JP2003130882A - Microcircuit for analyzing protein - Google Patents
Microcircuit for analyzing proteinInfo
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- JP2003130882A JP2003130882A JP2001328028A JP2001328028A JP2003130882A JP 2003130882 A JP2003130882 A JP 2003130882A JP 2001328028 A JP2001328028 A JP 2001328028A JP 2001328028 A JP2001328028 A JP 2001328028A JP 2003130882 A JP2003130882 A JP 2003130882A
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- protein
- circuit
- solution
- microcircuit
- adsorption
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Abstract
Description
【発明の詳細な説明】
【０００１】
【発明の属する技術分野】本発明は、蛋白質の構造・機
能解析及び蛋白質を使用した反応に用いられる装置・機
器の内、蛋白質溶液の流動または反応または分析を行な
う微小回路に関する。
【０００２】
【従来の技術】現在、種々の化学反応を行なう為の微小
回路は反応効率、速度、省試薬の観点から注目されつつ
ある技術であり、既に「ラボオンアチップ」と呼ばれる
数センチ角の硝子製チップ上に形成された回路の中で化
学反応・分析を行なう新しい分析方法に関する概念が一
般的に定着している。今後、バイオテクノロジーの進展
に伴い生化学分野においても微小回路の利用は不可欠な
技術であり、特に蛋白質の構造・機能解析及び蛋白質を
使用した反応への微小回路の応用が期待されている。
【０００３】蛋白質溶液を微小回路に流す際に大きな問
題となるのは回路表面への蛋白質の吸着であり、微量蛋
白質は吸着による減少及び構造変化の影響を大きく受け
るだけでなく、回路をくり返し使用する場合には吸着し
た蛋白質が履歴として残る点、更に回路自体が微小であ
るために吸着残留した蛋白質によって回路が閉塞する恐
れのある点も問題となる。また微小回路内で２種類の溶
液を流動させた状態で反応を行なう場合には、２種類の
溶液が１本の回路内で相流を形成する事が重要である
が、吸着残留した蛋白質によって回路壁面に凹凸が形成
され、その結果乱流が発生し反応効率が低下してしまう
点は微小回路にとって非常に大きな問題である。
【０００４】現在微小回路に使用されている基材の殆ど
は硝子（石英硝子）若しくはプラスチック類であるが、
それらの基材に対して蛋白質は高い吸着性を示す。また
微小回路の加工性に関する問題点として、基材上に微小
回路を形成した際に表面に微細な凹凸が発生する場合が
あり、その様な凹凸は前述の理由により好ましくない。
【０００５】
【発明が解決しようとする課題】本発明の目的は感度、
精度、再現性を向上させる蛋白質分析用微小回路の提供
であり、回路壁面への蛋白質の吸着及び回路表面の平滑
性の向上が前記目的を達成する為の課題であると考え
た。
【０００６】
【課題を解決するための手段】即ち本発明は、蛋白質溶
液が接触する断面積１ｍｍ2以下の微小回路において、
回路の少なくとも蛋白質溶液と接触する表面が超親水性
ポリマーで被覆されている蛋白質分析用微小回路であ
る。
【０００７】
【発明の実施の形態】従来の硝子又はプラスチックへの
蛋白質吸着は短時間の接触で発生し、低濃度領域（約１
ｎｇ〜１００ｕｇ／ｍｌ）においてその吸着率（接触さ
せた蛋白質溶液中の蛋白質の内吸着する蛋白質の割合）
は、最大約５０％にも達し、一度吸着した蛋白質は不可
逆な構造変化（変性）を起こし、変性した蛋白質は二次
的な蛋白質の吸着を誘引し、結果として蛋白質の多層吸
着層が形成される。
【０００８】そこで、本発明においては蛋白質溶液が接
触する表面を超親水性ポリマーで被覆する事によって、
蛋白質の吸着を引き起こす最も大きな要因である疎水性
相互作用を低減し、蛋白質の初期の吸着を防止してい
る。超親水性ポリマーとは水との親和性に非常に優れた
ポリマーで、水に浸漬する事により表面に均一な自由水
層を保持し、その水接触角が０〜１度となる高分子材料
を指す。
【０００９】超親水性ポリマーの例としては、ポリヒド
ロキシアルキルメタクリレート、ポリオキシＣ２−Ｃ４
アルキレン基含有メタクリレート重合体又はこれを含む
共重合体、あるいはポリビニルピロリドン、リン脂質・
高分子複合体（特開平５−１６１４９１号公報及び特開
平６−４６８３１号公報）、２−メタクリロイルオキシ
エチルホスホリルコリン共重合体（以下ＭＰＣと略す）
又はこれを含む共重合体（特開平９−３１３２号公報）
などが挙げられる。
【００１０】超親水性ポリマーの被覆は蛋白質の吸着防
止以外の効果も有している。超親水性ポリマーは液体と
接触する事で自由水を含み膨潤し、その表面は平滑にな
る。すなわち、回路表面の乱流の原因となる加工時の凹
凸が超親水性ポリマーの被覆及び膨潤により低減され
る。超親水性ポリマーを被覆する際に注意すべき点は被
覆層の厚みである。超親水性ポリマーの被覆層が厚すぎ
ると、蛋白質溶液と接触して膨潤した際に回路を閉塞し
てしまう可能性があり、また、蛋白質の吸着性能も低下
するため被覆層の厚みは５ｕｍ以下が好ましく、表面が
完全に覆われていれば可能な限り薄い層であることが好
ましい。
【００１１】超親水性ポリマーを被覆する方法としては
特に限定するものでは無いが、超親水性ポリマーを溶媒
に溶解した溶液を回路内に充填し、回路の開口端に吸引
ポンプに接続した吸引ノズルをあて、充填した親水性ポ
リマー溶液を吸引し、回路表面に残留した親水性ポリマ
ーが乾燥するまで吸引を続ける方法が好ましい。前記被
覆方法によって、回路が閉塞する事無く超親水性の被覆
層が形成され、その厚みは親水性ポリマー溶液の粘度に
よって容易に調節する事が出来る。また、エアレーショ
ン乾燥によって平滑な被覆表面を得る事が出来る。以
下、実施例によって本発明を更に具体的に説明する。
【００１２】
【実施例】（実施例１）厚さ２ｍｍ、２０×３０ｍｍ角
のポリスチレンプレートの長軸方向にドリルを用いて直
径０．５ｍｍ、長さ３０ｍｍの直線状の回路を形成し
た。回路の一方の開口端からポリヒドロキシエチルメタ
クリレート（ＳＩＧＭＡ製Ｐ−３９３２）の２．５Ｗｔ
／ｖｏｌ％メタノール溶液を注入した。注入の際、０．
５ｍｍφの鈍針を先端に取り付けた２．５ｍｌのシリン
ジを使用した。注入後開口端にテープでふたをして３０
分間静置した後に、吸引ポンプ−トラップに接続したノ
ズルを開口端に押し当て、１分間吸引を行なった。更に
一晩乾燥させた。乾燥後のコート層の厚みを回路部分を
切り出して電子顕微鏡にて測定したところ、厚みは約
１．３ｕｍであった。
【００１３】（実施例２）実施例１で使用した物と同一
の回路の一方の開口端からＭＰＣポリマーの０．５ｗｔ
／ｖｏｌ％エタノール溶液を注入した。ＭＰＣポリマー
は、「リン脂質類似構造を有するハイドロゲル膜からの
薬物放出 高分子論文集，４６，５９１−５９５（１９
８９）」の内容に従いＭＰＣとＢＭＡ（ブチルメタクリ
レート）比＝３／７の共重合体を合成し使用した。注入
後開口端にテープでふたをして３０分間静置した後に、
吸引ポンプ−トラップに接続したノズルを開口端に押し
当て、１分間吸引を行なった。更に一晩乾燥させ、実施
例２とした。乾燥後のコート層の厚みを回路部分を切り
出して電子顕微鏡にて測定したところ、厚みは約０．５
ｕｍであった。
【００１４】（比較例１）厚さ２ｍｍ、２０×３０ｍｍ
角のポリスチレンプレートの長軸方向にドリルを用いて
直径０．５ｍｍ、長さ３０ｍｍの直線状の回路を形成し
たものを比較例１とした。
【００１５】（比較例２）厚さ２ｍｍ、２０×３０ｍｍ
角のガラス製プレートの長軸方向にドリルを用いて直径
０．５ｍｍ、長さ３０ｍｍの直線状の回路を形成したも
のを比較例２とした。
【００１６】（蛋白質吸着性の比較）実施例１、実施例
２及び比較例１、比較例２の回路部分に５０ｎｇ／ｍｌ
のウシアルブミン（ＢＳＡ）溶液２００ｕｌをくり返し
３０回循環させた後にＢＳＡ溶液の濃度を測定し、濃度
の変化率を求めた。ＢＳＡ溶液の濃度の測定は以下の手
順で実施した。
【００１７】回収したＢＳＡ溶液をＥＬＩＳＡ用プレー
ト（住友ベークライト製 スミロンＥＬＩＳＡ用プレー
トＨ）に分注し、３７℃で１時間インキュベート。その
後プレートウォッシャーを用いて３回洗浄を繰り返し
た。尚、洗浄液には０．０５％Ｔｗｅｅｎ２０含有リン
酸緩衝液（日水製薬製 ダルベッコＰＢＳ−ｐＨ７．
４）を使用した。
【００１８】その後、３％スキムミルク（コスモバイオ
製）リン酸緩衝液溶液を２５０μＬ／ウェルで分注し、
３７℃で１時間インキュベートした。その後プレートウ
ォッシャーを用いて３回洗浄を繰り返した。次にペルオ
キシターゼ標識坑ウシアルブミン抗体（コスモバイオ
製）の１．５ｕｇ／ｍｌリン酸緩衝液溶液を１００ｕｌ
／ウェルで分注し、室温で３０分静置した後、プレート
ウォッシャーを用いて３回洗浄を繰り返した。
【００１９】次にＴＭＢＺ基質緩衝液（住友ベークライ
ト製 スミロンペルオキシターゼ用発色キットＴ）を用
いて発色させた後に、プレートリーダーにて吸光度を測
定、検量線から濃度を求め、初期濃度からの変化率を求
めた。結果は表１の通りで、実施例１、実施例２共に比
較例１、比較例２に比べて蛋白質溶液中の蛋白質の濃度
変化が大幅に抑えられている事を確認した。
【００２０】
【表１】
【００２１】（回路閉塞性の比較）回路に吸着残留した
蛋白質が与える影響を比較する為に、実施例１、比較例
１を用いて以下の検討を実施した。実施例１、比較例１
各々の回路の開口端にシリコン製チューブを接着接続
し、その中に５００ｍｇ／ｍｌのＢＳＡリン酸緩衝液溶
液を充填し、そのチューブをぺリスターポンプに接続す
る事でＢＳＡ溶液を回路に連続的に循環させる事の出来
る装置を作製した。
【００２２】実施例１、比較例１各々に接続したぺリス
ターポンプを１時間稼動させた後に純水洗浄後乾燥、更
にポンプに接続した後にＢＳＡ溶液を１時間循環させる
作業を５日間（３０回）くり返し実施した後の回路の内
腔面の状態を観察した。その結果、実施例１においては
ＢＳＡの吸着層は確認されず回路内腔面は検討開始時の
状態を保持していたが比較例１においては内腔面に一様
にＢＳＡが付着しており、所々で瘤状のかたまりが確認
され、回路内でのＢＳＡ溶液の流れが妨げられている事
は明らかであった。
【００２３】
【発明の効果】以上述べた如く、本発明によれば下記の
優れた効果が得られる。第一に、回路表面への蛋白質の
吸着が無く、分析を行なう蛋白質の減少及び変性が発生
しないために、微量かつ高精度な分析が可能となる。第
二に、回路表面への蛋白質の吸着が無く、使用した蛋白
質が回路内に履歴として残る事無く、くり返し精度の優
れた分析を行なう事ができる。第三に、回路表面への蛋
白質の吸着が無く、回路内の乱流の発生が抑えられ閉塞
の問題も無い。第四に、超親水性ポリマーのコーティン
グ及び膨潤により回路表面の平滑性向上も期待される。Description: BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus / equipment used for analyzing the structure and function of a protein and for performing a reaction using a protein, and the flow or reaction or analysis of a protein solution. The present invention relates to a microcircuit that performs. 2. Description of the Related Art At present, microcircuits for performing various chemical reactions are attracting attention from the viewpoints of reaction efficiency, speed, and saving of reagents, and are already called "lab-on-a-chips". The concept of a new analysis method for performing a chemical reaction / analysis in a circuit formed on a glass chip is generally established. In the future, the use of microcircuits will be indispensable in the field of biochemistry with the development of biotechnology. In particular, application of microcircuits to protein structure and function analysis and reactions using proteins is expected. A major problem when flowing a protein solution through a microcircuit is the adsorption of the protein to the surface of the circuit. Trace proteins are not only greatly affected by the decrease and structural change due to the adsorption, but are also used repeatedly. In this case, there is a problem in that the adsorbed protein remains as a history, and further, there is a possibility that the circuit may be blocked by the adsorbed residual protein because the circuit itself is minute. When the reaction is carried out in a state where two kinds of solutions are fluidized in a microcircuit, it is important that the two kinds of solutions form a phase flow in one circuit. Irregularities are formed on the circuit wall, and as a result, turbulence is generated and the reaction efficiency is reduced, which is a very serious problem for a microcircuit. Most of substrates currently used for microcircuits are glass (quartz glass) or plastics.
Proteins exhibit high adsorptivity to these substrates. In addition, as a problem related to workability of the microcircuit, when a microcircuit is formed on a base material, fine irregularities may be generated on the surface, and such irregularities are not preferable for the above-described reason. [0005] The object of the present invention is sensitivity,
It is an object of the present invention to provide a microcircuit for protein analysis which improves the accuracy and reproducibility, and considers that the adsorption of the protein to the circuit wall surface and the improvement of the smoothness of the circuit surface are the problems for achieving the above object. That is, the present invention provides a microcircuit having a cross-sectional area of 1 mm 2 or less, which is in contact with a protein solution.
A protein analysis microcircuit wherein at least the surface of the circuit that comes into contact with the protein solution is coated with a superhydrophilic polymer. DETAILED DESCRIPTION OF THE INVENTION Conventional protein adsorption to glass or plastic occurs in a short time contact, and occurs in a low concentration region (approximately 1%).
ng to 100 ug / ml) (the ratio of adsorbed protein in the protein in the contacted protein solution)
Reaches a maximum of about 50%, and once adsorbed protein undergoes irreversible structural change (denaturation), the denatured protein induces secondary protein adsorption, and as a result, a multilayer adsorption layer of protein is formed. You. Therefore, in the present invention, by covering the surface in contact with the protein solution with a superhydrophilic polymer,
It reduces hydrophobic interactions, the biggest factor that causes protein adsorption, and prevents early protein adsorption. A superhydrophilic polymer is a polymer with a very good affinity for water, a polymer material that maintains a uniform free water layer on the surface when immersed in water and has a water contact angle of 0 to 1 degree. Point to. Examples of the superhydrophilic polymer include polyhydroxyalkyl methacrylate, polyoxy C2-C4
Alkylene group-containing methacrylate polymer or copolymer containing the same, or polyvinylpyrrolidone, phospholipid
Polymer composite (JP-A-5-161492 and JP-A-6-46831), 2-methacryloyloxyethyl phosphorylcholine copolymer (hereinafter abbreviated as MPC)
Or a copolymer containing the same (JP-A-9-3132)
And the like. [0010] The coating of the superhydrophilic polymer has effects other than the prevention of protein adsorption. The superhydrophilic polymer swells with free water upon contact with a liquid, and its surface becomes smooth. That is, irregularities during processing that cause turbulence on the circuit surface are reduced by coating and swelling of the superhydrophilic polymer. A point to be considered when coating the superhydrophilic polymer is the thickness of the coating layer. If the coating layer of the superhydrophilic polymer is too thick, it may block the circuit when swollen by contact with the protein solution, and the adsorption performance of the protein also decreases, so the thickness of the coating layer is 5 μm or less. Preferably, the layer is as thin as possible if the surface is completely covered. The method of coating the superhydrophilic polymer is not particularly limited, but a solution obtained by dissolving the superhydrophilic polymer in a solvent is filled in a circuit, and a suction nozzle connected to a suction pump at an open end of the circuit. , The filled hydrophilic polymer solution is suctioned, and suction is continued until the hydrophilic polymer remaining on the circuit surface is dried. By the coating method, a superhydrophilic coating layer is formed without blocking the circuit, and the thickness can be easily adjusted by the viscosity of the hydrophilic polymer solution. In addition, a smooth coating surface can be obtained by aeration drying. Hereinafter, the present invention will be described more specifically with reference to examples. EXAMPLE 1 A straight circuit having a diameter of 0.5 mm and a length of 30 mm was formed by using a drill in the longitudinal direction of a polystyrene plate having a thickness of 2 mm and a size of 20 × 30 mm. 2.5 Wt of polyhydroxyethyl methacrylate (SIGMA P-3932) from one open end of the circuit
/ Vol% methanol solution was injected. Upon injection, 0.
A 2.5 ml syringe with a 5 mmφ blunt needle attached to the tip was used. After injection, cover the open end with tape.
After standing still for one minute, the nozzle connected to the suction pump-trap was pressed against the open end, and suction was performed for one minute. It was further dried overnight. When the thickness of the dried coat layer was measured by an electron microscope after cutting out a circuit portion, the thickness was about 1.3 μm. (Example 2) From one open end of the same circuit as that used in Example 1, 0.5 wt.
/ Vol% ethanol solution was injected. MPC polymers are described in “Release of Drugs from Hydrogel Membrane Having Phospholipid-Like Structure”, Journal of Polymers, 46, 591-595 (19)
89), a copolymer having a ratio of MPC to BMA (butyl methacrylate) = 3/7 was synthesized and used. After the injection, after covering the open end with tape and leaving it to stand for 30 minutes,
The nozzle connected to the suction pump-trap was pressed against the open end, and suction was performed for one minute. Further drying was carried out overnight to obtain Example 2. When the thickness of the dried coat layer was measured by an electron microscope after cutting out a circuit portion, the thickness was about 0.5.
um. Comparative Example 1 Thickness 2 mm, 20 × 30 mm
Comparative Example 1 was formed by forming a linear circuit having a diameter of 0.5 mm and a length of 30 mm using a drill in the longitudinal direction of the square polystyrene plate. Comparative Example 2 Thickness 2 mm, 20 × 30 mm
Comparative Example 2 was formed by forming a linear circuit having a diameter of 0.5 mm and a length of 30 mm using a drill in the major axis direction of a square glass plate. (Comparison of protein adsorption properties) The circuit portions of Example 1, Example 2, Comparative Example 1, and Comparative Example 2 were added at 50 ng / ml.
200 ul of bovine albumin (BSA) solution was repeatedly circulated 30 times, then the concentration of the BSA solution was measured, and the rate of change of the concentration was determined. The measurement of the concentration of the BSA solution was performed according to the following procedure. The collected BSA solution was dispensed into an ELISA plate (Sumitomo Bakelite Sumilon ELISA plate H) and incubated at 37 ° C. for 1 hour. Thereafter, washing was repeated three times using a plate washer. The washing solution was a phosphate buffer containing 0.05% Tween 20 (Dulbecco PBS-pH7.
4) was used. Thereafter, a 3% skim milk (Cosmo Bio) phosphate buffer solution was dispensed at 250 μL / well,
Incubated at 37 ° C. for 1 hour. Thereafter, washing was repeated three times using a plate washer. Next, 100 ul of a 1.5 ug / ml phosphate buffer solution of peroxidase-labeled anti-bovine albumin antibody (manufactured by Cosmo Bio) was added.
/ Well, and allowed to stand at room temperature for 30 minutes, and then repeated washing three times using a plate washer. Next, after the color was developed using a TMBZ substrate buffer (Sumitomo Bakelite Sumilon Peroxidase Coloring Kit T), the absorbance was measured with a plate reader, the concentration was determined from a calibration curve, and the rate of change from the initial concentration was determined. I asked. The results are as shown in Table 1. It was confirmed that the change in the protein concentration in the protein solution was significantly suppressed in both Examples 1 and 2 as compared with Comparative Examples 1 and 2. [Table 1] (Comparison of Circuit Blocking Property) In order to compare the influence of the protein adsorbed and remaining on the circuit, the following examination was conducted using Example 1 and Comparative Example 1. Example 1, Comparative Example 1
A silicone tube is adhesively connected to the open end of each circuit, and a 500 mg / ml BSA phosphate buffer solution is filled therein, and the tube is connected to a peristaltic pump to continuously connect the BSA solution to the circuit. A device that can be circulated through the system was manufactured. Example 1 and Comparative Example 1 The operation of circulating a BSA solution for 1 hour after washing the pure water, drying after connecting the Lister pump connected to each of the pumps for 1 hour, and connecting to the pump for 5 days (30 times) ) The state of the lumen surface of the circuit after the repetition was observed. As a result, the adsorbed layer of BSA was not confirmed in Example 1, and the inner surface of the circuit maintained the state at the start of the examination. However, in Comparative Example 1, BSA was uniformly attached to the inner surface of the circuit. Nodules were observed in some places, and it was clear that the flow of the BSA solution in the circuit was obstructed. As described above, according to the present invention, the following excellent effects can be obtained. First, since no protein is adsorbed on the circuit surface and the amount of the protein to be analyzed is not reduced or denatured, the analysis can be performed with a small amount and high accuracy. Second, no protein is adsorbed on the circuit surface, and the protein used does not remain in the circuit as a history, and an analysis with excellent repeatability can be performed. Third, there is no adsorption of proteins to the circuit surface, and the occurrence of turbulence in the circuit is suppressed and there is no problem of blockage. Fourth, improvement in the smoothness of the circuit surface is expected by coating and swelling of the superhydrophilic polymer.
Claims (1)
下の微小回路において、回路の少なくとも蛋白質溶液と
接触する表面が超親水性ポリマーで被覆されている蛋白
質分析用微小回路。Claims: 1. A microcircuit having a cross-sectional area of 1 mm 2 or less, which is in contact with a protein solution, wherein at least the surface of the circuit in contact with the protein solution is coated with a superhydrophilic polymer. .
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| JP2001328028A JP3865614B2 (en) | 2001-10-25 | 2001-10-25 | Microcircuit for protein analysis |
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| JP2001328028A JP3865614B2 (en) | 2001-10-25 | 2001-10-25 | Microcircuit for protein analysis |
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| JP2006158127A Division JP2006250953A (en) | 2006-06-07 | 2006-06-07 | Microcircuit for analyzing protein |
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| JP3865614B2 JP3865614B2 (en) | 2007-01-10 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2006025352A1 (en) | 2004-08-30 | 2006-03-09 | Toray Industries, Inc. | Fractionation apparatus |
| WO2008102744A1 (en) | 2007-02-20 | 2008-08-28 | Toray Industries, Inc. | Method for production of molded resin article |
| US7795010B2 (en) | 2004-01-21 | 2010-09-14 | Toray Industries, Inc. | Fractionator and method of fractionation |
| JP2014130065A (en) * | 2012-12-28 | 2014-07-10 | Dainippon Printing Co Ltd | Surface structure body for protein adsorption inhibition, micro flow path and microchip |
-
2001
- 2001-10-25 JP JP2001328028A patent/JP3865614B2/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7795010B2 (en) | 2004-01-21 | 2010-09-14 | Toray Industries, Inc. | Fractionator and method of fractionation |
| WO2006025352A1 (en) | 2004-08-30 | 2006-03-09 | Toray Industries, Inc. | Fractionation apparatus |
| WO2008102744A1 (en) | 2007-02-20 | 2008-08-28 | Toray Industries, Inc. | Method for production of molded resin article |
| JP2014130065A (en) * | 2012-12-28 | 2014-07-10 | Dainippon Printing Co Ltd | Surface structure body for protein adsorption inhibition, micro flow path and microchip |
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| Publication number | Publication date |
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| JP3865614B2 (en) | 2007-01-10 |
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