Wavelength-adjustable stimulated-emission nanoprobe, preparation method therefor and use thereof

Abstract

Disclosed is a wavelength-adjustable stimulated-emission nanoprobe, comprising an Au nanorod, a dye molecule, a mesoporous silica shell layer, and a hyaluronic acid encapsulation layer. The Au nanorod is used as a plasmonic resonant cavity of the stimulated-emission nanoprobe, and the dye molecule is used as a gain medium. By regulating and controlling the energy transfer between the gain medium and the plasmonic resonant cavity, a stimulated-emission nanoprobe having a pump threshold of 0.5 mJ / cm2, a stimulated-emission spectrum linewidth of 3-10 nm, and a wavelength that can be flexibly adjusted is prepared. The probe has the advantages of overcoming the limitation of a relatively wide spectral linewidth of traditional fluorescent probes , and solving the problem that the wavelength of a stimulated-emission nanoprobe is difficult to adjust, thus opening up new avenues for realizing multi-channel imaging in a complex biological environment.

Description

A wavelength-tunable lasing-luminescent nanoprobe, its preparation method and application Technical Field

[0001] This invention relates to the fields of nanomaterials and biodetection, specifically to a wavelength-tunable lasing-luminescent nanoprobe, its preparation method, and its applications. Background Technology

[0002] In the vast field of modern biological and medical research, the innovation and application of advanced fluorescent probes play an indispensable role. However, a common challenge faced by traditional fluorescent probes is their relatively broad emission spectrum (approximately 30-100 nm). This characteristic leads to significant spectral overlap and crosstalk problems in multichannel labeling and imaging applications, greatly limiting the number of parallel color labels that can be distinguished and restricting further technological development. Despite years of continuous exploration by scientists, the problem of spectral broadening has not been fundamentally solved due to the physical limitations of spontaneous emission in fluorescent materials.

[0003] Laser technology, with its extremely narrow emission linewidth, stands out as an ideal candidate to overcome this bottleneck. If the size of lasers could be miniaturized to the nanometer scale, the problem of spectral crosstalk would be completely eliminated. However, traditional optical resonators are limited by the diffraction limit, making it difficult to break through the micrometer scale, which is too large for labeling and imaging at the cellular and molecular levels. To address this, the concept of the surface plasmon laser (Spaser) has emerged. It cleverly uses surface plasmons to replace traditional photon oscillations, successfully compressing the laser size to the nanometer scale and ushering in a new era of nanophotonics. However, the practical application of Spaser technology still faces many challenges, especially how to realize a family of multi-wavelength tunable Spaser nanoprobes, which is crucial for promoting the development of multi-channel sensing and imaging technologies. Since the first successful demonstration of Spaser in 2009, although Spaser based on nanoparticle resonators has shown the potential for multi-band emission, the flexibility and breadth of wavelength tuning remain a major challenge, requiring further exploration and breakthroughs by researchers. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to address the shortcomings of existing fluorescent probes with broad emission spectra and lasing-luminescent nanoprobes with difficult wavelength adjustment, by providing a wavelength-tunable lasing-luminescent nanoprobe, its preparation method, and its application. This probe not only has a low pump threshold and narrow emission spectral linewidth, but also allows for flexible control of the wavelength of the lasing-luminescent nanoprobe, enabling simultaneous detection of multiple channels within tumor cells and opening up new avenues for multi-channel imaging in complex biological environments.

[0005] Technical Solution: To achieve the above objectives, the present invention proposes the following technical solution:

[0006] This application provides a wavelength-tunable lasing-luminescent nanoprobe, comprising Au nanorods, dye molecules, a mesoporous silica shell, and a hyaluronic acid encapsulation layer. The Au nanorods serve as the plasmon resonant cavity of the lasing-luminescent nanoprobe; the dye molecules serve as the gain medium; the mesoporous silica shell serves as a support layer, loading the dye molecules around the Au nanorods; the hyaluronic acid encapsulation layer covers the surface of the mesoporous silica shell to prevent leakage of the dye molecules; the construction of the lasing-luminescent nanoprobe needs to satisfy the energy level matching principle.

[0007] This application also provides a method for preparing the wavelength-tunable lasing-luminescent nanoprobe, comprising the following steps:

[0008] Step 1: Prepare Au nanorod aqueous solution. Using the seed growth-mediated method, first reduce chloroauric acid with a strong reducing agent to obtain gold seed solution, then obtain gold growth solution by using the synergistic effect of binary surfactants, and finally obtain Au nanorod aqueous solution by adding silver nitrate, hydrochloric acid and gold seed solution to gold growth solution.

[0009] Step 2: Prepare an aqueous solution of Au nanorods with a mesoporous silica shell. Using the Stober method, add 0.1M sodium hydroxide solution to the Au nanorod aqueous solution obtained in Step 1, stir for 15 min, then add methanol solution containing 20% ​​tetraethyl silicate in three portions with an interval of 30 min. Then stir for 24 h in a water bath at 28℃-30℃. After centrifugation and washing with water, an aqueous solution of Au nanorods with a mesoporous silica shell is obtained.

[0010] Step 3: Prepare an aqueous solution of nanoparticles doped with dye molecules. According to the principle of energy level matching, mix the aqueous solution of nanoparticles obtained in step 2 with the dye molecule solution in the dark for 48 hours to ensure that the dye molecules fully penetrate and absorb into the mesoporous silica shell. Remove the unloaded dye molecules by centrifugation and redispersion to obtain an aqueous solution of nanoparticles doped with dye molecules.

[0011] Step 4: Prepare wavelength-tunable lasing-luminescent nanoprobes. Hyaluronic acid is added to the aqueous solution of nanoparticles obtained in step 3, and the mixture is stirred overnight at room temperature in the dark. After centrifugation and washing, the wavelength-tunable lasing-luminescent nanoprobes are obtained.

[0012] This application also provides the application of the wavelength-tunable lasing-luminescent nanoprobe in multicolor imaging within tumor cells. The wavelength-tunable lasing-luminescent nanoprobe is used to test multicolor imaging within tumor cells using a laser confocal microscope. During testing, multiple detection channels are set, with the detection range of each channel set to 10 nm. The excitation wavelength of each detection channel is set to the maximum absorption wavelength of the gain medium used for the lasing-luminescent nanoprobe. The excitation light intensity of each detection channel is set to be higher than the pump threshold to obtain multicolor imaging within tumor cells. Beneficial effects

[0013] This application uses Au nanorods as a plasmonic resonant cavity. The resonant frequency of the Au nanorods is affected by their aspect ratio. Dye molecules are used as a gain medium. The energy level of the dye molecules matches the resonant frequency of the Au nanorods. By controlling the energy transfer between the plasmonic resonant cavity and the gain medium, a narrow-band emission, low pump threshold and wavelength-flexible lasing luminescent nanoprobe is constructed.

[0014] Compared with the prior art, the advantages of this application are: the probe overcomes the limitation of the wide spectral linewidth of traditional fluorescent probes, and solves the problem of the difficulty in adjusting the wavelength of lasing luminescent nanoprobes. It can realize the simultaneous detection of multiple channels in tumor cells, opening up a new way for multi-channel imaging in complex biological environments.

[0015] The wavelength-tunable lasing-luminescent nanoprobe of this application has a spectral linewidth of only 3 nm to 10 nm and a lasing threshold of 0.5 mJ / cm. 2 This lasing-luminescent nanoprobe not only possesses narrowband emission similar to lasers but can also be excited by commercial confocal microscopy systems. By controlling the energy transfer between the plasmon resonator and the gain medium, the emission wavelength of the lasing-luminescent nanoprobe can be flexibly adjusted, constructing a family of nanoprobes encompassing multiple lasing wavelengths within a wavelength window of 500 nm to 900 nm. This probe family successfully overcomes the influence of spectral crosstalk, enabling simultaneous multi-channel detection within tumor cells. The preparation method of the aforementioned wavelength-tunable lasing-luminescent nanoprobes disclosed in this application is safe, efficient, and suitable for large-scale synthesis applications. Attached Figure Description

[0016] Figure 1 is a schematic diagram of the structure of the wavelength-tunable lasing-luminescent nanoprobe of this application.

[0017] Figure 2 shows the transmission electron microscope (TEM) image and ultraviolet absorption spectrum of this application. In Figure 2, A to E are schematic diagrams of probe TEM; and F in Figure 2 is the ultraviolet absorption spectrum of Au nanorods with different aspect ratios.

[0018] Figure 3 shows the variation of the lasing emission spectrum of the present application with pump energy. In Figure 3, A represents the original lasing emission spectrum as the pump energy increases from 0.1 mJ / cm². 2 -0.5mJ / cm 2 The graph shows the change process. In Figure 3, B represents the lasing emission spectrum after baseline subtraction as the pump energy changes from 0.5 mJ / cm². 2 -1.0mJ / cm 2 The diagram shows the process of change.

[0019] Figure 4 shows the curve of the lasing spectrum of the present application as a function of pump energy.

[0020] Figure 5 shows all the lasing emission spectra of this application.

[0021] Figure 6 is a multicolor imaging diagram of cells involved in this application. In Figure 6, A is a bright-field image of the cell, B is an individual image of four lasing probes with different wavelengths in the same cell, and C is a simultaneous image of four lasing probes with different wavelengths in the same cell. Detailed Implementation

[0022] The preferred embodiments of this application will be described in detail below with reference to examples. It should be understood that the following embodiments are given for illustrative purposes only and are not intended to limit the scope of this application. Those skilled in the art can make various modifications and substitutions to this application without departing from the spirit and intent of this application.

[0023] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0024] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0025] The reagents used in the following examples were sourced from:

[0026] Tetrachloroauric acid trihydrate, hexadecyltrimethylammonium bromide, sodium borohydride, sodium oleate, silver nitrate, ascorbic acid, sodium hydroxide, tetraethyl silicate, dimethyl sulfoxide, and hyaluronic acid were purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.; Oregon green 488 and Alexa Fluor series dyes were purchased from Thermo Fisher Scientific (China) Co., Ltd.; LDS821 and IR140 dyes were purchased from Zhiyun Optoelectronics (Shanghai) Co., Ltd.

[0027] One embodiment of this application provides a wavelength-tunable lasing-luminescent nanoprobe, the probe comprising Au nanorods, dye molecules, a mesoporous silica shell, and a hyaluronic acid encapsulation layer, wherein the Au nanorods serve as the plasmon resonant cavity of the lasing-luminescent nanoprobe; the dye molecules serve as the gain medium; the mesoporous silica shell serves as a support layer, loading the dye molecules around the Au nanorods; the hyaluronic acid encapsulation layer covers the surface of the mesoporous silica shell to prevent leakage of dye molecules; the construction of the lasing-luminescent nanoprobe needs to meet the energy level matching principle.

[0028] In one embodiment, the aspect ratio of the Au nanorods ranges from 1.0 to 5.0. The resonant frequency of the Au nanorods is influenced by their aspect ratio.

[0029] In one embodiment, the dye molecules include Oregon green 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 790, LDS821, and IR140.

[0030] In one embodiment, the energy level matching principle is that the resonance absorption curve of the Au nanorod overlaps with the spontaneous emission curve of the dye molecule in spectral form.

[0031] In one embodiment, the wavelength-tunable lasing nanoprobe includes different lasing wavelengths, and the lasing wavelength range of the wavelength-tunable lasing nanoprobe is 500 nm to 900 nm.

[0032] In one embodiment, the lasing wavelength of the wavelength-tunable lasing nanoprobe can be flexibly adjusted within a wavelength window of 500–900 nm.

[0033] In one embodiment, the pump threshold of the wavelength-tunable lasing nanoprobe is 0.5 mJ / cm2, and the lasing spectral linewidth is 3 nm to 10 nm.

[0034] One embodiment of this application provides a method for preparing the wavelength-tunable lasing-luminescent nanoprobe, comprising the following steps:

[0035] Step 1: Prepare Au nanorod aqueous solution. Using the seed growth-mediated method, first reduce chloroauric acid with a strong reducing agent to obtain gold seed solution, then obtain gold growth solution by using the synergistic effect of binary surfactants, and finally obtain Au nanorod aqueous solution by adding silver nitrate, hydrochloric acid and gold seed solution to gold growth solution.

[0036] Step 2: Prepare an aqueous solution of Au nanorods with a mesoporous silica shell. Using the Stober method, add 0.1M sodium hydroxide solution to the Au nanorod aqueous solution obtained in Step 1, stir for 15 min, then add methanol solution containing 20% ​​tetraethyl silicate in three portions with an interval of 30 min. Then stir for 24 h in a water bath at 28℃-30℃. After centrifugation and washing with water, an aqueous solution of Au nanorods with a mesoporous silica shell is obtained.

[0037] Step 3: Prepare an aqueous solution of nanoparticles doped with dye molecules. According to the principle of energy level matching, mix the aqueous solution of nanoparticles obtained in step 2 with the dye molecule solution in the dark for 48 hours to ensure that the dye molecules fully penetrate and absorb into the mesoporous silica shell. Remove the unloaded dye molecules by centrifugation and redispersion to obtain an aqueous solution of nanoparticles doped with dye molecules.

[0038] Step 4: Prepare wavelength-tunable lasing-luminescent nanoprobes. Hyaluronic acid is added to the aqueous solution of nanoparticles obtained in step 3, and the mixture is stirred overnight at room temperature in the dark. After centrifugation and washing, the wavelength-tunable lasing-luminescent nanoprobes are obtained.

[0039] In one embodiment, in step 1, the strong reducing agent is sodium borohydride, and the binary surfactant includes hexadecyl ammonium bromide and sodium oleate.

[0040] In one embodiment, in step 2, the volume ratio of the Au nanorod solution, the sodium hydroxide solution, and the methanol solution containing 20% ​​tetraethyl silicate is 100:1:1.

[0041] In one embodiment, in step 3, the molar ratio of the Au nanorod particles to the dye molecules is 1:10. 4 Magnitude.

[0042] In one embodiment, in step 4, the molecular weight of the hyaluronic acid is 5000 Da.

[0043] One embodiment of this application provides the application of the wavelength-tunable lasing-luminescent nanoprobe in multicolor imaging within tumor cells. A laser confocal microscope is used to test the multicolor imaging of the wavelength-tunable lasing-luminescent nanoprobe within tumor cells. During the test, multiple detection channels are set, with the detection range of each channel set to 10 nm. The excitation wavelength of each detection channel is set to the maximum absorption wavelength of the gain medium used for the lasing-luminescent nanoprobe. The excitation light intensity of each detection channel is set to be higher than the pump threshold to obtain multicolor imaging within tumor cells.

[0044] Example 1

[0045] As shown in Figure 1, this embodiment provides a wavelength-tunable lasing-luminescent nanoprobe, which includes Au nanorods 1, dye molecules 2, a mesoporous silica shell 3, and a hyaluronic acid encapsulation layer 4. The preparation method of the wavelength-tunable lasing-luminescent nanoprobe is as follows:

[0046] (1) Preparation of Au nanorod (Au NRs) aqueous solution:

[0047] First, 2.5 mL of 0.5 mM chloroauric acid and an equal volume of 0.2 M hexadecyltrimethylammonium bromide solution were mixed in a beaker. Under vigorous stirring, 300 μL of 0.01 M sodium borohydride ice-water solution was added, stirred for 2 min, and aged at room temperature for 1 h to obtain a gold seed solution. Then, 0.7 g of hexadecyltrimethylammonium bromide and 0.1234 g of sodium oleate were dissolved in 25 mL of warm water, cooled to 30 °C, and 25 mL of 1 mM chloroauric acid solution was added. The mixture was stirred until the solution became colorless. 125 μL of 0.064 M ascorbic acid was added to the above mixed solution, and the mixture was stirred vigorously for 30 s to obtain a gold growth solution. Finally, 40 μL of the gold seed solution was added to the gold growth solution, stirred for 30 s, and allowed to stand at room temperature for 12 h to obtain an aqueous solution of Au nanorods (Au NRs(1.0)) with an aspect ratio of 1.0.

[0048] (2) Preparation of aqueous solution of Au nanorods (Au NRs@SiO2) coated with mesoporous silica shell:

[0049] Take 20 mL of the Au NRs(1.0) aqueous solution prepared in step (1), centrifuge and wash, then redisperse in 10 mL of 1 mM hexadecyltrimethylammonium bromide solution. Then, add 100 μL of 0.1 M sodium hydroxide solution and stir for 15 min. Next, under gentle stirring, add a total of 100 μL of methanol solution containing 20% ​​tetraethyl silicate in three portions, with an interval of 30 min. Finally, stir the reaction at 28-30 °C for 24 h. After centrifugation and washing, disperse the resulting product in 5 mL of ultrapure water to obtain the Au NRs(1.0)@SiO2 aqueous solution. Its transmission electron microscope image is shown in Figure 2A, and its ultraviolet absorption spectrum is shown in Figure 2F curve a.

[0050] (3) Preparation of aqueous solution of dye-doped Au NRs@SiO2 (Dye-Au NRs@SiO2) nanoparticles:

[0051] The aqueous solution of nanoparticles prepared in step (2) was added to an Oregon green 488 (OG488) dye solution with matching energy level under slight stirring. The mixture was stirred for 48 h to ensure that the dye molecules fully penetrated and absorbed into the mesoporous silica shell. Unloaded dye molecules were removed by centrifugation and redispersion to obtain an OG488-Au NRs(1.0)@SiO2 aqueous solution.

[0052] (4) Preparation of aqueous solution of hyaluronic acid-encapsulated Dye-Au NRs@SiO2 (Dye-Au NRs@SiO2-HA) nanoparticles:

[0053] Add 5 mg of hyaluronic acid to the aqueous solution of nanoparticles prepared in step (3), stir overnight at room temperature in the dark, and obtain lasing luminescent nanoprobes after centrifugation and washing.

[0054] Example 2

[0055] The preparation method of wavelength-tunable lasing-luminescent nanoprobes is as follows:

[0056] (1) Preparation of Au nanorod (Au NRs) aqueous solution:

[0057] First, 2.5 mL of 0.5 mM chloroauric acid and an equal volume of 0.2 M hexadecyltrimethylammonium bromide solution were mixed in a beaker. Under vigorous stirring, 300 μL of 0.01 M sodium borohydride ice-water solution was added, and the mixture was stirred for 2 min. After aging at room temperature for 1 h, a gold seed solution was obtained. Then, 0.7 g of hexadecyltrimethylammonium bromide and 0.1234 g of sodium oleate were dissolved in 25 mL of warm water. Subsequently, 1.2 mL and 2.4 mL of silver nitrate solution were added respectively, and after standing for 15 min, 25 mL of 1 mM chloroauric acid solution was added, and the mixture was stirred until the solution became colorless. Subsequently, 210 μL and 150 μL of hydrochloric acid solution were introduced respectively, and after stirring for 15 min, 125 μL of 0.064 M ascorbic acid was added to the above mixed solution. After stirring vigorously for 30 s, gold growth solution was obtained. Finally, 80 μL and 20 μL of gold seed solution were added to the gold growth solution respectively, and after stirring for 30 s, the solution was allowed to stand at room temperature for 12 h to obtain aqueous solutions of Au nanorods with aspect ratios of 1.9 and 2.7 (Au NRs (1.9) and Au NRs (2.7)).

[0058] (2) Preparation of aqueous solution of Au nanorods (Au NRs@SiO2) coated with mesoporous silica shell:

[0059] Take 20 mL of the aqueous solutions of Au NRs(1.9) and Au NRs(2.7) prepared in step (1), centrifuge and wash, and redisperse in 10 mL of 1 mM hexadecyltrimethylammonium bromide solution. Then, add 100 μL of 0.1 M sodium hydroxide solution and stir for 15 min. Then, under gentle stirring, add 100 μL of methanol solution containing 20% ​​tetraethyl silicate in three portions, with an interval of 30 min. Finally, stir and react at 28-30 °C for 24 h. After centrifugation and washing, disperse the obtained products in 5 mL of ultrapure water to finally obtain aqueous solutions of Au NRs(1.9)@SiO2 and Au NRs(2.7)@SiO2. The transmission electron microscope images are shown in Figure 2B and Figure 2C, and the ultraviolet absorption spectra are shown in Figure 2F curve b and curve c.

[0060] (3) Preparation of aqueous solution of dye-doped Au NRs@SiO2 (Dye-Au NRs@SiO2) nanoparticles:

[0061] The nanoparticle aqueous solution prepared in step (2) was added to Alexa Fluor 568 (AF568), Alexa Fluor 594 (AF594) (compatible with Au NRs (1.9)@SiO2), Alexa Fluor 660 (AF660), Alexa Fluor 680 (AF680), and Alexa Fluor 700 (AF700) (compatible with Au NRs (2.7)@SiO2) dye solutions with matching energy levels under gentle stirring. The mixture was stirred for 48 h to ensure that the dye molecules fully penetrated and absorbed into the mesoporous silica shell. Unloaded dye molecules were removed by centrifugation and redispersion to obtain AF568-Au NRs (1.9)@SiO2, AF594-Au NRs (1.9)@SiO2, AF660-Au NRs (2.7)@SiO2, and AF680-Au NRs (2.7)@SiO2. NRs(2.7)@SiO2 and AF700-Au NRs(2.7)@SiO2 aqueous solution.

[0062] (4) Preparation of aqueous solution of hyaluronic acid-encapsulated Dye-Au NRs@SiO2 (Dye-Au NRs@SiO2-HA) nanoparticles:

[0063] Add 5 mg of hyaluronic acid to the aqueous solution of nanoparticles obtained in step (3), stir overnight at room temperature in the dark, and then centrifuge and wash to obtain lasing luminescent nanoprobes.

[0064] Example 3

[0065] The preparation method of wavelength-tunable lasing-luminescent nanoprobes is as follows:

[0066] (1) Preparation of Au nanorod (Au NRs) aqueous solution:

[0067] First, 2.5 mL of 0.5 mM chloroauric acid and an equal volume of 0.2 M hexadecyltrimethylammonium bromide solution were mixed in a beaker. Under vigorous stirring, 300 μL of 0.01 M sodium borohydride ice-water solution was added, and the mixture was stirred for 2 min. After aging at room temperature for 1 h, a gold seed solution was obtained. Then, 0.7 g of hexadecyltrimethylammonium bromide and 0.1234 g of sodium oleate were dissolved in 25 mL of warm water. Subsequently, 2.4 mL and 1.8 mL of silver nitrate solution were added respectively, and after standing for 15 min, 25 mL of 1 mM chloroauric acid solution was added, and the mixture was stirred until the solution became colorless. Subsequently, 300 μL and 210 μL of hydrochloric acid solution were introduced respectively, and after stirring for 15 min, 125 μL of 0.064 M ascorbic acid was added to the above mixed solution. After stirring vigorously for 30 s, gold growth solution was obtained. Finally, 20 μL and 80 μL of gold seed solution were added to the gold growth solution respectively, and after stirring for 30 s, the solution was allowed to stand at room temperature for 12 h to obtain aqueous solutions of Au nanorods with aspect ratios of 4.1 and 5.0 (Au NRs (4.1) and Au NRs (5.0)).

[0068] (2) Preparation of aqueous solution of Au nanorods (Au NRs@SiO2) coated with mesoporous silica shell:

[0069] Take 20 mL of the aqueous solutions of Au NRs(4.1) and Au NRs(5.0) nanoparticles prepared in step (1), centrifuge and wash them, and then redisperse them in 10 mL of 1 mM hexadecyltrimethylammonium bromide solution. Then, add 100 μL of 0.1 M sodium hydroxide solution and stir for 15 min. Next, under gentle stirring, add 100 μL of methanol solution containing 20% ​​tetraethyl silicate in three portions, with an interval of 30 min. Finally, stir the reaction at 28-30 °C for 24 h. After centrifugation and washing, disperse the resulting products in 5 mL of ultrapure water to finally obtain aqueous solutions of Au NRs(4.1)@SiO2 and Au NRs(5.0)@SiO2. Their transmission electron microscope images are shown in Figure 2D and Figure 2E, and their ultraviolet absorption spectra are shown in Figure 2F curve d and curve e.

[0070] (3) Preparation of aqueous solution of dye-doped Au NRs@SiO2 (Dye-Au NRs@SiO2) nanoparticles:

[0071] The nanoparticle aqueous solution prepared in step (2) was added to the Alexa Fluor 700 (AF700), Alexa Fluor 790 (AF790), LDS821 (compatible with Au NRs (4.1)@SiO2) and IR140 (compatible with Au NRs (5.0)@SiO2) dye solutions with matching energy levels under slight stirring. The mixture was mixed for 48 h to ensure that the dye molecules fully penetrated and absorbed into the mesoporous silica shell. Unloaded dye molecules were removed by centrifugation and redispersion. The aqueous solutions of AF700-Au NRs (4.1)@SiO2, AF790-Au NRs (4.1)@SiO2, LDS821-Au NRs (4.1)@SiO2 and IR140-Au NRs (5.0)@SiO2 were then mixed.

[0072] (4) Preparation of aqueous solution of hyaluronic acid-encapsulated Dye-Au NRs@SiO2 (Dye-Au NRs@SiO2-HA) nanoparticles:

[0073] Add 5 mg of hyaluronic acid to the aqueous solution of nanoparticles prepared in step (3), stir overnight at room temperature in the dark, and then centrifuge and wash to obtain lasing luminescent nanoprobes.

[0074] Example 4

[0075] Detection of luminescence properties of wavelength-tunable lasing-luminescent nanoprobes:

[0076] The luminescence properties of the lasing-luminescent nanoprobes prepared in Examples 1-3 were determined using a self-built spectrometer system. The self-built spectrometer system used a pulsed laser (20Hz, Quanta-Ray INDI, Quanta-Ray INDI, Spectra-Physics, USA) as the pump source and a compact fiber optic spectrometer (CCS200, Thorlabs Inc., USA) to collect the emission spectra. Taking the prepared AF700-Au NRs(2.7)@SiO2-HA as an example, the relationship between the luminescence intensity of the lasing-luminescent nanoprobe and the pump energy is shown in Figures 3 and 4, with a pump threshold of approximately 0.5 mJ / cm². 2 As shown in Figure 5, by controlling the energy transfer between the gain medium and the plasmonic resonator, the wavelength of the lasing-luminescent nanoprobe was flexibly tunable. At the same time, a family of nanoprobes containing nine lasing wavelengths within a wavelength window of 500–900 nm was constructed, with lasing spectral linewidths between 3 and 10 nm.

[0077] Example 5

[0078] Wavelength-tunable lasing-luminescent nanoprobes for multicolor imaging within tumor cells:

[0079] The application of lasing-luminescent nanoprobes prepared in Examples 1-3 in multicolor imaging within tumor cells was determined using a laser confocal microscopy system. Taking four types of the prepared lasing-luminescent nanoprobes as examples, HeLa cells were first cultured in a confocal dish for 18 hours. Then, solutions of four luminescent nanoprobes with the same concentration (50 μg / mL) but different lasing wavelengths were added sequentially to the confocal dish and incubated at 37°C for 6 hours. Before testing, the confocal dish was washed three times with PBS solution at pH 7.4 to remove excess nanoparticles. During testing, four detection channels were set, with the detection range of each channel set to 10 nm. The excitation wavelength of each channel was set to the maximum absorption wavelength of the gain medium used, and the excitation light intensity of each detection channel was set higher than the pump threshold. As shown in Figure 6, Figure 6A is the bright-field image of the cell, Figure 6B is the individual imaging of the four lasing-luminescent probes with different wavelengths in the same cell, and Figure 6C is the simultaneous imaging of the four lasing-luminescent probes with different wavelengths in the same cell. This wavelength-tunable lasing-luminescent nanoprobe enabled simultaneous four-color imaging within tumor cells.

[0080] The above are merely preferred embodiments of this application. It should be noted that, for those skilled in the art, several improvements and modifications can be made without departing from the principles of this application, and these improvements and modifications should also be considered within the scope of protection of this application.

Claims

1. A wavelength-tunable lasing-luminescent nanoprobe, characterized in that, The probe comprises Au nanorods, dye molecules, a mesoporous silica shell, and a hyaluronic acid encapsulation layer. The Au nanorods serve as the plasmon resonant cavity of the lasing-luminescent nanoprobe; the dye molecules serve as the gain medium; the mesoporous silica shell serves as a support layer, loading the dye molecules around the Au nanorods; the hyaluronic acid encapsulation layer covers the surface of the mesoporous silica shell to prevent leakage of the dye molecules; the construction of the lasing-luminescent nanoprobe must satisfy the energy level matching principle.

2. The wavelength-tunable lasing-luminescent nanoprobe according to claim 1, characterized in that, The aspect ratio of the Au nanorods ranges from 1.0 to 5.

0.

3. The wavelength-tunable lasing-luminescent nanoprobe according to claim 1, characterized in that, The dye molecules include Oregon green 488, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 660, Alexa Fluor 680, Alexa Fluor 700, Alexa Fluor 790, LDS821, and IR140.

4. The wavelength-tunable lasing-luminescent nanoprobe according to claim 1, characterized in that, The energy level matching principle is that the resonance absorption curve of Au nanorods and the spontaneous emission curve of dye molecules overlap in spectral form.

5. The wavelength-tunable lasing-luminescent nanoprobe according to claim 1, characterized in that, The wavelength-tunable lasing-luminescent nanoprobe has a lasing wavelength range of 500 nm to 900 nm.

6. The wavelength-tunable lasing-luminescent nanoprobe according to claim 1, characterized in that, The pump threshold of the wavelength-tunable lasing-luminescent nanoprobe is 0.5 mJ / cm. 2 The lasing spectral linewidth is 3nm to 10nm.

7. A method for preparing a wavelength-tunable lasing-luminescent nanoprobe according to any one of claims 1 to 6, characterized in that, Includes the following steps: Step 1: Prepare Au nanorod aqueous solution. First, reduce chloroauric acid with a strong reducing agent to obtain gold seed solution. Then, obtain gold growth solution by using a binary surfactant synergistic effect. Finally, add silver nitrate, hydrochloric acid and gold seed solution to the gold growth solution to obtain Au nanorod aqueous solution. Step 2: Prepare an aqueous solution of Au nanorods with a mesoporous silica shell. Add 0.1M sodium hydroxide solution to the Au nanorod aqueous solution obtained in Step 1, stir for 15 min, then add methanol solution containing 20% ​​tetraethyl silicate in 3 portions with an interval of 30 min. Stir for 24 h in a water bath at 28℃-30℃, centrifuge and wash with water to obtain an aqueous solution of Au nanorods with a mesoporous silica shell. Step 3: Prepare an aqueous solution of nanoparticles doped with dye molecules. According to the principle of energy level matching, mix the aqueous solution of nanoparticles obtained in step 2 with the dye molecule solution in the dark for 48 hours. Remove the unloaded dye molecules by centrifugation and redispersion to obtain an aqueous solution of nanoparticles doped with dye molecules. Step 4: Prepare wavelength-tunable lasing-luminescent nanoprobes. Hyaluronic acid is added to the aqueous solution of nanoparticles obtained in step 3, and the mixture is stirred overnight at room temperature in the dark. After centrifugation and washing, the wavelength-tunable lasing-luminescent nanoprobes are obtained.

8. The method for preparing the wavelength-tunable lasing-luminescent nanoprobe according to claim 7, characterized in that, In step 1, the strong reducing agent is sodium borohydride, and the binary surfactant includes hexadecyl ammonium bromide and sodium oleate; in step 2, the volume ratio of the Au nanorod solution, sodium hydroxide solution, and methanol solution containing 20% ​​tetraethyl silicate is 100:1:

1.

9. The method for preparing the wavelength-tunable lasing-luminescent nanoprobe according to claim 7, characterized in that, In step 3, the molar ratio of Au nanorod particles to dye molecules is 1:

10. 4 In step 4, the molecular weight of the hyaluronic acid is 5000 Da.

10. An application of the wavelength-tunable lasing-luminescent nanoprobe according to any one of claims 1 to 6 in multicolor imaging within tumor cells, characterized in that, The wavelength-tunable lasing-luminescent nanoprobe was tested for multicolor imaging within tumor cells using laser confocal microscopy. During the test, multiple detection channels were set, with the detection range of each channel set to 10 nm. The excitation wavelength of each detection channel was set to the maximum absorption wavelength of the gain medium used for the lasing-luminescent nanoprobe. The excitation light intensity of each detection channel was set to be higher than the pump threshold to obtain multicolor imaging within tumor cells.

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