Fluorescent probe for detecting polyamine compounds based on methyl cucurbituril [6] / AIE molecules as well as preparation and application of fluorescent probe

Abstract

The invention relates to the technical field of fluorescent probes, and provides a fluorescent probe for detecting polyamine compounds based on methyl cucurbit [6] uril / AIE molecules and preparation and application of the fluorescent probe. The fluorescent probe is prepared from methyl cucurbit [6] uril and aggregation-induced emission molecules according to the molar ratio of 1: 1; the preparation method comprises the following steps: dissolving methyl cucurbit [6] uril in distilled water to obtain a solution A; dissolving aggregation-induced emission molecules in dimethyl sulfoxide to obtain a solution B; mixing the solution A and the solution B, and diluting; the fluorescent probe can be used for detecting spermine and spermidine, and determination is carried out through fluorescence emission intensity changes. The detection limits are 5.781 * 10 <-6 > mol / L and 6.143 * 10 <-6 > mol / L. The fluorescent probe shows sensitivity in the detection process and is suitable for real-time detection in a solution.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of fluorescent probe, in particular to a fluorescent probe for detecting polyamine compounds based on methyl cucurbit[6]uril / AIE molecule and its preparation and application. BACKGROUND

[0002] Polyamine compounds contain two or more amino groups, and their synthesis is mainly derived from ornithine and arginine, involving key enzymes such as ornithine decarboxylase and arginine decarboxylase. Common such compounds include putrescine, spermidine and spermine, which play a role in promoting tissue growth and maintaining normal membrane function. Spermine and spermidine as polyamine compounds have functions in cell growth, division and metabolism, although there are differences in structure and function, but they are involved in anti-aging, metabolic regulation and disease prevention processes.

[0003] Detecting spermine and spermidine helps to understand their physiological roles. Fluorescent detection technology has the characteristics of high selectivity and sensitivity in the analysis field, and is convenient to operate and has a faster detection speed. Aggregation-induced emission molecules emit light in an enhanced manner in an aggregated state, and can be applied to the development of fluorescent detectors. In the prior art, the research on fluorescent probes for spermine or spermidine in solution still needs to be further developed to achieve higher sensitivity detection. SUMMARY

[0004] This section aims to summarize some aspects of the embodiments of the present application and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the abstract and title of the specification to avoid obscuring the purpose of this section, abstract and title, and such simplifications or omissions cannot be used to limit the scope of the present application.

[0005] In view of the problems in the prior art, the present application is proposed.

[0006] Therefore, the purpose of the present application is to overcome the deficiencies in the prior art, and to provide a fluorescent probe for detecting polyamine compounds based on methyl cucurbit[6]uril / AIE molecule and its preparation and application.

[0007] To solve the need for detecting polyamine compounds in the prior art, the present application provides a fluorescent probe based on methyl cucurbit[6]uril and aggregation-induced emission molecule, which is used for detecting spermine and spermidine.

[0008] The fluorescent probe is made of methyl cucurbit[6]uril (OMeCB[6]) and aggregation-induced emission molecule (TPE-Py), and the molecular formula of OMeCB[6] and TPE-Py is C 44 H 52 N 24 O 12 and C35 H 30 The structural formulas of OMeCB[6] and TPE-Py are as follows:

[0009]

[0010] The molar ratio of OMeCB[6] and TPE-Py in the fluorescent probe is 1:1, and the structural formula of the fluorescent probe is as follows:

[0011]

[0012] The preparation method of the fluorescent probe comprises the following steps:

[0013] (1) methyl calix[6] urea is taken and dissolved in distilled water to obtain solution A;

[0014] (2) the aggregation-induced emission molecule is taken and dissolved in dimethyl sulfoxide to obtain solution B;

[0015] (3) solution A and solution B are mixed, and then distilled water is added for dilution to obtain a fluorescent probe solution.

[0016] The concentration of methyl calix[6] urea in solution A is 2.0×10 -3 mol / L; and the concentration of the aggregation-induced emission molecule in solution B is 2.0×10 -3 mol / L.

[0017] In step (3), solution A and solution B are mixed in a molar ratio of 1:1 of methyl calix[6] urea to the aggregation-induced emission molecule, and react at room temperature for 1 min, and then distilled water is added for dilution to a total concentration of 2×10 -5 mol / L.

[0018] The application of the fluorescent probe in detecting spermine and spermidine comprises that the fluorescent probe solution is used for detecting spermine and spermidine.

[0019] The detection method of the fluorescent probe solution on spermidine is the same as the detection method of spermine, and the detection method of spermine comprises the following steps:

[0020] 1) different molar ratios of spermine standard solution are added to the fluorescent probe solution, and stirring is performed for 10 s;

[0021] 2) the fluorescence emission spectrum is determined at a fixed excitation wavelength of 393 nm, and the fluorescence emission spectrum of the probe solution with different molar ratios of spermine is determined;

[0022] 3) the change characteristics of the fluorescence emission intensity are used to detect spermine.

[0023] The concentration of the spermine standard solution is 2×10 -2The detection limit of the fluorescent probe for spermine is 5.781*10 - 6 The concentration of the spermidine standard solution is 2*10 -2 The detection limit of the fluorescent probe for spermidine is 6.143*10 -6 mol / L.

[0024] The detection limit of spermidine is calculated in the same way as that of spermine, and the detection limit of spermine is calculated in the following steps:

[0025] 1) Add spermine standard solutions with different concentrations to the fluorescent probe solution, and stir for 10 seconds;

[0026] 2) Determine the fluorescence emission spectrum of the probe solution with different concentrations of spermine at a fixed excitation wavelength of 393 nm;

[0027] 3) Take the fluorescence emission intensity at 560 nm to calculate the detection limit.

[0028] The fluorescent probe provided by the application can be used for the detection of spermine or spermidine in solution, has high sensitivity, is convenient to operate, has low cost, and has high detection efficiency, and the like. Methyl cucurbit[6]uril and an aggregation-induced emission molecule form a supramolecular complex, when spermine or spermidine is added, these compounds are combined with methyl cucurbit[6]uril in the probe to form a new complex, the aggregation-induced emission molecule is separated, the fluorescence is weakened, and thus real-time detection of spermine or spermidine in solution is realized. Compared with high-performance liquid chromatography and gas chromatography, the detection cost of the application is lower, the operation is more convenient, rapid determination and real-time detection can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the embodiments of the application, the drawings needed in the embodiment description will be briefly introduced. Obviously, the drawings in the following description are only some embodiments of the application, and other drawings can be obtained by those skilled in the art without creative labor. Among them:

[0030] Figure 1 It is the ultraviolet absorption spectrum obtained by the mole ratio method.

[0031] Figure 2 It is the ultraviolet absorption spectrum obtained by the equimolar continuous transformation method.

[0032] Figure 3 It is the fluorescence spectrum of the spermine standard solution with different mole ratios.

[0033] Figure 4The fluorescence spectrum of the spermidine standard solution with different molar ratios is determined.

[0034] Figure 5 The detection limit of the fluorescence probe solution for spermine is determined.

[0035] Figure 6 The detection limit of the fluorescence probe solution for spermidine is determined. DETAILED DESCRIPTION

[0036] In order to make the above objectives, characteristics and advantages of the present application more obvious and easy to understand, the specific embodiments of the present application will be described in detail below with reference to the embodiments of the present application.

[0037] In the following description, a large number of specific details are set forth in order to facilitate a thorough understanding of the present application, but the present application can also be implemented in other ways different from the description, and those skilled in the art can make similar generalizations without departing from the concept of the present application, so the present application is not limited to the specific embodiments disclosed below.

[0038] Secondly, the "one embodiment" or "embodiment" referred to herein means that the specific features, structures or characteristics can be included in at least one implementation of the present application. "In one embodiment" appearing in different places in the specification does not mean the same embodiment, nor is it an embodiment that is independent of or mutually exclusive with other embodiments.

[0039] In the embodiments, methyl cucurbit[6]uril is abbreviated as OMeCB[6], and the aggregation-induced emission molecule is abbreviated as TPE-Py. The OMeCB[6] solution is prepared by using distilled water, and the TPE-Py solution is prepared by using dimethyl sulfoxide.

[0040] Embodiment 1

[0041] The present embodiment provides a fluorescence probe for detecting polyamine compounds based on methyl cucurbit[6]uril / AIE molecules, and preparation and application thereof.

[0042] Specifically, the molar ratio of the fluorescence probe formed by OMeCB[6] and TPE-Py is explored, and the preparation method of the fluorescence probe includes the following steps:

[0043] (1) OMeCB[6] is dissolved in distilled water to obtain solution A, wherein the concentration of OMeCB[6] is 2.0×10 -3 mol / L;

[0044] (2) TPE-Py is dissolved in dimethyl sulfoxide to obtain solution B, wherein the concentration of TPE-Py is 2.0×10 -3 mol / L;

[0045] (3) Solution A and Solution B were mixed according to the OMeCB[6] and TPE-Py molar ratio 1:1, reacted at room temperature for 1 min, and diluted with distilled water to a total concentration of 2×10 -5 mol / L to obtain a fluorescent probe solution.

[0046] To determine the interaction between OMeCB[6] and TPE-Py, UV absorption spectrum was used for detection:

[0047] First, the molar ratio method was used: the TPE-Py concentration was fixed at 2×10 -5 mol / L, the OMeCB[6] concentration was gradually changed, and solutions with [OMeCB[6]] / [TPE-Py] molar ratios of 0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2, 1.4, 1.6, 1.8, and 2.0 were formed, and the UV-visible absorption spectrum was measured at room temperature.

[0048] As shown in Figure 1 , with the increase of OMeCB[6] concentration, the absorption peak changed, indicating that a complex was formed between the two, and the equilibrium was reached at a molar ratio of 1:1.

[0049] Second, the equimolar continuous transformation method was used: the total concentration was fixed at 2×10 -5 mol / L, and the ratio of OMeCB[6] to TPE-Py was changed to 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, and 0.9, and the UV-visible absorption spectrum was measured.

[0050] As shown in Figure 2 , the Job's curve peaked at 0.5, confirming a molar ratio of 1:1.

[0051] Through these determinations, the fluorescent probe formed a stable complex at a molar ratio of 1:1, which helped to improve the stability of subsequent detection applications.

[0052] Further analysis showed that this complex remained uniformly distributed in solution, facilitating fluorescence measurement.

[0053] Compared with other ratios, the absorption characteristics of the complex at a molar ratio of 1:1 were more consistent, which helped to improve the repeatability of the detection. In practical applications, this ratio can ensure that the performance of the probe is similar when prepared in different batches.

[0054] Example 2

[0055] This example provides a fluorescent probe for detecting polyamine compounds based on methyl cucurbit[6]uril / AIE molecules, and its preparation and application, specifically for detecting spermine.

[0056] Specifically, the fluorescent probe solution prepared in Example 1 was used, and the total concentration was 2 x 10 -5 mol / L. Different molar ratios of spermine standard solution (spermine to probe molar ratio was 0, 0.5, 1, 1.5, 2) were added, stirred for 10 s, and the fluorescence emission spectrum was measured at an excitation wavelength of 393 nm, and the range of 400-700 nm was collected.

[0057] As shown in Figure 3 , with the increase of the molar ratio of spermine, the fluorescence intensity decreased, and the intensity at 560 nm was the strongest. The detection method: adding the test solution or spermine standard solution to the probe solution, stirring for 10 s; measuring the fluorescence intensity at 560 nm with 393 nm excitation; judging the presence of spermine according to the intensity change.

[0058] Analysis showed that spermine was complexed with OMeCB[6], which caused TPE-Py to detach, and the fluorescence decreased.

[0059] This mechanism shows a linear response at different molar ratios, which is convenient for quantitative analysis. Compared with the blank group without adding spermine, the fluorescence change after adding can be used to distinguish low concentration samples.

[0060] In repeated experiments, this method showed consistency and was suitable for solution detection. Further investigation showed that stirring for 10 s was sufficient to balance the reaction and avoid long-term exposure affecting stability.

[0061] Comparative Example 1

[0062] A single TPE-Py solution (concentration 2 x 10 -5 mol / L without OMeCB[6]) was used, and the same molar ratio of spermine was added, and the fluorescence was measured.

[0063] The results showed that there was no obvious change in fluorescence intensity, and spermine could not be detected. This comparative example illustrates the necessity of complex formation for detection.

[0064] Example 3

[0065] This example provides a fluorescent probe for detecting polyamine compounds based on methyl calix[6] urea / AIE molecules, and its preparation and application, specifically for the detection of spermidine.

[0066] Specifically, the fluorescent probe solution prepared in Example 1 was used, and the total concentration was 2 x 10 -5 mol / L. Different molar ratios of spermine standard solution (spermine to probe molar ratio was 0, 0.5, 1, 1.5, 2) were added, stirred for 10 s, and the fluorescence emission spectrum was measured at an excitation wavelength of 393 nm, and the range of 400-700 nm was collected.

[0067] As shown in Figure 4As shown, the fluorescence intensity decreases with increasing spermidine molar ratio, reaching its strongest intensity at 560 nm. Detection method: Add spermidine standard solution to the probe solution and stir for 10 s; excite at 393 nm and measure the fluorescence intensity at 560 nm; determine the presence of spermidine based on the intensity change.

[0068] Analysis showed that spermidine complexes with OMeCB[6] in a similar manner to spermine, resulting in decreased fluorescence. The response was linear within the molar ratio range, facilitating quantification.

[0069] Repeated assays confirmed the reliability of the method, making it suitable for real-time monitoring. Further observations showed that the fluorescence changed rapidly after the addition of spermidine and stabilized after 10 seconds of stirring, which is helpful for rapid detection.

[0070] Comparative Example 2

[0071] Using a single OMeCB[6] solution (concentration 2×10) -5 mol / L (without TPE-Py), add the same molar amount of spermine and measure fluorescence.

[0072] The result showed no fluorescence signal and could not be detected, highlighting the role of the AIE molecule in this comparison.

[0073] Example 4

[0074] Specifically, this involves the determination of the detection limit of spermine.

[0075] The fluorescent probe solution prepared in Example 1 had a total concentration of 2 × 10⁻⁶. -5 Add different concentrations of spermine standard solution (0, 0.18, 0.9, 1.8, 2.4, 3.3, 4.5, 5.7 μmol / L), stir for 10 s, excite at 393 nm, and measure the fluorescence intensity at 560 nm.

[0076] like Figure 5 As shown, the detection limit is calculated to be 5.781 × 10⁻⁶ based on the slope and standard deviation of the fitted curve. -6 mol / L.

[0077] Analysis showed that the detection limit was effective in the low concentration range, and the curve fitting showed good linearity (R²). 2 >0.95).

[0078] In actual samples, this limit can detect trace amounts of spermine. The experiment was repeated three times, and the deviation was less than 5%, confirming its reliability.

[0079] Further discussion showed that selecting a 560nm wavelength optimized the signal-to-noise ratio and improved measurement accuracy.

[0080] Comparative Example 3

[0081] The same detection was performed using OMeCB[6] and TPE-Py complex with a molar ratio of 2:1, and the detection limit was raised to about 1 x 10 -5 mol / L.

[0082] This comparative example illustrates the superiority of the 1:1 ratio.

[0083] Example 5

[0084] Detection limit determination of spermidine.

[0085] Using the fluorescent probe solution prepared in Example 1, the total concentration was 2 x 10 -5 mol / L, different concentrations of spermidine standard solution (concentration 0, 1.2, 2.7, 4.2, 5.7 μmol / L) were added, stirred for 10 s, excited at 393 nm, and the fluorescence intensity at 560 nm was determined.

[0086] As Figure 6 shown, the detection limit was calculated to be 6.143 x 10 -6 mol / L according to the slope of the fitted curve and the standard deviation.

[0087] Analysis shows that this detection limit is suitable for low concentration spermidine, the linear range is wide, and repeated experiments show consistency and small deviation.

[0088] Further investigation shows that the selection of concentration gradient ensures the accurate fitting of the curve, which is helpful for quantitative application in practice.

[0089] Comparative Example 4

[0090] The same detection was performed using OMeCB[6] and TPE-Py complex with a molar ratio of 1:2, and the detection limit was raised. This comparative example highlights the importance of the 1:1 ratio.

[0091] Example 6

[0092] Stability test of fluorescent probe.

[0093] Using the fluorescent probe solution prepared in Example 1, the total concentration was 2 x 10

[0094] The results show that the intensity change is less than 3%, indicating that the solution is stable.

[0095] Analysis shows that this stability is due to the supramolecular structure of the complex, which is convenient for long-term storage, and the performance after storage is similar to that of freshly prepared solution, which is helpful for the convenience in actual detection. Further tests show that the stability remains good under pH 7 conditions.

[0096] Example 7

[0097] Detection of mixed samples by the fluorescent probe.

[0098] The fluorescence intensity of the solution of Example 1 was measured after the addition of spermine and spermidine (1:1 molar ratio) and stirring for 10 s. The decrease in fluorescence intensity was additive, indicating no interference.

[0099] Analysis showed that the probe responded to polyamine compounds, but could be distinguished by a standard curve, and repeated measurements confirmed no cross-influence, which was helpful for the detection of complex samples.

[0100] Comparative Example 5

[0101] The use of non-AIE molecules instead of TPE-Py complexes to detect mixed samples showed no fluorescence change, indicating the necessity of AIE characteristics.

[0102] Example 8

[0103] Preparation batch repeatability of the fluorescent probe.

[0104] The preparation process of Example 1 was repeated three times, and the fluorescence response of each batch to spermine (1:1 molar ratio) was measured. The intensity deviation was less than 4%.

[0105] Analysis showed that the preparation method was simple and repeatable, and the concentration control of solutions A and B ensured consistency, which was convenient for scaling up. Further investigation of the effect of dilution step on uniformity confirmed that 1 min of reaction was sufficient.

[0106] Example 9

[0107] Detection of the fluorescent probe at different temperatures.

[0108] The probe of Example 1 was used to detect spermine (1:1 molar ratio) at 20℃, 25℃, and 30℃. The fluorescence decrease was similar, and the temperature had little effect.

[0109] Analysis showed that the probe was stable within the room temperature range, which was convenient for laboratory application. Temperature changes did not affect the complexation mechanism.

[0110] Comparative Example 6

[0111] Testing at 40℃ showed an increase in fluorescence baseline, which suggested room temperature operation.

[0112] Example 10

[0113] Response of the fluorescent probe to other polyamines.

[0114] The addition of putrescine (1:1 molar ratio) to the probe of Example 1 showed a smaller decrease in fluorescence, indicating selectivity.

[0115] The analysis shows that the response to spermine and spermidine is strong, and the response to other polyamines is weak, which helps specific detection. Further comparison shows that structural similarity affects the response strength.

[0116] It should be noted that the above examples are only used to illustrate the technical solutions of the present application but not limit the present application. Although the present application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present application can be modified or equivalently replaced without departing from the spirit and scope of the present application, and they should be covered in the scope of the claims of the present application.

Claims

1. A fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules, characterized in that: The fluorescent probe is made of methylcucurbita[6]urea (OMeCB[6]) and aggregation-induced emission molecule (TPE-Py), with the molecular formulas of OMeCB[6] and TPE-Py being C 44 H 52 N24O 12 and C 35 H 30 BrN, with the following structural formula:

2. The fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 1, characterized in that: The molar ratio of OMeCB[6] to TPE-Py in the fluorescent probe is 1:1, and the structural formula of the fluorescent probe is:

3. The fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 2, characterized in that: The preparation method of this fluorescent probe includes the following steps: (1) Dissolve methylcucurbita[6]urea in distilled water to obtain solution A; (2) Dissolve the aggregation-induced emission molecules in dimethyl sulfoxide to obtain solution B; (3) Mix solution A and solution B and dilute with distilled water to obtain fluorescent probe solution.

4. The fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 3, characterized in that: In step (1), the concentration of methylcucurbita[6]urea in solution A is 2.0 × 10⁻⁶. -3 mol / L; the concentration of aggregation-induced luminescence molecules in solution B in step (2) is 2.0 × 10⁻⁶ mol / L. -3 mol / L.

5. A fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 3, characterized in that: In step (3), solutions A and B are mixed at a molar ratio of methylcucurbita[6]urea to aggregation-induced emission molecules of 1:1, reacted at room temperature for 1 min, and then diluted with distilled water to a total concentration of 2×10⁻⁶. -5 mol / L.

6. The fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 2, characterized in that: This fluorescent probe is used to detect spermine and spermidine.

7. The application of a fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 6, characterized in that: The detection method for spermidine using fluorescent probe solution is the same as that for spermine; the spermine detection method includes the following steps: 1) Add spermine standard solutions of different molar ratios to the fluorescent probe solution and let it stand and stir for 10 seconds; 2) Fluorescence emission spectra were measured using a fixed excitation wavelength of 393 nm, and the fluorescence emission spectra of probe solutions with different molar ratios of spermine were measured respectively. 3) Detect spermine based on the changes in fluorescence emission intensity.

8. The application of a fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 6, characterized in that: The concentration of the spermine standard solution is 2 × 10⁻⁶. -2 The detection limit for spermine by the fluorescent probe is 5.781 × 10⁻⁶ mol / L. -6 mol / L; the concentration of spermidine standard solution is 2×10⁻⁶ mol / L. -2 mol / L; the detection limit for spermidine is 6.143 × 10⁻⁶ mol / L. -6 mol / L.

9. The application of a fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in claim 7, characterized in that: The method for calculating the detection limit of spermidine is the same as that for spermine. The method for calculating the detection limit of spermine includes the following steps: 1) Add spermine standard solutions of different concentrations to the fluorescent probe solution and let it stand and stir for 10 seconds; 2) Fluorescence emission spectra were measured using a fixed excitation wavelength of 393 nm. The fluorescence emission spectra of probe solutions with different concentrations of spermine were measured respectively. 3) The detection limit is calculated by fitting the fluorescence emission intensity at 560 nm.

10. A fluorescent probe for detecting polyamine compounds based on methylcucurbita[6]urea / AIE molecules as described in any one of claims 1-9, characterized in that: The fluorescent probe is formed through a supramolecular complex. When spermine or spermidine is added, it complexes with methylcucurbita[6]urea, causing the aggregation-induced luminescent molecules to detach, resulting in reduced fluorescence and thus enabling detection.

Images