Hybrid luminescent crystal prepared by in-situ dual-doping lanthanide complex, method and application thereof

Abstract

The present invention discloses a hybrid luminescent crystal prepared by in-situ dual-doping lanthanide complex, a method and an application thereof, and a configuration of Eu 3+ and Tb 3+ The mixed solution was transferred to the Eu 3+ and Tb 3+ After a period of time, the DNA crystals are removed, washed, and then transferred into a mixed solution of 2-thiophenecarboxylic acid trifluoroacetone (TTA), o-phenanthroline (phen) and acetylacetone (acac). After a period of time, the crystals are transferred to a buffer solution to maintain the morphology, and hybrid luminescent crystals prepared by in-situ dual-doped lanthanide complexes are prepared. By using DNA crystals to load two lanthanide complexes to prepare hybrid luminescent crystals and detecting drug concentrations based on the light color sensing of the crystals, the drug concentration can be quantitatively detected without being affected by environmental factors.

Description

Technical Field

[0001] The present invention relates to the field of nano functional materials, and in particular to a hybrid luminescent crystal prepared by in-situ doping of a DNA crystal with a lanthanide complex, and a method and application thereof. Background Art

[0002] As an important biological macromolecule, DNA has broad application prospects in the fields of life sciences, nanotechnology, and materials science. DNA crystals have also attracted widespread attention in optoelectronics, optical sensing, and drug delivery due to their highly ordered structure, excellent optical and electrical properties, and other characteristics. However, single DNA crystals have limitations in terms of luminescence performance. Therefore, by hybridizing DNA crystals with other substances, their luminescence performance and functions can be improved, broadening their application areas.

[0003] In recent years, the research on the preparation of new fluorescent sensing materials by hybridizing DNA crystals with fluorescent probes has attracted much attention. In particular, doping with lanthanide complexes with excellent fluorescent properties can significantly improve the luminescence efficiency and sensitivity of DNA crystals through fluorescence resonance energy transfer, thereby realizing fluorescent sensing. In addition, as a type of rare earth element, lanthanide complexes have good photostability and biocompatibility, and also have broad application prospects in fluorescent labeling and bioimaging. Summary of the invention

[0004] The purpose of the present invention is to provide a hybrid luminescent crystal prepared by in-situ doping of a DNA crystal with a lanthanide complex, and a method and application thereof. The hybrid luminescent crystal is prepared by loading two lanthanide complexes with a DNA crystal and drug concentration is detected based on the photochromic sensing of the crystal. The drug concentration can be quantitatively detected and will not be affected by environmental factors.

[0005] The method for preparing hybrid luminescent crystals by in-situ doping of lanthanide complexes comprises firstly adding different concentrations of Eu(TTA) 3 Phen and Tb (ACAC) 3 phen two complex solutions are mixed in a certain ratio, and the concentration ratio when the fluorescence intensity of the two complexes is similar is found, and Eu is prepared at this concentration ratio 3+ and Tb 3+ The mixed solution was transferred to the Eu 3+ and Tb 3+ After a period of time, the DNA crystals were taken out, washed, and then moved into a mixed solution of 2-thiopheneyltrifluoroacetone (TTA), o-phenanthroline (phen) and acetylacetone (acac). After a period of time, the crystals were transferred to a buffer solution to maintain the morphology, thereby preparing a hybrid luminescent crystal prepared by in situ dual-doped lanthanide complexes.

[0006] The method is characterized in that different concentrations of Eu(TTA) 3 Phen and Tb (ACAC) 3 The two complex solutions of phen are mixed in a ratio of 1:1-2.

[0007] In the method described above, in step (1), the complex mixed solution loaded on the crystal must first find a concentration ratio at which the fluorescence intensity remains constant, so that changes in drug concentration can be more easily reflected in the crystal color.

[0008] A hybrid luminescent crystal prepared by in-situ doping of a lanthanide complex prepared according to any of the methods.

[0009] The hybrid luminescent crystal prepared by in-situ dual-doping of lanthanide complexes is used to add drugs into fluorescent DNA crystals, and the drug concentration can be quantified by comparing the luminescent color of the crystal with the chromaticity diagram.

[0010] In the application, the drug should have different effects on the fluorescence of the two complexes, that is, one is enhanced and the other is quenched, and the drug includes glutathione, curcumin, emodin, camptothecin, doxorubicin, indirubin or daunorubicin.

[0011] The beneficial effects of the present invention are: DNA crystals can be used as carriers with good biocompatibility. By loading rare earth complexes, hybrid luminescent crystals are obtained. The hybrid luminescent crystals can effectively detect drug concentrations. As the drug concentration changes, the luminescent color of the crystals changes, and the characteristic peak intensities of the two complexes change simultaneously. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 Optical microscope images of crystals formed from DNA solutions of different concentrations;

[0013] Figure 2 SEM images of crystals before (a) and after (b) cross-linking.

[0014] Figure 3 The effect of drug concentration after adjusting the fluorescence intensity of the complex to a level;

[0015] Figure 4 The effect of different drug concentrations on the luminescent color of the crystal;

[0016] Figure 5 The crystals were photographed under a confocal microscope using a 405nm light source to obtain red (a) and green (b) dual-channel crystal images, which proved that the complex was relatively uniform inside the crystal. The distribution of the complex was observed along the Z axis at 2μm each time (2μm, 4μm, 6μm, 8μm, 10μm). DETAILED DESCRIPTION

[0017] The present invention is described in detail below in conjunction with specific embodiments.

[0018] The DNA chain sequences used (5-end to 3-end) are:

[0019] A:CCGTACA CGTACA CCGTACA

[0020] B:GAGCAGCCTGTACGG ACATCA

[0021] C:TCTGATGTGGCTGC

[0022] The DNA strand used needs to be phosphorylated at the 5' end.

[0023] DNA crystal synthesis method: Centrifuge the three DNA chains at 4000pm for 5 minutes and add pure water to dissolve. Prepare the solution used for culture (30mM sodium cacodylate, 55mM magnesium chloride, 40mM sodium chloride, 25mM HEPES) and dilute it 10 times. Add 15 microliters of the above prepared solution, 1.4 microliters of water, 2 microliters of A chain, 6 microliters of B chain, and 6 microliters of C chain to the growth plate well. Seal with transparent tape and place in a 60℃ oil bath for 10 minutes, then transfer to a crystal incubator and cool naturally to 22℃. Let the crystals grow completely after standing for 3 days.

[0024] Example 1

[0025] Two complexes were synthesized in situ inside the DNA crystals, and the luminescence color of the crystals was regulated by changing the drug concentration.

[0026] Step (1) uses 10 -2 M,10 -3 M,10 -4 M Eu(TTA) 3 Phen solution and 10 -2 M,10 -3 M,10 -4 MTb(ACAC) 3 Phen solution was mixed in a 1:1 ratio and the fluorescence intensity was tested. The concentration ratio at which the two characteristic peaks were equal (i.e., the fluorescence intensity of the two complexes was similar, so that when affected by the drug, the two characteristic peaks changed significantly, and the corresponding fluorescence color of the crystal also changed significantly) was Eu(TTA). 3 phen:Tb(ACAC) 3 phen=10 - 4 M: 10 -3 M, and this concentration was used for the in situ synthesis of the complex in the crystal.

[0027] Step (2) Use a crystal picker to pick out DNA crystals with good morphology after cross-linking, wash the crystals in buffer, and transfer them to Eu 3+ and Tb 3+ In the mixed solution (EuCL 3 and TbCL 3 Ethanol solution, the concentration ratio is 10 -4 M: 10 -3 M), after 20 to 30 minutes, the crystals are removed and washed, and then transferred into a mixed solution of 2-thenoyltrifluoroacetone (TTA), o-phenanthroline (phen) and acetylacetone (acac). After 10 to 20 minutes, the crystals are transferred to a buffer solution to maintain the morphology.

[0028] Step (3) uses anhydrous ethanol as solvent to prepare drugs of different concentrations of 1 to 10 mM, and the drugs used are glutathione, curcumin, emodin, camptothecin, doxorubicin, indirubin, daunorubicin, etc. The drugs are added to the rare earth complex solution (1 to 100 mM) respectively, and the fluorescence intensity and fluorescence lifetime are detected after 10 to 60 minutes. The drugs are added to the fluorescent DNA crystal from low concentration to high concentration (0.01 mM to 10 mM) by dropwise addition, immersion, etc., and the same position is observed under a microscope and the fluorescence detection of the microscopic area is performed.

[0029] Figure 3 The complex concentration is adjusted to the concentration at which the fluorescence intensity is flat, and the change in crystal fluorescence intensity after loading the crystal with different concentrations of drugs (glutathione GSH). It can be seen that when the intensity of the characteristic peaks of the two complexes is flat, as the drug concentration increases, Eu(TTA) 3 The characteristic peak of phen gradually increases, while Tb(ACAC) 3 The characteristic peak of phen gradually weakened.

[0030] Figure 4 The changes in the luminescent color of the crystals loaded with two complexes at different GSH concentrations (0.02mM~0.1mM), (a) chromaticity diagram corresponding to the fluorescence color of the crystal, (b) 0.02mM GSH, (c) 0.04mM GSH, (d) 0.06Mm GSH, (e) 0.08mM GSH, (f) 0.1mM GSH; it can be seen that different GSH concentrations lead to different color changes of the crystals. Specifically, with the increase of GSH concentration, the crystals gradually change from green to yellow-green, then to orange-yellow, and finally to red. By comparing the crystal color with the chromaticity diagram, the drug concentration can be quantified.

[0031] Example 2

[0032] DNA crystals were directly loaded with the two complexes, but the crystals did not show any fluorescent properties.

[0033] Use 10 -2 M,10 -3 M,10 -4 M Eu(TTA) 3 Phen solution and 10 -2 M,10 -3 M,10 -4 MTb(ACAC) 3 Phen solution was mixed in a 1:1 ratio and the fluorescence intensity was tested. The concentration ratio of the two characteristic peaks was Eu(TTA). 3 phen:Tb(ACAC) 3 phen=10 -4 M: 10 -3 M, and this concentration was used for the in situ synthesis of the complex in the crystal.

[0034] Use a crystal picker to pick out DNA crystals with good morphology after cross-linking, wash the crystals in buffer, and transfer them to Eu(TTA) 3 After 20 to 30 minutes in phen solution, remove the crystals, wash them, and then transfer them into Tb(ACAC) 3 After 10-20 minutes in phen solution, the crystals were transferred to buffer to maintain the morphology. Using 365nm ultraviolet excitation, it was found that the crystals did not have any fluorescent properties, indicating that the two complexes could not be synthesized inside the crystals. This is because the solvent channels of the crystals are small. After the complexes are synthesized, the efficiency of penetrating into the crystals through the channels is extremely low. Most of them are attached to the surface of the crystals. Once the crystals are washed, they no longer emit light. Therefore, it is necessary to synthesize the complexes in situ in the crystals in steps.

[0035] Example 3

[0036] By changing the ratio of the two complexes and synthesizing the complex in situ inside the crystal, the luminescent color of the crystal can be regulated by changing the drug concentration.

[0037] Use 10 -2 M,10 -3 M,10 -4 M Eu(TTA) 3 Phen solution and 10 -2 M,10 -3 M,10 -4 MTb(ACAC) 3 The phen solution was mixed in a ratio of 1:2 and the fluorescence intensity was tested. The concentration ratio of the two characteristic peaks was equal to Eu(TTA). 3 phen:Tb(ACAC) 3 phen=10 -3 M: 10-3 M, and this concentration was used for the in situ synthesis of the complex in the crystal.

[0038] Use a crystal picker to pick out DNA crystals with good morphology after cross-linking, wash the crystals in buffer, and transfer them to Eu 3+ and Tb 3+ In the mixed solution (EuCL 3 and TbCL 3 Ethanol solution, concentration ratio 10 -3 M: 10 -3 M), after 20 to 30 minutes, the crystals are removed and washed, and then transferred into a mixed solution of 2-thenoyltrifluoroacetone (TTA), o-phenanthroline (phen) and acetylacetone (acac). After 10 to 20 minutes, the crystals are transferred to a buffer solution to maintain the morphology.

[0039] Using anhydrous ethanol as solvent, different concentrations of drugs of 1 to 10 mM were prepared. The drugs used were glutathione, curcumin, emodin, camptothecin, doxorubicin, indirubin, daunorubicin, etc. The drugs were added to the rare earth complex solution (1 to 100 mM) respectively, and the fluorescence intensity and fluorescence lifetime were detected after 10 to 60 minutes. The drugs were added to the fluorescent DNA crystal from low concentration to high concentration (0.01 mM to 10 mM) by dropwise addition and immersion, and the same position was observed under a microscope and the fluorescence detection of the microscopic area was performed. The drug concentration was detected by comparing the crystal color with the chromaticity diagram.

[0040] Example 4

[0041] Environmental factors experiments: pH, temperature.

[0042] Referring to the method of Example 1, 10 -4 M Eu(TTA) 3 Luminescent crystals of phen-loaded europium complexes, 10 -3 M Tb(ACAC) 3 Phen-loaded terbium complex luminescent crystals and 10 -4 M Eu(TTA) 3 phen and 10 -3 MTb(ACAC) 3 The luminescent crystals of the two complexes were loaded with phen in a ratio of 1:1.

[0043] The three crystals were stored at 20°C, 40°C, and 60°C for 15 minutes, and fluorescence tests were performed. It was found that the fluorescence intensity of the crystal loaded with a single complex decreased during the temperature increase. This is because the increase in temperature will increase the vibration and collision of the molecules, which may lead to an increase in non-radiative energy dissipation, thereby reducing the fluorescence intensity. The crystal loaded with a double complex contains a variety of metal ions and ligands, and these components interact and have synergistic effects. This interaction and synergistic effect can enhance the stability of the complex, and the fluorescence intensity does not change significantly during the temperature increase.

[0044] The three crystals were stored in an environment with a pH of 6, 8, and 10 for 15 minutes (because the crystals are easily broken and dissolved in an acidic environment, the alkaline environment was selected for testing), and fluorescence tests were performed. The results showed that the emission peaks of the crystals loaded with a single complex all shifted to a certain extent. The emission peak of the crystal loaded with europium shifted from 612nm to 623nm, and the emission peak of the crystal loaded with terbium shifted from 545 to 553nm. The emission peak of the crystal loaded with a double complex did not shift.

[0045] It should be understood that those skilled in the art can make improvements or changes based on the above description, and all these improvements and changes should fall within the scope of protection of the appended claims of the present invention.

Claims

1. A method for preparing hybrid luminescent crystals by in-situ dual doping of lanthanide complexes, It is characterized in that First, different concentrations of Eu(TTA) 3 Phen and Tb (ACAC) 3 phen two complex solutions are mixed in a certain ratio, and the concentration ratio that makes the fluorescence intensity of the two complexes equal is found, and Eu is prepared at this concentration ratio 3+ and Tb 3+ The mixed solution was transferred to the Eu 3+ and Tb 3+ After a period of time, the DNA crystals were removed, washed, and then transferred into a mixed solution of 2-thenoyltrifluoroacetone TTA, o-phenanthroline phen and acetylacetone ACAC. After a period of time, the crystals were transferred to a buffer solution to maintain the morphology, and a hybrid luminescent crystal prepared by in-situ dual-doped lanthanide complex was prepared; the DNA crystal synthesis method described: the three DNA chains were centrifuged at 4000pm for 5 minutes, and pure water was added to dissolve; the solution used for culture was prepared: 30mM sodium dimethyl cacoate, 55mM magnesium chloride, 40mM sodium chloride, 25mM HEPES, and diluted 10 times; 15 microliters of the above-prepared solution, 1.4 microliters of water, 2 microliters of A chain, 6 microliters of B chain, and 6 microliters of C chain were added to the growth plate well. After sealing with transparent tape, it was placed in an oil bath at 60°C for 10 minutes, and then transferred to a crystal incubator to naturally cool down to 22°C; the crystals were left to grow completely after standing for 3 days; the sequences of the three DNA chains were: A: CCGTACA CGTACA CCGTACA; B: GAGCAGCC TGTACGG ACATCA; C: TCTGATGTGGCTGC; The DNA strand used needs to be phosphorylated at the 5' end.

2. The method according to claim 1, It is characterized in that Different concentrations of Eu(TTA) 3 Phen and Tb (ACAC) 3 The two complex solutions of phen are mixed in a ratio of 1:1-2.

3. A hybrid luminescent crystal prepared by in-situ dual-doped lanthanide complex prepared by the method according to claim 1 or 2.

4. According to the use of the hybrid luminescent crystal prepared by in situ doped lanthanide complex as described in claim 3, a drug is added to the fluorescent DNA crystal, and the drug concentration can be quantified by comparing the crystal luminescence color with the chromaticity diagram; the drug is glutathione.

Images