KR20200089575A - Nanopillar for cell activity analysis and method of preparing the same - Google Patents

Abstract

The present invention relates to a nanopillar for cell activity analysis and a manufacturing method thereof, and more specifically, to a nanopillar for cell activity analysis, capable of culturing cells on a nano-pillar structure etched with gold and analyzing cell activity through SEM or fluorescence microscopy, and a manufacturing method thereof. By providing nano pillars for cell activity analysis and a manufacturing method thereof, it is possible to observe living cells, fluorescent staining of cells is possible, and using the same, there is an effect of confirming the characteristics of the cells themselves by analyzing the activities of the cells.

Description

Nanopillar for cell activity analysis and method of preparing the same}

The present invention relates to a nano-pillar for cell activity analysis and a manufacturing method, and more specifically, a cell capable of culturing cells on a nano-pillar structure etched with gold and analyzing cell activity through SEM or fluorescence microscopy. It relates to a pillar and its manufacturing method.

Nanostructures or nanoscale structures are currently used in electronic materials (eg, electrodes, light emitting diodes (LEDs) and solar cells), semiconductor manufacturing processes, data storage devices, wearable devices, and various sensors (eg, flexible) Sensors, electrochemical sensors, and biosensors). In particular, nano-scale structures such as nano-rod, nanopillar, or nano-wire have been applied to various electronic and optical devices due to their unique optical and electrical properties. . In addition to increasing time and space efficiency, such a nano-scale fine pattern is an important factor for imparting new functions, and the demand for nanostructures corresponding thereto is increasing.

Until now, the method of manufacturing a nanostructure using a photolithography technique is the most widely used, but recently, a technique for manufacturing a nano-pattern structure in a simpler and lower-cost manner has been actively studied.

In the case of nanostructures that have been studied recently, their physical properties and behavior depend on the molecular composition and precise spatial positioning, and the unexpected properties due to the controlled morphology in large-scale devices. It is known that it can provide, and attempts are being made to apply nanostructures to new uses or new fields.

Accordingly, in the'Registration No. 10-1910851 Republic of Korea', various pathogens are fixed thereto using a complex nanostructure in which a nano web of a second polymer material is formed on the surface of a nano-pillar array of a first polymer material, and in a subsequent procedure. Although it discloses a method and a detection system capable of detecting this specifically or non-specifically, there is a problem in that only the use of a limited nanostructure is provided by using the nanopillar structure only as a fixed use of a pathogen.

KR 10-1878404 B1

Nano-micro pattern production and application using electron beam lithography, Jin-Tae Kim, Graduate School of Chonnam National University, Feb. 2010

In order to solve the above problems, an object of the present invention is to provide a method for preparing a nano-pillar for analyzing cell activity.

It is also an object of the present invention to provide a nano-pillar prepared according to a method of manufacturing a nano-pillar for cell activity analysis.

The present invention to solve the above object

A first step of manufacturing a quartz substrate coated with poly(methyl methacrylate), PMMA;

A second step of patterning the PMMA-coated quartz substrate to form a patterned substrate;

A third step of depositing chromium (Cr) on the etched portion of the patterned substrate to form a chromium-deposited substrate;

A fourth step of washing the substrate on which chromium is deposited to form a substrate from which residual PMMA is removed; And

And a fifth step of etching the substrate from which the residual PMMA is removed, and depositing gold through thermal evaporation on the etched region.

The second step is characterized by patterning a quartz substrate coated with PMMA using E-beam lithography.

The present invention to solve the above other object

It provides a nano-pillar for cell activity analysis prepared by the above manufacturing method.

The present invention provides a nano-pillar for cell activity analysis and a method of manufacturing the living cells to observe the cells, fluorescence staining of the cells is possible, and by using the cells to analyze the activity of the characteristics of the cell itself.

1 is a view showing a process of forming a nanostructure prepared according to an embodiment of the present invention.
2 is a photo of light scattering simulation results of a nanostructure prepared according to a comparative example of the present invention.
3 is a photo of light scattering simulation results of a nanostructure prepared according to an embodiment of the present invention.
4 is a diagram showing the structure of a fluorescence microscope for imaging cells on nanostructures prepared according to one embodiment and a comparative example of the present invention.
5 is a photograph of cells on a nanostructure prepared according to a comparative example of the present invention measured by a fluorescence microscope.
6 is a photograph of cells on a nanostructure prepared according to an embodiment of the present invention measured with a fluorescence microscope.
7 is an SEM photograph of cells on a nanostructure prepared according to an embodiment of the present invention.

Hereinafter, the present invention will be described in more detail.

In the present specification, when a part “includes” a certain component, it means that the component may further include other components, not to exclude other components, unless otherwise stated.

In the present specification, when a member is said to be positioned “on” another member, this includes not only the case where one member is in contact with the other member but also another member between the two members.

According to an aspect of the present invention, a first step of producing a poly(methyl methacrylate) (Poly(methyl methacrylate), PMMA) coated quartz substrate; A second step of patterning the PMMA-coated quartz substrate to form a patterned substrate; A third step of depositing chromium (Cr) on the etched portion of the patterned substrate to form a chromium-deposited substrate; A fourth step of washing the substrate on which chromium is deposited to form a substrate from which residual PMMA is removed; And a fifth step of etching the substrate from which the residual PMMA has been removed, and depositing gold through the thermal evaporation on the etched region.

The phenomenon that occurs inside the cell occurs at the nano-scale, and for this reason, super-resolution observation is required. The nano-pillar structure has a high aspect ratio, excellent surface area and adhesion to cells, can fix cells, and can infiltrate cells with a desired substance, thereby changing the mechanism of cell activity to observe the cell image of fluorescence. Can. However, in the nano-pillar structure, scattering of the optical signal occurs according to the filler, and thus only a low-resolution fluorescence image can be obtained, and thus it is difficult to observe an ultra-high resolution image.

Accordingly, in the present invention, to prevent scattering of the light signal in the nano-pillar structure, and to coat the gold on the existing nano-pillar in order to enable the cells to be cultured well, the light scattering loss is reduced as much as possible and the tip of the nano-pillar surface ( Pillar, the maximum height measurement part) provides a nanostructure that can be condensed light.

The second step of the manufacturing method of the nano-pillar for cell activity analysis is characterized by patterning a PMMA-coated quartz substrate using E-beam lithography.

Electron beam lithography (E-beam lithography) can produce a fine pattern in the correct position, because this fine patterning is because the spot size of the electron beam is very small. Lithography of nm size is possible and it can provide very good resolution among many existing patterning techniques.

According to another aspect of the present invention, there is provided a nano-pillar prepared by a method of manufacturing a nano-pillar for cell activity analysis.

The nano-pillar for cell activity analysis is a first step of manufacturing a poly(methyl methacrylate), a quartz substrate coated with PMMA; A second step of patterning the PMMA-coated quartz substrate to form a patterned substrate; A third step of depositing chromium (Cr) on the etched portion of the patterned substrate to form a chromium-deposited substrate; A fourth step of washing the substrate on which chromium is deposited to form a substrate from which residual PMMA is removed; And a fifth step of etching the substrate from which the residual PMMA has been removed, and depositing gold through the thermal evaporation on the etched region. Can.

The phenomenon that occurs inside the cell occurs at the nano-scale, and for this reason, super-resolution observation is required. The nano-pillar structure has a high aspect ratio, excellent surface area and adhesion to cells, can fix cells, and can infiltrate cells with a desired substance, thereby changing the mechanism of cell activity to observe the cell image of fluorescence. Can. However, in the nano-pillar structure, scattering of the optical signal occurs according to the filler, and thus only a low-resolution fluorescence image can be obtained, and thus it is difficult to observe an ultra-high resolution image.

Accordingly, in the present invention, to prevent scattering of the light signal in the nano-pillar structure, and to coat the gold on the existing nano-pillar in order to enable the cells to be cultured well, the light scattering loss is reduced as much as possible and the tip of the nano-pillar surface ( Pillar, the maximum height measurement part) provides a nanostructure that can be condensed light.

The second step of the manufacturing method of the nano-pillar for cell activity analysis is characterized by patterning a PMMA-coated quartz substrate using E-beam lithography.

Electron beam lithography (E-beam lithography) can produce a fine pattern in the correct position, because this fine patterning is because the spot size of the electron beam is very small. Lithography of nm size is possible and it can provide very good resolution among many existing patterning techniques.

Hereinafter, examples will be described in detail to specifically describe the present specification. However, the embodiments according to the present specification may be modified in various other forms, and the scope of the present specification is not construed as being limited to the embodiments described below. The embodiments of the present specification are provided to more fully describe the present specification to those skilled in the art.

<Example>

Example 1-Formation of nanostructures with gold deposited on the surface

E-beam lithography was used to form a nanostructure in the present invention. First, a resist sensitive to an electron beam (E-beam) is coated on a quartz substrate using spin coating. At this time, poly(methyl methacrylate), PMMA, is used as the resist. Thereafter, by irradiating the electron beam, the chemical bonds in the PMMA were cut off, and after patterning, only the part where the electron beam touched was developed through patterning, and after chromium (Cr) was deposited thereon, the remaining PMMA part was washed by washing. It was removed (lift-off). Here, the quartz substrate was etched through RIE etching, and gold was deposited on the surface portion of the pattern through thermal evaporation. This process is illustrated in FIG. 1.

Example 2-Cell culture

After preparing NIH3T3 cells in the medium, D ₁₀ (DMEM 90% + FBS 10% + Genramicin 500 μl) medium and cell separation solution trypsin were heated in a water bath at 37° C. for about 10 minutes. After disinfecting and arranging the cleanbench to turn on the alcohol lamp, cells were removed from the incubator to remove the old medium. Thereafter, 5 ml of PBS, a cell washing solution, was pipetted and gently shaken to remove waste products using a pipette, and 2 ml of trypsin was added and incubated for 2 minutes to separate cells. After centrifugation (2.5 rpm, 2 min) of the cell solution containing trypsin, only the supernatant (trypsin) of the centrifugation solution is removed, and a small medium plate is prepared by mixing the cells and a little medium by pipetting. The nanostructures of Example 1, D ₁₀ , 10 ml) were transferred and cultured.

Comparative Example 1-Preparation of nanostructures

A resist sensitive to an electron beam (E-beam) was coated on a quartz substrate using spin coating, and poly(methyl methacrylate), PMMA, was used as the resist. Thereafter, by irradiating the electron beam, the chemical bonds in the PMMA were cut off, and after patterning, only the part where the electron beam touched was developed through patterning, and after chromium (Cr) was deposited thereon, the remaining PMMA part was washed by washing. After removing (lift-off), the quartz substrate was etched through RIE etching to finally produce a nanostructure.

Comparative Example 2-Cell culture

After preparing NIH3T3 cells in the medium, D ₁₀ (DMEM 90% + FBS 10% + Genramicin 500 μl) medium and cell separation solution trypsin were heated in a water bath at 37° C. for about 10 minutes. After disinfecting and arranging the cleanbench to turn on the alcohol lamp, cells were removed from the incubator to remove the old medium. Thereafter, 5 ml of PBS, a cell washing solution, was pipetted and gently shaken to remove waste products using a pipette, and 2 ml of trypsin was added and incubated for 2 minutes to separate cells. After centrifugation (2.5 rpm, 2 min) of the cell solution containing trypsin, only the supernatant (trypsin) of the centrifugation solution is removed, and a small medium plate is prepared by mixing the cells and a little medium by pipetting. Nanostructures, D ₁₀ , 10 ml) and cultured.

<Evaluation method>

Experimental Example 1-Light Scattering Simulation

A simulation was performed to compare the degree of light scattering of the nanostructures prepared according to Example 1 and Comparative Example 1.

The simulation program used MATLAB, and was performed under a condition of 100 nm in diameter, 1000 nm in height, and 1 μm cycle. However, the nanostructure of Example 1 contains 80 nm of Au, and the nanostructure of Comparative Example 1 does not contain Au.

Experimental Example 2-Fluorescence Microscopy Measurement

Fluorescence solution was prepared by mixing 990 μl of DMSO (Dimethyl sulfoxide) and 10 μl of DiD solution. Thereafter, the medium was removed from each cell culture cultured according to Example 2 and Comparative Example 2, sprinkled with a fluorescent solution, and placed in an incubator for 5 minutes to suction the solution. Put the medium in the solution-suctioned cell and react for another 5 minutes in the incubator to attach fluorescent dye to the cell, and put the medium into each nanostructure (nanopillar) prepared according to Example 1 and Comparative Example 1 above. Fluorescently stained cells were sprinkled and cultured in an incubator for 5 minutes.

The cells cultured in the nanopillar were observed with a fluorescence microscope using an objective lens having a magnification of 100 and an NA of 0.9, and setting the wavelength of fluorescence excitation light to 633 nm.

Experimental Example 3-SEM image measurement

The experiment proceeded in the hood. 8% glutaraldehyde was diluted with PBS as a solvent to a concentration of 2.5%. The 2.5% glutaraldehyde solution diluted in the cells cultured on the nanostructures according to Example 2 was sprinkled and sealed and stored refrigerated for 1 hour. Through these steps, autolysis was prevented and the change in cell survival was minimized, and tissue was preserved upon dehydration and exposure to electron beam. Ethanol and 100% PBS for each concentration were prepared in advance and put in a hood, and then the refrigerated cell solution was washed three times with 100% PBS in the hood. After that, the cells were sprayed with ethanol for each concentration and dried at room temperature for 1 hour. Through this process, it prevented cell tissue deformation during drying and blocked the possibility of shrinkage of the sample. The SEM image of the sample produced in this way was measured.

<Evaluation and Results>

Result 1-nanostructure manufacturing result

According to Example 1 and Comparative Example 1, a nano structure (nano pillar) having gold deposited on the surface and a nano structure (nano pillar) having no gold deposited on the surface were prepared, respectively. In the future, when cells were cultivated on the nanostructures and several experiments were conducted, nanostructures of Examples and Comparative Examples were produced in the same process except for the gold deposition process in order to confirm the difference depending on whether the nanostructure surface was deposited or not. The size of the nano-pillar was 200 nm in diameter, the highest height was 820 nm, and the period was 1 μm. In the case of the nano-pillar on which gold was deposited on the surface, a diameter of 200 nm, a maximum height of 820 nm, and a period of 1 μm were exhibited, including a gold thickness of 100 nm.

Result 2-Light scattering simulation result

Example above The results of comparing the light scattering degree of the nano-pillars prepared according to 1 and Comparative Example 1 are shown in FIGS. 2 to 3.

As a result, the structure of the nano-pillar made of only quartz prepared according to Comparative Example 1, when the laser was incident, scattered and light was not collected at any one point, but the gold prepared according to Example 1 was coated. In the nano-pillar, it was possible to reduce the scattering loss as much as light could not pass through the gold-coated portion, and it was confirmed that light was condensed at the end of the nano-pillar and the gold portion due to the plasmon effect.

Therefore, it was found that the fluorescence analysis using a gold-coated nano-pillar focused the laser on a local area, so that the corresponding area could be observed with high resolution.

Result 3-Fluorescence microscopic measurement result

Example above After culturing the cells cultured according to Example 2 and Comparative Example 2 on the nano pillars prepared according to Example 1 and Comparative Example 1, the results of fluorescence microscopy are shown in FIGS. 5 to 6.

As a result, microscopic photographs of the nano-pillars that did not deposit gold on the surface confirmed that light scattered and the ends of the nano-pillars appeared relatively blurry, and in the case of nano-pillars deposited gold on the surface, the ends of the nano-pillars It was confirmed that this appeared more clearly. From these results, it was found that the nano-pillar coated with gold is clearly measured at the tip of the nano-pillar where light is collected by the plasmon effect.

Result 4-SEM image measurement result

The results of measuring the SEM image after culturing the cells on the nano pillars according to the above example are shown in FIG. 7. As a result, it was confirmed that the cells were well attached and cultured on the nano pillars.

Claims (3)

Step 1 of preparing a poly(methyl methacrylate), PMMA coated quartz substrate;
A second step of patterning the PMMA-coated quartz substrate to form a patterned substrate;
3 steps of forming a substrate on which chromium is deposited by depositing chromium (Cr) on an etched portion of the patterned substrate;
Washing the chromium-deposited substrate to form a substrate from which residual PMMA is removed; And
A method of manufacturing a nano-pillar for cell activity analysis, comprising etching the substrate from which the residual PMMA is removed, and depositing gold on the etched region through thermal evaporation. According to claim 1,
The second step is a method of manufacturing a nano-pillar for analyzing cell activity, characterized by patterning a quartz substrate coated with PMMA using electron beam lithography (E-beam lithography). Nanopillar for cell activity analysis prepared by the method according to any one of claims 1 or 2.

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