JP2014160060A - Water repellent frame for electric field agitation - Google Patents
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Abstract
Description
本発明は、診断対象となる検体や抗体を含んだ液滴又は洗浄液の形状及び寸法精度を整えることにより、電界印加による非接触撹拌を利用した迅速免疫組織染色反応の精度のばらつきを抑制し、再現性を高めることを可能とする電界撹拌用ならびに電界洗浄用はっ水フレーム及び、これを用いた電界非接触撹拌方法に関する。 The present invention suppresses variations in the accuracy of rapid immunohistochemical staining reaction using non-contact agitation by applying an electric field by adjusting the shape and dimensional accuracy of a droplet or washing liquid containing a specimen or antibody to be diagnosed, The present invention relates to a water-repellent frame for electric field stirring and electric field cleaning that can improve reproducibility, and an electric field non-contact stirring method using the same.
下記特許文献１及び特許文献２に提案するように、本願の発明者らは、非常に高精度な診断ができるが、一連の作業に時間がかかるという免疫組織染色反応の問題を、電界を印加し、液滴の内部までを非接触にコンタミネーションを抑制する撹拌技術で、該反応を迅速化することにより解決を図ってきた。これにより、例えば、肺がんにおける原発性または転移性などのがんの性状や脳腫瘍における悪性度など病理診断精度の向上が要求されていた術中迅速病理診断に、免疫組織染色反応が利用可能になり、その効果として確定診後に再手術の施工が不要になることが期待され、患者の負担軽減、さらには医療費の縮減に寄与することが期待されるに至っている。 As proposed in Patent Document 1 and Patent Document 2 below, the inventors of the present application can apply an electric field to the problem of the immunohistochemical staining reaction, which can be diagnosed with very high accuracy but takes a long time for a series of operations. However, it has been attempted to solve the problem by speeding up the reaction with a stirring technique that suppresses contamination in a non-contact manner up to the inside of the droplet. As a result, for example, immunohistochemical staining reactions can be used for intraoperative rapid pathological diagnosis that required improvement in pathological diagnosis accuracy such as primary or metastatic cancer characteristics in lung cancer and malignancy in brain tumors, As an effect, it is expected that re-operation will not be required after a definitive diagnosis, and it is expected to contribute to reducing the burden on the patient and further reducing medical costs.
上述のような電界撹拌技術で液滴を非接触撹拌するには、液滴に十分な液滴内空間が確保される必要がある。具体的には、スライドガラス上で液滴をドーム形状とする必要がある。しかし、液滴は通常、電界撹拌の場となるスライドガラスとの親和性が良く、スライドガラス上で薄く広がるので、接触角を有するドーム形状を保持することが困難である。このような問題に対して従来、はっ水機能を付与するはっ水ペンで、スライドガラスに円環を描画し、この描画した円環内に電界撹拌しようとする液滴を滴下することで、液滴に一定の高さ（重力方向の厚み）を形成して液滴内空間を確保していた。 In order to non-contact agitate a droplet by the electric field stirring technique as described above, it is necessary to secure a sufficient space in the droplet. Specifically, it is necessary to make the droplet into a dome shape on the slide glass. However, since the droplets usually have a good affinity with the slide glass serving as a field stirring field and spread thinly on the slide glass, it is difficult to maintain a dome shape having a contact angle. Conventionally, a water repellent pen that provides a water repellent function is used to draw a ring on a slide glass and drop a droplet to be stirred in an electric field into the drawn ring. , A certain height (thickness in the direction of gravity) was formed on the droplets to secure the space inside the droplets.
はっ水ペンでスライドガラス上に円環を描画する際の問題点として、描く円環の直径、真円度等にばらつきが生じるため、形成した液滴のドーム形状の底面の径寸法、ｚ軸方向の頂点位置（厚み）が、ロット毎に異なることが挙げられる。液滴の底面の径寸法や厚みがロット毎に異なると、電界を与える電極までの距離もばらつき、撹拌性がロット毎に異なる原因になる。そして、例えば、免疫組織染色反応に適用すれば、その染色の進行度合いがロット毎に異なるようになるため、診断に疑義が生じてしまう可能性がある。 As a problem when drawing an annulus on a slide glass with a water-repellent pen, the diameter of the annulus to be drawn, roundness, etc. vary. It is mentioned that the apex position (thickness) in the axial direction varies from lot to lot. If the diameter and thickness of the bottom surface of the droplet are different for each lot, the distance to the electrode to which the electric field is applied also varies, which causes the agitation to be different for each lot. For example, when applied to an immunohistochemical staining reaction, the degree of progress of the staining varies from lot to lot, which may cause doubt in diagnosis.
従来、このような問題に対して、液滴に対する撹拌性が均一になるように、液滴撹拌時の波面振動現象のばらつきが生じるとその都度、電圧を与える時の電極間距離の調整又は与える電圧や周波数の調整等を行い、反応系のばらつきを抑制してきたが、反応系の再現性を高めるためには、上述のようなばらつきを根本的に解決する手段の開発が求められる。さらに、電界撹拌ならびに電界洗浄によって液滴が活発に振動運動するため、はっ水ペンで形成した円環を乗り越え液滴が流れ出るのを防止する手段も必要となる。 Conventionally, with respect to such a problem, adjustment of the distance between the electrodes when applying a voltage is given or applied every time the fluctuation of the wavefront vibration phenomenon during stirring of the droplets occurs so that the stirring property for the droplets becomes uniform. Although the variation of the reaction system has been suppressed by adjusting the voltage and frequency, etc., in order to improve the reproducibility of the reaction system, development of a means for fundamentally solving the above-described variation is required. Furthermore, since the droplet vibrates actively by electric field stirring and electric field cleaning, a means for preventing the droplet from flowing out over the ring formed by the water-repellent pen is also required.
テンププレート等の治具を利用してはっ水ペンにより円環を描くことを試みたものの、ペンとテンププレートとの位置関係によっては、ペン先とスライドガラスとの接触角度にばらつきが生じること、操作者によってペンがスライドガラスに与える筆圧が異なること、さらには、ペン先端部が消耗してくると描かれる円環の形状寸法に変化が生じやすくなること等の問題があった。 Although we tried to draw a ring with a water-repellent pen using a jig such as a temp plate, the contact angle between the pen tip and the slide glass may vary depending on the positional relationship between the pen and the temp plate. The pen pressure applied to the slide glass by the operator differs depending on the operator. Further, when the pen tip portion is consumed, the shape of the circle drawn tends to change.
また、印刷技術を用い、均一なサークルをスライドガラス上に描くことも想定され得るが、免疫組織染色反応に適用することを鑑みれば、試料の固定等の工程でアセトン等の有機溶剤を用いるため、アセトン等の有機溶剤で溶出されない円環を印刷で描画できるか否かを検討する必要がある。なお、電界印加環境に影響を与え難い材質でサークルを均一に形成していく必要もある。 In addition, it may be assumed that a uniform circle is drawn on the slide glass using a printing technique. However, in view of applying to an immunohistochemical staining reaction, an organic solvent such as acetone is used in a process such as sample fixation. It is necessary to examine whether or not a ring that is not eluted with an organic solvent such as acetone can be drawn by printing. In addition, it is necessary to form the circle uniformly with a material that does not easily affect the electric field application environment.
本発明は、上記実情に鑑み提案され、電界印加環境に影響を与え難い材質で形成され、滴下形状並びに寸法精度に富んだ液滴に、非接触で均一な撹拌特性を与え、その反応系の再現性を高めることができる場をスライドガラス上に設置して構成される電界撹拌用ならびに電界洗浄用はっ水フレーム及び、これを用いた電界非接触撹拌方法を提供することを目的とする。 The present invention has been proposed in view of the above circumstances, is formed of a material that does not easily affect the electric field application environment, and provides a non-contact and uniform stirring characteristic to a droplet having excellent drop shape and dimensional accuracy. An object of the present invention is to provide a water-repellent frame for electric field stirring and electric field cleaning constituted by installing a field capable of enhancing reproducibility on a slide glass, and an electric field non-contact stirring method using the same.
上記目的を達成するため、本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームは、耐アセトン性を有し、電界印加環境において電界分布に影響を与えない樹脂からなり、貫通孔を有するフレーム体を形成し、このフレーム体を基板に設けて構成され、前記フレーム体の前記貫通孔に滴下された液滴が前記基板に対して４５度以下の接触角を与えることを特徴とする。また、前記フレーム体の前記貫通孔に滴下された液滴の厚みは前記フレーム体の厚みと同じ又は薄い構成であることが好ましい。なお、前記貫通孔に滴下された液滴の厚みは前記フレーム体の厚みよりも厚くても良い。 In order to achieve the above object, the water-repellent frame for electric field stirring and electric field cleaning according to the present invention has acetone resistance and is made of a resin that does not affect electric field distribution in an electric field application environment and has a through hole. A frame body is formed, the frame body is provided on a substrate, and a droplet dropped into the through hole of the frame body gives a contact angle of 45 degrees or less to the substrate. Moreover, it is preferable that the thickness of the droplet dropped into the through hole of the frame body is the same as or thinner than the thickness of the frame body. In addition, the thickness of the droplet dropped into the through hole may be larger than the thickness of the frame body.
上記フレーム体の材質が、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂であり、そのフレーム幅が０．５〜５．０ｍｍ、前記貫通孔の面積が１９〜６００ｍｍ2であり、前記基板上に形成する前記フレーム体の厚みは、前記基板に取り付ける樹脂そのものの厚みであって、０．１〜０．５ｍｍであり、前記貫通孔に滴下される液滴の容量は、５〜１０００μＬであることを特徴とする。また、液滴が撹拌されたか否かを視認可能として染色工程の正常性を確認可能なセンサー部材が基板上に設けられていることも特徴とする。 The material of the frame body is a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine. The frame width is 0.5 to 5.0 mm, and the area of the through hole is 19 to 600 mm. 2, the thickness of the frame body formed on said substrate, a thickness of the resin itself to be attached to the substrate is 0.1 to 0.5 mm, the volume of the droplet to be dropped into the through hole Is 5 to 1000 μL. In addition, it is also characterized in that a sensor member capable of visually confirming whether or not the droplet has been stirred and confirming the normality of the dyeing process is provided on the substrate.
また、本発明に係る上記電界撹拌用ならびに電界洗浄用はっ水フレームを用いた電界非接触撹拌方法は、前記貫通孔に液滴を滴下し、主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波を形成した電界を、周波数０．１〜３００Ｈｚで前記液滴に与えることにより、前記液滴を非接触で撹拌可能とすることを特徴とする。 In the electric field non-contact stirring method using the water repellent frame for electric field stirring and electric field cleaning according to the present invention, a droplet is dropped into the through hole, and the main voltage is set to 0.4-2. By adding an offset electric field strength of 0.2 to 1.0 kV / mm to this and applying an electric field that forms a repeating square wave biased to the plus side to the droplets at a frequency of 0.1 to 300 Hz. The liquid droplets can be stirred without contact.
本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームは、耐アセトン性を有し、電界印加環境において電界分布に影響を与えない樹脂からなり、貫通孔を有するフレーム体を形成し、このフレーム体を基板に設けて構成され、フレーム体の貫通孔に滴下された液滴が基板に対して４５度以下の接触角を与える作用を有する。このとき、貫通孔に滴下された液滴の液量によっては、その厚みがフレーム体の厚みより薄い構成であってもよい。フレーム体の貫通孔に滴下された液滴がこのような範囲であれば、本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームにより、当該液滴に対し、液滴の底面の径寸法のばらつきを抑制することができ、撹拌される液滴内空間を均一化することができる。 The water-repellent frame for electric field stirring and electric field cleaning according to the present invention is made of a resin that has acetone resistance and does not affect the electric field distribution in an electric field application environment, and forms a frame body having through holes. The frame body is provided on the substrate, and the liquid droplets dropped into the through holes of the frame body have an effect of giving a contact angle of 45 degrees or less to the substrate. At this time, depending on the amount of the liquid dropped into the through hole, the thickness may be smaller than the thickness of the frame body. If the droplet dropped into the through-hole of the frame body is in such a range, the diameter dimension of the bottom surface of the droplet with respect to the droplet by the water-repellent frame for electric field stirring and electric field cleaning according to the present invention. Variation can be suppressed, and the agitated droplet inner space can be made uniform.
これにより、撹拌される液滴内空間が均一化するので、その撹拌特性を均一化することができる。すなわち、主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波を形成した電界を、０．１〜３００Ｈｚの周波数で液滴に与えることにより、フレーム体の貫通孔に滴下された液滴の撹拌特性のばらつきを抑制し、反応系のばらつきを抑制して再現性のよい高い品位な電界撹拌を非接触で実現することができる。さらに、液滴の頂点位置と電極間の距離の調整作業並びに調整機構、与える電界強度の調整作業並びに調整機構及び、液滴高さの情報をセンシングする機構等が不要になって、本発明が適用される電界非接触撹拌装置の構成を簡略化することができる。 Thereby, since the space in the droplet to be stirred is made uniform, the stirring characteristics can be made uniform. That is, 0.4 to 2.0 kV / mm on the plus side as the main voltage, and an offset electric field strength of 0.2 to 1.0 kV / mm is added to this, and an electric field that forms a repetitive square wave biased to the plus side, By giving the droplets at a frequency of 0.1 to 300 Hz, it is possible to suppress variations in stirring characteristics of the droplets dropped into the through-holes of the frame body, and to suppress variations in the reaction system and to have high reproducibility and high quality. Electric field agitation can be achieved without contact. Further, the adjustment work and adjustment mechanism of the distance between the vertex position of the droplet and the electrode, the adjustment work and adjustment mechanism of the applied electric field strength, the mechanism for sensing the information on the height of the liquid droplet, and the like are no longer necessary. The configuration of the applied electric field non-contact stirring apparatus can be simplified.
なお、電界撹拌用ならびに電界洗浄用はっ水フレームは、フレーム体を基板に設けて構成されるので、ペンで枠を描く等の人手で作業する煩雑さも解消される。また、フレーム体は耐アセトン性を有し、電界印加環境において電界分布に影響を与えない樹脂から形成されるため、アセトン固定等を行うパラフィン切片を用いる免疫組織染色反応に適用するのに好ましい構成であり、かつ、フレーム体と液滴等との反応について電界付与に際して懸念する必要がない。 In addition, since the water-repellent frame for electric field stirring and electric field cleaning is configured by providing the frame body on the substrate, the troublesome work such as drawing a frame with a pen is eliminated. In addition, since the frame body is acetone-resistant and is formed from a resin that does not affect the electric field distribution in an electric field application environment, the frame body is preferable for application to an immunohistochemical staining reaction using a paraffin section that is fixed with acetone or the like. In addition, there is no need to worry about the reaction between the frame body and the droplets when applying an electric field.
ここで、電界撹拌用ならびに電界洗浄用はっ水フレームは、フレーム体の材質としての電界印加環境において電界分布に影響を与えない樹脂の例示として、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂を挙げることができる。また、フレーム体の貫通孔に滴下された液滴に対して４５度以下の接触角を与える条件の例示として、そのフレーム幅が０．５〜５．０ｍｍであることを挙げることができる。基板上に形成するフレーム体の厚みは、基板に取り付ける樹脂そのものの厚みであって、０．１〜０．５ｍｍであることが好ましい。厚みが０．１ｍｍ以下では非接触撹拌時に被撹拌液滴が決壊する虞があるためである。また、貫通孔の面積が１９〜６００ｍｍ2であること、フレーム体の貫通孔において滴下する液滴は５〜１０００μＬであることが撹拌の均一性の面から好ましい。 Here, the water repellent frame for electric field agitation and electric field cleaning is exemplified by resins that do not affect the electric field distribution in the electric field application environment as the frame body material, such as polyvinyl, polyvinyl chloride, silicone, or fluorine. One resin selected from the system can be mentioned. Moreover, as an example of conditions for giving a contact angle of 45 degrees or less to the droplets dropped in the through holes of the frame body, the frame width can be 0.5 to 5.0 mm. The thickness of the frame body formed on the substrate is the thickness of the resin itself attached to the substrate, and is preferably 0.1 to 0.5 mm. This is because if the thickness is 0.1 mm or less, the stirred droplet may break during non-contact stirring. It area of the through-hole is 19～600Mm 2, droplets dropping in the through hole of the frame body is preferably from the viewpoint of uniformity of agitation to be 5～1000MyuL.
さらに、本発明では、液滴が撹拌されたか否かを視認可能として染色工程の正常性を確認可能なセンサー部材が基板上に設けられている構成により、液滴が撹拌されたか否かを視認することができ、免疫組織染色反応の進行度合いを視覚的に確認することができる。例えば、撹拌されると発色するポジティブコントロール、撹拌しても発色しないネガティブコントロールをセンサー部材とすれば、このセンサー部材の存在が染色工程の正常性を確認することができる、すなわち免疫組織染色反応が行われたか否かの指標にすることができるので、免疫組織染色反応を術中迅速病理診断で採用することを促すことができる。また、ポジティブコントロール等が検体組織と反応しないことの確認が得られる場合には、１つのフレーム体の枠に検体試料とセンサー部材とをそれぞれ独立させる構成とすることも可能となる。 Furthermore, in the present invention, it is possible to visually check whether or not the droplet has been stirred by the configuration in which the sensor member that can check whether or not the droplet has been stirred and can check the normality of the dyeing process is provided on the substrate. And the degree of progress of the immunohistochemical staining reaction can be visually confirmed. For example, if the sensor member is a positive control that develops color when stirred and a negative control that does not develop color even when stirred, the presence of this sensor member can confirm the normality of the staining process. Since it can be used as an indicator of whether or not it has been performed, it is possible to encourage the use of an immunohistochemical staining reaction in rapid pathological diagnosis during the operation. In addition, when it is confirmed that the positive control or the like does not react with the specimen tissue, it is possible to adopt a configuration in which the specimen sample and the sensor member are independent from each other in the frame of one frame body.
本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームにより、基板として例示されるガラスプレート上に耐アセトン性を有し、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂からなる電界撹拌の良好な撹拌動作を生む反応場液滴形状並びに領域を確保する領域を形成し、ばらつきの少ない一定の量の液滴を滴下可能にすることによって、液滴反応場の高さのばらつきが抑制され、電極間と液滴頭頂部との距離長ばらつきが抑制されて、電界撹拌性のばらつきが抑制され、極めて簡易でありながら優れた液滴の反応場を形成することができる。 The water-repellent frame for electric field stirring and electric field cleaning according to the present invention has acetone resistance on a glass plate exemplified as a substrate, and is selected from polyvinyl, polyvinyl chloride, silicone, or fluorine. By forming a reaction field droplet shape and a region to ensure a good stirring operation of electric field stirring made of resin, and allowing a certain amount of droplets with little variation to be dropped, the droplet reaction field Variation in height is suppressed, variation in distance between the electrodes and the top of the droplet is suppressed, variation in electric field agitation is suppressed, and an extremely simple yet excellent droplet reaction field is formed. Can do.
以下、本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームについての一実施形態を、図面を参照しつつ説明する。具体的には、図１〜図２に示すような円環形状の電界撹拌用はっ水リング１について取り上げる。なお、本実施形態は、本発明の構成を具現化した例示に過ぎず、本発明は、特許請求の範囲に記載した事項を逸脱することがなければ、種々の設計変更を行うことができる。例えば、電界撹拌用ならびに電界洗浄用はっ水フレームにおけるフレーム体は、円環形状のリングのほか、ロット毎に同じ形状であって、滴下される液滴に対して均一な撹拌性が確保される限り、正方形、長方形の矩形状等の各種形状のフレーム体でよい。 Hereinafter, an embodiment of a water-repellent frame for electric field stirring and electric field cleaning according to the present invention will be described with reference to the drawings. Specifically, an annular water-repellent ring 1 for electric field stirring as shown in FIGS. The present embodiment is merely an example embodying the configuration of the present invention, and the present invention can be modified in various ways without departing from the matters described in the claims. For example, the frame body in the water-repellent frame for electric field agitation and electric field cleaning has the same shape for each lot in addition to an annular ring, and uniform agitation is ensured for the dropped droplets. As long as it is a frame body of various shapes such as a square and a rectangular rectangle.
電界撹拌用はっ水リング１は、図１に示すように、耐アセトン性を有し、電界印加環境において電界分布に影響を与えない樹脂からなり、円環状の貫通孔を有するフレーム体（以下、「リング体」と称する。）１０が形成され、このリング体１０が基板としての、スライドガラス状のプラスチック基板２（図２参照）に設けられて構成されている。そして、リング体１０の貫通孔部分であるリング内部１１に滴下された液滴に対して撹拌可能となるように、例えば、電界撹拌用はっ水リング１は、リング体１０のリング内部１１に滴下された液滴Ｌに対して４５度以下の接触角を与える構成である。また、図７に示すように、リング内部１１に滴下された液滴の厚みがリング体１０の厚みと同じ又は薄い構成としてもよい。 As shown in FIG. 1, the water-repellent ring 1 for electric field stirring is made of a resin that has acetone resistance and does not affect electric field distribution in an electric field application environment, and has a frame body (hereinafter referred to as an annular through hole). , 10 is formed, and this ring body 10 is provided on a slide glass plastic substrate 2 (see FIG. 2) as a substrate. Then, for example, the water repellent ring 1 for electric field stirring is placed in the ring interior 11 of the ring body 10 so that the liquid droplets dropped into the ring interior 11 that is the through hole portion of the ring body 10 can be stirred. In this configuration, a contact angle of 45 degrees or less is given to the dropped droplet L. In addition, as shown in FIG. 7, the thickness of the droplet dropped into the ring interior 11 may be the same as or thinner than the thickness of the ring body 10.
そして、所定の接触角を満たすために、リング体１０は、リング幅Ｗが０．５〜５．０ｍｍ、内部面積Ｓが１９〜６００ｍｍ2とされる。リング体１０の厚みは、プラスチック基板２に取り付ける樹脂そのものの厚みであって、０．１〜０．５ｍｍである。この構成のリング体１０において、リング内部１１に滴下される液滴Ｌの容量は、５〜１０００μＬである。リング幅Ｗが０．５ｍｍ未満、或いは内部面積Ｓが１９ｍｍ2未満となると、撹拌時に液滴Ｌがリング体１０の枠外に溢れる恐れがある。内部面積Ｓが６００ｍｍ2超となると、均一な撹拌が行われなくなる恐れがある。リング幅Ｗの上限は実施可能な範囲として特に制限されるものではないが、適用される電界非接触撹拌装置の電圧印加の場となる試料室の大きさ、基板の大きさ、材料の経済性等から合理的なリング幅（例えば、５ｍｍ程度まで）とすればよい。 In order to satisfy a predetermined contact angle, the ring body 10 has a ring width W of 0.5 to 5.0 mm and an internal area S of 19 to 600 mm 2 . The thickness of the ring body 10 is the thickness of the resin itself attached to the plastic substrate 2 and is 0.1 to 0.5 mm. In the ring body 10 having this configuration, the volume of the droplet L dropped into the ring interior 11 is 5 to 1000 μL. If the ring width W is less than 0.5 mm or the inner area S is less than 19 mm 2 , the droplet L may overflow outside the frame of the ring body 10 during stirring. If the internal area S exceeds 600 mm 2 , uniform stirring may not be performed. The upper limit of the ring width W is not particularly limited as a feasible range, but the size of the sample chamber, the size of the substrate, and the economics of the material used as the voltage application field of the applied electric field non-contact stirrer Therefore, a reasonable ring width (for example, up to about 5 mm) may be used.
リング体１０は、電界印加環境において電界分布に影響を与えない樹脂、例えば、ポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から１つ選ばれる樹脂から形成されている。「電界印加環境において電界分布に影響を与えない」とは、電界撹拌用はっ水リング１が電界印加環境下で、その他の物質（少なくとも、リング内部１１に滴下された液滴Ｌに含まれる物質等：披験物質）と一切、反応しないことをいう。また、プラスチック基板２から指等で着脱可能とするためのヘソ部１０ａをリング体１０に設けることが操作性の観点から好ましい構成である。 The ring body 10 is made of a resin that does not affect the electric field distribution in an electric field application environment, for example, a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine. “Does not affect the electric field distribution in an electric field application environment” means that the water stirring water repellent ring 1 for electric field agitation is included in other substances (at least in the droplet L dropped into the ring interior 11) in the electric field application environment. Substance etc .: Test substance) means no reaction at all. Moreover, it is a preferable structure from a viewpoint of operativity to provide the ring body 10 with the navel part 10a for making it detachable with a finger etc. from the plastic substrate 2. FIG.
本実施形態では、フッ素系のニトフロンテープ（登録商標、日東電工Ｎｏ．９０３−Ｔ）又はポリ塩化ビニル系のＰＶＣテープ（日東電工ＳＰＶ−２０２）を材質として形成したリング体１０を例示している。下記［表１］に本実施形態で採用したリング体１０の一覧（実施例１〜８）を示す。なお、比較例は、従来のはっ水ペンでサークルを描いた例である。 In the present embodiment, a ring body 10 formed by using a fluorine-based nitroflon tape (registered trademark, Nitto Denko No. 903-T) or a polyvinyl chloride-based PVC tape (Nitto Denko SPV-202) as an example is illustrated. Yes. Table 1 below shows a list (Examples 1 to 8) of the ring bodies 10 employed in the present embodiment. The comparative example is an example in which a circle is drawn with a conventional water-repellent pen.
なお、本発明を構成するプラスチック基板２は公知のものでよく、プラスチックに限らず、ガラス製のものでも構わない。本発明を構成するフレーム体は、上述したリング体１０の形状のほか、ロット毎に同じ形状であって、滴下される液滴に対して均一な撹拌性が確保される限り、矩形状等の各種の形状を採用することができる。 In addition, the plastic substrate 2 which comprises this invention may be a well-known thing, and may be not only a plastic but the thing made from glass. In addition to the shape of the ring body 10 described above, the frame body constituting the present invention has the same shape for each lot, and has a rectangular shape or the like as long as uniform stirring is ensured for the dropped droplet. Various shapes can be employed.
本発明に係る電界撹拌用はっ水リング１は、図２（ａ），（ｂ）に示すように、電界非接触撹拌装置Ｅの電圧印加の場となる試料室Ｅ１に設置されて使用される。電界非接触撹拌装置Ｅは、試料室Ｅ１の電極Ｅ２において、主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波の電界を形成することができる。また、その電界を０．１〜３００Ｈｚの範囲で液滴Ｌが特異的に振動する周波数により、試料室Ｅ１に設置される電界撹拌用はっ水リング１に含まれる液滴Ｌに与えることができる。電界を与えられた液滴Ｌはリング体１０のリング内部１１にて非接触で撹拌される。なお、２〜４０本の束としたバースト電界として、液滴に与えることもできる。 The water-repellent ring 1 for electric field stirring according to the present invention is installed and used in a sample chamber E1 that serves as a voltage application field of an electric field non-contact stirring device E as shown in FIGS. 2 (a) and 2 (b). The The electric field non-contact stirring device E adds 0.4 to 2.0 kV / mm as a main voltage to the electrode E2 in the sample chamber E1, and adds an offset electric field strength of 0.2 to 1.0 kV / mm to the plus side. A repeating square-wave electric field biased to the side can be formed. Further, the electric field can be applied to the droplet L contained in the water-repellent ring 1 for electric field stirring installed in the sample chamber E1 with a frequency at which the droplet L specifically vibrates in the range of 0.1 to 300 Hz. it can. The droplet L to which an electric field is applied is stirred in a non-contact manner inside the ring 11 of the ring body 10. In addition, it can also give to a droplet as a burst electric field made into the bundle | flux of 2-40 pieces.
以下、電界撹拌用はっ水リング（実施例１〜８）の性能について評価したので、これを説明していく。 Hereinafter, since the performance of the water repellent ring for electric field stirring (Examples 1 to 8) was evaluated, this will be described.
まず、実施例１〜８の電界撹拌用はっ水リング及び比較例について、１％ＢＳＡ（仔牛血清アルブミン）溶液を１５０μＬ滴下したときに形成される接触角を調べた。その結果を、図３に示す。 First, the contact angle formed when 150 μL of 1% BSA (calf serum albumin) solution was dropped on the water-repellent rings for electric field stirring of Examples 1 to 8 and the comparative example was examined. The result is shown in FIG.
図３に示すように、実施例１〜８の電界撹拌用はっ水リング１はすべて、滴下した液滴Ｌに対して４５度以下の接触角を与えた。特に、実施例１及び実施例８以外の例では、はっ水ペンでサークルを描いた比較例と比べ同等又はそれ以上の接触角を与えた。これにより、実施例１〜８の電界撹拌用はっ水リング１は、液滴Ｌに対して４５度以下の接触角をロット毎にばらつくことなく与えることができ、均一な撹拌特性を与えられることが分かった。 As shown in FIG. 3, all the water repellent rings 1 for electric field stirring in Examples 1 to 8 gave a contact angle of 45 degrees or less to the dropped liquid droplets L. In particular, in examples other than Example 1 and Example 8, a contact angle equal to or greater than that of the comparative example in which a circle was drawn with a water-repellent pen was given. Thereby, the water repellent ring 1 for electric field stirring of Examples 1-8 can give the contact angle of 45 degrees or less with respect to the droplet L without varying for every lot, and is given uniform stirring characteristics. I understood that.
また、実施例１〜８の電界撹拌用はっ水リング１を使って、一連の迅速診断プロトコルを、上述した電界非接触撹拌装置Ｅの試料室Ｅ１内で実施した。すなわち、リング体１０のリング内部１１に滴下した一次抗体を含む溶液、二次抗体を含む溶液のそれぞれの液滴Ｌに対し、試料室Ｅ１の電極Ｅ２において、主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波の電界を形成し、この電界を０．１〜３００Ｈｚの液滴Ｌが特異的に振動する周波数で与えて撹拌した。そして、滴下した試料（液滴）が、リング体１０の外に漏れないで保持されているかどうかを調べた。その結果を、図４に示す。 Moreover, using the water-repellent ring 1 for electric field stirring in Examples 1 to 8, a series of rapid diagnostic protocols was performed in the sample chamber E1 of the electric field non-contact stirring apparatus E described above. That is, with respect to the respective droplets L of the solution containing the primary antibody and the solution containing the secondary antibody dropped into the ring interior 11 of the ring body 10, the main voltage at the electrode E2 in the sample chamber E1 is 0.4 on the positive side. ~ 2.0kV / mm, offset electric field strength of 0.2 ~ 1.0kV / mm is added to this to form a repeating square wave electric field biased to the plus side. L was stirred at a frequency that specifically vibrates. And it was investigated whether the dripped sample (droplet) was hold | maintained without leaking out of the ring body 10. FIG. The result is shown in FIG.
上記迅速診断プロトコルとは、抗原抗体反応法の工程をいい、コンパウンド除去（３０秒）、アセトン固定（２分）、ＰＢＳ洗浄（３０秒）、内因性ＰＯＤ除去（１５秒）、ＰＢＳ洗浄（３０秒）、ブロッキング（５分）、一次抗体（５分）、ＰＢＳ洗浄（３０秒）、二次抗体（５分）の順に進める工程である。この試験では、一次抗体、二次抗体の代わりに、１％ＢＳＡ溶液を１５０μＬ、電界撹拌用はっ水リング１のリング内部１１に滴下して電界撹拌を行った。 The rapid diagnostic protocol refers to a process of antigen-antibody reaction method. Compound removal (30 seconds), acetone fixation (2 minutes), PBS washing (30 seconds), endogenous POD removal (15 seconds), PBS washing (30 Second), blocking (5 minutes), primary antibody (5 minutes), PBS washing (30 seconds), secondary antibody (5 minutes). In this test, 150 μL of 1% BSA solution was dropped into the ring interior 11 of the water-repellent ring 1 for electric field stirring instead of the primary antibody and the secondary antibody, and electric field stirring was performed.
図４に示すように、ニトフロン（登録商標）を材質とした実施例１〜４の電界撹拌用はっ水リング１では、リング幅Ｗが１ｍｍであるもの以外で、液滴Ｌがリング体１０の外に漏れないで保持されていた。一方、ＰＶＣを材質とした実施例５〜８の電界撹拌用はっ水リング１では、リング幅Ｗが１ｍｍであるもので、液滴Ｌがリング体１０の外に漏れないで保持されていた。したがって、所定の材質のリング体１０により本発明を構成すれば、免疫組織染色反応で一般的な５〜１０００μＬの容量の範囲において、そのリング体１０のリング幅Ｗを０．５〜５．０ｍｍ、特に、１．０〜４．０ｍｍとすることで、滴下した液滴Ｌを電界印加環境下であっても、リング体１０の外に漏らさないで保持できることが分かった。 As shown in FIG. 4, in the water-repellent ring 1 for electric field agitation of Examples 1 to 4 made of nitoflon (registered trademark), the liquid droplets L are formed in the ring body 10 except for the ring width W of 1 mm. Was kept leaking out. On the other hand, in the water-repellent ring 1 for electric field stirring of Examples 5 to 8 made of PVC, the ring width W is 1 mm, and the droplet L is held without leaking out of the ring body 10. . Therefore, if the present invention is constituted by the ring body 10 of a predetermined material, the ring width W of the ring body 10 is set to 0.5 to 5.0 mm in a volume range of 5 to 1000 μL which is general in an immunohistochemical staining reaction. In particular, it was found that by setting the thickness to 1.0 to 4.0 mm, the dropped liquid droplet L can be held without leaking out of the ring body 10 even in an electric field application environment.
なお、リング幅Ｗが０．５〜５．０ｍｍ程度であれば、リング体１０の材質は、実施例に用いたポリ塩化ビニル系、フッ素系の樹脂のほか、ポリビニル系又はシリコーン系等の電界印加環境において電界分布に影響を与えない樹脂であるかぎり、採用することができる。すなわち、そのような電界撹拌用はっ水リング１において、液滴Ｌをリング体１０の外に漏らさず、かつ、均一な非接触撹拌を行うことができる。 In addition, if the ring width W is about 0.5 to 5.0 mm, the material of the ring body 10 may be polyvinyl chloride or fluorine resin used in the embodiment, or polyvinyl or silicone electric field. Any resin that does not affect the electric field distribution in the applied environment can be used. That is, in such a water repellent ring 1 for electric field agitation, uniform non-contact agitation can be performed without causing the droplets L to leak out of the ring body 10.
ここで、本発明に係る電界撹拌用はっ水フレームは、貫通孔部分であるリング内部にセンサー部材を同梱して構成することができる。なお、センサー部材を基板上に設けるのであれば、リング外部に設けても構わない。 Here, the water-repellent frame for electric field stirring according to the present invention can be configured by enclosing a sensor member inside a ring which is a through-hole portion. If the sensor member is provided on the substrate, it may be provided outside the ring.
本発明に係る電界撹拌用はっ水リング１００は、図５に示すように、センサー部材として、電界が付与されて撹拌されることにより、免疫組織染色反応を通じて発色するポジティブコントロール部１０１と、電界が付与されて撹拌されることにより、免疫組織染色反応を通じて非発色となるネガティブコントロール部１０２とがリング内部１１に同梱されて構成されている。 As shown in FIG. 5, the water-repellent ring 100 for electric field stirring according to the present invention includes a positive control unit 101 that develops color through an immunohistochemical staining reaction when an electric field is applied and stirred as a sensor member, And a negative control part 102 that does not develop color through an immunohistochemical staining reaction is included in the ring interior 11.
具体的には、リング内部１１にニトロセルロースを粘着テープで固定し、このニトロセルロース上に、撹拌により発色するタンパク質（例えば、ＣＫ１９とその抗体の組み合わせであれば、ＣＫ１９）を所定量滴下してポジティブコントロール部１０１を形成する。同様に、ニトロセルロース上に、撹拌により非発色となるタンパク質（例えば、ＨＥＲ２とその抗体の組み合わせであれば、ＨＥＲ２）を滴下し、ネガティブコントロール部１０２を形成して構成する。リング内部に粘着テープで固定する部材としては、ニトロセルロースのほか、アガロースゲル、ゼラチン等を採用することができる。 Specifically, nitrocellulose is fixed to the inside 11 of the ring with an adhesive tape, and a predetermined amount of a protein that develops color by stirring (for example, CK19 in the case of a combination of CK19 and its antibody) is dropped on the nitrocellulose. A positive control unit 101 is formed. Similarly, a protein (for example, HER2 in the case of a combination of HER2 and its antibody) that is not colored by stirring is dropped onto nitrocellulose to form a negative control unit 102. In addition to nitrocellulose, agarose gel, gelatin or the like can be used as a member to be fixed inside the ring with an adhesive tape.
なお、図５において、電界撹拌用はっ水リング１００は、ポジティブコントロール部１０１及びネガティブコントロール部１０２の両方がリング内部１１にそれぞれ独立して固定されているが、いずれか一方がリング内部１１に固定される構成でもよい。また、リング内部１１で固定するニトロセルロースの大きさは特に限定されないが、それぞれ１〜２×１〜２ｍｍ程度が視認性の観点から好ましい。 In FIG. 5, in the water-repellent ring 100 for electric field stirring, both the positive control unit 101 and the negative control unit 102 are fixed independently in the ring interior 11, but either one is in the ring interior 11. It may be a fixed configuration. Moreover, although the magnitude | size of the nitrocellulose fixed in the ring inside 11 is not specifically limited, About 1-2x1-2mm is preferable from a viewpoint of visibility, respectively.
粘着テープの材質についても特に限定されないが、アセトンとの反応し難い材質として、アクリルベースの両面テープ（例えば、デクセリアルズ社製Ｇ９９５１Ｗ−１００）等を用いることが好ましい。電界撹拌用はっ水リング１００を用いて、免疫組織染色反応においてアセトン固定処理を行う際には、タンパク質の溶出の懸念を回避するために静置状態で行うことが好ましい。 The material of the adhesive tape is not particularly limited, but it is preferable to use an acrylic-based double-sided tape (for example, G9951W-100 manufactured by Dexerials) as a material that does not easily react with acetone. When performing the acetone fixation treatment in the immunohistochemical staining reaction using the water repellent ring 100 for electric field stirring, it is preferably performed in a stationary state in order to avoid concern about protein elution.
本発明では、センサー部材と検体試料とがコンタミネーションを生じないことを確認した上でリング内部１１にセンサー部材として同梱することや、本発明を構成するリング体１０を２枚用い、そのうち一方を検体試料用に、他方をセンサー部材用に用いることで、一枚のプラスチック基板２に検体試料とセンサー部材とを同梱させ、コンタミネーションを生じない構成とすることができる。そして、例えば、一連の迅速診断プロトコルが上述した電界非接触撹拌装置Ｅで実施された際に、リング内部１１に滴下された液滴Ｌが正常に撹拌されているか否かを、試料室Ｅ１内で知ることができる。例えば、何らかの原因によって停電が生じて電界撹拌が正常に動作しなかったことを、術中迅速病理診断の途中で知ることができる。すなわち、異常をいち早く察知し、迅速診断に支障をきたさないようにすることができることとなる。 In the present invention, after confirming that the sensor member and the specimen sample do not cause contamination, the sensor is enclosed in the ring interior 11 as a sensor member, or two ring bodies 10 constituting the present invention are used. Is used for the specimen sample and the other is used for the sensor member, so that the specimen sample and the sensor member can be packaged on one plastic substrate 2 so that no contamination occurs. Then, for example, when a series of rapid diagnosis protocols is performed by the electric field non-contact stirring device E described above, whether or not the droplet L dropped into the ring interior 11 is normally stirred is determined in the sample chamber E1. You can find out at For example, it is possible to know during an intraoperative rapid pathological diagnosis that a power failure has occurred for some reason and the electric field agitation did not operate normally. In other words, it is possible to detect an abnormality early and prevent a quick diagnosis from being hindered.
図６に、ＣＫ１９とその抗体の組み合わせで構成されるポジティブコントロール部１０１、ＨＥＲ２で構成されるネガティブコントロール部１０２を同封した本発明に係る電界撹拌用はっ水リング１００に、電界付与して免疫組織染色反応を進めた結果を示す。 In FIG. 6, an electric field is applied to the water-repellent ring 100 for electric field stirring according to the present invention in which a positive control unit 101 composed of a combination of CK19 and its antibody and a negative control unit 102 composed of HER2 are enclosed. The result of having advanced the tissue staining reaction is shown.
図６から、電界撹拌用はっ水リング１００のリング内部１１に滴下した液滴が電界撹拌され、ポジティブコントロール部１０１で発色され、ネガティブコントロール部１０２で発色していない様子が把握される。したがって、リング内部１１のセンサー部材の存在が、免疫組織染色反応が行われたか否かの指標となるので、本発明を用いた免疫組織染色反応を、術中迅速病理診断で活用することを促すことができる。 From FIG. 6, it can be seen that the droplet dropped into the ring interior 11 of the water repellent ring 100 for electric field stirring is electric field stirred and colored by the positive control unit 101 and not colored by the negative control unit 102. Therefore, since the presence of the sensor member inside the ring 11 serves as an index as to whether or not the immunohistochemical staining reaction has been performed, it is urged to utilize the immunohistochemical staining reaction using the present invention for rapid pathological diagnosis during the operation. Can do.
なお、電界撹拌用はっ水リング１００では、上述した実施例１の電界撹拌用はっ水リングにポジティブコントロール部１０１やネガティブコントロール部１０２をそれぞれ独立して固定されるようにして同梱した構成とした。本発明を構成するリング体１０を２枚用い、そのうち一方を検体試料用に、他方をセンサー部材用に採用することで、一枚のプラスチック基板２に検体試料とセンサー部材とを同梱させた構成を提供することもできる。電界付与の方法は上記実施例１〜８の電界撹拌用はっ水リング１を使って実施した一連の迅速診断プロトコルと同様である。ただし、この試験では、一次抗体の溶液（１５０μＬ）に、ＣＫ１９に結合する抗体が含まれている。二次抗体の溶液（１５０μＬ）には、一次抗体に結合する抗体が含まれている。ポジティブコントロール部１０１の発色には、発色剤として３，３’ジアミノベンジジンを用いた。このような一次抗体は、ネガティブコントロール部１０２のＨＥＲ２に対して結合しないので、二次抗体とも反応せず、したがって発色しない。 The electric field agitation water repellent ring 100 is configured such that the positive control unit 101 and the negative control unit 102 are independently fixed to the electric field agitation water repellent ring of Example 1 described above. It was. The two ring bodies 10 constituting the present invention are used, one of which is used for the sample and the other is used for the sensor member, so that the sample sample and the sensor member are bundled on one plastic substrate 2. A configuration can also be provided. The method for applying an electric field is the same as the series of rapid diagnosis protocols performed using the water-repellent ring 1 for electric field stirring in Examples 1 to 8 above. However, in this test, an antibody that binds to CK19 is contained in the primary antibody solution (150 μL). The secondary antibody solution (150 μL) contains an antibody that binds to the primary antibody. For the color development of the positive control unit 101, 3,3'diaminobenzidine was used as a color former. Since such a primary antibody does not bind to HER2 of the negative control unit 102, it does not react with the secondary antibody and therefore does not develop color.
ポジティブコントロール部１０１やネガティブコントロール部１０２に用いるタンパク質は、上述したＣＫ１９やＨＥＲ２に限定されず、ほとんどすべてのタンパク質を用いることができる。現状、ほとんどすべてのタンパク質において、そのアミノ酸配列の情報は明らかにされているので、この情報に基づいて様々なタンパク質を合成し、また、その抗体を製造することができるからである。例えば、がん関連の迅速診断において、本発明を構成するポジティブコントロール部１０１やネガティブコントロール部１０２を活用する際には、Ｋｉ６７、Ｐ５３、ＥＧＦＲ、ＣＤ５６、ＣＫ２０、ＣＡ１２５等のタンパク質を利用することができる。 Proteins used for the positive control unit 101 and the negative control unit 102 are not limited to the above-described CK19 and HER2, and almost all proteins can be used. At present, information on the amino acid sequence of almost all proteins has been clarified, and various proteins can be synthesized and antibodies can be produced based on this information. For example, when the positive control unit 101 and the negative control unit 102 constituting the present invention are used in rapid diagnosis related to cancer, proteins such as Ki67, P53, EGFR, CD56, CK20, and CA125 may be used. it can.
ここで、本発明は、図７に示すように、リング体１０ｘの貫通孔部分であるリング内部１１ｘに滴下された液滴Ｌの厚みが、リング体１０ｘの厚みと同じ又は薄くなる構成を含む。例えば、リング体１０ｘの厚みが０．２ｍｍで内径φ２０ｍｍ（内部面積Ｓは３１４ｍｍ2）の電界撹拌用はっ水リング１ｘの場合、滴下された液滴Ｌの容量が１００μＬ以下であるときにその厚みが、リング体１０ｘの厚みと同じになり、又はリング体１０ｘの厚みよりも薄くなる。また、図示を省略するが、リング体の厚みが０．２ｍｍで内部面積Ｓが１９ｍｍ2の電界撹拌用はっ水リングの場合、ポジティブコントロール部１０１やネガティブコントロール部１０２用で、滴下された液滴Ｌの容量が５μＬであるときにその厚みが、リング体の厚みと同じになる。そして、このような形態において、上述した一連の迅速診断プロトコルを、電界非接触撹拌装置Ｅの試料室Ｅ１内で、電極Ｅ２に主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波の電界を形成し、この電界を０．１〜３００Ｈｚの液滴Ｌが特異的に振動する周波数で与えて撹拌して実施した。その結果、滴下した液滴Ｌが電界印加環境下であっても、リング体１０ｘの外に漏れさないで保持できることが分かり、すなわち、フレーム体の枠内部に滴下された液滴の撹拌特性のばらつきを抑制し、反応系のばらつきを抑制して再現性のよい高い品位な電界撹拌を非接触で実現することができた。これにより、非常に高価な抗体試薬の節約を図ることが理解される。 Here, as shown in FIG. 7, the present invention includes a configuration in which the thickness of the droplet L dropped into the ring interior 11x, which is a through hole portion of the ring body 10x, is the same as or thinner than the thickness of the ring body 10x. . For example, in the case of a water repellent ring 1x for electric field stirring having a thickness of 0.2 mm and an inner diameter φ20 mm (internal area S is 314 mm 2 ), when the volume of the dropped droplet L is 100 μL or less, The thickness becomes the same as the thickness of the ring body 10x or becomes thinner than the thickness of the ring body 10x. Although not shown, in the case of a water-repellent ring for electric field stirring with a ring body thickness of 0.2 mm and an internal area S of 19 mm 2 , the liquid dropped for the positive control unit 101 and the negative control unit 102 When the volume of the droplet L is 5 μL, the thickness is the same as the thickness of the ring body. In such a form, the above-described series of rapid diagnosis protocols is performed in the sample chamber E1 of the electric field non-contact stirrer E by 0.4 to 2.0 kV / mm on the positive side as the main voltage for the electrode E2. Is applied with an offset electric field strength of 0.2 to 1.0 kV / mm to form a repetitive square wave electric field biased to the plus side, and the frequency at which the droplet L of 0.1 to 300 Hz specifically vibrates. Was carried out with stirring. As a result, it can be seen that the dropped liquid droplet L can be held without leaking out of the ring body 10x even under an electric field application environment, that is, the stirring characteristics of the liquid droplet dropped inside the frame of the frame body. It was possible to suppress non-uniformity and suppress non-uniformity of the reaction system and achieve high-quality electric field stirring with good reproducibility in a non-contact manner. This is understood to save on very expensive antibody reagents.
以上、本発明について出願人が最良であると信じる実施形態を詳述したが、本発明は、特許請求の範囲に記載された事項を逸脱することがなければ、上記実施形態に限定されることなく、種々の設計変更を行うことが可能である。上記実施形態では、電界撹拌用はっ水リングのリング内径について１２ｍｍのものを使用して説明したが、１９〜６００ｍｍ2のパラメータを満たせばよく、例えば、リング内径が５〜２０ｍｍのもので本発明を構成することができる。例えば、リング内径が２０ｍｍのものでは、本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームとして、ポジティブコントロール部やネガティブコントロール部を同梱させるほか、本発明を構成するフレーム体を２枚用い、そのうち一方を検体資料用に、他方をセンサー部材用に用いることで、一枚の基板に検体試料とセンサー部材とを同梱させた構成を提供することができる。すなわち迅速診断の対象である被診断試料を含めて同梱することが可能となる。 As mentioned above, although the embodiment which the applicant believes is the best about this invention was explained in full detail, this invention is limited to the said embodiment, unless it deviates from the matter described in the claim. It is possible to make various design changes. In the above embodiment, the ring inner diameter of the water-repellent ring for electric field agitation has been described using 12 mm. However, it is only necessary to satisfy the parameters of 19 to 600 mm 2 , for example, the ring inner diameter is 5 to 20 mm. The invention can be configured. For example, when the ring inner diameter is 20 mm, the positive control part and the negative control part are included as the water-repellent frame for electric field stirring and electric field cleaning according to the present invention, and two frame bodies constituting the present invention are included. By using one of them for the specimen material and the other for the sensor member, it is possible to provide a configuration in which the specimen sample and the sensor member are bundled on a single substrate. That is, it is possible to enclose a sample to be diagnosed that is a target of rapid diagnosis.
また、基板上に形成するリング体の上下方向の厚みは、基板に取り付ける樹脂そのものの厚みであり、０．１〜０．５ｍｍの範囲のうち、およそ０．２ｍｍ程度であることが好ましい。ちなみに本発明は、ポジティブコントロール部やネガティブコントロール部等のセンサー部材を同梱することを必須の構成要件とするものではない点に留意されたい。また、図２等に示すような基板に対する液滴の接触角が正の値であって４５度以下であるような場合は、免疫組織染色工程における洗浄時に適用することで洗浄効果を上げることができる。図７に示すような基板に対する液滴の接触角がゼロ又は負の値（リング内部に滴下された液滴の厚みが、リング体の厚みと同じ又は薄くなる構成）であるような場合は、免疫組織染色工程における抗原抗体反応時又は染色時に適用することで抗体試薬の節約、抗原抗体反応の効果を上げることができる。 Moreover, the thickness of the ring body formed on the substrate in the vertical direction is the thickness of the resin itself attached to the substrate, and is preferably about 0.2 mm within the range of 0.1 to 0.5 mm. Incidentally, it should be noted that the present invention does not require the inclusion of sensor members such as a positive control unit and a negative control unit as an essential component. In addition, when the contact angle of the liquid droplet with respect to the substrate as shown in FIG. 2 is a positive value and is 45 degrees or less, the cleaning effect can be improved by applying it at the time of cleaning in the immunohistochemical staining step. it can. When the contact angle of the droplet to the substrate as shown in FIG. 7 is zero or a negative value (a configuration in which the thickness of the droplet dropped inside the ring is the same as or thinner than the thickness of the ring body) Application during antigen-antibody reaction or staining in the immunohistochemical staining step can save antibody reagents and increase the effect of antigen-antibody reaction.
このほか、本発明に係る電界撹拌用ならびに電界洗浄用はっ水フレームは、上述した実施形態のような免疫組織染色とともに、核酸におけるハイブリダイゼーション工程、並びに一般的な抗原抗体反応等に適用可能である。また、組織切片等を試料とし、これに試薬等を含んだ液滴を滴下し、その反応から病理診断する等の手段としても有効である。電界撹拌用ならびに電界洗浄用はっ水フレームであるから、電界撹拌、電界洗浄のいずれの用途にも活用することができる。 In addition, the water-repellent frame for electric field agitation and electric field washing according to the present invention can be applied to the hybridization process in nucleic acids, general antigen-antibody reaction, and the like together with the immunohistochemical staining as in the above-described embodiment. is there. Further, it is also effective as a means for making a pathological diagnosis from the reaction by dropping a droplet containing a reagent or the like on a tissue slice or the like as a sample. Since it is a water-repellent frame for electric field stirring and electric field cleaning, it can be used for both electric field stirring and electric field cleaning applications.
１・・・電界撹拌用はっ水リング（本発明）
１ｘ・・電界撹拌用はっ水リング（本発明）
１００・電界撹拌用はっ水リング（本発明）
１０・・リング体（フレーム体）
１０ｘ・リング体（フレーム体）
１０ａ・ヘソ部
１１・・リング内部（貫通孔）
１１ｘ・リング内部（貫通孔）
１０１・ポジティブコントロール部
１０２・ネガティブコントロール部
２・・・プラスチック基板（基板）
Ｅ・・・電界非接触撹拌装置
Ｅ１・・試料室
Ｅ２・・電極
Ｌ・・・液滴
Ｗ・・・リング幅
Ｓ・・・リング内部（貫通孔）の内部面積
1. Water-repellent ring for electric field stirring (present invention)
1x ... Water-repellent ring for electric field stirring (invention)
100. Water repellent ring for electric field stirring (present invention)
10. Ring body (frame body)
10x ring body (frame body)
10a · Navel part 11 · · Inside the ring (through hole)
11x ring inside (through hole)
101 ・ Positive control part 102 ・ Negative control part 2 ・ ・ ・ Plastic substrate (substrate)
E ... Electric field non-contact stirrer E1 ... Sample chamber E2 ... Electrode L ... Droplet W ... Ring width S ... Inner area inside ring (through hole)
上記目的を達成するために、本発明に係る電界撹拌用はっ水フレームは、貫通孔を有するフレーム体を形成し、このフレーム体を基板に設けて構成され、フレーム体の貫通孔に滴下された液滴が基板に対して４５度以下の接触角を与え、フレーム体は、基板から着脱可能とするためにその外周端に外向き方向に突出させたヘソ部が設けられ、フレーム体の材質がポリビニル系、ポリ塩化ビニル系、シリコーン系又はフッ素系から選ばれる樹脂であり、フレーム体のフレーム幅が０．５〜５．０ｍｍであって、フレーム体の厚みが０．１〜０．５ｍｍであることを特徴とする。 To achieve the above object, the water frame Tsu is for a field stirrer according to the present invention is to form a frame body having a transmural hole, it is configured by providing the frame member to the substrate, transmural hole of the frame member giving 45 degrees or less contact angle with the droplet board dropped on the frame body, navel portion which projects outward direction is provided at its outer peripheral end to detachable from the substrate, The material of the frame is a resin selected from polyvinyl, polyvinyl chloride, silicone, or fluorine, the frame width of the frame is 0.5 to 5.0 mm, and the thickness of the frame is 0.1. It is -0.5 mm.
また、本発明に係る電界撹拌用はっ水フレームは、フレーム体の貫通孔に滴下された液滴が撹拌されたか否かを視認可能として染色工程の正常性を確認可能なセンサー部材が基板上に設けられていることを特徴とする。 In addition, the water-repellent frame for electric field stirring according to the present invention has a sensor member on the substrate that can visually check whether or not the droplets dropped in the through holes of the frame body have been stirred and can check the normality of the dyeing process. It is provided in .
また、本発明に係る電界撹拌用はっ水フレームは、主電源としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波を形成した電界を、周波数０．１〜３００Ｈｚでフレーム体の貫通孔に滴下された液滴に与えることにより、液滴を非接触で撹拌可能とすることを特徴とする。 Further, the water-repellent frame for electric field stirring according to the present invention has 0.4 to 2.0 kV / mm on the plus side as a main power source, and an offset electric field strength of 0.2 to 1.0 kV / mm is added to this plus side repeating electric field to form a square wave of biased, by providing a droplet that is dripped into the through hole of the frame body at a frequency 0.1～300Hz, and characterized in that a stirrable liquid droplets in a non-contact To do.
Claims (5)
前記フレーム体の前記貫通孔に滴下された液滴が前記基板に対して４５度以下の接触角を与える、
ことを特徴とする電界撹拌用ならびに電界洗浄用はっ水フレーム。 Made of a resin that has acetone resistance and does not affect the electric field distribution in an electric field application environment, forms a frame body having a through hole, and is configured by providing this frame body on a substrate,
A droplet dropped in the through hole of the frame body gives a contact angle of 45 degrees or less to the substrate;
A water-repellent frame for electric field stirring and electric field cleaning.
ことを特徴とする請求項１に記載の電界撹拌用ならびに電界洗浄用はっ水フレーム。 The thickness of the droplet dropped into the through hole of the frame body is the same as or thinner than the thickness of the frame body,
The water-repellent frame for electric field stirring and electric field cleaning according to claim 1.
前記貫通孔に滴下される前記液滴の容量は、５〜１０００μＬである、
ことを特徴とする請求項１又は請求項２に記載の電界撹拌用ならびに電界洗浄用はっ水フレーム。 The frame body is a resin whose material is selected from polyvinyl, polyvinyl chloride, silicone, or fluorine. The frame body has a frame width of 0.5 to 5.0 mm and an area of the through hole of 19. a ～600Mm 2, the thickness of the frame body formed on said substrate, a thickness of the resin itself to be attached to the substrate is 0.1 to 0.5 mm,
The volume of the droplet dropped into the through hole is 5 to 1000 μL.
The water-repellent frame for electric field stirring and electric field cleaning according to claim 1 or 2.
ことを特徴とする請求項１から請求項３の何れか１項に記載の電界撹拌用ならびに電界洗浄用はっ水フレーム。 A sensor member capable of visually confirming whether the liquid droplets have been agitated and confirming the normality of the staining process is provided on the substrate.
The water repellent frame for electric field stirring and electric field cleaning according to any one of claims 1 to 3.
主電圧としてプラス側に０．４〜２．０ｋＶ／ｍｍ、これにオフセット電界強度０．２〜１．０ｋＶ／ｍｍを加え、プラス側に偏った繰り返しの方形波を形成した電界を、周波数０．１〜３００Ｈｚで前記液滴に与えることにより、前記液滴を非接触で撹拌可能とする、
ことを特徴とする電界非接触撹拌方法。 Using the water-repellent frame for electric field stirring and electric field cleaning according to any one of claims 1 to 4, a droplet is dropped into the through hole,
The main voltage is 0.4 to 2.0 kV / mm on the plus side, and an offset electric field strength of 0.2 to 1.0 kV / mm is added to the plus side. . Giving the droplets agitated in a non-contact manner by giving them to the droplets at 1 to 300 Hz;
An electric field non-contact stirring method.
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