CN114854032B - Preparation and application of water-soluble lanthanide AIE fluorescent nanoparticle - Google Patents
Preparation and application of water-soluble lanthanide AIE fluorescent nanoparticle Download PDFInfo
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- CN114854032B CN114854032B CN202210574958.6A CN202210574958A CN114854032B CN 114854032 B CN114854032 B CN 114854032B CN 202210574958 A CN202210574958 A CN 202210574958A CN 114854032 B CN114854032 B CN 114854032B
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
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Abstract
The invention discloses a preparation method of water-soluble lanthanide AIE fluorescent nanoparticles, which effectively combines the advantages of AIE and rare earth ions, takes TPE small molecule derivative TPE-3N containing terpyridine structure as a first ligand, takes 11, 10-phenanthroline as a second ligand, takes a third ligand PAA as a water-soluble unit, coordinates with rare earth ions Eu to obtain lanthanide complexes TPNE, and finally obtains the water-soluble lanthanide AIE fluorescent nanoparticles TPNE NPs through self-assembly. The aqueous solution of TPNE NPs emits blue/red double fluorescence near 435 nm and 615 nm, resulting from the fluorescent emission of the AIE small molecule TPE and Eu rare earth complex, respectively. Meanwhile, TPNE NPs have temperature responsiveness and low cytotoxicity, have excellent membrane targeting imaging capability on MKN-45 cancer cells, and exhibit reversible bicolor fluorescence imaging effects. In addition, TPNE NPs also show excellent reversible bicolor fluorescence imaging effect in living organism zebra fish, and can be used as a potential reversible bicolor bioluminescence probe.
Description
Technical Field
The invention relates to a preparation method of water-soluble lanthanide AIE fluorescent nanoparticles, and simultaneously relates to application of the water-soluble lanthanide AIE fluorescent nanoparticles as dual-color fluorescent probes in fluorescent imaging, belonging to the technical fields of nanomaterial preparation and biological imaging.
Background
In recent years, AIE has been reported in a large number. The AIE effect results from the Restriction of Intramolecular Motion (RIM) and the restriction into the dark state (RADS). RIM occurs when the luminophore is in a poor solvent or viscous environment, and therefore, due to steric hindrance, rotation within the molecule is limited, which blocks relaxation pathways for non-radiative energy loss, facilitating the pathway for radiative decay. Over the years, AIE research has been extended from traditional organic compounds to metal complexes. Metal complexes having the AIE effect mainly include gold, zinc, and iridium, but this phenomenon is rarely seen in lanthanide structures or lanthanide-containing metal complexes. Indeed, until now, there has been limited research and development of lanthanide materials with AIE properties. In general, the mechanism behind the AIE effect is related to limiting intramolecular motion (vibration-RIV or rotation-RIR), which minimizes losses due to non-radiative energy. Molecules with structural freedom of movement (rotation or vibration) are more susceptible to loss of non-radiative energy, such as pyridine units. The development of the rare earth complex bioluminescence probe with the advantages of both organic small molecules and metal nanoparticles having AIE characteristics greatly enriches the variety of the fluorescent probe and promotes the rapid development of bioluminescence imaging.
Disclosure of Invention
The invention aims to provide a preparation method of water-soluble lanthanide AIE fluorescent nanoparticles;
another object of the invention is to provide the use of the water-soluble lanthanide AIE fluorescent nanoparticles as dual-color fluorescent probes in fluorescence imaging.
1. Preparation of water-soluble lanthanide AIE fluorescent nanoparticles
(1) Synthesis of 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene)
4,4' -Dimethoxybenzophenone, 4-bromobenzophenone and zinc powder were added to Tetrahydrofuran (THF), the mixture was cooled to 0℃under a nitrogen atmosphere, and titanium tetrachloride (TiCl) was added dropwise 4 ) Then, heating and refluxing the mixture at 60-80 ℃ and stirring for 10-15 hours; after cooling to room temperature, the reaction was quenched with potassium carbonate solution, the mixture was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to give 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene), labeled TPE-Br. Wherein the molar ratio of the 4,4' -dimethoxy benzophenone to the 4-bromo benzophenone is 1:1-1:2; molar ratio of 4,4' -Dimethoxybenzophenone to Zinc powder1:3 to 1:5; the molar ratio of the 4,4' -dimethoxy benzophenone to the titanium tetrachloride is 1:3-1:4.
(2) Synthesis of 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan
TPE-Br, bis (triphenylphosphine) palladium dichloride (Pd (PPh) 3 ) 2 Cl 2 )、K 2 CO 3 And bis (pinacolato) diboron ester is added to anhydrous 1, 4-dioxane, the mixture is heated to reflux at 80 ℃ under nitrogen atmosphere and stirred for 20 to 25 hours, water is added and the mixture is rinsed with dichloromethane, and the organic phase is combined with MgSO 4 The solvent was dried and evaporated under reduced pressure and the residue was purified by silica gel column chromatography to give 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan, labeled TPE-B. Wherein the molar ratio of TPE-Br to bis (pinacolato) diboron ester is 1:1-1:2; the molar ratio of TPE-Br to bis (triphenylphosphine) palladium dichloride is 25:1-28:1; the TPE-Br and K 2 CO 3 The molar ratio of (2) is 1:3-1:4.
(3) Synthesis of 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl ] -4-yl) -2,2':6',2' ' -terpyridine
TPE-B and 4'- (4-bromophenyl) -2,2':6',2' -terpyridine were dissolved in dioxane, then Na was added 2 CO 3 Tetrakis (triphenylphosphine) palladium (Pd (PPh) 3 ) 4 ) Stirring at 100-120deg.C for 20-25 hr, cooling to room temperature after the reaction is completed, and using MgSO 4 Drying, evaporating the solvent under reduced pressure, purifying the residue by silica gel column chromatography to obtain 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl)]-4-yl) -2,2':6',2 "-terpyridine, labeled TPE-3N. Wherein the molar ratio of TPE-B to 4' - (4-bromophenyl) -2,2':6',2' ' -terpyridine is 1:1-1:1.5; the molar ratio of TPE-B to tetraphenylphosphine palladium is 1:0.02-1:0.04.
(4) Synthesis of Eu-containing ligands
TPE-3N, euCl 3 Adding ethanol and THF into phenanthroline (Phen) and polyacrylic acid (PAA), mixing, and dissolvingHeating to 60-80 ℃ in the reaction agent for 20-25 hours; cooling the reaction solution to room temperature, adding normal hexane for precipitation, centrifuging, purifying, and vacuum drying to obtain a Eu-containing ligand TPNE; wherein, the TPE-3N and EuCl 3 The molar ratio of (2) is 1:5-1:6; the molar ratio of TPE-3N to phenanthroline is 1:5-1:6; the molar ratio of TPE-3N to polyacrylic acid is 1:100. In the ethanol and THF mixed solvent, the volume ratio of ethanol to THF is 3:1.
(5) Preparation of TPNE nanoparticles
TPNE NPs are prepared by nano precipitation. And dissolving the TPNE in anhydrous THF, adding the obtained TPNE solution into ultrapure water under the action of ultrasound, performing ultrasound for 5min, stirring at room temperature for 5-15 min, removing THF, and filtering by using a water-based filter head with the thickness of 0.22 mu m to obtain the water-soluble lanthanide AIE fluorescent nano-particles TPNE NPs. Wherein the concentration of the TPNE solution is 50 mug/mL; the volume ratio of the TPNE solution to the ultrapure water is 1:5.
Luminescence of near infrared lanthanoids is easily extinguished by high-energy c—h vibration bonds in molecules and solvents. In contrast to small molecules, aggregated particles aggregate hundreds or thousands of lanthanide ions and antenna ligands within one cavity, which to some extent isolates the effective vibrational quenching of the molecule from the solvent. This helps to increase the luminous power of the lanthanide.
Therefore, as shown in fig. 1, the preparation process of the TPNE NPs is that TPE-3N is used as a first ligand, phen is used as a second ligand, PAA is used as a third ligand, and the TPNE NPs are coordinated with rare earth ions Eu, and finally, the water-soluble lanthanide complex TPNE is synthesized through self-assembly.
The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of TPE-Br are shown in figures 2 and 3; the nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of TPE-Br are shown in figures 4 and 5; the nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of TPE-3N are shown in figures 6 and 7.
2. Characterization of Water-soluble lanthanide AIE fluorescent nanoparticles
1. Infrared spectra of TPNE
In the coordination compound, rare earth ion Eu 3+ Is coordinated to the pyridine ring on TPE-3N, and can be determined by the variation of the peak on the IR spectrum. FIG. 8 is a block diagram of TPE-3N before and after complexationAn IR spectrum of TPNE of (c). As can be seen from the graph, the hydrocarbon vibration peak of the pyridine ring on TPE-3N is located at 721.38 cm -1 At the point, the characteristic absorption peak is located at 1406.10 cm -1 、1442.76 cm -1 At the position of Eu 3+ After coordination, move to 806 cm respectively -1 、1417.68 cm -1 、1452.40 cm -1 At this point, and the peak shape becomes wider. The results show that TPE-3N and Eu 3+ Coordination is performed, electron-withdrawing induction effect exists between the coordination and the coordination, electron cloud density among bonds is reduced, bond force constant is reduced, vibration frequency is reduced, and a peak moves towards a high wave number direction and moves.
2. Zeta potential of TPNE NPs
We measured the Zeta potential of TPNE NPs, as shown in FIG. 9, which shows that the TPNE NPs system is stable with a Zeta potential value of-19.1 mV.
XPS data of 3 TPNE NPs
（1）TPNE
XPS was used to confirm the surface element composition and bonding state of TPNE NPs. From FIG. 10 (a), it can be seen that TPNE NPs exhibit a Eu-3d, eu-4d peak, compared to TPE-3N, indicating that Eu has been coordinated to TPE-3N. As can be seen from FIGS. 10 (b-d), the binding energy of TPE-3N and TPNE NPs was not significantly changed, but the binding energy of N, O was changed, thereby proving Eu 3+ Coordination with N, O may occur. To more intuitively observe the changes before and after bonding, the atomic bonding energy data for TPE-3N and TPNE NPs are shown in Table 1. By analyzing the result of XPS, it was found that there was some change in the chemical environment of the element. N1s binding energy on the C-N bond increases by 1.33 eV, O1s binding energy on the C=O bond increases by 4.87. 4.87 eV, and shift of binding energy indicates Eu 3+ Coordinates to N, O element. Without any change in the binding energy of the C element. This is mainly because the change in the out-of-core electron distribution affects the shielding effect of the inner electrons, and electrons of nitrogen of the pyridine ring and electrons of oxygen of PAA in the complex are transferred to Eu 3+ On the outer empty orbit of (2), the electron density of the outer layer is reduced, the shielding effect is weakened, the binding energy of the inner layer electrons is increased, thereby proving N-Eu 3+ And O.fwdarw.Eu 3+ Formation of coordination bonds. In addition, the electron binding energy of Eu-3d (1128.2 eV) and Eu-4d (135.6 eV) appeared in XPS spectrum of TPNE NPs, further explaining the formation of Eu complexes.
4. Characterization of topography
Since PAA is hydrophilic and TPE-3N is hydrophobic, TPNE is prone to self-priming in a suitable solution
Assembling to form nano-sphere TPNE NPs. Therefore, THF is used as a solvent, deionized water is used as a dispersion medium, TPNE NPs particles are synthesized by a nano precipitation method, and the microstructure and composition of the TPNE NPs are observed by TEM and EDS. As can be seen from fig. 11 (a), the TPNE NPs were monodisperse, spherical in structure, smooth in surface, and approximately 150 a nm a in diameter. In addition, dynamic Light Scattering (DLS) was used to study the distribution of TPNE NPs in deionized water. As shown in fig. 11 (b), the particle size of the TPNE NPs was mainly between 60 and 210 and nm, the average diameter was +138.5 and nm, and it was observed from the inset that the aqueous solution of the TPNE NPs had a tyndall effect, which demonstrated that the TPNE NPs had formed nanoparticles in the aqueous solution.
In addition, a spectral transmission electron microscope (TEM/EDS) can analyze the composition of the material. Thus, to further analyze the elemental composition of the TPNE NPs, we tested the TPNE NPs using TEM/EDS. EDS spectra of elemental distributions of TPNE NPs are shown in FIG. 12 (a), which shows the presence of C, N, O and Eu in TPNE NPs 3+ It was further confirmed that TPNE NPs were successfully obtained. The X-ray spectroscopy elemental imaging analysis technique (EDS-Mapping) of fig. 12 (a') demonstrates a distribution analysis of key elements, consistent with our designed elemental distribution. Wherein C, O, N are the main components of the complex, and the content of lanthanide ions is relatively low, indicating Eu 3+ Coordination with TPE-3N has been successfully performed to form TPNE NPs.
3. Properties of TPE-3N
The photoelectric properties of the organic functional material are often adjusted using a combination of electron donating groups (D) and electron withdrawing groups (a). The invention designs the synthesized TPE-3N, takes TPE as D and terpyridine as A, constructs a D-A electronic push-pull system, reduces intermolecular energy gaps, promotes Intramolecular Charge Transfer (ICT), and enables emission wavelength to be red-shifted.
1. Optical Property study of TPE-3N
The optical properties of TPE-3N were well studied by UV-visible absorption and fluorescence emission spectroscopy (FIG. 13). As shown in fig. 13 (b), the uv-vis spectrum of TPE-3N in DMSO shows maximum absorption wavelength at 340 nm. In addition, the fluorescence emission spectrum of TPE-3N in DMSO was studied, with a maximum emission wavelength at 475 nm under excitation of 340 nm. Push-pull systems with donor and acceptor units may exhibit ICT characteristics due to dipole-dipole interactions. Thus, the solvent effect of TPE-3N in various solvents was investigated. The emission spectrum of TPE-3N in strongly polar solvents is broad and shows a pronounced red shift from weakly polar solvent (425 nm) to strongly polar solvent (475 nm) (fig. 13 (a)), which can be attributed to the highly polarized ICT excited state, an important phenomenon for D-a compounds. Thus, TPE-3N with D-A push-pull effect was successfully constructed.
2. AIE Property study of TPE-3N
TPE is a classical AIE small molecule, so we examined the AIE properties of TPE-3N. DMSO is selected as a good solvent, and water (H) 2 O) as poor solvent, DMSO/H with different volume fractions were tested respectively 2 Fluorescence change of TPE-3N in O mixed solvent. As shown in FIG. 14 (a), poor solvent H was added dropwise to a DMSO solution of TPE-3N 2 In the course of O, it is observed that with H 2 The increase in the volume fraction of O, the fluorescence intensity of TPE-3N increases gradually in a nonlinear degree (FIG. 14 (b)), and when H 2 After the volume fraction of O is increased to 30%, the fluorescence intensity of the TPE-3N solution is obviously enhanced, and after the volume fraction reaches 60%, the fluorescence intensity of the TPE-3N solution is greatly enhanced again until the fluorescence intensity reaches the highest value when the volume fraction reaches 90%. The fluorescence intensity is increased by more than 24 times at maximum. This phenomenon may be due to molecules of TPE-3N in DMSO solventInternal rotation is active, which is a relaxation channel of decay of the excited state. However, when a large amount of water is added, aggregates of TPE-3N begin to form, and intramolecular rotation is limited due to physical constraints, and thus aggregation-induced emission occurs. This is consistent with the AIE luminescence properties of TPE molecules reported in the literature. Thus, TPE-3N has excellent AIE luminescence properties.
4. Properties of TPNE NPs
1. Optical Properties of TPNE NPs
FIG. 15 (b) is the UV-visible absorption and fluorescence emission spectra of TPNE NPs. Its uv-vis spectrum in THF shows a maximum absorption wavelength at 300 nm. Two emission peaks in 430 nm and 615 nm appear in the fluorescence emission spectrum, the emission peak at 430 nm being generated by the TPE derivative and the emission peak at 615 nm being Eu 3+ Is characterized by an emission peak. Wherein a small peak of 595, 595 nm is located beside the peak at 615, 615 nm, corresponding to Eu respectively 3+ Magnetic dipole transition 5 D 0 → 7 F 1 ) And Eu 3+ Transition of electric dipole 5 D 0 → 7 F 2 ) In particular, a sharp emission peak is shown at 615 nm, which is due to Eu 3+ The ultrasensitive transition of ions has better monochromaticity.
TPNE NPs can be dissolved in different solvents, and for the next step of experiment we selected acetic Acid (ACOH), methanol (CH 3 OH), THF, N-Dimethylformamide (DMF), acetone (DMK) as solvents. As shown in fig. 15 (a), TPNE NPs have the sharpest emission peak in THF under the same test conditions in different solvents, and the fluorescence intensity is the greatest, so the following experiments were all tested with THF as the solvent.
Fluorescence quantum yield (. Phi.) f ) Is a measure of the amount of fluorescence of the fluorescent substance. Phi of TPNE NPs was determined by reference method f The quinine sulfate aqueous solution (phi) is selected f =55%) as a reference for the fluorescent emission of TPNE NPs at 430 nm; rhodamine B (phi) f =97%) as a reference for the fluorescent emission of TPNE NPs at 615 nm. Such asFIG. 4 shows Φ of TPNE NPs at 430 nm, 615 nm f 8.8% and 17.1% respectively. Higher Φ of TPNE NPs f Is favorable for biological imaging experiments, phi f Calculation was performed using equation 1:
（1）Φ f =Φ s ×(F U /Fs)×(As/A U )
Φ f is the fluorescence quantum yield of the sample to be detected, phi s is the fluorescence quantum yield of the reference substance, F U Fs is the fluorescence integration area of the dilute solution of the sample to be tested and the reference sample, A U As is the maximum absorbance value of the excitation wavelength of the sample to be detected and the reference sample.
2. AIE Performance study of the Complex
We believe that when TPE-3N with AIE properties is used as ligand, the resulting complex should also have AIE activity. Based on this, we examined the AIE properties of TPNE NPs. THF is selected as a good solvent, CH 2 Cl 2 THF/CH with different volume fractions were tested as poor solvents 2 Cl 2 Double fluorescence change of TPNE NPs in mixed solvent. As shown in FIG. 16 (a), the poor solvent CH was added dropwise to the THF solution of TPNE NPs 2 Cl 2 In the course of (2), it can be observed that following CH 2 Cl 2 The fluorescence of TPNE NPs gradually increases in a nonlinear degree (FIG. 16 (b)) and when CH 2 Cl 2 After increasing the volume fraction to 70%, the fluorescence intensity of the TPNE NPs solution was significantly increased, with blue fluorescence being increased to a much greater extent than red fluorescence, up to 3.8-fold increase, without such a significant increase in red fluorescence. This demonstrates that the change in the aggregation level of TPNE NPs in the system has a significant effect on blue fluorescence. This is mainly because in good solvents, the TPNE NPs are in a stretched state, which gives the complex a large space for the TPE molecules to move, and TPE can vibrate through four benzene rings to make most of the energyThe amount is released as thermal energy resulting in a decrease in fluorescence emission. While when poor solvent CH is added into THF good solution of TPNE NPs 2 Cl 2 After that, as the solubility thereof decreases, the molecules gradually form aggregates, the rotation within the molecules is suppressed, and the non-radiative transition thereof is hindered, thereby enhancing the fluorescence intensity, and the fluorescence intensity also takes a form of continuous enhancement as the aggregation degree increases.
To further investigate the AIE properties of TPNE NPs, we again observed their fluorescence emission at different concentrations. As shown in FIG. 16 (c, d), with 1X 10 -4 The fluorescence of TPNE NPs increases gradually in a nonlinear fashion with increasing concentrations of mg/mL to 50 mg/mL, and after reaching a concentration of 1 mg/mL, the red fluorescence emission intensity is greater than the blue fluorescence emission intensity. As can be seen from FIG. 16 (d), the red fluorescence increases to a much greater extent than the blue fluorescence, with the red fluorescence increasing up to 390 times, much more than the blue fluorescence in THF/CH 2 Cl 2 Fold increase in solvent. The extent to which the red fluorescence increases by such a high factor means that this may not only be the AIE effect acting, but also the AE effect contributing to it. However, after reaching a concentration of 20 mg/mL, the blue fluorescence intensity of TPNE NPs decreased. This is probably because the size of the TPNE NPs molecule aggregates increases with increasing concentration. Only the molecules on the surface of the ligand can be excited and fluoresce. A large number of molecules of the ligand core are not excited or the fluorescence emitted by these molecules is reabsorbed by the outer molecules. Thus, when the concentration of TPNE NPs reached 20 mg/mL, the blue fluorescence intensity decreased.
TPNE NPs have excellent dual-color fluorescent AIE luminescence properties, which would be advantageous for subsequent imaging experimental applications in cells and zebra fish.
3. AE effect
To verify our hypothesis that there is an AE effect on TPNE NPs, we further performed a verification experiment. The control PPE was synthesized using the same experimental conditions (EuCl 3 (15.50 mg,0.06 mmol), phen (10.80 mg,0.06 mmol), PAA (1 mmol,2 g, mn=2000 g/mol) was added to the mixture containing 9.00In a round bottom flask of mL of a THF mixed solvent of EA and 3.00. 3.00 mL, then heated to 70 ℃, and the reaction was stopped after 24 hours. The reaction solution was cooled to room temperature, precipitated with 100.00 mL N-hexane, centrifuged and purified three times, and finally vacuum-dried in a vacuum oven for 24 hours to obtain a pink solid ligand PAA-Phen-Eu (PPE)), as shown in FIG. 17, after TPE-3N was introduced, the fluorescence of TPNE NPs at 615 nm was enhanced, and the fluorescence intensity was significantly higher than that of PPE and EuCl 3 . This is mainly due to Eu 3+ Is weak in light absorption capacity, and at the same time, its 4f-4f transition is suppressed, so Eu 3+ Is very weak. However, TPE-3N contains unsaturation in the molecule and pi electrons in the carbon-carbon double bond, which results in pi-pi transition. Since pi electrons are easily excited and pi orbitals have low energies, the excitation energy required for this transition is low. Furthermore, the molar absorption coefficient of pi-pi transition is very large. After TPE-3N is excited by ultraviolet light, free electrons in the molecule go from the ground state (S 0 ) Transition to the first excited singlet state (S 1 ) Then through intersystem crossing from S 1 Transition to the first electronically excited triplet (T 1 ). TPE-3N then will T 1 Energy transfer of state to Eu 3+ ，Eu 3+ Sufficient energy is available to transition its electrons from the ground state to the excited state. Electrons in the excited state return to the ground state in the form of a radiation transition, thereby emitting Eu 3+ Intrinsic fluorescence of (2). This demonstrates TPE-3N and Eu 3+ There is an antenna effect in between.
4. Fluorescence lifetime
Rare earth luminescent materials have the advantage of a longer fluorescence lifetime. This can be further confirmed by the following fluorescence lifetime experiments. In a different sensitization process, energy transfer from the ligand triplet excited state to the lanthanide excited state (T 1 →ln) is a "ligand S 1 Ligand T 1 The energy flow of Ln @, light is first absorbed into the singlet excited state of the antenna (S 0 →S 1 ) Then undergo intersystem crossing into a ligand triplet excited state (S 1 →T 1 ). Then energy is transferred from T of antenna 1 The state transitions to the excited state of the lanthanide (Ln) and eventually returns to the ground state of the lanthanide. The prohibition of 4f-4f conversion allows Eu to be converted from 3+ The relaxation of the excited state is slow. Thus, lanthanide complexes generally exhibit longer decay lifetimes, which can be as long as microseconds and milliseconds, compared to nanoseconds for organic molecules.
The fluorescence lifetime of TPNE NPs is shown in FIG. 18. The fluorescence lifetime of the TPNE NPs was 1.345. 1.345 ms, indicating the presence of an effective antenna effect within the system of TPNE NPs. The long lifetime of TPNE NPs enables time-gated and time-resolved fluorescence lifetime measurements, which can facilitate differentiation from cell autofluorescence (ns) and improve signal-to-noise ratio. Fluorescence lifetime was calculated using equation 2:
wherein B is 1 、B 2 And B 3 Is a constant; τ is time, in ns; τ 1 、τ 2 、τ 3 Is the fit lifetime in ns.
5. Double fluorescence emission
On the one hand, TPNE NPs comprise TPE small molecules which emit blue light and rare earth Eu which emits red light 3+ The two luminescent units of the luminescent complex can not generate FRET phenomenon if the two luminescent units have larger difference between emission wavelength and excitation wavelength and do not overlap, so that the TPNE NPs can have double fluorescence emission performance.
In order to observe the change of the double fluorescence more clearly, we select DMK as solvent here to reduce the red fluorescence emission at 615 and nm, so that the change of the blue fluorescence is more prominent. FIGS. 19 (a-c) are
Fluorescence spectra of 2 mg/mL TPNE NPs DMK solution at different excitation wavelengths within the range of 350-400 nm. As shown in fig. 19, excitation of 395 nm enables optimal sensitized metal center emission from Eu, which results in a characteristic red chromaticity. However, while maintaining all other parameters, the excitation wavelength varies between 350 and 400 nm, moving in the short wavelength direction with the excitation wavelengthResulting in reduced contribution from Eu, enhanced ligand-based emission, and a large variation in the observed chromaticity. At lambda ex At=395 nm, a source of origin is observed 5 D 0 → 7 F J The converted intense Eu emission, the ligand does not contribute significantly. Excitation at 380 nm causes the emission intensity of Eu to decrease and the emission contribution of the ligand to increase, and as the excitation wavelength continues to decrease to 350 nm, a further decrease in emission intensity of Eu is observed while the emission intensity of the ligand increases at 430 nm, rendering the system blue in color. FIG. 19 (d) shows the CIE chromaticity diagram of TPNE NPs, which chromaticity shifts from blue to red under excitation at 350-400 nm. Thus, TPNE NPs were demonstrated to have bi-color fluorescence emission properties.
6. Temperature responsiveness
In addition, to examine the condition of the double fluorescence of TPNE NPs with temperature, we performed a temperature-variable fluorescence spectrum test on DMF solution of TPNE NPs, and the results are shown in FIG. 20. It can be observed from the trend graph of the dual fluorescence intensity with temperature that the blue fluorescence intensity and the red fluorescence intensity both decrease linearly with increasing temperature. At a lower temperature, the TPE molecules are frozen, movement is limited, energy is difficult to release in a non-radiative transition mode, so that radiative transition is increased, fluorescence intensity is higher, rotation of the TPE molecules is accelerated after the temperature is increased, and at the moment, energy is released in a non-radiative transition mode, so that blue fluorescence is reduced. And AE effect existing in TPNE NPs system, eu after blue fluorescence intensity is reduced 3+ Sufficient energy is not available for excitation and therefore the red fluorescence intensity is also reduced. It can be seen that TPNE NPs have excellent temperature responsiveness.
5. Imaging experiments and cytotoxicity
Cell culture, cytotoxicity detection and cell imaging experiments were as follows:
(1) Cell culture
Cell lines: human gastric cancer cells (MKN-45) are supplied by the basic medical college of Gansu university of traditional Chinese medicine.
Cell resuscitation: taking out the frozen MKN-45 cancer cells, and placing in hot water at 37 ℃ for constant temperatureAfter thawing, the suspension was aspirated into a centrifuge tube, and 10.00 a mL a of cell culture solution, which had been prepared in advance, was added. Centrifuging at 1500 r/min for 5.00 min, discarding supernatant, adding 10.00 mL high sugar culture medium (DMEM), washing, discarding supernatant, adding mixed liquid containing 12% south America Fetal Bovine Serum (FBS) DMEM and 100 u/mL "double antibody" (penicillin/streptomycin), blowing and inoculating in culture medium, and standing at 37deg.C under wet CO 2 And (5) standing and culturing in an incubator.
Cell culture and passage: the required instrument is sterilized in autoclave for use. And observing the cell density under a microscope, and when the density reaches 80-90%, passaging is carried out. Pouring out the stock culture solution, washing with Phosphate Buffered Saline (PBS) for three times, immediately adding 2.00 mL DMEM culture solution containing 12% FBS after 0.25% trypsin digests for three minutes to stop digestion, blowing and centrifuging at 1500 r/min for 5min, discarding the solution, adding 2.00 mL DMEM culture solution containing 12% FBS, placing into culture medium, placing into 37 ℃ and wetting with CO 2 Culturing in an incubator.
Cell cryopreservation: taking cancer cells growing in logarithmic phase, removing stock culture solution, cleaning with PBS for three times, adding appropriate amount of trypsin, centrifuging at 1500 r/min for 5min, adding pre-prepared frozen stock solution, blowing, counting, and concentrating to a density of 5×10 6 ~1×10 7 And (3) transferring the mixture into a freezing storage tube and storing the mixture in a refrigerator at the temperature of-80 ℃ at the time of/mL.
(2) Cytotoxicity detection
For cell activity assays, samples were evaluated for cytotoxicity using the (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) (MTT) method. Trypsin digested MKN-45 cells were grown at 10 4 The MTT assay was performed after incubation for 24 hours in 96-well plates at a concentration of one/mL under conventional cell culture conditions. The experiment is divided into 5 experiment groups and 1 control group, the control group is added with DMEM, the experiment groups are respectively added with samples with different concentrations, and the samples are placed under the conventional cell culture condition for continuous culture for 24 h, and then added with the concentration of 5 mg/mL thiazole blue (MTT) solution was placed into incubator for continuous cultivation of 4 h, the solution was aspirated, and 150.00. Mu.L of dimethyl sulfoxide (DMSO) solution was added to each well and shaken for 10min. Finally, the cell activity was evaluated by a microplate reader based on absorbance (OD) values at 490 and nm in 3 experiments in parallel, the following formula was used to calculate the survival rate of cell growth:
cell survivinal% = ODr/ODc ×100% (ODr mean absorbance measured in experimental wells, ODc mean absorbance measured in control group)
(3) Cell imaging experiments
For cell imaging, the cell slide with 24-well plate was inoculated with the logarithmic phase pancreatin digested MKN-45 cells, and cultured under conventional cell culture conditions after DMEM medium was added thereto for 24 h. Samples of 100 μg/mL were added to each well, incubated for 24 h, and rinsed three times with PBS buffer. Finally, cell observations and photographs were taken under a zeiss Axio scope. A1 forward fluorescence microscope.
The cytotoxicity characteristics of fluorescent materials are critical to their use as luminescent biological probes, and cytotoxicity testing is an important prerequisite for successful or unsuccessful cell imaging experiments. To examine whether TPNE NPs are biocompatible, we performed a standard MTT assay to determine the data of the cellular activity assay of TPNE NPs in human gastric cancer cell MKN-45. As can be seen from FIG. 21, MKN-45 cancer cells still retain more than 90% of their activity at concentrations as high as 100. Mu.g/mL. Thus, TPNE NPs can be used in cell imaging experiments.
In addition, fluorescence microscopy photographs of cells at different excitation wavelengths were taken with a fluorescence microscope after 24 hours of incubation of MKN-45 cancer cells with 100. Mu.g/mL of TPNE NPs. As shown in fig. 22 (a), at λ ex =360 nm and λ ex When=395 nm, MKN-45 cancer cells displayed a blue fluorescence photograph under the blue channel and a red fluorescence photograph under the red channel, respectively. In the figure, it can be seen that TPNE NPs attach to cell membranes after uptake by MKN-45 cancer cells, and fluorescence intensity is stronger in the cell membrane region. While TPNE NPs are water-soluble and are pellets covered with hydrophilic groups, thus being in an aqueous environmentThe fluorescence of MKN-45 cell boundaries is likely due to the specific interaction of TPNE NPs with proteins on cell membranes by electrostatic attraction, showing negative charges. Meanwhile, good water solubility is also a necessary condition for avoiding cell absorption, and the TPNE NPs are ensured to be fully and firmly contacted with the cell membrane, so that the imaging capability of the cell membrane is further enhanced. In addition, in the test process, the intensity of the emitted blue/red double-color fluorescence on the cell membrane is still unchanged under the condition of long-time irradiation of the dyed cells, which shows that the prepared TPNE NPs have good photobleaching resistance and can perform long-time fluorescence imaging of the cell membrane. These results indicate that the obtained TPNE NPs with high photostability, low cytotoxicity and superior biocompatibility can be used as fluorescent probes for cell membrane imaging.
To verify the application of TPNE NPs in biology, we further performed zebra fish imaging experiments, as shown in fig. 22 (b), photographs of TPNE NPs after staining zebra fish also showed blue/red dual channel imaging under a fluorescence microscope, which is consistent with the results of cell imaging experiments. Thus, blue fluorescence and intense red fluorescence clearly demonstrate the potential use of TPNE NPs in high contrast bioimaging applications. I.e. organisms stained by TPNE NPs, have the ability to achieve blue/red bi-colour fluorescence imaging by adjusting the different excitation wavelengths. It can be seen that TPNE NPs are an ideal bioluminescent probe based on their low toxicity and good cell membrane imaging ability.
In summary, the TPE derivative (TPE-3N) with a terpyridine structure and coordination ability is used as a first ligand, 1, 10-phenanthroline is used as a second ligand, a third ligand PAA is used as a water-soluble unit, and coordination is carried out on the third ligand PAA and rare earth ions Eu, so that a lanthanide complex TPNE with water solubility is synthesized; finally, obtaining the water-soluble lanthanide AIE fluorescent nanoparticle TPNE NPs through self-assembly. TEM and DLS test results show that the TPNE NPs are pellets of 150 nm, and the particle size ranges from 120 to 180 nm. TPNE NPs are AIE-type NPs with fluorescence lifetimes as long as 1.345 ms, emitting blue/red bifluorescence around 430 nm and 615 nm with blue fluorescence quantum yields of about 8.8% and red fluorescence quantum yields of about 17.1%. In addition, TPNE NPs have temperature responsiveness, biocompatibility and low cytotoxicity. Biological imaging experimental results show that TPNE NPs have excellent membrane targeting imaging capability on MKN-45 cancer cells and exhibit excellent reversible bicolor fluorescent imaging effects. In addition, TPNE NPs also show excellent reversible bicolor fluorescence imaging effect in living organism zebra fish, and can be used as a potential reversible bicolor bioluminescence probe.
Drawings
FIG. 1 is a synthetic route map of TPNE and TPNN, and self-assembly of TPNE;
FIG. 2 shows the nuclear magnetic hydrogen spectrum (600 MHz, CDCl) of TPE-Br 3 ）；
FIG. 3 shows the nuclear magnetic carbon spectrum (151 MHz, CDCl) of TPE-Br 3 ）；
FIG. 4 shows the nuclear magnetic hydrogen spectrum (600 MHz, CDCl) of TPE-B 3 ）；
FIG. 5 shows the nuclear magnetic carbon spectrum (151 MHz, CDCl) of TPE-B 3 ）；
FIG. 6 shows a nuclear magnetic hydrogen spectrum (400 MHz, CDCl) of TPE-3N 3 ）；
FIG. 7 shows a nuclear magnetic carbon spectrum (151 MHz, CDCl) of TPE-3N 3 ）；
FIG. 8 is a Fourier infrared spectrum of TPNE;
FIG. 9 is a Zeta potential diagram of TPNE NPs;
FIG. 10 is a graph of the overall XPS binding energy profile (a), C1s profile (b), N1s (C) and O1s (d) for TPE-3N before complexation and TPNE NPs after complexation;
FIG. 11 is a TEM image (a) and DLS image (b) of TPNE NPs (b) inset: a photograph of the Tyndall effect of TPNE NPs under laser light irradiation;
FIG. 12 TEM-EDS element scan spectrum of TPNE NPs (a); (a') a face scan picture corresponding to the four elements C, O, N and Eu on TPNE NPs;
FIG. 13 (a) fluorescence spectra of TPE-3N in different solvents; (b) Ultraviolet absorption spectrum (left) and fluorescence emission spectrum (right) of TPE-3N. (solution: DMSO,. Lambda. ex =340 nm，1×10 -2 M)；
FIG. 14 (a) fluorescence spectra of TPE-3N at different water and DMSO volume ratios, (b) corresponding trend of fluorescence intensity for TPE-3N solutions at different water and DMSO volume ratios;
FIG. 15 (a) fluorescence spectra of TPNE NPs in different solvents; (b) Ultraviolet absorbance spectra (left) and fluorescence emission spectra (right) of TPNE NPs. (lambda) ex =360 nm，2 mg/mL)；
FIG. 16 (a) different volume fractions CH 2 Cl 2 Fluorescence spectrum (lambda) of TPNE NPs solution ex =360 nm,2 mg/mL) (inset: TPNE NPs in different CH 2 Cl 2 Volume fraction of CH 2 Cl 2 Photo of ultraviolet light (365, nm) in THF mixture, (b) different volume fractions CH 2 Cl 2 Corresponding trend of fluorescence intensity of TPNE NPs solution; (c) Fluorescence spectra (lambda) of TPNE NPs solutions of different concentrations ex =360 nm) (inset: photographs of TPNE NPs under uv light (365, nm) irradiation in different concentrations; (d) Corresponding change trend of fluorescence intensity of TPNE NPs solutions with different concentrations;
FIG. 17EuCl 3 Fluorescence spectrum of PPE, TPNE NPs (λex=360 nm,2 mg/mL) (inset: euCl) 3 Photographs of PPE, TPNE NPs under uv light (365 nm);
FIG. 18 fluorescence lifetime plot of TPNE NPs;
FIG. 19 (a, c) is a graph of dual fluorescence of TPNE NPs under different excitations, (b) is a graph of trend of dual fluorescence of TPNE NPs under different excitations; (d) The location of the bifluorescence color of the TPNE NPs solution on the standard colorimetric plate under different excitations;
FIG. 20 (a) fluorescence spectra of TPNE NPs solutions at different temperatures, (b) trend of variation of fluorescence intensity of TPNE NPs solutions at different temperatures;
FIG. 21 toxicity test of MKN-45 cancer cells treated with TPNE NPs at various concentrations for 48 hours;
FIG. 22 (a) blue/red dual channel fluorescence microscopy images (100 μg/mL) of MKN-45 cancer cells stained 24 h with TPNE NPs, and (b) zebra fish blue/red dual channel fluorescence microscopy images stained with TPNE NPs.
Detailed Description
The preparation method of the water-soluble lanthanide AIE fluorescent nanoparticle of the present invention is described in further detail below by way of specific examples.
The reagents used in the present invention are as shown in Table 3:
the apparatus used in the present invention is shown in Table 4:
examples
(1) Synthesis of 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene) (TPE-Br)
4,4' -Dimethoxybenzophenone (4.84 g,20.00 mmol), 4-bromobenzophenone (6.26 g,24.00 mmol) and zinc powder (5.20 g,80.00 mmol) were added to a 500.00 mL round bottom flask, which was evacuated under vacuum and purged three times with dry nitrogen. After addition of 250.00. 250.00 mL Tetrahydrofuran (THF), the mixture was cooled to 0deg.C and titanium tetrachloride (TiCl) was added dropwise 4 ) (7.70 mL,70.00 mmol). The mixture was then heated to reflux at 70 ℃ and stirred for 12 hours. After cooling to room temperature, potassium carbonate (K) 2 CO 3 ) The reaction was quenched with solution and the mixture was extracted three times with dichloromethane, the organic phases were combined, and dried over anhydrous magnesium sulfate (MgSO 4 ) And (5) drying. The solvent was evaporated under reduced pressure and the residue purified by silica gel column chromatography using ethyl acetate/hexane (v/v 1:40) as eluent to give 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene) (TPE-Br) as a white solid, yield: 76 Percent of the total weight of the composition.
1 H NMR (600 MHz, Chloroform-d) δ(ppm): 7.27-7.20 (m, 2H), 7.14-7.06 (m, 3H), 7.04-6.99 (m, 2H), 6.97-6.92 (m, 4H), 6.92-6.88 (m, 2H), 6.67 (dd, 2H), 6.66–6.61 (m, 2H), 3.74 (d, 6H).
13 C NMR (151 MHz, Chloroform-d) δ(ppm): 206.81, 158.31, 158.22, 143.81, 143.33, 140.82, 137.91, 136.01, 135.93, 133.06, 132.56, 132.53, 131.35, 130.86, 127.83, 126.32, 120.03, 113.23, 113.05, 55.11, 55.07.
ESI-MS: m/z calcd for C 28 H 23 O 2 Br [M] + , 470.0881; found 470.0876.
(2) Synthesis of 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan (TPE-B)
TPE-Br (3.77 g,8.00 mmol), bis (triphenylphosphine) palladium dichloride (Pd (PPh) 3 ) 2 Cl 2 )（0.21 g，0.30 mmol）、K 2 CO 3 (4.15 g,30.00 mmol) and bis (pinacolato) diboron ester (3.05 g, 12.00 mmol), anhydrous 1, 4-dioxane (40.00 mL) was added to a 100.00 mL two neck round bottom flask which was evacuated under vacuum and purged three times with dry nitrogen. The mixture was heated to reflux at 80 ℃ and stirred under nitrogen for 24 hours. Then, water was added, and the mixture was washed with dichloromethane (CH 2 Cl 2 ) And flushing for three times. The organic phases were combined with MgSO 4 Drying, evaporation of the solvent under reduced pressure, purification of the residue by silica gel column chromatography using dichloromethane/hexane (v/v 1:2) as eluent gave 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan (TPE-B) as a white solid, yield: 55 Percent of the total weight of the composition.
1 H NMR (600 MHz, Chloroform-d) δ(ppm): 7.57-7.52 (m, 2H), 7.12-7.05 (m, 3H), 7.05-6.98 (m, 4H), 6.96–6.91 (m, 4H), 6.65-6.60 (m, 4H), 3.73 (d, 6H), 1.32 (s, 12H).
13 C NMR (151 MHz, Chloroform-d) δ(ppm): 158.13, 158.10, 147.40, 144.12, 140.58, 139.12, 136.29, 136.17, 134.10, 132.58, 131.39, 130.89, 130.75, 128.83, 127.65, 126.08, 113.07, 112.98, 83.64, 55.05, 55.03, 24.89.
ESI-MS: m/z calcd for C 34 H 35 O 4 B [M] + , 518.2628; found 518.2634
(3) Synthesis of 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl ] -4-yl) -2,2':6',2' ' -terpyridine (TPE-3N)
TPE-B (518.26 mg,1 mmol) and 4' - (4-bromophenyl) -2,2':6',2 "-terpyridine (465.92 mg,1.20 mmol) were dissolved in 30.00 mL dioxane, then Na was added 2 CO 3 (2M, 3.00 mL), tetrakis (triphenylphosphine) palladium (Pd (PPh) 3 ) 4 ) (34.00 mg,0.03 mmol) stirring at 110deg.C for 24 hr, cooling to room temperature after the reaction, and using MgSO 4 Drying, evaporation of the solvent under reduced pressure and purification of the residue by silica gel column chromatography using methanol/dichloromethane (v/v 1:20) as eluent gave 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl) as a yellow solid]-4-yl) -2,2':6',2 "-terpyridine (TPE-3N), yield: 66 Percent of the total weight of the composition.
1 H NMR (400 MHz, Chloroform-d) δ(ppm): 7.62-7.49 (m, 4H), 7.41-7.33 (m, 6H), 7.18-7.03 (m, 11H), 7.02-6.91 (m, 5H), 6.70-6.60 (m, 5H), 3.75 (s, 6H).
13 C NMR (151 MHz, Chloroform-d) δ(ppm): 166.94, 160.17, 158.93, 155.94, 149.01 (2C), 147.11, 145.40, 143.04, 141.13, 140.05, 137.06, 136.57, 135.49, 134.20, 132.50, 132.10,132.00 (2C), 130.93 (2C), 130.43, 130.14, 129.01, 128.89 (2C), 128.56, 127.60, 127.15, 126.36, 125.86, 124.78, 123.83, 123.54, 122.75, 121.48, 121.46 (2C), 120.68, 119.76, 118.64, 118.02, 117.40, 114.41, 112.83 (2C), 65.56 (2C).
ESI-MS: m/z calcd for C 49 H 38 O 2 N 3 [M+H] + , 700.2826; found 700.2958
(4) Synthesis of Eu-containing Complex (TPNE)
Europium chloride (EuCl) 3 ) Is prepared from the following steps: europium oxide (Eu) 2 O 3 ) (3.51 g,1.00 mmol) was placed in a clean beaker, concentrated hydrochloric acid (HCl) was added dropwise to about 20.00. 20.00 mL, followed by a small amount of ammonium chloride (NH) 4 Cl) powder (with a small amount of reaction product water removed). Heating and boiling until the mixture is almost dry for standby.
TPE-3N (6.90 mg,0.01 mmol), euCl 3 (15.50 mg,0.06 mmol), phenanthroline (Phen) (10.80 mg,0.06 mmol) and polyacrylic acid (PAA) (1.00 mmol,2.00 g,Mn =2000 g/mol) were added to a round-bottomed flask containing a mixed solvent of ethanol 9.00 mL and THF 3.00 mL, followed by heating to 70 ℃ and stopping the reaction after 24 hours. After the reaction solution is cooled to room temperature, 100.00 mL normal hexane is added for precipitation, centrifugal purification is carried out for three times, and finally, the reaction solution is placed in a vacuum drying oven for vacuum drying for 24 hours to obtain pink solid TPNE, and the yield is: 64 Percent of the total weight of the composition.
(5) Preparation of TPNE Nanoparticles (NPs)
TPNE NPs are prepared by nano precipitation. TPNE was dissolved in anhydrous THF to give a solution with a concentration of 1 mg/mL. After diluting the solution to 50. Mu.g/mL, 5mL of the solution was poured into a cuvette containing 10.00/mL of ultrapure water under ultrasonic action and sonicated for 5min, followed by placing in a fume hood and stirring at room temperature for 10min. After THF was removed, the mixture was filtered through a 0.22 μm aqueous filter head to obtain TPNE NPs.
Claims (2)
1. A preparation method of water-soluble lanthanide AIE fluorescent nanoparticles, which comprises the following steps:
(1) Synthesis of 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene): adding 4,4' -dimethoxy benzophenone, 4-bromo-benzophenone and zinc powder into tetrahydrofuran, cooling the mixture to 0 ℃ under nitrogen atmosphere, dropwise adding titanium tetrachloride, and then heating and refluxing the mixture at 60-80 ℃ and stirring for 10-15 hours; after cooling to room temperature, the reaction was quenched with potassium carbonate solution, the mixture was extracted with dichloromethane, the organic phases were combined, dried over anhydrous magnesium sulfate, the solvent was evaporated under reduced pressure and the residue was purified by silica gel column chromatography to give 4,4' - (2- (4-bromophenyl) -2-phenylethene-1, 1-diyl) bis (methoxybenzene), labeled TPE-Br;
the molar ratio of the 4,4' -dimethoxy benzophenone to the 4-bromo benzophenone is 1:1-1:2; the molar ratio of the 4,4' -dimethoxy benzophenone to the zinc powder is 1:3-1:5; the molar ratio of the 4,4' -dimethoxy benzophenone to the titanium tetrachloride is 1:3-1:4;
(2) Synthesis of 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan: TPE-Br, bis (triphenylphosphine) palladium dichloride, K 2 CO 3 And bis (pinacolato) diboron ester is added to anhydrous 1, 4-dioxane, the mixture is heated to reflux at 80 ℃ under nitrogen atmosphere and stirred for 20 to 25 hours, water is added and the mixture is rinsed with dichloromethane, and the organic phase is combined with MgSO 4 Drying, evaporating the solvent under reduced pressure, and purifying the residue by silica gel column chromatography to obtain 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4, 5-tetramethyl-1, 3, 2-dioxaborolan, labeled TPE-B;
the molar ratio of TPE-Br to bis (pinacolato) diboron ester is 1:1-1:2; the molar ratio of TPE-Br to bis (triphenylphosphine) palladium dichloride is 25:1-28:1; the TPE-Br and K 2 CO 3 The molar ratio of (2) is 1:3-1:4;
(3) 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl ]]-synthesis of 4-yl) -2,2':6',2 "-terpyridine: TPE-B and 4'- (4-bromophenyl) -2,2':6',2' -terpyridine were dissolved in dioxane, then Na was added 2 CO 3 Stirring tetra (triphenylphosphine) palladium at 100-120 ℃ for 20-25 hours, cooling to room temperature after the reaction is finished, and using MgSO 4 Drying, evaporating the solvent under reduced pressure, purifying the residue by silica gel column chromatography to obtain 4' - (4 ' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenyl)]-4-yl) -2,2':6',2 "-terpyridine, labeled TPE-3N;
the molar ratio of TPE-B to 4'- (4-bromophenyl) -2,2':6',2' -terpyridine is 1:1-1:1.5; the molar ratio of TPE-B to tetra (triphenylphosphine) palladium is 1:0.02-1:0.04;
(4) Synthesis of Eu-containing ligands: TPE-3N, EuCl 3 Adding 1, 10-phenanthroline and polyacrylic acid into a mixed solvent of ethanol and THF, heating to 60-80 ℃ and reacting for 20-25 hours; cooling the reaction solution to room temperature, adding normal hexane for precipitation, centrifuging, purifying, and vacuum drying to obtain a Eu-containing ligand TPNE;
the TPE-3N and EuCl 3 The molar ratio of (2) is 1:5-1:6; the molar ratio of TPE-3N to 1, 10-phenanthroline is 1:5-1:6; the molar ratio of TPE-3N to polyacrylic acid is 1:100; in the ethanol and THF mixed solvent, the volume ratio of ethanol to THF is 3:1;
(5) Preparation of TPNE nanoparticles: dissolving TPNE in anhydrous THF, adding the obtained TPNE solution into ultrapure water under the action of ultrasound, performing ultrasound for 5min, stirring at room temperature for 5-15 min, removing THF, and filtering by using a water-based filter head with the thickness of 0.22 mu m to obtain water-soluble lanthanide AIE fluorescent nano-particles TPNE NPs; the concentration of the TPNE solution is 50 mug/mL; the volume ratio of the TPNE solution to the ultrapure water is 1:5.
2. Use of water-soluble lanthanide AIE fluorescent nanoparticles prepared according to the method of claim 1 as dual-color fluorescent probes in fluorescence imaging.
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