A symmetrical double Schiff base fluorescent probe and its preparation method and application

Abstract

The present invention relates to a symmetrical double Schiff base fluorescent probe and a preparation method and application thereof. The symmetrical double Schiff base fluorescent probe is prepared by adding anhydrous ethanol to thioformyl dihydrazide, dissolving the solution (solution A) by reflux under heating at 80°C, dissolving salicylaldehyde in an appropriate amount of anhydrous ethanol (solution B), dripping solution B into solution A, maintaining heating and reflux at 80°C and continuously stirring during the whole process, and obtaining a light yellow precipitate after reflux for 4-8 hours, decompressing and filtering the light yellow precipitate, washing the filter cake with anhydrous ethanol, and then filtering, repeating the above steps three times, and obtaining a light yellow solid, which is a thioformyl dihydrazide salicylaldehyde Schiff base ligand fluorescent probe. The fluorescent probe can specifically identify β-hydroxybutyrate (abbreviated as β-HB) under the interference of common ions, has strong anti-interference ability and high sensitivity, and can be used to detect β-HB in human body fluids (tears, tissue fluid, blood, urine or other body fluids).

Description

Technical Field

[0001] Symmetrical double Schiff base fluorescent probe can be used to detect the content of β-HB in human body fluids (tears, tissue fluid, blood, urine or other body fluids), which is of great significance for the early detection of diabetes. Background Art

[0002] As of 2022, the number of adult diabetes patients in the world will reach 537 million, and China ranks first in the number of diabetes patients, with 141 million patients, accounting for more than 26% of the total number of diabetes patients in the world. Diabetes can cause complications such as vascular and neurological lesions, which seriously threaten the life and health of patients. There are often no symptoms in the early stages of diabetes, and patients often find that they are already ill through physical examinations and blood sugar tests. Once they are ill, they have reached an irreversible stage and can only be treated for life. Studies have found that when the body lacks glucose or cannot use glucose normally, the decomposition of body fat increases, the fatty acid metabolism of the liver is correspondingly vigorous, and a large number of ketone bodies are produced, of which β-hydroxybutyrate (β-HB) accounts for about 78%, acetoacetate accounts for 20%, and acetone accounts for 2%. β-HB is the main ketone body and the most stable ketone body. In the early latent period of diabetes, there is no abnormality in blood sugar, but β-HB has obvious abnormal changes. Therefore, β-HB can be used as an important indicator for early detection of diabetes. The concentration of β-HB in a normal person is between 0.03-0.2 mmol / L, the concentration in people in the low-risk latent period is between 0.2-0.35 mmol / L, and the concentration in people in the high-risk latent period is between 0.35-0.5 mmol / L. If the concentration is higher than 0.5 mmol / L, it means the person has diabetes. Therefore, β-HB can be used as a marker for diabetes. Summary of the invention

[0003] In view of the shortcomings of existing market technologies, the present invention provides a symmetrical double Schiff base fluorescent probe and its preparation method and application, which can detect β-HB more efficiently and sensitively. The present invention uses thioformyl dihydrazide as a raw material to undergo a condensation reaction with salicylaldehyde, and connects salicylaldehyde to both ends of thiosemicarbazide to form a symmetrical double Schiff base fluorescent probe, which has symmetry and can be used to detect β-HB in human body fluids (tears, tissue fluid, blood, urine or other body fluids). Its structural formula is:

[0004]

[0005] The synthesis method of the symmetrical double Schiff base fluorescent probe (Probe) described in the invention comprises the following steps:

[0006] (1) Place thioformyl dihydrazide in a round-bottom flask and add anhydrous ethanol solution (heated at 80°C and refluxed) until thioformyl dihydrazide is completely dissolved. This is solution A.

[0007] (2) preparing corresponding salicylaldehyde and placing it in a beaker, and adding an appropriate amount of anhydrous ethanol to dissolve the salicylaldehyde into a homogeneous solution, which is referred to as solution B;

[0008] (3) adding solution B dropwise to solution A, heating and reflux at 80° C. with continuous stirring during the whole process, and obtaining a crude light yellow precipitate after reflux for 4-8 hours;

[0009] (4) The crude pale yellow precipitate is filtered under reduced pressure, the filter cake is placed in anhydrous ethanol for washing, and then filtered under reduced pressure. The above steps are repeated three times to obtain a pure pale yellow solid, which is the thioformyl dihydrazide salicylaldehyde Schiff base fluorescent probe.

[0010] The present invention provides a method for detecting β-HB in human body fluids (tears, tissue fluid, blood, urine or other body fluids) using a fluorescent probe. The symmetrical double Schiff base fluorescent probe of the present invention is prepared into a solution using DMF or DMSO, and the concentration is 5.0×10 -5 mol / L; as solution C, β-HB was prepared in deionized water with a concentration of 2.0×10 -2 mol / L; as solution D, mix solution D with solution C in a certain proportion, and use fluorescence spectroscopy to detect the mixed solution to compare whether the original solution has fluorescence.

[0011] The preparation method of the symmetrical double Schiff base fluorescent probe described in the present invention is extremely simple, the raw material cost is low, it can be synthesized in one step, the purity can reach 98%, and it can produce a fluorescent effect of specifically recognizing β-HB. It is more selective than ordinary unilateral fluorescent probes, has higher sensitivity and strong anti-interference ability.

[0012] In terms of its application, when β-HB is used as a marker for diabetes, the symmetrical double Schiff base fluorescent probe of the present invention can be used to qualitatively detect β-HB in human body fluids (tears, tissue fluid, blood, urine or other body fluids), which is of great significance for the early warning of diabetes. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 is the curve of the fluorescence value of the fluorescent probe probe changing with the concentration of β-HB;

[0014] Figure 2 It is the fluorescence emission spectrum of the fluorescent probe probe selectively recognizing β-HB in cations;

[0015] Figure 3 It is the fluorescence emission spectrum of the fluorescent probe probe selectively identifying β-HB in anions;

[0016] Figure 4 It is the fluorescence emission spectrum of the fluorescent probe probe selectively recognizing β-HB among amino acids;

[0017] Figure 5 It is the fluorescence emission spectrum of the fluorescent probe probe that selectively recognizes β-HB among biologically active molecules;

[0018] Figure 6 This is a standard curve diagram showing the change of fluorescence value of the fluorescent probe probe with the concentration of β-HB. Specific implementation plan

[0019] The present invention will be analyzed in more detail below in conjunction with the accompanying drawings and embodiments. Example 1

[0020] The present invention provides a symmetrical double Schiff base fluorescent probe (Probe), which is a thioformyl dihydrazide salicylaldehyde Schiff base ligand compound, and its structural formula is as follows:

[0021]

[0022] Thioformyl dihydrazide (0.106 g, 1 mmol / L) was heated and dissolved in 10 mL of anhydrous ethanol. 2 mL of salicylaldehyde (0.244 g, 2 mmol / L) solution was added dropwise under oil bath conditions (75-80°C). The whole process was carried out under magnetic stirring and refluxed for 4-8 hours to allow it to react fully. The reaction progress was monitored by TLC every hour until the reaction was complete and the reaction was stopped. The reaction solution was left overnight to produce a large amount of light yellow solid. The reaction solution was filtered under reduced pressure, the filter cake was taken out and washed with anhydrous ethanol, and the above operation was repeated 3-4 times to obtain a pure light yellow solid, which was a symmetrical double Schiff base fluorescent probe, with a yield of 89% and a melting point of 182-185°C. The reaction formula is as follows:

[0023] Example 2

[0024] The symmetrical double Schiff base fluorescent probe (Probe) described in the present invention can specifically recognize β-HB to produce a fluorescent effect. Figure 1 As shown, the detection method is that the above-mentioned symmetrical double Schiff base fluorescent probe is prepared into a solution with DMF at a concentration of 5.0×10 -5 mol / L; β-HB was prepared into solution with deionized water, the concentration was 2.0×10 -2 mol / L; using fluorescence spectroscopy, take 2mL of probe solution in a glass cuvette, add 10μL of quantitative β-HB solution each time, and detect 10 times in total. The results show that the fluorescence value of the fluorescent probe can increase with the increase of β-HB concentration. Example 3

[0025] The symmetrical double Schiff base fluorescent probe (Probe) described in the present invention can specifically recognize β-HB to produce a fluorescent effect. Figure 2 As shown, the detection method is that the above-mentioned symmetrical double Schiff base fluorescent probe is prepared into a solution with DMF at a concentration of 5.0×10 -5 mol / L; various metal cations were prepared into solutions with deionized water (all metal cations used were prepared from the corresponding chloride hydrates), namely Na + , K + , Li + , Ba 2+ 、Al 3+ 、Co 2+ Cr 3+ , Cu 2+ 、Ni 2+ , the concentration is 2.0×10 -2 mol / L; β-HB was prepared into solution with deionized water, the concentration was 2.0×10 -2 mol / L; using fluorescence spectroscopy, 2mL of the probe solution was taken into a glass cuvette, and 20 equivalents of metal cations and β-HB were added respectively. The results showed that under the common cation interference, the fluorescent probe could specifically identify β-HB without being interfered by the cation. Example 4

[0026] The symmetrical double Schiff base fluorescent probe (Probe) described in the present invention can specifically recognize β-HB to produce a fluorescent effect. Figure 3 As shown, the detection method is that the above-mentioned symmetrical double Schiff base fluorescent probe is prepared into a solution with DMF at a concentration of 5.0×10 -5 mol / L; various anions were prepared into solutions with deionized water (all anions were prepared from the corresponding sodium salt hydrates), namely Br - , Cl - 、H2PO4 - 、HSO3 - 、HSO4 - ,I - 、NO2 - 、NO3 - 、C2H2ClO2 - , the concentration is 2.0×10 -2 mol / L; β-HB was prepared into solution with deionized water, the concentration was 2.0×10 -2mol / L; using fluorescence spectroscopy, 2mL of the probe solution was taken into a glass cuvette, and 20 equivalents of anions and β-HB were added respectively. The results showed that under the interference of common anions, the fluorescent probe could specifically identify β-HB without being interfered by anions. Example 5

[0027] The symmetrical double Schiff base fluorescent probe (Probe) described in the present invention can specifically recognize β-HB to produce a fluorescent effect. Figure 4 As shown, the detection method is that the above-mentioned symmetrical double Schiff base fluorescent probe is prepared into a solution with DMF at a concentration of 5.0×10 -5 mol / L; various amino acids were prepared into solutions with deionized water, namely D-alanine, glucose, D-proline, glycine, L-proline, D-aspartic acid, D-glutamic acid, lactic acid, and pyruvic acid, with a concentration of 2.0×10 -2 mol / L; β-HB was prepared into solution with deionized water, the concentration was 2.0×10 -2 mol / L; using fluorescence spectroscopy, 2mL of the probe solution was taken into a glass cuvette, and 20 equivalents of amino acids and β-HB were added respectively. The results showed that under the interference of common amino acids, the fluorescent probe could specifically recognize β-HB without being interfered by amino acids. Example 6

[0028] The symmetrical double Schiff base fluorescent probe (Probe) described in the present invention can specifically recognize β-HB to produce a fluorescent effect. Figure 5 As shown, the detection method is that the above-mentioned symmetrical double Schiff base fluorescent probe is prepared into a solution with DMF at a concentration of 5.0×10 -5 mol / L; various bioactive molecules were prepared into solutions with deionized water, including α-ketoglutaric acid, fumaric acid, succinic acid, citric acid, and malic acid, with a concentration of 2.0×10 -2 mol / L; β-HB was prepared into solution with deionized water, the concentration was 2.0×10 -2 mol / L; using fluorescence spectroscopy, 2mL of the probe solution was taken into a glass cuvette and 20 equivalents of β-HB were added. The results showed that under the interference of biologically active molecules, the fluorescent probe could specifically identify β-HB without interference. Example 7

[0029] Fluorescence spectroscopy was used to draw the standard concentration curve of the probe and β-HB. Figure 6As shown, the minimum detection limit is 0.17 μmol / L. 200 urine samples from diabetic patients were obtained from the diabetes department of a tertiary hospital, recorded as sample A; 200 urine samples from healthy people without diabetes were obtained from a physical examination center, recorded as sample B. 50 μM probe solution was added to samples A and B respectively, and the fluorescence intensity of the samples was detected under a fluorescence spectrophotometer. According to the standard concentration curve of β-HB, the concentration of β-HB in the sample was obtained. A normal human body with a β-HB concentration higher than 0.5 mmol / L can basically be determined as ketoacidosis.

Claims

1. A symmetrical double Schiff base fluorescent probe and its application in the specific identification of β-hydroxybutyrate. The structural formula of the symmetrical fluorescent probe is as follows:

2. The use according to claim 1, characterized in that Selective recognition of β-hydroxybutyrate in cationic aqueous solutions.

3. The use according to claim 1, characterized in that Selective recognition of β-hydroxybutyrate in anionic aqueous solution.

4. The use according to claim 1, characterized in that Selective recognition of β-hydroxybutyrate in amino acid solution.

5. The use according to claim 1, characterized in that Selective recognition of β-hydroxybutyrate among biologically active molecules.

6. The use according to claim 1, characterized in that Used to detect β-hydroxybutyrate in human tears, tissue fluid, blood, urine or other body fluids.

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