KR102530499B1 - Method of detecting protein within a nucleus using plasmonic metal nanoparticle - Google Patents

Abstract

The present invention comprises the steps of processing a probe containing a conjugate of an antibody to the protein and a plasmonic metal nanoparticle to a cell containing proteins to be detected in the cell nucleus;
obtaining a scattering image of the probe for the cell and a spectrum according to a wavelength; and
It relates to a method for detecting proteins in cell nuclei, including obtaining distribution information of the proteins by analyzing the spectrum.

Description

Method of detecting protein within a nucleus using plasmonic metal nanoparticle}

The present invention relates to a method for detecting proteins in a cell nucleus using a plasmonic metal nanoparticle probe, and specifically, to a method for obtaining distribution information between proteins in a cell nucleus.

Histone modification in the cell nucleus is the main cause of rapid topological changes in chromatin. The 'histone code', which is a combination of various modifications of histones, is determined by the balance between various writer enzymes and eraser enzymes. Information in histone codes is recognized by reader proteins involved in almost all events in chromatin, such as transcription, repair, replication and chromatin topology. For example, the chromosomal region of heterochromatin protein-1 (HP1), a reader protein, recognizes and binds H3K9me3, the trimethyl of the 9th lysine residue of histone 3. After binding, HP1 oligomerizes itself, and oligomerization of HP1 makes the chromatin structure more compact. Thus, H3K9me3 is often found in heterochromatin, a condensed chromatin structure. Due to the interaction between HP1 and lamin A, a structural protein composing the inner membrane of the nuclear membrane, H3K9me3 is located close to the inner membrane of the nuclear membrane. Through this mechanism, H3K9me3 can determine the structure and three-dimensional localization of chromatin regions. Recent findings extending to other heterochromatin histone markers such as H3K27me3, H3K56me3, H3K64me3 and H4K20me3 complicate the relationship between histone code and chromatin topology. Genome-wide analysis of cancer mutations has identified dozens of genes encoding histones, DNA/histone modifying enzymes, deletion enzymes, and chromatin remodeling complexes as cancer-causing genes, suggesting that changes in the histone code and chromatin structure are closely related to cancer pathogenesis. suggests that there is Thus, visualizing the spatial distribution of histone modifications at the cellular level could allow for expanded applications for diagnosing aging as well as cancer or other disease-specific cellular phenotypes.

To develop a new imaging method to analyze the spatial distribution of histone codes using plasmonic metal nanoparticle probes, because the process of Oncogene-Induced Senescent (OIS) induced drastic changes in heterochromatin distribution, OIS Cells are used as a model system. Oncogenes such as Ras, Raf and Myc induce cellular senescence in the absence of additional mutations in tumor suppressor genes. Immunostaining of OIS cells with an antibody specific for H3K9me3 reveals a distinct clustered pattern of H3K9me3 termed senescence-associated heterochromatin foci (SAHF). In contrast, in growing cells H3K9me3 is detected in a dispersed pattern inside the nucleus and is concentrated along the inner membrane of the nuclear envelope. Spatial redistribution of several histone modifications, such as H3K9me3, H3K27me3 and corresponding reader proteins, during the OIS process forms SAHF formations. Thus, the precise spatial distribution of histone modifications in the nucleus may represent a unique feature of the epigenetic state of growing, senescent or tumor cells.

Existing histone imaging methods using organic fluorescent dyes have limitations such as photobleaching, lower resolution, and mandatory use of secondary antibodies. (Non-Patent Document 1, T Chandra, Molecular Cell 47, 203-214) In order to overcome the disadvantages of existing organic fluorophores, nano-sized inorganic probes including quantum dots and metal nanoparticles are attracting attention. Recently, plasmonic metal nanoparticles such as gold nanoparticles (GNPs) and silver nanoparticles (SNPs) have been utilized in various imaging and sensing applications due to their excellent optical properties, photostability, biocompatibility, and ease of synthesis and surface modification. The optical properties of the plasmonic metal nanoparticle particles can be tuned by controlling their shape, size and composition. In particular, when two or more plasmonic metal nanoparticles are placed in close proximity, a plasmon coupling effect that induces spectral shifts such as absorbance and scattering is exhibited. These spectral shifts are closely related to the interparticle distance, distribution and type of plasmonic metal nanoparticle particles. Despite the excellent optical properties of plasmonic metal nanoparticles, no attempt has been made to visualize histone modification in the nucleus so far.

Therefore, for the purpose of analyzing the spatial arrangement of proteins inside the cell nucleus through plasmonic metal nanoparticles, a histone marker in the nucleus of a living cell is labeled using a conjugate of an antibody against the histone marker and plasmonic metal nanoparticles inside the cell nucleus, and OIS is detected. The present invention was completed by confirming the dynamic color change and spectrum change of the plasmonic metal nanoparticle probe according to the spatial rearrangement of histone markers occurring during the aging process of cells upon induction.

One object of the present invention is to provide a method for obtaining information on the distance or distribution between proteins, eg, modified histones, in a cell nucleus.

Another object of the present invention is to provide a method for detecting proteins by using the plasmonic metal nanoparticles to determine changes in distance and spatial distribution between proteins in cell nuclei.

Another object of the present invention is to provide a method of utilizing plasmonic metal nanoparticles in determining the aging stage of a cell.

In one aspect, the present invention includes the steps of processing a probe containing a conjugate of an antibody to the protein and a plasmonic metal nanoparticle to a cell containing proteins to be detected in the cell nucleus;

obtaining a scattering image of the cell nucleus probe and a spectrum according to wavelength; and

Analyzing the spectrum to obtain distribution information of the proteins; provides a method for detecting proteins in the cell nucleus, including.

In another aspect, the present invention includes the steps of treating a cell containing proteins to be detected in the cell nucleus with a probe composed of a conjugate of an antibody to the protein and a plasmonic metal nanoparticle;

obtaining scattering images of the probe at the first and second time points of the cell and spectra according to wavelengths;

obtaining distribution information of proteins at the first time point and the second time point by analyzing the spectrum; and

It provides a method for detecting a change in protein distribution in the cell nucleus, comprising: determining a change in spatial distribution of the protein to be detected in the cell nucleus between the first time point and the second time point.

In another aspect, the present invention provides a probe for detecting distribution information of heterochromatin histone markers in a cell nucleus, including plasmonic metal nanoparticles bound to a heterochromatin histone marker antibody in a cell nucleus.

The method for detecting proteins in the cell nucleus according to the present invention uses plasmonic metal nanoparticles as a detection probe, so unlike conventionally used images using organic fluorescent molecules, it does not require the use of a secondary antibody and provides higher resolution. Imaging is possible, and information on the distance between proteins and their spatial distribution can be obtained by analyzing the spectral change according to the coupling of plasmonic metal nanoparticles.

1 shows an example of a method for binding a primary antibody to gold nanoparticles for preparing a plasmonic metal nanoparticle probe.
Figure 2 shows simulation results according to the distance or distribution of gold nanoparticles, part a of Figure 2 shows a schematic diagram of the plasmonic metal nanoparticle probe distributed in heterochromatin, part b of Figure 2 shows the distribution of the schematic diagram shows the scattering spectrum according to the distance between the plasmonic metal nanoparticles, and part c of FIG. 2 shows the tendency of the scattering spectrum peak (λ _max ) according to the distribution of the plasmonic metal nanoparticle probe.
3 shows darkfield scattering images of a gold nanoparticle probe bound to a single histone marker (H3K9me3) redistributed at each time point during the OIS process in the cell nucleus.
4 is a virtual view and dark field scattering images of plasmonic metal nanoparticle probes targeting histone markers of heterochromatin in growing cells and senescent cells. Scale bar is 10 μm.
5 shows fluorescence images of an organic fluorescent probe bound to a single histone marker (H3K9me3) redistributed at each time point during the OIS process in the cell nucleus.
Figure 6a shows the scattering color and spectrum by the redistribution of the gold nanoparticle probe bound to the histone marker (H3K9me3) measured at several points in the OIS cell nucleus;
Figure 6b shows the maximum scattering wavelength (λ _max ) distribution of 50 scattering spectra measured in 15 or more cells at each time point during the OIS process,
6c shows the maximum scattering wavelength change of the gold nanoparticle probe at each time point during the OIS process.
7 is a view of optical simulation according to the distribution of gold nanoparticles, where a part shows the 2D distribution from trimer to heptamer of gold nanoparticles, and part b shows the target maximum scattering wavelength (λ _max ) in the 2D distribution. shows the scattering spectrum according to , part c shows the interparticle distance according to the target maximum scattering wavelength (λ _max ) in the 2D distribution, part d shows the octamer, nonamer, dodecamer, tetradecamer of gold nanoparticles, Represents the 3D distribution up to icosomer, e part shows the scattering spectrum according to the target maximum scattering wavelength (λ _max ) in the 3D distribution, f part is the particle according to the target maximum scattering wavelength (λ _max ) in the 3D distribution represents the distance between
8 is a simulation of scattering spectra according to 2D and 3D distributions composed of gold nanoparticle probes, wherein part a is a trimer, part b is a tetramer, part c is a pentamer, part d is a hexamer, and part e is a Hexamer with central gold nanoparticle, f part is heptamer, g part is octamer, h part is nonamer, i part is dodecamer, j part is dodecamer with central gold nanoparticle, k part is tetradeca The l part represents an icosomer, and the m part represents an icosamer having a central gold nanoparticle.
9 is a schematic diagram of determining the distance between histone markers based on the distance between gold nanoparticle probes, assuming that all gold nanoparticle probes are attached in the same direction.
Figure 10a is a conceptual diagram when applying the analysis of dual histone markers (H3K9me3 and H3K27me3) using homogeneous gold nanoparticles and heterogeneous gold and silver nanoparticles (left: homogeneous nanoparticles, right: heterogeneous nanoparticles ),
10b is a dark field scattering image of cell nuclei at 144 hours after 4-OHT treatment for dual histone markers (left: homogeneous nanoparticles, right: heterogeneous nanoparticles);
FIG. 10C shows a pixel that classifies aggregated scattering colors into a single scattering color in a dark field scattering image (left: homogeneous nanoparticles, right: heterogeneous nanoparticles),
10d shows the observed scattering color and scattering spectrum (left: homogeneous nanoparticles, right: heterogeneous nanoparticles),
Figure 10e shows the scattering spectrum and the average spectrum at a single pixel classified in 10c (top: homogeneous nanoparticles, bottom: heterogeneous nanoparticles),
10f shows the maximum scattering wavelength (λ _max ) distribution of 50 scattering spectra measured from 15 or more cells (left: homogeneous nanoparticles, right: heterogeneous nanoparticles).
11 is a simulation diagram of scattering spectra according to 2D and 3D distributions composed of a gold nanoparticle probe (center) and a silver nanoparticle probe (satellite).
12 shows the scattering spectrum of gold nanoparticle probes bound to double histone markers (H3K9me3 and H3K27me3) analyzed by simulation and the variation range of the maximum scattering wavelength.
FIG. 13 shows the correlation between simulation and measured scattering spectra using a gold nanoparticle probe for a single histone marker (H3K9me3).
14 shows scattering spectra and maximum scattering wavelength fluctuation ranges of gold nanoparticle probes and silver nanoparticle probes bound to H3K9me3 and H3K27me3, respectively, analyzed by simulation.
15 shows the correlation between simulated and measured scattering spectra of homogeneous gold nanoparticle probes bound to dual histone markers.
16 shows correlations between simulated and measured scattering spectra of a gold nanoparticle probe and a silver nanoparticle probe bound to H3K9me3 and H3K27me3, respectively.
FIG. 17 shows an analysis of the distance between histone markers based on the correlation between the simulation and the measured scattering spectrum presented on the right side of FIG. 16 .

Hereinafter, the present invention will be described in detail.

Embodiments of the present invention may be modified in various forms, and the scope of the present invention is not limited to the embodiments described below. In addition, the embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Furthermore, "include" a certain component throughout the specification means that other components may be further included without excluding other components unless otherwise stated.

One aspect of the present invention,

treating cells containing proteins to be detected in the cell nucleus with a probe containing a conjugate of an antibody to the protein and plasmonic metal nanoparticles (step 1);

obtaining a scattering image of the probe for the cell and a spectrum according to wavelength (step 2); and

Analyzing the spectrum to obtain distribution information of the proteins (step 3); provides a method for detecting protein distribution in the cell nucleus.

The detection method may further include a step (step 4) of determining a distribution change through comparison with arbitrary distribution information, thereby providing a method of detecting a change in protein distribution in the cell nucleus.

In another aspect similar to the detection method, the present invention provides a cell containing proteins to be detected in the cell nucleus with a probe composed of a conjugate of an antibody to the protein and plasmonic metal nanoparticles (step 1');

obtaining scattering images of the probe at the first and second time points of the cell and spectra according to wavelengths (step 2′);

obtaining distribution information of proteins at the first time point and the second time point by analyzing the spectrum (step 3'); and

It provides a method for detecting a change in protein distribution in the cell nucleus, comprising: determining a change in spatial distribution of the protein to be detected in the cell nucleus between the first time point and the second time point (step 4').

The detection method is described.

Since the distribution information of the protein to be detected causes a spectrum change as a result of plasmon coupling according to the distance between the proteins to be detected, the distribution information means information that causes a change in spectrum, .

The distribution or aggregation pattern of the protein includes trimer, tetramer, pentamer, hexamer, hexamer*, heptamer, and the like. Information about distances between particles in the aggregation pattern may be included.

The first step is to process the probe (steps 1 and 1').

The cell nucleus includes both live cells and dead cells, and in the case of living cells, protein distribution information on living cells can be obtained momentarily.

The protein includes intranuclear and extranuclear proteins, particularly intranuclear proteins, and examples of the intranuclear proteins include histone proteins. The histone proteins include modified histones that can be markers of heterochromatin. . The protein may mean the entire protein or a specific domain of the protein.

One example of the modification is methylation, and modified histones include H3K9me3, H3K27me3, H3K56me3, H3K64me3, and H4K20me3.

The probe includes an antibody for the protein to be detected and plasmonic metal nanoparticles coupled to the antibody.

The antibody refers to an antibody that can specifically bind to a protein to be detected. The antibody can be used by selecting from known antibodies.

The plasmonic metal nanoparticles refer to metal nanoparticles capable of generating surface plasmon resonance. The particle size may be selected from the range of 5 to 100 nm and 30 to 50 nm.

At this time, if the average diameter of the plasmonic metal nanoparticles is less than 5 nm, a problem in that scattering color cannot be effectively visualized occurs, and if it exceeds 100 nm, a problem in that they cannot effectively penetrate cells occurs. In addition, the metal nanoparticles may use a metal exhibiting a plasmonic resonance effect, and specific examples include gold (Au) nanoparticles, silver (Ag) nanoparticles, or gold (Au) nanoparticles and silver ( Ag) nanoparticles can be used together. The plasmon resonance effect can be confirmed by absorbance and scattering spectra, and in particular, when two or more plasmonic metal nanoparticles are located close to each other, the absorbance and scattering spectra are shifted, which is called the plasmon coupling effect. Although the plasmon coupling effect occurs between metal nanoparticles of the same type, a bimetallic plasmon coupling effect that is differentiated from the plasmon coupling effect between metal nanoparticles of the same type appears between metal nanoparticles of the same type. Since the change in refractive index of silver with wavelength is larger than that of gold, the optical properties of silver vary more widely with incident wavelength. Therefore, the bimetallic plasmon effect spectrum between the gold and silver nanoparticles shows a broad and unique multi-peak spectrum. This broad and unique spectrum enables more accurate analysis of the distribution of nanoparticles than using a single type of metal nanoparticle.

In the above detection method, the second step is obtaining a spectrum (step 2; step 2').

The spectrum begins with obtaining a dark field microscope scattering image according to the surface plasmon resonance phenomenon. In addition, the image includes a scattering spectrum according to wavelength, and the spectrum may be a scattering image of a probe at a specific position and a spectrum according to wavelength. The specific position is a position of a specific metal nanoparticle, and this position can be interpreted as a position of a protein to be detected or a specific domain of a protein to which the corresponding probe is bound. The spectrum changes according to the distance from another plasmonic metal nanoparticle affecting the surroundings. The surrounding metal nanoparticles may be the same or different metal nanoparticles. The specific location may be a specific location in the cell nucleus as a reference regardless of the metal nanoparticle of the probe. By obtaining a spectrum according to the surface plasmon resonance at the corresponding position, the distribution of metal nanoparticles at the corresponding position, eventually the distribution of proteins to which the metal nanoparticles are bound, can be analyzed. Step 2' is to obtain spectra of the same cell at different viewpoints. In the step of obtaining the spectrum, after selecting a predetermined number of positions within the measurement area, a scattering spectrum at each position is obtained for each position, a wavelength at which a peak of the scattering spectrum appears at each position is obtained, and then a wavelength distribution ratio of each peak. Further comprising obtaining information about. The wavelength of each peak can be divided into a certain wavelength region, and the wavelength region can be arbitrarily set in units of 1 to 100 nm.

The distribution rate may include obtaining a time-sequential change with respect to the same measured cell nucleus. In the above detection method, the third step is obtaining protein distribution information (step 3; step 3').

The distribution information can be obtained by analysis from the previously obtained spectrum, and for analysis, a spectrum with known distribution information can be prepared in advance and analyzed through comparison with this spectrum.

The spectrum for which the distribution information is known includes one obtained through actual measurement or simulation, but includes one obtained through simulation in the present invention as an example. The simulation includes performing simulation for each distribution or aggregation pattern of trimers, tetramers, pentamers, hexamers, etc., in terms of the distance and distribution of the metal nanoparticles. Further, the method may include simulating a scattering spectrum according to a distance for each pattern, and may further include analyzing a scattering spectrum obtained through simulation for each pattern. Information on the distance between particles in each pattern can be generated for a specific peak wavelength. The simulation may be performed whenever probes containing the same kind of metal nanoparticles or probes containing different metal nanoparticles are used. It includes the case where the protein to be detected is one or more, and includes, for example, one or two proteins. The protein may refer to each of the different domains within one protein. These domains may be located in different proteins. Step 3' is to obtain protein distribution information from spectra obtained at different viewpoints for the same cell. The aging stage of the cell can be determined through the distribution information. The spatial distribution of proteins in the cell nucleus changes according to the aging stage of the cell. For example, modified histones, which are markers of heterochromatin, are rearranged in space according to the cell growth, that is, the aging stage. Therefore, by preparing cell distribution information and preliminary information on the cell growth or aging stage, the cell growth or aging stage can be determined by analyzing the cell distribution information.

In the above detection method, the fourth step is to determine the change in protein distribution (step 4; step 4').

This is a step of comparing the distribution information of the protein obtained in step 3 with arbitrary distribution information to determine the distribution change, and the arbitrary distribution information may be distribution information of the same cell nucleus at a certain point in time or distribution information of another cell nucleus. . Distribution change can be determined through comparison of the two distribution information. Distribution information of proteins can be obtained through spectra obtained at two random points in the same cell nucleus, and a change in the distribution of proteins in space can be determined by comparing the spectra (step 4').

Another aspect of the present invention is,

treating the histone markers of heterochromatin in the cell nucleus with a probe composed of a conjugate of an antibody against the histone marker and plasmonic metal nanoparticles;

obtaining a scattering image of the probe for the cell nucleus and a spectrum according to wavelength;

A method for detecting histone markers of heterochromatin in a cell nucleus is provided, which includes obtaining distribution information of the histone markers by analyzing the spectrum.

Components of the histone marker detection method are as described above.

Another aspect of the present invention is,

Provided is a probe for detecting information based on a distance between histone markers of heterochromatin in a cell nucleus, including plasmonic metal nanoparticles to which an antibody of a histone marker of heterochromatin in a cell nucleus is bound.

Components of the detection probe are as described above.

In another aspect of the present invention, the scattering spectrum of the plasmonic metal nanoparticles is shifted to a red wavelength according to a change in the distribution of the plasmonic metal nanoparticles as the distance between them becomes closer and the degree of aggregation increases. Therefore, when a shift to a longer wavelength is observed in the scattering spectrum, it means that the distance between the proteins to be detected becomes closer or the degree of aggregation increases, and a change in spatial distribution occurs.

In another aspect of the present invention, heterogeneous chromatin within a cell generally changes in the distribution ratio of a wavelength band within a measurement region according to cell aging. The degree of aging of the cells can be determined through the analysis of the distribution rate of the wavelength range through the analysis of the spectrum at each measurement position existing in the measurement area. At this time, according to cell aging, the distribution ratio in the wavelength range of 530 to 570 nm decreases, and the distribution ratio in the wavelength range of 570 to 640 nm increases.

Hereinafter, the present invention will be described in detail by production examples, examples and experimental examples.

However, the following preparation examples, examples and experimental examples are merely illustrative of the present invention, and the content and scope of the present invention are not limited to the following contents.

Experiment content:

1. Material preparation

Dulbecco's modified Eagle medium (DMEM) and penicillin/streptomycin (PenStrep) were purchased from Gibco-Life Technologies (Carlsbad, CA, USA). Fetal bovine serum (FBS) was purchased from MP Biomedicals (Irvine, CA, USA). Bovine serum albumin (BSA), phosphate buffered saline (PBS) and 40 nm silver nanoparticles (SNP) were purchased from Sigma-Aldrich (St Louis, MO, USA). 40 nm gold nanoparticles (GNP) were purchased from BBI Solutions (Crumlin, UK). Anti-histone H3 (tri methyl K9) antibody and anti-histone H3 (tri methyl K27) antibody were purchased from Abcam (Cambridge, MA, USA).

2. Combination of Antibodies with Plasmonic Metal Nanoparticles

As shown in FIG. 1, the biobinding of antibodies to plasmonic metal nanoparticles was performed using the following procedure: sulfosuccinimidyl 6-[3'-(2-pyridyldithio)-propionamido] hexanoate ( Sulfo-LC-SPDP, 21650, Thermo). A total of 100 μM Sulfo-LC-SPDP was prepared in ultrapure water immediately before use. Then, 25 μL of Sulfo-LC-SPDP was added to 10 μg IgG dissolved in 0.5 mL of PBS-EDTA (100 mM sodium phosphate, 150 mM NaCl, 1 mM EDTA, 0.02% sodium azide, pH 7.5). and incubated for 30 minutes at room temperature (RT). Bound antibodies were exchanged into 40 mM HEPES buffer using a 10K MWCO centrifugal filter (Millipore). Plasmonic metal nanoparticles (40 nm gold nanoparticles and 40 nm silver nanoparticles) were centrifuged (3000 x g, 15 min) and resuspended in 40 mM HEPES buffer prior to antibody binding. Plasmonic metal nanoparticles and antibody were mixed in a 1:250 molar ratio and incubated overnight at 4 °C.

3. Construction of retroviral expression vectors and retroviral production

The ΔB-Raf:ER fusion protein consists of a mutant form of the protein kinase domain of mouse B-Raf (449-804aa) and the hormone-binding domain of the mouse estrogen receptor (281-599aa, a single amino acid substitution from glycine to arginine at 525) and their expression is induced by 4-hydroxytamoxifen (4-OHT). 1 mM of 4-OHT was prepared in ethanol and diluted to a concentration of 100 nM.

4. OIS cell culture

Human lung fibroblasts, IMR-90 cells, were purchased from ATCC (#CCL-186). IMR90 cells were transduced with a retrovirus encoding ΔB-Raf:ER. Cells were cultured in Eagle's Minimum Essential Medium (MEM; #10-009-CV, Corning) supplemented with 10% fetal bovine serum (FBS), 100 IU/mL penicillin and 100 μg/mL streptomycin. All cells were cultured at 37°C under humidified air containing 5% CO ₂ .

5. Cell culture using plasmonic metal nanoparticle probes

Plasmonic metal nanoparticle probes were diluted in 0.1% BSA blocking solution before treating OIS cells. OIS cells were fixed with 4% paraformaldehyde (PFA) for 15 minutes and washed with PBS at room temperature. Permeabilization was performed with 0.4% Triton X-100 in PBS for 10 minutes and washed three times with PBST (0.05% Tween-20 in PBS) for 5 minutes. After blocking cells with 5% BSA in PBST for 1 h, they were treated with plasmonic metal nanoparticle probes and incubated overnight at 4 °C.

6. Imaging single OIS cells through dark field microscopy

Imaging of histone codes targeted by plasmonic metal nanoparticle probes using a dark field microscope (Olympus BX43, Tokyo, Japan) equipped with a hyperspectral imaging spectrophotometer (CytoViva, Auburn, AL, USA) and cells for various OIS times The scattering spectrum of was measured. A 40× objective lens was used for imaging, and the exposure time for collecting the scattering spectrum was 0.3 seconds.

7. Simulation analysis of scattering spectra of plasmonic metal nanoparticle probes

Wave optics simulations were performed to determine the optical behavior of plasmonic metal nanoparticles according to distance, number, and distribution. Planar electromagnetic waves with a wavelength of 400 to 800 nm were applied. All simulations were performed using commercial software (COMSOL Multiphysics 5.4).

8. Existing organic fluorescent probe using a secondary antibody in which a primary antibody and a fluorescent dye are combined

The cells are washed with PBS and fixed for 15 minutes at room temperature with PBS containing 4% paraformaldehyde. After permeabilization with PBS containing 0.4% Triton X-100 for 5 minutes, it was washed with PBST (PBS containing 0.05% Tween 20). Cells were blocked by treatment with PBST containing 5% BSA for 1 hour at room temperature, then treated with primary antibodies and incubated overnight (16 hours). After washing with PBST buffer, the cells were incubated with Alexa Fluor® 488 (Invitrogen A-11034)-conjugated secondary antibody (1:500) for 1 hour at room temperature. After washing again with PBST buffer, it was observed by placing it on a slide glass.

<Example 1> Spectrum simulation according to distribution and aggregation between gold nanoparticles

Simulations were performed for various distributions and inter-particle distances to investigate the optical properties according to the distribution and aggregation of gold nanoparticles. As shown in FIG. 2 , the scattering spectrum shows a red shift as the number of gold nanoparticles (GNP) approaches and the number of gold nanoparticles (N _GNP ) increases. A change in the spatial distribution of a target protein in a cell nucleus can be detected using a spectral change that varies depending on the distance and distribution between the gold particles.

<Example 2> Imaging of H3K9me3 redistribution in OIS cells using gold nanoparticle probes

Treatment of IMR90-ΔB-RAF:ER cells with 4-OHT stabilizes the ΔB-RAF:ER fusion protein and initiates the OIS process in IMR90-ΔB-RAF:ER cells. To visualize the redistribution of the inhibitory histone marker H3K9me3 in OIS cells, a plasmonic gold nanoparticle probe composed of a conjugate of H3K9me3 antibody and gold nanoparticles was used. Scattering colors and spectra of plasmonic nanoprobes in OIS cells were collected via dark field microscopy coupled with a spectrophotometer. Scattering spectra were measured at 50 random points per cell. As shown in FIG. 3, when using a plasmonic gold nanoparticle probe, a visualized image in which various scattering colors appear as the distance between target histone markers becomes closer during the OIS process due to the plasmonic coupling effect is obtained. Scattering images of cell nuclei using plasmonic gold nanoparticle probes show a gradual color change from green to yellow or orange during the OIS process. Formation of SAHF is also observed in senescent cells.

Considering the spatial distribution of heterochromatin in growing cells and senescent cells, Fig. 4 shows a virtual view and a dark field scattering image when a gold nanoparticle probe coupled with an antibody specific to heterochromatin binds to heterochromatin. The dark field scattering images of FIG. 4 correspond to 0 hour and 144 hours after 4-OHT treatment.

On the other hand, as shown in FIG. 5, the image obtained using the organic phosphor of Comparative Example 1, which will be described later, shows no change in fluorescence color, overall resolution is not high, and formation of SAHF is not clearly confirmed.

As shown in FIG. 6A, scattering color and spectrum by redistribution of the gold nanoparticle probe bound to the histone marker (H3K9me3) measured at several points in the OIS cell nucleus are measured. As shown in FIGS. 6B and 6C, after 4-OHT treatment, scattering spectra were obtained at random points for a specified time, and after obtaining scattering spectra corresponding to each point at 50 points, scattering patterns were measured at 10 nm wavelength intervals. Analyze systematically. Based on the scattering peak analysis as shown in FIGS. 6b and 6c, it can be seen that the distribution of the scattering peak dynamically changes during the OIS process. Most of the scattering points in the growing cells (before 4-OHT treatment) show peaks in the range of 530-560 nm, close to the scattering wavelength of the original gold nanoparticle probe. As OIS progresses, the peak in the wavelength range 530-570 nm decreases, but the peak in the 570-640 nm increases noticeably. A clear SAHF was formed at 144 hours after 4-OHT treatment, with the largest group of scattering peaks observed in the range of 570-580 nm. Scattering peaks with wavelengths above 600 nm (up to 640 nm) are frequently observed in yellow and orange in senescent cell nuclei. This peak wavelength change is caused by the strong plasmonic coupling between the gold nanoparticle probes caused by the proximity of the target H3K9me3 in SAHF during OIS.

<Comparative Example 1> Imaging H3K9me3 redistribution in OIS cells using a conventional organic fluorescent probe

In a similar manner to Example 2, the gold nanoparticle probe used was changed to a conventional organic fluorescent probe. As shown in FIG. 5 , in the absence of 4-OHT, a conventional fluorescent probe using an H3K9me3 primary antibody and a fluorescent dye-conjugated secondary antibody visualizes the diffusion pattern of H3K9me3 in the cell nucleus. After 48 h of treatment with 4-OHT, H3K9me3 clumps. After 144 hours, SAHF is evident, suggesting that OIS spatially redistributes heterochromatin. However, the resolution of closely distributed H3K9me3 in SAHF is too limited to investigate the spatial redistribution of H3K9me3 during OIS due to the smearing of emission from conventional organic fluorophores. In addition, the H3K9me3 formulation bound by the fluorescent probe shows the same green emission color regardless of the distribution change.

<Example 3> Simulation analysis of H3K9me3 redistribution in OIS cells using gold nanoparticle probes

As shown in FIG. 7 , computer simulation was performed to further understand the scattering signal of the gold nanoparticle probe according to the interparticle distance and OIS cell nucleus distribution. Various configurations including two-dimensional (2D) and three-dimensional (3D) are simulated considering different number of particles (N _GNP ) and distance between particles (d _GNP ). As shown in parts a and d of FIG. 7 , the optical simulation area is composed of water with a refractive index similar to that of the mounting solution while gold nanoparticles are placed on glass for imaging. A plane wave of incident light propagates along the z direction. For the 2D distribution of gold nanoparticles, six types of general structures are considered: trimer (N _GNP = 3), tetramer (N _GNP = 4), pentamer (N _GNP = 5), and hexamer (N _GNP = 6), hexamer (i.e. hexamer*, N _GNP = 7) and heptamer (N _GNP = 7) with a central gold nanoparticle. For the three-dimensional distribution of gold nanoparticles, five possible structures are considered: octamer (N _GNP = 8), nonamer (N _GNP = 9), dodecamer (N GNP = 12), and tetradecamer (N _GNP = 12). _GNP = 14) and icosomer (N _GNP = 20). In the case of octamers, dodecamers and icosomers, gold nanoparticles are placed at the vertices of these polyhedrons. For nonamers and tetradecamers, gold nanoparticles are added inside the octamers. To investigate the effect of interparticle distance, _dGNP , which is determined by the distance between the gold nanoparticle and the nearest particle, is considered to vary from 1 nm to 9 nm.

First, simulations of various distributions and interparticle distances are performed to characterize the optical properties of the gold nanoparticle-based structure. As shown in FIG. 8, the scattering spectrum shows a red shift as the gold nanoparticles get closer and N _GNP increases. In order to correlate with the experimental results observed in FIGS. 6a to 6c, the spectra in which λ _max is the main scattering peak (580 nm, 600 nm, 620 nm, and 640 nm) are shown in parts b and e of FIG. 7 . The results generally showed that the intensity of the scattering spectrum increased with increasing λ _max and increasing N _GNP .

Next, d _GNP of each distribution according to λ _max is shown in part c and part f of FIG. 7 . When λ _max is 580 nm, d _GNP between gold nanoparticle structures is distributed in a wide range, while the difference in d _GNP according to the gold nanoparticle distribution decreases as λ _max increases. Considering these simulation results, it is assumed that all the gold nanoparticle probes are attached in the same direction as shown in FIG. 9, and the distance between the histone marker H3K9me3 to which the gold nanoparticle probes are bound is determined. The distance between the histone markers is the sum of the distance between the gold nanoparticle probes and the diameter (40 nm) of the gold nanoparticle probe (ie, d _{histone marker} = d _GNP + 40 nm). In the experimental results after 144 hours after 4-OHT treatment in FIG. 6a, the highest ratio of scattering peaks appears at 580 nm. In the simulation analysis results of parts b, c, e, and f of FIG. 7, the distance between the gold nanoparticle probes at which the maximum scattering wavelength of 580 nm occurs in 2D and 3D distributions is 3.4 to 10.7 nm. Therefore, it means that several H3K9me3 markers were distributed within an average distance of 43.4 to 50.7 nm in the scattering area of OIS cells 144 hours after 4-OHT treatment. Similarly, the observation that the maximum peak of several scattering points in senescent cells is around 640 nm indicates that H3K9me3 bound by the gold nanoparticle probe is distributed within a short distance of 40.3 to 42.0 nm in the corresponding scattering region.

<Example 4> Simultaneous imaging of redistribution of H3K9me3 and H3K27me3 in OIS cells using gold nanoparticle probe and silver nanoparticle probe

Similar to Example 3, the single histone modification (H3K9me3) was changed to the simultaneous targeting of two histone modifications (H3K9me3 and H3K27me3). In addition, two different combinations of plasmonic metal nanoparticle probes coupled to H3K9me3 or H3K27me3 antibodies were performed (see Fig. 10a). First, as shown in FIG. 10A , probes in which gold nanoparticles were conjugated to both H3K9me3 and H3K27me3 antibodies and silver nanoparticles were conjugated to H3K27me3 antibodies were prepared.

Example 4-1:

Similar to Example 2, cell nuclei were imaged according to OIS using two types of probes in which gold nanoparticles were bound to both the H3K9me3 antibody and the H3K27me3 antibody.

Example 4-2:

Similar to Example 2, cell nuclei were imaged according to OIS using two types of probes in which gold nanoparticles were conjugated to the H3K9me3 antibody and silver nanoparticles were conjugated to the H3K27me3 antibody.

In both cases, larger scattering points (Figs. 10b, 10c) are frequently observed than in the case of targeting a single histone modification (Fig. 3). This is because H3K9me3 and H3K27me3 constitute small SAHFs and thus the number of plasmonic nanoparticle probes targeting histone markers in SAHFs has increased. Therefore, as shown in Fig. 10e, the spectrum is collected and averaged at a pixel occupying a single SAHF to collect the scattering signal at each point. Compared to the single targeting of H3K9me3, simultaneous targeting of H3K9me3 and H3K27me3 showed drastic changes in scattering color and intensity (Fig. 10d), which is an effect of the stronger plasmonic coupling effect between the coexisting plasmonic nanoparticle probes at the scattering point. . In addition, a larger red shift reaching 700 nm is observed when targeting H3K9me3 and H3K27me3 redistributed during the OIS process compared to single histone marker images (Fig. 6b). (FIG. 10F)

<Example 5> Simulation analysis of optical properties of nanoparticle probes distributed on H3K9me3 and H3K27me3 in OIS cells

When using different plasmonic nanoparticle probes, i.e., a 40 nm gold nanoparticle probe and a 40 nm silver nanoparticle probe, to target H3K9me3 and H3K27me3 performed in Example 4-2, the collected scattering patterns are varied and the peak shift range is wide. As shown in FIG. 11, in order to investigate the optical properties of the heterogeneous plasmonic nanoparticle probe distribution composed of a gold nanoparticle probe and a silver nanoparticle probe, spectral simulations are performed for possible 2D and 3D distributions by varying the distance between particles.

FIG. 12 shows the scattering spectrum and maximum scattering wavelength fluctuation range of gold nanoparticle probes bound to double histone markers (H3K9me3 and H3K27me3) analyzed by simulation, and FIG. The correlation between the simulation and the measured scattering spectra is shown, and FIG. 14 shows the scattering spectra and maximum scattering wavelength fluctuation ranges of gold nanoparticle probes and silver nanoparticle probes bound to H3K9me3 and H3K27me3, respectively, analyzed by simulation. FIG. 16 shows the correlation between simulation and measured scattering spectra of gold nanoparticle probes and silver nanoparticle probes bound to H3K9me3 and H3K27me3, respectively. , and FIG. 17 shows the analysis of the distance between histone markers based on the correlation between the simulation and the measured scattering spectrum presented on the right side of FIG. 16 .

Compared to the combined gold nanoparticle probe/gold nanoparticle probe structure (FIG. 12), the combined silver nanoparticle probe/gold nanoparticle probe structure (FIG. 14) shows a wider shift in the scattering spectrum when the interparticle distance changes. show Since the change in the refractive index of silver with wavelength is greater than that of gold, the optical properties of silver change more dynamically with respect to the incident wavelength. In addition, the 3D structure of the silver nanoparticle probe/gold nanoparticle probe combination shows unique multiple peaks as shown in the lower part of FIG. 14 . This unique spectrum arises from the bimetallic plasmon coupling effect between silver nanoparticles and gold nanoparticle probes. This broad and unique spectrum of silver nanoparticle probe/gold nanoparticle probe binding structures could be useful for analyzing the distribution of two different histone modifications. That is, the distribution of histone markers bound to different nanoparticle probes can be more accurately analyzed than using one type of nanoparticle probe.

15 shows scatter spectra for targeting of two types of histone markers (H3K9me3 and H3K27me3). As shown in FIG. 15, when the two biomarkers are labeled with the gold nanoparticle probe, the distance between the two markers labeled with the nanoparticle probe at the observed point is determined to be 46.0 to 47.0 nm. This result suggests that the target histone modifications, H3K9me3 and H3K27me3, can be located on different nucleosomes considering the size of each nucleosome (about 11-13 nm).

As shown in FIG. 16, when the gold nanoparticle probe and the silver nanoparticle probe are used together, there is a distinct difference from the spectrum of FIG. 15. Depending on the scattering point as shown in Fig. 16, the spectral features vary from a single dominant peak to an intense double peak. This significant difference can also be confirmed in the simulation results. A large amount of information is obtained with more diverse spectra from dual targeting using two different nanoparticle probes, and based on this, the spectra obtained from experiments and simulations can be accurately compared. The histone modifications in FIG. 16 are judged to be in the form of 2D nucleus* and 3D icosomers with central gold nanoparticle probes at distances of 9.0 nm and 3.0 nm, respectively. Based on the above judgment, it can be determined that the histone modification labeled with the nanoparticle probe was located within 43.0 nm to 49.0 nm in the nucleus as shown in FIG. 17 . In addition, based on the spectrum collected from the dual targeting image, the possibility of three-dimensional arrangement of the targeted histone modifications was confirmed. Therefore, the unique and diverse scattering spectra obtained using the two types of nanoparticle probes can be of great help in understanding the spatial distribution of histone modifications in the nucleus. Considering the diameter of the nanoparticle probe used (40 nm) and the size of the nucleosome (approximately 11–13 nm), it is impossible to detect multiple histone modifications on a single nucleosome and target all histone markers in the nucleus. However, the proposed specific nanoparticle probes for histone markers (i.e., primary antibody-coupled plasmonic metal nanoparticles) can successfully track dynamic changes in the distribution of target histone markers during OIS.

Claims (12)

Cells containing the proteins to be detected inside the cell nucleus are treated with a probe containing a conjugate of an antibody to the protein and plasmonic metal nanoparticles, so that the plasmonic metal nanoparticles bound to the protein through the antibody exhibit a plasmonic coupling effect positioning in close proximity;
Obtaining a spectrum according to a wavelength according to a scattering image of the probe in the cell and a plasmon coupling effect between the plasmonic metal nanoparticles; and
Analyzing the spectrum to obtain distribution information of the proteins;
Characterized in that the protein is a modified histone as a marker of heterochromatin that is redistributed during the aging process, a method for detecting protein in the cell nucleus.
Cells containing the proteins to be detected inside the cell nucleus are treated with a probe composed of a conjugate of an antibody to the protein and plasmonic metal nanoparticles, so that the plasmonic metal nanoparticles bound to the protein through the antibody are brought into proximity to exhibit a plasmonic coupling effect Positioning step;
Obtaining spectra according to wavelengths according to a plasmon coupling effect between the scattering images of the probe and the plasmonic metal nanoparticles at the first and second time points of the cell;
obtaining distribution information of proteins at the first time point and the second time point by analyzing the spectrum; and
Determining a change in spatial distribution of the protein to be detected in the cell nucleus between the first time point and the second time point;
Characterized in that the protein is a modified histone as a marker of heterochromatin that is redistributed during the aging process, a method for detecting changes in protein distribution in the cell nucleus.
According to claim 1 or 2,
Wherein the plasmonic metal nanoparticles are selected from gold nanoparticles, silver nanoparticles, and combinations thereof.
According to claim 1,
Wherein the spectrum comprises a scattering spectrum.
According to claim 1,
Wherein the analysis comprises analyzing a spectrum change according to a plasmon coupling effect.
According to claim 1,
The method further comprises preparing in advance a spectrum obtained by measurement or simulation of cells having different distributions of proteins.
According to claim 1,
The method further comprises determining a cellular senescence stage of the cell through the distribution information.
delete According to claim 1 or 2,
wherein the histone is selected from H3K9me3, H3K27me3, H3K56me3, H3K64me3 and H4K20me3.
delete delete delete

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