CN210269616U

CN210269616U - Nano probe based on surface plasma enhancement effect

Info

Publication number: CN210269616U
Application number: CN201920906184.6U
Authority: CN
Inventors: 王忆; 李旸晖; 李雨雪; 马超然
Original assignee: China Jiliang University
Current assignee: China Jiliang University
Priority date: 2019-06-17
Filing date: 2019-06-17
Publication date: 2020-04-07
Anticipated expiration: 2029-06-17

Abstract

The utility model discloses a nanoprobe based on surface plasma enhanced effect, including nanoparticle, fluorophore, nanometer antibody. The two ends of the nano antibody are respectively connected with the nano particles and the fluorophore in a non-covalent combination mode on the surface of the biotin and streptavidin system. The nano probe regulates and controls the oscillation frequency of the nano particles by changing the size of the nano particles, utilizes the surface plasma effect of the nano particles, enhances the fluorescence intensity, realizes the super-resolution imaging of low-loss light, and can sensitively detect the position of a biological sample and accurately position and image.

Description

Nano probe based on surface plasma enhancement effect

Technical Field

The utility model relates to an optical instrument field and biomedical formation of image field, concretely relates to nanoprobe based on surface plasma reinforcing effect.

Background

At present, the requirement of the research in the biomedical imaging field on the resolution is higher and higher, and researchers need to know the structural information of micro-morphological substances on various nanometer scales. Unlike electron microscope imaging, which can damage the sample itself, fluorescence microscopy imaging can provide physical, chemical, and biological information of the sample without damaging the sample. Stimulated emission depletion microscopy (STED) based on far-field imaging is widely applied to research of problems of cell biology and the like on a nanometer scale due to the characteristics of real time, rapidness, ultrahigh resolution and the like.

An article published in the journal of Chemical Science by Li Shang et al, entitled Protein-based fluorescent Nanoparticles for Super-resolution STED Imaging of Live Cells, proposes a method for Super-resolution Imaging using Nanoparticles (NPs) to prepare Protein-based fluorescent NPs that can be used as probes for Super-resolution microscopes. The method is simple and direct and is easy to popularize. Taking Atto647N-transferrinNPs as an example, the resolution was improved by approximately four times by using an STED super-resolution microscope. The protein-based NPs have good biocompatibility and good colloidal stability, and are ideal choices for biological research. Although the method can realize super-resolution imaging, the method still has obvious defects, such as high requirement on optical power loss and easy damage to a fluorescence sample, including photobleaching, phototoxicity and the like.

Disclosure of Invention

The utility model discloses to the too big problem of loss optical power that exists among the super-resolution microtechnology before, provided a nanoprobe based on surface plasma reinforcing effect. The probe regulates the oscillation frequency of the nano particles by changing the size of the nano particles, enhances the fluorescence intensity by utilizing the surface plasma effect, and realizes the super-resolution imaging of low-loss light.

Furthermore, the utility model discloses contain following technical scheme:

a nanoprobe based on surface plasmon enhancement effect comprises nanoparticles, nanobodies and fluorophores;

the method comprises the following steps that a nanometer probe composed of nanoparticles, a nanometer antibody and a fluorophore enters a biological sample under the endocytosis of cells, when a laser beam irradiates the nanometer probe in the biological sample, plasma is generated on the surface of the nanoparticles of the nanometer probe, an electric field around the nanoparticles is enhanced by the plasma, the fluorophore in the electric field absorbs photons in the light beam, fluorescence is emitted from an imaging part positioned by the nanometer antibody in the biological sample under the action of the enhanced electric field, and the fluorescence is collected by a stimulated emission fluorescence microscopic imaging system to realize imaging;

the nanoparticles are of a shell-core structure containing metal particles and inorganic materials, and the electric field distribution generated by the nanoparticles under the irradiation of incident light can be simulated by a Finite-Difference-Time-Domain (FDTD) method;

the fluorophore comprises an organic dye and a fluorescent protein;

the fluorophore, the nanobody and the nanoparticle are combined by the surface of a biotin-streptavidin system in a non-covalent way;

the nano antibody is used for specific binding reaction with antigen in a biological sample, and can be accurately positioned to an imaging part of the biological sample.

Preferably, the metal particle material is gold, the particle size of the metal particle is 10-70 nanometers, the inorganic material is silicon dioxide, and the thickness of the inorganic material shell layer is 5-20 nanometers.

Preferably, the fluorophore is the organic dye ATTO 488.

The size of the preferable nano antibody is about 2 nanometers, so that the fluorescence quenching effect of the nano particles on a fluorophore can be effectively isolated, and the fluorescence intensity of a fluorescent molecule can be greatly enhanced.

Preferably, the laser beam wavelength is 595 nm.

Compared with the prior art, the nanoprobe based on the surface plasma enhanced effect adopting the scheme has the following effective effects:

1. the utility model discloses a nanoparticle structure utilizes surface plasma resonance effect can realize more excellent super resolution formation of image, and simple structure need not high-power loss laser beam.

2. The utility model discloses a biological sample position can sensitively be surveyed to the nanoprobe, and accurate location formation of image.

Drawings

FIG. 1 is a schematic diagram of an embodiment of a nanoprobe based on surface plasmon enhancement effect according to the present invention; wherein: a. Nanoparticle B, fluorophore C, nanobody.

FIG. 2 is a schematic structural diagram of the nanoparticle A in FIG. 1; wherein: 1. streptavidin 2, a silicon dioxide layer 3 and gold nanoparticles.

FIG. 3 is a schematic diagram of the structure of fluorophore B in FIG. 1; wherein: 1. streptavidin 4, fluorescent molecule.

FIG. 4 is a schematic structural diagram of Nanobody C in FIG. 1; wherein: 5. immunoglobulin 6, biotin.

Fig. 5 is a graph of electric field strength around nanoparticles calculated using FDTD in an embodiment.

Fig. 6 is a calculated electric field distribution of the laser beam coupled through the nanoparticle structure in accordance with an embodiment.

Detailed Description

The present invention will be described in detail with reference to the accompanying drawings, but the present invention is not limited thereto.

In this embodiment, two ends of the nanobody C are respectively connected to the nanoparticle a and the fluorophore B, and the overall structure of the nanoprobe is shown in fig. 1. The components shown in fig. 2, 3 and 4 are connected by using non-covalent binding of biotin and streptavidin.

Fig. 2 shows a nanoparticle shell-core structure, a core-shell structure composed of a silica layer 2 and gold nanoparticles 3 determines a position of plasma resonance, and the silica layer 2 keeps a certain distance between fluorescent molecules 4 and the gold nanoparticles 3 to prevent fluorescence emitted by the fluorescent molecules 4 from being quenched. The nano-particle model is ABN-SG-20 of Baiohotai, the particle diameter of the gold nano-particle 3 is 20 nanometers, and the thickness of the silicon dioxide layer is 10 nanometers.

The electric field intensity around the nanoparticle a is calculated by using a Finite-Difference-Time-Domain (FDTD) method, and the sizes of the gold nanoparticle 3 and the silicon dioxide layer 2 are optimized to obtain the maximum electric field enhanced by the surface of the nanoparticle. Through calculation, the particle size of the gold nanoparticles 3 in this example is 20 nm, the thickness of the silicon dioxide layer 2 is 10 nm, and the result of the electric field intensity is shown in fig. 5.

The data in fig. 5 were analyzed, and the normalized value of the surface electric field strength of the metal particle structure under the above simulation conditions was 5.41191, and the average near field enhancement was about 1.4 times, so that super-resolution imaging based on the surface plasmon electric field enhancement was achieved, and the electric field distribution thereof is shown in fig. 6.

Under the irradiation of laser beams, plasmas are generated on the surface of a nano particle A, the plasmas enhance the electric field around the nano particle A, fluorescent molecules 4 in the electric field absorb photons in the light beams, fluorescence is emitted from an imaging part positioned by immunoglobulin 5 of a nano antibody in a biological sample under the action of the enhanced electric field, and the fluorescence is collected by a stimulated emission fluorescence microscopic imaging system to realize imaging.

Wherein, the fluorescent molecule is ATTO488 of American molecular probe company, and the nano antibody is anti- α tubulin antibody of TAKARA company.

Finally, it should be noted that the above embodiments are only used for illustrating the technical solutions of the present invention and are not limited, and those skilled in the art can also make a plurality of modifications and improvements without departing from the principles of the present invention, and these should also be regarded as the protection scope of the present invention.

Claims

1. A nanoprobe based on surface plasmon enhancement effect is characterized by comprising nanoparticles, a fluorophore and a nanobody;

the method comprises the following steps that a nanometer probe composed of nanoparticles, a nanometer antibody and a fluorophore enters a biological sample under the endocytosis of cells, when a laser beam irradiates on the nanometer probe in the biological sample, plasma is generated on the surface of the nanoparticles of the nanometer probe, an electric field around the nanoparticles is enhanced by the plasma, the fluorophore in the electric field absorbs photons in the light beam, fluorescence is emitted from an imaging part positioned by the nanometer antibody in the biological sample under the action of the enhanced electric field, and the fluorescence is collected by a stimulated emission fluorescence microscopic imaging system to realize imaging.

2. The nanoprobe based on the surface plasmon enhancement effect according to claim 1, wherein the two ends of the nanobody are respectively connected with a nanoparticle and a fluorophore; the attachment is by non-covalent binding of biotin to the surface of the streptavidin system.

3. The nanoprobe based on the surface plasmon enhancement effect as claimed in claim 1, wherein the nanoparticle is a core-shell structure comprising metal particles-inorganic materials; the metal particles are gold nanoparticles, and the particle size is 10-70 nanometers; the inorganic material is silicon dioxide, and the thickness of the inorganic material shell layer is 5-20 nanometers.

4. The nanoprobe based on the surface plasmon enhancement effect of claim 1, wherein the nanobody comprises immunoglobulin, which can specifically bind to an antigen in a biological sample and accurately locate to an imaging site of the biological sample; the size of the nano antibody is about 2 nanometers, so that the fluorescence quenching effect of the nano particles on a fluorophore can be effectively isolated, and the fluorescence intensity of a fluorescent molecule can be greatly enhanced.

5. The surface plasmon enhancement effect based nanoprobe of claim 1, wherein the laser beam has a wavelength of 595 nm, which can realize the plasmon enhancement electric field generated around the nanoparticle and the resonance coupling of the fluorophore.