JP2020105535A - Container, method for manufacturing container, and method for evaluating amorphous carbon film - Google Patents

Abstract

To provide a technology capable of evaluating more simply an effect of a membrane composition of an amorphous carbon membrane exerted on cell adhesion or protein adsorption.SOLUTION: A container includes a substrate having one or more storage parts for storing a sample, a conductive film formed on at least a part of the substrate, and an amorphous carbon membrane formed on at least a part of the conductive film, or on at least a part of the substrate. In the container, the stored sample can have a contact with the amorphous carbon membrane.SELECTED DRAWING: None

Description

The present invention relates to a container, a method for manufacturing the container, and a method for evaluating an amorphous carbon film.

The amorphous carbon film is an amorphous hard film formed of hydrocarbon or carbon allotrope. For example, Patent Document 1 describes a synthesis device and a synthesis method for amorphous carbon.

Conventionally, when evaluating the effect of the film composition of an amorphous carbon film on cell adhesion and protein adsorption, an amorphous carbon film having a different composition is formed on a flat plate substrate (substrate), and the flat plate substrate is placed in a predetermined container. It was common to house and evaluate. However, in this case, a step of processing the base material so that it can be housed in a container is required, and the test step is complicated.

JP-A-8-12492

The container according to one embodiment has a base material having one or more storage parts for storing a sample, a conductive film formed on at least a part of the base material, at least a part of the conductive film, or An amorphous carbon film formed on at least a part of a base material, and the contained sample can contact the amorphous carbon film.

A method for manufacturing a container according to one embodiment is the method for manufacturing a container described above, wherein a step of forming the conductive film on at least a part of the base material, applying a voltage to the conductive film, and physically Forming the amorphous carbon film by a vapor deposition method or a chemical vapor deposition method.

A method for evaluating an amorphous carbon film according to one embodiment evaluates an interaction between the sample and the amorphous carbon film, a step of accommodating the sample in the container described above and bringing the sample and the amorphous carbon film into contact with each other. And a process.

(A) And (b) is a schematic diagram explaining the structure of the container which concerns on one Embodiment. It is a schematic block diagram of a filtered cathodic vacuum arc (FCVA) apparatus. (A) is a graph which shows the result of having measured the cell adhesion rate in Experimental example 6. (B) is a graph showing the result of extracting only the standard deviation of the result of (a). (A) is a graph which shows the result of having measured the albumin adsorption amount in Experimental example 7. (B) is a graph showing the result of extracting only the standard deviation of the result of (a). (A) is a graph which shows the result of having measured the fibrinogen adsorption amount in Experimental example 8. (B) is a graph showing the result of extracting only the standard deviation of the result of (a).

Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings in some cases. In the drawings, the same or corresponding parts will be denoted by the same or corresponding reference numerals and redundant description will be omitted. Note that the dimensional ratios in the drawings are exaggerated for the sake of explanation, and do not necessarily match the actual dimensional ratios.

[container]
The container of the present embodiment includes a base material having one or more storage parts for storing a sample, a conductive film formed on at least a part of the base material, at least a part of the conductive film, or the base. An amorphous carbon film formed on at least a part of the material, and the contained sample can contact the amorphous carbon film.

By using the container of the present embodiment, it is not necessary to prepare a flat plate base material or the like separately from the container, and therefore the test process for evaluating the influence of the membrane composition on cell adhesion and protein adsorption is simple. Further, as described later in Examples, by using the container of the present embodiment, for example, when evaluating the effect of the film composition of the amorphous carbon film on cell adhesion and protein adsorption, with higher measurement accuracy, It can be evaluated easily. Further, by using the container of the present embodiment, it is possible to reduce the cost of cutting and polishing the base material, which has been conventionally required. Note that the amorphous carbon film in the present specification is a carbon film containing both carbon atoms of sp ² hybrid orbital and carbon atoms of sp ³ hybrid orbital. Regardless of the content of carbon atoms in the sp ² hybrid orbital and the carbon atom in the sp ³ hybrid orbital, if both carbon atoms are included, it is called an amorphous carbon film. Further, in the present specification, when the amorphous carbon film contains titanium atoms, it is referred to as a titanium-doped amorphous carbon film.

In the container of the present embodiment, the storage section stores the sample. The sample may be a substance to be evaluated for its interaction with the amorphous carbon film. Examples of the sample include biological samples such as proteins and cells.

The shape of the accommodating portion is not particularly limited, and examples thereof include a concave shape. For example, the bottom of the accommodating portion may be flat or U-shaped. More specifically, examples thereof include shapes such as petri dishes and well plates used in biological experiments and the like. The number of wells in the well plate is not particularly limited, and may be, for example, 6 wells, 12 wells, 24 wells, 48 wells, 96 wells, or the like. The base material 110 is made of a resin material, glass, or the like, and may be insulative. The insulating property in this specification means that the volume resistivity is 10 ¹¹ (Ω·cm) or more.

As described later, in order to form the amorphous carbon film on the base material 110, it is preferable to apply a bias voltage to the base material. However, when the base material 110 is insulative, the bias voltage cannot be applied. In such a case, the conductive film 120 may be formed on at least a part of the base material 110 so that a bias voltage can be applied. The conductive film 120 is not particularly limited as long as a bias voltage can be applied, and examples thereof include a titanium film, a chromium film, and a tantalum film. The thickness of the conductive film may be about 50 to 100 nm. In addition, the conductivity in this specification means that the volume resistivity is approximately 10 ⁻⁴ (Ω·cm) or less.

1A and 1B are schematic diagrams illustrating the structure of the container according to the embodiment. As shown in FIG. 1A, a container 100 is formed on a base material 110 having a housing, a conductive film 120 formed on at least a part of the base material 110, and at least a part of the conductive film 120. And the amorphous carbon film 130. In addition, the respective accommodating portions are connected via the base material 110. The film thickness of the amorphous carbon film 130 may be about 10 to 20 nm.

The amorphous carbon film 130 may be arranged at a position where the sample contained in the container contacts the amorphous carbon film 130. For example, the amorphous carbon film 130 may be formed in a region including at least a part of the bottom portion of the housing portion. In the present specification, the bottom part of the accommodation part refers to a region of the accommodation part which the sample comes into contact with when the sample is accommodated in the accommodation part. That is, the amorphous carbon film 130 may be formed in a region including at least a part of the bottom portion of the housing portion.

In FIG. 1A, a base material 110, a conductive film 120, and an amorphous carbon film 130 are laminated in this order. However, the order of disposing the base material 110, the conductive film 120, and the amorphous carbon film 130 is not limited to this. For example, as shown in FIG. , And the amorphous carbon film 130 may be laminated in this order.

The conductive film 120 may be arranged at a position where a bias voltage can be applied to the base material 110. Therefore, as shown in FIG. 1A, the conductive film 120 may be arranged on the surface of the base material 110 on which the amorphous carbon film 130 is formed. Alternatively, as shown in FIG. 1B, the conductive film 120 may be arranged on the surface of the base material 110 opposite to the surface on which the amorphous carbon film 130 is formed.

<<Amorphous carbon film>>
Here, the amorphous carbon film 130 will be described. The amorphous carbon film 130 is formed on the base material 110 or the conductive film 120 by a PVD method (physical vapor deposition method) or a CVD method (chemical vapor deposition method) using a carbon raw material as a raw material target. You can Examples of the PVD method include an ion beam evaporation method, an ion beam sputtering method, a magnetron sputtering method, a laser evaporation method, a laser sputtering method, an arc ion plating method, and a filtered cathodic vacuum arc (FCVA) method. Examples of the CVD method include a microwave plasma CVD method, a direct current plasma CVD method, a high frequency plasma CVD method, and a magnetic field plasma CVD method.

Among them, the FCVA method is preferable because it is a film forming method capable of performing uniform coating with high adhesion even at room temperature. The FCVA method is a film forming method in which ionized particles are generated by arc-discharging a raw material target and only the particles are guided to the substrate 110 or the conductive film 120 to form a film.

FIG. 2 is a schematic configuration diagram of the FCVA device 200. In the FCVA apparatus 200, the arc plasma generation chamber 201 in which the raw material target 202 is installed and the film formation chamber 206 are connected by the spatial filter 205.

The film forming chamber 206 includes a substrate holder 207 inside. The base material holder 207 fixes the base material 110, and the base material 110 can be tilted in the θX direction or rotated in the θY direction by a driving unit (not shown). The spatial filter 205 is double-bended in the −X axis direction and the Y axis direction. An electromagnet coil 203 is wound around the spatial filter 205, and an ion scan coil 204 is wound near a portion communicating with the film forming chamber 206.

The amorphous carbon film 130 can be formed by using a carbon raw material such as a graphite target as the raw material target. Moreover, by using a target such as a graphite sintered body containing a metal as the raw material target, the metal-doped amorphous carbon film 130 can be formed. For example, the titanium-doped amorphous carbon film 130 can be formed by using TiC as a raw material target. The metal to be doped is not limited to Ti, but Na, K, Ca, B, Mg, Cu, Sr, Ba, Zn, Hf, Al, Zr, Fe, Co, Ni, V, Cr, Mo, W, Mn, Re, Ag, Au, Pt, Nb, Ta, or an alloy of two or more of these metals can be used. The material is not limited to metal, and a semiconductor material such as Si or H, N, F, or the like may be doped.

When the amorphous carbon film 130 is a titanium-doped amorphous carbon film containing titanium atoms, the ratio of the number of titanium atoms to the number of carbon atoms in the film may be about 2 to 30 atom %. When the ratio of the number of titanium atoms to the number of carbon atoms is α (atomic %), α is represented by the following formula (1).
α (atomic %)=(the number of Ti atoms)/{(the number of sp ² −C atoms)+(the number of sp ³ −C atoms)}×100 (1)
[In the formula (1), (the number of Ti atoms) represents the number of Ti atoms occupied in the amorphous carbon film, (the number of sp ² -C atoms) represents the number of carbon atoms of the sp ² hybrid orbital occupied in the amorphous carbon film, The number of sp ³ -C atoms) represents the number of carbon atoms in the sp ³ hybrid orbital occupied in the amorphous carbon film. ]

In order to form the amorphous carbon film 130 or the titanium-doped amorphous carbon film 130 by the FCVA method, first, a DC voltage is applied to the target 202 in the arc plasma generation chamber 201 to cause arc discharge to generate arc plasma. ..

Neutral particles, C ⁺ ions, Ti ⁺ ions, Ti ²⁺ ions, Ti ³⁺ ions, Ti ⁴⁺ ions, and other ions in the generated arc plasma are transported to the spatial filter 205 and pass through the spatial filter 205. Then, the neutral particles are trapped by the electromagnet coil 203, and only C ⁺ ions, Ti ⁺ ions, Ti ²⁺ ions, Ti ³⁺ ions, Ti ⁴⁺ ions and other ions are introduced into the film forming chamber 206.

At this time, the ion scan coil 204 can move the flight direction of the ion flow in any direction. A negative bias voltage is applied to the conductive film 120 in the film forming chamber 206. C ⁺ ions, Ti ⁺ ions, Ti ²⁺ ions, Ti ³⁺ ions, Ti ⁴⁺ ions and other ions ionized by the arc discharge are accelerated by the bias voltage and deposited as a dense film on the substrate 110.

The amorphous carbon film 130 thus formed is a solid film composed of carbon atoms and is roughly classified into carbon atoms having sp ² hybrid orbitals and carbon atoms having sp ³ hybrid orbitals.

In the FCVA method, the content of carbon atoms of sp ² hybrid orbitals and sp ³ hybrid orbitals in the amorphous carbon film 130 or the titanium-doped amorphous carbon film 130 is controlled by adjusting the bias voltage during film formation. can do.

For example, by adjusting the bias voltage, the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital in the amorphous carbon film is 15 to 85 atoms. It can be %. Further, for example, by adjusting the bias voltage, the number of carbon atoms in the sp ² hybrid orbital, the number of carbon atoms in the sp ³ hybrid orbital, and the carbon in the sp ³ hybrid orbital with respect to the total number of titanium atoms in the titanium-doped amorphous carbon film are adjusted. The ratio of the number of atoms can be 40 atomic% or less. When the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital in the amorphous carbon film is β (atomic %), β is represented by the following formula. It is represented by (2).
β (atomic %)=(number of sp ³ −C atoms)/{(number of sp ² −C atoms)+(number of sp ³ −C atoms)}×100 (2)
[In Formula (2), (the number of sp ² -C atoms) represents the number of carbon atoms of the sp ² hybrid orbital occupied in the amorphous carbon film, and (the number of sp ³ -C atoms) is the sp ³ hybrid orbital occupied in the amorphous carbon film] Represents the number of carbon atoms of. ]

Further, the number of carbon atoms in sp ² hybrid orbitals, the number of carbon atoms in sp ³ hybrid orbitals, and the ratio of the number of carbon atoms in sp ³ hybrid orbitals to the total number of titanium atoms in the titanium-doped amorphous carbon film are expressed as γ (atomic %). Then, γ is represented by the following equation (3).
γ (atomic %)=(number of sp ³ -C atoms)/{(number of sp ² -C atoms)+(number of sp ³ -C atoms)+(number of Ti atoms)}×100 (3)
[In the formula (3), (the number of sp ² -C atoms) represents the number of carbon atoms of the sp ² hybrid orbital occupied in the amorphous carbon film, and (the number of sp ³ -C atoms) is the sp ³ hybrid orbital occupied in the amorphous carbon film] Represents the number of carbon atoms, and (the number of Ti atoms) represents the number of Ti atoms in the amorphous carbon film. ]

In the FCVA method, only C ⁺ ions, Ti ⁺ ions, Ti ²⁺ ions, Ti ³⁺ ions, Ti ⁴⁺ ions, and other ions having uniform flight energies are introduced into the film forming chamber 206 and applied to the conductive film 120. By controlling the bias voltage, it is possible to control the ion bombardment energy of various ion particles incident on the substrate 110. Therefore, it is possible to uniformly form a film even on the substrate 110 having a complicated shape.

Functional groups such as a hydroxyl group and a carboxyl group may be formed on the surface of the amorphous carbon film 130. The amorphous carbon film 130 formed by the FCVA method or the like has a pure water contact angle of about 50° measured by the droplet method, regardless of the ratio of the number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital. That is all.

By forming a functional group such as a hydroxyl group or a carboxyl group on the surface of the amorphous carbon film 130, the contact angle of pure water can be reduced. The degree of decrease in the contact angle of pure water varies depending on the amount of functional groups formed, and can be adjusted to, for example, 10° or less, for example 5° or less, for example 4° or less.

The method for forming the functional group on the amorphous carbon film 130 is not particularly limited, and it can be performed by irradiating the amorphous carbon film 130 with light including ultraviolet rays, for example. The wavelength of the ultraviolet ray and the irradiation amount of the ultraviolet ray can be appropriately adjusted, and examples thereof include a condition of irradiating the light containing the ultraviolet ray having a wavelength of 185 nm for about 20 minutes. At this time, for example, the irradiated light may include ultraviolet rays having a wavelength of 254 nm or the like.

The same applies when the amorphous carbon film 130 is a titanium-doped amorphous carbon film, and a functional group such as a hydroxyl group or a carboxyl group may be formed.

In the titanium-doped amorphous carbon film 130 formed by the FCVA method or the like, the contact angle of pure water measured by the droplet method is approximately 60° or more when the ratio of the number of titanium atoms to the number of carbon atoms is 2 atomic% or more. ..

By forming a functional group such as a hydroxyl group or a carboxyl group on the surface of the titanium-doped amorphous carbon film 130, the contact angle of pure water can be reduced. The degree of decrease in the contact angle of pure water varies depending on the amount of functional groups formed, and can be adjusted to, for example, 10° or less, for example 5° or less, for example 4° or less.

The method for forming the functional group on the titanium-doped amorphous carbon film 130 is not particularly limited, and it can be performed, for example, by irradiating the titanium-doped amorphous carbon film 130 with light including ultraviolet rays. The wavelength of the ultraviolet ray and the irradiation amount of the ultraviolet ray can be appropriately adjusted, and examples thereof include a condition of irradiating the light containing the ultraviolet ray having a wavelength of 185 nm for about 20 minutes.

《Middle layer》
In the amorphous carbon film 130 is deposited, for example, when the ratio of the number of carbon atoms of the sp ³ hybrid orbital to the total number of carbon atoms of the carbon atoms and sp ³ hybrid orbital of sp ² hybrid orbital is 50 to 85 atomic%, The amorphous carbon film 130 has high film stress and tends to peel off. In such a case, an intermediate layer having a low film stress may be formed between the amorphous carbon film 130 and the conductive film 120 or between the amorphous carbon film 130 and the base material 110. Thereby, the adhesion of the amorphous carbon film 130 is improved and peeling can be suppressed. A suitable thickness of the intermediate layer is about 30-40 nm.

An amorphous carbon film having a low film stress can be used as the intermediate layer. For example, an amorphous carbon film in which the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital is 15 atomic% or more and less than 50 atomic% is Since it is low, it is suitable as an intermediate layer.

A titanium-doped amorphous carbon film is also suitable as the intermediate layer. By containing titanium, the film stress tends to be smaller. Therefore, by using the titanium-doped amorphous carbon film, further improvement in adhesion can be expected.

[Method of manufacturing container]
The container manufacturing method of the present embodiment is the above-described container manufacturing method, and includes a step of forming the conductive film 120 on at least a part of the base material, and a voltage is applied to the conductive film 120 to perform physical vapor deposition. Forming the amorphous carbon film 130 by a chemical vapor deposition method or a chemical vapor deposition method.

The physical vapor deposition method for forming the amorphous carbon film 130 may be the FCVA method. That is, the manufacturing method of the present embodiment can be suitably implemented by the FCVA device described above. When the base material 110 is insulative, a bias voltage cannot be applied, and thus it is difficult to form the amorphous carbon film 130 by the FCVA method. However, according to the manufacturing method of this embodiment, the amorphous carbon film can be preferably formed by the FCVA method even if the base material 110 has an insulating property.

As a result, for example, when evaluating the influence of the film composition of the amorphous carbon film on cell adhesion and protein adsorption, it is possible to manufacture a container that can be evaluated more easily with higher measurement accuracy.

In the manufacturing method of the present embodiment, the position where the amorphous carbon film 130 is formed differs depending on the arrangement of the conductive film 120.

For example, as shown in FIG. 1A, when the base material 110, the conductive film 120, and the amorphous carbon film 130 are formed in this order, a bias voltage is applied to the conductive film 120 to form the amorphous carbon film. When the film 130 is formed, the amorphous carbon film 130 is formed on at least a part of the conductive film 120.

Alternatively, for example, as shown in FIG. 1B, when the conductive film 120, the base material 110, and the amorphous carbon film 130 are stacked in this order, a bias voltage is applied to the conductive film 120 to make the amorphous film. When the carbon film 130 is formed, the amorphous carbon film 130 is formed on at least a part of the base material 110.

[Amorphous carbon film evaluation method]
The method for evaluating an amorphous carbon film according to the present embodiment includes a step of accommodating a sample in the container described above and bringing the sample into contact with the amorphous carbon film, and a step of evaluating the interaction between the sample and the amorphous carbon film. And

Here, the sample is a substance that is a target for evaluating the interaction with the amorphous carbon film. Examples of the sample include biological samples such as proteins and cells.

The interaction between the sample and the amorphous carbon film can be evaluated by an evaluation method appropriately selected according to the intended evaluation content. For example, in the case of evaluating the cell adhesiveness to the amorphous carbon film, it can be evaluated by bringing the cells into contact with the amorphous carbon film and then measuring the number of cells adhered to the amorphous carbon film.

Alternatively, in the case of evaluating the protein adsorption on the amorphous carbon film, it can be evaluated by bringing the protein into contact with the amorphous carbon film and then measuring the amount of the protein adhered to the amorphous carbon film.

As will be described later in Examples, the evaluation method of the present embodiment makes it possible to evaluate the influence of the film composition of the amorphous carbon film on cell adhesion and protein adsorption with higher measurement accuracy and more easily.

Next, the present embodiment will be described with reference to examples, but the present invention is not limited to the following examples.

[Experimental Example 1]
(Manufacture of containers)
A container was manufactured by processing a 24-well plate for cell culture made of polystyrene. First, titanium was formed into a film on the surface of a 24-well plate to give conductivity. The thickness of the titanium film was 100 nm. As a result, although polystyrene has an insulating property, it is possible to apply a voltage using a conductive titanium film.

Subsequently, a bias voltage was applied to the titanium film, and an amorphous carbon film was formed on the titanium film by the FCVA method. Next, the bias voltage was changed to form a plurality of amorphous carbon films. Then, an amorphous carbon film was obtained in which the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital was 50 to 85 atom %.

The ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital in the amorphous carbon film can be controlled by adjusting the bias voltage during film formation, Moreover, it was confirmed by this experiment that the amorphous carbon film can be formed on the insulating container. As described in the embodiment, by using the container in which the amorphous carbon film is formed, it is possible to more easily evaluate the influence of the film composition on cell adhesion and protein adsorption.

[Experimental Example 2]
(Study of film composition of amorphous carbon film 1)
An amorphous carbon film was formed on the surface of a 24-well plate as in Experimental Example 1, and the influence of the position on the 24-well plate on the uniformity of the film composition was examined.

First, a polystyrene base material cut into about 1 cm×1 cm was fixed to the well bottom of a 24-well plate similar to that of Experimental Example 1 with a conductive tape. Subsequently, a titanium film was formed on this base material. The thickness of the titanium film was 100 nm.

Then, an amorphous carbon film was formed on the formed titanium film by the FCVA method. The conditions of the FCVA device were that the arc current value was set to 40A and the substrate bias was set to -66V.

Subsequently, by X-ray photoelectron spectroscopy (XPS) measurement, of the formed amorphous carbon film surface, sp ² carbon atoms hybrid orbitals and sp ³ sp ³ carbon atoms hybridized to the total number of carbon atoms of the hybrid orbitals Was measured. As a result, it was 85 atom% at the edge of the 24-well plate (position of well D1) and was 83 atom% near the center of the 24-well plate (position of well B4). Further, the uniformity calculated by the following formula (4) was calculated to be 1.2%.
Uniformity (%)=(maximum film thickness-minimum film thickness)/(maximum film thickness+minimum film thickness)×100 (4)

From this result, it has been clarified that when the amorphous carbon is formed by the above method, the amorphous carbon film having high uniformity can be obtained regardless of the position on the 24-well plate.

[Experimental Example 3]
(Examination of film composition of amorphous carbon film 2)
An amorphous carbon film was formed by the FCVA method on the titanium film formed on the 24-well plate in the same manner as in Experimental Example 2. The conditions of the FCVA device were that the arc current value was 40 A and the substrate bias was -1980 V.

Subsequently, the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the number of carbon atoms in the sp ³ hybrid orbital on the surface of the formed amorphous carbon film was measured by XPS measurement. As a result, it was 57 atom% at the edge of the 24-well plate (position of well A1) and 57 atom% near the center of the 24-well plate (position of well B4). Further, the uniformity calculated by the above formula (4) was calculated to be 0%.

This result further supports that when the amorphous carbon is formed by the above method, an amorphous carbon film having high uniformity can be obtained regardless of the position on the 24-well plate.

[Experimental Example 4]
(Study of film composition of titanium-doped amorphous carbon film 1)
A titanium-doped amorphous carbon film was formed on the titanium film formed on the 24-well plate in the same manner as in Experimental Example 2 by the FCVA method using a carbon target containing 4.0 atom% of titanium as a raw material. As the conditions of the FCVA device, the arc current value was set to 60 A and the substrate bias was set to 0V.

Subsequently, the ratio of the number of titanium atoms to the number of carbon atoms on the surface of the titanium-doped amorphous carbon film was measured by Rutherford backscattering spectroscopy (RBS) measurement.

As a result, the ratio of the number of titanium atoms to the number of carbon atoms was 23.9 to 26.6 atom% at the edge of the 24-well plate (positions of wells A1, A6, D1, and D6), and the center of the 24-well plate was In the vicinity (the positions of wells B4, B5, C2, and C4), it was 23.9 to 25.3 atom %. Further, the uniformity calculated by the following formula (5) was calculated to be 5.3%.
Uniformity (%)=(maximum value of the ratio of the number of titanium atoms−minimum value of the ratio of the number of titanium atoms)/(maximum value of the ratio of titanium atom+minimum value of the ratio of titanium atom)×100 (5 )

From this result, it was clarified that when the titanium-doped amorphous carbon film was formed by the above method, the titanium-doped amorphous carbon film with high uniformity was obtained regardless of the position on the 24-well plate.

[Experimental Example 5]
(Study of film composition of titanium-doped amorphous carbon film 2)
A titanium-doped amorphous carbon film was formed on the titanium film formed on the 24-well plate in the same manner as in Experimental Example 2 in the same manner as in Experimental Example 4. Regarding the conditions of the FCVA device, the arc current value was set to 60 A and the substrate bias was set to -1980V.

Subsequently, the ratio of the number of titanium atoms to the number of carbon atoms on the surface of the titanium-doped amorphous carbon film was measured by RBS measurement.

As a result, the ratio of the number of titanium atoms to the number of carbon atoms was 23.8 to 26.4 atom% at the edge of the 24-well plate (positions of wells A1, A6, D1 and D6), and the center of the 24-well plate was In the vicinity (the position of well C4), it was 27.1 atom %. Further, the uniformity calculated by the above formula (5) was calculated to be 6.5%.

This result further supports that when the titanium-doped amorphous carbon film is formed by the above method, a titanium-doped amorphous carbon film having high uniformity can be obtained regardless of the position on the 24-well plate.

[Experimental Example 6]
(Cell adhesion test)
A cell adhesion test was performed using a container in which an amorphous carbon film was formed on the inner surface of the well of a 24-well plate as a sample. Human normal umbilical vein endothelial cells (HUVEC) were used as cells. Further, as a control material, there was used a material in which an amorphous carbon film was formed on a flat plate base material (1 cm×1 cm, whole surface mirror-polished) made of stainless steel (SUS316L).

As the amorphous carbon film, seven kinds of films shown in Table 1 below were used. In Table 1, "Ti/C (at %)" represents the ratio (atomic %) of the number of titanium atoms to the number of carbon atoms. In the case of amorphous carbon, “sp ² —C (at %)” means the ratio of the number of carbon atoms in the sp ² hybrid orbital to the total number of carbon atoms in the sp ² hybrid orbital and the sp ³ hybrid orbital (atomic% In the case of titanium-doped amorphous carbon, the number of carbon atoms in the sp ² hybrid orbital, the number of carbon atoms in the sp ³ hybrid orbital, and the ratio of the number of carbon atoms in the sp ² hybrid orbital to the total number of titanium atoms (atomic %) Represents. ^"Sp 3 -C (at%)" in the case of amorphous carbon, sp ² carbon atoms hybrid orbitals and sp ³ ratio of the number of carbon atoms of the sp ³ hybrid orbital to the total number of carbon atoms of the hybrid orbitals (atomic% In the case of titanium-doped amorphous carbon, the number of carbon atoms in the sp ² hybrid orbital, the number of carbon atoms in the sp ³ hybrid orbital, and the ratio of the number of carbon atoms in the sp ³ hybrid orbital to the total number of titanium atoms (atomic %) Represents.

<Cell adhesion test using a container>
HUVECs were seeded at 5×10 ⁴ cells/well in the wells of each sample container, and cultured at 37° C. in an environment of 5% CO ₂ for 24±2 hours. Subsequently, the cells in each well were fixed, the nuclei were stained, and observed with a fluorescence microscope to measure the cell adhesion rate.

<Cell adhesion test using flat plate substrate>
Also, each plate material as a control material was placed in the well of the cell culture plate, HUVECs were seeded at 5×10 ⁴ cells/well, and cultured at 37° C. for 24±2 hours. Subsequently, the cells in each well were fixed, the nuclei were stained, and observed with a fluorescence microscope to measure the cell adhesion rate.

"result"
FIG. 3( a) is a graph showing the results of measuring the cell adhesion rate for each sample and the control material. The graph is shown as a relative value with the cell adhesion rate when HUVECs were seeded on a culture plate on which amorphous carbon was not formed as a film, which was taken as 100%. Further, FIG. 3B is a graph showing the result of extracting only the standard deviation of the result of FIG.

As a result, it was confirmed that the cell adhesion test can be carried out without problems even when the container in which the amorphous carbon film is formed is used.

[Experiment 7]
(Albumin adsorption test)
A protein adsorption test was performed using a container in which an amorphous carbon film was formed on the inner surface of the well of a 24-well plate as a sample. Albumin was used as the protein. Further, as a control material, a material in which an amorphous carbon film was formed on a flat plate base material made of stainless steel (SUS316L) was used. As the amorphous carbon film, seven kinds of films similar to those used in Experimental Example 6 were used.

<<Albumin adsorption test using a container>>
A phosphate buffer (PBS) solution containing albumin (30 mg/mL) was placed in the well of the container of each sample, and left in a CO ₂ incubator (37°C, 5% CO ₂ ) for 24 hours. Subsequently, the PBS solution was removed, and the unadsorbed albumin was removed by washing with pure water (500 μL). Subsequently, a surfactant solution (PBS containing 2% Triton-X) was added and shaken at 37° C. for 30 minutes to remove albumin adsorbed on the amorphous carbon film. Subsequently, the surfactant solution was recovered, and the amount of adsorbed albumin was quantified by the BCA method.

<< Albumin adsorption test using a flat plate substrate >>
Also, each plate material as a control material was placed in the well of the cell culture plate, a PBS solution containing albumin (30 mg/mL) was placed therein, and the plate was placed in a CO ₂ incubator (37° C., 5% CO ₂ ) for 24 hours. I put it. Subsequently, the PBS solution was removed, and the unadsorbed albumin was removed by washing with pure water (500 μL). Then, each plate substrate was transferred to a well of a new cell culture well plate and washed again with pure water to remove unadsorbed albumin.

Subsequently, a surfactant solution (PBS containing 2% Triton-X) was added and shaken at 37° C. for 30 minutes to remove albumin adsorbed on the amorphous carbon film. Subsequently, the surfactant solution was recovered, and the amount of adsorbed albumin was quantified by the BCA method.

"result"
FIG. 4A is a graph showing the results of measuring the amount of albumin adsorbed on each sample and the control material. Further, FIG. 4B is a graph showing the result of extracting only the standard deviation of the result of FIG.

As a result, it was confirmed that the albumin adsorption test can be carried out without problems even when the container in which the amorphous carbon is formed is used. Further, it was revealed that the standard deviation of the measured albumin adsorption amount was remarkably reduced by using the container in which the amorphous carbon film was formed, as compared with the control material. This result further supports that by using the container in which the amorphous carbon film is formed, it is possible to obtain the experimental result with less variation and improved measurement accuracy.

[Experimental Example 8]
(Fibrinogen adsorption test)
As a protein, a container in which an amorphous carbon film was formed on the inner surface of a well of a 24-well plate was used as a sample in the same manner as in Experimental Example 7 except that fibrinogen at a concentration of 3 mg/mL was used instead of albumin. An adsorption test was conducted. Further, as a control material, a material in which an amorphous carbon film was formed on a flat plate base material made of stainless steel (SUS316L) was used. As the amorphous carbon film, seven kinds of films similar to those used in Experimental Example 6 were used.

FIG. 5( a) is a graph showing the results of measuring the amount of fibrinogen adsorbed on each sample and the control material. Further, FIG. 4B is a graph showing the result of extracting only the standard deviation of the result of FIG.

As a result, it was confirmed that the fibrinogen adsorption test can be performed without any problem even when the container in which the amorphous carbon film is formed is used. Further, it was observed that the standard deviation of the measured fibrinogen adsorption amount tended to be smaller than that of the control material by using the container in which the amorphous carbon film was formed. This result further supports that by using the container in which the amorphous carbon film is formed, it is possible to obtain the experimental result with less variation and improved measurement accuracy.

100... Container, 110... Base material, 120... Conductive film, 130... Amorphous carbon film, 200... FCVA device, 201... Arc plasma generating chamber, 202... Raw material target, 203... Electromagnetic coil, 204... Ion scan coil, 205 ... Spatial filter, 206... Film forming chamber, 207... Base material holder.

Claims (16)

A container,
A base material having at least one storage portion for storing a sample;
A conductive film formed on at least a part of the base material,
At least a part of the conductive film, or an amorphous carbon film formed on at least a part of the substrate,
A container in which the contained sample can contact the amorphous carbon film. The container according to claim 1, wherein the base material is made of an insulating material. The container according to claim 1, wherein the base material is made of a resin material. The container according to claim 1, wherein the sample is a biological sample. The container according to claim 1, wherein the base material, the conductive film, and the amorphous carbon film are laminated in this order. The container according to claim 5, wherein the amorphous carbon film is formed in a region including at least a part of a bottom portion of the housing portion. The container according to claim 1, wherein the conductive film, the base material, and the amorphous carbon film are laminated in this order. The container according to claim 7, wherein the amorphous carbon film is formed in a region including at least a part of a bottom portion of the housing portion. The amorphous carbon film, the ratio of the number of carbon atoms of the sp ³ hybrid orbital to the total number of carbon atoms of the carbon atoms and sp ³ hybrid orbital of sp ² hybrid orbital is 15 to 85 atomic%, claims 1-8 The container according to any one of 1. The amorphous carbon film is a titanium-doped amorphous carbon film containing titanium atoms,
In the titanium-doped amorphous carbon film, the ratio of the number of titanium atoms to the number of carbon atoms is 2 to 30 atom %,
The container according to any one of claims 1 to 9. In the titanium-doped amorphous carbon film, the ratio of the number of carbon atoms in sp ² hybrid orbitals, the number of carbon atoms in sp ³ hybrid orbitals, and the number of carbon atoms in sp ³ hybrid orbitals to the total number of titanium atoms is 40 atomic% or less. The container according to claim 10. A method of manufacturing a container according to any one of claims 1 to 11,
Forming the conductive film on at least a portion of the substrate,
Applying a voltage to the conductive film, a step of forming the amorphous carbon film by physical vapor deposition or chemical vapor deposition,
A method of manufacturing a container, comprising: The method for producing a container according to claim 12, wherein the physical vapor deposition method is a filtered cathodic vacuum arc method. The manufacturing method according to claim 12, wherein in the step of forming the amorphous carbon film, the amorphous carbon film is formed on at least a part of the conductive film. The manufacturing method according to claim 12 or 13, wherein in the step of forming the amorphous carbon film, the amorphous carbon film is formed on at least a part of the base material. Storing a sample in the container according to any one of claims 1 to 11 and bringing the sample into contact with the amorphous carbon film;
Evaluating the interaction between the sample and the amorphous carbon film,
A method for evaluating the amorphous carbon film, comprising:

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