WO2011001597A1 - Substrate for immobilizing biological substance and method for producing same - Google Patents

Substrate for immobilizing biological substance and method for producing same Download PDF

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Publication number
WO2011001597A1
WO2011001597A1 PCT/JP2010/003693 JP2010003693W WO2011001597A1 WO 2011001597 A1 WO2011001597 A1 WO 2011001597A1 JP 2010003693 W JP2010003693 W JP 2010003693W WO 2011001597 A1 WO2011001597 A1 WO 2011001597A1
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group
sio
coupling agent
silane coupling
substrate
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PCT/JP2010/003693
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French (fr)
Japanese (ja)
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川太規之
神谷晋司
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日本板硝子株式会社
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent

Definitions

  • the present invention relates to a biological material fixing base material and a method for producing the same.
  • Biological substances such as nucleic acids, sugar chains, peptides, proteins, enzymes, antibodies, antigens, viruses, bacteria, cells, etc. can be used on various carriers (substrates for immobilizing biological substances) for diagnosis, treatment, culture, substance production, etc. Used fixed.
  • a carrier a substrate in which a synthetic polymer, a cell adhesive protein, a silane coupling agent, a noble metal or the like is coated on a substrate such as glass, silicon wafer, resin, ceramics, or metal is known.
  • a glass substrate with high light transmittance is suitable for the analysis and measurement of biological materials using light.
  • a cover glass that matches the objective lens characteristics.
  • the cells are cultured and used for microscopic observation as they are.
  • glass substrate glass containing an alkali component such as soda lime glass is generally used because it is inexpensive.
  • this alkali component is eluted from the glass, and the siloxane bond or the bond between the glass and the silane coupling agent is not cut, so that there is a problem that the immobilized biological substance is detached from the base material.
  • Patent Document 1 proposes a biological material immobilization substrate in which the surface of a glass substrate is coated with a surface active layer such as a SiO 2 layer by a sol-gel method in order to suppress elution of alkali components.
  • a surface active layer such as a SiO 2 layer by a sol-gel method.
  • the biological material immobilization substrate described in Patent Document 1 has room for improvement, although the stability of immobilization of the biological material is improved.
  • An object of the present invention is to provide a biological material immobilization substrate that uses a glass substrate containing an alkali component, suppresses elution of the alkali component, and can immobilize the biological material stably.
  • the present invention that has solved the above problems is a glass substrate containing an alkali component, A biological material fixing base material comprising a SiO 2 sputtered film formed on the glass base material and a silane coupling agent layer bonded to the sputtered film.
  • the present invention also includes a step of forming a SiO 2 sputtered film by sputtering SiO 2 on the surface of a glass substrate containing an alkali component, and the surface of the obtained SiO 2 sputtered film is converted into a silane cup. It is a manufacturing method of the base material for biological material fixation including the process processed with a ring agent.
  • the biological material can be stably fixed to the substrate for a long period of time. Therefore, for example, more cells can be grown during cell culture.
  • the base material for immobilizing a biological material of the present invention is excellent in light transmittance, and is therefore suitable for analysis and measurement of biological material using light.
  • a glass substrate is used from the viewpoint of optical properties, and among them, a glass substrate containing an alkali component is used.
  • the alkali component include Li, Na, K, and the like, and these are included in the glass as Li 2 O, Na 2 O, K 2 O.
  • the glass containing an alkali component include soda lime glass, borosilicate glass, alumino borosilicate glass, and alumino silicate glass. From the cost aspect, soda lime glass is preferable, from the viewpoint of chemical durability. Of these, borosilicate glass is preferable, and borosilicate glass having a refractive index of 1.5235 is preferable from an optical surface such as microscopic observation.
  • the shape of the glass substrate is not particularly limited, and may be appropriately determined according to the handling method, analysis method, and measurement method of the biological material. From the viewpoint of versatility and ease of SiO 2 sputtering, it is preferably a plate shape. In view of ease of analysis, the shape of the cover glass is more preferable.
  • a SiO 2 sputtered film is formed on the glass substrate. Since the SiO 2 film is excellent in optical characteristics such as transmittance, it is very suitable for analysis and measurement of biological materials using light.
  • an SiO 2 film (passivation film) is formed by a sol-gel method in order to suppress elution of alkali components.
  • the SiO 2 film formed by the sol-gel method lacks the denseness of the SiO 2 network structure, leaving room for alkali components to pass through the film.
  • SiO 2 film formed by sputtering since the network structure of SiO 2 is formed densely, it is possible to suppress the permeation of the membrane of the alkali component. As a result, the siloxane bond or the bond between the glass-silane coupling agent due to the alkali component is less likely to occur.
  • the thickness of the SiO 2 sputtered film is not particularly limited as long as the elution of alkali components can be suppressed.
  • a high alkali component elution suppressing ability can be exhibited with a film thickness smaller than the thickness of the SiO 2 film formed by the sol-gel method.
  • the thickness of the SiO 2 sputtered film is 1 to 100 nm. 10 to 30 nm is more preferable. If the film thickness is too small, the alkaline component elution suppression effect may not be sufficiently obtained. On the other hand, if the film thickness is too large, it may adversely affect optically due to the difference in refractive index with the substrate.
  • the SiO 2 sputtered film only needs to be formed at least in the region where the biological material is analyzed or measured on the glass substrate surface.
  • the biological material fixing base material of the present invention has a silane coupling agent layer bonded to the sputtered film.
  • a silane coupling agent is a silicon-containing compound having a functional group capable of reacting or interacting with an organic group or the like in one molecule and a hydrolyzable group.
  • Y—R—Si— Represented by Z 3 (wherein Y is a functional group capable of reacting or interacting with an organic group or the like, R is a divalent hydrocarbon group, Z is a hydrolyzable group, Z May be a lower alkyl group).
  • the silane coupling agent is bonded to the sputtered film through a covalent bond formed by the reaction between the hydrolyzable group of the silane coupling agent and the surface hydroxyl group of the SiO 2 sputtered film.
  • the silane coupling agent is bonded to the sputtered film through a hydrogen bond formed by the hydroxyl group generated by hydrolysis of the hydrolyzable group of the silane coupling agent and the surface hydroxyl group of the SiO 2 sputtered film.
  • the functional group capable of reacting or interacting with an organic group or the like of the silane coupling agent the functional group capable of reacting or interacting with a biological substance, or reacting with a substance having adhesiveness or reactivity with a biological substance or There is no particular limitation as long as it is a functional group capable of interacting.
  • the functional group include amino group, SH group, aldehyde group, carboxyl group, halogen atom (eg, iodine atom, bromine atom, chlorine atom) and the like.
  • the hydrolyzable group of the silane coupling agent include an alkoxyl group (eg, methoxy group, ethoxy group, etc.).
  • silane coupling agent examples include those having an amino group such as 2-aminoethyltrimethoxysilane, 2-aminoethyltriethoxysilane, 2-aminoethylmethyldimethoxysilane, 2-aminoethylmethyldiethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropylmethyldiethoxysilane, 4-aminobutyltrimethoxysilane, 4-aminobutyltriethoxysilane, 4 -Aminobutylmethyldimethoxysilane, 4-aminobutylmethyldiethoxysilane, 2-aminoundecyltrimethoxysilane, 2-aminoundecyltriethoxysilane, aminophenyltrimethoxysilane, aminophenyltri
  • a substrate capable of immobilizing a compound having an SH group is one of advantageous embodiments.
  • a biological substance having an SH group such as a protein having an SH group
  • a thiol compound capable of adhering the biological substance is fixed, and a layer for bonding the biological substance to be observed is formed. It can also be formed.
  • the silane coupling agent component of the silane coupling agent layer comprises a functional group that is reactive with the SH group.
  • the functional group having reactivity with the SH group refers to a functional group that can react with the SH group to form a covalent bond (for example, a disulfide bond or a thioether bond).
  • the functional group having reactivity with the SH group include a halogen atom (preferably, the halogen atom is included in the form of a halogenated alkyl group, a halogenated aryl group, a haloacetyl group, an acid halide group, etc.), keto Groups, aldehyde groups, epoxy groups, maleimide groups and the like.
  • a halogen atom is preferable from the viewpoint of availability, and a bromine atom and an iodine atom are more preferable from the viewpoint of reactivity.
  • the silane coupling agent containing a halogen atom is as exemplified above.
  • the base material for biological material fixation of this embodiment is particularly excellent in optical characteristics, and has an advantage that it can be applied to base materials of various shapes.
  • the silane coupling agent component of the silane coupling agent layer contains an amino group and / or SH group, and a noble metal colloid is bonded to the amino group and / or SH group.
  • noble metal colloids include gold colloids, platinum colloids, silver colloids and the like, and are not particularly limited as long as they can be combined with amino groups and / or SH groups, but gold colloids having no cytotoxicity are preferable.
  • the silane coupling agent having an amino group and an SH group is as exemplified above. Since the amino group has a positive charge, it can hold a noble metal colloid. The SH group can retain the noble metal colloid by reacting with the noble metal to form a bond. The noble metal colloid has a high reaction with the SH group. For this reason, the base material for immobilizing a biological material according to this embodiment is particularly excellent in reactivity with the SH group.
  • the biological material fixing base material of the present invention includes, for example, a step of sputtering SiO 2 on the surface of a glass base material containing an alkali component to form a SiO 2 sputtered film (sputtering step), and the obtained SiO 2
  • the surface of the sputtered film can be manufactured by carrying out a step (silane coupling agent treatment step) of treating with a silane coupling agent.
  • a method of sputtering SiO 2 on a glass substrate is known, and the sputtering step can be performed according to a known method.
  • SiO 2 can be used as a target and RF magnetron sputtering can be performed.
  • the sputtering conditions may be appropriately set so that a SiO 2 sputtered film having a desired thickness can be obtained.
  • the silane coupling agent treatment step can be performed according to a known method.
  • the biological material fixing base material of the present invention the elution of the alkaline component from the glass base material is suppressed, so that the siloxane bond or the bond between the glass-silane coupling agent is less likely to be broken by the alkaline component. Moreover, the adverse effect on the biological material by the eluted alkali component is reduced. Therefore, the biological material can be fixed to the base material stably for a long period of time. Therefore, for example, more cells can be grown during cell culture. Moreover, since the base material for biological material fixation of this invention is excellent in the light transmittance, it is suitable for the analysis and measurement of the biological material using light, and can also be comprised as a cover glass. In order to configure the cover glass, a glass substrate suitable for the cover glass (eg, D263 manufactured by Schott, Microsheet 0211 manufactured by Corning, etc.) may be selected.
  • a glass substrate suitable for the cover glass eg, D263 manufactured by Schott, Microsheet 0211 manufactured by Corning
  • Reference Example SiO 2 film evaluating the Na elution amount due to the difference in the method for forming the first, with the difference alkali components by (Na) effect of suppressing the elution method of forming the SiO 2 film, is not provided with a silane coupling agent layer substrate And evaluated.
  • SiO 2 film was formed by RF magnetron sputtering method using a SiO 2 target on a clean glass substrate washed with an aqueous potassium hydroxide solution and pure water.
  • the back pressure is 3.0 ⁇ 10 ⁇ 4 Pa or less
  • the film formation temperature is room temperature
  • the sputtering pressure is 0.4 Pa
  • the argon gas flow rate is 95 sccm
  • the oxygen gas flow rate is 5 sccm
  • the incident power is 1.5 kW. did.
  • sol-gel coating solution was flow-coated on a clean glass substrate washed with an aqueous potassium hydroxide solution and pure water.
  • the sol-gel coating solution was prepared by adding 4 g of tetraethylorthosilicate to a mixed solvent of 40 g of ethanol and 2.6 g of water, and further adding 0.25 g of hydrochloric acid.
  • the flow coating was performed by standing the glass substrate vertically and hanging about 1 ml of the sol-gel coating solution on the glass substrate. After coating, baking was performed at 200 ° C. for 10 minutes to form a SiO 2 film on the glass substrate.
  • SiO 2 film thickness was formed on the film thickness and the Na elution amount measured each glass substrate was measured by a contact type step meter.
  • the amount of Na elution from each glass substrate was such that 30 ml of pure water was brought into contact with a SiO 2 film (a glass substrate for a sample without a SiO 2 film) at a contact area of 28 cm 2 and kept at that temperature at 95 ° C. for 24 hours.
  • the amount of Na eluted in pure water was quantified by flame photometry.
  • a biological material fixing base material provided with a silane coupling agent layer was prepared, and a cell culture experiment was performed.
  • Example 1 In the same manner as in Reference Example (1), a SiO 2 film was produced on a glass substrate. The obtained glass substrate was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the SiO 2 film. Thus, a biological material fixing base material of Example 1 was obtained.
  • Comparative Example 1 A SiO 2 film was produced on a glass substrate in the same manner as in Reference Example (2). The obtained glass substrate was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the SiO 2 film. In this way, a biological material fixing base material of Comparative Example 1 was obtained.
  • Comparative Example 2 A clean glass substrate washed with an aqueous potassium hydroxide solution and pure water was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the glass substrate. In this way, a biological material fixing base material of Comparative Example 2 was obtained.
  • Example 1 and Comparative Examples 1 and 2 prepared above were cut into a size of 15 mm ⁇ 15 mm so that the coating surface would be on the well of a resin microwell plate having 12 wells. And sterilized with 70% ethanol.
  • NIH3T3 mouse fibroblasts adjusted to a concentration of 1 ⁇ 10 4 cells / ml with a medium were seeded in each well containing a substrate for immobilizing a biological material, and cultured under conditions of 37 ° C. and 5% CO 2 .
  • the substrate for immobilizing a biological material was washed with PBS to remove unadhered cells, and then nuclear staining of the cells was performed with DAPI. This was observed with a fluorescence microscope with a 20 ⁇ objective lens, and the number of cells in the same visual field area was counted.
  • Table 2 The results are shown in Table 2.
  • the alkali component breaks the siloxane bond or the bond between the glass and the silane coupling agent, and a part of the cells are detached from the substrate together with the silane coupling agent.
  • the eluted alkaline component reduces the activity of the cells, and the cells are detached from the substrate due to death or the like. Therefore, the more the alkaline component is eluted, the fewer the number of cells to be cultured. In the results of Table 2, the number of cells increases in the order of Example 1> Comparative Example 1> Comparative Example 2. According to the biological material immobilizing substrate of the present invention, the elution of alkaline components is small, It can be seen that the cells can be cultured.

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Abstract

Disclosed is a substrate for immobilizing a biological substance which comprises a glass substrate containing an alkali component, wherein the elution of the alkali component is prevented and thus a biological substance can be stably immobilized. Specifically disclosed is a substrate for immobilizing a biological substance which comprises a glass substrate containing an alkali component, a sputtered SiO2 film formed on the glass substrate, and a silane coupling agent layer bonded to the sputtered film.

Description

生体物質固定用基材およびその製造方法Biological substance fixing substrate and method for producing the same
　本発明は、生体物質固定用基材およびその製造方法に関する。 The present invention relates to a biological material fixing base material and a method for producing the same.
　核酸、糖鎖、ペプチド、蛋白質、酵素、抗体、抗原、ウイルス、細菌、細胞等の生体物質は、診断、治療、培養、物質生産等のために様々な担体（生体物質固定用基材）に固定されて用いられる。担体としては、ガラス、シリコンウェーハ、樹脂、セラミックス、金属等の基材に、合成高分子、細胞接着性蛋白、シランカップリング剤、貴金属等をコーティングしたものが知られている。 Biological substances such as nucleic acids, sugar chains, peptides, proteins, enzymes, antibodies, antigens, viruses, bacteria, cells, etc. can be used on various carriers (substrates for immobilizing biological substances) for diagnosis, treatment, culture, substance production, etc. Used fixed. As the carrier, a substrate in which a synthetic polymer, a cell adhesive protein, a silane coupling agent, a noble metal or the like is coated on a substrate such as glass, silicon wafer, resin, ceramics, or metal is known.
　生体物質を固定化した担体を用いた分析、測定等には、発光、蛍光、吸光、透過、反射、表面プラズモン共鳴などを利用する、光を用いた測定が多く行われている。このような測定に対しては、シリコンウェーハ、セラミックス、金属基材を用いた担体、および貴金属をコーティングした担体は、光透過性の面から不向きである。また、樹脂基材の中には透明なものもあるが、透過率が不十分であり、高感度（高倍率）の測定には不向きである。さらに、樹脂基材は、蛍光顕微鏡観察では、樹脂由来の自家蛍光によって蛍光観察が妨げられるという問題もある。以上のことから、光を用いた生体物質の分析および測定には、光透過性の高いガラス基材が好適であり、例えば、特に高倍率顕微鏡観察においては、対物レンズ特性に合致したカバーガラス上で細胞を培養し、そのままそれを顕微鏡観察に供するということが行われている。 For analysis, measurement, etc. using a carrier on which a biological substance is immobilized, measurement using light using luminescence, fluorescence, absorption, transmission, reflection, surface plasmon resonance, etc. is often performed. For such measurement, silicon wafers, ceramics, a carrier using a metal substrate, and a carrier coated with a noble metal are unsuitable for light transmittance. Some resin base materials are transparent, but the transmittance is insufficient, which is unsuitable for high sensitivity (high magnification) measurement. Furthermore, the resin base material also has a problem that the fluorescence observation is hindered by the autofluorescence derived from the resin in the fluorescence microscope observation. From the above, a glass substrate with high light transmittance is suitable for the analysis and measurement of biological materials using light. For example, in the observation with a high magnification microscope, on a cover glass that matches the objective lens characteristics. In this method, the cells are cultured and used for microscopic observation as they are.
　ガラス基材を用いた担体への生体物質の固定化には、ガラス基材上へ生体物質を直接物理吸着させることも可能であるが、この場合、ガラスと生体物質間の結合が弱く安定性に乏しい。そこで、ガラス基材の表面に、アミノ基やカルボキシル基等の官能基をシランカップリング剤により導入し、これらの官能基と生体物質との間で安定な共有結合を形成させて、生体物質を固定することが行われている。 For immobilization of biological materials on a carrier using a glass substrate, it is possible to physically adsorb the biological material directly on the glass substrate, but in this case, the bond between the glass and the biological material is weak and stable. It is scarce. Therefore, functional groups such as amino groups and carboxyl groups are introduced on the surface of the glass substrate by a silane coupling agent, and a stable covalent bond is formed between these functional groups and the biological material, thereby It has been fixed.
　ガラス基材には、安価であることからソーダライムガラス等のアルカリ成分を含有するガラスが一般に用いられている。しかし、このアルカリ成分がガラスから溶出し、シロキサン結合またはガラス－シランカップリング剤間の結合を切断していまい、固定化された生体物質が基材から脱離するという問題があった。 As the glass substrate, glass containing an alkali component such as soda lime glass is generally used because it is inexpensive. However, this alkali component is eluted from the glass, and the siloxane bond or the bond between the glass and the silane coupling agent is not cut, so that there is a problem that the immobilized biological substance is detached from the base material.
　そこで、特許文献１では、アルカリ成分の溶出を抑制するために、ガラス基板表面を、ゾルゲル法によりＳｉＯ2層などの表面活性層でコーティングした生体物質固定用基板が提案されている。しかし、特許文献１に記載の生体物質固定用基板では、生体物質の固定化の安定性は向上しているものの、未だ改善の余地があった。 Therefore, Patent Document 1 proposes a biological material immobilization substrate in which the surface of a glass substrate is coated with a surface active layer such as a SiO 2 layer by a sol-gel method in order to suppress elution of alkali components. However, the biological material immobilization substrate described in Patent Document 1 has room for improvement, although the stability of immobilization of the biological material is improved.
特開２００３－１７７１２９号公報JP 2003-177129 A
　本発明は、アルカリ成分を含有するガラス基材を用い、アルカリ成分の溶出が抑制され、安定して生体物質を固定することが可能な生体物質固定用基材を提供することを目的とする。 An object of the present invention is to provide a biological material immobilization substrate that uses a glass substrate containing an alkali component, suppresses elution of the alkali component, and can immobilize the biological material stably.
　上記課題を解決した本発明は、アルカリ成分を含有するガラス基材、
　前記ガラス基材上に形成されたＳｉＯ2スパッタ膜、および
　前記スパッタ膜と結合したシランカップリング剤層
を含む生体物質固定用基材である。 The present invention that has solved the above problems is a glass substrate containing an alkali component,
A biological material fixing base material comprising a SiO 2 sputtered film formed on the glass base material and a silane coupling agent layer bonded to the sputtered film.
　本発明はまた、別の側面から、アルカリ成分を含有するガラス基材の表面にＳｉＯ2をスパッタリングして、ＳｉＯ2スパッタ膜を形成する工程、および
　得られたＳｉＯ2スパッタ膜の表面をシランカップリング剤で処理する工程を含む生体物質固定用基材の製造方法である。 In another aspect, the present invention also includes a step of forming a SiO 2 sputtered film by sputtering SiO 2 on the surface of a glass substrate containing an alkali component, and the surface of the obtained SiO 2 sputtered film is converted into a silane cup. It is a manufacturing method of the base material for biological material fixation including the process processed with a ring agent.
　本発明によれば、ガラス基材からのアルカリ成分の溶出が抑制されるため、長期間安定に生体物質を基材に固定することができる。従って、例えば、細胞培養時には、細胞をより多く増殖させることができる。本発明の生体物質固定用基材は、光透過性に優れるため、光を用いた生体物質の分析および測定に好適である。 According to the present invention, since the elution of the alkaline component from the glass substrate is suppressed, the biological material can be stably fixed to the substrate for a long period of time. Therefore, for example, more cells can be grown during cell culture. The base material for immobilizing a biological material of the present invention is excellent in light transmittance, and is therefore suitable for analysis and measurement of biological material using light.
　本発明においては、光学特性の観点から、ガラス基材が用いられ、中でも、アルカリ成分を含有するガラス基材が用いられる。アルカリ成分としては、Ｌｉ、Ｎａ、Ｋ等が挙げられ、これらはガラス中にＬｉ2Ｏ、Ｎａ2Ｏ、Ｋ2Ｏとして含まれる。アルカリ成分を含有するガラスとしては、ソーダライムガラス、ほう珪酸塩ガラス、アルミノほう珪酸塩ガラス、アルミノ珪酸塩ガラス等が挙げられ、コスト面からはソーダライムガラスが好ましく、化学的耐久性の面からはほう珪酸ガラスが好ましく、顕微鏡観察などの光学面からは屈折率が１．５２３５であるほう珪酸ガラスが好ましい。 In the present invention, a glass substrate is used from the viewpoint of optical properties, and among them, a glass substrate containing an alkali component is used. Examples of the alkali component include Li, Na, K, and the like, and these are included in the glass as Li 2 O, Na 2 O, K 2 O. Examples of the glass containing an alkali component include soda lime glass, borosilicate glass, alumino borosilicate glass, and alumino silicate glass. From the cost aspect, soda lime glass is preferable, from the viewpoint of chemical durability. Of these, borosilicate glass is preferable, and borosilicate glass having a refractive index of 1.5235 is preferable from an optical surface such as microscopic observation.
　ガラス基材の形状としては特に制限がなく、生体物質の取扱い方法、分析方法および測定方法に応じて適宜決定すればよく、汎用性およびＳｉＯ2スパッタリングの容易さの観点から、好ましくは板状であり、分析の容易さの観点から、より好ましくはカバーガラスの形状である。 The shape of the glass substrate is not particularly limited, and may be appropriately determined according to the handling method, analysis method, and measurement method of the biological material. From the viewpoint of versatility and ease of SiO 2 sputtering, it is preferably a plate shape. In view of ease of analysis, the shape of the cover glass is more preferable.
　本発明においては、当該ガラス基材上にはＳｉＯ2スパッタ膜が形成される。ＳｉＯ2膜は、透過率等の光学特性に優れるため、光を用いた生体物質の分析および測定には非常に好適である。従来の技術では、アルカリ成分の溶出を抑制するために、ゾルゲル法により、ＳｉＯ2膜（パッシベーション膜）が形成されていた。しかし、ゾルゲル法により形成されたＳｉＯ2膜は、ＳｉＯ2のネットワーク構造の緻密さに欠け、アルカリ成分が膜を透過する余地が残っていた。一方、スパッタリングにより形成されたＳｉＯ2膜は、ＳｉＯ2のネットワーク構造が緻密に形成されているため、アルカリ成分の膜の透過をより抑制することができる。その結果、アルカリ成分による、シロキサン結合またはガラス－シランカップリング剤間の結合の切断がより起こりにくくなる。 In the present invention, a SiO 2 sputtered film is formed on the glass substrate. Since the SiO 2 film is excellent in optical characteristics such as transmittance, it is very suitable for analysis and measurement of biological materials using light. In the conventional technique, an SiO 2 film (passivation film) is formed by a sol-gel method in order to suppress elution of alkali components. However, the SiO 2 film formed by the sol-gel method lacks the denseness of the SiO 2 network structure, leaving room for alkali components to pass through the film. On the other hand, SiO 2 film formed by sputtering, since the network structure of SiO 2 is formed densely, it is possible to suppress the permeation of the membrane of the alkali component. As a result, the siloxane bond or the bond between the glass-silane coupling agent due to the alkali component is less likely to occur.
　ＳｉＯ2スパッタ膜の厚さとしては、アルカリ成分の溶出が抑制できる限り特に制限はない。本発明においては、ゾルゲル法により形成されたＳｉＯ2膜の厚さよりも小さい膜厚で、高いアルカリ成分の溶出抑制能を発揮することができ、ＳｉＯ2スパッタ膜の厚さとしては、１～１００ｎｍが好ましく、１０～３０ｎｍがより好ましい。膜厚が小さすぎると、アルカリ成分の溶出抑制効果が十分に得られないおそれがある。一方、膜厚が大きすぎると、基材との屈折率差により光学的に悪影響を及ぼす場合がある。 The thickness of the SiO 2 sputtered film is not particularly limited as long as the elution of alkali components can be suppressed. In the present invention, a high alkali component elution suppressing ability can be exhibited with a film thickness smaller than the thickness of the SiO 2 film formed by the sol-gel method. The thickness of the SiO 2 sputtered film is 1 to 100 nm. 10 to 30 nm is more preferable. If the film thickness is too small, the alkaline component elution suppression effect may not be sufficiently obtained. On the other hand, if the film thickness is too large, it may adversely affect optically due to the difference in refractive index with the substrate.
　ＳｉＯ2スパッタ膜は、少なくともガラス基材表面の生体物質の分析または測定を行う領域に形成されていればよい。 The SiO 2 sputtered film only needs to be formed at least in the region where the biological material is analyzed or measured on the glass substrate surface.
　本発明の生体物質固定用基材は、前記スパッタ膜と結合したシランカップリング剤層を有する。シランカップリング剤は、一分子中に、有機基等と反応または相互作用が可能な官能基と、加水分解性基とを有する含ケイ素化合物であり、一般的には、Ｙ－Ｒ－Ｓｉ－Ｚ3で表される（式中、Ｙは、有機基等と反応または相互作用が可能な官能基であり、Ｒは２価の炭化水素基であり、Ｚは加水分解性基であり、Ｚの一部が低級アルキル基である場合もある）。シランカップリング剤の加水分解性基と、ＳｉＯ2スパッタ膜の表面水酸基との反応により形成された共有結合を介して、シランカップリング剤は、前記スパッタ膜に結合する。あるいは、シランカップリング剤の加水分解性基が加水分解して生成した水酸基と、ＳｉＯ2スパッタ膜の表面水酸基とが形成する水素結合を介して、シランカップリング剤は、前記スパッタ膜に結合する。 The biological material fixing base material of the present invention has a silane coupling agent layer bonded to the sputtered film. A silane coupling agent is a silicon-containing compound having a functional group capable of reacting or interacting with an organic group or the like in one molecule and a hydrolyzable group. Generally, Y—R—Si— Represented by Z 3 (wherein Y is a functional group capable of reacting or interacting with an organic group or the like, R is a divalent hydrocarbon group, Z is a hydrolyzable group, Z May be a lower alkyl group). The silane coupling agent is bonded to the sputtered film through a covalent bond formed by the reaction between the hydrolyzable group of the silane coupling agent and the surface hydroxyl group of the SiO 2 sputtered film. Alternatively, the silane coupling agent is bonded to the sputtered film through a hydrogen bond formed by the hydroxyl group generated by hydrolysis of the hydrolyzable group of the silane coupling agent and the surface hydroxyl group of the SiO 2 sputtered film. .
　シランカップリング剤の、有機基等と反応または相互作用が可能な官能基としては、生体物質と反応または相互作用可能である官能基、あるいは生体物質と接着性または反応性を有する物質と反応または相互作用可能である官能基である限り、特に制限はない。当該官能基の例としては、アミノ基、ＳＨ基、アルデヒド基、カルボキシル基、ハロゲン原子（例、ヨウ素原子、臭素原子、塩素原子）等が挙げられる。シランカップリング剤の加水分解性基としては、アルコキシル基（例、メトキシ基、エトキシ基など）が挙げられる。 As the functional group capable of reacting or interacting with an organic group or the like of the silane coupling agent, the functional group capable of reacting or interacting with a biological substance, or reacting with a substance having adhesiveness or reactivity with a biological substance or There is no particular limitation as long as it is a functional group capable of interacting. Examples of the functional group include amino group, SH group, aldehyde group, carboxyl group, halogen atom (eg, iodine atom, bromine atom, chlorine atom) and the like. Examples of the hydrolyzable group of the silane coupling agent include an alkoxyl group (eg, methoxy group, ethoxy group, etc.).
　シランカップリング剤の具体例としては、アミノ基を有するものとして、２－アミノエチルトリメトキシシラン、２－アミノエチルトリエトキシシラン、２－アミノエチルメチルジメトキシシラン、２－アミノエチルメチルジエトキシシラン、３－アミノプロピルトリメトキシシラン、３－アミノプロピルトリエトキシシラン、３－アミノプロピルメチルジメトキシシラン、３－アミノプロピルメチルジエトキシシラン、４－アミノブチルトリメトキシシラン、４－アミノブチルトリエトキシシラン、４－アミノブチルメチルジメトキシシラン、４－アミノブチルメチルジエトキシシラン、２－アミノウンデシルトリメトキシシラン、２－アミノウンデシルトリエトキシシラン、アミノフェニルトリメトキシシラン、アミノフェニルトリエトキシシラン、Ｎ－（２－アミノエチルアミノプロピル）トリメトキシシラン、Ｎ－（２－アミノエチルアミノプロピル）トリエトキシシラン、Ｎ－（２－アミノエチルアミノプロピル）メチルジメトキシシラン、Ｎ－（２－アミノエチルアミノプロピル）メチルジエトキシシラン、３－フェニルアミノプロピルトリメトキシシラン、３－フェニルアミノプロピルトリエトキシシランなど；ＳＨ基を有するものとして、３－メルカプトプロピルトリメトキシシラン、３－メルカプトプロピルトリエトキシシラン、３－メルカプトプロピルメチルジメトキシシラン、３－メルカプトプロピルメチルジエトキシシランなど；アルデヒド基を有するものとして、４－トリメトキシシリルブタナール、４－トリエトキシシリルブタナールなど；カルボキシル基を有するものとして、カルボキシメチルトリメトキシシラン、カルボキシメチルトリエトキシシランなど；ヨウ素原子を有するものとして、ヨードメチルトリメトキシシラン、ヨードメチルトリエトキシシラン、ヨードメチルメチルジメトキシシラン、ヨードメチルメチルジエトキシシラン、２－ヨードエチルトリメトキシシラン、２－ヨードエチルトリエトキシシラン、２－ヨードエチルメチルジメトキシシラン、２－ヨードエチルメチルジエトキシシラン、３－ヨードプロピルトリメトキシシラン、３－ヨードプロピルトリエトキシシラン、３－ヨードプロピルメチルジメトキシシラン、３－ヨードプロピルメチルジエトキシシラン、ｎ－ヨードブチルトリメトキシシラン、ｎ－ヨードブチルトリエトキシシラン、ｎ－ヨードブチルメチルジメトキシシラン、ｎ－ヨードブチルメチルジエトキシシラン、（ｐ－ヨードメチル）フェニルトリメトキシシラン、（ｐ－ヨードメチル）フェニルトリエトキシシラン、（ｐ－ヨードメチル）フェニルメチルジメトキシシラン、（ｐ－ヨードメチル）フェニルメチルジエトキシシラン、（ｍ－ヨードメチル）フェニルトリメトキシシラン、（ｍ－ヨードメチル）フェニルトリエトキシシラン、（ｍ－ヨードメチル）フェニルメチルジメトキシシラン、（ｍ－ヨードメチル）フェニルメチルジエトキシシラン、〔（ヨードメチル）フェニルメチル〕トリメトキシシラン、〔（ヨードメチル）フェニルメチル〕トリエトキシシラン、〔（ヨードメチル）フェニルメチル〕メチルジメトキシシラン、〔（ヨードメチル）フェニルメチル〕メチルジエトキシシラン、〔（ヨードメチル）フェニルエチル〕トリメトキシシラン、〔（ヨードメチル）フェニルエチル〕トリエトキシシラン、〔（ヨードメチル）フェニルエチル〕メチルジメトキシシラン、〔（ヨードメチル）フェニルエチル〕メチルジエトキシシラン、２－（ヨードメチル）アリルトリメトキシシラン、２－（ヨードメチル）アリルトリエトキシシラン、２－（ヨードメチル）アリルメチルジメトキシシラン、２－（ヨードメチル）アリルメチルジエトキシシラン、２－（４－ヨードスルホニルフェニル）エチルトリメトキシシラン、２－（４－ヨードスルホニルフェニル）エチルトリエトキシシラン、２－（４－ヨードスルホニルフェニル）エチルメチルジメトキシシラン、２－（４－ヨードスルホニルフェニル）エチルメチルジエトキシシラン、３－（４－ヨードスルホニルフェニル）プロピルトリメトキシシラン、３－（４－ヨードスルホニルフェニル）プロピルトリエトキシシラン、３－（４－ヨードスルホニルフェニル）プロピルメチルジメトキシシラン、３－（４－ヨードスルホニルフェニル）プロピルメチルジエトキシシランなど；臭素原子を有するものとして、ブロモメチルトリメトキシシラン、ブロモメチルトリエトキシシラン、ブロモメチルメチルジメトキシシラン、ブロモメチルメチルジエトキシシラン、２－ブロモエチルトリメトキシシラン、２－ブロモエチルトリエトキシシラン、２－ブロモエチルメチルジメトキシシラン、２－ブロモエチルメチルジエトキシシラン、３－ブロモプロピルトリメトキシシラン、３－ブロモプロピルトリエトキシシラン、３－ブロモプロピルメチルジメトキシシラン、３－ブロモプロピルメチルジエトキシシラン、ｎ－ブロモブチルトリメトキシシラン、ｎ－ブロモブチルトリエトキシシラン、ｎ－ブロモブチルメチルジメトキシシラン、ｎ－ブロモブチルメチルジエトキシシラン、（ｐ－ブロモメチル）フェニルトリメトキシシラン、（ｐ－ブロモメチル）フェニルトリエトキシシラン、（ｐ－ブロモメチル）フェニルメチルジメトキシシラン、（ｐ－ブロモメチル）フェニルメチルジエトキシシラン、（ｍ－ブロモメチル）フェニルトリメトキシシラン、（ｍ－ブロモメチル）フェニルトリエトキシシラン、（ｍ－ブロモメチル）フェニルメチルジメトキシシラン、（ｍ－ブロモメチル）フェニルメチルジエトキシシラン、〔（ブロモメチル）フェニルメチル〕トリメトキシシラン、〔（ブロモメチル）フェニルメチル〕トリエトキシシラン、〔（ブロモメチル）フェニルメチル〕メチルジメトキシシラン、〔（ブロモメチル）フェニルメチル〕メチルジエトキシシラン、〔（ブロモメチル）フェニルエチル〕トリメトキシシラン、〔（ブロモメチル）フェニルエチル〕トリエトキシシラン、〔（ブロモメチル）フェニルエチル〕メチルジメトキシシラン、〔（ブロモメチル）フェニルエチル〕メチルジエトキシシラン、２－（ブロモメチル）アリルトリメトキシシラン、２－（ブロモメチル）アリルトリエトキシシラン、２－（ブロモメチル）アリルメチルジメトキシシラン、２－（ブロモメチル）アリルメチルジエトキシシラン、２－（４－ブロモスルホニルフェニル）エチルトリメトキシシラン、２－（４－ブロモスルホニルフェニル）エチルトリエトキシシラン、２－（４－ブロモスルホニルフェニル）エチルメチルジメトキシシラン、２－（４－ブロモスルホニルフェニル）エチルメチルジエトキシシラン、３－（４－ブロモスルホニルフェニル）プロピルトリメトキシシラン、３－（４－ブロモスルホニルフェニル）プロピルトリエトキシシラン、３－（４－ブロモスルホニルフェニル）プロピルメチルジメトキシシラン、３－（４－ブロモスルホニルフェニル）プロピルメチルジエトキシシランなど；塩素原子を有するものとして、クロロメチルトリメトキシシラン、クロロメチルトリエトキシシラン、クロロメチルメチルジメトキシシラン、クロロメチルメチルジエトキシシラン、２－クロロエチルトリメトキシシラン、２－クロロエチルトリエトキシシラン、２－クロロエチルメチルジメトキシシラン、２－クロロエチルメチルジエトキシシラン、３－クロロプロピルトリメトキシシラン、３－クロロプロピルトリエトキシシラン、３－クロロプロピルメチルジメトキシシラン、３－クロロプロピルメチルジエトキシシラン、ｎ－クロロブチルトリメトキシシラン、ｎ－クロロブチルトリエトキシシラン、ｎ－クロロブチルメチルジメトキシシラン、ｎ－クロロブチルメチルジエトキシシラン、（ｐ－クロロメチル）フェニルトリメトキシシラン、（ｐ－クロロメチル）フェニルトリエトキシシラン、（ｐ－クロロメチル）フェニルメチルジメトキシシラン、（ｐ－クロロメチル）フェニルメチルジエトキシシラン、（ｍ－クロロメチル）フェニルトリメトキシシラン、（ｍ－クロロメチル）フェニルトリエトキシシラン、（ｍ－クロロメチル）フェニルメチルジメトキシシラン、（ｍ－クロロメチル）フェニルメチルジエトキシシラン、〔（クロロメチル）フェニルメチル〕トリメトキシシラン、〔（クロロメチル）フェニルメチル〕トリエトキシシラン、〔（クロロメチル）フェニルメチル〕メチルジメトキシシラン、〔（クロロメチル）フェニルメチル〕メチルジエトキシシラン、〔（クロロメチル）フェニルエチル〕トリメトキシシラン、〔（クロロメチル）フェニルエチル〕トリエトキシシラン、〔（クロロメチル）フェニルエチル〕メチルジメトキシシラン、〔（クロロメチル）フェニルエチル〕メチルジエトキシシラン、２－（クロロメチル）アリルトリメトキシシラン、２－（クロロメチル）アリルトリエトキシシラン、２－（クロロメチル）アリルメチルジメトキシシラン、２－（クロロメチル）アリルメチルジエトキシシラン、２－（４－クロロスルホニルフェニル）エチルトリメトキシシラン、２－（４－クロロスルホニルフェニル）エチルトリエトキシシラン、２－（４－クロロスルホニルフェニル）エチルメチルジメトキシシラン、２－（４－クロロスルホニルフェニル）エチルメチルジエトキシシラン、３－（４－クロロスルホニルフェニル）プロピルトリメトキシシラン、３－（４－クロロスルホニルフェニル）プロピルトリエトキシシラン、３－（４－クロロスルホニルフェニル）プロピルメチルジメトキシシラン、３－（４－クロロスルホニルフェニル）プロピルメチルジエトキシシランなどが挙げられる。 Specific examples of the silane coupling agent include those having an amino group such as 2-aminoethyltrimethoxysilane, 2-aminoethyltriethoxysilane, 2-aminoethylmethyldimethoxysilane, 2-aminoethylmethyldiethoxysilane, 3-aminopropyltrimethoxysilane, 3-aminopropyltriethoxysilane, 3-aminopropylmethyldimethoxysilane, 3-aminopropylmethyldiethoxysilane, 4-aminobutyltrimethoxysilane, 4-aminobutyltriethoxysilane, 4 -Aminobutylmethyldimethoxysilane, 4-aminobutylmethyldiethoxysilane, 2-aminoundecyltrimethoxysilane, 2-aminoundecyltriethoxysilane, aminophenyltrimethoxysilane, aminophenyltrie Xisilane, N- (2-aminoethylaminopropyl) trimethoxysilane, N- (2-aminoethylaminopropyl) triethoxysilane, N- (2-aminoethylaminopropyl) methyldimethoxysilane, N- (2-amino Ethylaminopropyl) methyldiethoxysilane, 3-phenylaminopropyltrimethoxysilane, 3-phenylaminopropyltriethoxysilane, etc .; those having SH groups such as 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane 3-mercaptopropylmethyldimethoxysilane, 3-mercaptopropylmethyldiethoxysilane, etc .; those having an aldehyde group such as 4-trimethoxysilylbutanal, 4-triethoxysilylbutanal, etc .; Carboxymethyltrimethoxysilane, carboxymethyltriethoxysilane, etc. having a sil group; iodomethyltrimethoxysilane, iodomethyltriethoxysilane, iodomethylmethyldimethoxysilane, iodomethylmethyldiethoxy having iodine atoms Silane, 2-iodoethyltrimethoxysilane, 2-iodoethyltriethoxysilane, 2-iodoethylmethyldimethoxysilane, 2-iodoethylmethyldiethoxysilane, 3-iodopropyltrimethoxysilane, 3-iodopropyltriethoxysilane 3-iodopropylmethyldimethoxysilane, 3-iodopropylmethyldiethoxysilane, n-iodobutyltrimethoxysilane, n-iodobutyltriethoxysilane, n-iodobuty Rumethyldimethoxysilane, n-iodobutylmethyldiethoxysilane, (p-iodomethyl) phenyltrimethoxysilane, (p-iodomethyl) phenyltriethoxysilane, (p-iodomethyl) phenylmethyldimethoxysilane, (p-iodomethyl) phenyl Methyldiethoxysilane, (m-iodomethyl) phenyltrimethoxysilane, (m-iodomethyl) phenyltriethoxysilane, (m-iodomethyl) phenylmethyldimethoxysilane, (m-iodomethyl) phenylmethyldiethoxysilane, [(iodomethyl) Phenylmethyl] trimethoxysilane, [(iodomethyl) phenylmethyl] triethoxysilane, [(iodomethyl) phenylmethyl] methyldimethoxysilane, [(iodomethyl) phenylmethyl] Tildiethoxysilane, [(iodomethyl) phenylethyl] trimethoxysilane, [(iodomethyl) phenylethyl] triethoxysilane, [(iodomethyl) phenylethyl] methyldimethoxysilane, [(iodomethyl) phenylethyl] methyldiethoxysilane, 2- (iodomethyl) allyltrimethoxysilane, 2- (iodomethyl) allyltriethoxysilane, 2- (iodomethyl) allylmethyldimethoxysilane, 2- (iodomethyl) allylmethyldiethoxysilane, 2- (4-iodosulfonylphenyl) Ethyltrimethoxysilane, 2- (4-iodosulfonylphenyl) ethyltriethoxysilane, 2- (4-iodosulfonylphenyl) ethylmethyldimethoxysilane, 2- (4-iodosulfonylphenyl) Tilmethyldiethoxysilane, 3- (4-iodosulfonylphenyl) propyltrimethoxysilane, 3- (4-iodosulfonylphenyl) propyltriethoxysilane, 3- (4-iodosulfonylphenyl) propylmethyldimethoxysilane, 3- (4-iodosulfonylphenyl) propylmethyldiethoxysilane and the like; those having a bromine atom include bromomethyltrimethoxysilane, bromomethyltriethoxysilane, bromomethylmethyldimethoxysilane, bromomethylmethyldiethoxysilane, 2-bromoethyl Trimethoxysilane, 2-bromoethyltriethoxysilane, 2-bromoethylmethyldimethoxysilane, 2-bromoethylmethyldiethoxysilane, 3-bromopropyltrimethoxysilane, 3-bromopropiyl Lutriethoxysilane, 3-bromopropylmethyldimethoxysilane, 3-bromopropylmethyldiethoxysilane, n-bromobutyltrimethoxysilane, n-bromobutyltriethoxysilane, n-bromobutylmethyldimethoxysilane, n-bromobutylmethyl Diethoxysilane, (p-bromomethyl) phenyltrimethoxysilane, (p-bromomethyl) phenyltriethoxysilane, (p-bromomethyl) phenylmethyldimethoxysilane, (p-bromomethyl) phenylmethyldiethoxysilane, (m-bromomethyl) Phenyltrimethoxysilane, (m-bromomethyl) phenyltriethoxysilane, (m-bromomethyl) phenylmethyldimethoxysilane, (m-bromomethyl) phenylmethyldiethoxysilane, [(bromine Methyl) phenylmethyl] trimethoxysilane, [(bromomethyl) phenylmethyl] triethoxysilane, [(bromomethyl) phenylmethyl] methyldimethoxysilane, [(bromomethyl) phenylmethyl] methyldiethoxysilane, [(bromomethyl) phenylethyl] Trimethoxysilane, [(bromomethyl) phenylethyl] triethoxysilane, [(bromomethyl) phenylethyl] methyldimethoxysilane, [(bromomethyl) phenylethyl] methyldiethoxysilane, 2- (bromomethyl) allyltrimethoxysilane, 2- (Bromomethyl) allyltriethoxysilane, 2- (bromomethyl) allylmethyldimethoxysilane, 2- (bromomethyl) allylmethyldiethoxysilane, 2- (4-bromosulfonylphenyl) Ethyltrimethoxysilane, 2- (4-bromosulfonylphenyl) ethyltriethoxysilane, 2- (4-bromosulfonylphenyl) ethylmethyldimethoxysilane, 2- (4-bromosulfonylphenyl) ethylmethyldiethoxysilane, 3- (4-bromosulfonylphenyl) propyltrimethoxysilane, 3- (4-bromosulfonylphenyl) propyltriethoxysilane, 3- (4-bromosulfonylphenyl) propylmethyldimethoxysilane, 3- (4-bromosulfonylphenyl) propyl Methyldiethoxysilane, etc .; those having a chlorine atom, such as chloromethyltrimethoxysilane, chloromethyltriethoxysilane, chloromethylmethyldimethoxysilane, chloromethylmethyldiethoxysilane, 2-chloroethyl Rutrimethoxysilane, 2-chloroethyltriethoxysilane, 2-chloroethylmethyldimethoxysilane, 2-chloroethylmethyldiethoxysilane, 3-chloropropyltrimethoxysilane, 3-chloropropyltriethoxysilane, 3-chloropropylmethyl Dimethoxysilane, 3-chloropropylmethyldiethoxysilane, n-chlorobutyltrimethoxysilane, n-chlorobutyltriethoxysilane, n-chlorobutylmethyldimethoxysilane, n-chlorobutylmethyldiethoxysilane, (p-chloromethyl) ) Phenyltrimethoxysilane, (p-chloromethyl) phenyltriethoxysilane, (p-chloromethyl) phenylmethyldimethoxysilane, (p-chloromethyl) phenylmethyldiethoxysilane, (m-chloromethyl) ) Phenyltrimethoxysilane, (m-chloromethyl) phenyltriethoxysilane, (m-chloromethyl) phenylmethyldimethoxysilane, (m-chloromethyl) phenylmethyldiethoxysilane, [(chloromethyl) phenylmethyl] trimethoxy Silane, [(chloromethyl) phenylmethyl] triethoxysilane, [(chloromethyl) phenylmethyl] methyldimethoxysilane, [(chloromethyl) phenylmethyl] methyldiethoxysilane, [(chloromethyl) phenylethyl] trimethoxysilane , [(Chloromethyl) phenylethyl] triethoxysilane, [(chloromethyl) phenylethyl] methyldimethoxysilane, [(chloromethyl) phenylethyl] methyldiethoxysilane, 2- (chloromethyl) allyltrimeth Silane, 2- (chloromethyl) allyltriethoxysilane, 2- (chloromethyl) allylmethyldimethoxysilane, 2- (chloromethyl) allylmethyldiethoxysilane, 2- (4-chlorosulfonylphenyl) ethyltrimethoxysilane, 2- (4-chlorosulfonylphenyl) ethyltriethoxysilane, 2- (4-chlorosulfonylphenyl) ethylmethyldimethoxysilane, 2- (4-chlorosulfonylphenyl) ethylmethyldiethoxysilane, 3- (4-chlorosulfonyl) Phenyl) propyltrimethoxysilane, 3- (4-chlorosulfonylphenyl) propyltriethoxysilane, 3- (4-chlorosulfonylphenyl) propylmethyldimethoxysilane, 3- (4-chlorosulfonylphenyl) propylmethyldiethoxy Sisilane etc. are mentioned.
　本発明の生体物質固定用基材において、ＳＨ基を有する化合物を固定可能な基材は有利な実施態様の一つである。この実施態様では、ＳＨ基を有する蛋白質等のＳＨ基を有する生体物質を固定することができ、また、生体物質を接着可能なチオール化合物を固定し、観察対象の生体物質の接着用の層を形成することもできる。 In the biological material immobilizing substrate of the present invention, a substrate capable of immobilizing a compound having an SH group is one of advantageous embodiments. In this embodiment, a biological substance having an SH group, such as a protein having an SH group, can be fixed, a thiol compound capable of adhering the biological substance is fixed, and a layer for bonding the biological substance to be observed is formed. It can also be formed.
　よって、有利な実施態様においては、シランカップリング剤層のシランカップリング剤成分は、ＳＨ基と反応性を有する官能基を含む。ＳＨ基と反応性を有する官能基とは、ＳＨ基と反応して共有結合（例えば、ジスルフィド結合またはチオエーテル結合）を生じ得る官能基を指す。ＳＨ基と反応性を有する官能基としては、例えば、ハロゲン原子（好適には、ハロゲン原子は、ハロゲン化アルキル基、ハロゲン化アリール基、ハロアセチル基、酸ハライド基等の形態で含まれる）、ケト基、アルデヒド基、エポキシ基、マレイミド基などが挙げられる。なかでも、入手の容易さの観点からは、ハロゲン原子が好ましく、反応性の観点から、臭素原子、ヨウ素原子がさらに好ましい。ハロゲン原子を含むシランカップリング剤については上記例示した通りである。なお、この実施形態の生体物質固定用基材は、光学特性に特に優れており、様々な形状の基材に適用可能であるという利点を有する。 Thus, in an advantageous embodiment, the silane coupling agent component of the silane coupling agent layer comprises a functional group that is reactive with the SH group. The functional group having reactivity with the SH group refers to a functional group that can react with the SH group to form a covalent bond (for example, a disulfide bond or a thioether bond). Examples of the functional group having reactivity with the SH group include a halogen atom (preferably, the halogen atom is included in the form of a halogenated alkyl group, a halogenated aryl group, a haloacetyl group, an acid halide group, etc.), keto Groups, aldehyde groups, epoxy groups, maleimide groups and the like. Among these, a halogen atom is preferable from the viewpoint of availability, and a bromine atom and an iodine atom are more preferable from the viewpoint of reactivity. The silane coupling agent containing a halogen atom is as exemplified above. In addition, the base material for biological material fixation of this embodiment is particularly excellent in optical characteristics, and has an advantage that it can be applied to base materials of various shapes.
　あるいは、別の有利な実施態様においては、シランカップリング剤層のシランカップリング剤成分が、アミノ基および／またはＳＨ基を含み、当該アミノ基および／またはＳＨ基に貴金属コロイドが結合する。貴金属コロイドの例としては、金コロイド、白金コロイド、銀コロイド等が挙げられ、アミノ基および／またはＳＨ基と結合できれば特にこれらに限定されないが、細胞毒性が無い金コロイドが好適である。アミノ基およびＳＨ基を有するシランカップリング剤については上記例示した通りである。アミノ基は、正電荷を有するために、貴金属コロイドを保持することが可能である。ＳＨ基は、貴金属と反応して結合を形成することにより、貴金属コロイドを保持することが可能である。そして、貴金属コロイドは、ＳＨ基と高い反応を有する。このため、この実施形態の生体物質固定用基材は、ＳＨ基との反応性が特に優れたものとなる。 Alternatively, in another advantageous embodiment, the silane coupling agent component of the silane coupling agent layer contains an amino group and / or SH group, and a noble metal colloid is bonded to the amino group and / or SH group. Examples of noble metal colloids include gold colloids, platinum colloids, silver colloids and the like, and are not particularly limited as long as they can be combined with amino groups and / or SH groups, but gold colloids having no cytotoxicity are preferable. The silane coupling agent having an amino group and an SH group is as exemplified above. Since the amino group has a positive charge, it can hold a noble metal colloid. The SH group can retain the noble metal colloid by reacting with the noble metal to form a bond. The noble metal colloid has a high reaction with the SH group. For this reason, the base material for immobilizing a biological material according to this embodiment is particularly excellent in reactivity with the SH group.
　本発明の生体物質固定用基材は、例えば、アルカリ成分を含有するガラス基材の表面にＳｉＯ2をスパッタリングして、ＳｉＯ2スパッタ膜を形成する工程（スパッタリング工程）、および得られたＳｉＯ2スパッタ膜の表面をシランカップリング剤で処理する工程（シランカップリング剤処理工程）を実施して製造することができる。 The biological material fixing base material of the present invention includes, for example, a step of sputtering SiO 2 on the surface of a glass base material containing an alkali component to form a SiO 2 sputtered film (sputtering step), and the obtained SiO 2 The surface of the sputtered film can be manufactured by carrying out a step (silane coupling agent treatment step) of treating with a silane coupling agent.
　ガラス基材にＳｉＯ2をスパッタリングする方法は公知であり、スパッタリング工程は、公知方法に準じて行うことができ、例えば、ＳｉＯ２をターゲットに用い、ＲＦマグネトロンスパッタリング法により行うことができる。スパッタリング条件は、所望の厚さのＳｉＯ2スパッタ膜が得られるように適宜設定すればよい。 A method of sputtering SiO 2 on a glass substrate is known, and the sputtering step can be performed according to a known method. For example, SiO 2 can be used as a target and RF magnetron sputtering can be performed. The sputtering conditions may be appropriately set so that a SiO 2 sputtered film having a desired thickness can be obtained.
　シランカップリング剤処理工程は、公知方法に準じて行うことができる。 The silane coupling agent treatment step can be performed according to a known method.
　本発明の生体物質固定用基材では、ガラス基材からのアルカリ成分の溶出が抑制されているため、アルカリ成分による、シロキサン結合またはガラス－シランカップリング剤間の結合の切断がより起こりにくい。また、溶出したアルカリ成分による生体物質への悪影響が低減されている。従って、長期間安定に生体物質を基材に固定することができる。よって、例えば、細胞培養時には、細胞をより多く増殖させることができる。また、本発明の生体物質固定用基材は、光の透過性に優れるため、光を用いた生体物質の分析および測定に好適であり、カバーガラスとして構成することもできる。カバーガラスを構成するには、ガラス基材に、カバーガラスに適したもの（例、ショット社製Ｄ２６３、コーニング社製マイクロシート０２１１等）を選択すればよい。 In the biological material fixing base material of the present invention, the elution of the alkaline component from the glass base material is suppressed, so that the siloxane bond or the bond between the glass-silane coupling agent is less likely to be broken by the alkaline component. Moreover, the adverse effect on the biological material by the eluted alkali component is reduced. Therefore, the biological material can be fixed to the base material stably for a long period of time. Therefore, for example, more cells can be grown during cell culture. Moreover, since the base material for biological material fixation of this invention is excellent in the light transmittance, it is suitable for the analysis and measurement of the biological material using light, and can also be comprised as a cover glass. In order to configure the cover glass, a glass substrate suitable for the cover glass (eg, D263 manufactured by Schott, Microsheet 0211 manufactured by Corning, etc.) may be selected.
　以下、実施例および比較例を挙げて本発明を詳細に説明するが、本発明は、これら実施例に限定されるものではない。 Hereinafter, the present invention will be described in detail with reference to examples and comparative examples, but the present invention is not limited to these examples.
参考例　ＳｉＯ2膜の形成法の違いによるＮａ溶出量の評価
　まず、ＳｉＯ2膜の形成方法の違いによるアルカリ成分（Ｎａ）溶出抑制効果を、シランカップリング剤層を設けていない基材を用いて評価した。 Reference Example SiO 2 film evaluating the Na elution amount due to the difference in the method for forming the first, with the difference alkali components by (Na) effect of suppressing the elution method of forming the SiO 2 film, is not provided with a silane coupling agent layer substrate And evaluated.
（１）スパッタリング法によるＳｉＯ2膜の形成
　水酸化カリウム水溶液および純水により洗浄した清浄なガラス基板上に、ＳｉＯ２ターゲットを用いてＲＦマグネトロンスパッタリング法にてＳｉＯ2膜を形成した。成膜条件として、背圧は３．０×１０-4Ｐａ以下、成膜温度は室温、スパッタ圧力は０．４Ｐａ、アルゴンガス流量は９５ｓｃｃｍ、酸素ガス流量は５ｓｃｃｍ、入射電力は１．５ｋＷとした。 (1) Formation of SiO 2 film by sputtering method An SiO 2 film was formed by RF magnetron sputtering method using a SiO 2 target on a clean glass substrate washed with an aqueous potassium hydroxide solution and pure water. As film formation conditions, the back pressure is 3.0 × 10 −4 Pa or less, the film formation temperature is room temperature, the sputtering pressure is 0.4 Pa, the argon gas flow rate is 95 sccm, the oxygen gas flow rate is 5 sccm, and the incident power is 1.5 kW. did.
（２）ゾルゲル法によるＳｉＯ2膜の形成
　水酸化カリウム水溶液および純水により洗浄した清浄なガラス基板に、ゾルゲルコーティング液をフローコートした。ゾルゲルコーティング溶液は、テトラエチルオルトシリケート４ｇを、エタノール４０ｇおよび水２．６ｇの混合溶媒に加えた後、さらに塩酸０．２５ｇを添加して調製した。フローコートは、前記ガラス基板を垂直に立て掛け、ガラス基板上部に前記ゾルゲルコーティング溶液１ｍｌ程度を垂らす事により行った。コート後、２００℃で１０分間焼成して、ガラス基板上にＳｉＯ2膜を形成した。 (2) Formation of SiO 2 film by sol-gel method A sol-gel coating solution was flow-coated on a clean glass substrate washed with an aqueous potassium hydroxide solution and pure water. The sol-gel coating solution was prepared by adding 4 g of tetraethylorthosilicate to a mixed solvent of 40 g of ethanol and 2.6 g of water, and further adding 0.25 g of hydrochloric acid. The flow coating was performed by standing the glass substrate vertically and hanging about 1 ml of the sol-gel coating solution on the glass substrate. After coating, baking was performed at 200 ° C. for 10 minutes to form a SiO 2 film on the glass substrate.
（３）膜厚およびＮａ溶出量測定
　各ガラス基板上に形成したＳｉＯ2膜厚は、接触式段差計にて測定した。各ガラス基板からのＮａ溶出量は、純水３０ｍｌをＳｉＯ2膜（ＳｉＯ2膜がない試料についてはガラス基板）に接触面積２８ｃｍ2で接触させ、その状態で温度９５℃で２４時間保持し、純水に溶出したＮａ量を炎光光度法により定量した。 (3) SiO 2 film thickness was formed on the film thickness and the Na elution amount measured each glass substrate was measured by a contact type step meter. The amount of Na elution from each glass substrate was such that 30 ml of pure water was brought into contact with a SiO 2 film (a glass substrate for a sample without a SiO 2 film) at a contact area of 28 cm 2 and kept at that temperature at 95 ° C. for 24 hours. The amount of Na eluted in pure water was quantified by flame photometry.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
　表１より、スパッタリングにより形成したＳｉＯ2膜によれば、ゾルゲル法により形成したＳｉＯ2膜に比べ、小さい膜厚でＮａの溶出を驚くほどに抑制できることがわかる。 As can be seen from Table 1, according to the SiO 2 film formed by sputtering, elution of Na can be suppressed surprisingly with a small film thickness as compared with the SiO 2 film formed by the sol-gel method.
　次に、シランカップリング剤層を設けた生体物質固定用基材を作製して、細胞培養実験を行った。 Next, a biological material fixing base material provided with a silane coupling agent layer was prepared, and a cell culture experiment was performed.
実施例１
　参考例（１）と同様にして、ガラス基板上にＳｉＯ2膜を作製した。得られたガラス基板を、０．５ｗｔ％ヨードプロピルトリメトキシシランエタノール溶液に浸漬することにより、ＳｉＯ2膜上にシランカップリング剤層を設けた。こうして実施例１の生体物質固定用基材を得た。 Example 1
In the same manner as in Reference Example (1), a SiO 2 film was produced on a glass substrate. The obtained glass substrate was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the SiO 2 film. Thus, a biological material fixing base material of Example 1 was obtained.
比較例１
　参考例（２）と同様にして、ガラス基板上にＳｉＯ2膜を作製した。得られたガラス基板を、０．５ｗｔ％ヨードプロピルトリメトキシシランエタノール溶液に浸漬することにより、ＳｉＯ2膜上にシランカップリング剤層を設けた。こうして比較例１の生体物質固定用基材を得た。 Comparative Example 1
A SiO 2 film was produced on a glass substrate in the same manner as in Reference Example (2). The obtained glass substrate was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the SiO 2 film. In this way, a biological material fixing base material of Comparative Example 1 was obtained.
比較例２
　水酸化カリウム水溶液および純水により洗浄した清浄なガラス基板を、０．５ｗｔ％ヨードプロピルトリメトキシシランエタノール溶液に浸漬することにより、ガラス基板上にシランカップリング剤層を設けた。こうして比較例２の生体物質固定用基材を得た。 Comparative Example 2
A clean glass substrate washed with an aqueous potassium hydroxide solution and pure water was immersed in a 0.5 wt% iodopropyltrimethoxysilane ethanol solution to provide a silane coupling agent layer on the glass substrate. In this way, a biological material fixing base material of Comparative Example 2 was obtained.
　上記作製した実施例１並びに比較例１および２の生体物質固定用基材を１５ｍｍ×１５ｍｍの大きさに切断し、ウェル数１２の樹脂製マイクロウェルプレートのウェルにコーティング面が上となるように入れ、７０％エタノールで滅菌を行った。生体物質固定用基材が入った各ウェルに、培地で１×１０4個／ｍｌの濃度に調整したＮＩＨ３Ｔ３マウス繊維芽細胞を播種し、３７℃、５％ＣＯ2の条件下で培養した。培養後１１日目に生体物質固定用基材をＰＢＳで洗浄し、接着していない細胞を除いた後、ＤＡＰＩで細胞の核染色を行った。２０倍の対物レンズの蛍光顕微鏡でこれを観察し、同一視野面積内の細胞数を計測した。結果を表２に示す。 The biological material fixing base materials of Example 1 and Comparative Examples 1 and 2 prepared above were cut into a size of 15 mm × 15 mm so that the coating surface would be on the well of a resin microwell plate having 12 wells. And sterilized with 70% ethanol. NIH3T3 mouse fibroblasts adjusted to a concentration of 1 × 10 4 cells / ml with a medium were seeded in each well containing a substrate for immobilizing a biological material, and cultured under conditions of 37 ° C. and 5% CO 2 . On the 11th day after culturing, the substrate for immobilizing a biological material was washed with PBS to remove unadhered cells, and then nuclear staining of the cells was performed with DAPI. This was observed with a fluorescence microscope with a 20 × objective lens, and the number of cells in the same visual field area was counted. The results are shown in Table 2.
Figure JPOXMLDOC01-appb-T000002
Figure JPOXMLDOC01-appb-T000002
　ガラス基材よりアルカリ成分が溶出すると、アルカリ成分によりシロキサン結合またはガラス－シランカップリング剤間の結合の切断が起こり、基材から細胞の一部がシランカップリング剤ごと脱離する。また、溶出したアルカリ成分によって、細胞の活性が低下し、細胞が死滅などにより基材から脱離する。よって、アルカリ成分の溶出が多いほど、培養される細胞数は少なくなる。表２の結果では、細胞数は、実施例１＞比較例１＞比較例２という順に多くなっており、本発明の生体物質固定用基材によれば、アルカリ成分の溶出が少なく、多くの細胞を培養できることがわかる。 When the alkali component elutes from the glass substrate, the alkali component breaks the siloxane bond or the bond between the glass and the silane coupling agent, and a part of the cells are detached from the substrate together with the silane coupling agent. In addition, the eluted alkaline component reduces the activity of the cells, and the cells are detached from the substrate due to death or the like. Therefore, the more the alkaline component is eluted, the fewer the number of cells to be cultured. In the results of Table 2, the number of cells increases in the order of Example 1> Comparative Example 1> Comparative Example 2. According to the biological material immobilizing substrate of the present invention, the elution of alkaline components is small, It can be seen that the cells can be cultured.

Claims (7)

  1. 　アルカリ成分を含有するガラス基材、
    　前記ガラス基材上に形成されたＳｉＯ2スパッタ膜、および
    　前記スパッタ膜と結合したシランカップリング剤層
    を含む生体物質固定用基材。     A glass substrate containing an alkali component,
    A biological material immobilizing substrate comprising: a SiO 2 sputtered film formed on the glass substrate; and a silane coupling agent layer bonded to the sputtered film.
  2. 　前記スパッタ膜の厚さが１～１００ｎｍである請求項１に記載の生体物質固定用基材。 2. The substrate for immobilizing a biological material according to claim 1, wherein the thickness of the sputtered film is 1 to 100 nm.
  3. 　前記シランカップリング剤層のシランカップリング剤成分が、ＳＨ基と反応性を有する官能基を含む請求項１に記載の生体物質固定用基材。 The base material for immobilizing a biological material according to claim 1, wherein the silane coupling agent component of the silane coupling agent layer contains a functional group reactive with an SH group.
  4. 　前記ＳＨ基と反応性を有する官能基が、ハロゲン原子である請求項３に記載の生体物質固定用基材。 4. The biological material immobilizing substrate according to claim 3, wherein the functional group having reactivity with the SH group is a halogen atom.
  5. 　前記シランカップリング剤層のシランカップリング剤成分が、アミノ基および／またはＳＨ基を含み、当該アミノ基および／またはＳＨ基に貴金属コロイドが結合している請求項１に記載の生体物質固定用基材。 The biological material immobilization according to claim 1, wherein the silane coupling agent component of the silane coupling agent layer contains an amino group and / or an SH group, and a noble metal colloid is bonded to the amino group and / or the SH group. Base material.
  6. 　カバーガラスである、請求項１に記載の生体物質固定用基材。 The biological material fixing substrate according to claim 1, which is a cover glass.
  7. 　アルカリ成分を含有するガラス基材の表面にＳｉＯ2をスパッタリングして、ＳｉＯ2スパッタ膜を形成する工程、および
    　得られたＳｉＯ2スパッタ膜の表面をシランカップリング剤で処理する工程を含む生体物質固定用基材の製造方法。     A biological material comprising a step of sputtering SiO 2 on the surface of a glass substrate containing an alkali component to form a SiO 2 sputtered film, and a step of treating the surface of the obtained SiO 2 sputtered film with a silane coupling agent A method for producing a fixing substrate.
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