JP2010266218A - Immunological sensor chip and immunological sensor device equipped with the same - Google Patents
Immunological sensor chip and immunological sensor device equipped with the same Download PDFInfo
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Abstract
Description
本発明は、液体試料中の標的物質の分析、特に免疫学的反応を用いた分析装置に関する。 The present invention relates to analysis of a target substance in a liquid sample, and particularly to an analysis apparatus using an immunological reaction.
医療などの分野において、生体中の標的物質の存在及びその量を分析することは有益である。中でも、標的物質に特異的に反応する抗体を用いた免疫学的分析法は最もよく用いられる方法の一つである。免疫学的分析法には、反応の検出方法により様々な測定法が存在する。例えば、標識物質を抗体に連結させた試薬を用い抗原抗体反応が生じたかどうかを標識物質により検知する標識免疫測定法が知られている。本方法は、標識物質に酵素、蛍光物質、放射性同位体を用いた方法が知られている。これらはそれぞれ、酵素免疫測定法、蛍光物質標識免疫測定法、放射線免疫測定法と呼ばれている。また、抗体を担持させた粒子状試薬を用い、抗原抗体反応が生じたかどうかを粒子状試薬の凝集状態により検知する、凝集反応に基づく免疫測定法が知られている。この代表的なものとしてラテックス粒子を用いるラテックス凝集法等がある。標識免疫測定法は高精度の分析が可能であるが、用いる試薬が不安定であることや、測定時間が比較的長いという問題がある。一方、凝集反応にもとづく免疫測定法であるラテックス凝集法は、分析精度はあまり高くないものの、測定時間が比較的短いという利点を有している。 In fields such as medicine, it is useful to analyze the presence and amount of a target substance in a living body. Among them, an immunological analysis method using an antibody that specifically reacts with a target substance is one of the most frequently used methods. There are various measurement methods for immunological analysis depending on the detection method of the reaction. For example, a labeled immunoassay method is known in which a reagent in which a labeling substance is linked to an antibody is used to detect whether or not an antigen-antibody reaction has occurred using the labeling substance. As this method, a method using an enzyme, a fluorescent substance, and a radioisotope as a labeling substance is known. These are called enzyme immunoassay, fluorescent substance-labeled immunoassay, and radioimmunoassay, respectively. Further, an immunoassay method based on an agglutination reaction is known in which a particulate reagent carrying an antibody is used to detect whether an antigen-antibody reaction has occurred or not based on the agglutination state of the particulate reagent. A typical example is a latex agglomeration method using latex particles. Although the labeled immunoassay can perform highly accurate analysis, there are problems that the reagent used is unstable and the measurement time is relatively long. On the other hand, the latex agglutination method, which is an immunoassay based on the agglutination reaction, has the advantage that the measurement time is relatively short, although the analysis accuracy is not so high.
ラテックス凝集法は次のように行われる。まず、標的物質に特異的に反応する抗体をラテックス粒子に担持（固定）する。当該ラテックス粒子と試料を反応させ、試料中に含まれる標的物質とラテックス粒子に担持された抗体を反応させる（抗原抗体反応）。当該反応の進行に従いラテックス粒子は凝集するので、その凝集塊を光学的に検出して分析対象物の濃度を測定する。 The latex agglomeration method is performed as follows. First, an antibody that specifically reacts with a target substance is supported (fixed) on latex particles. The latex particles are reacted with the sample, and the target substance contained in the sample is reacted with the antibody supported on the latex particles (antigen-antibody reaction). Latex particles aggregate as the reaction proceeds, so the aggregate is optically detected and the concentration of the analyte is measured.
ラテックス凝集法における測定感度・測定時間は、用いるラテックス粒子の粒子径によって異なり、ラテックス粒子径が大きければ測定感度は高くなるが、測定時間が長くなる。粒子径が小さければ測定感度は低くなるが、測定時間が短くなる。通常、当該方法で用いられるラテックス粒子の粒子径は数百ｎｍであり、測定可能な領域はｎＭ程度である。ラテックス凝集法はいくつかの改良方法が報告されており、特許文献１には、粒径０．５μｍ〜１０μｍのラテックス粒子を用い、試料液に交流電圧を印加して電場をかけることにより、粒子を一直線上に並べる方法が開示されている。この方法によれば、１分で全粒子の９０％を凝集させることが可能であり、比較的大きなラテックス粒子を用いて測定感度を高めつつ、測定時間を短くすることができる。 The measurement sensitivity and measurement time in the latex agglutination method vary depending on the particle size of the latex particles used. If the latex particle size is large, the measurement sensitivity is high, but the measurement time is long. If the particle size is small, the measurement sensitivity is low, but the measurement time is short. Usually, the particle diameter of latex particles used in the method is several hundred nm, and the measurable region is about nM. Several improved methods for the latex agglutination method have been reported. Patent Document 1 uses latex particles having a particle diameter of 0.5 μm to 10 μm, and applies an electric field by applying an AC voltage to the sample liquid. A method of arranging the lines in a straight line is disclosed. According to this method, 90% of all particles can be aggregated in one minute, and the measurement time can be shortened while increasing the measurement sensitivity using relatively large latex particles.
しかしながら、ラテックス凝集法及び特許文献１で示したラテックス凝集改良法は、単一の標的物質を検出するために用いられるものの、複数項目を同時に検出することには適していない。仮に検出手段によって分類可能な複数種類のラテックス粒子を同時に用い、ラテックス粒子毎に異なる項目を検出しようとしても、種類の異なるラテックス粒子が同種ラテックス粒子間の凝集反応を邪魔する物理的な障害となるため、凝集効率が落ちてしまう。そのため、通常は検査前に検体を分注し、複数種類のラテックス粒子と各々混合するという煩雑な作業を行い、複数項目を個別に検査する必要があった。 However, the latex agglutination method and the latex agglutination improvement method shown in Patent Document 1 are used for detecting a single target substance, but are not suitable for detecting a plurality of items simultaneously. Even if multiple types of latex particles that can be classified by the detection means are used at the same time and an attempt is made to detect different items for each latex particle, different types of latex particles are a physical obstacle that disturbs the agglutination reaction between the same types of latex particles. Therefore, the aggregation efficiency is lowered. Therefore, it is usually necessary to individually inspect a plurality of items by performing a complicated operation of dispensing a specimen before mixing and mixing with a plurality of types of latex particles.
本発明は上記の課題を解決するためのもので、ラテックス凝集反応において複数項目を同時に検出するための免疫センサチップおよびそれを備えた免疫センサ装置を提供することを目的とする。 An object of the present invention is to provide an immunosensor chip for simultaneously detecting a plurality of items in a latex agglutination reaction and an immunosensor device including the same.
本発明の免疫センサチップは、標的物質が含まれる検体と、前記標的物質に特異的に結合する生体親和性分子が担持された粒子とを含む試料が配置される試料収納部と、粒径に基づいて前記粒子を分離する粒子分離手段と、前記試料に電圧を印加して前記粒子を凝集させるための一対の電極とを備える。粒径に基づいて粒子を分離する粒子分離手段を備えるので、粒径ごとに分離させて粒子を凝集させることができる。従って、それらの粒子の凝集状態を観察すれば、一度の測定で複数種類の標的物質の検出が可能となる。 The immunosensor chip of the present invention includes a sample storage portion in which a sample including a specimen containing a target substance and particles carrying biocompatible molecules that specifically bind to the target substance is disposed, And a pair of electrodes for aggregating the particles by applying a voltage to the sample. Since the particle separation means for separating the particles based on the particle diameter is provided, the particles can be separated for each particle diameter to aggregate the particles. Therefore, by observing the aggregation state of these particles, it is possible to detect a plurality of types of target substances with a single measurement.
前記粒子分離手段は階段状の段差であることが好ましい。粒径の違いによって、高い精度で粒子を分離することができる。 The particle separation means is preferably a stepped step. Depending on the difference in particle size, the particles can be separated with high accuracy.
前記試料収納部は、試料注入口、空気口、および前記試料注入口から前記空気口に向かって伸びる流路を有し、前記階段状の段差は、前記注入口から前記空気口の方向に連続的に占有面積を増すように形成されていることが好ましい。粒径の違いによって、より高い精度で粒子を分離することができる。 The sample storage unit has a sample injection port, an air port, and a flow path extending from the sample injection port toward the air port, and the stepped step is continuous from the injection port toward the air port. It is preferable to form so as to increase the occupied area. Depending on the difference in particle size, the particles can be separated with higher accuracy.
他の一対の電極をさらに有するのが好ましい。一対の電極は前記流路の伸びる方向に対して平行に形成され、他の一対の電極は前記流路の伸びる方向に対して垂直方向に形成されるのが好ましい。粒径の違いによって、より高い精度で粒子を分離することができる。 It is preferable to further include another pair of electrodes. Preferably, the pair of electrodes are formed in parallel to the direction in which the flow path extends, and the other pair of electrodes are formed in a direction perpendicular to the direction in which the flow path extends. Depending on the difference in particle size, the particles can be separated with higher accuracy.
本発明の免疫センサ装置は、前記免疫センサチップと、前記粒子分離手段によって分離された前記粒子の分布状態を観察する試料観察手段とを有する。試料観察手段によって、粒径ごとに分離された粒子の分布状態（凝集状態）を観察できるので、一度の測定で複数種類の標的物質の検出が可能となる。 The immunosensor device of the present invention includes the immunosensor chip and sample observation means for observing the distribution state of the particles separated by the particle separation means. Since the sample observation means can observe the distribution state (aggregation state) of the particles separated for each particle size, a plurality of types of target substances can be detected by one measurement.
本発明の免疫センサ装置は、前記免疫センサチップと、電圧印加手段を有する。前記電圧印加手段は、前記他の一対の電極に電圧を印加した後に、前記一対の電極に電圧を印加してもよい。より高い精度で、粒径の違いによって粒子を分離させた状態で凝集させることができる。 The immunosensor device of the present invention includes the immunosensor chip and voltage application means. The voltage applying means may apply a voltage to the pair of electrodes after applying a voltage to the other pair of electrodes. It is possible to agglomerate the particles in a state of being separated by the difference in particle size with higher accuracy.
本発明の免疫センサチップは、粒径に基づいて粒子を分離する粒子分離手段を備えるので、粒径ごとに分離させて粒子を凝集させることができる。従って、それらの粒子の凝集状態を観察すれば、一度の測定で複数種類の標的物質の検出が可能となる。 Since the immunosensor chip of the present invention includes particle separation means for separating particles based on the particle size, the particles can be separated for each particle size to aggregate the particles. Therefore, by observing the aggregation state of these particles, it is possible to detect a plurality of types of target substances with a single measurement.
以下本発明を実施形態を図面を参照しながら説明する。
（実施の形態１）
図１は、本発明の第一の態様における免疫センサ装置の模式図である。本発明の免疫センサ装置１１１は、センサチップ１１２と、センサチップ１１２に形成された電極４に連結された交流電圧印加装置１１４、光学検出手段（試料観察手段）１１３からなる。センサチップ１１２の詳細は後述するが、少なくとも１つの流路１３が設けられ、光学検出手段１１３により流路１３（試料収納部）内部の複数個所が観察できるように構成されている。
Embodiments of the present invention will be described below with reference to the drawings.
(Embodiment 1)
FIG. 1 is a schematic diagram of an immunosensor device according to the first embodiment of the present invention. The immunosensor device 111 of the present invention includes a sensor chip 112, an AC voltage application device 114 connected to an electrode 4 formed on the sensor chip 112, and an optical detection means (sample observation means) 113. Although details of the sensor chip 112 will be described later, at least one flow path 13 is provided, and the optical detection unit 113 is configured to be able to observe a plurality of locations inside the flow path 13 (sample storage unit).
図２は、本発明の第一の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、少なくとも１対の電極４が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 2 is an exploded perspective view of the sensor chip in the first embodiment of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower base 3 is provided with at least one pair of electrodes 4, part of which is exposed to the hollow structure forming the flow path 13.
図３は、本発明の第一の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある階段状の段差である。 FIG. 3 is an inverted view of the upper cover of the sensor chip in the first embodiment of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a stepped step between the inlet 11 and the air port 12.
以下に図面を参照し本発明について詳細に説明する。
前記の通り、本発明のセンサ装置に含まれるセンサチップは中空構造、すなわち試料を充填するための空間を有する。センサチップは単に「チップ」とも呼ばれる。チップの形状には制限はないが、直方体であることが好ましい。
Hereinafter, the present invention will be described in detail with reference to the drawings.
As described above, the sensor chip included in the sensor device of the present invention has a hollow structure, that is, a space for filling the sample. The sensor chip is also simply called “chip”. The shape of the chip is not limited, but is preferably a rectangular parallelepiped.
当該空間の内部には少なくとも１つの流路が形成されている。流路の数を増減することも可能である。空間の形状は制限されないが、例えば直方体であることが好ましい。当該流路には注入口と空気口が形成される必要があるが、その場所は流路内にその一部が露出している電極をはさむように形成されることが望ましい。当該流路への試料の充填は毛細管力を用いて行うことができる。また、ポンプ圧によって行わうことも可能であり、注入口へポンプを接続し、空気口は排気口として用いることで実現できる。ポンプの種類に制限はない。当該流路の内壁を形成する材料は、ＰＥＴ（ポリエチレンテレフタレート）やポリカーボネート等のポリマーフィルム、ガラスなどを用いることができる。上記材料は、より良くは表面性質が親水性であればよく、界面活性剤、リン脂質、蛋白質で処理されていてもよい。これらには公知のものが用いられる。また、センサチップの内部空間に存在する粒子像を顕微鏡で観察できる程度に透明性の高いものを用いる。スペーサーは、接着剤、光硬化樹脂、両面粘着テープなどを用いて形成することができ、その材質による制限はない。電極の作製は、スパッタリングや蒸着を用いることができ、電極素材には、金や銀などを用いることができる。本発明のセンサチップの上カバーに形成される粒子分離手段は注入口と空気口の間にある階段状の段差であり、ポリマーの射出成型やガラス切削により作製することができる。この段差と下基盤との間に形成される流路高は、標的物質検出に使用する微粒子群のうち、より大きな粒子群の平均直径よりも小さいことが必要であるが、検出に使用する微粒子の種類により適宜設定することができる。 At least one flow path is formed inside the space. It is also possible to increase or decrease the number of flow paths. The shape of the space is not limited, but is preferably a rectangular parallelepiped, for example. An inlet and an air port need to be formed in the flow path, but the place is preferably formed so as to sandwich an electrode that is partially exposed in the flow path. Filling the channel with the sample can be performed using capillary force. It can also be performed by pump pressure, and can be realized by connecting a pump to the injection port and using the air port as an exhaust port. There are no restrictions on the type of pump. As a material for forming the inner wall of the flow path, polymer films such as PET (polyethylene terephthalate) and polycarbonate, glass, and the like can be used. The above-mentioned material only needs to have a hydrophilic surface property, and may be treated with a surfactant, phospholipid, or protein. Known materials are used for these. Further, a particle having such a high transparency that a particle image existing in the internal space of the sensor chip can be observed with a microscope is used. The spacer can be formed using an adhesive, a photo-curing resin, a double-sided adhesive tape, and the like, and is not limited by the material. The electrode can be produced by sputtering or vapor deposition, and gold or silver can be used as the electrode material. The particle separation means formed on the upper cover of the sensor chip of the present invention is a stepped step between the injection port and the air port, and can be produced by polymer injection molding or glass cutting. The flow path height formed between the step and the lower substrate needs to be smaller than the average diameter of the larger particles among the particles used for target substance detection. It can be set appropriately depending on the type of the above.
本発明のセンサチップの分析対象は液体試料であり、より詳しくは液体試料中に存在する標的物質である。液体試料は特に限定されないが、その例には生体から採取した血液等が含まれる。本発明のセンサチップの分析対象である液体試料には、不溶性担体を分析試薬として混合して用いる。不溶性担体（単に「担体粒子」「粒子」ともいう。）には、市販の生化学用ビーズを用いることができるが、好ましくはラテックスである。粒子の直径に関しては、光学検出手段により粒子を観察できる径であればよく、好ましくは１〜１０μｍである。本発明においては少なくとも２種類の異なる粒径の粒子を用いるが、好ましくは直径差が２倍となるように選択することが好ましい。粒子表面には、液体試料中の標的物質に特異的に結合する物質が担持されている。「液体試料中の標的物質に特異的に結合する物質」とは、液体試料中の標的物質（分析対象物）の特定部位を認識して当該部位に結合できる物質をいう。このような物質の例には抗体、抗原、受容体、酵素、核酸、ペプチドなどの生体高分子が含まれる。本発明においては、「液体試料中の特定成分に特異的に結合する物質」は「抗体」であることが好ましい。抗体には公知のものを用いることができる。その例には抗アルブミン抗体、抗ＨＣＧ抗体、抗ＩｇＡ抗体、抗ＩｇＭ抗体、抗ＩｇＥ抗体、抗ＩｇＤ抗体、抗ＡＦＰ抗体、抗ＤＮＴ抗体、抗プロスタグランジン抗体、抗ヒト凝固ファクター抗体、抗ＣＲＰ抗体、抗ＨＢｓ抗体、抗ヒト成長ホルモン抗体、抗ステロイドホルモン抗体等が含まれる。抗原の例にはアルブミン、ＨＣＧ、ＩｇＡ、ＩｇＭ、ＩｇＥ、ＩｇＤ、ＡＦＰ、ＤＮＴ、プロスタグランジン、ヒト凝固ファクター、ＣＲＰ、ＨＢｓ、ヒト成長ホルモン、ステロイドホルモン等が含まれる。 The analysis target of the sensor chip of the present invention is a liquid sample, and more specifically, a target substance present in the liquid sample. The liquid sample is not particularly limited, but examples thereof include blood collected from a living body. In the liquid sample to be analyzed by the sensor chip of the present invention, an insoluble carrier is mixed and used as an analysis reagent. As the insoluble carrier (also simply referred to as “carrier particles” or “particles”), commercially available biochemical beads can be used, but latex is preferred. The diameter of the particles may be any diameter as long as the particles can be observed by an optical detection means, and is preferably 1 to 10 μm. In the present invention, at least two kinds of particles having different particle diameters are used, but it is preferable to select such that the difference in diameter is doubled. A substance that specifically binds to the target substance in the liquid sample is supported on the particle surface. “A substance that specifically binds to a target substance in a liquid sample” refers to a substance that can recognize a specific part of a target substance (analyte) in the liquid sample and bind to the part. Examples of such substances include biopolymers such as antibodies, antigens, receptors, enzymes, nucleic acids, peptides. In the present invention, the “substance that specifically binds to a specific component in a liquid sample” is preferably an “antibody”. Known antibodies can be used. Examples include anti-albumin antibody, anti-HCG antibody, anti-IgA antibody, anti-IgM antibody, anti-IgE antibody, anti-IgD antibody, anti-AFP antibody, anti-DNT antibody, anti-prostaglandin antibody, anti-human coagulation factor antibody, anti-CRP Antibodies, anti-HBs antibodies, anti-human growth hormone antibodies, anti-steroid hormone antibodies and the like are included. Examples of antigens include albumin, HCG, IgA, IgM, IgE, IgD, AFP, DNT, prostaglandin, human clotting factor, CRP, HBs, human growth hormone, steroid hormone and the like.
「粒子表面に粒子に液体試料中の標的物質に特異的に結合する物質を担持する」とは、粒子表面に化学的あるいは物理的に前記物質を結合させることをいう。例えば、前記物質と粒子を物理的な吸着反応により結合して得る方法や、シランカップリング剤等を用いて化学的に結合して得る方法がある。 “Supporting a substance that specifically binds to a target substance in a liquid sample on a particle surface” means that the substance is chemically or physically bound to the particle surface. For example, there are a method in which the substance and particles are bonded by a physical adsorption reaction, and a method in which the substance and particles are chemically bonded using a silane coupling agent or the like.
本粒子を含む分析試薬は、「液体試料中の標的物質に特異的に結合する物質」の保存性を高めるために他の試薬を含んでいてもよい。このような試薬の例には糖類等が含まれる。また、本粒子を含む分析試薬には、測定に適した条件に調節するための緩衝液成分を含んでいても良い。 The analysis reagent containing the present particles may contain other reagents in order to enhance the storage stability of the “substance that specifically binds to the target substance in the liquid sample”. Examples of such reagents include saccharides and the like. Further, the analysis reagent containing the present particles may contain a buffer component for adjusting to conditions suitable for measurement.
次に本発明の実施の形態１による免疫センサチップを用いた、標的物質を検出する方法を述べる。図４（ａ）、（ｂ）および（ｃ）は免疫センサチップにおける交流電圧印加時の粒子挙動を説明するための模式図である。図４（ａ）は免疫センサチップを上から見た図であり、（ｂ）は（ａ）のＡ部分の拡大図で、（ｃ）は（ａ）のＢ−Ｂ断面図である。
検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。試料混合物が流路内を流れる際、より径の大きなＢ抗体担持粒子１６は粒子分離手段部分で流れが妨げられるため、図４(ａ)に示すように粒子分離手段よりも注入口側の流路にとどまる。一方で、Ａ抗体担持粒子１５は粒子分離手段を通過し空気口側まで流れることができる。これにより、注入口側と空気口側でＡ抗体担持粒子１５とＢ抗体担持粒子１６の局在化が起こる。この試料混合物の充填と粒子分離操作後、流路内の電極に交流電圧を印加し、粒子を誘電泳動力により直列させると図４（ｂ）、（ｃ）に示すように、異なる粒径の粒子それぞれが凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。
Next, a method for detecting a target substance using the immunosensor chip according to Embodiment 1 of the present invention will be described. FIGS. 4A, 4B, and 4C are schematic views for explaining particle behavior when an alternating voltage is applied in the immunosensor chip. 4A is a view of the immunosensor chip as viewed from above, FIG. 4B is an enlarged view of a portion A in FIG. 4A, and FIG. 4C is a cross-sectional view taken along line BB in FIG.
The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. This sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. When the sample mixture flows in the flow path, the B antibody-carrying particles 16 having a larger diameter are prevented from flowing in the particle separation means, so that the flow on the inlet side of the particle separation means as shown in FIG. Stay on the road. On the other hand, the A antibody-carrying particles 15 can pass through the particle separation means and flow to the air port side. Thereby, the localization of the A antibody-carrying particles 15 and the B antibody-carrying particles 16 occurs on the injection port side and the air port side. After filling the sample mixture and separating the particles, an AC voltage is applied to the electrodes in the flow path, and the particles are connected in series by the dielectrophoretic force. As shown in FIGS. Each particle forms an aggregate. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように、本実施形態によると、粒子分離手段によって粒径の異なるＡ抗体担持粒子１５とＢ抗体担持粒子１６とを分離し、電圧を印加するので、Ａ抗体担持粒子１５の凝集体とＢ抗体担持粒子１６の凝集体とを形成できる。したがって一度の測定で２種類の粒径の粒子の分布を測定できる。すなわち、一度の測定で複数項目の検出が可能となる。 As described above, according to the present embodiment, the A antibody-carrying particles 15 and the B antibody-carrying particles 16 having different particle diameters are separated by the particle separation means and a voltage is applied. And an aggregate of B antibody-carrying particles 16 can be formed. Therefore, it is possible to measure the distribution of particles having two types of particle diameters in a single measurement. That is, a plurality of items can be detected by a single measurement.
（実施の形態２）
実施の形態２について、詳細を説明する。但し、本発明は実施の形態１で述べた免疫センサ装置とほぼ同一であるため、違いを中心に記載する。
(Embodiment 2)
Details of the second embodiment will be described. However, since the present invention is almost the same as the immunosensor device described in the first embodiment, the differences will be mainly described.
図５は、本発明の第二の態様における免疫センサ装置の模式図である。本発明の免疫センサ装置１１１は、センサチップ１１２と、センサチップ１１２に形成された電極５、電極６、電極７、電極８に連結された交流電圧印加装置１１４、光学検出手段１１３からなる。センサチップ１１２の詳細は後述するが、少なくとも１つの流路１３が設けられ、光学検出手段１１３により流路１３内部の複数個所が観察できるように構成されている。 FIG. 5 is a schematic diagram of the immunosensor device according to the second embodiment of the present invention. The immunosensor device 111 of the present invention includes a sensor chip 112, an electrode 5, an electrode 6, an electrode 7 and an electrode 8 connected to the electrode 8 formed on the sensor chip 112, and an optical detection means 113. Although details of the sensor chip 112 will be described later, at least one flow path 13 is provided, and a plurality of locations inside the flow path 13 can be observed by the optical detection means 113.
図６は、本発明の第二の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、２対の電極（ａ電極５とｂ電極６、ｃ電極７とｄ電極８）が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 6 is an exploded perspective view of the sensor chip in the second embodiment of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower substrate 3 is provided with two pairs of electrodes (a electrode 5 and b electrode 6, c electrode 7 and d electrode 8), part of which is exposed to the hollow structure forming the flow path 13.
図７は、本発明の第一の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある階段状の段差である。
本発明による免疫センサ装置、およびセンサチップは実施の形態１と同様に作製することができる。
FIG. 7 is an inverted view of the upper cover of the sensor chip in the first embodiment of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a stepped step between the inlet 11 and the air port 12.
The immunosensor device and sensor chip according to the present invention can be manufactured in the same manner as in the first embodiment.
次に本発明の実施の形態２によるセンサチップを用いた、標的物質を検出する方法を述べる。図８は免疫センサチップにおける交流電圧印加時の粒子挙動を説明するための模式図である。図８（ａ）は免疫センサチップを上から見た図であり、（ｂ）および（ｃ）は（ａ）のＡ部分の拡大図で、（ｄ）は（ａ）のＢ−Ｂ断面図である。図８（ｂ）はｃ電極７およびｄ電極８に電圧を印加した場合を示すものであり、図８（ｃ）はａ電極５およびｂ電極６に電圧を印加した場合を示すものである。図８（ｄ）は、ｃ電極７およびｄ電極８に電圧を印加した場合を示すものである。 Next, a method for detecting a target substance using the sensor chip according to the second embodiment of the present invention will be described. FIG. 8 is a schematic diagram for explaining the particle behavior when an AC voltage is applied in the immunosensor chip. 8A is a view of the immunosensor chip as viewed from above. FIGS. 8B and 8C are enlarged views of a portion A of FIG. 8A, and FIG. 8D is a cross-sectional view taken along the line BB of FIG. It is. FIG. 8B shows a case where a voltage is applied to the c electrode 7 and the d electrode 8, and FIG. 8C shows a case where a voltage is applied to the a electrode 5 and the b electrode 6. FIG. 8D shows a case where a voltage is applied to the c electrode 7 and the d electrode 8.
検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。次に電極ｃ、電極ｄに交流電圧を印加した状態で、この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。試料混合物が流路内を流れる際、図８（ｂ）、（ｄ）に示すように、粒子は流れに対して垂直方向に直列に凝集し、流れに逆った力を受けるため、粒子分離手段よりも空気口側に流れにくくなる。さらに径の大きなＢ抗体担持粒子１６はさらに粒子分離手段部分で流れが妨げられるため、粒子分離手段よりも注入口側の流路にとどまりやすくなる。一方で、Ａ抗体担持粒子１５は粒子分離手段を通過し空気口側まで流れることができる。これにより、注入口側と空気口側でＡ抗体担持粒子１５とＢ抗体担持粒子１６の局在化がよりよく起こる。さらに、試料混合物をセンサチップの注入口に点着する際に、電極ｃ，電極ｄにではなく電極ａと電極ｃあるいは電極ｂと電極ｄに対して交流電圧を印加した場合には、図８（ｃ）に示すように、粒子は斜め方向に直列に凝集し、流れに逆らった力を受ける。そのため電極ｃ，電極ｄに対して交流電圧を印加するのと同様の粒子分離を行うことができると共に、粒子を斜めに並べることでＢ抗体担持粒子１６に衝突することでＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がよりよく起こる。すなわち、２対の電極を用いることにより、１対の電極を用いる場合よりも高い精度で、粒径ごとに粒子を分離することができる。 The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. Next, in a state where an AC voltage is applied to the electrodes c and d, this sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. When the sample mixture flows in the flow path, as shown in FIGS. 8B and 8D, the particles aggregate in series in a direction perpendicular to the flow and receive a force against the flow. It becomes difficult to flow to the air port side than the means. Further, since the flow of the B antibody-carrying particles 16 having a larger diameter is further hindered by the particle separation means, the B antibody-carrying particles 16 are more likely to stay in the flow channel on the inlet side than the particle separation means. On the other hand, the A antibody-carrying particles 15 can pass through the particle separation means and flow to the air port side. As a result, the localization of the A antibody-carrying particles 15 and the B antibody-carrying particles 16 occurs more favorably on the injection port side and the air port side. Furthermore, when an AC voltage is applied to the electrodes a and c or the electrodes b and d instead of the electrodes c and d when spotting the sample mixture on the inlet of the sensor chip, FIG. As shown in (c), the particles aggregate in series in an oblique direction and receive a force against the flow. Therefore, it is possible to perform the same particle separation as when an AC voltage is applied to the electrodes c and d, and the particles of the A antibody-carrying particles 15 collide with the B antibody-carrying particles 16 by arranging the particles obliquely. Particle localization occurs better because the probability of hindering flow is reduced. That is, by using two pairs of electrodes, it is possible to separate particles for each particle size with higher accuracy than when using a pair of electrodes.
この試料混合物の充填、粒子分離操作後、流路内のａ電極とｂ電極、ｃ電極とｄ電極に対して交流電圧を印加し、粒子を誘電泳動力により直列させ、粒子の凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。 After filling the sample mixture and separating the particles, an AC voltage is applied to the a and b electrodes and the c and d electrodes in the flow path, and the particles are connected in series by the dielectrophoretic force to form particle aggregates. To do. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように本実施形態によると、２対ある電極のうち１対に対し電圧を印加しながら試料を充填し、流れに逆らった力を粒子に与えることで、粒子分離手段による粒径の異なるＡ抗体担持粒子１５とＢ抗体担持粒子１６の分離をより精度よく行うことができるという効果が得られる。さらに電圧を印加して、Ａ抗体担持粒子１５の凝集体とＢ抗体担持粒子１６の凝集体とを形成することで、一度の測定で２種類の粒径の粒子の分布を測定でき、一度の測定で複数項目の検出をより精度よく行うことが可能となる。 As described above, according to this embodiment, the sample is filled while applying a voltage to one of the two pairs of electrodes, and a force against the flow is applied to the particles, so that the particle size of the particles by the particle separation means can be increased. The effect that the different A antibody-carrying particles 15 and B antibody-carrying particles 16 can be separated with higher accuracy is obtained. Further, by applying a voltage to form an aggregate of the A antibody-carrying particles 15 and an aggregate of the B antibody-carrying particles 16, the distribution of particles having two types of particle sizes can be measured at one time. A plurality of items can be detected more accurately by measurement.
（実施の形態３）
実施の形態３について、詳細を説明する。但し、本発明は実施の形態１で述べた免疫センサ装置とほぼ同一であるため、違いを中心に記載する。
(Embodiment 3)
Details of the third embodiment will be described. However, since the present invention is almost the same as the immunosensor device described in the first embodiment, the differences will be mainly described.
図９は、本発明の第三の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、少なくとも１対の電極４が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 9 is an exploded perspective view of the sensor chip in the third embodiment of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower base 3 is provided with at least one pair of electrodes 4, part of which is exposed to the hollow structure forming the flow path 13.
図１０は、本発明の第三の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある段差である。前記段差は注入口１１から空気口１２の方向に、連続的に占有面積を増すように形成されている。本発明によるセンサチップは図１で示した免疫センサ装置で使用する事ができる。また、本発明による免疫センサ装置、およびセンサチップは実施の形態１と同様に作製することができる。 FIG. 10 is an inverted view of the upper cover of the sensor chip according to the third embodiment of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a step between the inlet 11 and the air port 12. The step is formed so as to continuously increase the occupied area in the direction from the inlet 11 to the air inlet 12. The sensor chip according to the present invention can be used in the immunosensor device shown in FIG. Further, the immunosensor device and the sensor chip according to the present invention can be manufactured in the same manner as in the first embodiment.
次に本発明の実施の形態３によるセンサチップを用いた、標的物質を検出する方法を述べる。検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。試料混合物が流路内を流れる際、より径の大きなＢ抗体担持粒子１６は粒子分離手段の段差部分で流れが妨げられるが、段差が連続的に占有面積を増すように形成されているために段差に当った粒子は流路の端側に流れる。これにより、粒子分離手段近傍において、Ｂ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がよりよく起こる。この試料混合物の充填、粒子分離操作後、流路内電極に交流電圧を印加、粒子を誘電泳動力により直列させ、粒子の凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。 Next, a method for detecting a target substance using the sensor chip according to Embodiment 3 of the present invention will be described. The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. This sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. When the sample mixture flows in the flow path, the B antibody-carrying particles 16 having a larger diameter are prevented from flowing at the step portion of the particle separation means, but the step is formed so as to continuously increase the occupied area. The particles that hit the step flow to the end side of the flow path. Thereby, in the vicinity of the particle separation means, the probability of collision with the B antibody-carrying particles 16 and hindering the flow of the A antibody-carrying particles 15 is reduced, so that the localization of the particles occurs better. After filling the sample mixture and separating the particles, an AC voltage is applied to the electrode in the flow path, and the particles are serially connected by a dielectrophoretic force to form an aggregate of particles. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように本実施形態によると、粒子分離手段の段差を連続的に占有面積を増すように形成することで、段差に当った径の大きなＢ抗体担持粒子１６を流路の端側に流す。これにより、Ｂ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がより精度よく行うことができるという効果が得られる。 As described above, according to the present embodiment, the step of the particle separation means is formed so as to continuously increase the occupied area, so that the B antibody-carrying particles 16 having a large diameter hitting the step are placed on the end side of the flow path. Shed. As a result, the probability that the A antibody-carrying particles 15 collide with the B antibody-carrying particles 16 and the flow of the A antibody-carrying particles 15 is hindered is reduced, so that the effect that the particles can be localized more accurately is obtained.
（実施の形態４）
実施の形態４について、詳細を説明する。但し、本発明は実施の形態２で述べた免疫センサ装置とほぼ同一であるため、違いを中心に記載する。
(Embodiment 4)
Details of the fourth embodiment will be described. However, since the present invention is almost the same as the immunosensor device described in the second embodiment, the differences will be mainly described.
図１１は、本発明の第四の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、２対の電極（ａ電極５とｂ電極６、ｃ電極７とｄ電極８）が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 11 is an exploded perspective view of the sensor chip according to the fourth embodiment of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower substrate 3 is provided with two pairs of electrodes (a electrode 5 and b electrode 6, c electrode 7 and d electrode 8), part of which is exposed to the hollow structure forming the flow path 13.
図１２は、本発明の第四の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある段差である。前記段差は注入口１１から空気口１２の方向に、連続的に占有面積を増すように形成されている。本発明によるセンサチップは図５で示した免疫センサ装置で使用する事ができる。また、本発明による免疫センサ装置、およびセンサチップは実施の形態１と同様に作製することができる。 FIG. 12 is an inverted view of the upper cover of the sensor chip according to the fourth aspect of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a step between the inlet 11 and the air port 12. The step is formed so as to continuously increase the occupied area in the direction from the inlet 11 to the air inlet 12. The sensor chip according to the present invention can be used in the immunosensor device shown in FIG. Further, the immunosensor device and the sensor chip according to the present invention can be manufactured in the same manner as in the first embodiment.
次に本発明の実施の形態４によるセンサチップを用いた、標的物質を検出する方法を述べる。検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。次に電極ｃ，電極ｄに交流電圧を印加した状態で、この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。粒子は実施の形態２と同様に、図８の本発明によるセンサチップ断面図における粒子挙動の模式図で示したように、流れに対して垂直方向に直列に凝集し、流れに逆った力を受けるため、粒子分離手段よりも空気口側に流れにくくなる。そのため、注入口側と空気口側でＡ抗体担持粒子１５とＢ抗体担持粒子１６の局在化がよりよく起こる。さらに、より径の大きなＢ抗体担持粒子１６は粒子分離手段の段差部分で流れが妨げられるが、段差が連続的に占有面積を増すように形成されているために段差に当った粒子は流路の端側に流れる。これにより、粒子分離手段近傍において、Ｂ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がよりよく起こる。 Next, a method for detecting a target substance using the sensor chip according to the fourth embodiment of the present invention will be described. The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. Next, in a state where an AC voltage is applied to the electrodes c and d, this sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. As in the second embodiment, the particles are aggregated in series in the direction perpendicular to the flow as shown in the schematic diagram of the particle behavior in the sensor chip cross-sectional view of the present invention in FIG. Therefore, it is more difficult to flow to the air port side than the particle separation means. For this reason, localization of the A antibody-carrying particles 15 and the B antibody-carrying particles 16 occurs more favorably on the injection port side and the air port side. Further, the flow of the B antibody-carrying particles 16 having a larger diameter is hindered at the step portion of the particle separation means, but the particles hitting the step are formed in the flow path because the step is formed so as to continuously increase the occupied area. It flows to the end side. Thereby, in the vicinity of the particle separation means, the probability of collision with the B antibody-carrying particles 16 and hindering the flow of the A antibody-carrying particles 15 is reduced, so that the localization of the particles occurs better.
試料混合物をセンサチップの注入口に点着する際に、電極ｃ，電極ｄにではなく電極ａと電極ｃあるいは電極ｂと電極ｄに対して交流電圧を印加した場合の効果については、実施の形態２で述べたとおりであるが、段差の形状によって、Ｂ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率が減るという効果が追加された分、さらに粒子の局在化をよりよく起こすことができる。 Regarding the effect when an AC voltage is applied not to the electrodes c and d but to the electrodes a and c or the electrodes b and d when the sample mixture is spotted on the inlet of the sensor chip, As described in the second embodiment, the effect of reducing the probability that the flow of the A antibody-carrying particles 15 is prevented from colliding with the B antibody-carrying particles 16 due to the shape of the step is further increased. Can make it better.
この試料混合物の充填、粒子分離操作後、流路内のａ電極とｂ電極、ｃ電極とｄ電極に対して交流電圧を印加し、粒子を誘電泳動力により直列させ、粒子の凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。 After filling the sample mixture and separating the particles, an AC voltage is applied to the a and b electrodes and the c and d electrodes in the flow path, and the particles are connected in series by the dielectrophoretic force to form particle aggregates. To do. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように本実施形態によると、２対ある電極のうち１対に対し電圧を印加しながら試料を充填し、流れに逆らった力を粒子に与える。さらに粒子分離手段の段差を連続的に占有面積を増すように形成することで、段差に当った径の大きなＢ抗体担持粒子１６を流路の端側に流す。これによりＢ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率が減る。これらの相乗効果により、粒子の局在化がより精度よく行うことができるという効果が得られる。 As described above, according to this embodiment, the sample is filled while applying a voltage to one of the two pairs of electrodes, and a force against the flow is applied to the particles. Further, by forming the step of the particle separation means so as to continuously increase the occupied area, the B antibody-carrying particles 16 having a large diameter hitting the step are caused to flow toward the end of the channel. As a result, the probability of collision with the B antibody-carrying particles 16 and hindering the flow of the A antibody-carrying particles 15 is reduced. By these synergistic effects, the effect that the localization of the particles can be performed with higher accuracy is obtained.
（実施の形態５）
実施の形態５について、詳細を説明する。但し、本発明は実施の形態２で述べた免疫センサ装置とほぼ同一であるため、違いを中心に記載する。
(Embodiment 5)
Details of the fifth embodiment will be described. However, since the present invention is almost the same as the immunosensor device described in the second embodiment, the differences will be mainly described.
図１３は、本発明の第五の態様におけるセンサ装置の模式図である。本発明の免疫センサ装置１１１は、センサチップ１１２と、センサチップ１１２に形成された電極５、電極６、電極７、電極８に連結された交流電圧印加装置１１４、光学検出手段１１３からなる。センサチップ１１２の詳細は後述するが、少なくとも１つの流路１３が設けられ、光学検出手段１１３により流路１３内部の複数個所が観察できるように構成されている。図１４は、本発明の第五の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、２対の電極（ａ電極５とｂ電極６、ｃ電極７とｄ電極８）が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 13 is a schematic diagram of a sensor device according to the fifth aspect of the present invention. The immunosensor device 111 of the present invention includes a sensor chip 112, an electrode 5, an electrode 6, an electrode 7 and an electrode 8 connected to the electrode 8 formed on the sensor chip 112, and an optical detection means 113. Although details of the sensor chip 112 will be described later, at least one flow path 13 is provided, and a plurality of locations inside the flow path 13 can be observed by the optical detection means 113. FIG. 14 is an exploded perspective view of the sensor chip according to the fifth aspect of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower substrate 3 is provided with two pairs of electrodes (a electrode 5 and b electrode 6, c electrode 7 and d electrode 8), part of which is exposed to the hollow structure forming the flow path 13.
図１５は、本発明の第五の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある階段状の段差である。
本発明による免疫センサ装置、およびセンサチップは実施の形態１と同様に作製することができる。
FIG. 15 is an inverted view of the upper cover of the sensor chip according to the fifth aspect of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a stepped step between the inlet 11 and the air port 12.
The immunosensor device and sensor chip according to the present invention can be manufactured in the same manner as in the first embodiment.
次に本発明の実施の形態５によるセンサチップを用いた、標的物質を検出する方法を述べる。図１６は免疫センサチップにおける交流電圧印加時の粒子挙動を説明するための模式図である。図１６（ａ）は免疫センサチップを上から見た図であり、（ｂ）および（ｃ）は（ａ）のＡ部分の拡大図で、（ｄ）は（ａ）のＢ−Ｂ断面図である。図１６（ｂ）はｃ電極７およびｄ電極８に電圧を印加した場合を示すものであり、図１６（ｃ）はａ電極５およびｂ電極６に電圧を印加した場合を示すものである。図１６（ｄ）は、ｃ電極７およびｄ電極８に電圧を印加した場合を示すものである。検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。次に電極ｃ，電極ｄに交流電圧を印加した状態で、この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。試料混合物が流路内を流れる際、図１６（ｂ）、（ｄ）に示すように、粒子は流れに対して平行方向に直列に凝集し、流れに逆った力を受けるため、粒子分離手段よりも空気口側に流れにくくなる。さらに径の大きなＢ抗体担持粒子１６はさらに粒子分離手段部分で流れが妨げられるため、粒子分離手段よりも注入口側の流路にとどまりやすくなる。一方で、Ａ抗体担持粒子１５は粒子分離手段を通過し空気口側まで流れることができる。これにより、注入口側と空気口側でＡ抗体担持粒子１５とＢ抗体担持粒子１６の局在化がよりよく起こる。このとき、粒子は流れに対して平行方向に並ぶため、Ｂ抗体担持粒子１６に衝突することでＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がよりよく起こる。 Next, a method for detecting a target substance using the sensor chip according to the fifth embodiment of the present invention will be described. FIG. 16 is a schematic diagram for explaining the particle behavior when an AC voltage is applied in the immunosensor chip. 16 (a) is a view of the immunosensor chip as viewed from above, (b) and (c) are enlarged views of portion A of (a), and (d) is a cross-sectional view taken along line BB of (a). It is. FIG. 16B shows the case where a voltage is applied to the c electrode 7 and the d electrode 8, and FIG. 16C shows the case where a voltage is applied to the a electrode 5 and the b electrode 6. FIG. 16D shows a case where a voltage is applied to the c electrode 7 and the d electrode 8. The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. Next, in a state where an AC voltage is applied to the electrodes c and d, this sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. When the sample mixture flows in the flow path, as shown in FIGS. 16 (b) and 16 (d), the particles aggregate in series in a direction parallel to the flow and receive a force against the flow. It becomes difficult to flow to the air port side than the means. Further, since the flow of the B antibody-carrying particles 16 having a larger diameter is further hindered by the particle separation means, the B antibody-carrying particles 16 are more likely to stay in the flow channel on the inlet side than the particle separation means. On the other hand, the A antibody-carrying particles 15 can pass through the particle separation means and flow to the air port side. As a result, the localization of the A antibody-carrying particles 15 and the B antibody-carrying particles 16 occurs more favorably on the injection port side and the air port side. At this time, since the particles are arranged in a direction parallel to the flow, the probability that the flow of the A antibody-carrying particles 15 is hindered by colliding with the B antibody-carrying particles 16 is reduced, and thus the localization of the particles occurs better.
この試料混合物の充填、粒子分離操作後、ｃ電極とｄ電極への交流電圧の印加を停止し、図１６（ｃ）に示すように流路内のａ電極とｂ電極に交流電圧を印加する。これにより、粒子を誘電泳動力により直列させ、粒子の凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。 After filling the sample mixture and separating the particles, the application of the AC voltage to the c and d electrodes is stopped, and the AC voltage is applied to the a and b electrodes in the flow path as shown in FIG. . As a result, the particles are connected in series by the dielectrophoretic force to form an aggregate of particles. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように本実施形態によると、２対ある電極のうち１対に対し電圧を印加しながら試料を充填し、流れに逆らった力を粒子に与えることで、粒子分離手段による粒径の異なるＡ抗体担持粒子１５とＢ抗体担持粒子１６の分離をより精度よく行うことができる。さらに粒子は流れに対して平行方向に並ぶため、Ｂ抗体担持粒子１６に衝突することでＡ抗体担持粒子１５の流れが妨げられる確率が減ることから、粒子の局在化をより精度よく行うことができるという効果が得られる。 As described above, according to this embodiment, the sample is filled while applying a voltage to one of the two pairs of electrodes, and a force against the flow is applied to the particles, so that the particle size of the particles by the particle separation means can be increased. Different A antibody-carrying particles 15 and B antibody-carrying particles 16 can be separated more accurately. Furthermore, since the particles are arranged in a direction parallel to the flow, the probability of hindering the flow of the A antibody-carrying particles 15 by colliding with the B antibody-carrying particles 16 is reduced, so that the particles can be localized more accurately. The effect of being able to be obtained.
（実施の形態６）
実施の形態６について、詳細を説明する。但し、本発明は実施の形態２で述べた免疫センサ装置とほぼ同一であるため、違いを中心に記載する。
(Embodiment 6)
Details of the sixth embodiment will be described. However, since the present invention is almost the same as the immunosensor device described in the second embodiment, the differences will be mainly described.
図１７は、本発明の第六の態様におけるセンサチップの分解斜視図である。センサチップ１１２は上カバー１、接着層２、下基盤３からなり、上カバー１と下基盤３との間は、接着層２により中空構造となり流路１３が形成されている。流路１３の一端には注入口１１が形成され、また注入口１１とは異なる他端には空気口１２が形成されている。下基盤３には、２対の電極（ａ電極５とｂ電極６、ｃ電極７とｄ電極８）が設けられ、その一部は流路１３を形成する中空構造に露出している。 FIG. 17 is an exploded perspective view of the sensor chip according to the sixth aspect of the present invention. The sensor chip 112 includes an upper cover 1, an adhesive layer 2, and a lower base 3, and the flow path 13 is formed between the upper cover 1 and the lower base 3 with a hollow structure by the adhesive layer 2. An inlet 11 is formed at one end of the flow path 13, and an air port 12 is formed at the other end different from the inlet 11. The lower substrate 3 is provided with two pairs of electrodes (a electrode 5 and b electrode 6, c electrode 7 and d electrode 8), part of which is exposed to the hollow structure forming the flow path 13.
図１８は、本発明の第六の態様におけるセンサチップの上カバーの反転図である。上カバーには注入口１１、空気口１２、少なくとも一つの粒子分離手段１４が形成される。粒子分離手段１４は注入口１１と空気口１２の間にある階段状の段差である。前記段差は注入口１１から空気口１２の方向に、連続的に占有面積を増すように形成されている。
本発明による免疫センサ装置、およびセンサチップは実施の形態１と同様に作製することができる。
FIG. 18 is an inverted view of the upper cover of the sensor chip according to the sixth aspect of the present invention. The upper cover is formed with an injection port 11, an air port 12, and at least one particle separation means. The particle separation means 14 is a stepped step between the inlet 11 and the air port 12. The step is formed so as to continuously increase the occupied area in the direction from the inlet 11 to the air inlet 12.
The immunosensor device and sensor chip according to the present invention can be manufactured in the same manner as in the first embodiment.
次に本発明の実施の形態６によるセンサチップを用いた、標的物質を検出する方法を述べる。検体試料を少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子（Ａ抗体担持粒子１５、Ｂ抗体担持粒子１６）を含む試料と混合し、試料混合物を調整する。次にｃ電極，ｄ電極に交流電圧を印加した状態で、この試料混合物をセンサチップの注入口に点着し、毛細管力により送液する。粒子は実施の形態５と同様に、図１６（ｂ）で示したように流れに対して平行方向に直列に凝集し、流れに逆った力を受けるため、粒子分離手段よりも空気口側に流れにくくなる。さらに径の大きなＢ抗体担持粒子１６はさらに粒子分離手段部分で流れが妨げられるため、粒子分離手段よりも注入口側の流路にとどまりやすくなる。一方で、Ａ抗体担持粒子１５は粒子分離手段を通過し空気口側まで流れることができる。これにより、注入口側と空気口側でＡ抗体担持粒子１５とＢ抗体担持粒子１６の局在化がよりよく起こる。このとき、粒子は流れに対して平行方向に並ぶため、Ｂ抗体担持粒子１６に衝突することでＡ抗体担持粒子１５の流れが妨げられる確率が減るため、粒子の局在化がよりよく起こる。 Next, a method for detecting a target substance using the sensor chip according to the sixth embodiment of the present invention will be described. The sample sample is mixed with a sample containing fine particles having different particle diameters (A antibody-carrying particles 15 and B antibody-carrying particles 16) each carrying at least two types of biocompatible molecules, thereby preparing a sample mixture. Next, in a state where an AC voltage is applied to the c electrode and the d electrode, this sample mixture is spotted on the inlet of the sensor chip and fed by capillary force. As in the fifth embodiment, the particles are aggregated in series in the direction parallel to the flow as shown in FIG. 16B, and receive a force opposite to the flow. It becomes difficult to flow. Further, since the flow of the B antibody-carrying particles 16 having a larger diameter is further hindered by the particle separation means, the B antibody-carrying particles 16 are more likely to stay in the flow channel on the inlet side than the particle separation means. On the other hand, the A antibody-carrying particles 15 can pass through the particle separation means and flow to the air port side. As a result, the localization of the A antibody-carrying particles 15 and the B antibody-carrying particles 16 occurs more favorably on the injection port side and the air port side. At this time, since the particles are arranged in a direction parallel to the flow, the probability that the flow of the A antibody-carrying particles 15 is hindered by colliding with the B antibody-carrying particles 16 is reduced, and thus the localization of the particles occurs better.
さらに、より径の大きなＢ抗体担持粒子１６は粒子分離手段の段差部分で流れが妨げられるが、段差が連続的に占有面積を増すように形成されているために段差に当った粒子は流路の端側に流れる。これにより、粒子分離手段近傍において、Ｂ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率がさらに減るため、粒子の局在化をよりよく起こすことができる。 Further, the flow of the B antibody-carrying particles 16 having a larger diameter is hindered at the step portion of the particle separation means, but the particles hitting the step are formed in the flow path because the step is formed so as to continuously increase the occupied area. It flows to the end side. This further reduces the probability that the flow of the A antibody-carrying particles 15 will be hindered by colliding with the B antibody-carrying particles 16 in the vicinity of the particle separation means, so that the localization of the particles can be improved.
この試料混合物の充填、粒子分離操作後、ｃ電極、ｄ電極への交流電圧の印加を停止し、流路内のａ電極、ｂ電極に交流電圧を印加する。これにより、粒子を誘電泳動力により直列させ、粒子の凝集体を形成する。粒子表面には標的物質に対する抗体が担時されており、標的物質が存在すれば標的物質を介して、静電的相互作用、水素結合、ファンデルワールス力、疎水的相互作用などの相互作用により、交流電圧の印加を停止した後も、凝集体を維持する。一方で、標的物質が存在しなければ凝集体は解消される。この粒子動態を光学検出手段により検出し、検体中の標的物質に関する情報を得る。 After filling the sample mixture and separating the particles, application of AC voltage to the c and d electrodes is stopped, and AC voltage is applied to the a and b electrodes in the flow path. As a result, the particles are connected in series by the dielectrophoretic force to form an aggregate of particles. The surface of the particle carries an antibody against the target substance. If the target substance is present, it interacts with the target substance through interactions such as electrostatic interaction, hydrogen bonding, van der Waals force, and hydrophobic interaction. Even after the application of the AC voltage is stopped, the aggregate is maintained. On the other hand, if the target substance is not present, the aggregate is eliminated. This particle dynamics is detected by an optical detection means to obtain information on the target substance in the specimen.
以上説明したように本実施形態によると、２対ある電極のうち１対に対し電圧を印加しながら試料を充填し、流れに逆らった力を粒子に与えることで、粒子分離手段による粒径の異なるＡ抗体担持粒子１５とＢ抗体担持粒子１６の分離をより精度よく行う。このとき、粒子は流れに対して平行方向に並んでおり、さらに粒子分離手段の段差を連続的に占有面積を増すように形成しているため段差に当った径の大きなＢ抗体担持粒子１６は流路の端側に流れる。これら２つの要因がＢ抗体担持粒子１６に衝突してＡ抗体担持粒子１５の流れが妨げられる確率を減らすため、粒子の局在化がより精度よく行うことができるという効果が得られる。 As described above, according to this embodiment, the sample is filled while applying a voltage to one of the two pairs of electrodes, and a force against the flow is applied to the particles, so that the particle size of the particles by the particle separation means can be increased. Different A antibody-carrying particles 15 and B antibody-carrying particles 16 are separated with higher accuracy. At this time, the particles are arranged in a direction parallel to the flow, and further, the step of the particle separating means is formed so as to continuously increase the occupied area. It flows to the end side of the flow path. Since these two factors collide with the B antibody-carrying particles 16 and reduce the probability that the flow of the A antibody-carrying particles 15 is hindered, the effect that the particles can be localized more accurately is obtained.
（実施例１）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態１なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。
Example 1
A glass substrate on which a flow path having a width of 600 μm, a depth of 20 μm, and a step portion having a depth of 5 μm was bonded to a glass substrate on which an electrode was formed by sputtering, and a sensor chip according to Embodiment 1 was manufactured. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope.
Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した結果、粒子分離手段である段差部分において８μｍ粒子がとどまり、３μｍ粒子は段差部分を越えて空気口側まで到達し、粒径の違いによる粒子分離が見られた。また、その後、２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると粒子凝集が起こり、免疫反応による標的物質の検出ができた。 Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. As a result of spotting the sample mixture on the inlet of the sensor chip and observing the particle behavior with a microscope, the 8 μm particles stay at the stepped portion, which is the particle separation means, and the 3 μm particles reach the air port side beyond the stepped portion. Particle separation due to the difference in particle size was observed. After that, when an AC voltage of 20 V (p / p) and 100 kHz was applied, particle aggregation occurred and the target substance could be detected by an immune reaction.
（実施例２）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態２なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。
(Example 2)
A glass substrate on which a flow path having a width of 600 μm, a depth of 20 μm, and a step portion having a depth of 5 μm was bonded to a glass substrate on which an electrode was formed by sputtering, and a sensor chip according to the second embodiment was manufactured. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope.
Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した。粒子点着時に電極ｃ，電極ｄに２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加することで、粒子分離手段である段差部分においてよりよく粒子分離ができる様子が見られた。また、その後、ａ電極、ｂ電極にも２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると流路のほぼ全域で粒子凝集が起こり、免疫反応による標的物質の検出ができた。 Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. The sample mixture was spotted on the inlet of the sensor chip, and the particle behavior was observed with a microscope. By applying an AC voltage of 20 V (p / p) and 100 kHz to the electrodes c and d when the particles were spotted, it was observed that the particles could be separated more satisfactorily at the stepped portion as the particle separating means. After that, when an AC voltage of 20 V (p / p) and 100 kHz was applied to the a electrode and the b electrode, particle aggregation occurred in almost the entire area of the flow path, and the target substance was detected by immune reaction.
（実施例３）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態３なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した結果、粒子分離手段である段差部分において８μｍ粒子がとどまり、３μｍ粒子は段差部分を越えて空気口側まで到達し、粒径の違いによる粒子分離が見られた。その際、段差部分に到達した８μｍ粒子が流路壁側に流れる様子が確認でき、３μｍ粒子の流れを阻害しにくい様子が見られた。その後、２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると粒子凝集が起こり、免疫反応による標的物質の検出ができた。
(Example 3)
A sensor substrate according to Embodiment 3 was fabricated by bonding a glass substrate having a flow path having a width of 600 μm, a depth of 20 μm, and a stepped portion having a depth of 5 μm to a glass substrate on which an electrode was formed by sputtering with a UV curable resin. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope. Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. As a result of spotting the sample mixture on the inlet of the sensor chip and observing the particle behavior with a microscope, the 8 μm particles stay at the stepped portion, which is the particle separation means, and the 3 μm particles reach the air port side beyond the stepped portion. Particle separation due to the difference in particle size was observed. At that time, it was confirmed that the 8 μm particles that reached the step portion flow toward the channel wall side, and it was difficult to inhibit the flow of the 3 μm particles. Thereafter, when an AC voltage of 20 V (p / p) and 100 kHz was applied, particle aggregation occurred, and the target substance could be detected by an immune reaction.
（実施例４）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態４なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した結果、粒子分離手段である段差部分において８μｍ粒子がとどまり、３μｍ粒子は段差部分を越えて空気口側まで到達し、粒径の違いによる粒子分離を見られた。粒子点着時に電極ｃ，電極ｄに２０Ｖ（ｐ／ｐ）、１００ｋＨｚの電圧を印加することで、粒子分離手段である段差部分においてよりよく粒子分離し、さらに段差部分に到達した８μｍ粒子が流路壁側に流れることで、３μｍ粒子の流れを阻害しにくい様子が見られた。その後、ａ電極、ｂ電極にも２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると流路のほぼ全域で粒子凝集が起こり、免疫反応による標的物質の検出ができた。
Example 4
A sensor substrate according to the fourth embodiment was fabricated by bonding a glass substrate having a flow path having a width of 600 μm, a depth of 20 μm, and a stepped portion having a depth of 5 μm to a glass substrate on which an electrode was formed by sputtering with a UV curable resin. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope. Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. As a result of spotting the sample mixture on the inlet of the sensor chip and observing the particle behavior with a microscope, the 8 μm particles stay at the stepped portion, which is the particle separation means, and the 3 μm particles reach the air port side beyond the stepped portion. Particle separation due to the difference in particle size was observed. By applying a voltage of 20 V (p / p) and 100 kHz to the electrodes c and d when the particles are spotted, the particles are separated better at the stepped portion as the particle separating means, and the 8 μm particles that have reached the stepped portion flow. It seemed that it was difficult to inhibit the flow of 3 μm particles by flowing to the road wall side. Thereafter, when an AC voltage of 20 V (p / p) and 100 kHz was applied to the a electrode and the b electrode, particle aggregation occurred in almost the entire area of the flow path, and the target substance was detected by an immune reaction.
（実施例５）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態５なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した。粒子点着時に電極ｃ，電極ｄに２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加することで、粒子分離手段である段差部分においてよりよく粒子分離ができる様子が見られた。また、その後、ｃ電極とｄ電極への交流電圧の印加を停止し、ａ電極とｂ電極に２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると粒子凝集が起こり、免疫反応による標的物質の検出ができた。
(Example 5)
A sensor substrate according to the fifth embodiment was manufactured by bonding a glass substrate having a width of 600 μm, a depth of 20 μm, a stepped portion having a depth of 5 μm, and a glass substrate on which an electrode was formed by sputtering with a UV curable resin. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope. Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. The sample mixture was spotted on the inlet of the sensor chip, and the particle behavior was observed with a microscope. By applying an AC voltage of 20 V (p / p) and 100 kHz to the electrodes c and d when the particles were spotted, it was observed that the particles could be separated more satisfactorily at the stepped portion as the particle separating means. After that, when application of AC voltage to the c electrode and d electrode is stopped, and AC voltage of 20 V (p / p) and 100 kHz is applied to the a electrode and b electrode, particle aggregation occurs, and the target substance caused by immune reaction I was able to detect it.
（実施例６）
幅６００μｍ、深さ２０μｍの流路、深さ５μｍの段差部分を作製したガラス基板と、スパッタリングにより電極を作製したガラス基盤とをＵＶ硬化樹脂で接着し、実施の形態６なるセンサチップを作製した。このセンサチップの電極と交流電圧印加装置とを接続し、流路部分を顕微鏡で観察した。Ｂａｎｇｓ Ｌａｂｏｒａｔｏｒｉｅｓ，Ｉｎｃ製の直径３μｍのラテックス粒子に抗ヒトＣＲＰ抗体を、直径８μｍのラテックス粒子に抗ヒトＡＦＰ抗体を担持し、粒子を調整した。この粒子０．４〜０．５％（ｗ／ｗ）、５％トレハロースを含む０．０２Ｍグリシン緩衝液（ｐＨ８．６）を検体試料と混合し、試料混合物とした。試料混合物をセンサチップの注入口に点着し、粒子挙動を顕微鏡にて観察した結果、粒子分離手段である段差部分において８μｍ粒子がとどまり、３μｍ粒子は段差部分を越えて空気口側まで到達し、粒径の違いによる粒子分離を見られた。粒子点着時に電極ｃ，電極ｄに２０Ｖ（ｐ／ｐ）、１００ｋＨｚの電圧を印加することで、粒子分離手段である段差部分においてよりよく粒子分離し、さらに段差部分に到達した８μｍ粒子が流路壁側に流れることで、３μｍ粒子の流れを阻害しにくい様子が見られた。その後、ｃ電極とｄ電極への交流電圧の印加を停止し、ａ電極とｂ電極に２０Ｖ（ｐ／ｐ）、１００ｋＨｚの交流電圧を印加すると粒子凝集が起こり、免疫反応による標的物質の検出ができた。
(Example 6)
A sensor substrate according to Embodiment 6 was manufactured by bonding a glass substrate having a width of 600 μm, a depth of 20 μm, a stepped portion having a depth of 5 μm, and a glass substrate on which an electrode was formed by sputtering with a UV curable resin. . The electrode of this sensor chip and the AC voltage application device were connected, and the flow path portion was observed with a microscope. Anti-human CRP antibody was supported on latex particles with a diameter of 3 μm manufactured by Bangs Laboratories, Inc., and anti-human AFP antibody was supported on latex particles with a diameter of 8 μm to prepare particles. A 0.02 M glycine buffer solution (pH 8.6) containing 0.4 to 0.5% (w / w) and 5% trehalose of the particles was mixed with a specimen sample to obtain a sample mixture. As a result of spotting the sample mixture on the inlet of the sensor chip and observing the behavior of the particles with a microscope, the 8 μm particles stay in the stepped portion, which is the particle separation means, and the 3 μm particles reach the air port side beyond the stepped portion. Particle separation due to the difference in particle size was observed. By applying a voltage of 20 V (p / p) and 100 kHz to the electrodes c and d when the particles are spotted, the particles are separated better at the stepped portion as the particle separating means, and the 8 μm particles that have reached the stepped portion flow. It seemed that it was difficult to inhibit the flow of 3 μm particles by flowing to the road wall side. After that, application of AC voltage to the c and d electrodes is stopped, and application of an AC voltage of 20 V (p / p) and 100 kHz to the a and b electrodes causes particle aggregation and detection of the target substance by immune reaction. did it.
本発明は、ラテックス凝集反応において複数項目を同時に検出する方法において、少なくとも２種類の生体親和性分子がそれぞれ担持された、異なる粒径の微粒子を用いることで、同一チップ内での複数項目同時検出を可能とするものであり、液体試料中の特定物質の分析、特に免疫学的反応を用いた分析装置等に有用である。 In the method of simultaneously detecting a plurality of items in the latex agglutination reaction, the present invention simultaneously detects a plurality of items in the same chip by using fine particles having different particle diameters each carrying at least two types of biocompatible molecules. It is useful for analysis of a specific substance in a liquid sample, particularly for an analysis apparatus using an immunological reaction.
１ 上カバー
２ 接着層
３ 下基盤
４ 電極
５ ａ電極
６ ｂ電極
７ ｃ電極
８ ｄ電極
１１ 注入口
１２ 空気口
１３ 流路
１４ 粒子分離手段
１５ Ａ抗体担持粒子
１６ Ｂ抗体担持粒子
１１１ 免疫センサ装置
１１２ センサチップ
１１３ 光学検出手段
１１４ 交流電圧印加装置
DESCRIPTION OF SYMBOLS 1 Upper cover 2 Adhesive layer 3 Lower base 4 Electrode 5 A electrode 6 b Electrode 7 c Electrode 8 d Electrode 11 Inlet 12 Air inlet 13 Channel 14 Particle separation means 15 A Antibody carrying particle 16 B Antibody carrying particle 111 Immunosensor device 112 sensor chip 113 optical detection means 114 AC voltage application device
Claims (6)
粒径に基づいて前記粒子を分離する粒子分離手段と、
前記試料に電圧を印加して前記粒子を凝集させるための一対の電極と、を備える免疫センサチップ。 A sample storage unit in which a sample including a specimen containing a target substance and particles carrying a bioaffinity molecule that specifically binds to the target substance is disposed;
Particle separation means for separating the particles based on particle size;
An immunosensor chip comprising: a pair of electrodes for aggregating the particles by applying a voltage to the sample.
前記階段状の段差は、前記注入口から前記空気口の方向に連続的に占有面積を増すように形成されている、請求項２に記載の免疫センサチップ。 The sample storage unit has a sample inlet, an air port, and a flow path extending from the sample inlet toward the air port,
The immunosensor chip according to claim 2, wherein the stepped step is formed so as to continuously increase an occupation area in a direction from the injection port to the air port.
前記一対の電極は前記流路の伸びる方向に対して平行に形成され、前記他の一対の電極は前記流路の伸びる方向に対して垂直方向に形成される、請求項１から３のいずれかに記載の免疫センサチップ。 It further has another pair of electrodes,
The pair of electrodes are formed in parallel to a direction in which the flow path extends, and the other pair of electrodes are formed in a direction perpendicular to the direction in which the flow path extends. The immunosensor chip according to 1.
前記粒子分離手段によって分離された前記粒子の分布状態を観察する試料観察手段とを有する、請求項１から４のいずれかに記載の免疫センサ装置。 The immunosensor chip;
The immunosensor device according to claim 1, further comprising a sample observation unit that observes a distribution state of the particles separated by the particle separation unit.
電圧印加手段を有し、
前記電圧印加手段は、前記他の一対の電極に電圧を印加した後に、前記一対の電極に電圧を印加する、請求項４に記載の免疫センサ装置。
The immunosensor chip;
Having voltage applying means,
The immunosensor device according to claim 4, wherein the voltage applying unit applies a voltage to the pair of electrodes after applying a voltage to the other pair of electrodes.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2013113627A (en) * | 2011-11-25 | 2013-06-10 | Ulvac Seimaku Kk | Solution component sensor, method for manufacturing the same, solution component analysis system, solution component analysis kit, and method for analyzing analyte solution |
| KR20200097531A (en) * | 2019-02-08 | 2020-08-19 | 고려대학교 산학협력단 | Specimen analysis method based on a LAMB-Wave and device for specimen analysis |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| JP2013113627A (en) * | 2011-11-25 | 2013-06-10 | Ulvac Seimaku Kk | Solution component sensor, method for manufacturing the same, solution component analysis system, solution component analysis kit, and method for analyzing analyte solution |
| KR20200097531A (en) * | 2019-02-08 | 2020-08-19 | 고려대학교 산학협력단 | Specimen analysis method based on a LAMB-Wave and device for specimen analysis |
| KR102185652B1 (en) | 2019-02-08 | 2020-12-02 | 고려대학교 산학협력단 | Specimen analysis method based on a LAMB-Wave and device for specimen analysis |
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