Micro-molecular fluorescent probe with CS as fluorophore, preparation method and application thereof

Abstract

The invention discloses a micro-molecular fluorescent probe taking CS as a fluorophore, a preparation method and application thereof. Coumarin is used as a fluorophore and salicyloyl hydrazine is taken as a basic substance, so that the fluorescent probe has very strong conjugated [Pi] electrons; wherein salicyloyl hydrazine and aldehyde groups are condensed to form chelation sites of metal ions. After the micromolecular fluorescent probe is combined with copper ions, the fluorescence is quenched, and electron transfer in probe molecules is hindered by the micromolecular fluorescent probe in the presence of the copper ions, so that the fluorescence of the micromolecular fluorescent probe is changed from existence to absence, the ultraviolet absorption peak red shift occurs, and new absorption occurs at the wavelength of 470 nm. Along with the increase of the concentration of the copper ions, the absorption peak is gradually increased and transferred, which indicates that the metal ionsare strongly combined with the probe. According to the technical scheme, the copper ions are accurately detected through the fluorescence technology, and the indigenous copper ions and the indigenouscopper ions of living cells can be detected, so that the method has a good application prospect in the aspect of copper ion detection. The synthesis method disclosed by the invention is simple and convenient to operate, and does not need rigorous conditions.

Description

technical field

[0001] The invention relates to the field of fluorescent imaging molecular probes, in particular to a probe for detecting copper ions using fluorescent imaging technology, and in particular to a molecular probe, its preparation method and application. Background technique

[0002] In living organisms, copper ions are the third most essential trace element after iron ions and zinc ions. A certain amount of copper ions plays a vital role in the basic physiological activities of plants and animals. However, excessive copper ions can generate reactive oxygen species, disrupt the balance of cell metabolism, and become toxic substances in biological systems. For example, excessive intake of copper ions may cause severe damage to the gastrointestinal tract, kidneys, and liver; some difficult-to-treat diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Menkes syndrome, Wilson's disease, and There is some association with excess copper ions. What...

Examples

Embodiment 1

[0036] Example 1 Synthesis of Small Molecule Fluorescent Probes

[0037] Synthesis of small molecule fluorescent probes with CS as fluorophore: Dissolve 7-(diethylamino)coumarin-3-carbaldehyde (500mg, 2.04mmol) in 30mL ethanol solution, and add salicylic acid at room temperature Hydrazide (310mg, 1.83mmol) was stirred, stirred at room temperature for 20h, and a yellow solid was obtained, which was washed with ethanol and dried. The small molecule fluorescent probe can be recrystallized with absolute ethanol to obtain a sample with higher purity, and the yield of the final sample is: 80%. The route diagram for synthesizing the small molecule fluorescent probe is shown in figure 1 as shown, figure 1 Represents the roadmap for the synthesis of small molecule fluorescent probes, where EtOH is ethanol and RT is room temperature.

[0038] It can be determined that the product is the target small molecule fluorescent probe by mass spectrometry, NMR and spectroscopic methods, and it...

Embodiment 2

[0045] Example 2 Ultraviolet and fluorescence spectra of small molecule fluorescent probes in response to copper ions

[0046] Prepare 1.0mL small molecule fluorescent probe (1.0×10 -5 mol / L) of DMSO / H 2 O (v / v=1:1) solution. 0.5 times the normal concentration of copper ion solution was added dropwise to the probe solution, such as Figure 5 As shown in (a), after copper ions are added to the CS probe solution, the absorption bands at 316nm and 460nm gradually decrease, and there is a new absorption peak at 400nm-500nm, and the maximum absorption peak appears at 470nm. The intensity gradually increases with the ion concentration, and the final stoichiometric ratio of the two is 2:1.

[0047] In the fluorescence titration experiment, prepare 3.0mL small molecule probe (5.0×10 -6 mol / L) of DMSO / H 2 O (v / v=1:1) solution. 0.5 times the equivalent concentration of copper ion solution was added dropwise to the probe solution. For the CS probe, 460nm was used as the excitation ...

Embodiment 3

[0048] Example 3 verifies the selectivity and competitiveness of small molecule fluorescent probes for copper ions.

[0049] Prepare 5.0mL molecular probe (5.0×10 -6 mol / L) of DMSO / H 2 O (v / v=1:1) solution. Various cationic solutions [Fe(III), Al(III), Cr(III), Zn(II), Ni(II), Co(II), Mg(II) were prepared by dissolving the corresponding salts in deionized water , Ca(II), Cd(II), Mn(II), Ag(I), K(I) and Na(I)] (1.0×10 -3 mol / L). Subsequently, an equivalent equivalent of the metal ion solution was added to the probe solution. Detection is carried out by fluorescence spectroscopy, the experimental results are shown in Figure 6 (a). Take the fluorescence maximum absorption wavelength for comparison, such as Figure 6 As shown in (b), ions include Fe(III), Al(III), Cr(III), Zn(II), Ni(II), Co(II), Mg(II), Ca(II), Cd( II), Mn(II), Ag(I), K(I) and Na(I). Except for Cu(II), none of these metal ions produced significant changes in the fluorescence of the probes. After adding...

technical field

[0001] The invention relates to the field of fluorescent imaging molecular probes, in particular to a probe for detecting copper ions using fluorescent imaging technology, and in particular to a molecular probe, its preparation method and application. Background technique

[0002] In living organisms, copper ions are the third most essential trace element after iron ions and zinc ions. A certain amount of copper ions plays a vital role in the basic physiological activities of plants and animals. However, excessive copper ions can generate reactive oxygen species, disrupt the balance of cell metabolism, and become toxic substances in biological systems. For example, excessive intake of copper ions may cause severe damage to the gastrointestinal tract, kidneys, and liver; some difficult-to-treat diseases, including Alzheimer's disease, amyotrophic lateral sclerosis, Menkes syndrome, Wilson's disease, and There is some association with excess copper ions. What...

Examples

Embodiment 1

[0036] Example 1 Synthesis of Small Molecule Fluorescent Probes

[0037] Synthesis of small molecule fluorescent probes with CS as fluorophore: Dissolve 7-(diethylamino)coumarin-3-carbaldehyde (500mg, 2.04mmol) in 30mL ethanol solution, and add salicylic acid at room temperature Hydrazide (310mg, 1.83mmol) was stirred, stirred at room temperature for 20h, and a yellow solid was obtained, which was washed with ethanol and dried. The small molecule fluorescent probe can be recrystallized with absolute ethanol to obtain a sample with higher purity, and the yield of the final sample is: 80%. The route diagram for synthesizing the small molecule fluorescent probe is shown in figure 1 as shown, figure 1 Represents the roadmap for the synthesis of small molecule fluorescent probes, where EtOH is ethanol and RT is room temperature.

[0038] It can be determined that the product is the target small molecule fluorescent probe by mass spectrometry, NMR and spectroscopic methods, and it...

Embodiment 2

[0045] Example 2 Ultraviolet and fluorescence spectra of small molecule fluorescent probes in response to copper ions

[0046] Prepare 1.0mL small molecule fluorescent probe (1.0×10 -5 mol / L) of DMSO / H 2 O (v / v=1:1) solution. 0.5 times the normal concentration of copper ion solution was added dropwise to the probe solution, such as Figure 5 As shown in (a), after copper ions are added to the CS probe solution, the absorption bands at 316nm and 460nm gradually decrease, and there is a new absorption peak at 400nm-500nm, and the maximum absorption peak appears at 470nm. The intensity gradually increases with the ion concentration, and the final stoichiometric ratio of the two is 2:1.

[0047] In the fluorescence titration experiment, prepare 3.0mL small molecule probe (5.0×10 -6 mol / L) of DMSO / H 2 O (v / v=1:1) solution. 0.5 times the equivalent concentration of copper ion solution was added dropwise to the probe solution. For the CS probe, 460nm was used as the excitation ...

Embodiment 3

[0048] Example 3 verifies the selectivity and competitiveness of small molecule fluorescent probes for copper ions.

[0049] Prepare 5.0mL molecular probe (5.0×10 -6 mol / L) of DMSO / H 2 O (v / v=1:1) solution. Various cationic solutions [Fe(III), Al(III), Cr(III), Zn(II), Ni(II), Co(II), Mg(II) were prepared by dissolving the corresponding salts in deionized water , Ca(II), Cd(II), Mn(II), Ag(I), K(I) and Na(I)] (1.0×10 -3 mol / L). Subsequently, an equivalent equivalent of the metal ion solution was added to the probe solution. Detection is carried out by fluorescence spectroscopy, the experimental results are shown in Figure 6 (a). Take the fluorescence maximum absorption wavelength for comparison, such as Figure 6 As shown in (b), ions include Fe(III), Al(III), Cr(III), Zn(II), Ni(II), Co(II), Mg(II), Ca(II), Cd( II), Mn(II), Ag(I), K(I) and Na(I). Except for Cu(II), none of these metal ions produced significant changes in the fluorescence of the probes. After adding...

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