Preparation method and application of a supramolecular fluorescent probe for detecting aliphatic biogenic amines in water

Abstract

The application discloses a preparation method and application of a supramolecular fluorescent probe for detecting aliphatic biological amine in water, wherein the supramolecular fluorescent probe is constructed by taking symmetrical tetramethyl six-membered cucurbituril (TMeQ[6]) as a host and 2-(2-pyridyl)benzimidazole as a guest, and the supramolecular fluorescent probe is used for recognizing aliphatic biological amine. The host-guest supramolecular fluorescent probe with a molar ratio of 1:1 is formed by TMeQ[6] and 2-(2-pyridyl)benzimidazole, and the specific recognition of aliphatic biological amine (putrescine, cadaverine, spermine and spermidine) is proved by ultraviolet-visible spectroscopy, fluorescence spectroscopy and nuclear magnetic titration under the condition of water solution medium, so that the supramolecular fluorescent probe can be applied to the detection of biological amine in water solution. The supramolecular fluorescent probe has the characteristics of low analysis cost, simple operation, high sensitivity and rapidness, strong anti-interference ability and the like.

Description

A method for preparing a supramolecular fluorescent probe for detecting aliphatic biogenic amines in water and its application. Technical Field

[0001] This invention belongs to the field of analytical chemistry technology, specifically relating to a method for preparing and applying a supramolecular fluorescent probe for detecting aliphatic biogenic amines in water. Background Technology

[0002] Biogenic amines (BAs) are a class of low-molecular-weight nitrogenous compounds formed by the decarboxylation of amino acids in organisms. They are ubiquitous in all cell types of plants and animals and contain two or more amino groups with highly effective biological activity. Based on their structure, biogenic amines can be divided into three parts: aliphatic amines such as putrescine, cadaverine, spermine, and spermidine; aromatic amines such as tyramine and phenylethylamine; and heterocyclic amines such as histamine and tryptamine. Food spoilage is a significant risk to human health. Putrescine and cadaverine are found in protein-rich foods such as fish, shrimp, meat, and cheese. Microbial contamination and improper food storage conditions can lead to the accumulation of cadaverine and putrescine, and their high concentrations can cause food spoilage and food poisoning. Therefore, BAs are considered biomarkers for assessing food quality and freshness.

[0003] Meanwhile, biogenic amines are normal active components in living organisms, playing important physiological roles in the human body. Appropriate amounts of biogenic amines are beneficial to human health, but excessive amounts can lead to poisoning, causing symptoms such as headaches, changes in blood pressure, respiratory disturbances, palpitations, and vomiting. In particular, high concentrations of spermine and spermidine in urine and plasma are associated with malignant tumors such as breast cancer, lung cancer, and pancreatic cancer. Elevated levels of spermine and spermidine in urine can serve as a tool for early diagnosis and assessment of cancer treatment effectiveness. Therefore, the quantification of these aliphatic biogenic amines can be a useful indicator for diagnosing various cancers.

[0004] Currently, analytical techniques for detecting biogenic amines include gas chromatography, high-performance liquid chromatography, capillary electrophoresis, colorimetric sensing, electrochemical sensors, and fluorescence spectroscopy. Among these, fluorescence-based optical sensors offer a viable option for detecting biogenic amines in low-concentration foods and for disease diagnosis due to their advantages such as portability, cost-effectiveness, ease of operation, and high sensitivity. Summary of the Invention

[0005] This invention provides a method for preparing a supramolecular fluorescent probe for detecting aliphatic biogenic amines in water and its application. The supramolecular fluorescent probe is prepared from a symmetrical tetramethyl hexamembered cucurbita ring and 2-(2-pyridyl)benzimidazole and can identify and detect common aliphatic biogenic amines.

[0006] The technical solution of the present invention is: a supramolecular fluorescent probe for detecting aliphatic biogenic amines in water, wherein the fluorescent probe is prepared by symmetrical tetramethyl hexamembered cucurbita and 2-(2-pyridyl)benzimidazole.

[0007] The fluorescent probe has the following structural formula:

[0008]

[0009] The method for preparing the fluorescent probe for detecting aliphatic biogenic amines in water involves preparing the fluorescent probe from an aqueous solution of 2-(2-pyridyl)benzimidazole and an aqueous solution of symmetrical tetramethylhexacyclic cucurbitacin.

[0010] The fluorescent probe is composed of an aqueous solution of 2-(2-pyridyl)benzimidazole and an aqueous solution of symmetrical tetramethyl hexa-membered cucurbitacinium in a molar ratio of 1:1.

[0011] The method for preparing the fluorescent probe for detecting aliphatic biogenic amines in water includes the following steps:

[0012] (1) Dissolve the symmetrical tetramethyl hexacyclic cucurbitacin in deionized water to prepare a solution with a concentration of 1×10⁻⁶. -4 Solution A is obtained by reacting a symmetrical tetramethyl hexacyclic cucurbitacinium ring M with an aqueous solution.

[0013] (2) Dissolve 2-(2-pyridyl)benzimidazole in deionized water to prepare a solution with a concentration of 1×10⁻⁶. -3 Solution B is obtained by dissolving M in an aqueous solution of 2-(2-pyridyl)benzimidazole.

[0014] (3) Mix solution A and solution B at a molar ratio of 1:1, dilute with deionized water, and obtain a concentration of 3 × 10⁻⁶. -5 M represents the host-guest supramolecular fluorescent probe.

[0015] The supramolecular fluorescent probe for detecting aliphatic biogenic amines in water is described in its application.

[0016] The supramolecular fluorescent probe is used for the detection of aliphatic biogenic amines in water as follows:

[0017] (1) Prepare a fluorescent probe standard solution, fix the excitation wavelength at 298 nm and measure the fluorescence emission spectrum, and plot the change curve of the excitation fluorescence intensity; (2) Add the aqueous solution of the sample to be tested to the fluorescent probe standard solution, let it stand for 10-20 min, fix the excitation wavelength at 298 nm and measure the fluorescence emission spectrum, and plot the change curve of the excitation fluorescence intensity; (3) According to the change curve of the fluorescence spectrum in steps (1) and (2), calculate the change value ΔI of the fluorescence emission spectrum intensity at 365 nm before and after the aqueous solution of the sample to be tested, so that the aliphatic biogenic amine in the aqueous solution of the sample to be tested can be detected.

[0018] The beneficial effects of the present invention are as follows: 1. The present invention provides a novel host-guest supramolecular fluorescent probe constructed from a symmetrical tetramethyl hexamembered cucurbit ring and 2-(2-pyridyl)benzimidazole, which can be used to quantitatively detect aliphatic biogenic amines in water based on changes in fluorescence intensity.

[0019] 2. The fluorescent probe detection method of the present invention has the advantages of high sensitivity, simple sample processing, convenient operation, rapid measurement and real-time detection.

[0020] 3. This invention investigates, through anti-interference experiments, whether the fluorescent probe can selectively detect aliphatic biogenic amines in the presence of 16 different amines. The 16 amines are: n-propylamine, tyramine, trimethylamine, spermidine, ethylamine, dimethylamine, putrescine, phenylethylamine, tryptamine, triethylamine, ammonia, cadaverine, histamine, spermine, hydroxylamine hydrochloride, and ethylamine. The results show that the supramolecular fluorescent probe of this invention exhibits strong anti-interference ability when detecting aliphatic biogenic amines. Attached Figure Description

[0021] Figure 1 shows the supramolecular fluorescent probe formed by the symmetrical tetramethyl hexamembered cucurbit ring and 2-(2-pyridyl)benzimidazole and its recognition of aliphatic biogenic amines;

[0022] Figure 2 shows the UV spectra of TMeQ[6] and 2-(2-pyridyl)benzimidazole;

[0023] Figure 3 shows the fluorescence spectra of TMeQ[6] and 2-(2-pyridyl)benzimidazole;

[0024] Figure 4 shows the NMR titration and reaction mode diagram of TMeQ[6] and 2-(2-pyridyl)benzimidazole; where: (a) pure TMeQ[6]; (b) 0.7 equivalents of 2-(2-pyridyl)benzimidazole; (c) 1.0 equivalents of 2-(2-pyridyl)benzimidazole; (d) 1.2 equivalents of 2-(2-pyridyl)benzimidazole; (e) pure 2-(2-pyridyl)benzimidazole;

[0025] Figure 5 shows the fluorescence spectra and detection limits when the fluorescent probe standard solution is added to spermine solution of different concentrations.

[0026] Figure 6 shows the fluorescence spectra and detection limits of the fluorescent probe standard solution when different concentrations of spermidine solution are added; Figure 7 shows the fluorescence spectra and detection limits of the fluorescent probe standard solution when different concentrations of putrescine solution are added.

[0027] Figure 8 shows the fluorescence spectra and detection limits of the fluorescent probe standard solution when different concentrations of cadaverine solution are added. Detailed Implementation

[0028] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0029] Embodiments of the present invention

[0030] Example 1:

[0031] 1. Preparation method of fluorescent probe:

[0032] (1) Accurately weigh 0.0105 g of symmetrical tetramethyl hexacyclic cucurbitacin, dissolve it in deionized water, and dilute to 100 mL in a volumetric flask to obtain a concentration of 1 × 10⁻⁶ g. -4 M's TMeQ[6] solution;

[0033] (2) Accurately weigh 0.0098 g of 2-(2-pyridyl)benzimidazole, dissolve it in deionized water, and dilute to a volumetric flask with a volume of 50 mL to obtain a concentration of 1×10⁻⁶. -3 M in a 2-(2-pyridyl)benzimidazole solution;

[0034] (3) Transfer the above 900uL (1×10) -4 M) TMeQ[6] solution and 90uL (1×10 -3 Mix the 2-(2-pyridyl)benzimidazole solution of M) in a 3 mL centrifuge tube, dilute to the mark, and shake well to obtain a concentration of 3 × 10⁻⁶. -5 M supramolecular fluorescent probe.

[0035] 2. The detection method for aliphatic biogenic amines in water is as follows:

[0036] (1) Take 3×10 -5 After placing the fluorescent probe M for 10 min, fluorescence emission spectra were measured under an excitation wavelength of 298 nm, and the change curve of the excitation fluorescence intensity was plotted.

[0037] (2) To 3×10 -5 Add the aqueous solution to be detected to the fluorescent probe M, let it stand for 10 min, and then perform fluorescence emission spectroscopy under the condition of excitation wavelength of 298 nm to plot the change curve of the excitation fluorescence intensity.

[0038] (3) Based on the fluorescence intensity change curves of steps (1) and (2), calculate the fluorescence emission spectrum intensity change value ΔI at 365nm before and after the detection of the aqueous solution, and then detect the aliphatic biogenic amine in the aqueous solution.

[0039] To verify the beneficial effects of this invention, the inventors conducted extensive experimental research, the process and results of which are as follows:

[0040] 1. Reagent materials: Symmetric tetramethyl hexamembered cucurbita; 2-(2-pyridyl)benzimidazole; amines (n-propylamine, tyramine, trimethylamine, spermidine, ethylamine, dimethylamine, putrescine, phenethylamine, tryptamine, triethylamine, ammonia, cadaverine, histamine, spermine, hydroxylamine hydrochloride, ethylamine); deionized water, deuterated water (D2O).

[0041] 2. Experimental Methods:

[0042] 2.1 Preparation method of fluorescent probe:

[0043] (1) Accurately weigh 0.0105 g of symmetrical tetramethyl hexacyclic cucurbitacin, dissolve it in deionized water, and dilute to 100 mL in a volumetric flask to obtain a concentration of 1 × 10⁻⁶ g. -4 M's TMeQ[6] solution;

[0044] (2) Accurately weigh 0.0098 g of 2-(2-pyridyl)benzimidazole, dissolve it in deionized water, and dilute to a volumetric flask with a volume of 50 mL to obtain a concentration of 1×10⁻⁶. -3 M in a 2-(2-pyridyl)benzimidazole solution;

[0045] (3) Transfer the above 900uL (1×10) -4 M) TMeQ[6] solution and 90uL (1×10 -3 Mix the 2-(2-pyridyl)benzimidazole solution of M) in a 3 mL centrifuge tube, dilute to the mark, and shake well to obtain a concentration of 3 × 10⁻⁶. -5 M supramolecular fluorescent probe.

[0046] 2.2 Preparation of amine standard solutions:

[0047] Accurately weigh the required analytical grade standards of 16 amines (n-propylamine, tyramine, trimethylamine, spermidine, ethylamine, dimethylamine, putrescine, phenylethylamine, tryptamine, triethylamine, ammonia, cadaverine, histamine, spermine, hydroxylamine hydrochloride, and ethylamine), dissolve them in a double-concentration aqueous solution to obtain a concentration of 1.00 × 10⁻⁶. -2 M is an amine standard solution.

[0048] 2.3 Determination of the standard curve:

[0049] (1) Determination of the standard curve: Take eight 10mL volumetric flasks, and add 300μL of a 1.0×10⁻⁶ solution to each flask. -3 M in a 2-(2-pyridyl)benzimidazole solution and 3000 μL of a 1.0 × 10 -4 Mix the M TMeQ[6] solution evenly to form a fluorescent probe, and then add 1.0×10 -2 0 μL, 3.0 μL, 6.0 μL, 9.0 μL, 12.0 μL, 15.0 μL, 18.0 μL, and 21.0 μL of aliphatic biogenic amine standard solutions M were prepared and diluted to volume with deionized water, then shaken well before use. Fluorescence emission spectroscopy was performed with a fixed excitation wavelength of 298 nm. A standard curve was plotted with the concentration of aliphatic biogenic amine on the x-axis and the difference between the fluorescence emission intensity of the probe at 365 nm (I0) and the fluorescence emission intensity (I) of different concentrations of aliphatic biogenic amine added (I0-I) on the y-axis. The detection limit (LOD) of the fluorescent probe for aliphatic biogenic amines was calculated using the slope s of the standard curve and the standard deviation σ of 11 blank measurements, with the formula LOD = 3σ / s.

[0050] 2.4 Sample Testing:

[0051] Take an aqueous solution containing an unknown concentration of aliphatic biogenic amines, and add the prepared 3×10 -5 If a standard solution of fluorescent probe M is added to the sample and its concentration is controlled to not exceed the linear range, and a change in fluorescence intensity is observed at 365 nm under an excitation wavelength of 298 nm, it indicates that the water sample contains aliphatic biogenic amines.

[0052] 3. Results:

[0053] The fluorescent probe was added with n-propylamine, tyramine, trimethylamine, spermidine, ethylamine, dimethylamine, putrescine, phenylethylamine, tryptamine, triethylamine, ammonia, cadaverine, histamine, spermine, hydroxylamine hydrochloride, and ethylamine (concentration 5 times that of the fluorescent probe). The emission spectrum of the solution was measured at an excitation wavelength of 298 nm. Only putrescine, cadaverine, spermine, and spermidine significantly reduced the fluorescence intensity of the probe solution at the maximum emission wavelength of 365 nm, as shown in Figure 1 and Table 1.

[0054] Table 1 shows the fluorescence intensity changes of the probe interacting with different amines at an emission wavelength of 365 nm.

[0055]

[0056]

[0057] 4. Conclusion:

[0058] The concentration obtained in this invention is 3.0 × 10⁻⁶.-5 Different volumes of aliphatic biogenic amine solution were added to the fluorescent probe M for detection. A standard curve can be constructed using this graph, and the detection results are shown in Figure 5-8. The spermine concentration ranges from (0.3 to 1.8) × 10⁻⁶. -5 The detection limit calculated within the M range is 4.54 × 10⁻⁶. -7 M, spermidine concentration in the range of (0.6–2.1) × 10 -5 The detection limit calculated within the M range is 4.74 × 10⁻⁶. -7 M, putrescine concentration in the range of (0.9–3.0) × 10 -5 The detection limit calculated within the M range is 7.17 × 10⁻⁶. -7 M, cadaverine concentration in the range of (0.3–2.4) × 10 -5 The detection limit calculated within the M range is 6.82 × 10⁻⁶. -7 M.

[0059] The above description is merely a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A supramolecular fluorescent probe for detecting aliphatic biogenic amines in water, characterized in that: The fluorescent probe is prepared from a symmetrical tetramethylhexamembered cucurbita ring and 2-(2-pyridyl)benzimidazole; the structural formula of the fluorescent probe is: 。 2. The method for preparing a fluorescent probe for detecting aliphatic biogenic amines in water as described in claim 1, characterized in that: The fluorescent probe was prepared from an aqueous solution of 2-(2-pyridyl)benzimidazole and an aqueous solution of a symmetrical tetramethylhexacyclic cucurbitacin.

3. The method for preparing a fluorescent probe for detecting aliphatic biogenic amines in water according to claim 2, characterized in that: The fluorescent probe is composed of an aqueous solution of 2-(2-pyridyl)benzimidazole and an aqueous solution of symmetrical tetramethyl hexa-membered cucurbitacinium in a molar ratio of 1:

1.

4. The method for preparing a fluorescent probe for detecting aliphatic biogenic amines in water according to claim 2, characterized in that: Includes the following steps: (1) Dissolve the symmetrical tetramethyl hexacyclic cucurbitacin in deionized water to prepare a solution with a concentration of 1×10⁻⁶. -4 (2) Take 2-(2-pyridyl)benzimidazole, dissolve it in deionized water, and prepare a solution with a concentration of 1×10 -3 (3) Mix solution A and solution B in a molar ratio of 1:1 and dilute with deionized water to obtain a concentration of 3×10⁻⁶. -5 M represents the host-guest supramolecular fluorescent probe.

5. The application of the supramolecular fluorescent probe for detecting aliphatic biogenic amines in water as described in claim 1.

6. The application according to claim 5, characterized in that: The method for detecting aliphatic biogenic amines in water using the supramolecular fluorescent probe is as follows: (1) Prepare a fluorescent probe standard solution, fix the excitation wavelength at 298 nm and measure the fluorescence emission spectrum, and plot the change curve of the excitation fluorescence intensity; (2) Add the aqueous solution of the sample to be tested to the fluorescent probe standard solution, let it stand for 10-20 min, fix the excitation wavelength at 298 nm and measure the fluorescence emission spectrum, and plot the change curve of the excitation fluorescence intensity; (3) Calculate the change value ΔI of the fluorescence emission spectrum intensity at 365 nm before and after the aqueous solution of the sample to be tested, based on the change curve of the fluorescence spectrum in steps (1) and (2), and then the aliphatic biogenic amine in the aqueous solution of the sample to be tested can be detected.

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