bulky targeting groups required by traditional probes.
[0018] Rapid self-assembly synthesis: The nanoprobe (CySO3NO2@ZIF-90) can be prepared by stirring at room temperature in just 5 minutes. The process is efficient and reproducible.
[0019] 4. Intelligent response and controlled release ATP-triggered release: The ZIF-90 structure dissociates in the presence of ATP through Zn²⁺ coordination, precisely releasing the probe molecule to the damaged area.
[0020] H2S-specific response: After the probe molecule reacts with H2S, the ether group is converted into a hydroxyl group, activating the near-infrared fluorescence signal and achieving "double lock" regulation (requiring the coexistence of ATP and H2S).
[0021] 5. Wide application potential Compatibility between cells and living organisms: Experiments have confirmed that it can effectively distinguish between normal and damaged states in liver cells (such as L02 cells), and the fluorescence signal is positively correlated with the degree of pathology.
[0022] Clinical translation prospects: The simple preparation process and stable nanostructure provide it with advantages in the development of clinical diagnostic reagents. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 Schematic diagram of the application of near-infrared nanofluorescent probe targeting ATP and responding to H2S in acute liver injury; Figure 2 (a) is the infrared spectrum of CySO3NO2@ZIF-90; (b) is the X-ray diffraction spectrum of CySO3NO2@ZIF-90; (c) is the thermogravimetric analysis diagram of CySO3NO2@ZIF-90; Figure 3 Time dependence of CySO3NO2@ZIF-90; Figure 4 Selectivity of CySO3NO2@ZIF-90; Figure 5 CySO3NO2@ZIF-90 imaging of acute liver injury cells. DETAILED DESCRIPTION The technical solution of the present invention is further described in detail below in conjunction with several preferred embodiments and the accompanying drawings. This embodiment is implemented on the premise of the technical solution of the invention, and provides a detailed implementation method and specific operation process, but the protection scope of the present invention is not limited to the following embodiments.
[0024] Unless otherwise specified, the experimental materials used in the following examples can be purchased from conventional biochemical reagent companies.
[0025] Example 1: Preparation of CySO3NO2@ZIF-90 (1) Synthesis of H2S-responsive compounds CySO3OH (625.0 mg, 1.0 mmol), 1-fluoro-2,4-dinitrobenzene (205.0 mg, 1.1 mmol), and K2CO3 (207.0 mg, 1.5 mmol) were added to dry DMF and stirred at 50.0°C. After the reaction was completed (TLC monitoring, approximately 0.5 to 1.0 h), the product was diluted with CH2Cl2 and washed with water three times. The organic phase was dried over anhydrous Na2SO4 and purified by column chromatography using CH2Cl2:CH3OH (100:1 to 20:1, v / v) as the eluent to obtain granular or flaky purple-red solid CySO3NO2. 1 The H NMR and high-resolution mass spectrometry data are as follows: 1 H NMR (400 MHz, DMSO- d 6) δ 8.96 (s, 1H), 8.56 (t, J = 20.1 Hz, 1H), 7.97 – 7.63 (m, 2H), 7.46 (dd, J = 52.4, 26.5 Hz, 1H), 7.19(s, 1H), 6.94 (d, J = 16.1 Hz, 1H), 5.41 (s, 2H), 4.65 (s, 2H), 4.11 (s, 3H), 2.50 (s, 8H), 2.04(d, J = 41.8 Hz, 2H), 1.79 (d, J = 39.4 Hz, 2H), 1.20 (d, J =26.0 Hz, 6H). High-resolution mass spectrometry theoretical calculated value C 34 H 32 N3NaO9S: 681.1854; measured value, 681.1732.
[0026] (2) Preparation of CySO3NO2@ZIF-90 A 2 mL solution of zinc acetate dihydrate (0.2 M) in N,N-dimethylformamide was poured into a 2 mL solution of CySO₃NO₂ (5 mg) and 2-methylimidazole (0.4 M). The mixture was stirred vigorously at room temperature for 5 minutes, after which N,N-dimethylformamide (6 mL) was added to stabilize the structure. The resulting nanoparticles were purified by centrifugation (10,000 rpm, 5 minutes) and washed twice with N,N-dimethylformamide. The nanoparticles were then collected and freeze-dried under vacuum for 3 hours to obtain the final product, CySO₃NO₂@ZIF-90.
[0027] The preparation method of ZIF-90 is as follows: A 2 mL solution of zinc acetate dihydrate (0.2 M) in N,N-dimethylformamide was poured into a 2 mL solution of 2-methylimidazole (0.4 M) in N,N-dimethylformamide. The mixture was stirred vigorously at room temperature for 5 minutes, and then N,N-dimethylformamide (6 mL) was added to stabilize the structure. The resulting nanoparticles were purified by centrifugation (10,000 rpm, 5 minutes) and washed twice with N,N-dimethylformamide. The nanoparticles were then collected and freeze-dried in a vacuum for 3 hours to obtain ZIF-90.
[0028] The successful preparation of near-infrared nanofluorescent probes was studied by thermogravimetry, X-ray diffraction and infrared spectroscopy. Specifically, thermal analysis was performed using a synchronous thermal analyzer STA449F3 under nitrogen, X-ray diffractometer was used for X-ray diffractometer analysis, and Fourier transform infrared spectrometer V70 was used for infrared spectroscopy analysis. Figure 2 a and Figure 2 b It can be seen that the thermogravimetric curves of ZIF-90 and CySO3NO2@ZIF-90 have similar trends and CySO3NO2@ZIF-90 has a greater weight loss. At the same time, the X-ray diffraction spectra of the two also show the same peak, which indicates that CySO3NO2@ZIF-90 has been successfully prepared and the crystal structure of ZIF-90 has not been destroyed. Figure 2 As shown in Figure c, both ZIF-90 and CySO3NO2@ZIF-90 showed characteristic peaks at 1676 nm, but there was no characteristic peak of the H2S-responsive fluorescent probe, which once again demonstrated that CySO3NO2@ZIF-90 was successfully prepared.
[0029] Example 2: Time dependence of CySO3NO2@ZIF-90 The nano fluorescent probe provided in this application has a certain time dependence in vitro. This example studies the time dependence of CySO3NO2@ZIF-90 prepared in Example 1 in vitro. The CySO3NO2@ZIF-90 synthesized in Example 1 was dissolved in a buffer solution to prepare 1 mg / mL and stored at room temperature. The time dependence of CySO3NO2@ZIF-90 was tested using fluorescence spectroscopy. The test used a quartz cuvette as the sample pool, mixed CySO3NO2@ZIF-90 with ATP (250 μM) and H2S (100 μM) and incubated at 37°C, and measured the changes in its fluorescence spectrum every 2 minutes. The results are as follows. Figure 3 As shown, from Figure 3It can be seen that the fluorescence intensity of CySO3NO2@ZIF-90 increases within 25 minutes and the increment decreases continuously, which indicates that CySO3NO2@ZIF-90 reaches the maximum fluorescence intensity at 30 minutes.
[0030] Example 3: Selectivity of CySO3NO2@ZIF-90 The CySO3NO2@ZIF-90 provided in this application can highly selectively target ATP and respond to two microenvironmental characteristics of H2S to light up near-infrared fluorescence signals. Therefore, this example studies the selectivity of CySO3NO2@ZIF-90 prepared in Example 1 in the presence of ATP and H2S. Specifically, the CySO3NO2@ZIF-90 synthesized in Example 1 was configured with a buffer solution to form a 1 mg / mL suspension. Divided into 2 groups, (i) no substance was added to the first group of suspensions, and incubated at 37°C for 25 minutes; (ii) ATP (250 μM) and H2S (100 μM) were added to the second group of suspensions, and incubated at 37°C for 25 minutes. After incubating the above solutions, the solution was tested at λ using a fluorescence spectrometer. ex = Fluorescence spectrum changes at 645 nm and at λ ex / em = Fluorescence intensity change at 660 / 750 nm. Figure 4 It can be seen that the fluorescence signal of the solution of CySO3NO2@ZIF-90 is weak when no treatment is performed. However, when ATP and H2S are present, the fluorescence signal of CySO3NO2@ZIF-90 is weak at λ em A strong near-infrared fluorescence signal was observed at 705 nm. This result indicates that CySO₃NO₂@ZIF-90 can only be illuminated when stimulated by both ATP and H₂S. This reduces background signal interference during the detection process, improves the reliability of the detection results, and facilitates its application in bioimaging.
[0031] Example 4: Application of CySO3NO2@ZIF-90 in Cell Imaging of Acute Liver Injury The CySO3NO2@ZIF-90 provided in this application can highly selectively target ATP and respond to two microenvironmental characteristics of H2S to illuminate the near-infrared fluorescence signal. Therefore, this example studies the application of CySO3NO2@ZIF-90 prepared in Example 1 in fluorescence imaging of acute liver injury cells.
[0032] Human normal liver cells L02 were selected as the research objects, and the imaging performance of the probe in human normal liver cells L02 was studied using a laser scanning confocal microscope (AxioImager.Z2). After culturing L02 cells in a cell culture incubator at 37°C and 5% carbon dioxide (volume percentage) for 24 hours, the cells were digested with trypsin and transferred to a cell imaging dish (cell concentration 1*10). The cells in the imaging dish were then divided into three groups: (i) the first group of cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide (volume percentage) for 24 hours, and then CySO3NO2@ZIF-90 was added and incubated for 30 minutes; (ii) the second group of cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide (volume percentage) for 24 hours, and then LPS (1 μg / mL) was pretreated for 12 hours, and then CySO3NO2@ZIF-90 (10 μg / mL) was added and incubated for 30 minutes; (iii) the third group of cells were cultured in a cell culture incubator at 37°C and 5% carbon dioxide (volume percentage) for 24 hours, and then the positive drugs aspirin (10 μM) and LPS (1 L02 cells were pretreated with CySO3NO2@ZIF-90 (10 μg / mL) for 12 hours, and then incubated for another 30 minutes. Finally, the culture medium in the imaging dish was washed away with PBS buffer, and the cells were imaged using a laser scanning confocal microscope with the 650-710 nm wavelength as the output signal channel. The results are shown in Figure 2. Figure 5 As shown. Figure 5 As can be seen, only the second group of cells exhibited a strong fluorescence signal; no near-infrared fluorescence signal was observed in the first or third groups of cells. This is because, among the experimental cells, the first group of cells did not suffer acute liver damage, while the third group of cells had enhanced resistance to liver damage due to aspirin. Only the second group of cells experienced acute liver damage due to LPS, leading to abnormal changes in intracellular ATP and H2S levels. These results demonstrate that CySO3NO2@ZIF-90 can enter hepatocytes and react with intracellular ATP and H2S, generating a strong fluorescence signal, which is suitable for specific fluorescence imaging of cells with acute liver damage.
Claims
1. A hydrogen sulfide near-infrared nanofluorescent probe targeting ATP, characterized in that: The probe is composed of the nanomaterial ZIF-90 and an H2S-responsive compound encapsulated within the ZIF-90. The chemical structure of the H2S-responsive compound is as follows: 。 2. The near-infrared nano-fluorescent probe according to claim 1, characterized in that The H2S-responsive compound was encapsulated in ZIF-90 by self-assembly of nanomaterials to form a stable nanofluorescent probe; in the presence of ATP, Zn 2+ The coordination with ATP destroys the structure of ZIF-90 and releases the H2S-responsive compound. At the same time, H2S reacts with the H2S-responsive compound to convert its ether group into a hydroxyl group, thereby targeting ATP and responding to H2S at the same time, generating a strong near-infrared fluorescence signal.
3. The method for preparing the near-infrared nano-fluorescent probe according to claim 1 or 2, characterized in that: The following steps are involved: (1) Mixing zinc acetate dihydrate, 2-methylimidazole, and H2S-responsive compound in N,N-dimethylformamide solution; (2) Stir at room temperature for 4-6 minutes and add N,N-dimethylformamide to stabilize the structure; (3) The nanoparticles were purified by centrifugation, washed and freeze-dried to obtain the near-infrared nanofluorescent probe CySO3NO2@ZIF-90.
4. The preparation method according to claim 3, characterized in that The molar ratio of the zinc acetate dihydrate to 2-methylimidazole is 1:2; the concentration of the H2S-responsive compound in the reaction system is 1-2 mg / mL.
5. The preparation method according to claim 3, characterized in that The centrifugation was performed at 10,000 rpm for 5 minutes, and the freeze-drying time was 3 hours.
6. Use of the near-infrared nanofluorescent probe according to claim 1 in the preparation of a fluorescent imaging detection reagent for acute liver injury.
7. The use according to claim 6, characterized in that The nanofluorescent probe can highly selectively target ATP and respond to two microenvironmental characteristics of H2S, and can only light up the near-infrared fluorescence signal when stimulated by ATP and H2S.
8. The use according to claim 6, characterized in that: The fluorescence imaging detection reagent is used for co-activation detection of ATP and H2S in acute liver injury cells, and the near-infrared fluorescence signal emission wavelength is 650-710 nm.