CN114854032A

CN114854032A - Preparation and application of water-soluble lanthanide AIE fluorescent nanoparticles

Info

Publication number: CN114854032A
Application number: CN202210574958.6A
Authority: CN
Inventors: 关晓琳; 张家铭; 来守军; 丁媛媛; 王康; 唐莹莹; 王涵; 李靖瑜
Original assignee: Northwest Normal University
Current assignee: Northwest Normal University
Priority date: 2022-05-25
Filing date: 2022-05-25
Publication date: 2022-08-05
Anticipated expiration: 2042-05-25
Also published as: CN114854032B

Abstract

The invention discloses a preparation method of water-soluble lanthanide AIE fluorescent nanoparticles, which is characterized by effectively combining the advantages of AIE and rare earth ions, taking TPE micromolecule derivative TPE-3N containing a terpyridine structure as a first ligand, 11, 10-phenanthroline as a second ligand, taking a third ligand PAA as a water-soluble unit, carrying out coordination with rare earth ions Eu to obtain a lanthanide complex TPNE, and finally obtaining the water-soluble lanthanide AIE fluorescent nanoparticles TPNE NPs through self-assembly. The aqueous solution of TPNE NPs emits blue/red double fluorescence at 435 nm and 615 nm, which are respectively derived from the fluorescence emission of AIE micromolecule TPE and Eu rare earth complex. Meanwhile, the TPNE NPs have temperature responsiveness and low cytotoxicity, have excellent membrane-targeted imaging capability on MKN-45 cancer cells, and show reversible bicolor fluorescence imaging effect. And TPNE NPs also show excellent reversible two-color fluorescence imaging effect in the living biological zebra fish, and can be used as a potential reversible two-color bioluminescent probe.

Description

Preparation and application of water-soluble lanthanide AIE fluorescent nanoparticles

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 a two-color fluorescent probe in fluorescence imaging, belonging to the technical fields of nano material preparation and biological imaging.

Background

In recent years, AIE has been reported in large numbers. The AIE effect arises from Restriction of Intramolecular Movement (RIM) and restriction into the dark state (RADS). RIM occurs when the luminophore is in a poor solvent or viscous environment, and thus intramolecular rotation is restricted due to steric hindrance, which blocks the relaxation channel of non-radiative energy loss and facilitates radiative decay pathways. Over the years of development, AIE research has 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, research and development of lanthanide materials with AIE characteristics has been limited to date. Generally, the mechanism behind the AIE effect is related to limiting intramolecular motion (vibration-RIV or rotation-RIR), which minimizes the loss of 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. Development of rare earth complex bioluminescent probes which have the advantages of both small organic molecules and metal nanoparticles with AIE characteristics can greatly enrich the types of the fluorescent probes and promote rapid development of bioluminescent imaging.

Disclosure of Invention

The invention aims to provide a preparation method of water-soluble lanthanide AIE fluorescent nanoparticles;

the invention also provides application of the water-soluble lanthanide AIE fluorescent nanoparticles as a two-color fluorescent probe in fluorescence imaging.

Preparation of water-soluble lanthanide series AIE fluorescent nano particle

(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 (THF), cooling the mixture to 0 deg.C under nitrogen atmosphere, and adding titanium tetrachloride (TiCl) 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 and 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; 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) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane

TPE-Br, bis (triphenylphosphine) palladium dichloride (Pd (PPh) ₃ ) ₂ Cl ₂ )、K ₂ CO ₃ And bis (pinacolato) diboron were added to anhydrous 1, 4-dioxane, the mixture was heated to reflux and stirred under nitrogen at 80 ℃ for 20-25 hours, water was added and the mixture was washed with dichloromethane, the combined organic phases were MgSO ₄ Drying, evaporation of the solvent under reduced pressure and purification of the residue by silica gel column chromatography gave 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane, labeled TPE-B. Wherein the molar ratio of the TPE-Br to the bis (pinacol) diboron ester is 1: 1-1: 2; the molar ratio of the TPE-Br to the bis (triphenylphosphine) palladium dichloride is 25: 1-28: 1; the TPE-Br and K ₂ CO ₃ The molar ratio of (a) to (b) is 1:3 to 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 the mixture for 20 to 25 hours at the temperature of between 100 and 120 ℃, cooling the mixture to room temperature after the reaction is finished, and adding MgSO ₄ Drying, evaporating the solvent under reduced pressure, and 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, labelled TPE-3N. Wherein the molar ratio of the TPE-B to the 4' - (4-bromophenyl) -2,2':6',2' ' -terpyridine is 1: 1-1: 1.5; the molar ratio of the TPE-B to the palladium tetratriphenylphosphine is 1: 0.02-1: 0.04.

(4) Synthesis of Eu-containing Complex

TPE-3N, EuCl ₃ Adding phenanthroline (Phen) and polyacrylic acid (PAA) into a mixed solvent of ethanol and THF, and heating to 60-80 ℃ for reacting for 20-25 hours; cooling the reaction liquid to room temperature, adding n-hexane for precipitation, carrying out centrifugal purification, and carrying out vacuum drying to obtain Eu-containing coordination compound TPNE; wherein, the TPE-3N and the EuCl ₃ The molar ratio of (A) to (B) is 1: 5-1: 6; the mol ratio of the TPE-3N to the phenanthroline is 1: 5-1: 6; the molar ratio of the TPE-3N to the polyacrylic acid is 1: 100. In the mixed solvent of ethanol and THF, the volume ratio of ethanol to THF is 3: 1.

(5) Preparation of TPNE nanoparticles

TPNE NPs were prepared by a nanoprecipitation method. Dissolving TPNE in anhydrous THF, adding the obtained TPNE solution into ultrapure water under the action of ultrasound, performing ultrasound for 5min, stirring for 5-15 min at room temperature, removing THF, and filtering with a 0.22-micrometer water-based filter head to obtain the water-soluble lanthanide AIE fluorescent nanoparticle 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.

The luminescence of the near infrared lanthanide is easily quenched by high energy C — H vibrational bonds in the molecule and solvent. In contrast to small molecules, the aggregate particles aggregate hundreds of lanthanide ions and antenna ligands within a cavity, which to some extent isolates the molecules from the effective vibrational quenching of the solvent. This helps to improve the luminous power of the lanthanide.

Therefore, the preparation process of the TPNE NPs is shown in figure 1, the TPNE NPs are formed by taking TPE-3N as a first ligand, Phen as a second ligand and PAA as a third ligand, and coordinating with rare earth ions Eu, and finally, the lanthanide complex TPNE with water solubility is synthesized through self-assembly.

The nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the TPE-Br are shown in the figure 2 and the figure 3; the nuclear magnetic hydrogen spectrum and the nuclear magnetic carbon spectrum of the TPE-Br are shown in the figure 4 and the figure 5; the nuclear magnetic hydrogen spectrum and nuclear magnetic carbon spectrum of TPE-3N are shown in FIGS. 6 and 7.

Characterization of Water-soluble lanthanide AIE fluorescent nanoparticles

1. Infrared Spectrum of TPNE

In a complex, rare earth ion Eu ³⁺ Since the ligand is coordinated to the pyridine ring of TPE-3N, it can be judged from the change of the peak on the IR spectrum. FIG. 8 is an IR spectrum of TPE-3N before and TPNE after coordination. As can be seen, the peak of hydrocarbon vibration of the pyridine ring on TPE-3N is 721.38 cm ^-1 The characteristic absorption peak is located at 1406.10 cm ^-1 、1442.76 cm ^-1 Is in contact with Eu ³⁺ After the coordination, the reaction solution was transferred to 806 cm ^-1 、1417.68 cm ^-1 、1452.40 cm ^-1 And the peak shape becomes wider. The above results show that TPE-3N and Eu ³⁺ When coordination is performed, an electron-withdrawing induction effect exists between them, the density of electron clouds between bonds decreases, the bond force constant becomes small, the vibration frequency decreases, and the peak moves in the direction of a high wave number, and the peak moves.

2. Zeta potential of TPNE NPs

We determined the Zeta potential of TPNE NPs, as shown in FIG. 9, which is-19.1 mV, indicating that the TPNE NPs system is stable.

XPS data for 3 TPNE NPs

（1）TPNE

XPS was used to confirm the surface elemental composition and bonding state of TPNE NPs. From FIG. 10 (a), it can be seen that TPE-3N is compared with TPETPNE NPs show Eu-3d, Eu-4d peaks, indicating that Eu has coordinated to TPE-3N. As can be seen from FIGS. 10 (b-d), the binding energies of the C elements of TPE-3N and TPNE NPs before and after coordination were not significantly changed, but the binding energy of the N, O element was changed, thus demonstrating that Eu ³⁺ Coordination with N, O is possible. In order to more visually observe the changes before and after the binding, the binding energy data of each atom in TPE-3N and TPNE NPs are shown in Table 1. By analyzing the results of XPS, some changes in the chemical environment of the elements can be found. The binding energy of N1s on the C-N bond is increased by 1.33 eV, the binding energy of O1s on the C = O bond is increased by 4.87 eV, and the movement of the binding energy indicates that Eu is Eu ³⁺ Coordinated to N, O element. Without any change in the binding energy of the C element. This is mainly because the electron distribution change outside the core affects the shielding effect of the inner layer electrons, and electrons of nitrogen of pyridine ring and electrons of oxygen of PAA in the complex are transferred to Eu ³⁺ The electron density of the outer layer is reduced, the shielding effect is weakened, and the binding energy of the inner layer electrons is increased, thereby proving that N → Eu ³⁺ And O → Eu ³⁺ And forming a coordinate bond. Furthermore, the XPS spectra of TPNE NPs show electron binding energies of Eu-3d (1128.2 eV) and Eu-4d (135.6 eV), further illustrating the formation of Eu complexes.

4. Topography characterization

Since PAA is hydrophilic and TPE-3N is hydrophobic, TPNE is easily self-assembled in a suitable solution

Assembling to form nano-small ball TPNE NPs. Therefore, the TPNE NPs particles are synthesized by adopting THF as a solvent and deionized water as a dispersion medium through a nano-precipitation method, and the microstructure and composition of the TPNE NPs are observed by adopting TEM and EDS. As can be seen from FIG. 11 (a), TPNE NPs are monodisperse, spherical in structure, smooth in surface, and approximately 150 nm 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 TPNE NPs is mainly between 60 and 210 nm, the average diameter is +138.5 nm, and it can be observed from the inset that an aqueous solution of TPNE NPs has a Tyndall effect, which demonstrates that TPNE NPs have formed nanoparticles in an aqueous solution.

Furthermore, spectral transmission electron microscopy (TEM/EDS) allows the analysis of the composition of the material. Therefore, to further analyze the elemental composition of TPNE NPs, we tested TPNE NPs using TEM/EDS. EDS spectra of element distributions of TPNE NPs are shown in FIG. 12 (a), and the results show the presence of C, N, O and Eu in TPNE NPs ³⁺ Further, successful acquisition of TPNE NPs was confirmed. The X-ray energy spectral elemental imaging analysis technique (EDS-Mapping) of fig. 12 (a') demonstrates the analysis of the distribution of key elements, which is consistent with the element distribution we have designed. Wherein C, O and N are main components of the complex, and the content of lanthanide ions is relatively low, which indicates that Eu is used as a main component ³⁺ The TPNE NPs have been successfully formed by coordination with TPE-3N.

Thirdly, the properties of TPE-3N

The photoelectric properties of organic functional materials are often tuned using a combination of electron donating groups (D) and electron withdrawing groups (a). The TPE-3N synthesized by the design of the invention takes TPE as D and terpyridine as A, so that a D-A electronic push-pull system is constructed, the energy gap between molecules is reduced, the charge transfer (ICT) in the molecules is promoted, and the emission wavelength is red-shifted.

1. Study of optical Properties 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-visible spectrum of TPE-3N in DMSO shows a 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 at 340 nm. Push-pull systems with donor and acceptor units may exhibit ICT characteristics due to dipole-dipole interactions. Therefore, the solvent effect of TPE-3N in various solvents was investigated. TPE-3N has a broad emission spectrum in strongly polar solvents and shows a significant red shift from weakly polar solvents (425 nm) to strongly polar solvents (475 nm) (fig. 13 (a)), which can be attributed to the highly polarized ICT excited state, which is an important phenomenon for D-a compounds. Therefore, TPE-3N with a D-A push-pull effect was successfully constructed.

2. AIE Property study of TPE-3N

TPE is a classical small molecule of AIE, so we examined the AIE properties of TPE-3N. DMSO is selected as a good solvent, and water (H) ₂ O) as poor solvent, different volume fractions of DMSO/H were tested separately ₂ Fluorescence change of TPE-3N in O mixed solvent. As shown in FIG. 14 (a), a poor solvent H was added dropwise to a DMSO solution of TPE-3N ₂ In the process of O, it can be observed to accompany H ₂ The fluorescence intensity of TPE-3N gradually increased to a non-linear degree as the volume fraction of O increased (FIG. 14 (b)), and as H increased ₂ After the volume fraction of O is increased to 30%, the fluorescence intensity of the TPE-3N solution is obviously enhanced, 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 most. This phenomenon is probably due to the fact that intramolecular spin of TPE-3N is active in DMSO solvent, which is a relaxation channel for excited state decay. However, when large amounts of water were added, aggregates of TPE-3N began to form, intramolecular rotation was limited due to physical constraints, and therefore, aggregation-induced emission occurred. This is consistent with the AIE luminescence properties of TPE molecules reported in the literature. Therefore, TPE-3N has excellent AIE luminescence property.

IV, 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-visible spectrum in THF exhibits a maximum absorption wavelength at 300 nm. Two emission peaks of 430 nm and 615 nm appear in a fluorescence emission spectrogram, the emission peak at 430 nm is generated by TPE derivatives, and the emission peak at 615 nm is Eu ³⁺ Characteristic emission peak of (1). Wherein a 595 nm small peak is beside the 615 nm peak and respectively corresponds to Eu ³⁺ Magnetic dipole transition ( ⁵ D ₀ → ⁷ F ₁ ) And Eu ³⁺ Electric dipole transition ( ⁵ D ₀ → ⁷ F ₂ ) In particular a sharp emission peak at 615 nm, which is attributed to Eu ³⁺ The ultrasensitive transition of ions has better monochromaticity.

TPNE NPs can be dissolved in different solvents, and for the next experiment, acetic Acid (ACOH) and methanol (CH) are selected ₃ OH), THF, N-Dimethylformamide (DMF), acetone (DMK) as solvents for fluorescence tests. As shown in fig. 15 (a), under the same test conditions, TPNE NPs have the sharpest emission peak in THF and the greatest fluorescence intensity among different solvents, so that 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 Selecting aqueous solution (phi) of quinine sulfate _f = 55%) as reference for the fluorescence emission of TPNE NPs at 430 nm; selecting rhodamine B (phi) _f = 97%) as a reference for the fluorescence emission of TPNE NPs at 615 nm. As shown in Table 4, phi at 430 nm, 615 nm for TPNE NPs _f Respectively 8.8 percent and 17.1 percent. Higher Φ of TPNE NPs _f Is favorable for biological imaging experiment and phi _f The calculation is 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 measured,. phi.s is the fluorescence quantum yield of the reference, F _U Fs is the integrated area of fluorescence of the dilute solution of the sample to be measured and the reference sample, A _U And As is the maximum absorbance value of the excitation wavelength of the sample to be detected and the reference sample.

2. Study of the AIE Properties of the complexes

It is believed that when TPE-3N having AIE properties is used as a 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 As poor solvents, THF/CH were tested in different volume fractions ₂ Cl ₂ Double fluorescence change of TPNE NPs in mixed solvents. As shown in FIG. 16 (a), a poor solvent CH was added dropwise to a THF solution of TPNE NPs ₂ Cl ₂ In the process of (2), it can be observed to accompany CH ₂ Cl ₂ The fluorescence of TPNE NPs gradually increases to a non-linear degree (FIG. 16 (b)) as the volume fraction of CH increases ₂ Cl ₂ The fluorescence intensity of the TPNE NPs solution was significantly increased after increasing the volume fraction of (a) to 70%, wherein the increase in blue fluorescence was much greater than the increase in red fluorescence, which was increased by up to 3.8-fold, while the increase in red fluorescence was not so significant. This indicates that changes in the degree of aggregation of TPNE NPs in the system have a significant effect on blue fluorescence. This is mainly because in a good solvent, the TPNE NPs are in a stretched state, which gives a large movement space for the TPE molecules in the complex, and the TPE can release most of the energy in the form of heat energy by the vibration of four benzene rings, resulting in the reduction of fluorescence emission. When a poor solvent CH is added into a good THF solution of TPNE NPs ₂ Cl ₂ Then, as the solubility of the fluorescent material is reduced, the molecules gradually form aggregates, the rotation in the molecules is inhibited, the nonradiative transition of the molecules is blocked, and the fluorescence intensity is enhanced, and the fluorescence intensity also shows a continuously enhanced form along with the increasing degree of the aggregation.

To further examine the AIE properties of TPNE NPs, we observed its fluorescence emission at different concentrations. As shown in fig. 16 (c, d), with 1 × 10 ^-4 The increase in the concentration of from mg/mL to 50 mg/mL, the fluorescence of TPNE NPs gradually increased in a non-linear manner, and the intensity of the red fluorescence emission was greater than the intensity of the blue fluorescence emission after the concentration reached 1 mg/mL. From FIG. 16 (d), it can be seen that the increase in red fluorescence is much greater than that of blue fluorescence, with the highest increase in red fluorescence being up to 390-fold, much greater than that of blue fluorescence in THF/CH ₂ Cl ₂ Fold increase in solvent. Such a high fold increase in red fluorescence means that this may not only be the effect of the AIE, but also thatThe AE effect also contributes to it. However, after the concentration reached 20 mg/mL, the blue fluorescence intensity of TPNE NPs decreased. This is probably because the size of molecular aggregates of TPNE NPs increases with increasing concentration. Only molecules on the surface of the ligand can be excited and fluoresce. Either a large number of molecules in the core of the ligand are not excited or the fluorescence emitted by these molecules is reabsorbed by the outer molecules. Therefore, when the concentration of TPNE NPs reaches 20 mg/mL, the blue fluorescence intensity decreases.

TPNE NPs have excellent bicolor fluorescent AIE luminescence property, which is beneficial to the subsequent application of imaging experiments in cells and zebra fish.

3. AE Effect

To verify our guess about the presence of AE effects of TPNE NPs, we further performed verification experiments. Control PPE (EuCl) was synthesized using the same experimental conditions ₃ (15.50 mg, 0.06 mmol), Phen (10.80 mg, 0.06 mmol), PAA (1 mmol, 2 g, Mn =2000 g/mol) were added to a round bottom flask containing 9.00 mL of EA and 3.00 mL of THF mixed solvent, followed by heating to 70 ℃ and reaction was stopped after 24 hours. Cooling the reaction solution to room temperature, adding 100.00 mL N-hexane for precipitation, centrifuging and purifying for three times, and finally placing the reaction solution in a vacuum drying oven for vacuum drying for 24 hours to obtain a pink solid coordination compound PAA-Phen-Eu (PPE), wherein as shown in FIG. 17, after TPE-3N is introduced, the fluorescence of TPNE NPs at 615 nm is enhanced, and the fluorescence intensity is obviously higher than that of PPE and EuCl ₃ . This is mainly because Eu ³⁺ Is weak in light absorption ability and, at the same time, its 4f-4f conversion is suppressed, so that Eu is ³⁺ The luminescence of (2) is weak. However, TPE-3N contains unsaturated bonds in the molecule and pi electrons in the carbon-carbon double bonds, which results in the occurrence of pi-pi transition. The excitation energy required for this transition is low, since pi electrons are easily excited and the energy of the pi orbitals is low. In addition, the molar absorption coefficient of the pi-pi transition is very large. After TPE-3N is excited by ultraviolet light, free electrons in the molecules are from the ground state (S) ₀ ) Transition to the first excited singlet state (S) ₁ ) Then from S by crossing between systems ₁ Transition to the first electronically excited triplet state (T) ₁ ). TPE-3N then adds T ₁ Energy transfer of states to Eu ³⁺ ，Eu ³⁺ Sufficient energy is obtained to transition its electrons from the ground state to the excited state. The excited electrons return to the ground state in the form of radiative transition, thereby emitting Eu ³⁺ Intrinsic fluorescence of (a). This demonstrates TPE-3N and Eu ³⁺ There is an antenna effect in between.

4. Lifetime of fluorescence

The rare earth luminescent material has the advantage of longer fluorescence lifetime. This can be further confirmed by the following fluorescence lifetime experiment. During the different sensitization processes, a shift of energy from the ligand triplet excited state to the lanthanide excited state (T) is generally observed ₁ → Ln), is a "ligand S ₁ → ligand T ₁ Energy flow of → Ln ″, light is first absorbed into the singlet excited state (S) of the antenna ₀ →S ₁ ) Then undergoes intersystem crossing to enter a ligand triplet excited state (S) ₁ →T ₁ ). Then energy from T of the antenna ₁ The state is transferred to the excited state (Ln) of the lanthanide and finally returns to the ground state of the lanthanide. The inhibition of the 4f-4f conversion is such that the Eu is switched from ³⁺ Relaxation of the excited state is slow. Thus, lanthanide complexes generally exhibit longer decay lifetimes, which can be as long as microseconds and milliseconds, than organic molecules have lifetimes on the nanosecond scale.

The fluorescence lifetime of TPNE NPs is shown in FIG. 18. The fluorescence lifetime of the TPNE NPs was 1.345 ms, indicating that there was an effective antenna effect in the system of TPNE NPs. The long lifetime of the TPNE NPs enables time-gated and time-resolved fluorescence lifetime measurements, which can facilitate differentiation from cellular 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; tau is ₁ 、τ ₂ 、τ ₃ Is the fitted lifetime in ns.

5. Dual fluorescence emission

On one hand, TPNE NPs comprise TPE micromolecules emitting blue light and rare earth Eu emitting red light ³⁺ Two luminescent units of a luminescent complex, if the difference between the emission wavelength and the excitation wavelength of the two luminescent units is large and the two luminescent units do not overlap, the FRET phenomenon will not occur, and the TPNE NPs may have double fluorescence emission performance.

In order to observe the change of the dual fluorescence more clearly, DMK is selected as a solvent to reduce the red fluorescence emission at 615 nm, so that the blue fluorescence change is more prominent. FIG. 19 (a-c) is a drawing

Fluorescence spectra of 2 mg/mL TPNE NPs DMK solution under different excitation wavelengths in the range of 350-400 nm. As shown in fig. 19, excitation at 395 nm enables the best sensitized metal center emission from Eu to be achieved, which results in a characteristic red chromaticity. However, while maintaining all other parameters, the excitation wavelength was varied between 350 and 400 nm, resulting in a decrease in Eu contribution, enhanced ligand-based emission, and a large change in observed chromaticity as the excitation wavelength was shifted toward short wavelengths. At λ _ex =395 nm, observed to originate from ⁵ D ₀ → ⁷ F _J The strong Eu emission of the conversion, the ligand does not contribute significantly. Excitation at 380 nm decreases the Eu emission intensity, the ligand emission contribution increases, and as the excitation wavelength continues to decrease to 350 nm, a further decrease in Eu emission intensity is observed, while the ligand emission intensity increases at 430 nm, giving the system a blue chromaticity profile. FIG. 19 (d) shows the CIE chromaticity diagram of TPNE NPs, with a shift in chromaticity from blue to red upon excitation at 350-400 nm. Thus, TPNE NPs were demonstrated to have two-color fluorescence emission properties.

6. Temperature responsiveness

In addition, to examine the variation of the dual fluorescence of TPNE NPs with temperature, the temperature-variable fluorescence spectrum of DMF solution of TPNE NPs was tested, and the results are shown in FIG. 20. It can be observed from the trend graph of the dual fluorescence intensity with temperature, and the blue fluorescence intensity and the red fluorescence intensity both decrease linearly with the increase of the temperature. At lower temperatures, the TPE molecules are frozenAnd the fluorescence intensity is higher, the rotation of TPE molecules is accelerated after the temperature is increased, and the energy is released in a non-radiative transition mode, so that the blue fluorescence is reduced. The AE effect existing in the TPNE NPs system is that Eu is reduced after the blue fluorescence intensity is reduced ³⁺ Insufficient energy is available for excitation and the intensity of the red fluorescence decreases accordingly. It can be seen that the TPNE NPs have excellent temperature responsiveness.

Imaging experiment and cytotoxicity

The experimental procedures of cell culture, cytotoxicity detection and cell imaging are as follows:

(1) cell culture

Cell lines: human gastric cancer cells (MKN-45) were provided by the basic medical college of Gansu university of traditional Chinese medicine.

Cell recovery: after frozen MKN-45 cancer cells are taken out and placed in hot water at 37 ℃ for thawing at constant temperature, the suspension is sucked out and transferred into a centrifuge tube, and then 10.00 mL of cell culture solution prepared in advance is added. Centrifuging at 1500 r/min for 5.00 min in a centrifuge, discarding supernatant, adding 10.00 mL of high sugar medium (DMEM), washing, discarding supernatant, adding mixed solution of DMEM containing 12% south American Fetal Bovine Serum (FBS) and 100 u/mL of "double antibody" (penicillin/streptomycin), blowing, inoculating in culture medium, and standing at 37 deg.C under wet CO ₂ And (5) standing and culturing in an incubator.

Cell culture and passage: the desired apparatus is sterilized in an autoclave for use. And (5) observing the cell density under a microscope, and passaging when the density reaches 80-90%. Pouring out original culture solution, washing with Phosphate Buffered Saline (PBS) for three times, digesting with 0.25% trypsin for three minutes, immediately adding 2.00 mL DMEM containing 12% FBS to stop digestion, blowing, centrifuging at 1500 r/min for 5min, discarding solution, adding 2.00 mL DMEM containing 12% FBS, placing in culture medium, placing in 37 deg.C, and moistening CO ₂ Culturing in an incubator.

Freezing and storing cells: removing stock culture solution from cancer cells growing in logarithmic phase, washing with PBS for three times, adding appropriate amount of trypsin, centrifuging at 1500 r/min for 5min, adding prepared freezing solution, blowing, beating, and counting when density reaches 5 × 10 ⁶ ~1×10 ⁷ When the concentration is/mL, the mixture is transferred into a freezing storage tube and stored in a refrigerator at the temperature of minus 80 ℃.

(2) Cytotoxicity assays

For the cellular activity assay, the toxicity of the samples on the cells was evaluated using the (3- (4, 5-dimethylthiazol-2-yl) -2, 5-diphenyltetrazolium bromide) (MTT) method. The trypsin digested MKN-45 cells were treated with 10 ⁴ The MTT assay was performed after seeding in 96-well plates at one/mL concentration and culturing for 24 hours under conventional cell culture conditions. The experiment is divided into 5 experimental groups and 1 control group, DMEM is added into the control group, samples with different concentrations are respectively added into the experimental groups, the samples are placed under the conventional cell culture condition for continuous culture for 24 hours, then thiazole blue (MTT) solution with the concentration of 5 mg/mL is added, the samples are placed into an incubator for continuous culture for 4 hours, the solution is completely absorbed, 150.00 mu L of dimethyl sulfoxide (DMSO) solution is added into each hole, and the oscillation is carried out for 10 min. Finally, the cell viability was assessed by a microplate reader, the measurement being based on the absorbance (OD) at 490 nm, 3 experiments in parallel, the following formula being used to calculate the viability of the cell growth:

cell survivval% = ODr/ODc × 100% (ODr is average absorbance measured for experimental well, ODc is average absorbance measured for control group)

(3) Cell imaging experiments

For cell imaging, MKN-45 cells after log phase pancreatic digestion were seeded on a cell-slide that had been placed in a 24-well plate, and cultured for 24 h under conventional cell culture conditions after addition of DMEM medium. Then, 100 mug/mL of sample was added to each well, and the mixture was washed three times with PBS buffer after 24-hour culture. Finally, cell observation and photo shooting are carried out under a Zeiss Axio scope.A1 upright fluorescence microscope.

The cytotoxicity characteristic of the fluorescent material is important for the application of the fluorescent material as a luminescent biological probe, and a cytotoxicity test is an important prerequisite for successful cell imaging experiments. To examine whether TPNE NPs are biocompatible, we performed a standard MTT assay to determine data for cellular activity assays of TPNE NPs on human gastric carcinoma cells MKN-45. As can be seen in FIG. 21, at concentrations as high as 100. mu.g/mL, MKN-45 cancer cells still retained more than 90% of their activity. Thus, the TPNE NPs can be used in cell imaging experiments.

In addition, 100. mu.g/mL TPNE NPs were incubated with MKN-45 cancer cells for 24 hours, and then fluorescence microscopy was performed to take a fluorescent microscopic photograph of the cells at different excitation wavelengths. As shown in FIG. 22 (a), at λ _ex =360 nm and λ _ex MKN-45 cancer cells showed blue fluorescence photographs at blue channel and red fluorescence photographs at red channel, respectively, when =395 nm. As can be seen from the figure, TPNE NPs are taken up by MKN-45 cancer cells and then attached to cell membranes, and the fluorescence intensity is strong in the cell membrane region. TPNE NPs are water-soluble, are small spheres covered with hydrophilic groups on the surfaces, and therefore show negative charges in an aqueous environment, and therefore, the fluorescence of MKN-45 cell boundaries is probably due to the fact that TPNE NPs specifically interact with proteins on cell membranes through electrostatic attraction. Meanwhile, good water solubility is also a necessary condition for avoiding cellular absorption, and TPNE NPs are ensured to be in full and firm contact with cell membranes, so that the cell membrane imaging capability is further enhanced. In addition, in the test process, the intensity of blue/red double-color fluorescence emitted on the cell membrane is still kept unchanged under the condition that the stained cells are irradiated for a long time, which shows that the prepared TPNE NPs have good photobleaching resistance and can carry out long-time fluorescence imaging on the cell membrane. These results indicate that the obtained TPNE NPs having high light stability, low cytotoxicity and superior biocompatibility can be used as fluorescent probes for cell membrane imaging.

To verify the application of the TPNE NPs in the organisms, we further performed zebrafish imaging experiments, and as shown in fig. 22 (b), the photographs of the zebrafish stained with the TPNE NPs under a fluorescence microscope also showed blue/red double-channel imaging, which is consistent with the results of the cell imaging experiments. Thus, blue fluorescence and intense red fluorescence clearly indicate the potential use of TPNE NPs in high contrast biological imaging applications. That is, organisms stained with TPNE NPs have the ability to achieve blue/red dual-color fluorescence imaging by adjusting 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 conclusion, TPE derivatives (TPE-3N) containing a terpyridine structure and having coordination capacity are 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 is coordinated with rare earth ions Eu to synthesize a water-soluble lanthanide complex TPNE; and finally, obtaining the water-soluble lanthanide series AIE fluorescent nano particles TPNE NPs through self-assembly. TEM and DLS test results show that TPNE NPs are 150 nm spheres, and the particle size range is between 120 and 180 nm. TPNE NPs are AIE type NPs having a fluorescence lifetime as long as 1.345 ms, emitting blue/red dual fluorescence at around 430 nm and 615 nm, with blue fluorescence quantum yield of about 8.8% and red fluorescence quantum yield of about 17.1%. In addition, TPNE NPs are temperature responsive, biocompatible and low cytotoxic. Biological imaging experiment results show that the TPNE NPs have excellent membrane targeted imaging capability on MKN-45 cancer cells and show excellent reversible two-color fluorescence imaging effect. And TPNE NPs also show excellent reversible two-color fluorescence imaging effect in the living organism zebra fish, and can be used as a potential reversible two-color bioluminescent probe.

Drawings

FIG. 1 is a synthetic roadmap for TPNE and TPNN and the self-assembly of TPNE;

FIG. 2 shows the nuclear magnetic hydrogen spectrum (600 MHz, CDCl) of TPE-Br ₃ ）；

FIG. 3 shows 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 the nuclear magnetic hydrogen spectrum (400 MHz, CDCl) of TPE-3N ₃ ）；

FIG. 7 shows 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 change in XPS binding energy for TPE-3N before and TPNE NPs after complexation (a), C1s (b), N1s (C) and O1s (d);

FIG. 11 is a TEM image (a) and a DLS image (b) of TPNE NPs (b) (inset: Tyndall effect photograph of TPNE NPs under laser irradiation);

FIG. 12 TEM-EDS elemental scan spectrum of TPNE NPs (a); (a') surface scanning pictures corresponding to C, O, N and Eu four elements on TPNE NPs;

FIG. 13 (a) fluorescence spectra of TPE-3N in different solvents; (b) UV 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 in different volume ratios of water to DMSO and (b) the trend of the fluorescence intensity of TPE-3N solutions in different volume ratios of water to DMSO;

FIG. 15 (a) fluorescence spectra of TPNE NPs in different solvents; (b) UV absorption 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 (A) _ex =360 nm, 2 mg/mL) (inset: TPNE NPs in different CHs ₂ Cl ₂ Volume fraction of CH ₂ Cl ₂ Photograph under irradiation with ultraviolet light (365 nm) in THF mixed solvent) and (b) different volume fractions CH ₂ Cl ₂ The variation trend of the corresponding fluorescence intensity of the 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) in different concentrations); (d) the variation trend of the corresponding fluorescence intensity of TPNE NPs solution with different concentrations;

FIG. 17EuCl ₃ Fluorescence spectra of PPE, TPNE NPs (. lamda.ex =360 nm, 2 mg/mL) (inset: EuCl) ₃ Photographs under UV (365 nm) irradiation of PPE, TPNE NPs);

FIG. 18 fluorescence lifetime plots of TPNE NPs;

FIG. 19 (a, c) dual fluorescence plots of TPNE NPs under different excitations, (b) dual fluorescence plot trend plots of TPNE NPs under different excitations; (d) the position of the bifluorescent 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 change in fluorescence intensity of TPNE NPs solutions at different temperatures;

FIG. 21 toxicity testing of MKN-45 cancer cells treated with different concentrations of TPNE NPs for 48 hours;

FIG. 22 (a) blue/red two-channel fluorescence microscopy images (100. mu.g/mL) of MKN-45 cancer cells stained with TPNE NPs for 24 h, and (b) zebrafish blue/red two-channel fluorescence microscopy images stained with TPNE NPs.

Detailed Description

The preparation method of the water-soluble lanthanide AIE fluorescent nanoparticles of the present invention is further detailed by the following specific examples.

The reagents used in the present invention are 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 charged 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 mL of Tetrahydrofuran (THF), the mixture was cooled to 0 deg.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, the reaction mixture is cooled with potassium carbonate (K) ₂ CO ₃ ) The reaction was quenched with solution and the mixture was extracted three times with dichloromethane, the organic phases combined and washed with anhydrous magnesium sulfate (MgSO) ₄ ) And (5) drying. The solvent was evaporated under reduced pressure and the residue was 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 in yield: 76 percent.

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) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (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 (pinacol) diboron ester (3.05 g, 12.00 mmol), anhydrous 1, 4-dioxane (40.00 mL) were added to a 100.00 mL two-necked 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 hThen (c) is performed. Then, water was added, and the mixture was extracted with dichloromethane (CH) ₂ Cl ₂ ) Rinsing three times. The combined organic phases were MgSO ₄ Drying, evaporation of the solvent under reduced pressure and 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) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane (TPE-B) as a white solid in yield: and 55 percent.

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) and stirred at 110 ℃ for 24 hours, after the reaction was complete, cooled to room temperature over MgSO ₄ Drying, evaporating the solvent under reduced pressure, and purifying the residue by silica gel column chromatography using methanol/dichloromethane (v/v 1: 20) as eluent to give 4' - (4' - (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) - [1,1' -biphenylyl ] as a yellow solid]-4-yl) -2,2':6',2 "-terpyridine (TPE-3N), yield: 66 percent.

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 ) The preparation of (1): europium oxide (Eu) ₂ O ₃ ) (3.51 g, 1.00 mmol) was placed in a clean beaker and concentrated hydrochloric acid (HCl) approximately 20.00 mL was added dropwise followed by a little ammonium chloride (NH) ₄ Cl) powder (to remove a small amount of reaction product water). Heating and boiling to near dryness for use.

TPE-3N (6.90 mg, 0.01 mmol), EuCl ₃ (15.50 mg, 0.06 mmol), phenanthroline (Phen) (10.80 mg, 0.06 mmol), polyacrylic acid (PAA) (1.00 mmol, 2.00 g, Mn =2000 g/mol) were added to a round bottom flask containing a mixed solvent of 9.00 mL of ethanol and 3.00 mL of THF, followed by heating to 70 ℃, and the reaction was stopped after 24 hours of reaction. Cooling the reaction solution to room temperature, adding 100.00 mL of n-hexane for precipitation, carrying out centrifugal purification for three times, and finally placing the reaction solution in a vacuum drying oven for vacuum drying for 24 hours to obtain pink solid TPNE, wherein the yield is as follows: and 64 percent.

(5) Preparation of TPNE Nanoparticles (NPs)

TPNE NPs were prepared by a nanoprecipitation method. TPNE was dissolved in anhydrous THF to give a solution having a concentration of 1 mg/mL. After diluting the solution to 50. mu.g/mL, 5mL of the solution was sonicated and poured into a cuvette containing 10.00 mL of ultrapure water and sonicated for 5min, followed by stirring in a fume hood at room temperature for 10 min. After THF was removed, TPNE NPs were obtained by filtration through a 0.22 μm aqueous filter head.

Claims

1. A preparation method of water-soluble lanthanide AIE fluorescent nanoparticles 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-bromobenzophenone and zinc powder into tetrahydrofuran, cooling the mixture to 0 ℃ in a nitrogen atmosphere, dropwise adding titanium tetrachloride, 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 and 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), label TPE-Br;

(2) synthesis of 2- (4- (2, 2-bis (4-methoxyphenyl) -1-phenylvinyl) phenyl) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane: TPE-Br, bis (triphenylphosphine) palladium dichloride and K ₂ CO ₃ And bis (pinacolato) diboron were added to anhydrous 1, 4-dioxane, the mixture was heated to reflux and stirred under nitrogen at 80 ℃ for 20-25 hours, water was added and the mixture was washed with dichloromethane, the combined organic phases were 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) -4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolane, labeled TPE-B;

(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 the palladium tetratriphenylphosphine at 100-120 ℃ for 20-25 hours, cooling to room temperature after the reaction is finished, and reacting with MgSO ₄ Drying, evaporating the solvent under reduced pressure, and 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, labelled TPE-3N;

(4) synthesis of Eu-containing complexes: TPE-3N, EuCl ₃ Adding phenanthroline and polyacrylic acid into a mixed solvent of ethanol and THF, and heating to 60-80 ℃ to react for 20-25 hours; cooling the reaction liquid to room temperature, adding n-hexane for precipitation, carrying out centrifugal purification, and carrying out vacuum drying to obtain Eu-containing coordination compound TPNE;

(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 for 5-15 min at room temperature, removing THF, and filtering with a 0.22-micrometer water-based filter head to obtain the water-soluble lanthanide AIE fluorescent nanoparticle TPNE NPs.

2. The method for preparing water-soluble lanthanide AIE fluorescent nanoparticles as claimed in claim 1, wherein: in the step (1), 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.

3. The method for preparing water-soluble lanthanide AIE fluorescent nanoparticles as claimed in claim 1, wherein: in the step (2), the molar ratio of the TPE-Br to the bis (pinacol) diboron ester is 1: 1-1: 2; the molar ratio of the TPE-Br to the bis (triphenylphosphine) palladium dichloride is 25: 1-28: 1; the TPE-Br and K ₂ CO ₃ The molar ratio of (a) to (b) is 1:3 to 1: 4.

4. The method for preparing water-soluble lanthanide AIE fluorescent nanoparticles as claimed in claim 1, wherein: in the step (3), the molar ratio of the TPE-B to the 4' - (4-bromophenyl) -2,2':6',2' ' -terpyridine is 1: 1-1: 1.5; the molar ratio of the TPE-B to the palladium tetratriphenylphosphine is 1: 0.02-1: 0.04.

5. The method for preparing water-soluble lanthanide AIE fluorescent nanoparticles as claimed in claim 1, wherein: in the step (4), the TPE-3N and the EuCl are ₃ The molar ratio of (A) to (B) is 1: 5-1: 6; the mol ratio of the TPE-3N to the phenanthroline is 1: 5-1: 6; the mol ratio of the TPE-3N to the polyacrylic acid is 1: 100; in the mixed solvent of ethanol and THF, the volume ratio of ethanol to THF is 3: 1.

6. The method for preparing water-soluble lanthanide AIE fluorescent nanoparticles as claimed in claim 1, wherein: in the step (5), the concentration of the TPNE solution is 50 mug/mL; the volume ratio of the TPNE solution to the ultrapure water is 1: 5.

7. The use of water-soluble lanthanide AIE fluorescent nanoparticles prepared according to the method of claim 1 as bi-color fluorescent probes in fluorescence imaging.