Hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease

Abstract

The invention discloses a hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease, and belongs to the technical field of biomedical detection. The probe is a product formed by combining chloroplatinic acid or a salt thereof with bipyrazine or bipyridine or a derivative thereof through a Pt-N bond, and can specifically form a Pt-S bond with H2S through platinum ions to trigger the electron energy level change of a ligand so as to change the fluorescence intensity. In a Parkinson's disease model, the fluorescence intensity of the probe is enhanced due to increase of the H2S level of the intestinal tract. The invention solves the problems of high cost and strong hysteresis of the existing diagnosis technology, is suitable for early screening of Parkinson's disease, and is beneficial to reducing the risk of illness and reducing the treatment cost.

Description

Technical Field

[0001] This invention belongs to the field of biomedical detection technology, specifically relating to a hydrogen sulfide (H2S) fluorescent probe, its preparation method, and its application in the auxiliary diagnosis of Parkinson's disease. Background Technology

[0002] Parkinson's disease (PD) is a progressive neurodegenerative disease characterized by tremor and bradykinesia. Globally, there are over 10 million people with PD, including nearly 3 million in China. Currently, clinical diagnosis of PD primarily relies on brain imaging, such as positron emission tomography (PET-CT) and magnetic resonance imaging (MRI). These methods can visually reveal changes in brain structure and function, providing crucial evidence for diagnosis. However, their limitations cannot be ignored. On the one hand, imaging diagnostics are expensive and time-consuming, making them unsuitable for large-scale screening. On the other hand, they are mainly used to observe existing central nervous system damage, and by the time abnormalities are observed on imaging, PD has often progressed to a late stage, making it difficult to cure.

[0003] Hydrogen sulfide (H2S), a key gaseous signaling molecule, not only participates in regulating intestinal barrier function and neuroinflammation but may also influence the central nervous system by affecting the gut-brain axis. Abnormal H2S levels are closely related to intestinal dysfunction and neurodegenerative diseases in Parkinson's disease patients (Research progress on the role of endogenous hydrogen sulfide in Parkinson's disease, Ye Haonan et al.). Parkinson's patients have higher intestinal H2S levels than healthy individuals. Previous analysis by the applicant, based on metabolomics and microbiome, revealed that H2S metabolites in the feces of early-stage PD patients were significantly downregulated. Sulfur-related bacteria decompose metabolites to produce H2S, leading to increased H2S generation. Therefore, detecting hydrogen sulfide can be used to diagnose the risk of Parkinson's disease.

[0004] Fluorescent probes can monitor gaseous signaling molecules in vivo in real time. They cause little or no damage to biological samples and are one of the core methods for studying gaseous signaling molecules in vivo. H2S fluorescent probes can be designed based on various principles such as exchange reactions, reduction reactions, nucleophilic addition reactions, and ring-opening reactions. However, they have drawbacks such as low sensitivity, poor specificity, slow reaction speed, and low biosafety, and are still not suitable for rapid diagnosis under complex in vivo environments (Research progress on fluorescent probes for detecting hydrogen polysulfides, Wang Maolin et al.).

[0005] Given the urgent need for clinical diagnosis of Parkinson's disease and the limitations of existing in vivo fluorescent probes, there is an urgent need to develop a new hydrogen sulfide fluorescent probe to provide technical support for the accurate prediction of Parkinson's disease. Summary of the Invention

[0006] The purpose of this invention is to provide a hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease, in order to solve the problems of high cost, long time consumption, and lag in existing disease diagnosis.

[0007] A hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease is formed by the combination of chloroplatinic acid or its salt with bipyrazine or bipyridine or its derivatives via a Pt-N bond; wherein the chloroplatinic acid salt includes sodium chloroplatinate, ammonium chloroplatinate or potassium chloroplatinate; and wherein the derivative is a product derived by conventional substitution of the pyrazine or pyridine ring.

[0008] The conventionally substituted substituents include carboxyl, nitro, alkyl, or halogen groups.

[0009] This invention uses bipyrazine or bipyridine or their derivatives as fluorescent groups and chloroplatinic acid as a responsive group. After H2S coordinates with platinum ions in the responsive group, it affects the electronic coupling and energy level distribution of the fluorescent group, resulting in enhanced emission of the fluorescent group. By detecting the intensity of the fluorescence signal, H2S can be quantified, thereby assisting in the diagnosis of Parkinson's disease.

[0010] The present invention further provides a method for preparing the hydrogen sulfide fluorescent probe, comprising the following steps: dissolving chloroplatinic acid or its salt and bipyrazine or bipyridine or its derivative in DMF at a molar ratio of 1:1, controlling the concentration of both to be 2-5 mg / mL, sonicating, reacting at 160-185℃ in the dark for 30-80 min, centrifuging, washing, and then lyophilizing.

[0011] Furthermore, the reaction is carried out under stirring conditions at a stirring speed of 300-800 rpm.

[0012] This invention provides the application of the hydrogen sulfide fluorescent probe in the preparation of diagnostic reagents for Parkinson's disease.

[0013] The present invention also provides a Parkinson's disease diagnostic kit, comprising the aforementioned hydrogen sulfide fluorescent probe and instructions for use, the instructions for use specifying the administration method of the probe and the fluorescence detection time.

[0014] The beneficial effects of this invention are: When H2S is present in the intestines, the fluorescent probe can specifically recognize and interact with H2S, triggering changes in intestinal fluorescence intensity. Detecting the level of hydrogen sulfide in the intestines by monitoring changes in fluorescence intensity provides an auxiliary reference for diagnosing Parkinson's disease. This invention enables early screening of the disease and facilitates early intervention. The fluorescent probe provided by this invention has advantages such as simple production process, high biosafety, high detection sensitivity, and strong specificity. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of fluorescent probe synthesis.

[0016] Figure 2 This is a scanning electron microscope image of a fluorescent probe.

[0017] Figure 3 This is the Fourier transform infrared spectrum of the fluorescent probe.

[0018] Figure 4 This is the UV-Vis spectrophotometric image of the fluorescent probe.

[0019] Figure 5 This is a bar chart showing the results of a cytotoxicity assay using a fluorescent probe.

[0020] Figure 6 These are in vivo imaging images of mice after the fluorescent probe has entered them. The left side represents the healthy group, and the right side represents the PD group.

[0021] Figure 7 It is the rate of change in fluorescence intensity after the fluorescent probes in Examples 1-6 are mixed with H2S.

[0022] Figure 8 It represents the fluorescence intensity after the fluorescent probe is mixed with different concentrations of H2S.

[0023] Figure 9 This is the result of a specificity test on the fluorescent probe. Detailed Implementation

[0024] The technical solution of the present invention will be further described in detail below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and not for limiting the scope of protection of the present invention. Various modifications or equivalent substitutions made by those skilled in the art based on the following embodiments should also be considered to fall within the scope of protection of the present invention. Experimental methods in the following embodiments that do not specify specific conditions are generally implemented according to conventional conditions or reference books. Materials in the embodiments that do not specify their source are all commonly used materials in the art and can be obtained commercially.

[0025] This invention provides a hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease. The probe is a product formed by the combination of chloroplatinic acid or its salt with bipyrazine or bipyridine or its derivatives via a Pt-N bond. In this invention, the chloroplatinic acid salt includes sodium chloroplatinate, ammonium chloroplatinate, potassium chloroplatinate, etc., and the bipyrazine (pyridine) derivative includes products derived from the conventional substitution of the pyrazine (pyridine) ring.

[0026] like Figure 1 As shown, chloroplatinic acid is bound to 2,2'-bipyrazine via a Pt-N bond to obtain a fluorescent probe. This fluorescent probe can bind to hydrogen sulfide in the intestine. Platinum ions, due to their unique d-orbital electron configuration and high electrophilicity, can undergo a highly selective coordination reaction with H₂S, thereby binding sulfur (S₂S) in H₂S. 2- or HS -It can coordinate with platinum ions through lone pair electrons to form stable platinum-sulfur bonds (Pt-S), thereby affecting the electronic coupling and energy level distribution of 2,2'-bipyrazine ligands, disrupting the vibrational degrees of freedom of the ligands, reducing intramolecular nonradiative transitions, and leading to enhanced fluorescence emission.

[0027] Example 1 The preparation method of hydrogen sulfide fluorescent probe is as follows: chloroplatinic acid and 2,2'-bipyrazine are dissolved in DMF, with a molar ratio of chloroplatinic acid to 2,2'-bipyrazine of 1:1. The concentration of both in DMF is 3 mg / mL. After sonication for 5 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 180℃ and the stirring speed is 600 rpm. After reacting for 60 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0028] Example 2 The preparation method of hydrogen sulfide fluorescent probe is as follows: chloroplatinic acid and 2,2'-bipyrazine are dissolved in DMF, with a molar ratio of chloroplatinic acid to 2,2'-bipyrazine of 1:1. The concentration of both in DMF is 5 mg / mL. After sonication for 3 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 165℃, and the stirring speed is 800 rpm. After reacting for 50 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0029] Example 3 The preparation method of hydrogen sulfide fluorescent probe is as follows: chloroplatinic acid and 2,2'-bipyrazine are dissolved in DMF, with a molar ratio of chloroplatinic acid to 2,2'-bipyrazine of 1:1. The concentration of both in DMF is 2 mg / mL. After sonication for 5 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 170℃ and the stirring speed is 500 rpm. After reacting for 80 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0030] Example 4 The preparation method of hydrogen sulfide fluorescent probe is as follows: chloroplatinic acid and 2,2'-bipyrazine are dissolved in DMF, with a molar ratio of chloroplatinic acid to 2,2'-bipyrazine of 1:1. The concentration of both in DMF is 4 mg / mL. After sonication for 6 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 185℃, and the stirring speed is 300 rpm. After reacting for 50 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0031] Example 5 The preparation method of hydrogen sulfide fluorescent probe is as follows: Ammonium chloroplatinate and 2,2'-bipyridine-4,4'-dicarboxylic acid are dissolved in DMF, with a molar ratio of 1:1 between ammonium chloroplatinate and 2,2'-bipyridine-4,4'-dicarboxylic acid. The concentration of both in DMF is 4 mg / mL. After sonication for 5 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 175℃ and the stirring speed is 400 rpm. After reacting for 30 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0032] Example 6 The preparation method of hydrogen sulfide fluorescent probe is as follows: Potassium chloroplatinate and 4,4'-dinitro-2,2'-bipyridine are dissolved in DMF, with a molar ratio of potassium chloroplatinate to 4,4'-dinitro-2,2'-bipyridine of 1:1. The concentration of both in DMF is 3 mg / mL. After sonication for 5 min, the mixture is placed in an oil bath and reacted under light-protected conditions. The reaction temperature is controlled at 160℃ and the stirring speed is 600 rpm. After reacting for 45 min, the mixture is centrifuged. The crystalline precipitate is washed three times each with deionized water and anhydrous ethanol, and finally freeze-dried in a vacuum freeze dryer.

[0033] Test case 1. Morphology and Characterization of Fluorescent Probes The probe prepared in Example 1 was characterized by scanning electron microscopy, transmission electron microscopy, ultraviolet-spectrophotometer, and Fourier transform infrared spectrometer.

[0034] Experimental results: From Figure 2 The scanning electron microscope image shown indicates that the particles have a diameter in the micrometer range and a cuboid shape. Figure 3 The Fourier infrared spectrum shown and Figure 4 As shown in the UV spectrophotometer, the fluorescent probe was successfully synthesized.

[0035] 2. Cytotoxicity assay of fluorescent probes Caco-2 cells were seeded into 96-well plates and cultured. Culture media containing different concentrations (0, 62.5, 125, 250, 500, 1000 μg / mL) of fluorescent probes were prepared. After Caco-2 cells adhered to the plates, the drug-containing culture medium was added to the 96-well plates and incubated for 24 h. The drug-containing culture medium was then discarded, and MTT solution was added and incubated for 4 h. The supernatant was then discarded, and DMSO was added to dissolve formazan. The absorbance was measured at 490 nm using a microplate reader to calculate the cell viability.

[0036] Experimental results: such as Figure 5As shown, the survival rate of Caco-2 cells decreased with increasing probe concentration, but the cell survival rate remained above 90% when the probe concentration was 1000 μg / mL. This indicates that the fluorescent probe has almost no toxicity to Caco-2 cells in the range of 0-1000 μg / mL and has good biosafety.

[0037] 3. Evaluation of the diagnostic efficacy of fluorescent probes In vivo imaging experiments were conducted on mice. Twenty male C57 mice weighing 20g were purchased. Ten mice were injected intraperitoneally with 30mg / kg MPTP for seven consecutive days to establish a PD model, while the other ten mice received no treatment. Both groups of mice were given normal food and water. Healthy mice and PD mice were each given 200μL of probe solution (10mg / kg) by gavage. In vivo imaging was performed at 3h, 6h, 9h, 12h, 24h, and 48h. The excitation wavelength was 543nm and the emission wavelength was 680nm. Changes in abdominal fluorescence intensity and differences in abdominal fluorescence intensity between healthy and PD mice were analyzed.

[0038] Experimental results: The fluorescence intensity reached its peak at about 12 hours after gavage and then decreased, indicating that the material was gradually excreted from the intestine after 12 hours. At each time point, the abdominal fluorescence of PD mice was significantly enhanced compared with that of healthy mice. Figure 6 These are representative in vivo images of the two groups of mice.

[0039] 4. Sensitivity of fluorescent probes for H2S detection The sensitivity of the fluorescent probes was characterized by the rate of change in fluorescence intensity after mixing the fluorescent probes synthesized in Examples 1-6 with H2S. 1 mg / mL of the fluorescent probes synthesized in Examples 1-6 was prepared, along with a 10 mmol / L H2S solution. 2 mL of the fluorescent probe was mixed thoroughly with 2 mL of ultrapure water and 2 mL of H2S solution, respectively. The fluorescence intensity was detected using a fluorescence spectrometer, and the rate of change in fluorescence intensity after mixing with H2S was calculated.

[0040] Result: As Figure 7 As shown, the fluorescence intensity change rates synthesized in Examples 1-6 were 56.55%, 46.94%, 48.01%, 49.07%, 31.44%, and 33.03%, respectively. Among them, the probe prepared in Example 1 had a larger fluorescence intensity change rate, indicating that it had higher detection sensitivity.

[0041] Next, H2S solutions of different concentrations were prepared, namely 0.625, 1.25, 2.5, 5, and 10 µmol / L. A 1 mg / L fluorescent probe synthesized in Example 1 was also prepared. 2 mL of the fluorescent probe solution was mixed with 2 mL of the above-mentioned H2S solutions of different concentrations and the fluorescence intensity was detected using a fluorescence spectrometer. 0 was the control group (2 mL of fluorescent probe was mixed with 2 mL of ultrapure water). The fluorescence intensity of each group was also detected.

[0042] Result: As Figure 8 As shown, when the H2S concentration ranges from 0.625 to 10 µmol / L, the fluorescence intensity of the fluorescent probe is significantly enhanced after mixing with it, and the detection limit of the probe is 0.625 µmol / L.

[0043] 5. Specificity of fluorescent probes for H2S detection To simulate the gastrointestinal environment, the specificity of the fluorescent probe was tested. 10 μmol / L solutions of H₂S, KCl, NaCl, glucose, cholesterol, hydrochloric acid, NaHCO₃, and mixtures thereof were prepared. Then, 1 mg / L of the fluorescent probe synthesized in Example 1 was prepared. 2 mL of the fluorescent probe was mixed thoroughly with 2 mL of each of the above solutions, and the fluorescence intensity was detected using a fluorescence spectrometer. Blank was used as the blank control. The fluorescence intensity of each group was analyzed.

[0044] Result: As Figure 9 As shown, compared with the blank group and K + Na + Compared with the groups containing glucose, cholesterol, hydrochloric acid, and sodium bicarbonate, the fluorescence intensity of the mixture group and the H2S group after mixing with the fluorescent probe was significantly enhanced, indicating that the fluorescent probe can eliminate interference from substances in vivo and has good specificity, making it suitable for in vivo detection of hydrogen sulfide.

Claims

1. A hydrogen sulfide fluorescent probe for diagnosing Parkinson's disease, characterized in that: The probe is formed by the combination of chloroplatinic acid or its salt with bipyrazine or bipyridine or its derivatives via a Pt-N bond; the chloroplatinic acid salt includes sodium chloroplatinate, ammonium chloroplatinate or potassium chloroplatinate; the derivative is a product derived from the conventional substitution of the pyrazine ring or pyridine ring.

2. The hydrogen sulfide fluorescent probe according to claim 1, characterized in that: The substituents include carboxyl, nitro, alkyl, or halogen groups.

3. A method for preparing the hydrogen sulfide fluorescent probe according to claim 1 or 2, characterized in that... Includes the following steps: Chloroplatinic acid or its salt and bipyrazine or bipyridine or its derivatives are dissolved in DMF at a 1:1 molar ratio, and the concentration of both is controlled at 2-5 mg / mL. After sonication, the mixture is reacted at 160-185℃ in the dark for 30-80 min, centrifuged, washed, and then freeze-dried.

4. The preparation method according to claim 3, characterized in that: The reaction is carried out under stirring conditions at a speed of 300-800 rpm.

5. The use of the hydrogen sulfide fluorescent probe according to claim 1 or 2 in the preparation of diagnostic reagents for Parkinson's disease.

6. The application according to claim 5, characterized in that: The reagent can determine the H2S content based on the fluorescence intensity in the intestine, thereby diagnosing whether a person has Parkinson's disease. The fluorescence excitation wavelength of the reagent is 543 nm, and the emission wavelength is 680 nm.

7. A Parkinson's disease diagnostic kit, comprising the hydrogen sulfide fluorescent probe as described in claim 1 or 2 and instructions for use, wherein the instructions specify the administration method of the probe and the fluorescence detection time.

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