KR100307807B1 - Surface-Modified Polymer Culture Dishes Using Ion Beam and Method for Surface Modification - Google Patents

Abstract

The present invention relates to a polymer culture plate surface-modified hydrophilically by using an ion beam and a method for surface modification thereof, wherein the polymer culture plate is irradiated with an ion beam having an energy of 2000 eV or less while introducing a reactive gas into the surface of the polymer culture plate The present invention provides a method for surface modification of a polymer culture plate that reacts an activated polymer culture plate surface with the reactive gas to form a hydrophilic group on the surface thereof while activating a surface of the culture plate, and thereby provides a surface-modified polymer culture plate.

Description

Surface-Modified Polymer Culture Plate Using Ion Beam and Surface Modification Method

The present invention relates to surface modification of a polymer culture dish, and more particularly, to a polymer culture dish that has been hydrophilically surface-modified so that cells adhere well to the culture dish using an ion beam, and a surface modification method thereof.

The conventionally used polymer culture dish material is polystyrene (PS), which is commonly used because of its low cost, transparency in the visible region, and ease of processing. : Polymer culture plates made of PE, polyprophylene (PP), polyethylene terephtalate (PET), etc. are also widely used.

However, the polymer compounds as described above exhibit hydrophobicity, so that when the cells are cultured in the culture dish prepared therefrom, the cells are hardly adhered to the surface of the culture dish. Among them, the PS culture plate is widely used for culturing bacteria that do not need to adhere to the surface of the culture plate in order to proliferate because of its low cost, transparency and easy molding.

However, in order to cultivate cells that can grow only by adhering to the culture dish, such as tissue cells, it is necessary to change the surface of the culture dish to be hydrophilic so that the tissue cells adhere well to the surface of the culture dish.

The hydrophilicity of the culture dish is confirmed by the wetting angle with water. The contact angle is defined as the angle formed by the contact between the surface of the culture dish and the surface of the water droplet when contacting the contact point (contact point) to the surface of the water droplet. According to the above definition, the small contact angle means that the droplets spread widely and thinly on the surface of the culture dish, and thus, the adsorption to the water of the culture dish, ie, the hydrophilicity, is large. The contact angle is set to an average value of the measured values at four positions by dropping 0.025 ml of the third distilled water drop to four positions on the surface of the culture dish using a contact angle measuring instrument.

The contact angle was measured for the culture dish before and after the surface modification by the method described above, and it was confirmed that the surface of the polymer culture dish was hydrophilically modified by decreasing the contact angle after the surface modification. It means that a hydrophilic group is formed.

Thus, attempts to modify the surface to form hydrophilic groups on the surface of the PS culture dish in order to ensure that the tissue cells adhere well to the culture dish has been attempted in a variety of ways, in the actual production process using a corona discharge or plasma treatment In addition, UV and ozone are used to form hydrophilic groups on the surface of the PS culture dish.

Among them, the method using the corona discharge fills the reaction gas to the atmospheric pressure in the vacuum chamber and applies a voltage at the electrode to ionize the reaction gas by the corona discharge to form a plasma including electrons, cations, and anions. That is, a method of modifying the surface to be hydrophilic by forming a hydrophilic group by reacting the plasma with the sample surface.

In addition, the general glow discharge process is similar to the corona discharge, but the reaction gas in the vacuum chamber is filled with 0.01-5 torr, which is much smaller than the atmospheric pressure of 76Orr, and the DC voltage or the radio frequency (RF) or the microwave ( A plasma is applied to form a plasma through the glow discharge generated at this time, and the plasma is used.

Another method of modifying the surface of the polymer is a method of improving the hydrophilicity by supplying ozone while irradiating ultraviolet light to the surface of the polymer using ultraviolet and ozone.

However, the surface-modified culture dish using these methods did not have a large decrease in the contact angle and had a problem of poor reproducibility.

In the case of glow discharge, when some high energy ions in the plasma are irradiated onto the polymer surface, the polymer surface is damaged, resulting in a rough surface and a drop in transparency of the culture dish.

In particular, in the case of corona discharge, the corona discharge helps to form a polar bond by converting a non-polar carbon-carbon chain bond into a short chain radical, but such a short chain bond is washed out when put in water. This problem is the same when treated with ultraviolet and ozone.

In order to solve the above problems, the present invention is a polymer culture plate made of one of polystyrene, polyethylene, polyprophylene, and polyethylene terephtalate, which is 2000 eV or less. The purpose of the present invention is to provide a polymer culture plate having a hydrophilic group that is not washed out by water by reacting an activated gas with a reactive gas and an active gas while irradiating an ion beam having an energy of.

In order to achieve the above object, the surface modification method of the polymer culture plate according to the present invention is a surface of the polymer culture plate with the ion beam by irradiating an ion beam having an energy of 2000 eV or less while introducing a reactive gas into the surface of the polymer culture plate. It is made by reacting the surface of the activated polymer culture plate with the reactive gas while activating to form a hydrophilic group on the surface.

Figure 1 is a longitudinal cross-sectional view showing a schematic configuration of the surface modification apparatus of the polymer culture plate according to the present invention.

2a to 2c are graphs showing the results of measuring contact angles of the PS culture dish with respect to the number of irradiated ions when the acceleration voltages of argon ions are 600, 1000, and 1200 V, respectively.

3a to 3d are graphs showing the results of the XPS measurement on the surface of the PS culture dish, and FIGS. 3a to 3b are the carbon and oxygen concentrations of 1s of the carbon on the surface of the PS culture dish without surface treatment with ion beams, respectively. spectrum.

3c to 3d are carbon and oxygen 1s spectra for the surfaces of PS culture dishes each surface-treated with an ion beam.

4 is a graph showing the surface energy of the PS culture dish versus the number of irradiated ions.

5 is a graph showing the results of measuring the contact angle of the PS culture dish with respect to time.

Figure 6 is a graph showing the number of cells for the elapsed days when the cells are grown in the surface-modified PS culture dish according to the present invention.

<Description of the symbols for the main parts of the drawings>

10: vacuum chamber 20: gas inlet

30: ion source 40: substrate holder

50: vacuum pump

Hereinafter, the surface-modified polymer culture plate and its surface modification method according to the present invention will be described in detail with reference to FIGS. 1 to 6.

First, Figure 1 is a longitudinal cross-sectional view showing a schematic configuration of the surface modification apparatus of the polymer culture plate according to the present invention, the apparatus is a gas inlet 20 for introducing a reactive gas into the interior of the vacuum chamber 10, It consists of an ion source 30 for generating an ion beam having energy, and a substrate holder 40 for mounting a sample opposite thereto, in order to maintain a constant degree of vacuum in the vacuum chamber 10, the vacuum pump 50 Is provided.

In this apparatus, ion assisted reaction (IAR: Ion Assist Reaction, hereinafter referred to as IAR) is used. That is, the ion beam is irradiated onto the surface of the culture dish to activate the surface to facilitate the reaction with the reactive gas, thereby helping the ion beam react with the surface of the polymer culture dish with the reactive gas. Important experimental variables at this time are the ionization rate of the ion source 30, the control of the ion beam energy and the amount of reactive gas. In particular, the ion source 30, first, the ionization rate should be adjustable, second, because the bond in the polymer is a variety of types must be adjustable from a low acceleration voltage to a high acceleration voltage, and third, must operate in a stable state for a long time Fourth, in order to increase the reliability of the surface-modified polymer culture dish, the ion beam should be generated uniformly.

One example of the ion source 30 in the present invention that satisfies the above conditions is a cold hall cathode ion source, which independently and precisely controls the energy of the ion beam and the number of ions reaching the substrate, respectively. There are features that can be done. At this time, the energy of the ion beam is obtained by applying an acceleration voltage to the formed ion beam, and the number of irradiated ions can be adjusted by adjusting the current of the ion beam. In addition to the cold hollow cathode ion source, there is a Kaufman type ion source, a radio frequency (RF) ion source, or a high frequency (HF) ion source. The ion beam density can be obtained in a low energy region. It is done. The ion generating gas used in the ion source as described above is selected from an inert gas and oxygen, nitrogen, carbon dioxide, carbon monoxide and mixed gas thereof.

In addition, the reactive gas may be selected from oxygen, carbon dioxide, carbon monoxide, ozone, and mixtures thereof.

According to an embodiment of the present invention, a method of surface modification using the apparatus as described above with the PS culture dish will be described in detail.

First, the surface of the commercially available PS culture dish is washed with an organic solvent to remove impurities, and then mounted on the substrate holder 40 in the vacuum chamber 10, and the degree of vacuum in the vacuum chamber 10 is adjusted to 1 × 10- ^. Keep at ⁵ Torr.

Next, oxygen is introduced into the reactive gas through the gas inlet 20 around the PS culture dish, argon is introduced into the ion source 30 as an ion generating gas, and the discharge voltage is 400-500 V. After generating the ion beam, the energized ion beam is irradiated onto the PS culture dish while changing the acceleration voltage in the range of 300 to 2000 V. At this time, the number of ions irradiated to the substrate by adjusting the current of the ion beam is set in the range of 1 × 10 ¹⁴ to 1 × 10 ¹⁷ pieces per 1 cm 2 area. The experimental variables, that is, the degree of vacuum, the discharge voltage, the acceleration voltage, and the number of irradiated ions are obtained through experiments with values suitable for surface modification.

2A to 2C show the number of ions to be irradiated at acceleration voltages of 600, 1000 and 1200 V, respectively, when the number of irradiated ions is changed within the above range and the amount of oxygen is 0, 4 and 8 ml / min. Change in contact angle.

The contact angle is measured in the same manner as mentioned above. However, the surface energy, the polar energy, and the dispersion energy were calculated from the values of the two forms of formamide, which is a polar solvent, in addition to water, which will be described later.

As shown in FIGS. 2b to 2c, when the ion beam is irradiated without oxygen, the contact angle is 36 ° to 50 °, and the contact angle reduction of this degree is used in the case of using a high energy ion beam of tens of thousands of eV or more and has been used conventionally. Similar to using corona discharge or plasma.

On the other hand, as shown in Figs. 2a to 2c, the contact angle when oxygen is introduced around the PS culture dish and irradiated with an ion beam is almost always smaller than that when no oxygen is introduced, from 2 ° to 20 °. It makes a difference. Therefore, the minimum contact angle when the ion beam is scanned while oxygen is introduced is when the acceleration voltage is 120000 V, that is, in FIG. 2C, the amount of oxygen introduced is 8 ml / min and the number of 5 × 10 ¹⁶ ions / cm 2. In this case, 19 ° was measured.

In addition, when the number of ions irradiated in FIGS. 2A to 2C is 1 × 10 ¹⁷ or more / cm 2, the contact angle increases, but is lower than the contact angle before surface treatment with the ion beam, so that the contact angle of the PS culture plate subjected to the surface treatment with the ion beam is It decreased in the full range of 1x10 ¹⁴ -1x10 ¹⁷ pieces / cm <2>.

In addition, in each of Figs. 2A, 2B and 2C, the reduction in contact angle is larger when the amount of oxygen introduced is 8 ml / min than when 4 ml / min. This means that in each case where the amount of oxygen introduced is 4 ml / min and 8 ml / min, even if the acceleration voltages of ions change to 600, 1000 and 1200 V, respectively, that is, there is no significant difference in FIGS. 2A to 2C. In comparison with this, it can be seen that the decrease in contact angle is more closely related to the amount of oxygen introduced than the energy of the ion beam due to the acceleration voltage.

2A to 2C show acceleration voltages of 600, 1000, and 1200 V and oxygen inflow amounts of 0, 4, and 8 ml / min, but experiments show acceleration voltages of 2000 V or less and 10 ml / min or less. It was confirmed that the contact angle was reduced in the amount of oxygen inflow. At this time, the oxygen inflow amount is a value that is determined according to the size of the vacuum chamber and the size of the specimen, which means that the optimal value is 10 ml / min or less in this embodiment. If larger vacuum chambers and specimens are used, an oxygen inflow of 10 ml / min or more will be required.

As mentioned above, the change in contact angle is closely related to the formation of hydrophilic groups on the surface of the polymer. The hydrophilic group was analyzed by X-ray photoelectron spectroscopy (hereinafter referred to as XPS) measurement.

3 is an XPS measurement result of the surface of the PS culture dish, wherein FIGS. 3A to 3B are carbon 1s and oxygen 1s spectra on the surface of the PS culture dish without surface treatment with ion beams, respectively, and FIGS. 3C to 3D The carbon 1s and oxygen 1s spectra of the surface of the PS culture dish surface-treated with 5 × 10 ¹⁶ pieces / cm 2 argon ions while introducing oxygen in an amount of 8 ml / min, respectively.

First, in FIG. 3A, the peak shown at 284.7 eV is a peak due to a carbon-to-carbon bond (hereinafter referred to as a C-C bond) or a bond of carbon to hydrogen (hereinafter referred to as a C-H bond). Therefore, in the case of PS which is not surface-treated with an ion beam, most of the carbon is formed of such C-C bond or C-H bond.

In addition, the peak of oxygen shown in FIG. 3b is a peak due to a single bond of carbon and oxygen (hereinafter referred to as a CO bond) and oxygen adsorbed in air, and the amount of CO bond is influenced by an additive that enters the manufacturing process. The very small 286.1 eV peak, which is a peak by carbon participating in this CO bond, is not observed in FIG. 3A.

Next, in FIG. 3C, which is the XPS result of carbon on the surface of the PS culture dish irradiated with ion beams, the peak due to CC bonds at 284.7 eV is reduced, and 286.1 eV peaks due to CO bonds, and carbon and oxygen double bonds (hereinafter, , 287.6 eV peak by the C = O bond, and 288.9 eV peak by the bond of C = O and oxygen (hereinafter referred to as O- (C = O) bond) were newly formed.

In addition, in FIG. 3D, which is the XPS result of oxygen on the surface of the PS culture dish irradiated with ion beams, the intensity of the peak was significantly increased as compared with FIG.

Therefore, when the ion beam is irradiated on the surface of the PS, it can be seen that CO bonds, C═O bonds, and O— (C═O) bonds are formed by chemical bonds of atoms on the surface. Since the distribution is polarized to one side, it shows hydrophilicity, and the formation of this hydrophilic group decreases the contact angle.

As described above, the fact that the formation of the hydrophilic group reduces the contact angle can be seen by obtaining the surface energy through the change of the contact angle. As mentioned above, the surface energy is calculated by mathematical calculation from the contact angle measured using water and formamide. The surface energy is composed of polar energy and dispersed energy. As shown in FIG. 4, the surface energy increases as the ion beam is irradiated, and the increase of the surface energy is caused by the increase of the polar energy. Since the polar energy is due to the hydrophilic group having affinity with the polar solvent, it can be seen that the increase of the surface energy is directly related to the increase of the hydrophilic group on the PS surface.

Therefore, hydrophilic groups such as C—O bonds, C═O bonds, and O— (C═O) bonds are formed on the surface of PS modified by irradiating the ion beam according to the present invention to reduce the contact angle. In this case, when a gas containing nitrogen is used as the reactive gas, hydrophilic groups such as a single bond of carbon and nitrogen (C-N) and a double bond of carbon and nitrogen (C = N) are formed.

The problem with the surface modification method of the conventional PS culture dish was that the hydrophilic group could be formed on the surface through surface modification, but when put into water, the hydrophilic group was washed away and no longer exhibited the surface-modified characteristics.

In the surface-modified PS culture dish according to the present invention, in order to verify the effect that the formed hydrophilic group is not washed out in water, the contact angle of the surface-modified PS culture dish by the ion assist reaction in the following time is changed according to the change of time. The change measured is shown in FIG. 5.

FIG. 5 shows two graphs of the surface-modified PS culture dish exposed by air and the case where it is placed in water. As shown in FIG. 5, when exposed to air, the contact angle increased with time, which is a phenomenon caused by the rotation of the binder chain in the polymer, and when it is put into water, the contact angle decreases again. When stored in water, the contact angle remained almost unchanged from the initial value even though the time was increased up to 1000 minutes, which means that the hydrophilic group formed by the ion assist reaction remained on the surface without being washed out by the water. Therefore, the PS culture plate surface-modified by the ion assist reaction provides superior conditions for cell culture than the surface plate cultured plate by the conventional method.

In fact, the result of proliferating cells in the PS culture dish surface-modified by the ion assist reaction is shown in FIG. In order to obtain the growth curve of FIG. 6, PS modified with argon ions of 1 × 10 ¹⁵ ion counts / cm 2 and 5 × 10 ¹⁶ ion counts / cm 2 by commercial PS culture dishes without surface treatment and ion assist reactions Rat chromophilized cells 12 (pheochromocytoma cell 12, hereinafter referred to as PC 12) were grown in a culture dish. At this time, PC 12 was selected because PC 12, a type of neuron, is more sensitive to environmental changes than other cells and has a low affinity for the surface, that is, it is difficult to culture because it is difficult to adhere to the surface. After seeding six PC 12s as described above, the growth curve of PC 12 measured for 6 days by counting the number of cells under a microscope every 24 hours is shown in FIG. 6.

As shown in FIG. 6, PC 12, which is difficult to cultivate, is almost completely killed in a commercial PS culture dish, whereas it is well cultured in a PS culture dish surface-modified by the ion assist reaction, thereby providing excellent conditions for cell culture. You can see the zoom.

In FIG. 6, the number of cells on the first day is smaller than that on day 0. Among the cells seeded on day 0, only the cells adhered to the surface of the PS culture dish grow and all remaining cells that do not adhere to the surface die. Because In addition, after 2 to 3 days, the number of cells grown by adhering to the surface was almost doubled to the initial number of cells adhered, and the number of cells gradually decreased from day 4 when the cells were almost saturated.

Since the cells are cultured at the bottom of the culture dish, the adhesion of the side surfaces is not important, but since the ion beam spreads outward from the center, the ion beam is irradiated to the entire surface of the polymer culture dish including the side surface perpendicular to the bottom surface. In this way, the entire surface of the polymer culture dish can be modified.

As described above, an example of surface modification of a PS culture plate generally used in a polymer culture plate by an ion assist reaction is disclosed, but the present invention is not limited thereto, and a polymer made of polyethylene, polypropylene, polyethylene terephthalate, etc. other than PS is used. It relates to the surface modification of the culture dish.

As described above, the PS culture plate surface-modified by the ion assist reaction is much lower in contact angle than the commercial PS culture plate or the surface-modified PS culture plate by the conventional method, so that the adhesion strength is very low and the cells are difficult to culture. In this case, it is effective to provide excellent conditions for cell culture enough to be well cultured.

In addition, by irradiating ions with a low energy of about 1 keV, the binder in the polymer is not interrupted, so that the hydrophilic groups formed on the surface are not washed out in water.

In addition, when the experiment under the same experimental variable in the same experimental device, there is an effect excellent in reproducibility showing the same result.

Claims (7)

By irradiating an ion beam having an energy of 2000 eV or less while introducing a reactive gas to the surface of the polymer culture plate, the surface of the polymer culture plate and the reactive gas are reacted with the activated polymer culture plate while activating the surface of the polymer culture plate with the ion beam. Surface modification method of the polymer culture plate, characterized in that to form a. The method of claim 1, wherein the reactive gas is one of oxygen, nitrogen, carbon dioxide, carbon monoxide, ozone, and a mixture thereof. The method of claim 1, wherein the ion source is one of a cold hallow cathode ion source, a Kaufman type ion source, a radio frequency (RF) ion source, and a high frequency (HF) ion source. A method for surface modification of a polymer culture dish, characterized in that a high ion beam density can be obtained in a low energy region. The method of claim 1, wherein the number of ions irradiated to the polymer culture dish is 1 × 10 ¹⁴ to 1 × 10 ¹⁷ per 1 cm 2 area. The surface of the polymer culture plate according to claim 1, wherein the hydrophilic group comprises one or two or more of a CO bond, a C═O bond, an O— (C═O) bond, a CN bond, and a C═N bond. Reforming method. The surface of the polymer culture plate of claim 1, wherein the polymer culture plate is made of one of polystyrene, polyethylene, polyprophylene, and polyethylene terephtalate. Reforming method. A polymer culture plate made of one of polystyrene, poly ethylene, poly prophylene, and polyethylene terephtalate. The surface of the polymer plate is irradiated with an ion beam having an energy of 2000 eV or less. A polymer culture plate, characterized in that a hydrophilic group is not washed out in water by reacting an activated gas with a reactive gas surface while activating the plate.