Near-infrared fluorescent probe for detecting and marking micro-plastics as well as preparation method and application of near-infrared fluorescent probe

Abstract

The invention relates to the technical field of organic molecular fluorescent probes, in particular to a near-infrared fluorescent probe for detecting and marking micro-plastics as well as a preparation method and application of the near-infrared fluorescent probe HCY-PS. The near-infrared fluorescent probe HCY-PS is prepared by taking hemicyanine dye as a framework and introducing 4-dimethylamine-1-naphthaldehyde. The fluorescent probe HCY-PS is combined with the micro-plastic through electrostatic adsorption, pi-pi accumulation and hydrophobic interaction, and the micro-plastic is marked through a dyeing method. The fluorescent probe HCY-PS disclosed by the invention has a wide linear response range on the micro-plastic and high detection sensitivity, and the detection limit is 0.153 mg / L. The fluorescent probe HCY-PS provided by the invention can be applied to cell level detection and microplastic marking. The fluorescent probe HCY-PS disclosed by the invention can be applied to horizontally marking micro-plastics in an organism. The fluorescent probe HCY-PS is obtained through one-step condensation, harsh reaction conditions are not needed, and mass production is possible.

Description

Technical Field

[0001] The present invention relates to the technical field of organic molecular fluorescent probes, and in particular to a near-infrared fluorescent probe for detecting and marking microplastics, and a preparation method and application thereof. Background Art

[0002] Micro- and nanoplastic particles (MnP) pollution has become an environmental issue of global concern. Every year, more than 80,000 tons of plastic waste drifts into the ocean and degrades to form MnP under physical and biological effects. MnP has a small particle size and low density, and can migrate and spread under the drive of dynamic processes such as ocean currents, wind and waves, and tsunamis, so it is widely present in nature. Multiple independent studies have detected the presence of MnP in the lungs, blood, breast milk, placenta, feces, and even the human reproductive system, indicating that MnP has been transmitted through the food chain and enriched in the human body. Due to its small size and high penetration into biological tissues, MnP has a high biological toxicity. Studies have shown that MnP pollution can cause cardiovascular diseases such as atherosclerosis, immune and inflammatory reactions, and reproductive abnormalities, which has a serious impact on human health.

[0003] Currently, the methods used to analyze and detect MnP include Fourier-transform infrared spectroscopy (FTIR), Raman spectroscopy (RM), scanning electron microscopy (SEM), and transmission electron microscopy (TEM). FTIR can determine the chemical composition of unknown plastic fragments, but it has limitations in identifying MnP smaller than 20 μm. RM can analyze MnP in aqueous samples, but it has low sensitivity, is susceptible to interference, and has low credibility. SEM and TEM are usually used to analyze the distribution of MnP at the subcellular level in plants and animals, but samples prepared by slices are not suitable for living cell observation.

[0004] Therefore, it is of great significance to detect and label MnP at the cellular and animal levels. Summary of the invention

[0005] One of the purposes of the present invention is to provide a near-infrared fluorescent probe for detecting and labeling microplastics. The near-infrared fluorescent probe HCY-PS can bind to MnP through electrostatic adsorption, π-π stacking and hydrophobic interaction, and label MnP by staining.

[0006] A second object of the present invention is to provide a method for preparing a near-infrared fluorescent probe for detecting and marking microplastics, which is simple and easy to adjust.

[0007] The third object of the present invention is to provide an application of a near-infrared fluorescent probe for detecting and marking microplastics.

[0008] A fourth object of the present invention is to provide another application of a near-infrared fluorescent probe for detecting and labeling microplastics.

[0009] A fifth object of the present invention is to provide another application of a near-infrared fluorescent probe for detecting and labeling microplastics.

[0010] The solution adopted by the present invention to achieve one of the purposes is: a near-infrared fluorescent probe for detecting and marking microplastics, wherein the near-infrared fluorescent probe is prepared by using a hemicyanine dye as a skeleton and introducing 4-dimethylamine-1-naphthaldehyde.

[0011] Preferably, the near-infrared fluorescent probe has a compound of formula 1,

[0012]

[0013] The solution adopted by the present invention to achieve the second purpose is: a method for preparing a near-infrared fluorescent probe for detecting and marking microplastics, wherein at a certain temperature, 4-dimethylamine-1-naphthaldehyde and 1,2,3,3-tetramethyl-3H-indole iodide are subjected to a one-step condensation reaction to generate the near-infrared fluorescent probe.

[0014] Preferably, the equivalent ratio of the 4-dimethylamine-1-naphthaldehyde to the 1,2,3,3-tetramethyl-3H-indole iodide is 1:1-1.5.

[0015] Preferably, the equivalent ratio of the 4-dimethylamine-1-naphthaldehyde to the 1,2,3,3-tetramethyl-3H-indole iodide is 1:1.

[0016] Preferably, the reaction temperature is 80-90° C., and the time required for the reaction to be complete is 10-12 hours.

[0017] Preferably, the reaction is carried out under the protection of an inert gas.

[0018] Specifically, 4-dimethylamine-1-naphthaldehyde and 1,2,3,3-tetramethyl-3H-indole iodide are dissolved in an ethanol solution, and a condensation reaction is carried out under the protection of an inert gas to generate HCY-PS. The reaction temperature is 80 to 90° C., and the reaction takes 10 to 12 hours to complete.

[0019] The solution adopted by the present invention to achieve the third purpose is: an application of a near-infrared fluorescent probe for detecting and marking microplastics, and applying the near-infrared fluorescent probe to detect and mark microplastics in an aqueous solution.

[0020] The solution adopted by the present invention to achieve the fourth purpose is: an application of a near-infrared fluorescent probe for detecting and labeling microplastics, and applying the near-infrared fluorescent probe to detect and label microplastics at the cellular level.

[0021] The solution adopted by the present invention to achieve the fifth purpose is: the application of a near-infrared fluorescent probe for detecting and marking microplastics, and the application of the near-infrared fluorescent probe to detect and mark microplastics at the biological level.

[0022] The HCY-PS of the present invention binds to MnP through electrostatic adsorption, π-π stacking and hydrophobic interaction, which can inhibit twisted intramolecular charge transfer (TICT) and reduce non-radiative transitions, thereby generating an enhanced fluorescence signal. The fluorescence signal intensity of HCY-PS has a good linear relationship with the concentration of MnP.

[0023] The present invention has the following advantages and beneficial effects:

[0024] The near-infrared fluorescent probe HCY-PS of the present invention can be combined with MnP through electrostatic adsorption, π-π stacking and hydrophobic interaction, and MnP can be labeled by a staining method.

[0025] The near-infrared fluorescent probe HCY-PS of the present invention has a wide linear response range to MnP, high detection sensitivity, and a detection limit of 0.153 mg / L.

[0026] The near-infrared fluorescent probe HCY-PS of the present invention is obtained by one-step condensation, does not require harsh reaction conditions, and has the possibility of mass production.

[0027] The near-infrared fluorescent probe HCY-PS of the present invention can perform in-situ real-time imaging of intracellular microplastics in living cells. At the same time, HCY-PS can label MnP in vitro, and the labeled MnP is successfully used to observe the absorption and distribution of MnP in A549, HepG2 cells and zebrafish larvae after exposure.

[0028] The near-infrared fluorescent probe HCY-PS of the present invention can detect and image MnP in water environment, at the cell level and at the animal level, thereby realizing the visualization of MnP. At the same time, HCY-PS can label MnP in vitro, which can be used to further study the biological toxicity of MnP. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1The fluorescence responsiveness of HCY-PS to different analytes, including (a) fluorescence emission spectra of HCY-PS after incubation with different concentrations of carboxyl polystyrene MnP (PS-COOH, 100 nm, negative charge); (b) fluorescence emission spectra of HCY-PS after incubation with different concentrations of amino polystyrene MnP (PS-NH2-100, 100 nm, positive charge); (c) fluorescence emission spectra of HCY-PS after incubation with different concentrations of amino polystyrene MnP (PS-NH2-766, 766 nm, positive charge); (d) Fluorescence emission spectra of HCY-PS after incubation with different concentrations of polyethylene terephthalate MnP (PET, 100 nm, negative charge); (e) Fluorescence emission spectra of HCY-PS after incubation with different concentrations of polypropylene MnP (PP, 100 nm, negative charge); Inset: Linear relationship between different types of MnP concentrations and the fluorescence intensity of HCY-PS at 645 nm; (f) Selective study of HCY-PS for MnP, 1: NaCl, 2: KCl, 3: CaCl2, 4: MgCl2,

[0030] 5: NaHCO3, 6: KH2PO4, 7: K2CO3, 8: Na2SO4, 9: lysine, 10: serine, 11: histidine, 12: alanine, 13: PP, 14: PS-NH2-766, 15: PS-NH2-100, 16: PS-COOH, 17: PET;

[0031] Figure 2 Fluorescence imaging of MnP in HepG2 cells by HCY-PS;

[0032] Figure 3 To study the labeling ability of HCY-PS on MnP;

[0033] Figure 4 The distribution of MnP taken up by A549 cells after exposure to MnP;

[0034] Figure 5 The distribution of MnP taken up by HepG2 cells after exposure to MnP;

[0035] Figure 6 The distribution of MnP ingested by zebrafish larvae after exposure to MnP;

[0036] Figure 7 HRMS (m / z) analysis of Formula 1;

[0037] Figure 8 is the hydrogen nuclear magnetic resonance spectrum of formula 1;

[0038] Fig. 9 is the carbon NMR spectrum of formula 1. DETAILED DESCRIPTION

[0039] For a better understanding of the present invention, the following examples are provided to further illustrate the present invention, but the present invention is not limited to the following examples.

[0040] Example 1

[0041] This embodiment relates to the synthesis of a near-infrared fluorescent probe HCY-PS for detecting and labeling MnP. The specific synthesis route is as follows: 4-Dimethylamine-1-naphthaldehyde (100 mg, 0.50 mmol) and 1,2,3,3-tetramethyl-3H-indole iodide (152 mg, 0.50 mmol) were added to ethanol (10 mL). The solution was heated to 80-90 ° C and stirred in the dark under the protection of an inert gas. After 10-12 hours, the reaction was cooled to room temperature and the solvent was removed under reduced pressure. Purification was performed by a silica gel column (using a mixed solution of DCM / MeOH prepared in a volume ratio of 200: 1-100: 1 for gradient elution) to obtain a yellow-brown solid compound (160 mg, 90%). 1 HNMR(400MHz,MeOD)δ9.05(d,J=15.6Hz,1H),8.52(d,J=8.6Hz,1H),8.36(d,J=8.4Hz,1H),8.29-8.19(m, 1H),7.79-7.69(m,3H),7.64-7.53(m,4H),7.21(d,J=8.6Hz,1H),4.11(s,3H),3.21(s,6H),1.91(s,6H). 13 C NMR (101MHz, DMSO) δ180.07,157.12,147.88,142.84,142.07,133.73,130.49,128.88,128.61,128.31,126.17,125 .73,125.26,123.31,122.79,122.40,114.30,112.55,109.45,51.39,44.28,33.86,26.40.HRMS(m / z):calcd.forC 25 H 27 N2[M] + 355.2169, found 355.2163.

[0042] Figure 7 HRMS (m / z) analysis of Formula 1; Figure 8 is the hydrogen nuclear magnetic resonance spectrum of formula 1; Fig. 9 is the carbon NMR spectrum of formula 1.

[0043] Example 2

[0044] Fluorescence responsiveness of fluorescent probe HCY-PS to MnP. We selected 5 representative MnPs to explore the fluorescence responsiveness of HCY-PS to MnPs of different types, charges and sizes. The 5 MnPs selected are: carboxyl polystyrene MnP (PS-COOH, 100nm, negative charge), 100nm amino polystyrene MnP (PS-NH2-100, 100nm, positive charge), 766nm amino polystyrene MnP (PS-NH2-766, 766nm, positive charge), polyethylene terephthalate MnP (PET, 100nm, negative charge), polypropylene MnP (PP, 100nm, negative charge). We continuously increased the concentration of MnP in phosphate buffered saline (PBS, pH = 7.4), and it can be observed that the fluorescence intensity of HCY-PS gradually increased with the increase of MnP concentration. At the same time, the fluorescence intensity and the concentration of MnP showed a wide linear response range ( Figure 1 Inset). By calculation, the detection limit of HCY-PS for MnP in aqueous solution was 0.153 mg / L. All fluorescence emission spectra were obtained under 615 nm excitation. Although there were some differences in the changes in fluorescence intensity caused by the binding of the five MnPs to the probe, this was mainly due to the differences in the types, sizes and surface charges of the five MnPs, which led to different binding abilities with the probe. Figure 1 As shown in Figures 1a and 1b, for MnPs of the same type and size, the MnP with negative surface charge (PS-COOH) is easier to bind to the HCY-PS probe (with positive charge) than the MnP with positive surface charge (PS-NH2-100), thus producing stronger fluorescence responsiveness. Figure 1 As shown in Figures 1b and 1c, for MnPs of the same type and charge, the larger the size of the MnP, the worse its water solubility, the easier it is to precipitate, and the smaller its specific surface area, which is not conducive to its binding with the probe. Therefore, the fluorescence signal generated by the binding of PS-NH2-766MnP to the probe is weaker than that of PS-NH2-100. It is worth noting that the concentrations of several MnPs show a linear relationship with the fluorescence intensity of HCY-PS, indicating that HCY-PS can be used for quantitative research on MnP.

[0045] Example 3

[0046] Determine the selectivity of the fluorescent probe HCY-PS for MnP. Selectivity is an important indicator of the probe. This example measures the common ions and amino acids in organisms (NaCl, KCl, CaCl2, MgCl2, NaHCO3, KH2PO4, K2CO3, Na2SO4, lysine, serine, histidine, alanine). The results show that among all the analytes, HCY-PS only produces fluorescence responsiveness to 5 MnPs, indicating that HCY-PS has good selectivity for MnP ( Figure 1 f).

[0047] Example 4

[0048] HCY-PS can be used to perform fluorescence imaging of MnP in living cells. Figure 2 As shown, HepG2 cells were first incubated with different concentrations of MnP overnight, and then the culture medium was replaced to remove the MnP that was not taken up by the cells. At the same time, the uptake of MnP by HepG2 cells was confirmed by biological transmission electron microscopy ( Figure 2 b, the red arrow indicates the MnP taken up by the cells). Then, 10 μM of the probe was added and incubated with HepG2 cells. When no MnP was added (control group), only a weak red fluorescence was seen, which came from the fluorescence emission of the probe HCY-PS itself. As the concentration of MnP increased, the red fluorescence gradually increased, and the fluorescence intensity of the probe was positively correlated with the concentration of MnP, indicating that HCY-PS can perform real-time imaging and detection of MnP in cells ( Figure 2 a).

[0049] Example 5

[0050] HCY-PS was used to label MnP in vitro to study the uptake and distribution of cells and zebrafish larvae after exposure to MnP. First, the MnP was stained with a probe using a staining method, and the probe that was not bound to MnP was removed by centrifugation. The labeled MnP was freeze-dried and then fluorescently imaged using a laser confocal microscope. It can be seen that the labeled MnP has a strong red fluorescence ( Figure 3 ), indicating that the probe has good labeling ability for MnP. The labeled MnP was co-incubated with A549 and HepG2 cells. As the concentration of labeled MnP increased, it was found that the fluorescence intensity of the red channel in A549 and HepG2 cell imaging also gradually increased ( Figure 4 and Figure 5 In addition, the labeled MnP was co-cultured with zebrafish larvae for 30 hours. In subsequent imaging experiments, it was found that MnP was mainly distributed in the yolk sac of zebrafish larvae, and its fluorescence intensity also increased with the increase of MnP concentration ( Figure 6 ). These experimental results all show that the probe HCY-PS mentioned in Example 1 has good detection and labeling capabilities for MnP, and can realize in situ and real-time detection of MnP in water environment, cell level and animal level, and realize visualization of microplastics in vivo and in vitro. At the same time, MnP labeled with the probe can provide a research tool for further studying the biological toxicity of MnP.

[0051] The above is only a preferred embodiment of the present invention, which certainly cannot be used to limit the scope of rights of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and changes can be made without departing from the principle of the present invention, and these improvements and changes are also regarded as the protection scope of the present invention.

Claims

1. A near-infrared fluorescent probe for detecting and labeling microplastics, characterized in that: The near-infrared fluorescent probe is prepared by using a hemicyanine dye as a skeleton and introducing 4-dimethylamine-1-naphthaldehyde.

2. The near-infrared fluorescent probe for detecting and marking microplastics according to claim 1, characterized in that: The near-infrared fluorescent probe has a compound of formula 1, 3. The method for preparing a near-infrared fluorescent probe for detecting and marking microplastics according to claim 1 or 2, characterized in that: At a certain temperature, 4-dimethylamine-1-naphthaldehyde and 1,2,3,3-tetramethyl-3H-indole iodide undergo a one-step condensation reaction to generate the near-infrared fluorescent probe.

4. The method for preparing a near-infrared fluorescent probe for detecting and marking microplastics according to claim 3, characterized in that: The equivalent ratio of the 4-dimethylamine-1-naphthaldehyde to the 1,2,3,3-tetramethyl-3H-indole iodide is 1:1-1.

5.

5. The method for preparing a near-infrared fluorescent probe for detecting and marking microplastics according to claim 4, characterized in that: The equivalent ratio of the 4-dimethylamine-1-naphthaldehyde to the 1,2,3,3-tetramethyl-3H-indole iodide is 1:

1.

6. The method for preparing a near-infrared fluorescent probe for detecting and marking microplastics according to claim 3, characterized in that: The reaction temperature is 80-90°C and the time required for the reaction to be complete is 10-12 hours.

7. The method for preparing a near-infrared fluorescent probe for detecting and marking microplastics according to claim 3, characterized in that: The reaction is carried out under the protection of inert gas.

8. A near-infrared fluorescent probe for detecting and marking microplastics according to claim 1 or 2 or a near-infrared fluorescent probe for detecting and marking microplastics prepared by the preparation method of any one of claims 3 to 7, characterized in that: The near-infrared fluorescent probe is used to detect and label microplastics in aqueous solution.

9. A near-infrared fluorescent probe for detecting and marking microplastics according to claim 1 or 2 or a near-infrared fluorescent probe for detecting and marking microplastics prepared by the preparation method of any one of claims 3 to 7, characterized in that: The near-infrared fluorescent probe is applied to detect and label microplastics at the cellular level.

10. A near-infrared fluorescent probe for detecting and marking microplastics according to claim 1 or 2 or a near-infrared fluorescent probe for detecting and marking microplastics prepared by the preparation method of any one of claims 3 to 7, characterized in that: The near-infrared fluorescent probe is used to detect and label microplastics at the biological level.

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