A near-infrared fluorescent probe for detecting and labeling microplastics, a preparation method and application thereof

Abstract

The present application relates to the technical field of organic molecule fluorescent probe, and particularly relates to a near-infrared fluorescent probe for detecting and marking microplastics, and a preparation method and application thereof, the near-infrared fluorescent probe HCY-PS takes a hemicyanine dye as a skeleton, and 4-dimethylamine-1-naphthaldehyde is introduced to complete preparation.The fluorescent probe HCY-PS of the present application is combined with microplastics through electrostatic adsorption, pi-pi stacking and hydrophobic interaction, and microplastics are marked through a dyeing method.The fluorescent probe HCY-PS of the present application has a wide linear response range for microplastics, high detection sensitivity, and a detection limit of 0.153 mg / L.The fluorescent probe HCY-PS of the present application can be applied to cell level detection and marking of microplastics.The fluorescent probe HCY-PS of the present application can be applied to biological level marking of microplastics.The fluorescent probe HCY-PS of the present application is obtained through one-step condensation, does not require harsh reaction conditions, and has the possibility of mass production.

Description

Technical Field

[0001] This invention relates to the technical field of fluorescent probes for organic molecules, and specifically to a near-infrared fluorescent probe for detecting and labeling microplastics, its preparation method, and its application. Background Technology

[0002] Microplastic and nanoplastic (MnP) pollution has become a global environmental concern. More than 80,000 tons of plastic waste drift into the ocean annually, degrading into MnP through physical and biological processes. Due to their small size and low density, MnP particles can migrate and diffuse under the influence of ocean currents, waves, and tsunamis, making them widespread in nature. Multiple independent studies have detected MnP in the lungs, blood, breast milk, placenta, feces, and even the human reproductive system, indicating that MnP has been transferred and accumulated in the human body through the food chain. Because of their small size and high penetrability into biological tissues, MnP exhibits significant biotoxicity. Studies have shown that MnP pollution can lead to cardiovascular diseases such as atherosclerosis, immune and inflammatory responses, and reproductive abnormalities, seriously impacting human health.

[0003] Currently, methods used for analyzing and detecting MnP include Fourier transform infrared spectroscopy (FTIR), Raman microscopy (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 its sensitivity is low, it is susceptible to interference, and its reliability is low. SEM and TEM are commonly used to analyze the distribution of MnP at the subcellular level in plants and animals, but samples prepared from sections are not suitable for live cell observation.

[0004] Therefore, the detection and labeling of MnP at the cellular and animal levels is of great significance. Summary of the Invention

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

[0006] The second objective of this invention is to provide a method for preparing a near-infrared fluorescent probe for detecting and labeling microplastics, which is simple and easy to adjust.

[0007] The third objective of this invention is to provide an application of a near-infrared fluorescent probe for detecting and labeling microplastics.

[0008] The fourth objective of this invention is to provide another application of near-infrared fluorescent probes for detecting and labeling microplastics.

[0009] The fifth objective of this invention is to provide yet another application of a near-infrared fluorescent probe for detecting and labeling microplastics.

[0010] One of the solutions adopted to achieve the purpose of this invention is: a near-infrared fluorescent probe for detecting and labeling microplastics, wherein the near-infrared fluorescent probe is prepared by introducing 4-dimethylamine-1-naphthaldehyde as a backbone of hemicyanine dye.

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

[0012]

[0013] The second objective of this invention is achieved through a method for preparing a near-infrared fluorescent probe for detecting and labeling microplastics, wherein 4-dimethylamine-1-naphthaldehyde and 1,2,3,3-tetramethyl-3H-indole iodide are reacted in a one-step condensation reaction at a certain temperature to generate the near-infrared fluorescent probe.

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

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

[0016] Preferably, the reaction temperature is 80–90°C, and the reaction time is 10–12 hours.

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

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

[0019] The solution adopted to achieve the third objective of this invention is: the application of a near-infrared fluorescent probe for detecting and labeling microplastics, wherein the near-infrared fluorescent probe is applied to detect and label microplastics in an aqueous solution.

[0020] The solution adopted to achieve the fourth objective of this invention is: the application of a near-infrared fluorescent probe for detecting and labeling microplastics, wherein the near-infrared fluorescent probe is applied to detect and label microplastics at the cellular level.

[0021] The solution adopted to achieve the fifth objective of this invention is: an application of a near-infrared fluorescent probe for detecting and labeling microplastics, wherein the near-infrared fluorescent probe is applied to detect and label microplastics at the biological level.

[0022] The HCY-PS of this invention binds to MnP through electrostatic adsorption, π-π stacking, and hydrophobic interactions. This binding suppresses distorted intramolecular charge transfer (TICT) and reduces nonradiative transitions, thereby generating an enhanced fluorescence signal. The fluorescence signal intensity of HCY-PS exhibits 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 bind to MnP through electrostatic adsorption, π-π stacking and hydrophobic interaction, and MnP can be labeled by 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, which does not require harsh reaction conditions and has the potential for mass production.

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

[0028] The near-infrared fluorescent probe HCY-PS of this invention enables the detection and imaging of MnP in aquatic environments, at the cellular level, and in animals, thereby achieving visualization of MnP. Simultaneously, HCY-PS can label MnP in vitro, which can be used for further studies on the biotoxicity of MnP. Attached Figure Description

[0029] Figure 1The fluorescence response of HCY-PS to different analytes is shown in (a) the fluorescence emission spectra of HCY-PS after incubation with different concentrations of carboxylated polystyrene MnP (PS-COOH, 100 nm, negative charge); (b) the fluorescence emission spectra of HCY-PS after incubation with different concentrations of aminopolystyrene MnP (PS-NH2-100, 100 nm, positive charge); and (c) the fluorescence emission spectra of HCY-PS after incubation with different concentrations of aminopolystyrene 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, negatively charged); (e) Fluorescence emission spectra of HCY-PS after incubation with different concentrations of polypropylene MnP (PP, 100 nm, negatively charged); Inset: Linear relationship between different types of MnP concentrations and the fluorescence intensity of HCY-PS at 645 nm; (f) Selectivity study of HCY-PS for MnP, with 1: NaCl, 2: KCl, 3: CaCl2, 4: MgCl2 in the x-axis.

[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 using HCY-PS;

[0032] Figure 3 For the study of HCY-PS's ability to label 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 juvenile zebrafish after exposure to MnP;

[0036] Figure 7 For the HRMS (m / z) analysis of Equation 1;

[0037] Figure 8 The 1H NMR spectrum is shown in Equation 1.

[0038] Figure 9 The NMR carbon spectrum is shown in Equation 1. Detailed Implementation

[0039] To better understand the present invention, the following embodiments are further illustrations of the present invention, but the content of the present invention is not limited to the following embodiments.

[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-naphthal (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 an inert gas atmosphere. After 10–12 hours, the reaction was cooled to room temperature, and the solvent was removed under reduced pressure. The solution was purified by silica gel column chromatography (using gradient elution with DCM / MeOH solutions prepared at volume ratios of 200:1–100:1) to obtain a yellowish-brown solid compound (160 mg, 90%). 1 H NMR(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) δ180.07,157.12,147.88,142.84,142.07,133.73,130.49,128.88,128.61,128.31,126.17,12 5.73,125.26,123.31,122.79,122.40,114.30,112.55,109.45,51.39,44.28,33.86,26.40.HRMS(m / z):calcd.for C 25 H 27 N2[M] + 355.2169, found 355.2163.

[0042] Figure 7 For the HRMS (m / z) analysis of Equation 1; Figure 8 The 1H NMR spectrum is shown in Equation 1. Figure 9The NMR carbon spectrum is shown in Equation 1.

[0043] Example 2

[0044] Fluorescence responsiveness of the fluorescent probe HCY-PS to MnP. We selected five representative MnPs to investigate the fluorescence responsiveness of HCY-PS to MnPs of different types, charges, and sizes. The five selected MnPs were: carboxylated polystyrene MnP (PS-COOH, 100 nm, negative charge), aminopolystyrene MnP (PS-NH2-100, 100 nm, positive charge), aminopolystyrene MnP (PS-NH2-766, 766 nm, positive charge), polyethylene terephthalate MnP (PET, 100 nm, negative charge), and polypropylene MnP (PP, 100 nm, negative charge). By continuously increasing the concentration of MnP in phosphate-buffered saline (PBS, pH = 7.4), we observed that the fluorescence intensity of HCY-PS gradually increased with increasing MnP concentration, and the fluorescence intensity exhibited a relatively wide linear response range with MnP concentration. Figure 1 (Illustration). Calculations showed that 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 fluorescence intensity changes induced by the binding of the five MnPs to the probe, this was mainly due to differences in the type, size, and surface charge of the five MnPs, resulting in different binding abilities to the probe. Figure 1 As shown in a and 1b, for MnP of the same type and size, negatively charged MnP (PS-COOH) binds more readily to the HCY-PS probe (which carries a positive charge) than positively charged MnP (PS-NH2-100), resulting in a stronger fluorescence response. Furthermore, as... Figure 1 As shown in b and 1c, for MnPs of the same type and charge, larger MnPs have poorer water solubility, are more prone to precipitation, and have smaller specific surface areas, which is unfavorable for their binding with the probe. Therefore, the fluorescence signal generated by PS-NH2-766MnP binding to the probe is weaker than that of PS-NH2-100. It is noteworthy that the concentrations of several MnPs showed a linear relationship with the fluorescence intensity of HCY-PS, indicating that HCY-PS can be used for quantitative studies of MnPs.

[0045] Example 3

[0046] The selectivity of the fluorescent probe HCY-PS for MnP was determined. Selectivity is an important indicator of a probe. In this example, the selectivity was measured for common ions and amino acids found in organisms (NaCl, KCl, CaCl2, MgCl2, NaHCO3, KH2PO4, K2CO3, Na2SO4, lysine, serine, histidine, and alanine). The results showed that HCY-PS exhibited fluorescence response to only five MnP molecules among all analytes, indicating that HCY-PS has good selectivity for MnP. Figure 1 f).

[0047] Example 4

[0048] HCY-PS can perform fluorescence imaging of MnP in live cells. For example... Figure 2 As shown, HepG2 cells were first incubated overnight with different concentrations of MnP, and then the culture medium was changed to remove untaken MnP. Simultaneously, the uptake of MnP by HepG2 cells was confirmed by transmission electron microscopy. Figure 2 (b, the red arrow indicates MnP taken up by the cells). Subsequently, 10 μM of the probe was added and HepG2 cells were incubated. In the absence of MnP (control group), only weak red fluorescence was observed, which originated from the fluorescence emission of the HCY-PS probe itself. As the MnP concentration increased, the red fluorescence gradually intensified, and the probe fluorescence intensity was positively correlated with the MnP concentration, 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 MnP in cells and zebrafish juveniles after exposure. First, MnP was stained using a staining method with probes. Unbound probes were removed by centrifugation. The labeled MnP was then lyophilized and subjected to fluorescence imaging using a laser confocal microscope. The labeled MnP exhibited strong red fluorescence. Figure 3 This indicates that the probe has good labeling ability for MnP. The labeled MnP was co-incubated with A549 and HepG2 cells. With increasing MnP concentration, the fluorescence intensity of the red channel in A549 and HepG2 cell imaging gradually increased. Figure 4 and Figure 5 Furthermore, when labeled MnP was co-cultured with zebrafish juveniles for 30 hours, subsequent imaging experiments revealed that MnP was mainly distributed in the yolk sac of the zebrafish juveniles, and its fluorescence intensity increased with increasing MnP concentration. Figure 6 These experimental results all demonstrate that the probe HCY-PS mentioned in Example 1 has excellent detection and labeling capabilities for MnP, enabling in-situ, real-time detection of MnP in aquatic environments, at the cellular level, and in animals, thus achieving in vitro and in vivo visualization of microplastics. Furthermore, probe-labeled MnP can provide a research tool for further studies on the biotoxicity of MnP.

[0051] The above description is merely a preferred embodiment of the present invention, and should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various improvements and modifications without departing from the principles of the present invention, and these improvements and modifications are also considered to be within the scope of protection of the present invention.

Claims

1. A near-infrared fluorescent probe for detecting and labeling microplastics, characterized in that, The near-infrared fluorescent probe has a compound with the structure of Formula 1. ， Formula 1, The anion of the compound is an iodide ion.

2. A method for preparing a near-infrared fluorescent probe for detecting and labeling microplastics according to claim 1, characterized in that, The near-infrared fluorescent probe is generated by a one-step condensation reaction of 4-dimethylamine-1-naphthaldehyde and 1,2,3,3-tetramethyl-3H-indole iodide.

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

5.

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

1.

5. The method for preparing a near-infrared fluorescent probe for detecting and labeling microplastics according to claim 2, characterized in that, The reaction temperature is 80~90 ℃, and the reaction time is 10~12 hours to complete.

6. The method for preparing a near-infrared fluorescent probe for detecting and labeling microplastics according to claim 2, characterized in that, The reaction is carried out under the protection of an inert gas.

7. An application of the near-infrared fluorescent probe of claim 1 for detecting and labeling microplastics, characterized in that: The near-infrared fluorescent probe was used to detect and label microplastics in aqueous solution.

8. An application of the near-infrared fluorescent probe of claim 1 for detecting and labeling microplastics, characterized in that: The near-infrared fluorescent probe is used to detect and label microplastics at the cellular level, for non-diagnostic and non-therapeutic purposes.

9. An application of the near-infrared fluorescent probe of claim 1 for detecting and labeling microplastics, characterized in that: The near-infrared fluorescent probe is used to detect and label microplastics at the biological level, for non-diagnostic and non-therapeutic purposes.

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