Supramolecular fluorescent probe as well as preparation method and application thereof

Abstract

The invention discloses a supramolecular fluorescent probe and a preparation method and application thereof, preparation raw materials comprise pyrene pyridinium salt derivatives and cucurbit [7] uril, an assembly is formed by electrostatic interaction of the pyrene pyridinium salt derivatives and macrocyclic cucurbit [7] uril, and visual detection is carried out by utilizing fluorescence color change generated by damage of heparin to the assembly. The system can directly identify the color change through naked eyes under the high concentration of heparin, and shows a good linear relationship under the low concentration. The probe has high sensitivity, can provide visual color change at 0.1 mu g / mL, and can be evaluated under the condition of no instrument. The supermolecular fluorescent probe disclosed by the invention is simple in synthesis route, can realize visual recognition of heparin response, has remarkable advantages compared with a traditional fluorescent probe, and has a wide biological application prospect.

Description

Technical Field

[0001] This invention relates to the field of testing or analyzing materials, specifically to a supramolecular fluorescent probe, its preparation method, and its application. Background Technology

[0002] Heparin is a naturally occurring, highly sulfated glycosaminoglycan with potent anticoagulant properties, rapidly inhibiting thrombus formation. Traditional heparin detection methods are based on coagulation principles; however, these tests are slow, relatively insensitive, lack specificity, and rely on expensive instruments in clinical settings. Currently, colorimetric methods relying on the colorimetric reaction between the analyte and test reagents have been developed; however, their sensitivity is limited, reproducibility is poor, and they are susceptible to cross-reactivity and matrix interference. Therefore, developing a rapid, accurate, and low-concentration heparin detection method is crucial.

[0003] Chinese invention patent CN110963911B discloses an AIE fluorescent probe for heparin detection and pH response, its synthesis method, and its application. The synthesized fluorescent probe BDA-4COOH exhibits strong binding ability to protamine under certain conditions. Heparin can antagonize protamine, thus achieving a heterogeneous response to heparin. However, this method requires protamine binding, resulting in high detection costs. Chinese invention patent CN115825019B discloses a method and application for heparin sodium detection based on a water-soluble cationic AIE fluorescent molecule. By synthesizing the fluorescent probe TPA-3Py, the quenching phenomenon caused by aggregation is overcome, exhibiting a significant AIE effect. This method provides high sensitivity, high fluorescence intensity, rapid response, and stable signal for heparin sodium detection. However, it has a high detection limit for heparin. Summary of the Invention

[0004] In order to develop a detection method that can quickly and accurately detect and achieve low concentrations of heparin, the first aspect of the present invention provides a supramolecular fluorescent probe, the raw materials of which include pyrene pyridinium salt derivatives and cucurbita[7]urea.

[0005] This invention utilizes a supramolecular fluorescent probe, synthesized from a pyrene-based pyridinium salt derivative and cucurbita[7]urea, to detect heparin. The addition of heparin disrupts this assembly structure, causing fluorescence to change with concentration. This invention enables quantitative detection of heparin over a very wide concentration range using colorimetry and fluorescence methods. At high concentrations of heparin, color changes can be directly identified by the naked eye, while at low concentrations, it shows good linear dependence. The probe has high sensitivity, with a calculated detection limit (LOD) of 0.1 μg / mL, and provides visual color changes that can be evaluated without instruments.

[0006] As one embodiment, the molar ratio of the pyrene-pyridinium salt derivative to cucurbit[7]urea is (1-3):(1-3).

[0007] As one embodiment, the molar ratio of the pyrene-based pyridinium salt derivative to cucurbit[7]urea is (1-2):(1-2).

[0008] In one embodiment, the molar ratio of the pyrene-based pyridinium salt derivative to cucurbit[7]urea is 1:1.

[0009] As one embodiment, the structural formula of the pyrene-based pyridinium salt derivative is as follows (I): In formula (Ⅰ), R includes one of -CH3O, -CHO, -COOCH3, -COCH3, -CH(CH3)2, -C(CH3)3, -COOH, -SO3H, -CH3, -CHF3O, -CH2OH or -CH2CH3.

[0010] In one embodiment, the pyrene pyridinium salt derivative includes at least one of PPOB (methoxypyrene pyridinium salt), PPFB (aldehyde pyrene pyridinium salt), PPMB (methyl pyrene pyridinium salt), PPCB (acetyl pyrene pyridinium salt), PPIB (isopropyl pyrene pyridinium salt), PPTB (tert-butyl pyrene pyridinium salt), PPCBB (carboxyl pyrene pyridinium salt), PPSB (sulfonic acid pyrene pyridinium salt), PPMTB (methyl pyrene pyridinium salt), PPTMB (trifluoromethoxypyrene pyridinium salt), PPBAB (hydroxymethyl pyrene pyridinium salt), or PPEB (ethyl pyrene pyridinium salt).

[0011] As one embodiment, the chemical structural formula of the pyrene-based pyridinium salt derivative is shown in Table 1 below: Table 1

[0012] As one embodiment, the preparation method of the pyrene-based pyridinium salt derivative includes the following steps: In an inert gas atmosphere, tetrakis(triphenylphosphine)palladium, 4-pyridylboronic acid, 1-bromopyrene and potassium carbonate were mixed, dissolved and stirred at 90-110°C for 20-25 h. After cooling to room temperature, the mixture was washed with water, extracted and dried to obtain intermediate I. Intermediate I and dinitrochlorobenzene were dissolved in acetone and heated under reflux in an inert atmosphere. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, washed with hexane, and dried to obtain intermediate II. Intermediate II and the aniline derivative were dissolved in an alcohol solvent, heated under reflux in an inert gas atmosphere, and the solvent was evaporated under reduced pressure to obtain a solution. The solution was then added dropwise to diethyl ether, filtered, and dried to obtain a pyrene-based pyridinium salt derivative.

[0013] The second aspect of the present invention provides a method for preparing a supramolecular fluorescent probe, comprising the following steps: forming a supramolecular assembly by electrostatic interaction between a pyrene-based pyridinium salt derivative and cucurbit[7]urea to obtain a supramolecular fluorescent probe.

[0014] As one embodiment, the preparation method of the supramolecular fluorescent probe includes the following steps: dissolving a pyrene-based pyridinium salt derivative in water to form a 10 -5 A solution of M's pyrene pyridinium salt derivatives, in which cucurbita[7]urea is dissolved in water to form 10 -5 The cucurbit[7]urea solution of M was mixed with the pyrene pyridinium salt derivative solution and the cucurbit[7]urea solution to form a supramolecular assembly, thus obtaining a supramolecular fluorescent probe.

[0015] A third aspect of the present invention provides an application of a supramolecular fluorescent probe for the detection of heparin.

[0016] As one implementation method, the method applied to heparin detection includes the following steps: The supramolecular fluorescent probe was prepared into a standard solution of the supramolecular fluorescent probe; Different concentrations of heparin aqueous solutions were added to the standard solution of the supramolecular fluorescent probe, and ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy were performed. A concentration-dependent standard curve was plotted based on the maximum absorption wavelength and the change in fluorescence intensity before and after assembly to show the responsiveness of the supramolecular fluorescent probe to the fluorescence change, thereby enabling the concentration detection of the heparin solution to be tested.

[0017] In one implementation, the maximum absorption wavelength is 400 nm.

[0018] As one implementation method, the detection limit of the heparin detection method is 0.1 μg / mL.

[0019] Compared with the prior art, the present invention has the following beneficial effects: (1) The supramolecular fluorescent probe of the present invention uses pyrene-based pyridinium salt derivatives and cucurbit[7]urea as raw materials. The addition of heparin will destroy the electrostatic adsorption of the supramolecular fluorescent probe, which will lead to changes in fluorescence intensity and color, thereby achieving high sensitivity and specificity detection of heparin.

[0020] (2) The supramolecular fluorescent probe of the present invention uses a pyrene-based pyridinium salt derivative and cucurbit[7]urea in a 1:1 molar ratio to form a stable supramolecular structure, produce specific fluorescence characteristics, and show obvious changes from yellow to green fluorescence. It can be directly identified by the naked eye and evaluated without instruments.

[0021] (3) The supramolecular fluorescent probe described in this invention is applied to heparin detection. In the concentration range of 0.1 μg / mL to 45 μg / mL, the fluorescence intensity is linearly related to the heparin concentration, which can achieve high sensitivity detection of heparin and is suitable for accurate detection of trace to high concentration heparin.

[0022] (4) The supramolecular fluorescent probe described in this invention is applied to heparin detection. Compared with traditional covalent fluorescent probes, the supramolecular fluorescent probe based on non-covalent interaction has the significant advantages of reversible binding and real-time dynamic monitoring of analyte concentration.

[0023] (5) The supramolecular fluorescent probe described in this invention is applied to heparin detection without the need for washing or incubation steps, and can achieve rapid mixed reading detection. It has the advantages of high sensitivity, real-time performance and convenience, and can be used for real-time monitoring. Attached Figure Description

[0024] Figure 1 This is a diagram illustrating the experimental mechanism for synthesizing pyrene-based pyridinium salt derivatives according to the present invention. Figure 2 The following is a graph showing the detection results of ultraviolet-visible absorption light during the synthesis of the supramolecular fluorescent probe of the present invention: the large graph shows the ultraviolet-visible absorption spectrum of pyrene-based pyridinium salt derivatives assembled with different concentrations of cucurbit[7]urea, and the small graph shows the linear fitting graph of different concentrations of cucurbit[7]urea and absorbance values. Figure 3 The following is a graph showing the fluorescence detection results during the synthesis of the supramolecular fluorescent probe of the present invention: the large graph shows the steady-state intensity spectrum of fluorescence detection of pyrene-based pyridinium salt derivatives assembled with different concentrations of cucurbit[7]urea, and the small graph shows the linear fitting graph of different concentrations of cucurbit[7]urea and fluorescence intensity. Figure 4 The following is a diagram of the detection results during the synthesis of the supramolecular fluorescent probe of the present invention: The large diagram is a CIE coordinate diagram of the fluorescence detection results of the assembly of pyrene-based pyridinium salt derivatives with different concentrations of cucurbita[7]urea, and the small diagram is a photograph of the cuvette, showing yellow fluorescence before assembly and green fluorescence after assembly.

[0025] Figure 5 This is a comparison of chemical shifts in 1H NMR spectra; the top image shows pyrene-based pyridinium salt derivatives. 1 The 1H NMR spectrum is shown below, with the pyrene-based pyridinium salt derivatives after the addition of cucurbita[7]urea. 1 H NMR spectrum.

[0026] Figure 6 This is a schematic diagram of the assembly of pyrene-based pyridinium salt derivatives with cucurbita[7]urea.

[0027] Figure 7 The figures show the experimental results of detecting heparin at concentrations of 0-14 μg / mL using a supramolecular fluorescent probe. The left figure shows the UV absorption spectrum of the supramolecular fluorescent probe, and the right figure shows the linear fitting curve of heparin concentration versus absorbance.

[0028] Figure 8 The figures show the experimental results of detecting heparin at concentrations of 0-1.4 μg / mL using a supramolecular fluorescent probe. The left figure shows the UV absorption spectrum of the supramolecular fluorescent probe, and the right figure shows the linear fitting curve of heparin concentration versus absorbance.

[0029] Figure 9 The figures show the experimental results of detecting heparin at concentrations of 0-140 μg / mL using supramolecular fluorescent probes. The left figure shows the fluorescence emission spectrum of the supramolecular fluorescent probes, and the right figure shows the dependence curve of heparin concentration on fluorescence intensity.

[0030] Figure 10 The figures show the experimental results of detecting heparin at concentrations of 0-7.2 μg / mL using a supramolecular fluorescent probe. The left figure shows the fluorescence emission spectrum of the supramolecular fluorescent probe, and the right figure shows the dependence curve of heparin concentration on fluorescence intensity.

[0031] Figure 11 The figures show the experimental results of detecting heparin at concentrations of 0-0.7 μg / mL using a supramolecular fluorescent probe. The left figure shows the fluorescence emission spectrum of the supramolecular fluorescent probe, and the right figure shows the dependence curve of heparin concentration on fluorescence intensity. Detailed Implementation

[0032] A schematic diagram of the assembly of pyrene pyridinium salt derivatives with cucurbita[7]urea is shown below. Figure 4 .

[0033] Example 1 A supramolecular fluorescent probe is prepared by means of pyrene-based pyridinium salt derivatives and cucurbita[7]urea in a molar ratio of 1:1.

[0034] The pyrene-based pyridinium salt derivative is PPOB.

[0035] The experimental mechanism diagram for the synthesis of pyrene-based pyridinium salt derivatives is shown below. Figure 1 The method for preparing the PPOB includes the following steps: Under a nitrogen atmosphere, 0.15 mmol tetraphenylphosphine palladium, 5.1 mmol 4-pyridylboronic acid, 5.0 mmol 1-bromopyrene and 25 mmol potassium carbonate were mixed, dissolved, and stirred at 100°C for 24 h. After cooling to room temperature, the mixture was washed with water, extracted, and dried to obtain intermediate I. 5.1 mmol of intermediate I and 5.1 mmol of 2,5-dinitrochlorobenzene were dissolved in acetone and heated under nitrogen atmosphere at 50 °C for 16 h. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, washed with hexane, and dried to obtain intermediate II. 0.208 mmol of intermediate II and 1.453 mmol of methoxyaniline were dissolved in methanol and heated under nitrogen atmosphere at 60 °C for 24 h. After evaporating the solvent under reduced pressure, a concentrated solution was obtained. The concentrated solution was added dropwise to diethyl ether (volume ratio of concentrated solution to diethyl ether was 1:5), filtered, washed, and dried to obtain 72 mg of pyrene-based pyridinium salt derivative PPOB. The yield was 83%.

[0036] A method for preparing a supramolecular fluorescent probe includes the following steps: dissolving the prepared PPOB in water to form a 10 -5 M's PPOB solution, cucurbitacin[7] dissolved in water, forms 10 -5 M cucurbit[7]urea solution, PPOB solution and cucurbit[7]urea solution are mixed to form supramolecular assembly, and supramolecular fluorescent probe is obtained.

[0037] The detection results during the synthesis of the supramolecular fluorescent probe are shown in the figure. Figure 2 .

[0038] The π-conjugated framework of this invention endows pyrene-based pyridinium salt derivatives with strong intermolecular π-π interactions, driving pyrene to form an ordered assembly structure and produce unique fluorescence. First, after gradually adding cucurbita[7]urea solution to PPOB solution, the UV-Vis absorption spectrum and steady-state fluorescence emission spectrum of PPOB were measured (PPOB=50μM; CB[7]=1.25 mM; H2O, 298K) to study the self-assembly behavior of the molecules. Figure 2 It can be seen that PPOB exhibits a maximum characteristic absorption peak at 410 nm. Figure 2 (Left), maximum emission wavelength is 550 nm (λ) ex =400 nm) Figure 2Concentration-dependent studies showed that absorption and emission were linear, indicating that no molecular aggregation occurred even at high concentrations. Then, cucurbita[7]urea solution was gradually added to a fixed concentration of PPOB solution. As the concentration of cucurbita[7]urea solution increased, the absorption peaks at 275 nm and 300-400 nm continued to decrease, reaching a constant value at 1.2 eq. of cucurbita[7]urea solution. Similarly, in the emission spectrum, the peak intensity at 550 nm disappeared after the addition of cucurbita[7]urea solution, and a new broad peak in the 505 nm range also increased, reaching a maximum of 1.2 eq. and then tended to flatten. The CIE coordinates of the fluorescence spectrum clearly showed the change from yellow to green fluorescence before and after assembly. Figure 2 (Right). These results indicate that the optimal complexation molar ratio of PPOB and cucurbita[7]urea is 1:1. To further confirm the formation of the supramolecular fluorescent probe, further investigation was conducted. 1 H NMR studies. From Figure 3 It can be seen that after adding cucurbita[7]urea solution to PPOB solution, the chemical shift of the aromatic region shows a significant low-field shift, while the chemical shifts at the benzylic and methyl sites shift to the high-field, confirming the formation of the supramolecular fluorescent probe.

[0039] An application of a supramolecular fluorescent probe for the detection of heparin, the detection method including the following steps: The supramolecular fluorescent probe was prepared into a standard solution of the supramolecular fluorescent probe; Different concentrations of heparin aqueous solutions were added to the standard solution of the supramolecular fluorescent probe (10). -5 In M), ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy were performed; A concentration-dependent standard curve was plotted based on the maximum absorption wavelength and the change in fluorescence intensity before and after assembly. Corresponding fluorescence quenching curves were plotted according to different concentration ranges to show the responsiveness of the fluorescence change of the supramolecular fluorescent probe, thereby realizing the concentration detection of the heparin solution to be tested.

[0040] The maximum absorption wavelength is 400 nm.

[0041] The UV-Vis absorption spectra of the supramolecular fluorescent probe at different concentrations of heparin were measured to investigate the probe's responsiveness. The test results are shown below. Figure 5-6 .

[0042] Depend on Figure 5As shown, with the increase of heparin concentration, the characteristic peaks of 250-300 nm from the local state and 350-450 nm from the conjugated π–π* both decreased, especially the 350-450 nm peak, which showed the original absorption peak before PPOB assembly. This indicates that after the addition of heparin, the negatively charged heparin destroys and gradually weakens the electrostatic adsorption between PPOB and cucurbita[7]urea. The strong negative charge of heparin will form a stronger electrostatic bond with the pyridine N of PPOB, and the gradual red shift of the absorption peak also illustrates this phenomenon. In addition, when the heparin concentration is 0-14 μg / mL and 0-1.4 μg / mL ( Figure 5 and 6 The UV spectra of all samples showed good responsiveness. A standard curve plotted at the maximum absorption peak revealed a concentration-dependent relationship between absorption and emission: y = -1122.97x + 12112.42 (i.e., fluorescence emission decreased with increasing heparin concentration, indicating that no special aggregates or clusters formed in the solution, as such aggregates would exhibit complex fluorescence peaks. In this application, fluorescence quenching was proportional to the addition of heparin. R) 2 (This confirmed the accuracy of the linearity), and molecular aggregation did not occur even at high concentrations of heparin.

[0043] To further illustrate the color change of the solution, the fluorescence emission spectra of the supramolecular fluorescent probe at different concentrations of heparin were measured at an excitation wavelength of 400 nm.

[0044] Depend on Figure 7 It can be seen that when heparin is added, the fluorescence intensity of the assembly characteristic peak of PPOB and cucurbita[7]urea at 505 nm decreases rapidly. As the concentration of heparin increases, the emission peak at this point exhibits a phenomenon of first blue shift and then red shift. The blue shift is because heparin disrupts the electrostatic interaction between PPOB and cucurbita[7]urea, resulting in heterogeneous emission. The red shift is due to the fact that PPOB... + It was slowly detached from cucurbita[7]urea and gradually bound to negatively charged heparin. Notably, the initial emission peak of PPOB was at 550 nm, while the supramolecular fluorescent probe showed a maximum emission wavelength of 580 nm after the fluorescence of heparin was added, which indirectly confirmed that the emission in the final solution came from PPOB. + It binds to heparin. Furthermore... Figure 7 It was also shown that when the concentration of added heparin reached 20 μg / mL, the fluorescence change of the probe was very small when added again. This means that at this concentration, the electrostatic interaction between the original PPOB and cucurbita[7]urea reached the critical point of destruction. It also shows that the supramolecular fluorescent probe has an extremely sensitive response to low concentrations of heparin. Therefore, the response of heparin at low concentrations was studied.

[0045] Further reduce the detection concentration of heparin ( Figure 8 and 9 The concentrations were 0-7.5 μg / mL and 0-0.75 μg / mL. First, at both low concentrations, no blue shift was observed in the emission spectra of the added heparin. This indicates that low concentrations of heparin do not strongly disrupt the assembly of the supramolecular fluorescent probe. Unlike before, the fluorescence change of the probe at extremely low concentrations showed a good linear relationship with concentration, and was almost linear, indicating that the supramolecular fluorescent probe has a unique linear response to extremely low concentrations of heparin. In particular, when the concentration of heparin was extremely low (… Figure 9 The fluorescence emission spectrum showed a good linear change (y = -2.11x + 6.60), and its fluorescence intensity linear constant was -1.55 × 10⁻⁶. 6 (R) 2 =0.992).

[0046] The fluorescence emission spectra of all tested supramolecular fluorescent probe solutions showed a single emission peak for the corresponding compound, therefore, it was concluded that no aggregates were formed in the solution.

[0047] Since the degree of fluorescence quenching in the steady-state spectrum can be fitted to a curve with heparin concentration, it can be used to quantitatively analyze the concentration of heparin to be detected. Furthermore, the detection limit can be determined from trace levels of 0.1 μg / mL to 45 μg / mL through fitting. Therefore, this detection method has been shown to have significant sensitivity to heparin and can accurately detect low concentrations.

Claims

1. A supramolecular fluorescent probe, characterized in that, The raw materials for preparation include pyrene-based pyridinium salt derivatives and cucurbita[7]urea.

2. The supramolecular fluorescent probe according to claim 1, characterized in that, The molar ratio of the pyrene-based pyridinium salt derivative to cucurbit[7]urea is (1-3):(1-3).

3. The supramolecular fluorescent probe according to claim 1, characterized in that, The structural formula of the pyrene-based pyridinium salt derivative is as follows (I): In formula (Ⅰ), R includes one of -CH3O, -CHO, -COOCH3, -COCH3, -CH(CH3)2, -C(CH3)3, -COOH, -SO3H, -CH3, -CHF3O, -CH2OH or -CH2CH3.

4. The supramolecular fluorescent probe according to claim 1, characterized in that, The pyrene-based pyridinium salt derivatives include at least one of PPOB, PPFB, PPMB, PPCB, PPIB, PPTB, PPCBB, PPSB, PPMTB, PPTMB, PPBAB, or PPEB.

5. The supramolecular fluorescent probe according to claim 1, characterized in that, The preparation method of the pyrene-based pyridinium salt derivative includes the following steps: In an inert gas atmosphere, tetrakis(triphenylphosphine)palladium, 4-pyridylboronic acid, 1-bromopyrene and potassium carbonate were mixed, dissolved and stirred at 90-110°C for 20-25 h. After cooling to room temperature, the mixture was washed with water, extracted and dried to obtain intermediate I. Intermediate I and dinitrochlorobenzene were dissolved in acetone and heated under reflux in an inert atmosphere. After the reaction was completed, the mixture was cooled to room temperature, filtered to collect the precipitate, washed with hexane, and dried to obtain intermediate II. Intermediate II and the aniline derivative were dissolved in an alcohol solvent, heated under reflux in an inert gas atmosphere, and the solvent was evaporated under reduced pressure to obtain a solution. The solution was then added dropwise to diethyl ether, filtered, and dried to obtain a pyrene-based pyridinium salt derivative.

6. A method for preparing a supramolecular fluorescent probe according to any one of claims 1-5, characterized in that, Includes the following steps: A supramolecular fluorescent probe was obtained by electrostatically combining a pyrene-based pyridinium salt derivative with cucurbit[7]urea to form a supramolecular assembly.

7. The application of a supramolecular fluorescent probe according to any one of claims 1-5, characterized in that, It is used for the detection of heparin.

8. The application of the supramolecular fluorescent probe according to claim 7, characterized in that, The method for heparin detection includes the following steps: The supramolecular fluorescent probe was prepared into a standard solution of the supramolecular fluorescent probe; Different concentrations of heparin aqueous solutions were added to the standard solution of the supramolecular fluorescent probe, and ultraviolet-visible absorption spectroscopy and fluorescence emission spectroscopy were performed. A concentration-dependent standard curve was plotted based on the maximum absorption wavelength and the change in fluorescence intensity before and after assembly to show the responsiveness of the supramolecular fluorescent probe to the fluorescence change, thereby enabling the concentration detection of the heparin solution to be tested.

9. The application of the supramolecular fluorescent probe according to claim 8, characterized in that, The maximum absorption wavelength is 400 nm.

10. The application of the supramolecular fluorescent probe according to claim 8, characterized in that, The detection limit of the heparin detection method is 0.1 μg / mL.

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