A near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, preparation method and application thereof

Abstract

The present invention discloses a near-infrared cyanine dye-sensitized rare earth up-conversion luminescent nanoprobe, a preparation method and an application thereof; the probe of the present invention comprises a near-infrared cyanine dye with a coordination group sulfonate group and carboxylate group directly connected to a conjugated structure and a rare earth up-conversion nanoparticle with a core-shell structure, and the cyanine dye is coordinated to the nanoparticle to realize up-conversion luminescence enhancement. The preparation method of the present invention is simple, and the cyanine dye can be coordinated to the rare earth nanoparticle. The cyanine dye of the present invention shortens the distance between the luminescent center of the dye and the lanthanide ion by modifying the coordination group on the conjugated part of the dye instead of connecting the conjugated part of the dye through a long carbon chain structure, thereby improving the efficiency of short-range triplet energy transfer, realizing stronger up-conversion luminescence, and then being used in the field of single particle imaging.

Description

Technical Field

[0001] The invention belongs to the technical field of luminescent materials, and in particular relates to a near-infrared cyanine dye-sensitized rare earth up-conversion luminescent nanoprobe, a preparation method and an application thereof. Background Art

[0002] Lanthanide upconversion nanoparticles (UCNPs) have attracted widespread interest due to their narrow emission, non-blinking and excellent photostability. Using UCNPs as imaging probes can eliminate the interference of spontaneous background fluorescence of biological samples and produce images with high contrast. 3+ The ions act as sensitizers to absorb infrared radiation and transfer energy to the activator ions Er non-radiatively. 3+ 、Tm 3+ Or Ho 3+ , to produce visible or ultraviolet emission. However, the absorption cross section of lanthanide ions is much lower than that of organic dyes. Using organic dyes as antenna molecules can significantly improve the ability of UCNPs to capture photons and increase their upconversion luminescence brightness.

[0003] However, the coordination groups of conventional near-infrared dyes are usually connected to the fluorescent luminescent center, i.e., the conjugated part, through a long carbon chain structure, and the distance of energy transfer between the dye and the lanthanide ion is extended, and the energy transfer efficiency is reduced. The present invention aims to shorten the energy transfer distance by designing the structure of the near-infrared dye, and improve the efficiency of short-range triplet energy transfer through the heavy atom effect, thereby improving the brightness of a single nanoparticle. Summary of the invention

[0004] In view of the deficiencies of the above-mentioned prior art, the purpose of the present invention is to provide a near-infrared dye-sensitized rare earth upconversion nanoprobe with high brightness, a preparation method and its application. The present invention achieves upconversion luminescence enhancement by coordinating near-infrared cyanine dyes to core-shell structure upconversion nanoparticles; in the near-infrared cyanine dyes of the present invention, the coordination group is directly modified on the conjugated part of the dye, which can achieve stronger upconversion luminescence; further, the high-brightness near-infrared dye-sensitized rare earth upconversion nanoprobe of the present invention can be used in single particle imaging. The technical solution of the present invention is specifically described as follows.

[0005] The present invention provides a near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, which comprises a near-infrared cyanine dye and a core-shell structure upconversion nanoparticle, wherein the surface coordination of the near-infrared cyanine dye and the core-shell structure upconversion nanoparticle realizes upconversion luminescence enhancement; wherein the conjugated system of the near-infrared cyanine dye is directly modified with a coordination group R, and the structure of the near-infrared cyanine dye is shown in Formula 1) or Formula 2):

[0006]

[0007]

[0008] Wherein: n is an integer between 1 and 20, R1 and R2 are independently selected from C1-C6 alkyl, aryl-substituted C1-C6 alkyl, alkynyl-substituted C1-C6 alkyl, alkoxy-substituted C1-C6 alkyl, carboxyl-substituted C1-C6 alkyl, sulfonic acid-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, hydroxybenzoyloxy-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, amide-substituted C1-C6 alkyl or cyano-substituted C1-C6 alkyl; R is sulfonate or carboxylate.

[0009] In the present invention, n is an integer between 2-5.

[0010] In the present invention, the near-infrared cyanine dye is selected from any one of sulfonated-ICG dye and sulfonated-Cy7 dye.

[0011] In the present invention, the near-infrared cyanine dye has an absorption wavelength of 600-900 nm and an emission wavelength of 650-950 nm.

[0012] In the present invention, the number ratio of the core-shell structure upconversion nanoparticles to the near-infrared cyanine dye molecules is between 1:1 and 1:1000; preferably, the number ratio is between 1:10 and 1:100.

[0013] In the present invention, the core-shell structure upconversion nanoparticles are NaYbF4:x%RE@NaLuF4:y%Yb, x=0.5-100, y=0.01-100, and the total particle size is less than 100nm, wherein the particle size of the core is <100nm, and the thickness of the shell is <50nm; RE doped in the core can be Er 3+ 、Tm 3+ and Ho 3+ Any emitter ion in the ion, with RE ion as the emitter, and Yb doped in the outermost layer 3+ Acts as an energy acceptor for cyanine dyes and transfers energy to RE.

[0014] The present invention also provides a method for preparing the above-mentioned near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, comprising the following steps:

[0015] (1) Synthesis of core-shell upconversion nanoparticles;

[0016] (2) Near-infrared cyanine dyes are coordinated to the surface of core-shell conversion nanoparticles.

[0017] In the present invention, in step (2), the core-shell structure upconversion nanoparticles are pretreated by NOBF4 method or the rare earth upconversion nanoparticles are directly dispersed by organic solvent, and then combined with near-infrared cyanine dye solution to coordinate the near-infrared cyanine dye to the core-shell structure upconversion nanoparticles through a ligand exchange process.

[0018] Furthermore, the present invention provides an application of the above-mentioned near-infrared cyanine Cy7 dye-sensitized rare earth upconversion luminescent nanoprobe in single particle imaging. When used, the power density used is: 1.43-11.6kW / cm 2 , the excitation wavelength is 600-900nm near-infrared light source excitation irradiation; the specific embodiment of the present invention provides a scheme for using 730nm and 808nm lasers for excitation for two systems respectively; the most preferred power density is 1.43kW / cm 2 , 808nm laser excitation, the luminescence brightness is significantly enhanced.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] (1) The present invention provides a novel near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, which realizes dye-sensitized luminescence enhancement by exchange coordination of ligands directly connected to the conjugated structure in the novel near-infrared cyanine dye to the surface of rare earth upconversion nanoparticles; under 808nm excitation, the laser power is 37.5W / cm 2 Under the test conditions, the luminescence spectrum enhancement of the sulfonated-ICG dye-sensitized upconversion luminescent nanoprobe can reach 2413 times compared with UCNPs; in contrast, traditional cyanine dyes, such as IR806, can only achieve a 73-fold upconversion luminescence brightness enhancement; the brightness of ICG-sensitized core-shell structure upconversion nanoparticles is similar to that of UCNPs under 980nm excitation.

[0021] (2) Compared with traditional cyanine dyes, this design strategy can also be extended to Cy7 dye excited at 730 nm. The sulfonated-Cy7 dye-sensitized rare earth upconversion luminescent nanoprobe can achieve an upconversion luminescence enhancement of 52 times relative to UCNPs.

[0022] (3) The near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe of the present invention can be applied to single particle imaging. Compared with rare earth upconversion nanoparticles (UCNPs), the near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe can be applied to single particle imaging. 2 By excitation with 808 nm laser and enhancing the photostability of the dye by adding the reducing agent ascorbic acid, the single-particle upconversion luminescence brightness of near-infrared cyanine dye-sensitized UCNPs can be more than 143 times higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The transmission electron microscope photo and X-ray diffraction pattern of the NaYbF4:8%Er@NaLuF4:70%Yb core-shell rare earth upconversion nanoparticles in Example 1 of the present invention. The sub-images are: transmission electron microscope image and corresponding size distribution (a) core structure: NaYbF4:Er and (b) core-shell structure: NaYbF4:Er@NaLuF4:Yb. (c) High-resolution transmission electron microscope image of the core-shell structure. (d) Crystallographic pattern of standard hexagonal NaYbF4 and NaLuF4 and XRD data of core NaYbF4:Er and core-shell NaYbF4:Er@NaLuF4:Yb.

[0024] Figure 2 The molecular structure, absorption spectrum and up-conversion emission spectrum of IR806, ICG and sulfonated-ICG in the 808nm sensitization system in Example 2. The sub-figures are: (a) the structural formula of IR806, ICG and sulfonated-ICG. (b) the absorption spectra of the three dyes, from left to right: ICG, sulfonated-ICG, IR806. The up-conversion spectrum of UCNPs when excited at 980nm and the up-conversion spectrum of dye-sensitized UCNPs when excited at 808nm, including (c) IR806 (d) ICG and (e) sulfonated-ICG. (f) the relationship between the integrated intensity of the up-conversion spectrum and the number of dyes. The dye is dispersed in a dimethyl sulfoxide solution with a concentration of 0.125mM. From top to bottom: IR806, ICG, sulfonated-ICG. (g) the integrated intensity of the up-conversion spectrum of UCNPs and dye-sensitized UCNPs when excited at 980 and 808nm. The power density at 980 and 808 nm excitation was 37.5 W cm -2 .

[0025] Figure 3 The molecular structure, absorption spectrum and up-conversion spectrum of sulfonated-Cy7 and traditional carboxylated-Cy7 in the 730nm sensitization system in Example 3. The sub-figures are: (a) Structural formula of the two dyes. (b) Absorption spectra of the two dyes, from left to right: carboxylated-Cy7, sulfonated-Cy7. (c) Absorption spectra of dye-modified UCNPs before and after centrifugation. (d) Up-conversion spectra of UCNPs when excited at 980nm and up-conversion spectra of two dye-sensitized UCNPs when excited at 730nm. (e) Integrated intensity of up-conversion spectra of UCNPs and dye-sensitized UCNPs when excited at 980 and 730nm. The power density at 980 and 730nm excitation is 37.5W cm -2 . .

[0026] Figure 4The single particle upconversion luminescence results of NaYbF4:8% Er@NaLuF4:70% Yb core-shell rare earth upconversion nanoparticles sensitized by sulfonation-ICG in Example 4. (a) Single particle brightness distribution histogram of sulfonation-ICG-sensitized UCNPs under 976 nm and 808 nm laser excitation (11.6 kW cm -2 ). (b) The enhancement factor of dye-sensitized UCNPs at different power densities. DETAILED DESCRIPTION

[0027] The technical solution of the present invention is described in detail below with reference to the accompanying drawings and embodiments.

[0028] Figure 1 The working principle diagram of the near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe in the embodiment of the present invention; the present invention modifies the coordination group: sulfonate or carboxylate in the conjugated part of the cyanine dye, rather than connecting it to the conjugated part of the dye through a long carbon chain structure, thereby shortening the distance between the luminescent center of the dye and the rare earth lanthanide ion, improving the efficiency of short-range triplet energy transfer, and achieving stronger upconversion luminescence. Near-infrared dye for dye sensitization and sensitizer Yb in the core-shell structure of upconversion nanoparticles 3+ Although there is a certain energy mismatch in the absorption of Yb ions, by improving the structure of the dye, that is, the position of the coordination group, the energy can still be more effectively transferred to Yb by enhancing the short-range triplet population under near-infrared light excitation. 3+ ions, effectively enhancing emitter ions such as Er 3+ 、Tm 3+ 、Ho 3+ Upconversion luminescence brightness.

[0029] Example 1

[0030] Synthesis of NaYbF4:8%Er@NaLuF4:70%Yb core-shell rare earth upconversion nanoparticles with a particle size of 14 nm.

[0031] A mixture of 1 mmol (1 mL, 1 M) rare earth chloride aqueous solution (YbCl3: 0.92 mmol, ErCl3: 0.08 mmol) was added to a 100 mL flask containing 10 mL oleic acid and 10 mL octadecene. The mixture was stirred under a nitrogen atmosphere, heated to 160 ° C, and evaporated open for 40 min to remove moisture; after cooling to 110-120 ° C, 0.34 g ammonium fluoride and 2.03 g sodium oleate were quickly added, dissolved at 110-120 ° C for 30 min, and then vacuumed for 30 min; heated to 290 ° C under nitrogen protection, and maintained for 50 min, and then cooled to room temperature; 20 mL ethanol was added to precipitate nanoparticles, and the mixture was centrifuged (15000 rpm, 10 min) to separate, and the supernatant was discarded. After washing the precipitate with a mixture of cyclohexane and ethanol (20 mL, 1:1, v / v), the obtained (core) nanoparticles were dispersed in 10 mL cyclohexane for storage;

[0032] A mixture of 0.1 mmol (0.1 mL, 1 M) of an aqueous solution of rare earth chlorides (LuCl 3 : 0.03 mmol, YbCl 3 : 0.07 mmol) was added to a 100 mL flask containing 3 mL of oleic acid and 8 mL of octadecene. The mixture was stirred under an inert atmosphere, heated to 160°C, and evaporated open for 40 minutes to remove moisture, then cooled to room temperature to obtain a shell precursor solution; 1 mL of the cyclohexane dispersion of the core nanoparticles obtained in the previous step and 1.25 mL of a methanol solution in which ammonium fluoride (0.75 mmol) and sodium hydroxide (0.5 mmol) were dissolved were added, stirred under nitrogen protection, and heated at 110-120°C for 30 minutes to remove low-boiling solvents and part of the water; the temperature was raised to 290°C under nitrogen protection, and the reaction was maintained for 30 minutes, and then cooled to room temperature; 10 mL of ethanol was added to precipitate the nanoparticles, the mixture was centrifuged (15000 rpm, 10 min) to separate, the supernatant was discarded, the precipitate was washed with a mixed solution of cyclohexane and ethanol (10 mL, 1:1, v / v), and the obtained (core-shell rare earth upconversion) nanoparticles were dispersed in 10 mL of cyclohexane for storage. The transmission electron microscopy photos and X-ray diffraction patterns of the NaYbF4:8%Er@NaLuF4:70%Yb core-shell rare earth upconversion nanoparticles in Example 1 are shown in FIG. Figure 1 shown.

[0033] Example 2

[0034] The upconversion luminescence spectrum of the sulfonated-ICG-sensitized NaYbF4:8%Er@NaLuF4:70%Yb system changes with the concentration of the added dye sulfonated-ICG.

[0035] Take 0.5mL of NaYbF4:8% Er@NaLuF4:70% Yb core-shell rare earth upconversion nanoparticles cyclohexane dispersion (0.02mM) and mix with 0.5mL of NOBF4 saturated solution, gently shake for about 3min until precipitation occurs, centrifuge (14000rpm, 10min), wash with 1mL of dichloromethane, centrifuge (14000rpm, 10min), and disperse in 4mL of dimethyl sulfoxide solution. Take 1.5mL in a quartz cuvette and mix with 0, 2, 4, 6, ... 50μL of near-infrared sulfonated-ICG dye (purchased from Xi'an Kangfuno Biotechnology Co., Ltd.) solution (0.125mM), stir for 2min, use 808nm external laser as excitation light source, and test the upconversion luminescence spectrum on FLS1000 fluorescence spectrometer, the laser power is 37.5W / cm 2 . Obtain fluorescence spectra of the system as the dye concentration changes. Integrate the spectrum of each spectrum in the range of 500-680nm to obtain the integrated up-conversion luminescence intensity corresponding to different dye concentrations. Draw a curve of luminescence intensity changing with the dye quantity, with the dye quantity (μL) as the horizontal axis and the integrated luminescence intensity as the vertical axis.

[0036] Figure 2 The molecular structure (a), absorption spectrum (b) and up-conversion emission spectrum (ce) of IR806 (purchased from Sigma-Aldrich Co., Ltd.), ICG (purchased from Duoying Biotechnology Co., Ltd.), and sulfonated-ICG in the 808nm sensitization system in Example 2. The molecular structure of sulfonated-ICG dye is as follows:

[0037]

[0038] The results showed that the 2 Under the condition of 808nm laser excitation, the upconversion luminescence integrated intensity of sulfonated-ICG-sensitized UCNPs was enhanced by 2413 times compared with UCNPs excited by 980nm at the same power density, and the sensitization effect was enhanced by 33 and 2351 times compared with the traditional near-infrared dyes IR806 and ICG excited by 808nm, respectively. Figure 2 f, g).

[0039] Example 3

[0040] Upconversion luminescence spectrum of sulfonated-Cy7-sensitized NaYbF4:8%Er@NaLuF4:70%Yb system.

[0041] 90 μL of cyclohexane dispersion (0.02 mM) of NaYbF4:8% Er@NaLuF4:70% Yb core-shell rare earth upconversion nanoparticles was taken and ultrasonically mixed with 1410 μL of chloroform. The mixture was placed in a quartz cuvette and mixed with 0, 1, 2, 3, ... 35 μL of near-infrared sulfonated-Cy7 solution (0.125 mM) respectively. After stirring for 2 min, a 730 nm external laser was used as the excitation light source. The upconversion luminescence spectrum was tested on an FLS1000 fluorescence spectrometer with a laser power of 37.5 W / cm 2 .

[0042] Figure 3 The molecular structure (a), absorption spectra (b, c) and up-conversion emission spectra (d) of sulfonated-Cy7 and traditional carboxylated-Cy7 (both purchased from Duoying Biotechnology Co., Ltd.) in the 730nm sensitization system in Example 3. The molecular structure of sulfonated-Cy7 is as follows:

[0043]

[0044] When the ratio of core-shell upconversion nanoparticles to near-infrared cyanine dyes was 1:30, the upconversion integrated intensity of sulfonated-Cy7-sensitized UCNPs was enhanced by 52 times compared with UCNPs excited at 980 nm at the same power density, and the sensitization effect was enhanced by 162 times compared with the traditional design of Cy7 dyes ( Figure 3 e).

[0045] Example 4

[0046] Single particle imaging of sulfonated-ICG-sensitized NaYbF4:8%Er@NaLuF4:70%Yb.

[0047] About 500 μL of 1% (w / v) poly-L-lysine aqueous solution was pre-dropped onto a clean glass-bottomed culture dish. After 6 hours, the excess poly-L-lysine was washed off with distilled water. When the distilled water evaporated and the glass-bottomed culture dish was dry, about 500 μL of dimethyl sulfoxide solution (6.67 nM) of sulfonated-ICG-sensitized UCNPs was dropped onto the glass-bottomed culture dish, and ascorbic acid was added to improve the photostability of the dye.

[0048] The prepared single particle sample was placed on the stage of a wide-field microscope and excited by an 808 nm laser. The emission light of the sample passed through a Nikon 100x objective lens (NA 1.49), and the upconversion luminescence signal was detected by an EMCCD camera. Each sample was imaged at 1430, 6500, and 11600 W / cm 2 Data from approximately 10 fields of view were collected at a power of .

[0049] The emission signal of each particle is displayed as a point spread function. The point spread function of each particle is fitted with a two-dimensional Gaussian function. The luminescence intensity of each particle is obtained by dividing the integral of the Gaussian fitting function on the whole plane by the exposure time and subtracting the background signal. The signals of hundreds of particles at different power densities are counted and statistically averaged to obtain the single particle luminescence intensity of the sample. 2 Under laser excitation, the single particle upconversion luminescence intensity of sulfonated-ICG-sensitized UCNPs under 808nm laser excitation is 143.3, 14.4, and 2.7 times that of 976nm laser excitation, respectively. Figure 4 a, b), Dye-sensitized luminescence enhancement was obtained under single particle, and the power dependence of dye sensitization was demonstrated.

Claims

1. A near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe, characterized in that: It includes a near-infrared cyanine dye with a coordination group directly connected to a conjugated structure and a core-shell structure upconversion nanoparticle, and the surface coordination of the near-infrared cyanine dye and the core-shell structure upconversion nanoparticle realizes upconversion luminescence enhancement; wherein the conjugated system of the near-infrared cyanine dye is directly modified with a coordination group R, and the structure of the near-infrared cyanine dye is shown in Formula 1) or Formula 2): ， 1） ， 2） In the formula: n is an integer between 1 and 20, R1 and R2 are independently selected from C1-C6 alkyl, aryl-substituted C1-C6 alkyl, alkynyl-substituted C1-C6 alkyl, alkoxy-substituted C1-C6 alkyl, carboxyl-substituted C1-C6 alkyl, sulfonic acid-substituted C1-C6 alkyl, hydroxyl-substituted C1-C6 alkyl, hydroxybenzoyloxy-substituted C1-C6 alkyl, halogen-substituted C1-C6 alkyl, amino-substituted C1-C6 alkyl, amide-substituted C1-C6 alkyl or cyano-substituted C1-C6 alkyl; R is sulfonate or carboxylate; The core-shell structure upconversion nanoparticles are NaYbF4:x%RE@NaLuF4:y%Yb, x=0.5-100, y=0.01-100, the total particle size is less than 100 nm, the particle size of the core is <100 nm, and the thickness of the shell is <50 nm; the RE doped in the core is selected from Er 3 + 、Tm 3+ and Ho 3+ Any emitter ion of ions, with RE ions as emitters, and Yb doped in the outermost layer 3+ Acts as an energy acceptor for cyanine dyes and transfers energy to RE.

2. The near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1, characterized in that: n is an integer between 2 and 5.

3. The near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1, characterized in that: The near-infrared cyanine dye is selected from any one of sulfonated-ICG dye and sulfonated-Cy7 dye.

4. The near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1, characterized in that: Near-infrared cyanine dyes absorb at wavelengths between 600-900 nm and emit at wavelengths between 650-950 nm.

5. The near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1, characterized in that: The number ratio of core-shell structure upconversion nanoparticles to near-infrared cyanine dye molecules is between 1:1 and 1:1000.

6. A method for preparing the near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1, characterized in that: The following steps are involved: (1) Synthesis of core-shell upconversion nanoparticles; (2) Near-infrared cyanine dyes are coordinated to the surface of core-shell structured upconversion nanoparticles.

7. The preparation method according to claim 6, characterized in that: In step (2), the core-shell structure upconversion nanoparticles are pretreated by NOBF4 method or the rare earth upconversion nanoparticles are directly dispersed by organic solvent, and then combined with near-infrared cyanine dye solution to coordinate the near-infrared cyanine dye to the core-shell structure upconversion nanoparticles through a ligand exchange process.

8. Use of the near-infrared cyanine dye-sensitized rare earth upconversion luminescent nanoprobe according to claim 1 in single particle imaging.

9. The use according to claim 8, characterized in that: When applied, use power density: 1.43-11.6 kW / cm 2 , excitation wavelength is 600-900nm near-infrared light source.

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