KR20120046603A - The detection method of protein-coated metal nanoparticle inside a single cell by z-depth dependent confocal surface-enhanced raman scattering combined with dark-field microscopy technique - Google Patents

Abstract

PURPOSE: A method for detecting a protein-coated metal nanoparticle inside a single cell using a z-depth dependent confocal surface-enhanced Raman scattering and a dark-field microscopy technique informs absorption inside a cell of a metal nano particle within an A549 cell and behavioral characteristic thereof by using a super spectral imaging technique and a SERS(Surface Enhanced Raman Scattering) combined with a DFM(Dark-Field Microscopy). CONSTITUTION: A method for detecting a protein-coated metal nanoparticle inside a single cell is as follows. A protein-coated metal nanoparticle is manufactured. The nanoparticle is absorbed inside a cell and detects the absorbed nanoparticle by using a dark-field microscope technique and a z-depth dependent confocal surface-enhanced Raman scattering.

Description

The detection method of protein-coated metal nanoparticles inside a single cell by z-depth dependent confocal surface- using darkfield microscopy and ｚ-ｄｅｐｔｈ-dependent confocal surface enhancement Raman scattering enhanced Raman scattering combined with dark-field microscopy technique}

The present invention relates to a method for the detection of single cell protein coated metal nanoparticles using dark field microscopy and z-depth dependent confocal surface enhanced Raman scattering.

Nanoparticles (NPs) are used in a variety of applications, including targeted drug / gene delivery, intracellular imaging, in vivo biosensing, and biomedical diagnostics or therapies (X. Michalet, et. Al., Science 307 (2005) ) 538-544./ W. Russ Algar, et al., Anal. Chim.Acta 673 (2010) 1-25./CR Patra, et.al., Adv. Drug.Deliv. Rev. 62 (2010) 346 -361./AS de Dios & ME D, Anal. Chim.Acta 666 (2010) 1-22.). Since there are a number of regions that can bind NPs in biological systems, it is possible to understand the complex process of determining cellular uptake and intracellular localization through NP (W. Qian, et. Al., J. Biomed. Opt. 15 (2010) 046025./AM Schrand, et.al., Nat. Protoc. 5 (2010) 744-757.). Gold nanoparticles have attracted much attention over the last decade because of their stability and optical properties (AN Shipway, et. Al., Chem Phys Chem. 1 (2000) 18-52./R. Jin, Nanoscale 2 (2010). ) 343-362.). Recent reports on the biological application of gold nanoparticles have focused on the effects of biocompatibility, uptake, and subcellular distribution of gold nanoparticles (T organelle levels) (T. Mironava, et. Al., Nanotoxicology 4 (2010) 120-37.).

In order to obtain three-dimensional localization information on the absorption of nanoparticles, confocal microscopy with sufficient resolution in the Z-height direction is generally indispensable (JB Pawley, Handbook of Biological Confocal Microscopy, Springer, New York, USA). 3rd Ed. 2006.). Conventional confocal microscopy techniques have been widely applied to locate intracellular fluorescence staining labeled proteins (RD Goldman, et. Al., Live Cell Imaging, Cold Spring Harbor Laboratory Press, New York, USA 2nd Ed. 2010. Xu Hun, et.al., Anal. Chim.Acta 625 (2008) 201-206.). Despite its excellent sensitivity, fluorescence-quenching (P. Sharma, et. Al., Anal. Chim. Acta 676 (2010) 87-92.) And photobleaching (JR Lakowicz, Principles of Fluorescence Spectroscopy, Springer, New York, USA 3rd Ed. 2006.) Due to the effects, it is not easy to monitor the intracellular location of nanoparticles for a long time.

Darkfield microscopy (DFM) utilizes oblique illumination and collects Rayleigh scattering of light rather than spatially direct irradiation light (G. Wang, et. Al. ., Analyst 135 (2010) 215-221.). The sample will appear as a bright image superimposed on a dark background. The dark field irradiation has a very low background, so even small signals can adequately detect each non-fluorescent nanoparticle. Darkfield hyperspectral imaging has recently been used to confirm receptor regulatory status using the optical properties of single cell-bound gold nanoparticles (J. Aaron, et. Al., Nano Lett. 9 (2009) 3612-3618.).

SERS, an ultra-sensitive spectroscopic tool for interface research, has been widely used for molecular imaging or biological sensing of surface areas of nanostructured conjugates since it was discovered (Yu-Chuan Liu, et. Al., Anal. Chim.Acta 636 (2009) 13-18./C. Schlucker, Chem Phys Chem. 10 (2009) 1344-1354.). The SERS method has a narrow bandwidth that avoids overlapping spectra without the photobleaching effect, providing many advantages in monitoring various biomolecules including proteins in intracellular processes due to other Raman signals.

In the past decades, interest in the biocombination of proteins on metal platform surfaces has increased due to the potential applications for diagnosis and treatment in nano-level medicine (DW Romanini and MB Francis, Bioconjugate Chem. 19 (2008) 153-157.). One of various biobinding methods can be achieved using 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), which is an amine-reactive crosslinker (GT Hermanson, Bioconjugate Techniques). , Academic Press, San Diego, USA 1996.). Transferrin (Tf) has been recently studied as a model system of potent ligands that enable drug targeting and delivery of therapeutic substances (ZM Qian, et. Al., Pharma. Rev. 54 (2002) 561.). NPs-Tf conjugates are known to be transported intracellularly by receptor-mediated endocytosis (BD Chithrani and WCW Chan, Nano Lett. 7 (2007) 15421550. / CHJ Choi, et. Al., Proc. Natl Acad.Sci. US A. 107 (2010) 1235-1240.).

To the best of the inventors' knowledge, there has been no confocal Raman spectroscopy study combining DFM with single cells to date.

Thus, the inventors were motivated by little report on the accumulation behavior of non-fluorescent nanoparticles at the single cell level using DFM or confocal Raman spectroscopy, and the gold nanoparticles were absorbed into A549 human lung carcinoma cells. The present invention was completed by conducting confocal SERS and DFM studies, confirming that better monitoring of their uptake mechanisms within cells and their location in mammalian cells is possible.

It is an object of the present invention to provide a method capable of detecting the intracellular presence of various proteins in a single cell and their positions.

More specifically, it is an object of the present invention to provide a method for detecting intracellular behavioral properties of each protein using protein coated metal nanoparticles.

In order to achieve the above object, the present invention comprises the steps of iii) preparing protein coated metal nanoparticles; Ii) absorbing the nanoparticles into cells and detecting them with dark field microscopy (DFM); And iii) detecting nanoparticles using z-depth dependent confocal surface enhanced Raman scattering (SERS).

Hereinafter, the present invention will be described in detail.

In the method for detecting protein-coated metal nanoparticles in a single cell of the present invention, the preparation of the protein-coated metal nanoparticles of step iii) includes a protein and a metal nanoparticle by a dye having a protein binding site and a SERS labeling site. It is preferred that the particles are connected, wherein the dye is more preferably a non-fluorescent material.

In addition, in the method for detecting protein-coated metal nanoparticles in a single cell of the present invention, the metal used for the metal nanoparticles is preferably gold or silver, and the intracellular uptake of the nanoparticles of step ii) is nanoparticles. It is preferable that the cells are cultured in the added medium, wherein the intracellular uptake of the nanoparticles of step ii) is more preferably confirmed using a transmission electron microscope (TEM).

In addition, in the method for detecting protein-coated metal nanoparticles in a single cell of the present invention, it is preferable that a hyperspectral imaging technique is combined with the technique of step ii), wherein the DFM technique is a slide glass. Mounting the cells thereon and sandwiching the cover glass thereon; Ii) injecting metal nanoparticles coated with Raman dye to the cells and culturing the cells; And iii) taking the cells under a conventional dark field microscope equipped with a CCD camera.

In addition, in the method for detecting single-cell protein-coated metal nanoparticles of the present invention, the SERS technique of step iii) is preferably detected at 180 ° using a Raman confocal spectrometer equipped with a Peltier-cooled CCD camera. Do.

In addition, in the method for detecting protein-coated metal nanoparticles in a single cell of the present invention, the cell is preferably a mammalian cell line, and the cell line is more preferably a cell line selected from A549, HeLa, and K562. .

We have prepared a colloidal dispersion of gold nanoparticles with negative charge by the citrate reduction method. To study the surface characterization of nanoparticles, UV-Vis absorption spectroscopy was used, and transmission electron microscopy was used to observe the morphology of gold nanoparticle aggregates. According to TEM measurements, the average diameter of the gold particles is from about 20 ㎚, the surface area of a single particle is calculated to be about ² 1260 ㎚, single particles 3.4x10 consists ^{of 5} gold atoms, the concentration of the gold nanoparticle is from about 3.8x10 ^- Calculated at ⁹ M, a concentration of about 10 ^-5 M is required for complete coverage of the monolayer.

The DLS method indicates that the hydraulic radii of the original gold nanoparticles and Tf-coated gold nanoparticles are 29.5 (± 3.9) and 345.8 (± 18.0) nm, respectively, and the humans used in the present invention having a molecular weight of 80 kDa Serum Tf protein samples have a, b, and c crystal axes of 4.50, 5.78, and 13.56 nm, respectively. Considering the measured surface areas of single gold particles of ˜1260 nm ² and single Tf proteins of 20-176 nm ² , respectively, it is theoretically predicted that 8-83 Tf proteins can bind to single gold nanoparticles. The Tf bound to the gold nanoparticles is believed to have a larger form of aggregation than the monomers from the TEM image and DLS measurements (see FIG. 5 ).

Tf was bound to the gold nanoparticles, first the color of the clean red gold nanoparticles first changed to MBA and then turned blue (see FIG. 6 ). In the TEM measurement, the colloidal solution is redispersed after Tf is bound to MBA-added gold nanoparticles (see FIG. 7 ). The IR and Raman data carried out by the inventors show the formation of amide bonds due to the coupling reaction between the COOH group of MBA and the amino group of Tf bound to the gold surface.

To confirm TEM images of cellular uptake of nanoparticles, uptake of gold nanoparticles in A549 cells was investigated using TEM. Gold nanoparticles are loaded into the endosome of A549 cells (see FIG. 3 ). In addition, gold nanoparticles are present in both the cytoplasm and nucleus of A549 cells (see FIG. 4 ). Hyperspectral images of gold nanoparticles were confirmed (see FIG. 5 ).

DFM and SERS of protein coated gold nanoparticles were investigated in single A549 cells using DFM, hyperspectral imaging and SERS methods. Various device configurations were used for this investigation (see FIG. 2 ). After absorption of MBA-modified gold nanoparticles, DFM images and SERS spectra corresponding to certain points in A549 single cells were identified (see FIG. 8 ). As a result, the internalization of nanoparticles was confirmed by Z-depth dependent SERS combined with DFM. Some of the MBA coated gold nanoparticles were absorbed into the intracellular zone. Tf-coated gold nanoparticles are found in single cells (see FIG. 9 ).

The inventors identified hyperspectral imaging scans with a horizontal resolution of about 30 nm to confirm that gold nanoparticles (Au NP) were embedded in the endosome of A549 cells. In addition, the uptake of gold nanoparticles in single cells can be confirmed first by DFM and then by z-depth dependent SERS at micrometer resolution. After the approximate location of the gold nanoparticles has been confirmed by DFM, it is possible to measure the distribution inside the cell using a Raman dye having a specific vibration marker band, thereby obtaining three-dimensional localization within a single cell by obtaining specific spectral characteristics. three-dimensional localization). We use a 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) reaction to produce a 4-mer having a -COOH surface functional group for binding to transferrin (Tf) protein. Gold nanoparticles modified with captobenzoic acid (MBA) were used. The protein binding reaction on the gold surface was examined by color change, UV-Vis absorption spectroscopy and SERS. The results of the present invention demonstrate that DFM techniques combined with SERS can have great potential for monitoring biological processes with protein binding at the single cell level. As such, we were able to distinguish nuclei from other cellular parts with partial help by color differences in the DFM image without using any fluorescent dye. In conclusion, the Z-depth dependent SERS method combined with our DFM imaging technology has a high potential for monitoring uptake or intracellular processes at the single cell level.

As described above, the present invention uses SERS combined with hyperspectral imaging technology and DFM to inform intracellular uptake and behavioral behavior of gold nanoparticles in A549 cells. Vibration bands of aromatic adsorbates gathered on gold nanoparticles provide inherent information about their presence and location within the cell without any interference from any organic component in the single cell, so these vibrations have aromatic stretching bands within the single cell. Fingerprinting can be used to confirm the intracellular distribution of protein coated gold nanoparticles using SERS combined with DFM. In addition, the detection method of the present invention may be useful for use as an intracellular imaging tool for non-fluorescent protein attached nanoparticles.

1 is a schematic showing protein preparation sample preparation.
2 is a schematic of the device configuration of a z-depth dependent SERS in combination with a DFM, with the slit to the right of the holographic notch filter can provide confocality of the Raman spectrometer.
FIG. 3 is a TEM image of gold nanoparticles comprising A549 cells, with an “E” arrow indicating the position of the gold nanoparticles in the endosome.
4 is a DFM image of gold nanoparticles containing A549 cells, with the "C" and "N" arrows pointing to the location of the gold nanoparticles in the cytoplasm and nucleus, respectively.
Fig. 5 shows an enlarged portion of the portion marked in the red box as a hyperspectral image of gold nanoparticles. Red indicates superimposed images from the reflectance spectrum of gold at 500-700 nm. To obtain the image, a nominal horizontal resolution of ˜30 nm was used. The pixel size is 31 nm X 31 nm. The arrow “E” points to the location of the gold nanoparticles in the endosome, consistent with the TEM image, and “Au” points to the gold nanoparticles.
Figure 6 is a photograph of pure citrate-reduced gold nanoparticles (left), the color change photograph after addition of MBA (middle), and the color recovery of gold nanoparticles after binding of proteins to MBA coated gold nanoparticles. Photo (right)
FIG. 7 shows the change in UV-Vis absorption spectra of citrate reduced gold colloidal nanoparticles before and after addition of MBA to citrate reduced gold nanoparticles and addition of Tf to MBA-coated gold nanoparticles. Recovery after one and corresponding TEM images obtained for MBA coated gold nanoparticles before and after binding of Tf.
8 is a DFM and z-depth dependent SERS spectrum of MBA-coated gold nanoparticles embedded in A549 cells. The arrow points to the “MBA” position of the gold nanoparticles determined according to the SERS spectrum. MBA coated gold nanoparticles were found located approximately ˜7 μm from the cell surface at the “MBA” site.
9 is DFM and z-depth dependent SERS spectra of Tf-bound MBA coated gold nanoparticles in single A549 cells. Arrows indicate the positions of "A" and "B" of the gold nanoparticles determined according to the SERS spectrum. Tf-bound MBA coated gold nanoparticles were found located approximately 3 μm from the cell surface at two different points, “A” and “B”.

Hereinafter, the present invention will be described in more detail based on the following examples. It should be noted, however, that the following examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The present invention is not limited to the following examples. Will be apparent to those skilled in the art to which the present invention pertains.

< Example  1> Sample Preparation

Colloidal dispersions of gold nanoparticles with negative charges were prepared by citrate reduction (PC Lee and D. Meisel, J. Phys. Chem. 86 (1982) 3391-3395.). To prepare a gold nanoparticle sample, 30 ml of an aqueous 1.4 mM HAuCl ₄ (99.9 +%, Sigma Aldrich) solution was first boiled. Then, approximately 3 ml of 1% sodium citrate solution was added to the HAuCl ₄ solution under vigorous stirring, and further boiled for about 1 hour. Tf (human, minimum ≥98%), 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC), 4-mercaptobenzoic acid (MBA, 90%) and benzylamide (99%) were purchased from Sigma Aldrich. , 4-aminobenzenethiol (ABT, 4-aminothiophenol,> 96%) and 4-hydroxyl benzenethiol (HBT, 4-mercaptophenol,> 95%) were purchased from Tokyo Kasei and used as is. Human lung carcinoma cells (A549, ATCC CCL-185) were cultured in RPMI1640 medium supplemented with 10% FBS and antibiotics at 37 ° C. temperature in a 5% CO ₂ atmosphere incubator. FBS (fetal bovine serum) was purchased from WelGene (Seoul, Korea).

< Example  2> Nanoparticles  Characterization-Surface Characterization

In order to investigate the surface plasmon band, UV-Vis absorption spectroscopy (Mecasys UV-3220 spectrophotometer) was used. In addition, transmission electron microscopy (TEM) was used to observe the morphology of the gold nanoparticle aggregates. The hydrodynamic radius and surface potential of the particles were monitored using quasi-elastic light scattering and zeta potential measurements. The Otsuka ELSZ-2 analyzer was used for this purpose. The gold-containing percentage of nanoparticle solution was measured at 139.3 ppm using Perkin-Elmer OPTIMA 4300DV ICP-AES. The morphology of gold nanoparticles having a diameter of about 20 nm was investigated using JEOL JEM-1010 TEM.

According to the TEM measurement, the average diameter of the gold particles was about 20 nm. The surface area of a single particle is calculated to be about 1260 nm ² . The single particle consisted of 3.4 × 10 ⁵ gold atoms and the concentration of gold nanoparticles was calculated to be about 3.8 × 10 ⁻⁹ M. A concentration of about 10 ⁻⁵ M is required for complete coverage of the monolayer film. Under our experimental conditions, the adsorbent concentrations of 10 ⁻⁵ to 10 ⁻³ M used for MBA binding on gold nanoparticles are higher than what is needed for its monolayer membrane coverage. Instead of gold nanoparticles, we tested silver nanoparticles and obtained similar results, confirming that the method of the present invention can be applied to a wide variety of other SERS active nanostructures.

Independent dynamic light scattering (DLS) measurements showed that the hydraulic radii of the original gold nanoparticles and Tf-coated gold nanoparticles were 29.5 (± 3.9) and 345.8 (± 18.0) nm, respectively. Our human serum Tf protein samples with a molecular weight of 80 kDa have a, b, and c crystal axes of 4.50, 5.78, and 13.56 nm, respectively (HW Yang, et. Al., Protein Sci. 9 (2000) 49 -52.). Considering the measured surface areas of single gold particles of ˜1260 nm ² and single Tf proteins of 20-176 nm ² , respectively, it is theoretically predicted that 8-83 Tf proteins can bind to single gold nanoparticles. The Tf bound to the gold nanoparticles is believed to have a larger form of aggregation than the monomers from the TEM image and DLS measurements of FIG. 5 . In addition, Tf is likely to form a rather bulky structure instead of a structure that is closely bonded to the gold surface. In aqueous solution, on the other hand, it will show a somewhat flexible protein structure, so a finite number of Tf is expected to be linked onto the gold surface. This may be for some reason whether a strong peak due to MBA is still present in an overwhelming way after the binding of Tf. Some of the gold MBA adsorbates are unlikely to react with Tf. Considering the bulky protein structure, it is still a matter of guessing how the Raman spectra change after binding of Tf on the MBA-coated gold nanoparticle surface, because the SERS intensity is very sensitive to distance from the surface. (C. Schlucker, Chem Phys Chem. 10 (2009) 1344-1354.).

< Example  3> On nanoparticles Tf Bond of-gold Nanoparticle phase  Protein binding

40 μl volume of MBA ( ^10-3 M) in anhydrous ethanol was added to a 200 μl volume of aqueous gold solution. Surfaces of the unreacted gold nanoparticles were covered with excess 2-mercaptoethanol (≥99%, Sigma Aldrich) to prevent nonspecific binding. After 5 minutes there was a color change from red to dark blue. A 200 μl volume of MBA-added gold nanoparticles solution was mixed with 1 ml of Tf ( ^10-6 M) and EDC ( ^10-6 M) in a 1: 1 mixture of buffer (10 mM potassium phosphate and The coupling reaction was maintained overnight under 0.15 M NaCl) or without buffer. A sample of micromolar concentration was prepared by dissolving 0.016 g of Tf having a molecular weight of 80 kDa in 20 ml of distilled water. Prior to the addition of the buffer, Au NPs were measured at pH 5-6 using a Thermoelectron Orion 3 star bench top pH meter. The remaining chemicals and Tf were centrifuged and the unreacted NHS group on the Au NP surface was quenched with ethanolamine (≥99.5%, Sigma Aldrich) to prevent further binding of serum proteins. 1 illustrates an experimental schematic of the present invention of the protein binding process for MBA-added Au NPs.

As shown in FIG. 6 , the color of the clean red gold nanoparticles first changed to blue after modifying with MBA. The color returned to red upon binding to the protein. We recently examined the binding behavior of amino acids on the surface of modified gold nanoparticles to observe changes in color, ATR-IR and SERS data (JH Park, et. Al., Chem Comm. (2009) 7354-7356. ). Proteins like bovine serum albumin showed similar behavior. In these figures, gold nanoparticle solutions generally show a change in color from red to blue because the surface plasmon bands shift to longer wavelengths when the nanoparticles are in an aggregated state. Conversely, redispersion of aggregated gold nanoparticles results in a color recovery from blue to red upon binding to the protein.

TEM measurements also showed that the colloidal solution should be redispersed after Tf is bound to the MBA-added gold nanoparticles, as in the UV spectrum in FIG. 7 . The spectroscopic study of the inventors clearly showed that there is a recovery of the surface plasmon band due to the increase in the interparticle distance after binding of Tf.

To further investigate the binding reaction of the gold nanoparticle surface, the inventors performed vibration spectroscopic means using surface-enhanced Raman scattering (SERS) and attenuated total-reflection Fourier-transform infrared (ATR-FTIR). Our IR and Raman data also support the formation of amide bonds due to the coupling reaction between the COOH group of MBA bound to the gold surface and the amino group of Tf.

< Example  4> Nanoparticles  Cell uptake TEM  Image-A549 Intracellular  gold Nanoparticles  absorption

The absorption of gold nanoparticles was investigated using TEM. A549 cells were placed on a 100 mm culture dish (SPL, Korea) at a concentration of 1 × 10 ⁴ cells per dish containing growth medium before contacting the nanoparticles. Nanoparticles were added and cells were incubated at 37 ° C., 5% CO ₂ concentration. After 24 hours, cells were washed twice with DPBS, fixed for 24 hours with Karnovsky fixative, and finally fixed in 0.5 M osmiumtetroxide. After fixation, samples were washed and dehydrated with 30, 50, 70, 80, 90% ethanol continuously for 15 minutes each, and this was done three more times with 100% ethanol. Samples were immersed in a resin mixture in propylene oxide polymerized at 80 ° C. Ultrathins were prepared for the TEM using a diamond knife. Samples were analyzed using transmission electron microscopy.

3 shows TEM images of A549 cells loaded with gold nanoparticles. From the TEM images, we were able to observe the particles loaded on the significant endosomes. Since fluorescence-quenching effects of gold nanoparticles interfere with colocalization experiments, experiments using organelle-specific markers to confirm the results are undesirable. Therefore, gold nanoparticles inserted into cells in their aggregated state could be identified using DFM instead. Specifically, a slide glass with A549 cells distributed widely apart on a microstage in a Raman spectrometer with a high resolution adapter for DFM at room temperature was placed. When the Raman laser shutter was closed, a DFM image was obtained. DFM image of the inventors of the present invention is, as shown in Figure 4, the gold particles of the A549 cell cytoplasm and nucleus (PV AshaRani, et. Al. , ACS Nano 3 (2009) 279-290.) Indicate that the presence in both It was. 5 shows a hyperspectral image of gold nanoparticles in an enlarged photograph of a portion marked with a red box. Red represents superimposed images from the reflectance spectra of gold at 500-700 nm, consistent with the conventional report (J. Aaron, et. Al., Nano Lett. 9 (2009) 36123618.). In order to obtain the image, a nominal horizontal resolution of about 30 nm was achieved. Arrow “E” indicates the position of the gold nanoparticles in the endosome, consistent with the TEM image.

< Example  5> DFM , Hyperspectral Imaging  And SERS  Method-Protein Coated Gold in Single A549 Cells Nanoparticles DFM  And SERS

Hyperspectral imaging was performed using a CytoViva (Auburn, USA) VNIR hyperspectral imager. To obtain the imaging pattern, a power state with a minimum spacing of 10 nm was used. An image with a horizontal resolution of ˜30 nm was obtained. In addition, intracellular uptake of nanoparticles was also monitored using DFM with a Leica DL LM vertical microscope and a high-resolution CytoViva 150 adapter. Approximately 10 ⁵ cells were mounted on gelatin-coated slide glass, sandwiched with cover glass using a DAKO fluorescent mounting medium, and incubated in nanoparticle solution for 24 hours. Sterile cover glass was prepared using an alcohol lamp and placed in the center of the cell culture Petri dish. 0.8 ml volume of gelatin solution (0.2%) was smeared on the cover glass, soaked for 30-45 minutes and then removed. A 10 mL volume of RPMI solution was poured into a cover glass covered culture dish in which the A549 cells were plated and incubated for 24 hours. Then, about 5 ppm of Raman dye-coated gold nanoparticles were injected using a micropipette and incubated for 24 hours. The cover glass was lifted off and placed in another clean slide glass treated with a mounting solution prepared for the dark field spectrometer. Images were obtained using an Infinite II color CCD camera using an objective lens (Leica, x40) with a corrected thickness for the cover glass.

Raman spectra were obtained using a Raman confocal system model 1000 spectrometer (Renishaw) equipped with an integrated microscope (Leica DM LM). Spontaneous Raman scattering was detected by 180 ° geometry using a Peltier cooled (70 ° C.) CCD camera (400 × 600 pixels). A suitable holographic supernotch filter was set in the spectrometer for 632.8 nm from a 20 mW air cooled helium-neon laser (Melles Griots Model 25 LHP 928) with a plasma line rejection filter. 2 illustrates such a device configuration.

FIG. 8 shows the DFM image after absorption of MBA-modified gold nanoparticles and the SERS spectrum corresponding to any particular point in A549 single cell. Internalization of nanoparticles was confirmed by Z-depth dependent SERS coupled with DFM. SERS spectra are consistent with previous reports (J. Kneipp, et. Al., Nano Lett. 7 (2007) 2819-2823.). Raman spectra for most of the points indicate that they do not contain any indication of the vibration band of the MBA indicating the absence of gold nanoparticles at these locations. It was confirmed that some of the MBA coated gold nanoparticles were taken up into intracellular zones as shown in FIG . 8 .

When Tf adheres to the surface of gold nanoparticles, receptor-mediated endocytosis can occur and the nanoparticles are transported intracellularly (BD Chithrani and WCW Chan, Nano Lett. 7 (2007) 15421550./CHJ Choi, et. Al., Proc. Natl. Acad. Sci. US A. 107 (2010) 1235-1240. Attached Tf molecules are still active and are predicted to be recognized by receptors at the cell surface. As shown in FIG . 9 , Tf-coated gold nanoparticles were found in single cells. For the SERS spectra at points “A” and “B”, we found very significant properties of Tf-bound MBAs attesting the presence of gold nanoparticles at those locations. The arrow indicates the position of the gold nanoparticles determined according to the SERS spectrum. The numbers inserted are z-depth positions that are relatively steered in micrometers.

We were able to distinguish nuclei from other cellular parts with partial help by color differences in the DFM image without using any fluorescent dye. However, because the fluorescence-quenching effect of gold prevents co-culture experiments using conventional organelle-specific organic label dyes (JB Pawley, Handbook of Biological Confocal Microscopy, Springer, New York, USA 3rd Ed. 2006 .), The location of nanoparticles in certain organelles of cells under our experimental conditions is still a problem to be improved.

< Example  6> Other Test

When we did not use Raman dye, we could not find the weak properties of DNA and protein in the intracellular environment (HW Tang, et. Al., Anal. Chem. 79 (2007) 3646-3653.). In addition to the MBA used as the Raman dye, 4-aminothiophenol (ABT) and 4-mer containing NH ₂ and OH surface functional groups on gold nanoparticles, respectively, to identify their 3-dimensional position in single cells. Other aromatic thiols of captophenol (HBT) as well as 1,4-phenylenediisocyanide (PDIC) were tested. Most aromatic adsorbents such as ABT and HBT are known to exhibit a similar spectral pattern as MBA. By introducing PDIC, which exhibits a unique NC stretching band at ˜2188 cm ⁻¹ , clearer distinctions in the Raman spectrum were possible. By introducing appropriate binding reactions of proteins or DNA, we can achieve complex analysis by differences in their spectra on the basis of DFM and SERS.

It has been suggested that the interaction of other particles with nanoparticles with the cell affects the charge of the cell membrane, thereby changing the shape and function of the cell membrane (P. Thevenot, et. Al, Nanomedicine 4 (2008) 226). -236.). Absorption of nanoparticles into cells, including phagocytosis, clathrin-mediated endocytosis, caveolae-mediated inclusion, and non-clatrine, non-cabeleol inclusion There are many estimation mechanisms for (K. Unfried, et. Al., Nanotoxicology 1 (2007) 52-71.). Gold nanoparticle-membrane interactions have been suggested to be cell line-dependent phenomena (HK Patra, et. Al., Nanomedicine 3 (2007) 111-119.). We obtained similar results using HeLa cells, another mammalian cell line. To better explain the cellular uptake mechanism and intracellular uptake mechanisms in mammalian cells, we plan to perform experiments on different proteins using different types of cell lines. The Z-depth dependent SERS method in combination with our DFM imaging technology has a high potential for monitoring uptake or intracellular processes at the single cell level.

As such, we studied the intracellular uptake of gold nanoparticles in mammalian A549 single cells by hyperspectral imaging and DFM bound SERS. Vibration bands of aromatic adsorbents bound to gold nanoparticles have been shown to provide unique information about the presence and location of cells within the cell, without interfering with other organic components. Such vibrational fingerprinting with aromatic stretching bands in single cells has been shown to be used for intracellular distribution of protein-coateddine gold nanoparticles using DFM bound SERS. As demonstrated by our combined data of color change, UV-Vis adsorption and SERS, protein binding by MBA with -COOH surface functional group via an EDC coupling reaction has been shown to be effective. The results of the present inventors show that the method can be used as a useful cell imaging tool for non-fluorescent protein-attached nanoparticles.

On the other hand, the specific scope of the present invention is defined by the claims rather than the embodiments described above, all changes and modifications derived from the meaning and scope and equivalent concepts of the claims to the scope of the invention Should be interpreted to include

Claims (11)

Iii) preparing protein coated metal nanoparticles;
Ii) absorbing the nanoparticles into cells and detecting them with dark field microscopy (DFM); And
Iii) detecting nanoparticles using z-depth dependent confocal surface enhanced Raman scattering (SERS).
According to claim 1, wherein the step of the protein-coated metal nanoparticles of the step iii) the protein and the metal nanoparticles are characterized in that the protein and metal nanoparticles are linked by a dye having a SERS labeling site (dye) Method for Detection of Coated Metal Nanoparticles.
The method of claim 2, wherein the dye is a non-fluorescent material.
The method of claim 1, wherein the metal used for the metal nanoparticles is gold or silver.
The method of claim 1, wherein the intracellular uptake of the nanoparticles of step ii) is performed by culturing the cells in a medium containing the nanoparticles.
The method of claim 1 or 5, wherein the intracellular uptake of the nanoparticles of step ii) is confirmed using a transmission electron microscope (TEM).
The method of claim 1, wherein the method of step ii) is combined with a hyperspectral imaging technique.
8. The method of claim 1 or 7, wherein the DFM technique is
Iii) mounting the cells on a slide glass and sandwiching the cover glass thereon;
Ii) injecting metal nanoparticles coated with Raman dye to the cells and culturing the cells; And
Iii) photographing the cells with a conventional dark field microscope equipped with a CCD camera.
The method of claim 1, wherein the SERS technique of step iii) is detected by a 180 ° geometry using a Raman confocal spectrometer equipped with a Peltier-cooled CCD camera. .
The method of claim 1, wherein the cell is a mammalian cell line.
The method of claim 10, wherein the cell line is a cell line selected from A549, HeLa, and K562.

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