JP2006132943A - Method of manufacturing biochip - Google Patents
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Abstract
Description
本発明は、生体試料中の多数の蛋白質、核酸等の並列検出および分析に用いられるバイオチップの製造方法に関する技術であり、さらに詳しくは、プロテオミクス、ならびに遺伝子活性の細胞内蛋白質レベルでの測定に用いられるバイオチップの製造方法に関するものである。 The present invention relates to a technique for producing a biochip used for parallel detection and analysis of a large number of proteins and nucleic acids in a biological sample. More specifically, the present invention relates to proteomics and measurement of gene activity at the intracellular protein level. The present invention relates to a method for producing a biochip to be used.
遺伝子活性の評価や、薬物効果の分子レベルでの生理的プロセスを解読するための試みは、伝統的にゲノミクスに焦点が当てられてきたが、プロテオミクスは、細胞の生物学的機能についてより詳細な情報を提供する。プロテオミクスは、遺伝子レベルというよりもむしろ、蛋白質レベルでの発現を検出しそして定量することによる、遺伝子活性の定性的かつ定量的な測定を含む。また、蛋白質の翻訳後修飾、蛋白質間の相互作用など遺伝子にコードされない事象の研究を含む。 Attempts to decipher the genetic activity and the molecular processes of drug effects at the molecular level have traditionally focused on genomics, but proteomics is more detailed about the biological functions of cells. Provide information. Proteomics involves the qualitative and quantitative measurement of gene activity by detecting and quantifying expression at the protein level rather than at the gene level. It also includes studies of events that are not encoded by genes such as post-translational modifications of proteins and interactions between proteins.
「生命の設計図」であるゲノムの構造が明らかにされ、膨大なゲノム情報の入手が可能となった今日、プロテオミクス研究はますます盛んになっており、それに伴って生理活性物質検出の迅速高効率（ハイスループット）化が求められている。この目的の分子アレイとして、ＤＮＡチップが開発され、実用化されつつある。一方、生体機能において最も複雑で多様性の高い蛋白質の検出に関してはプロテインチップが提唱され、近年研究が進められている。プロテインチップとは、蛋白質、またはそれを捕捉する分子をチップ（微小な基板）表面に固定化したものを総称する。 Now that the structure of the genome, which is the “blueprint of life”, has been clarified and a large amount of genome information has become available, proteomics research is becoming increasingly popular, and as a result, rapid detection of physiologically active substances is increasing. There is a need for efficiency (high throughput). A DNA chip has been developed and put into practical use as a molecular array for this purpose. On the other hand, protein chips have been proposed for the detection of the most complex and highly diverse proteins in biological functions, and research has been promoted in recent years. The protein chip is a general term for a protein or a molecule that captures the protein immobilized on the surface of the chip (micro substrate).
しかし、現状のプロテインチップは一般にＤＮＡチップの延長線上に位置付けられて開発がなされているため、ガラス基板上に蛋白質、またはそれを捕捉する分子をチップ表面に固定化する検討がなされている（例えば特許文献１参照）。 However, since the current protein chip is generally developed on the extension line of the DNA chip, studies have been made to immobilize a protein or a molecule for capturing the protein on the glass substrate on the surface of the chip (for example, Patent Document 1).
蛋白質を捕捉する分子（以下、捕捉分子と略す）を基板上に固定化した後、例えばサンドイッチ法のように該表面上で他の蛋白質（抗原抗体反応の場合、抗原に相当）と反応させ、更に、標識された蛋白質を反応させ最終的に検出機等で検出する場合、捕捉分子が固定されていない部分に該分子以外の蛋白質、即ち、抗原や標識された蛋白質が固定されると、検出時にノイズとなり正確な評価ができなくなる。 After immobilizing a protein-capturing molecule (hereinafter abbreviated as a capturing molecule) on a substrate, it is reacted with another protein (corresponding to an antigen in the case of an antigen-antibody reaction) on the surface, for example, as in the sandwich method, Furthermore, when a labeled protein is reacted and finally detected by a detector or the like, if a protein other than the molecule, that is, an antigen or a labeled protein is immobilized on a portion where the capture molecule is not immobilized, detection is performed. Sometimes it becomes noise and accurate evaluation is impossible.
また、すべての蛋白質（プロテオーム）の変動をプロファイリングする技術面では、超微量の蛋白質や数ナノリットルというような超微量の溶液の操作を可能とするマイクロフルイディクスの技術や、チップ上での前処理、分離、検出を目標とする「ラボ・オン・チップ」の概念が重要となってくる。この技術においては、サンプルである蛋白質などの生理活性物質が、流路内に固定化されたキャプチャーと特異的に反応し、かつキャプチャー部以外の流路の内壁への非特異吸着を抑制することが必要となる。
本発明は、蛋白質、またはそれを捕捉する分子等の生理活性物質を基板表面の任意の位置に固定化し、それ以外の部分には不要な生理活性物質が吸着しない、高感度でハイスループットな生理活性物質の検出ができるバイオチップの製造方法を提供することを目的とする。 The present invention is a highly sensitive and high-throughput physiological system in which a physiologically active substance such as a protein or a molecule that captures the protein is immobilized at an arbitrary position on the surface of the substrate, and an unnecessary physiologically active substance is not adsorbed to other parts. An object of the present invention is to provide a biochip manufacturing method capable of detecting an active substance.
すなわち本発明は、
（１） 基板表面に、生理活性物質を特異的に捕捉する物質であるキャプチャーをスポット状に固定化してなるバイオチップの製造方法であって、
（1）基板表面のキャプチャーを固定化する部分を予めマスキング物質で覆う工程、
（2）マスキング物質で覆われた部分を含む基板表面を生理活性物質を結合又は吸着を抑制するする物質でコートする工程、
（3）マスキング物質を除去する工程、
（4）マスキング物質を除去した部分にキャプチャーを含む溶液を接触する工程、を含むことを特徴とするバイオチップの製造方法、
（２） 生理活性物質を結合又は吸着を抑制する物質がホスホリルコリン基を有する高分子、ポリペプチドを有する高分子、スキムミルク、ウシ血清アルブミンより選択される少なくとも１種類である（１）記載のバイオチップの製造方法、
（３） キャプチャーが蛋白質である（１）又は（２）記載のバイオチップの製造方法、
（４） 更に、（5）基板表面処理としてガンマ線照射、プラズマ処理、紫外線照射の少なくとも１種からなる処理工程、を有する（１）〜（３）いずれか記載のバイオチップの製造方法、
（５） 基板の材質がプラスチックである（１）〜（４）いずれか記載のバイオチップの製造方法、
（６） プラスチックがポリカーボネート、ポリエチレン、ポリプロピレン、ポリスチレン、飽和環状ポリオレフィン、ポリペンテン、ポリアミド、及びそれらの共重合体よりなる群より選択された少なくとも１種である（５）記載のバイオチップの製造方法、
（７） 基板形状がスライドグラス状である（１）〜（６）いずれか記載のバイオチップの製造方法、
（８） 基板表面に流路を有し、流路内にキャプチャーを固定化することを特徴とする（１）〜（７）いずれか記載のバイオチップの製造方法、
である。
That is, the present invention
(1) A biochip manufacturing method in which a capture, which is a substance that specifically captures a physiologically active substance, is immobilized in a spot shape on a substrate surface,
(1) A step of previously covering a portion to fix the capture on the substrate surface with a masking substance,
(2) coating the surface of the substrate including the portion covered with the masking substance with a substance that binds or suppresses the adsorption of the physiologically active substance;
(3) a step of removing the masking substance,
(4) a step of contacting a solution containing a capture with the portion from which the masking substance has been removed,
(2) The biochip according to (1), wherein the substance that binds or adsorbs a physiologically active substance is at least one selected from a polymer having a phosphorylcholine group, a polymer having a polypeptide, skim milk, and bovine serum albumin. Manufacturing method,
(3) The method for producing a biochip according to (1) or (2), wherein the capture is a protein,
(4) Furthermore, (5) The manufacturing method of the biochip as set forth in any one of (1) to (3), further including (5) a processing step comprising at least one of gamma ray irradiation, plasma processing, and ultraviolet irradiation as substrate surface treatment,
(5) The biochip manufacturing method according to any one of (1) to (4), wherein the substrate material is plastic.
(6) The biochip production method according to (5), wherein the plastic is at least one selected from the group consisting of polycarbonate, polyethylene, polypropylene, polystyrene, saturated cyclic polyolefin, polypentene, polyamide, and copolymers thereof,
(7) The biochip manufacturing method according to any one of (1) to (6), wherein the substrate shape is a slide glass shape,
(8) The biochip manufacturing method according to any one of (1) to (7), wherein the substrate surface has a channel and the capture is immobilized in the channel.
It is.
本発明のバイオチップの製造方法によれば、蛋白質、またはそれを捕捉する分子等の生理活性物質を基板表面の任意の位置に固定化し、それ以外の部分には不要な生理活性物質が吸着しない、高感度でハイスループットな生理活性物質の検出ができるバイオチップを製造することが可能となる。 According to the method for producing a biochip of the present invention, a physiologically active substance such as a protein or a molecule that captures the protein is immobilized at an arbitrary position on the surface of the substrate, and an unnecessary physiologically active substance is not adsorbed to other portions. It is possible to produce a biochip capable of detecting a physiologically active substance with high sensitivity and high throughput.
本発明の製造方法は、基板作製工程、マスキング工程、基板表面処理（修飾）工程を含む。基板表面処理工程は省いても使用可能であるが、より強固に捕捉物を固定化する為に用いる事が好ましい。 The manufacturing method of the present invention includes a substrate manufacturing step, a masking step, and a substrate surface treatment (modification) step. Although it can be used even if the substrate surface treatment step is omitted, it is preferable to use it in order to immobilize the captured substance more firmly.
（基板の素材）
本発明に使用する基板の素材は、通常ガラス、金属その他を用いることができるが、本発明に使用する基板の素材としては、表面処理の容易性、量産性の観点から、プラスチックを使用し、特に熱可塑性樹脂が好ましい。熱可塑性樹脂としては、蛍光発生量の少ないものが好ましい。例えばポリエチレン、ポリプロピレン、ポリペンテン等の直鎖状ポリオレフィン、ポリカーボネート、ポリスチレン、ポリアミド、飽和環状ポリオレフィン、含フッ素樹脂等を用いることが好ましく、耐熱性、耐薬品性、低蛍光性、成形性に特に優れる飽和環状ポリオレフィンを用いることがより好ましい。ここで飽和環状ポリオレフィンとは、環状オレフィン構造を有する重合体単独または環状オレフィンとα−オレフィンとの共重合体を水素添加した飽和重合体等を指す。
本発明に使用する基板の形状は、特に限定しないが、スライドグラス状であることが好ましい。その他、基板表面に流路を有し、流路内にキャプチャーを固定化することも好ましい実施形態の一つである。
(Substrate material)
The material of the substrate used in the present invention can usually be glass, metal or the like, but as the material of the substrate used in the present invention, from the viewpoint of ease of surface treatment and mass productivity, plastic is used, A thermoplastic resin is particularly preferable. As a thermoplastic resin, a thing with little fluorescence generation amount is preferable. For example, it is preferable to use linear polyolefin such as polyethylene, polypropylene, polypentene, polycarbonate, polystyrene, polyamide, saturated cyclic polyolefin, fluorine-containing resin, etc., and saturation that is particularly excellent in heat resistance, chemical resistance, low fluorescence, and moldability. It is more preferable to use a cyclic polyolefin. Here, the saturated cyclic polyolefin refers to a polymer having a cyclic olefin structure or a saturated polymer obtained by hydrogenating a copolymer of a cyclic olefin and an α-olefin.
The shape of the substrate used in the present invention is not particularly limited, but is preferably a slide glass shape. In addition, it is one of preferred embodiments to have a channel on the surface of the substrate and immobilize the capture in the channel.
（基板のマスキング）
本発明の基板のマスキングに使用する物質としては、基板に固定化でき、マスキング部分を含む基板表面を生理活性物質を結合および又は吸着を抑制する物質でコートした後マスキング物質を除去したときに、マスキング部分に前記マスキング物質が存在しない状態を作り出すことができる物質であればよく、例えばゴム系の物質が挙げられ、特にシリコーンゴムが好ましい。
(Board masking)
As a substance used for masking the substrate of the present invention, when the masking substance is removed after coating the surface of the substrate including the masking portion with a substance that binds and / or adsorbs a physiologically active substance, Any material can be used as long as it can create a state in which the masking material is not present in the masking portion. For example, a rubber-based material can be used, and silicone rubber is particularly preferable.
本発明においては、基板表面のキャプチャーを固定化する部分を予めマスキング物質でスポット状に覆い、次いでマスキング物質で覆われた部分を含む基板表面を生理活性物質を結合又は吸着を抑制する物質でコートし、その後マスキング物質を除去することが好ましい。
生理活性物質を結合又は吸着を抑制する物質としては、ホスホリルコリン基を有する高分子、ポリペプチドを有する高分子、スキムミルク、ウシ血清アルブミンより選択される少なくとも１種類であることが好ましい。
In the present invention, the portion of the substrate surface where the capture is immobilized is covered in advance with a masking substance in the form of a spot, and then the substrate surface including the portion covered with the masking substance is coated with a substance that inhibits binding or adsorption of the physiologically active substance. Thereafter, it is preferable to remove the masking substance.
The substance that binds or suppresses the adsorption of a physiologically active substance is preferably at least one selected from a polymer having a phosphorylcholine group, a polymer having a polypeptide, skim milk, and bovine serum albumin.
（基板の表面処理）
本発明において、基板の表面処理（修飾）を行うことが好ましい。表面処理方法としては種々の方法が用いられるが、ガンマ線処理を行うと基板表面が改質され、生理活性物質がより強固に固定されるので好ましい。その他にはプラズマ処理、紫外線処理等が挙げられる。
基板の表面処理は、マスキング物質を除去した後に行うことが好ましい。
(Surface treatment of substrate)
In the present invention, it is preferable to perform surface treatment (modification) of the substrate. Although various methods are used as the surface treatment method, it is preferable to perform gamma ray treatment since the substrate surface is modified and the physiologically active substance is more firmly fixed. Other examples include plasma treatment and ultraviolet treatment.
The substrate surface treatment is preferably performed after removing the masking substance.
以下、実施例を挙げて本発明を更に具体的に説明するが、この発明の技術的範囲はこれら実施例に限定されるものではない。
（実施例）
ポリスチレン樹脂をスライドガラス形状（寸法：７６ｍｍ×２６ｍｍ×１ｍｍ）の基板に加工した。この基板表面に、主剤と硬化剤からなる液状シリコーンゴムを混合させ、即座にスポット状に塗布して硬化させたのち、２−メタクリロイルオキシエチルホスホリルコリン−ブチルメタクリレート共重合体の０．５重量％エタノール溶液に浸漬することにより、基板のシリコン塗布部以外に蛋白無吸着処理を施した。次いでシリコーンゴムを取り除いた。
次に、この基板に１０ｋＧｙのガンマ線を照射して表面の改質を行った。
続いて、基板のシリコン塗布部に一次抗体として抗マウスＩｇＧ２ａ抗体をスポットし、室温４℃、飽和状態の湿度の環境下に２４時間静置して固定化させた。固定化後、０．０５％Ｔｗｅｅｎ２０含有のＰＢＳで洗浄を行った。その後、抗原であるマウスＩｇＧ２ａ抗体および血清蛋白の混合物をＣｙ３標識したものを反応させ、各スポットについて蛍光量測定を行い、その際のＳ／Ｎ比（Signal/noise ratio）を計算した。結果を表１に示す。
EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples. However, the technical scope of the present invention is not limited to these examples.
(Example)
Polystyrene resin was processed into a glass substrate (size: 76 mm × 26 mm × 1 mm). A liquid silicone rubber composed of a main agent and a curing agent was mixed on the surface of the substrate, and immediately applied in a spot shape and cured, and then 0.5 wt% ethanol of 2-methacryloyloxyethyl phosphorylcholine-butyl methacrylate copolymer. By immersing in the solution, a protein non-adsorption treatment was performed in addition to the silicon coated portion of the substrate. The silicone rubber was then removed.
Next, the surface was modified by irradiating the substrate with 10 kGy of gamma rays.
Subsequently, an anti-mouse IgG2a antibody was spotted as a primary antibody on the silicon-coated portion of the substrate, and was allowed to stand for 24 hours in an environment of room temperature 4 ° C. and saturated humidity to be immobilized. After immobilization, washing with PBS containing 0.05% Tween 20 was performed. Thereafter, a mixture of an antigen mouse IgG2a antibody and serum protein labeled with Cy3 was reacted, the amount of fluorescence was measured for each spot, and the S / N ratio (Signal / noise ratio) at that time was calculated. The results are shown in Table 1.
（比較例）
飽和環状ポリオレフィン樹脂をスライドガラス形状（寸法：７６ｍｍ×２６ｍｍ×１ｍｍ）に加工した。基板表面に親水化処理を施したのち、アミノ基含有アルキルシランの２％水溶液中に浸漬後、熱処理を施して表面にアミノ基を導入した。これを１％グルタルアルデヒド水溶液中に浸漬することにより、表面のアミノ基とグルタルアルデヒドを反応させ、アルデヒド基を導入した。
次に、該基板に一次抗体として抗マウスＩｇＧ２ａ抗体をスポットし、室温４℃、飽和状態の湿度の環境下に２４時間静置して固定化させた。固定化後、非特異吸着防止の為に大日本製薬（株）製免疫実験用ブロッキング剤「ブロックエース」を純水で４倍希釈した溶液に該基板を浸し、室温で１時間静かに振とうした。その後、０．０５％Ｔｗｅｅｎ２０含有のＰＢＳで洗浄を行った。続いて、抗原であるマウスＩｇＧ２ａ抗体および血清蛋白の混合物をＣｙ３標識したものを反応させ、各スポットについて蛍光量測定を行い、その際のＳ／Ｎ比（Signal/noise ratio）を計算した。結果を表１に示す。
(Comparative example)
The saturated cyclic polyolefin resin was processed into a slide glass shape (dimensions: 76 mm × 26 mm × 1 mm). After subjecting the substrate surface to hydrophilization, the substrate was immersed in a 2% aqueous solution of an amino group-containing alkylsilane and then subjected to heat treatment to introduce amino groups on the surface. This was immersed in a 1% glutaraldehyde aqueous solution to react the surface amino groups with glutaraldehyde to introduce aldehyde groups.
Next, an anti-mouse IgG2a antibody was spotted on the substrate as a primary antibody, and was allowed to stand for 24 hours in a saturated humidity environment at room temperature of 4 ° C. to be immobilized. After immobilization, the substrate is immersed in a solution obtained by diluting Blocking Ace, a blocker for immunity experiments manufactured by Dainippon Pharmaceutical Co., Ltd., with pure water 4 times to prevent nonspecific adsorption, and gently shaken at room temperature for 1 hour. did. Thereafter, washing was performed with PBS containing 0.05% Tween20. Subsequently, a mixture of an antigen mouse IgG2a antibody and serum protein labeled with Cy3 was reacted, the amount of fluorescence was measured for each spot, and the S / N ratio (Signal / noise ratio) at that time was calculated. The results are shown in Table 1.
実施例における蛍光量の測定には、Packard BioChip Technologies社製バイオチップスキャナー「ScanArray」を用いた。測定条件は、レーザー出力９０％、ＰＭＴ感度５０％、励起波長５５０ｎｍ、測定波長５７０ｎｍ、解像度３０μｍであった。
実施例は、蛍光強度では比較例より高く、バックグラウンド値は比較例に比べて格段に低く、Ｓ／Ｎ比も大きかった。すなわち、高感度な生理活性物質の検出ができたことが確認された。
A biochip scanner “ScanArray” manufactured by Packard BioChip Technologies was used to measure the amount of fluorescence in the examples. The measurement conditions were laser output 90%, PMT sensitivity 50%, excitation wavelength 550 nm, measurement wavelength 570 nm, and resolution 30 μm.
In the examples, the fluorescence intensity was higher than that of the comparative example, the background value was much lower than that of the comparative example, and the S / N ratio was also large. That is, it was confirmed that a highly sensitive physiologically active substance could be detected.
本発明のバイオチップの製造方法によれば、蛋白質、またはそれを捕捉する分子等の生理活性物質を基板表面の任意の位置に固定化し、それ以外の部分には不要な生理活性物質が吸着しない、高感度でハイスループットな生理活性物質の検出が可能なバイオチップを提供することができるので、マイクロフルイディクスを含む各種バイオチップの製造に適用できる。 According to the method for producing a biochip of the present invention, a physiologically active substance such as a protein or a molecule that captures the protein is immobilized at an arbitrary position on the surface of the substrate, and an unnecessary physiologically active substance is not adsorbed to other portions. Since a biochip capable of detecting a bioactive substance with high sensitivity and high throughput can be provided, it can be applied to the production of various biochips including microfluidics.
Claims (8)
（１）基板表面のキャプチャーを固定化する部分を予めマスキング物質で覆う工程、
（２）マスキング物質で覆われた部分を含む基板表面を生理活性物質を結合又は吸着を抑制する物質でコートする工程、
（３）マスキング物質を除去する工程、
（４）マスキング物質を除去した部分にキャプチャーを含む溶液を接触する工程、を含むことを特徴とするバイオチップの製造方法。 A biochip manufacturing method in which a capture, which is a substance that specifically captures a physiologically active substance, is immobilized in a spot shape on a substrate surface,
(1) A step of previously covering a portion for fixing the capture on the substrate surface with a masking substance,
(2) A step of coating a substrate surface including a portion covered with a masking substance with a substance that binds or adsorbs a physiologically active substance,
(3) a step of removing the masking substance;
(4) A method for producing a biochip, comprising a step of contacting a solution containing a capture with a portion from which a masking substance has been removed.
The biochip manufacturing method according to claim 1, wherein a flow path is provided on a substrate surface, and a capture is immobilized in the flow path.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012511718A (en) * | 2008-12-11 | 2012-05-24 | コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ | Detection device for detecting a target component in a fluid |
| JP2012108035A (en) * | 2010-11-18 | 2012-06-07 | Sumika Chemical Analysis Service Ltd | Carrier for immunoreaction measurement, method for producing the same, device for immunoreaction measurement using the same, immunoreaction measuring kit, and immunoreaction measuring method |
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2004
- 2004-11-02 JP JP2004318752A patent/JP2006132943A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2012511718A (en) * | 2008-12-11 | 2012-05-24 | コーニンクレッカ フィリップス エレクトロニクス エヌ ヴィ | Detection device for detecting a target component in a fluid |
| JP2012108035A (en) * | 2010-11-18 | 2012-06-07 | Sumika Chemical Analysis Service Ltd | Carrier for immunoreaction measurement, method for producing the same, device for immunoreaction measurement using the same, immunoreaction measuring kit, and immunoreaction measuring method |
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