WO2018193991A1

WO2018193991A1 - Container, method for producing container, and method for evaluating amorphous carbon film

Info

Publication number: WO2018193991A1
Application number: PCT/JP2018/015568
Authority: WO
Inventors: 佑来高橋; 真樹森山
Original assignee: 株式会社ニコン
Priority date: 2017-04-21
Filing date: 2018-04-13
Publication date: 2018-10-25
Also published as: JP2020105535A

Abstract

A container which comprises: a base material that has one or more containing parts, in each of which a sample is to be contained; a conductive film that is formed on at least a part of the base material; and an amorphous carbon film that is formed on at least a part of the conductive film or on at least a part of the base material. At least a part of the amorphous carbon film is exposed to the inside of the containing parts.

Description

Container, container manufacturing method, and amorphous carbon film evaluation method

The present invention relates to a container, a method for manufacturing the container, and a method for evaluating an amorphous carbon film.
This application claims priority based on Japanese Patent Application No. 2017-084213 filed in Japan on April 21, 2017, the contents of which are incorporated herein by reference.

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

Conventionally, when evaluating the influence of the composition of an amorphous carbon film on cell adhesion or protein adsorption, an amorphous carbon film having a different composition is formed on a flat substrate (substrate), and the flat substrate is used as a predetermined container. It was common to house and evaluate. However, in this case, a process such as processing the base material so as to be accommodated in the container is necessary, and the test process is complicated.

JP-A-8-12492

A container according to an 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 An amorphous carbon film formed on at least a part of the substrate, and the accommodated sample can contact the amorphous carbon film.

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

An evaluation method of an amorphous carbon film according to an embodiment includes a step of accommodating a sample in the container described above and bringing the sample and the amorphous carbon film into contact with each other, and evaluating an interaction between the sample and the amorphous carbon film. 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. FIG. (B) is a graph which shows the result of having taken out 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. FIG. (B) is a graph which shows the result of having taken out only the standard deviation of the result of (a). (A) is a graph which shows the result of having measured the amount of fibrinogen adsorption in Experimental Example 8. (B) is a graph which shows the result of having taken out 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 as the case may be. In the drawings, the same or corresponding parts are denoted by the same or corresponding reference numerals, and redundant description is omitted. Note that the dimensional ratio in each drawing is exaggerated for the sake of explanation, and does not necessarily match the actual dimensional ratio.

[container]
The container of the present embodiment includes a base material having one or more storage portions 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. In other words, at least a part of the amorphous carbon film is exposed in the housing portion.

If the container of this embodiment is used, since it is not necessary to prepare a flat base material etc. separately from a container, the test process in the case of evaluating the influence which a film composition has on cell adhesion and protein adsorptivity is simple. Further, as will be described later in the examples, by using the container of the present embodiment, for example, when evaluating the influence of the film composition of the amorphous carbon film on cell adhesion and protein adsorption, with higher measurement accuracy, It can be easily evaluated. Moreover, if the container of this embodiment is used, the cost of the cutting | disconnection of a base material, grinding | polishing, etc. which were conventionally required can be reduced. Note that the amorphous carbon film in this specification refers to a carbon film containing both carbon atoms of sp ² hybrid orbitals and carbon atoms of sp ³ hybrid orbitals. Regardless of the content of carbon atoms in sp ² hybrid orbitals and carbon atoms in sp ³ hybrid orbitals, an amorphous carbon film is referred to as containing both carbon atoms. In the present specification, when the amorphous carbon film contains titanium atoms, it is called a titanium-doped amorphous carbon film.

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

The shape of the housing portion is not particularly limited, and examples thereof include a concave shape. For example, the bottom of the housing part may be planar or U-shaped. More specifically, for example, shapes such as petri dishes and well plates used in biological experiments and the like can be mentioned. 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 substrate 110 is made of a resin material, glass, or the like, and may be insulating. In addition, the insulating property in this specification indicates that the volume resistivity is 10 ¹¹ (Ω · cm) or more.

As will be described later, in order to form an amorphous carbon film on the substrate 110, it is preferable to apply a bias voltage to the substrate. However, when the substrate 110 is insulative, a bias voltage cannot be applied. In such a case, the conductive film 120 may be formed on at least a part of the substrate 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 film thickness of the conductive film may be about 50 to 100 nm. In this specification, the term “conductivity” means that the volume resistivity is approximately 10 ⁻⁴ (Ω · cm) or less.

FIGS. 1A and 1B are schematic views for explaining the structure of a container according to an embodiment. As shown in FIG. 1A, the container 100 is formed on a base material 110 having an accommodating portion, 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. The amorphous carbon film 130 is provided. Moreover, each accommodating part is 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 disposed at a position where the sample accommodated in the accommodating portion comes into contact with 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 of the housing portion. In this specification, the bottom part of a storage part means the area | region where a sample contacts, when a sample is stored in a storage part among storage parts. 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 substrate 110, a conductive film 120, and an amorphous carbon film 130 are laminated in this order. However, the order of arrangement of the base 110, the conductive film 120, and the amorphous carbon film 130 is not limited to this, and for example, as shown in FIG. 1B, the conductive film 120, the base 110, The amorphous carbon film 130 may be laminated in this order.

The conductive film 120 only needs to be disposed at a position where a bias voltage can be applied to the substrate 110. For this reason, as shown to Fig.1 (a), the electroconductive film 120 may be arrange | positioned on the surface on the base material 110 in which the amorphous carbon film 130 is formed. Alternatively, as shown in FIG. 1B, the conductive film 120 may be disposed on the surface of the base 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 substrate 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. Can do. Examples of the PVD method include an ion beam deposition method, an ion beam sputtering method, a magnetron sputtering method, a laser deposition 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.

Above all, the FCVA method is preferable because it is a film forming method capable of uniformly coating with high adhesive force even at room temperature. The FCVA method is a film forming method in which ionized particles are generated by causing arc discharge to a raw material target, and only the particles are guided on 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, an arc plasma generation chamber 201 in which a raw material target 202 is installed and a film formation chamber 206 are connected by a spatial filter 205.

The film forming chamber 206 includes a substrate holder 207 therein. The base material holder 207 fixes the base material 110, and can tilt the base material 110 in the θX direction or rotate it in the θY direction by a driving means (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 the communication portion 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 a raw material target. Moreover, the amorphous carbon film 130 doped with metal can be formed by using a target such as a graphite sintered body containing metal as a raw material target. For example, the titanium-doped amorphous carbon film 130 can be formed by using TiC as a raw material target. The doped metal 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. Moreover, it is not restricted to a metal, You may dope semiconductor materials, such as Si, H, N, F, etc.

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 atomic%. In addition, 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%) = (Ti atom number) / {(sp ² −C atom number) + (sp ³ −C atom number)} × 100 (1)
[In Formula (1), (Ti atom number) represents the number of Ti atoms in the amorphous carbon film, and (sp ² -C atom number) represents the number of carbon atoms in the sp ² hybrid orbital in the amorphous carbon film. (sp ³ -C atom number) represents the number of carbon atoms in the sp ³ hybrid orbital 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, an arc discharge is generated by applying a DC voltage to the target 202 in the arc plasma generation chamber 201 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. Thus, 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 guided into the deposition chamber 206.

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

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 in the sp ² hybrid orbital and sp ³ hybrid orbit in the amorphous carbon film 130 and the titanium-doped amorphous carbon film 130 is controlled by adjusting the bias voltage at the time of 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. %. Further, for example, by adjusting the bias voltage, the number of carbon atoms of the sp ² hybrid orbital of titanium-doped amorphous carbon film, sp ³ carbon atoms hybrid orbitals, and the sp ³ hybrid orbital to the total number of titanium atoms of carbon 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 orbit in the amorphous carbon film is β (atomic%), β is 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), (sp ² -C atom number) represents the carbon atom number of sp ² hybrid orbital in the amorphous carbon film, and (sp ³ -C atom number) represents the sp ³ hybrid orbital in the amorphous carbon film. Represents the number of carbon atoms. ]

Further, the number of carbon atoms of sp ² hybrid orbital of titanium-doped amorphous carbon film, sp ³ carbon atoms hybrid orbitals, and the sp ³ hybrid orbital to the total number of titanium atoms to the percentage of the number of carbon atoms gamma (atomic%) Then, γ is expressed by the following formula (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), (sp ² -C atom number) represents the carbon atom number of sp ² hybrid orbital in the amorphous carbon film, and (sp ³ -C atom number) represents the sp ³ hybrid orbital in the amorphous carbon film. (Ti atom number) 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 with uniform flight energy are introduced into the deposition chamber 206 and applied to the conductive film 120. By controlling the bias voltage, the ion bombardment energy of various ion particles incident on the substrate 110 can be controlled. Therefore, it is possible to form a uniform film even on the substrate 110 having a complicated shape.

A functional group such as a hydroxyl group or 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 measured by the droplet method of about 50 ° regardless 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's it.

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 the functional group to be 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 can be performed, for example, by irradiating the amorphous carbon film 130 with light including ultraviolet rays. The wavelength of ultraviolet light and the amount of ultraviolet light irradiation can be adjusted as appropriate, and examples include conditions of irradiating light containing ultraviolet light 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 to the case where 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 atom% 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 the functional group to be 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 can be performed, for example, by irradiating the titanium-doped amorphous carbon film 130 with light containing ultraviolet rays. The wavelength of ultraviolet light and the amount of ultraviolet light irradiation can be adjusted as appropriate, and examples include conditions of irradiating light containing ultraviolet light 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 a 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 substrate 110. Thereby, the adhesiveness of the amorphous carbon film 130 is improved, and peeling can be suppressed. The thickness of the intermediate layer is suitably about 30 to 40 nm.

As the intermediate layer, an amorphous carbon film having a low film stress can be used. For example, the 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 orbit is 15 atomic% or more and less than 50 atomic% is a film stress. 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, further improvement in adhesion can be expected by using a titanium-doped amorphous carbon film.

[Manufacturing method of container]
The manufacturing method of the container of this embodiment is the manufacturing method of the container mentioned above, the step of forming the conductive film 120 on at least a part of the base material, the voltage is applied to the conductive film 120, and the physical vapor deposition is performed. Forming an 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 an FCVA method. In other words, the manufacturing method of the present embodiment can be preferably implemented by the FCVA apparatus described above. When the substrate 110 is insulative, it is difficult to form the amorphous carbon film 130 by the FCVA method because a bias voltage cannot be applied. However, with the manufacturing method of the present embodiment, an amorphous carbon film can be suitably formed by the FCVA method even if the base material 110 has insulating properties.

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

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 a substrate 110, a conductive film 120, and an amorphous carbon film 130 are formed in this order, a bias voltage is applied to the conductive film 120 to form amorphous carbon. 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 a conductive film 120, a substrate 110, and an amorphous carbon film 130 are laminated in this order, a bias voltage is applied to the conductive film 120 to form an amorphous film. When the carbon film 130 is formed, the amorphous carbon film 130 is formed on at least a part of the substrate 110. In other words, the amorphous carbon film is formed on the other surface side of the substrate.

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

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

Evaluation of the interaction between the sample and the amorphous carbon film can be performed by an evaluation method appropriately selected according to the target evaluation content. For example, when cell adhesion to an amorphous carbon film is evaluated, the cell can be contacted with the amorphous carbon film, and then the number of cells adhered to the amorphous carbon film can be measured.

Alternatively, when evaluating protein adsorption to the amorphous carbon film, the protein can be brought into contact with the amorphous carbon film, and then the amount of protein adhered to the amorphous carbon film can be measured.

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

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 24-well plate for cell culture made of polystyrene was processed to produce a container. First, titanium was deposited on the surface of a 24-well plate to impart conductivity. The thickness of the titanium film was 100 nm. As a result, although polystyrene is insulative, a voltage can be applied 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, a plurality of amorphous carbon films were formed by changing the bias voltage. Thus, 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 atomic%.

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 orbit in the amorphous carbon film can be controlled by adjusting the bias voltage during film formation, In addition, this experiment confirmed that an amorphous carbon film can be formed on an insulating container. As described in the embodiment, by using a container in which an amorphous carbon film is formed, the influence of the film composition on cell adhesion and protein adsorption can be more easily evaluated.

[Experiment 2]
(Examination of film composition of amorphous carbon film 1)
An amorphous carbon film was formed on the surface of a 24-well plate similar to 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 to about 1 cm × 1 cm was fixed to the well bottom of a 24-well plate similar to Experimental Example 1 with a conductive tape. Subsequently, a titanium film was formed on this substrate. The thickness of the titanium film was 100 nm.

Subsequently, an amorphous carbon film was formed on the formed titanium film by the FCVA method. The conditions of the FCVA apparatus were an arc current value of 40 A and a base material bias of −66 V.

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 The proportion of was measured. As a result, it was 85 atomic% at the end of the 24-well plate (the position of the well D1) and 83 atomic% near the center of the 24-well plate (the position of the well B4). Moreover, the uniformity calculated by the following formula (4) was calculated to be 1.2%.
Uniformity (%) = (maximum value of film thickness−minimum value of film thickness) / (maximum value of film thickness + minimum value of film thickness) × 100 (4)

From this result, it has been clarified that when 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.

[Experiment 3]
(Examination of film composition of amorphous carbon film 2)
In the same manner as in Experimental Example 2, an amorphous carbon film was formed on the titanium film formed on the 24-well plate by the FCVA method. The conditions for the FCVA apparatus were an arc current value of 40 A and a base material bias of −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 atomic% at the end of the 24-well plate (position of the well A1) and 57 atomic% near the center of the 24-well plate (position of the well B4). Further, the uniformity calculated by the above formula (4) was calculated to be 0%.

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

[Experimental Example 4]
(Examination of film composition of titanium-doped amorphous carbon film 1)
A titanium-doped amorphous carbon film was formed on a titanium film formed on a 24-well plate in the same manner as in Experimental Example 2 by the FCVA method using a carbon target containing 4.0 atomic% titanium as a raw material. The conditions of the FCVA apparatus were an arc current value of 60 A and a base material bias of 0 V.

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 is 23.9 to 26.6 atomic% at the end of the 24-well plate (positions of wells A1, A6, D1, and D6). In the vicinity (positions of wells B4, B5, C2, and C4), it was 23.9 to 25.3 atomic%. Moreover, the uniformity calculated by the following formula (5) was calculated to be 5.3%.
Uniformity (%) = (maximum value of the proportion of titanium atoms−minimum value of the proportion of titanium atoms) / (maximum value of the proportion of titanium atoms + minimum value of the proportion of titanium atoms) × 100 (5) )

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

[Experimental Example 5]
(Investigation of film composition of titanium-doped amorphous carbon film 2)
A titanium-doped amorphous carbon film was formed on a titanium film formed on a 24-well plate in the same manner as in Experimental Example 2 in the same manner as in Experimental Example 4. The conditions of the FCVA apparatus were an arc current value of 60 A and a base material bias of −1980 V.

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 is 23.8 to 26.4 atomic% at the end of the 24-well plate (positions of wells A1, A6, D1, and D6), and the center of the 24-well plate In the vicinity (position of well C4), it was 27.1 atomic%. Further, the uniformity calculated by the above formula (5) was calculated to be 6.5%.

This result further supports that when a titanium-doped amorphous carbon film is formed by the above method, a highly uniform titanium-doped amorphous carbon film 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 a well of a 24-well plate as a sample. As cells, human normal umbilical vein endothelial cells (HUVEC) were used. Further, as a control material, an amorphous carbon film formed on a flat plate substrate (1 cm × 1 cm, full mirror polishing) made of stainless steel (SUS316L) was used.

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

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

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

"result"
FIG. 3A is a graph showing the results of measuring cell adhesion rates for each sample and control material. The graph represents a relative value in which the cell adhesion rate is 100% when HUVEC is seeded on a culture plate on which amorphous carbon is not formed. FIG. 3B is a graph showing the result of taking out only the standard deviation of the result of FIG.

As a result, it was confirmed that the cell adhesion test can be performed without any problem even when a container formed with amorphous carbon is used.

[Experimental Example 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 a well of a 24-well plate as a sample. Albumin was used as the protein. Further, as a control material, an amorphous carbon film formed on a stainless steel (SUS316L) flat plate base material was used. As the amorphous carbon film, seven types of films similar to those used in Experimental Example 6 were used.

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

《Albumin adsorption test using flat plate substrate》
Further, each flat substrate is a control material, placed in cell culture plate wells placed PBS solution containing albumin (30mg / mL), CO ₂ incubator _{(37 ℃, 5% CO 2} ) 24 hours in static I put it. Subsequently, the PBS solution was removed and washed with pure water (500 μL) to remove unadsorbed albumin. Subsequently, each plate base material was transferred to each well of a new cell culture well plate, and washed with pure water again to remove unadsorbed albumin.

Subsequently, a surfactant solution (PBS containing 2% Triton-X) was added, and the albumin adsorbed on the amorphous carbon film was peeled off by shaking at 37 ° C. for 30 minutes. 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 control material. 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 performed without problems even when a container formed with amorphous carbon is used. Moreover, it became clear that the standard deviation of the measured amount of adsorbed albumin was significantly reduced by using a container in which amorphous carbon was formed as compared with the control material. This result further supports that it is possible to obtain an experimental result with small variations and improved measurement accuracy by using a container formed with amorphous carbon.

[Experimental Example 8]
(Fibrinogen adsorption test)
As a protein, using a container in which an amorphous carbon film was formed on the inner surface of a well of a 24-well plate as a sample, except that fibrinogen at a concentration of 3 mg / mL was used instead of albumin as a protein. An adsorption test was performed. Further, as a control material, an amorphous carbon film formed on a stainless steel (SUS316L) flat plate base material was used. As the amorphous carbon film, seven types 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 adsorbed fibrinogen for each sample and control material. 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 problems even when a container formed with amorphous carbon is used. In addition, by using a container on which amorphous carbon was formed, the standard deviation of the measured fibrinogen adsorption amount tended to be smaller than that of the control material. This result further supports that it is possible to obtain an experimental result with small variations and improved measurement accuracy by using a container formed with amorphous carbon.

DESCRIPTION OF SYMBOLS 100 ... Container, 110 ... Base material, 120 ... Conductive film, 130 ... Amorphous carbon film, 200 ... FCVA apparatus, 201 ... Arc plasma generation chamber, 202 ... Raw material target, 203 ... Electromagnetic coil, 204 ... Ion scan coil, 205 ... Spatial filter, 206 ... Deposition chamber, 207 ... Substrate holder.

Claims

A base material having one or more storage portions for storing samples;
A conductive film formed on at least a portion of the substrate;
Including at least a part of the conductive film, or an amorphous carbon film formed on at least a part of the base material,
A container in which at least a part of the amorphous carbon film is exposed in the housing portion.
The container according to claim 1, wherein the base material is made of an insulating material.
The container according to claim 1 or 2, wherein the substrate is made of a resin material.
The container according to any one of claims 1 to 3, wherein the substrate is a petri dish or a well plate.
The container according to any one of claims 1 to 4, wherein the sample is a biological sample.
The container according to any one of claims 1 to 5, wherein the base material, the conductive film, and the amorphous carbon film are laminated in this order.
The container according to any one of claims 1 to 5, wherein the conductive film, the base material, and the amorphous carbon film are laminated in this order.
The container according to claim 6 or 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 to 8, The container according to any one of the above.
The amorphous carbon film is a titanium-doped amorphous carbon film containing titanium atoms,
The titanium-doped amorphous carbon film has a ratio of the number of titanium atoms to the number of carbon atoms of 2 to 30 atomic%.
The container according to any one of claims 1 to 9.
The titanium-doped amorphous carbon film, sp 2 hybrid orbital of carbon atoms, sp 3 carbon atoms hybrid orbitals, and the ratio of the number of carbon atoms of the sp 3 hybrid orbital to the total number of titanium atoms is 40 atomic% or less The container according to claim 10.
A method for producing 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 and forming the amorphous carbon film by physical vapor deposition or chemical vapor deposition;
A method for manufacturing a container.
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 or 13, wherein, in the step of forming the amorphous carbon film, the amorphous carbon film is laminated and formed on at least a part of the conductive film.
In the step of forming the conductive film, the conductive film is formed on one surface side of the base material, and in the step of forming the amorphous carbon film, the amorphous carbon film is formed on the other surface side of the base material. The manufacturing method according to claim 12 or 13.
Storing the 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.