A Rayleigh scattering-induced fluorescence enhancement method

Abstract

This invention proposes a method for fluorescence enhancement induced by Rayleigh scattering. First, a nanomaterial with Rayleigh scattering properties is prepared into a solution of a certain concentration to serve as a Rayleigh scattering source. The nanomaterial and the fluorescent substance to be enhanced are then dispersed in the solution and mixed uniformly. Finally, an optical instrument is used to collect the fluorescence signal from the mixed solution. This method utilizes the Rayleigh scattering properties of the nanomaterial to improve the fluorescence excitation efficiency and luminescence collection efficiency, thereby achieving fluorescence enhancement.

Description

Technical Field

[0001] The present invention relates to a fluorescence enhancement method induced by Rayleigh scattering, and in particular to a method for enhancing the luminous efficiency of nanoluminescent materials and fluorescent dyes by utilizing Rayleigh scattering (RS) of nanoparticles, belonging to the technical field of nanoparticles and optical composites. Background Art

[0002] Fluorescence is the phenomenon in which a substance absorbs energy and emits light after being irradiated by laser light. It has a wide range of important applications in medicine, biology, materials science, environmental testing, food safety, information storage / display, and lasers.

[0003] Improving the luminous intensity and efficiency of fluorescence is of great significance and value in scientific research, medical diagnosis, biology, and materials science. First, increasing the intensity of the fluorescence signal can more easily detect low-solubility target substances, which is of great value for high-sensitivity detection in fields such as biology, medicine, and environmental monitoring. Secondly, increasing the fluorescence intensity helps to improve spatial resolution. In microscopy and imaging technology, it can make images clearer, thereby more accurately observing and studying microstructures and biological processes, which is crucial for a deep understanding of life activities and pathogenic mechanisms. In addition, fluorescence enhancement also has a positive effect on reducing costs. By improving the efficiency of the fluorescence signal, the required fluorescent markers and light source energy can be reduced, thereby reducing experimental costs and achieving better results while reducing the side effects of strong light sources. Finally, the development of a universal fluorescence enhancement method is also of great significance to the research and development of high-precision optical devices and display equipment.

[0004] Currently, commonly used fluorescence enhancement methods include aggregation-induced fluorescence enhancement (AIE), molecular resonance energy transfer-based fluorescence enhancement, and noble metal SPR-based fluorescence enhancement. AIE has very high requirements for molecular structure and requires the formation of aggregates to achieve enhancement, which greatly limits its scope of application. Fluorescence enhancement based on molecular resonance energy transfer requires spectral overlap between the donor and acceptor, which is not universal and universal. Fluorescence enhancement based on noble metal SPR is often accompanied by quenching, and the relative intensity of the two is affected by the distance between the metal particles and the luminophore. To achieve a good enhancement effect, the distance between the nanoparticles and the fluorophore must be precisely controlled. The assembly technology is difficult, and the high price of noble metals is not conducive to popularization. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a fluorescence enhancement method induced by Rayleigh scattering, which utilizes the Rayleigh scattering characteristics of nanoparticles to enhance the luminescence of fluorescent nanomaterials and fluorescent dyes.

[0006] The present invention provides a method for fluorescence enhancement induced by Rayleigh scattering, comprising the following steps:

[0007] Step 1: preparing a solution of a certain concentration of a nanomaterial having a Rayleigh scattering effect as a Rayleigh scattering source;

[0008] Step 2: Dispersing the nanomaterial and the fluorescent substance to be enhanced in the solution and mixing them evenly;

[0009] Step 3: Use an optical instrument to collect the fluorescence signal of the mixed solution.

[0010] The present invention provides a convenient and versatile fluorescence enhancement method that can be achieved simply by mixing a nanomaterial with the fluorescent substance to be enhanced. This method utilizes the Rayleigh scattering properties of the nanomaterial to improve the efficiency of fluorescence excitation and luminescence collection, thereby achieving fluorescence enhancement. The nanoparticles used for fluorescence enhancement in the present invention do not require special materials, shapes, or structures; they only need to exhibit Rayleigh scattering. Therefore, this method is simple to operate, cost-effective, and highly suitable for widespread application.

[0011] The technical solution further optimized as the present invention is as follows:

[0012] Preferably, the nanomaterial is any one of metal nanomaterial, inorganic non-metal nanomaterial and organic polymer nanomaterial; the fluorescent substance is any one of nanoluminescent material and fluorescent dye.

[0013] Preferably, the metal nanomaterial is a noble metal nanomaterial, the inorganic non-metallic nanomaterial is nano-silica, nano-cerium dioxide, carbon nanomaterial or magnetic nano-microspheres, etc., the organic polymer nanomaterial is polyethylene microspheres, etc.; the nano-luminescent material is any one of quantum dots, fluorescent microspheres, gold nanoclusters, and upconversion nanoparticles.

[0014] Preferably, the particle size of the nanomaterial is 2 to 10,000 nm.

[0015] Preferably, the quantum dots are carbon quantum dots, CdSe / ZnS core-shell quantum dots 585 or CdSe / ZnS core-shell quantum dots 625, the noble metal nanomaterials are gold nanoparticles with a particle size of 13 nm, and the fluorescent microspheres are polystyrene fluorescent microspheres.

[0016] In step 2, the solution is water or a buffer solution.

[0017] Preferably, the buffer solution is one of Tris-HCl buffer, HEPES buffer, PBS buffer, and MPOS buffer, and the pH of the buffer solution is 1-12.

[0018] In step 2, the nanomaterial and the fluorescent substance are sealed, and the sealing agent is SuperBlock purchased from ThermoScientific TM , Tween, BSA, or any one of the sulfhydryl small molecules.

[0019] In order to prevent nonspecific binding, the present invention can selectively block the nanomaterials and fluorescent substances. The blocking agent used is SuperBlock purchased from Thermo Scientific. TM The blocking agent can be omitted or replaced with Tween, BSA, thiol small molecules, etc. according to the specific experiment.

[0020] Compared with the existing technology, the present invention has no special requirements for the fluorescent substance to be enhanced, and can achieve good enhancement effects on various luminescent nanomaterials and fluorescent dyes. The universality and convenience of the present invention provide a more flexible and efficient fluorescence enhancement solution for various fields. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is a schematic diagram of the principle of Rayleigh scattering (RS) enhanced fluorescence in the present invention.

[0022] Figure 2 This is the Rayleigh scattering enhanced excitation (EX) diagram in the present invention.

[0023] Figure 3 This is the Rayleigh scattering enhanced emission (EM) image of the present invention.

[0024] Figure 4 This is a diagram of the fluorescence enhancement of quantum dots by gold nanoparticles in the present invention.

[0025] Figure 5 This is a graph showing the results of a study on enhanced versatility in the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and specific operation process are given, but the protection authority of the present invention is not limited to the following embodiments.

[0027] The gold nanoparticles used in the present invention were prepared according to the literature [Nature Protocols 2006, 1, 246-252]. CdSe / ZnS core-shell quantum dots 585 (QDs-585) and CdSe / ZnS core-shell quantum dots 655 (QDs-655) were purchased from Thermo Fisher Scientific, CdSe / ZnS core-shell quantum dots 625 (QDs-625) were purchased from Wuhan Jiayuan, nano-cerium dioxide was purchased from Roan's Reagent, polystyrene fluorescent microspheres were purchased from Bangs Laboratories, Inc., and SuperBlock TM Purchased from Thermo Scientific. Example 1

[0028] Place 300 μL of 13 nm gold nanoparticles (10 nM) in a centrifuge tube and disperse the gold nanoparticles in 600 μL of HEPES buffer (10 mM, pH 7.6).

[0029] Make as Figure 2 The cuvette shown in A is divided into two unequal areas in the middle by a transparent quartz plate, which are marked as area B (Block) and area E (Excite). Area B is pasted with black opaque tape on the side facing the incident light as the Block area; and area E is the direct irradiation area.

[0030] 500 μL of gold nanoparticle solution was placed in area E of the separating cuvette, and 200 μL of 250 pM QDs-625 was placed in area B of the cuvette. The two were not in contact with each other, as the experimental group.

[0031] 500 μl of HEPES buffer solution (10 mM, pH 7.6) was placed in area E of the separation cuvette, and 200 μl of 250 pM QDs-625 was placed in area B of the cuvette. The two were not in contact with each other, serving as a blank control group.

[0032] Place the experimental group cuvette and the control group cuvette in the molecular fluorescence spectrophotometer respectively, and collect the corresponding fluorescence emission curves. The results are as follows: Figure 2 As shown in B.

[0033] like Figure 2 As shown in Figure B, when the excitation light illuminates the control cuvette, the luminescent light signal of the quantum dots is extremely weak (see Figure 2, blank). However, under the same conditions, when the experimental cuvettes were illuminated with excitation light, a significant increase in the quantum dot luminescence signal was observed. This difference arises from the extremely weak scattering effect of a simple solution system. However, when region E contains a gold nanoparticle solution, the excitation light is scattered into region B due to the scattering effect of the gold nanoparticles, exciting the quantum dots and significantly enhancing the fluorescence emission signal. Figure 2 C is the normalized QDs fluorescence intensity change diagram and the Rayleigh scattering intensity change diagram of gold particles in this process obtained by experiments and theoretical simulations. It can be seen from the figure that the change trend of the quantum dots' luminescence intensity is consistent with the change trend of the Rayleigh scattering intensity, thus confirming that this fluorescence enhancement is caused by Rayleigh scattering. Example 2

[0034] 300 μL of 13 nm gold nanoparticles (10 nM) were placed in a centrifuge tube and dispersed into 600 μL of HEPES buffer (10 mM, pH 7.6).

[0035] 500 μL of 13 nm gold nanoparticle solution was placed in area B of the separated cuvette, and 200 μL of 250 pM QDs-625 was placed in area E of the cuvette. The two were not in contact with each other, as the experimental group, in which area E was placed before area B and the detector.

[0036] 500 μL of HEPES buffer solution (10 mM, pH 7.6) was placed in area B of the separating cuvette, and 200 μL of 250 pM QDs-625 was placed in area E of the cuvette. The two were not in contact with each other, serving as a blank control group. Area E was placed before area B and the detector.

[0037] Place the cuvettes containing the experimental group and the control group in the molecular fluorescence spectrophotometer respectively, ensuring that the excitation light only illuminates the E area containing the luminescent substance (QDs-625), and collect the corresponding fluorescence emission curves. The results are as follows: Figure 3 shown.

[0038] like Figure 3 As shown in Figures A to C, at the same quantum dot concentration, the fluorescence signal obtained when gold nanoparticles are present in area B is significantly stronger. During the excitation light irradiation experiment, the quantum dots can be directly excited, and their light is further scattered after hitting the gold particles, resulting in an increase in the amount of emitted light entering the collection device. At the same quantum dot concentration, the fluorescence is enhanced. Example 3

[0039] 300 μL of 13 nm gold nanoparticles (10 nM) were placed in a centrifuge tube and dispersed into 600 μL of HEPES buffer (10 mM, pH 7.6).

[0040] 500 μL of 13 nm gold nanoparticle solution was placed in area B of the separating cuvette, and 200 μL of 250 pM QDs-625 was placed in area E of the cuvette. The two were not in contact with each other, as the experimental group, in which area B was placed between area E and the detector.

[0041] 500 μL of HEPES buffer solution (10 mM, pH 7.6) was placed in area B of the separating cuvette, and 200 μL of 250 pM QDs-625 was placed in area E of the cuvette. The two were not in contact with each other, serving as a blank control group. Area B was placed between area E and the detector.

[0042] Place the cuvettes containing the experimental group and the control group in the molecular fluorescence spectrophotometer respectively, ensuring that the excitation light only illuminates the E area containing the luminescent substance (QDs-625), and collect the corresponding fluorescence emission curves. The results are as follows: Figure 3 shown.

[0043] like Figure 3 As shown in D to F, at the same quantum dot concentration, the fluorescence signal obtained when the gold particles are in area B is significantly stronger. During the excitation light irradiation experiment, although a significant enhancement of the quantum dot luminescence was observed, the enhancement effect was lower than that of Example 2. This is because when the gold particles are placed between the quantum dots and the detector, their absorption of the quantum dot luminescence is enhanced, and the inner filter effect is enhanced. Figure 2 and Figure 3 It can be seen that the scattering characteristics of nanomaterials can enhance the amount of excitation light and the amount of emitted light collected, thereby significantly enhancing fluorescence.

[0044] Examples 1 to 3 are the verification of the mechanism of Rayleigh scattering enhancement of fluorescence. Figure 1 As shown, the Rayleigh scattering characteristics of nanomaterials are used to improve the fluorescence excitation efficiency and luminescence collection efficiency, thereby achieving fluorescence enhancement. Example 4

[0045] Different amounts of 13 nm gold nanoparticles (10 nM) were placed in a centrifuge tube and dispersed into 400 μL of HEPES buffer (10 mM, pH 7.6) to prepare gold nanoparticle solutions of different concentrations.

[0046] 20 μL of the above 13 nm gold nanoparticle solution was mixed with 1 μL of 5 nM quantum dots QDs-625, and then dispersed into HEPES buffer to a volume of 100 μL. The mixture was vortexed and used as the experimental group.

[0047] 99 μL of HEPES buffer solution (10 mM, pH 7.6) was mixed with 1 μL of quantum dots QDs-625 (5 nM) and vortexed to mix. This was used as the control group.

[0048] 90 μl of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. The results are as follows: Figure 4 As shown in AC, after quantum dots (QDs) are mixed with gold nanoparticles of different concentrations, the luminescence of QDs changes with the concentration of gold particles. When the gold particle concentration is in the range of 0-3nM, the luminescence of quantum dots is significantly enhanced, with the maximum enhancement factor being 5.7. Example 5

[0049] Take 20 μL of gold nanoparticles (10 nM) with a particle size of 13 nm and place them in a centrifuge tube. Disperse the gold nanoparticles in 400 μL of HEPES buffer (10 mM, pH 7.6).

[0050] 20 μL of 13 nm gold nanoparticle solution was mixed with 1 μL of 5 nM QDs-655, dispersed into HEPES buffer to a volume of 100 μL, and vortexed to mix. This was used as the experimental group.

[0051] 99 μL of HEPES buffer solution (10 mM, pH 7.6) was mixed with 1 μL of 5 nM quantum dots QDs-655 and vortexed to mix, which served as the control group.

[0052] 90 μL of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. Figure 4 D. Example 6

[0053] In order to rule out the possibility that the fluorescence enhancement is caused by nonspecific adsorption between gold particles and quantum dots, single-stranded DNA (ssDNA)-modified gold particles and quantum dots were prepared respectively to increase the negative charge on the surface of the nanoparticles and enhance the repulsion between the particles. The modification of ssDNA on the surface of quantum dots utilizes the binding effect of streptavidin and biotin. The modification of ssDNA on the surface of gold particles can be found in the reference [JACS, 2021,143, 3065-3069].

[0054] Take 20 μl of ssDNA-gold nanoparticles (10 nM) with a particle size of 13 nm and place them in a centrifuge tube, add 1 μl of blocking agent (superblock TM The blocking agent was removed by centrifugation, and the cells were washed twice with HEPES buffer (10 mM, pH 7.6). The ssDNA-gold nanoparticles were then dispersed in 400 μL of HEPES buffer (10 mM, pH 7.6).

[0055] Take 20 μL of blocked 13 nm ssDNA-gold nanoparticle solution and mix it with 1 μL of ssDNA-modified QDs-585 (ssDNA-QDs-585) (5 nM), disperse it into HEPES buffer to a volume of 100 μL, and vortex to mix it. This is used as the experimental group.

[0056] 99 μL of HEPES buffer solution (10 mM, pH 7.6) was mixed with 1 μL of quantum dot ssDNA-QDs-585 (5 nM) and vortexed to mix. This was used as a control group.

[0057] 90 μL of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. Figure 4 E.

[0058] Examples 4 to 6 are verifications of the fluorescence enhancement effect. Example 7

[0059] 20 μL of gold nanoparticles (10 nM) with a particle size of 13 nm were placed in a centrifuge tube, and the nanomaterials were dispersed in 400 μL of HEPES buffer (10 mM, pH 7.6).

[0060] 40 μL of 13 nm gold nanoparticles were mixed with 1 μL of 3.8 mg / mL fluorescent dye rhodamine B (purchased from Xishen Reagent Company), dispersed into HEPES buffer to a volume of 100 μL, and vortexed to mix. This was used as the experimental group.

[0061] Take 99 μl of HEPES buffer solution (10 mM, pH 7.6) and mix it with 1 μl of fluorescent dye with a concentration of 3.8 mg / ml. Vortex and mix well. This is used as the control group.

[0062] 90 μl of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. The results are as follows: Figure 5 As shown in A. Example 8

[0063] 20 μL of gold nanoparticles (10 nM) with a particle size of 13 nm were placed in a centrifuge tube, and the nanomaterials were dispersed in 400 μL of HEPES buffer (10 mM, pH 7.6).

[0064] 40 μL of 13 nm gold nanoparticles were mixed with 1 μL of 0.2 mg / ml polystyrene fluorescent microspheres, dispersed into HEPES buffer (10 mM, pH 7.6) to a volume of 100 μL, and vortexed to mix. This was used as the experimental group.

[0065] Take 99 μl of HEPES buffer solution (10 mM, pH 7.6) and mix it with 1 μl of 0.2 mg / ml polystyrene fluorescent microspheres, vortex and mix thoroughly, as the control group.

[0066] 90 μl of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. The results are as follows: Figure 5 As shown in B.

[0067] Figure 5 The enhancement curves of 13 nm gold nanoparticles to (A) fluorescent dyes and (B) fluorescent microspheres are shown. Figure 5 It can be seen that 13nm gold nanoparticles also have an enhancing effect on fluorescent dyes and fluorescent microspheres. Example 9

[0068] Take 10 μL of 0.32 mg / mL CeO2 nanoparticles and mix them with 1 μL of 5 nM QDs-625, disperse them into HEPES buffer to a volume of 100 μL, and vortex to mix them. This is used as the experimental group.

[0069] 99 μL of HEPES buffer (10 mM, pH 7.6) was mixed with 1 μL of 5 nM quantum dots QDs-625 and vortexed to mix, which served as the control group.

[0070] 90 μl of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. The results are shown in the table. Figure 5 C. Example 10

[0071] 10 μL of 0.3 nM upconversion nanoparticles were mixed with 1 μL of 5 nM QDs-625, dispersed into HEPES buffer to a volume of 100 μL, and vortexed to mix. This was used as the experimental group.

[0072] 99 μL of HEPES buffer (10 mM, pH 7.6) was mixed with 1 μL of 5 nM quantum dots QDs-625 and vortexed to mix, which served as the control group.

[0073] 90 μl of the solution from the experimental group and the control group were placed in quartz cuvettes respectively, and the corresponding emission curves were collected under the same conditions. The results are shown in the table. Figure 5 D.

[0074] Examples 7 to 10 are studies on the versatility of Rayleigh scattering enhanced fluorescence.

[0075] The above description is only a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any person familiar with the technology can understand and think of any changes or replacements within the technical scope disclosed by the present invention, which should be included in the scope of the present invention. Therefore, the scope of protection of the present invention should be based on the scope of protection of the claims.

Claims

1. A method for fluorescence enhancement induced by Rayleigh scattering, characterized in that: The following steps are involved: Step 1: preparing a nanomaterial having a Rayleigh scattering effect into a solution of a certain concentration for standby use as a Rayleigh scattering source; the nanomaterial is any one of a metal nanomaterial, an inorganic non-metallic nanomaterial, and an organic polymer nanomaterial; the metal nanomaterial is a noble metal nanomaterial, the inorganic non-metallic nanomaterial is nano-silicon dioxide, nano-cerium dioxide, carbon nanomaterial, or magnetic nano-microspheres, and the organic polymer nanomaterial is polyethylene microspheres; Step 2: Dispersing the nanomaterial and the fluorescent substance to be enhanced in the solution and mixing them evenly; The solution is water or a buffer solution, wherein the buffer solution is at least one of a Tris-HCl buffer solution, a HEPES buffer solution, and a PBS buffer solution; the fluorescent substance is any one of a nanoluminescent material and a fluorescent dye; Step 3: Use an optical instrument to collect the fluorescence signal of the mixed solution.

2. The method for fluorescence enhancement induced by Rayleigh scattering according to claim 1, characterized in that: The nano-luminescent material is any one of quantum dots, fluorescent microspheres, gold nanoclusters, and up-conversion nanoparticles.

3. The method of inducing fluorescence enhancement by utilizing the Rayleigh scattering effect of nanoparticles according to claim 2, characterized in that: The quantum dots are carbon quantum dots, CdSe / ZnS core-shell quantum dots 585, CdSe / ZnS core-shell quantum dots 625 or CdSe / ZnS core-shell quantum dots 655, the noble metal nanomaterials are gold nanoparticles with a particle size of 13 nm, and the fluorescent microspheres are polystyrene fluorescent microspheres.

4. The method for fluorescence enhancement induced by Rayleigh scattering according to claim 1, characterized in that: In step 2, the nanomaterial and the fluorescent substance are sealed, and the sealing agent is SuperBlock TM , Tween, BSA, or any one of the sulfhydryl small molecules.

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