Cationic fluorescent probe, preparation method and application

Abstract

The invention discloses a cationic fluorescent probe with long wavelength emission as well as a preparation method and application of the cationic fluorescent probe. The fluorescent probe disclosed by the invention has the characteristic of emitting red fluorescence, the probe is combined with heparin through electrostatic interaction, and the fluorescence is quenched, so that the heparin is detected; furthermore, the protamine is specifically combined with the heparin, probe molecules are released, fluorescence is recovered, and the protamine is detected; the long-wavelength fluorescent probe provided by the invention has good selectivity and sensitivity on heparin and protamine, and can be applied to a serum dilution environment.

Description

Technical Field

[0001] This invention belongs to the field of functional materials technology, and relates to fluorescent probe materials, specifically to a terthiophene-derived long-wavelength emitting cationic fluorescent probe, its preparation method, and its application. Background Technology

[0002] Biomolecules play a vital role in human physiological processes, disease detection, and treatment. Heparin, an anticoagulant, prevents thrombus formation and disseminated intravascular coagulation. In the treatment of cardiovascular and cerebrovascular diseases, it is necessary to quantify the heparin content in the patient's blood or plasma to prevent complications such as bleeding. Protamine sulfate has high nutritional and functional value and can be used as a heparin detoxifier, as well as for lowering blood pressure and promoting digestion. Their concentrations need to be controlled within a reasonable range to achieve their physiological and medical effects while avoiding side effects.

[0003] Fluorescence sensing methods, with their advantages of simplicity, high sensitivity, and rapid response, are widely used in the detection of various substances. Currently, most fluorescent probes reported for heparin detection have relatively short emission wavelengths, falling within the blue to green light spectrum. These are susceptible to interference from autoluminescence in serum samples (around 510 nm), leading to reduced sensitivity. Red light, on the other hand, has strong tissue penetration, making it advantageous for probe applications in biological tissues or living organisms. Therefore, long-wavelength fluorescent probes, such as those emitting red or infrared light, can improve their anti-interference capabilities, laying the foundation for their application in biological tissue detection. Developing long-wavelength fluorescent probes is a crucial direction in the field of fluorescence sensing and holds significant research value. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention provides a cationic fluorescent probe molecule with long-wavelength emission, its preparation method, and its applications. Using this probe, highly selective and sensitive detection of heparin and protamine can be achieved.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a long-wavelength emitting cationic fluorescent probe, the chemical structure of which is shown in Formula I: (Formula I).

[0006] This invention also provides a method for preparing the above-mentioned fluorescent probe, comprising the following steps: dissolving compound 1 and compound 2 in ethanol, and adding piperidine. Plugging with nitrogen gas and refluxing. Removing the solvent by rotary evaporation to obtain a crude product. Washing the crude product with ethyl acetate. Recrystallizing from methanol / petroleum ether to obtain the probe molecule.

[0007] The chemical structural formula of compound 1 is shown in Formula II: (Formula II); The chemical structural formula of compound 2 is shown in Formula III: (Formula III).

[0008] In the steps described, the molar ratio of compound 1 to compound 2 is 1:1 to 1:1.3; the molar ratio of piperidine to compound 1 is 0.05:1 to 0.5:1; and the volume ratio of methanol to petroleum ether is 1:2 to 1:5.

[0009] In the above steps, the reflux time is 6-10 h; the reflux temperature is 75-85 ℃; the nitrogen flow rate is 10-50 mL / min; the amount of ethyl acetate used is 3-5 mL each time, and the number of washings is 2-5 times; the pressure during vacuum distillation is 20-80 kPa, and the temperature is 25-40 ℃.

[0010] The present invention also provides the application of the fluorescent probe in the detection of heparin.

[0011] The present invention also provides the application of the electrostatic complex system of the fluorescent probe and heparin in the detection of protamine.

[0012] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a cationic fluorescent probe molecule with long-wavelength emission. Through electrostatic binding, this probe binds to heparin, "turning off" the fluorescence signal and enabling quantitative detection of heparin. Furthermore, protamine specifically binds to heparin, causing fluorescence emission to recover, thus enabling quantitative detection of protamine.

[0013] This probe exhibits good selectivity and sensitivity for the quantitative detection of heparin and protamine, and has the potential to be applied in complex media.

[0014] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of the present invention, nor is it intended to limit the scope of the invention. Other features of the invention will become readily apparent from the following description. Attached Figure Description

[0015] The above and other objects, features, and advantages of exemplary embodiments of the present invention will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of the invention are illustrated in the drawings by way of example and not limitation, wherein: In the accompanying drawings, the same or corresponding reference numerals indicate the same or corresponding parts.

[0016] Figure 1 The UV-Vis absorption and fluorescence emission spectra of the probe molecules are shown in the image (λ). ex=470 nm); the inset is a photograph of the solution under a UV lamp; Figure 2 a) shows the fluorescence emission spectrum when heparin (0~4 μg / mL) is added to the probe solution (13.3 μM); b) shows the linear relationship between fluorescence intensity at 670 nm and heparin concentration. Figure 3 Figure a shows the selectivity of the probe to heparin before and after the addition of common interfering substances; Figure b shows the effect of heparin and two analogues on the fluorescence of the probe at different concentrations. Figure 4 The graph shows the linear response of the probe (13.3 μM) to heparin (0~3 μg / mL) in 10% serum. Figure 5 Figure a shows the change in fluorescence spectrum after adding protamine (0~5 μg / mL) to the probe-heparin system (13.3 μM -4 μg / mL); Figure b shows the linear fit between the maximum emission intensity and the concentration of protamine. Figure 6 The graph shows the changes in maximum emission intensity when various interfering agents (4 μg / mL) are added to the probe-heparin-protamine system (13.3 μM - 4 μg / mL - 4.5 μg / mL); Figure 7 The graph shows the relationship between the maximum emission intensity of the probe-heparin complex system (13.3 μM - 3 μg / mL) and the concentration of protamine (0~4 μg / mL) in a 10% serum solution. Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the following examples will further illustrate the specific steps and features of the invention. These examples are for illustrative purposes only and are not intended to limit the invention. Unless otherwise specified, the methods used in this invention are conventional methods in the art. Unless otherwise specified, all reagents and materials involved in this invention are commercially available.

[0018] Example 1 Synthesis of Fluorescent Probe The synthesis route is shown in the following formula:

[0019] Compound 1 (0.027 g, 0.1 mmol) and compound 2 (0.046 g, 0.13 mmol) were dissolved in 10 mL of ethanol and added to a 50 mL three-necked flask. 5 μL of piperidine was added. Nitrogen gas was purged, and the mixture was refluxed at 80 °C for 8 h. The solvent was removed by vacuum distillation (80 kPa, 40 °C) to obtain the crude product. The crude solid was washed with ethyl acetate (3 × 5 mL), and then recrystallized from methanol (5 mL) and petroleum ether (15 mL) to obtain the probe molecule. The yield was 67%.

[0020] The product was characterized by 1H NMR, 1C NMR, and high-resolution mass spectrometry. 1 H NMR (600 MHz, DMSO- d 6): δ8.97 (d, J = 6.5 Hz, 2H), 8.29 (d, J = 16.0 Hz, 2H), 8.26 (d, J = 6.3 Hz, 1H), 8.15(dt, J = 21.2, 10.0 Hz, 1H), 7.59 (d, J = 5.1 Hz, 1H), 7.54-7.50 (m, 1H), 7.49-7.43 (m, 1H), 7.42-7.37 (m, 1H), 7.36 (d, J = 3.9 Hz, 1H), 7.21 (d, J = 16.0 Hz, 1H), 7.14 (d, J = 4.6 Hz, 1H), 4.59 (dt, J = 28.6, 6.9 Hz, 2H), 3.45-3.40 (m,2H), 3.10 (s, 9H), 2.44 (m, 2H). 13 C NMR (150 MHz, CD3OD): δ 155.71, 145.13,142.86, 140.67, 139.48, 137.67, 136.21, 136.03, 135.19, 129.23, 127.32,126.50, 126.05, 125.82, 125.47, 124.90, 122.23, 63.87, 57.89, 54.01, 53.98,53.96, 26.07. HR-MS (ESI): m / z calcd. for C 25 H 28 N2S32+ : 226.0702 [M-2Br] 2+ ;found: 226.0715.

[0021] Example 2: Fluorescence response of probe molecules to heparin Figure 1 The UV-Vis absorption spectrum and fluorescence emission spectrum (λ) of the probe molecule in aqueous solution. ex =470 nm). The inset shows a photograph of the probe solution under UV light. The maximum absorption wavelength of the probe molecule is at 470 nm, the maximum emission wavelength is at 670 nm, and the Stokes shift is 200 nm. HEPES buffer solution (10 mM, pH 7.4) was selected for subsequent experiments. Heparin (0~4 μg / mL) was gradually added to the probe solution (13.3 μM), and the fluorescence intensity gradually decreased, as shown in the figure. Figure 2 As shown in Figure a, the fluorescence intensity at 670 nm exhibits a linear relationship with heparin concentration in the range of 0–3.5 μg / mL (R0). 2 =0.9924), such as Figure 2 Figure b shows the standard curve for measuring heparin concentration using the fluorescence intensity of the probe molecule. Based on this standard curve, heparin concentration can be measured. Under these conditions, the detection limit of the probe for heparin is 9.10 ng / mL. The recovery results are shown in Table 1. When 1, 2, and 3 μg / mL of heparin were added to the solution, the heparin concentrations were calculated to be 1.05, 2.18, and 3.27 μg / mL, respectively, based on the standard curve. These results indicate that the probe molecule has the potential to serve as a heparin probe.

[0022] Table 1. Heparin recovery rate in buffer solution.

[0023] Example 3: Specificity of the probe molecule to heparin Specificity and interference resistance are important indicators of probe performance. Common heparin interfering agents were selected to study the selectivity and interference resistance of the probe molecules of this invention. Figure 3 As shown in a, ATP, hyaluronic acid (HA), glucose, bovine serum albumin (BSA), chondroitin sulfate (Chs), vitamin C (VC), sodium citrate (SC), and inorganic salt ions (PO4) are present. 3- Ca 2+ SO4 2- Zn 2+Common interfering agents (4 μg / mL), whether present alone or coexisting with heparin (4 μg / mL), did not significantly affect the selectivity of the probe. We conducted a detailed study on the effects of two heparin analogues (chondroitin sulfate and hyaluronic acid), such as... Figure 3 As shown in b, heparin caused the greatest degree of fluorescence quenching, and the higher the concentration, the more obvious the difference between the three.

[0024] Example 4: Fluorescence detection of heparin by probe molecules in serum solution The response of the probe to heparin in serum medium was further investigated. A buffer solution containing 10% serum was used for the experiment. Figure 4 As shown, the probe (13.3 μM) exhibited a linear response to heparin in the range of 0–3 μg / mL (R0). 2 =0.9901), and the limit of detection is 64.56 ng / mL. Compared with buffer solutions, the serum environment is more complex and contains more interfering substances, resulting in a higher limit of detection. The recovery results in Table 2 show that the probe has good accuracy in 10% serum. The fluorescent probe of this invention can be used in a 10% serum environment, indicating its potential for application in complex media.

[0025] Table 2. Heparin recovery rate of probe detection in buffer solution containing 10% serum.

[0026] Example 5: Fluorescence response and specificity of the probe-heparin complex system to protamine. Protamine is an antidote for heparin. In a buffer solution, the addition of protamine to a probe-heparin complex system results in a recovery of fluorescence intensity. Figure 5 As shown in Figure a, with the addition of protamine (0–5 μg / mL), the fluorescence intensity of the probe-heparin complex system (13.3 μM – 4 μg / mL) gradually increased. This is attributed to the binding of protamine to heparin, which causes the probe molecules to leave heparin, thus restoring fluorescence. The linear range is 1–4.5 μg / mL. Figure 5 (b) The limit of detection was 33.62 ng / mL. The selectivity of the probe-heparin complex system for protamine was also investigated. Figure 6 As shown, arginine (L-Arginine), lysine (L-lysine), ovalbumin, hemoglobin, bovine serum albumin (BSA), pepsin, and Ca were added to the probe-heparin-protamine system (13.3 μM - 4 μg / mL - 4.5 μg / mL). 2 + Zn2+ Interfering substances such as glucose (4 μg / mL) did not significantly affect the fluorescence spectrum. This indicates that the probe-heparin complex system has good selectivity and anti-interference ability for protamine. Table 3 shows the recovery results of the probe-heparin complex system for protamine detection. It can be seen that the probe-heparin complex system has good sensitivity for protamine.

[0027] Table 3. Recovery rate of protamine in buffer solution using probe-heparin system.

[0028] Example 6: Application of the probe-heparin system for detecting protamine in serum solution The response of the probe-heparin complex system to protamine in serum solution was further investigated. In a 10% serum solution, the fluorescence intensity of the probe-heparin complex system (13.3 μM - 3 μg / mL) gradually increased with the addition of protamine (0–4 μg / mL). This is because protamine competitively binds to heparin, causing the probe to gradually dissociate from the heparin. The intensity at its maximum fluorescence wavelength showed a linear relationship with the concentration in the range of 0.5–3.5 μg / mL (R0). 2 =0.9912)( Figure 7 The detection limit was 56.66 ng / mL. Recovery results are shown in Table 4. These results indicate that the probe-heparin complex system can be used to detect protamine in 10% serum solution.

[0029] Table 4. Recovery rate of protamine in 10% serum using the probe-heparin system.

[0030] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. A cationic fluorescent probe, characterized by, The structural formula of the probe is shown as Formula I: Formula I.

2. A method for preparing the cationic fluorescent probe according to claim 1, characterized by, The method comprises the following steps: Compound 1 and compound 2 are dissolved in ethanol, piperidine is added, nitrogen is introduced, and reflux is carried out; pressure reduction distillation is carried out to remove the solvent, and the product is washed with ethyl acetate; Then methanol / petroleum ether recrystallization is carried out to obtain the cationic fluorescent probe with long wavelength emission as claimed in claim 1; The chemical structural formula of the compound 1 is shown as Formula II: Formula II; The chemical structural formula of the compound 2 is shown as Formula III: Formula III.

3. The preparation method according to claim 2, characterized in that, The molar ratio of the compound 1 and the compound 2 is 1:1-1:1.3; the molar ratio of the piperidine and the compound 1 is 0.05:1-0.5:1; and the volume ratio of the methanol and the petroleum ether is 1:2-1:

5.

4. The preparation method according to claim 2, characterized in that, The reflux time is 6-10 h.

5. The preparation method according to claim 2, characterized in that, The reflux temperature is 75-85 DEG C.

6. The preparation method according to claim 2, characterized in that, The flow rate of the nitrogen is 10-50 mL / min.

7. The preparation method according to claim 2, characterized in that, The amount of the ethyl acetate is 3-5 mL each time, and the washing times are 2-5 times.

8. The preparation method according to claim 2, characterized in that, The pressure during the pressure reduction distillation is 20-80 kPa, and the temperature is 25-40 DEG C.

9. Application of the cationic fluorescent probe as claimed in claim 1 to detection of heparin.

10. Application of the complex of the cationic fluorescent probe as claimed in claim 1 and heparin to detection of protamine.

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