JP2017219394A - Measuring method, and reaction container used in the same - Google Patents
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Abstract
Description
本発明は、測定方法とそれに用いる反応容器に関する。 The present invention relates to a measurement method and a reaction vessel used therefor.
現在、タンパク質や遺伝子などの分析には、複数のウェルが形成されたマイクロプレートが使用されている（たとえば、特許文献１参照）。このようなマイクロプレートを用いた分析方法としては、ウェル内に細胞などの試料や試薬を注入し、抗原抗体反応や酵素反応など目的に応じた化学反応を実施し、反応液から放射される発光または蛍光の強度を測定して、目的物質を検出または定量する方法が挙げられる。一度に複数の分析を行うため、近年では、ウェルの容積が小さく数が多いマイクロプレートが使用されるようになってきている。このマイクロプレートでは、少量の試料や試薬を用いて一度に複数の化学反応を実施し、複数の目的物質を検出または定量することができる。 Currently, microplates in which a plurality of wells are formed are used for analysis of proteins and genes (see, for example, Patent Document 1). As an analysis method using such a microplate, a sample or reagent such as a cell is injected into a well, a chemical reaction according to the purpose such as an antigen-antibody reaction or an enzyme reaction is performed, and light emitted from the reaction solution is emitted. Alternatively, a method of detecting or quantifying a target substance by measuring the intensity of fluorescence can be mentioned. In recent years, microplates having a small well volume and a large number have been used to perform a plurality of analyzes at a time. In this microplate, a plurality of chemical reactions can be performed at once using a small amount of sample or reagent, and a plurality of target substances can be detected or quantified.
しかし、ウェルの容積が小さいマイクロプレートを用いると、反応液からの発光または蛍光の絶対量が低下するため、発光または蛍光の検出感度が低下するという問題がある。 However, when a microplate having a small well volume is used, there is a problem that the absolute amount of luminescence or fluorescence from the reaction solution is lowered, and the detection sensitivity of luminescence or fluorescence is lowered.
本発明は上記のような課題を解決するためになされたものであり、発光または蛍光の検出感度が低下しない測定方法とそれに用いる反応容器を提供することを目的とする。 The present invention has been made to solve the above-described problems, and an object of the present invention is to provide a measurement method in which the detection sensitivity of luminescence or fluorescence does not decrease and a reaction vessel used therefor.
以下に、課題を解決するための手段として複数の態様を説明する。これら態様は必要に応じて任意に組み合わせることができる。 Hereinafter, a plurality of modes will be described as means for solving the problems. These embodiments can be arbitrarily combined as necessary.
本発明の測定方法は、
複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなる反応容器のウェル内に少なくとも２種の液体を注入して反応させ、反応液から生じ透明な鉱油を通過した発光を検出し、発光の強度を測定することを特徴とするものである。
The measurement method of the present invention includes:
Transparent mineral oil produced from the reaction liquid by injecting and reacting at least two liquids into the wells of a reaction vessel comprising a microplate having a plurality of wells and a transparent mineral oil injected into the wells The light emission that has passed through is detected, and the intensity of the light emission is measured.
また、本発明の測定方法は、
複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなる反応容器のウェル内に少なくとも１種の液体を注入し、液体に励起光を照射して液体から蛍光を生じさせ、透明な鉱油を通過した蛍光を検出し、蛍光の強度を測定することを特徴とするものである。
Moreover, the measuring method of the present invention comprises:
At least one liquid is injected into a well of a reaction vessel including a microplate having a plurality of wells and a transparent mineral oil injected into the well, and the liquid is irradiated with excitation light from the liquid. Fluorescence is generated, fluorescence passing through a transparent mineral oil is detected, and the intensity of the fluorescence is measured.
また、本発明の測定方法は、
複数のウェルが形成されたマイクロプレートのウェル内に少なくとも２種の液体を注入して反応させ、反応直後に透明な鉱油を注入し、反応液から生じ透明な鉱油を通過した発光を検出し、発光の強度を測定することを特徴とするものである。
Moreover, the measuring method of the present invention comprises:
Injecting and reacting at least two kinds of liquids into the wells of the microplate in which a plurality of wells are formed, injecting a transparent mineral oil immediately after the reaction, and detecting light emitted from the reaction liquid and passing through the transparent mineral oil; It measures the intensity of light emission.
また、本発明の測定方法は、
複数のウェルが形成されたマイクロプレートのウェル内に、少なくとも１種の液体と透明な鉱油を注入し、液体に励起光を照射して液体から蛍光を生じさせ、透明な鉱油を通過した蛍光を検出し、蛍光の強度を測定することを特徴とするものである。
Moreover, the measuring method of the present invention comprises:
At least one kind of liquid and transparent mineral oil are injected into the well of the microplate in which a plurality of wells are formed, and the liquid is irradiated with excitation light to generate fluorescence from the liquid, and the fluorescence that has passed through the transparent mineral oil is emitted. It detects and measures the intensity | strength of fluorescence.
また、本発明の反応容器は、
複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなることを特徴とするものである。
The reaction container of the present invention is
It comprises a microplate in which a plurality of wells are formed, and a transparent mineral oil injected into the wells.
本発明の測定方法は、複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなる反応容器のウェル内に少なくとも２種の液体を注入して反応させ、反応液から生じ透明な鉱油を通過した発光を検出し、発光の強度を測定するように構成した。 In the measurement method of the present invention, at least two kinds of liquids are injected into a well of a reaction vessel containing a microplate in which a plurality of wells are formed and a transparent mineral oil injected into the wells, and reacted. Light emission generated from the reaction solution and passing through transparent mineral oil was detected, and the intensity of light emission was measured.
また、本発明の測定方法は、複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなる反応容器のウェル内に少なくとも１種の液体を注入し、液体に励起光を照射して液体から蛍光を生じさせ、透明な鉱油を通過した蛍光を検出し、蛍光の強度を測定するように構成した。 Further, the measurement method of the present invention injects at least one liquid into a well of a reaction vessel including a microplate in which a plurality of wells are formed and a transparent mineral oil injected into the well. The liquid was irradiated with excitation light to generate fluorescence from the liquid, the fluorescence that passed through the transparent mineral oil was detected, and the intensity of the fluorescence was measured.
また、本発明の測定方法は、複数のウェルが形成されたマイクロプレートのウェル内に少なくとも２種の液体を注入して反応させ、反応直後に透明な鉱油を注入し、反応液から生じ透明な鉱油を通過した発光を検出し、発光の強度を測定するように構成した。 In the measurement method of the present invention, at least two kinds of liquids are injected into the wells of a microplate in which a plurality of wells are formed and reacted, and transparent mineral oil is injected immediately after the reaction to produce a transparent liquid. The luminescence that passed through the mineral oil was detected and the intensity of the luminescence was measured.
また、本発明の測定方法は、複数のウェルが形成されたマイクロプレートのウェル内に、少なくとも１種の液体と透明な鉱油を注入し、液体に励起光を照射して液体から蛍光を生じさせ、透明な鉱油を通過した蛍光を検出し、蛍光の強度を測定するように構成した。 In the measurement method of the present invention, at least one liquid and transparent mineral oil are injected into a well of a microplate having a plurality of wells, and the liquid is irradiated with excitation light to generate fluorescence from the liquid. The fluorescence passing through the transparent mineral oil was detected, and the intensity of the fluorescence was measured.
したがって、本発明の測定方法によれば、発光または蛍光の検出感度が低下することがない。 Therefore, according to the measurement method of the present invention, the detection sensitivity of luminescence or fluorescence does not decrease.
また、本発明の反応容器は、複数のウェルが形成されたマイクロプレートと、ウェル内に注入された透明な鉱油とを含んでなるように構成した。したがって、本発明によれば、発光または蛍光の検出感度が低下することがない反応容器を得ることができる。 Moreover, the reaction container of the present invention was configured to include a microplate in which a plurality of wells were formed and a transparent mineral oil injected into the wells. Therefore, according to the present invention, it is possible to obtain a reaction vessel in which the detection sensitivity of luminescence or fluorescence does not decrease.
以下、本発明の加飾品の製造方法について、実施形態の一例を説明する。 Hereinafter, an example of an embodiment is explained about a manufacturing method of a decoration article of the present invention.
＜第１実施形態＞
本発明の測定方法は、複数のウェル１０ａが形成されたマイクロプレート１０と、ウェル１０ａ内に注入された透明な鉱油１１とを含んでなる反応容器１のウェル１０ａ内に少なくとも２種の液体２、３を注入して反応させ、反応液４から生じ透明な鉱油１１を通過した発光を検出し、発光の強度を測定することを特徴とするものである（図１参照）。
<First Embodiment>
In the measurement method of the present invention, at least two kinds of liquids 2 are contained in a well 10a of a reaction vessel 1 including a microplate 10 in which a plurality of wells 10a are formed and a transparent mineral oil 11 injected into the well 10a. 3 is injected and reacted, and the luminescence generated from the reaction solution 4 and passing through the transparent mineral oil 11 is detected, and the intensity of the luminescence is measured (see FIG. 1).
マイクロプレート１０は、長さ０．１〜１３０ｍｍ、幅０．１〜９０ｍｍ、高さ０．１〜３０ｍｍであり、材質はポリエチレン、ポリスチレン、ポリプロピレンなどの樹脂やガラスなどを用いることができる。マイクロプレート１０には複数のウェル１０ａが形成されている。ウェル１０ａは、深さ０．０１ｍｍ〜２０ｍｍ、直径０．０１ｍｍ〜８０ｍｍ、容積０．８ｐＬ〜１００ｍＬ、数１〜４２４０００００、ウェル間距離１５μｍ〜１４５ｍｍであるマイクロプレート１０を用いることができる。なお、ウェル１０ａの底は平らでもよいし、丸くてもよい。 The microplate 10 has a length of 0.1 to 130 mm, a width of 0.1 to 90 mm, and a height of 0.1 to 30 mm. The material can be a resin such as polyethylene, polystyrene, or polypropylene, or glass. The microplate 10 has a plurality of wells 10a. As the well 10a, the microplate 10 having a depth of 0.01 mm to 20 mm, a diameter of 0.01 mm to 80 mm, a volume of 0.8 pL to 100 mL, a number of 1 to 42400000, and a distance between wells of 15 μm to 145 mm can be used. The bottom of the well 10a may be flat or round.
ウェル１０ａ内にはあらかじめ透明な鉱油１１を注入しておく（図１（ａ）、図３参照）。透明な鉱油１１は、精製されたものであり、不揮発性を有し、水相よりも比重が低いものを用いる。たとえば、流動パラフィンなどの炭素数が６以上の炭化水素化合物（直鎖飽和炭化水素、分岐鎖飽和炭化水素、環状飽和炭化水素）、エチルエーテル、石油エーテル、ジフェニルエーテル、イオン液体、フッ素油、大豆油、シリコーンオイル、植物油などを用いることができる。これらは、単体または混合物として用いることができる。中でも、流動パラフィンまたはミネラルオイルと呼ばれるものが好ましい。透明であり不純物が少ないため、反応液４から生じた発光を吸収または減衰しにくい。 A transparent mineral oil 11 is poured into the well 10a in advance (see FIGS. 1A and 3). The transparent mineral oil 11 is refined, non-volatile, and has a specific gravity lower than that of the aqueous phase. For example, hydrocarbon compounds having 6 or more carbon atoms such as liquid paraffin (linear saturated hydrocarbons, branched saturated hydrocarbons, cyclic saturated hydrocarbons), ethyl ether, petroleum ether, diphenyl ether, ionic liquid, fluorine oil, soybean oil Silicone oil, vegetable oil, etc. can be used. These can be used alone or as a mixture. Among these, what is called liquid paraffin or mineral oil is preferable. Since it is transparent and contains few impurities, it is difficult to absorb or attenuate the light emitted from the reaction solution 4.
上記のマイクロプレート１０のウェル１０ａ内に透明な鉱油１１を注入した反応容器１に、液体２を注入する（図１（ｂ）参照）。注入には、たとえばディスペンサ８を用いることができる。液体２はたとえば、細胞組織、抗体、ＤＮＡ、ＲＮＡなどの試料を用いることができる。このとき、ウェル１０ａ内には透明な鉱油１１が注入されているため、透明な鉱油１１によって液体２の蒸発を防ぐことができる。 The liquid 2 is injected into the reaction vessel 1 in which the transparent mineral oil 11 is injected into the well 10a of the microplate 10 (see FIG. 1B). For the injection, for example, a dispenser 8 can be used. As the liquid 2, for example, a sample such as a cell tissue, an antibody, DNA, or RNA can be used. At this time, since the transparent mineral oil 11 is injected into the well 10a, the transparent mineral oil 11 can prevent the liquid 2 from evaporating.
次に、ウェル１０ａ内に液体３を注入する（図１（ｃ）参照）。注入には、たとえばディスペンサ８を用いることができる。液体３はたとえば、試薬、抗原、基質などを用いることができる。 Next, the liquid 3 is injected into the well 10a (see FIG. 1C). For the injection, for example, a dispenser 8 can be used. As the liquid 3, for example, a reagent, an antigen, a substrate, or the like can be used.
液体２、３をウェル１０ａ内に注入した後、化学反応が開始される（図１（ｄ）参照）。反応液４から発光が生じ、その光が透明な鉱油１１を通過して、測定器９の検出部９ａに届く（図１（ｅ）参照）。検出部９ａで光を検出し、発光強度を測定する。測定器９としては、マイクロプレートリーダ、マイクロアレイスキャナ、ルミノメーター、ＣＣＤイメージャーなどを用いることができる。 After injecting the liquids 2 and 3 into the well 10a, a chemical reaction is started (see FIG. 1 (d)). Luminescence is generated from the reaction solution 4, and the light passes through the transparent mineral oil 11 and reaches the detection unit 9a of the measuring instrument 9 (see FIG. 1 (e)). The detection unit 9a detects light and measures the emission intensity. As the measuring instrument 9, a microplate reader, a microarray scanner, a luminometer, a CCD imager, or the like can be used.
反応液４から生じた発光は、透明な鉱油１１を通過して検出部９ａへ集められる。透明な鉱油１１がない従来の方法においては、反応液からの発光が拡散してしまう（図２（ｂ）参照）。一方、反応液４の液面に透明な鉱油１１が存在している本発明においては（図２（ａ）参照）、透明な鉱油１１が凸メニスカスレンズを形成している。ここで、マイクロプレート１０は上記したように樹脂やガラスでできており、反応液４は水溶液である。そのため、反応液４がウェル１０ａの内壁を濡らし、反応液４の液面は下に凸のメニスカスとなる。その液面上にある透明な鉱油１１も同様にウェル１０ａの内壁を濡らすため、下に凸のメニスカスを形成する。したがって、反応液４から生じた発光は凸メニスカスレンズ状の透明な鉱油１１によって集光され、従来の方法と比較して発光の検出感度を高めることができる。凸メニスカスレンズによる効果を得るためには、透明な鉱油１１の注入量は反応液４の体積の半分以下であることが好ましい。 The luminescence generated from the reaction solution 4 passes through the transparent mineral oil 11 and is collected in the detection unit 9a. In the conventional method without the transparent mineral oil 11, light emission from the reaction solution diffuses (see FIG. 2B). On the other hand, in the present invention in which the transparent mineral oil 11 exists on the liquid surface of the reaction solution 4 (see FIG. 2A), the transparent mineral oil 11 forms a convex meniscus lens. Here, the microplate 10 is made of resin or glass as described above, and the reaction solution 4 is an aqueous solution. Therefore, the reaction solution 4 wets the inner wall of the well 10a, and the liquid surface of the reaction solution 4 becomes a downward meniscus. The transparent mineral oil 11 on the liquid surface similarly wets the inner wall of the well 10a, and forms a downward meniscus. Therefore, the luminescence generated from the reaction solution 4 is collected by the convex meniscus lens-like transparent mineral oil 11, and the detection sensitivity of the luminescence can be increased as compared with the conventional method. In order to obtain the effect of the convex meniscus lens, the injection amount of the transparent mineral oil 11 is preferably half or less of the volume of the reaction solution 4.
なお、透明な鉱油１１があらかじめ注入されたウェル１０ａ内に反応液４を注入し、直後に液体２、３とは異なる試料をさらに注入する形態であってもよい。この形態においても、透明な鉱油の凸メニスカスレンズ効果によって、発光の検出感度を高めることができる。 In addition, the form which inject | pours the reaction liquid 4 in the well 10a in which the transparent mineral oil 11 was previously injected, and inject | pours the sample different from the liquids 2 and 3 immediately after that may be sufficient. Also in this embodiment, the detection sensitivity of light emission can be increased by the convex meniscus lens effect of transparent mineral oil.
反応容器１は、複数のウェル１０ａが形成されたマイクロプレート１０と、ウェル１０ａ内に注入された透明な鉱油１１とを含んでなる（図３参照）。なお、ウェル１０ａは角穴であってもよい。 The reaction vessel 1 includes a microplate 10 in which a plurality of wells 10a are formed, and a transparent mineral oil 11 injected into the wells 10a (see FIG. 3). The well 10a may be a square hole.
ウェル１０ａ内には透明な鉱油１１があらかじめ注入されている。したがって、反応容器１を保管する際は、透明な鉱油１１がウェル１０ａから漏れないようにする必要がある。そのような例としては、たとえば、図４（ａ）〜（ｃ）に示すような形態が挙げられる。図４（ａ）は、マイクロプレート１０と同じ大きさの蓋材１２を用いる形態である。蓋材１２の材質は、たとえばマイクロプレート１０と同じものにすることができる。四隅に突起を設けた蓋材１２と、突起に対応した箇所に穴を形成したマイクロプレート１０とを用いて蓋材１２を固定することができる。また、蓋材１２は粘着剤などでマイクロプレート１０に密着させて固定することもできる。蓋材１２を固定すると、ウェル１０ａから透明な鉱油１１が漏れることを防止できる。なお、蓋材１２はマイクロプレート１０に固定しなくてもよい。蓋材１２を固定しない場合は、反応容器を使用したい時にすぐ使用することができる。 A transparent mineral oil 11 is injected into the well 10a in advance. Therefore, when storing the reaction vessel 1, it is necessary to prevent the transparent mineral oil 11 from leaking from the well 10a. As such an example, the form as shown to FIG.4 (a)-(c) is mentioned, for example. FIG. 4A shows a form in which a lid member 12 having the same size as the microplate 10 is used. The material of the lid member 12 can be the same as that of the microplate 10, for example. The lid member 12 can be fixed using the lid member 12 provided with projections at the four corners and the microplate 10 having holes formed at locations corresponding to the projections. Further, the lid member 12 can be fixed by being brought into close contact with the microplate 10 with an adhesive or the like. When the lid 12 is fixed, the transparent mineral oil 11 can be prevented from leaking from the well 10a. The lid member 12 may not be fixed to the microplate 10. When the lid member 12 is not fixed, it can be used immediately when it is desired to use the reaction vessel.
図４（ｂ）は、マイクロプレート１０と同じ大きさのシール材１３を用いる形態である。シール材１３の材質は、たとえば樹脂フィルムなどを用いることができる。シール材１３は、マイクロプレート１０に粘着剤で密着させて固定する。蓋材１２を固定すると、ウェル１０ａから透明な鉱油１１が漏れることを防止できる。 FIG. 4B shows a form in which the sealing material 13 having the same size as the microplate 10 is used. As the material of the sealing material 13, for example, a resin film can be used. The sealing material 13 is fixed in close contact with the microplate 10 with an adhesive. When the lid 12 is fixed, the transparent mineral oil 11 can be prevented from leaking from the well 10a.
図４（ｃ）は、真空包装材１４を用いてマイクロプレート１０を密封する形態である。真空包装材１４の材質は、たとえば、ガスバリア性を有する樹脂フィルムなどを用いることができる。透明な鉱油１１をウェル１０ａ内に注入したマイクロプレート１０を真空包装材１４で覆い、脱気シーラーなどを用いて密封する。このようにすると、図４（ａ）や（ｂ）と比較してより確実に、ウェル１０ａから透明な鉱油１１が漏れることを防止できる。なお、上記した蓋材１２やシール材１３を用いた上で、真空包装材１４を用いて密封する形態も好ましい。このようにすると、さらに確実にウェル１０ａから透明な鉱油１１が漏れることを防止できる。 FIG. 4C shows a form in which the microplate 10 is sealed using the vacuum packaging material 14. As the material of the vacuum packaging material 14, for example, a resin film having a gas barrier property can be used. The microplate 10 in which the transparent mineral oil 11 is injected into the well 10a is covered with a vacuum packaging material 14, and sealed using a degassing sealer or the like. If it does in this way, it can prevent that the transparent mineral oil 11 leaks from the well 10a more reliably compared with Fig.4 (a) and (b). In addition, after using the above-mentioned cover material 12 and the sealing material 13, the form sealed with the vacuum packaging material 14 is also preferable. In this way, it is possible to more reliably prevent the transparent mineral oil 11 from leaking from the well 10a.
＜第２実施形態＞
次に、本発明の第２実施形態について説明する。本発明の測定方法は、複数のウェル１０ａが形成されたマイクロプレート１０と、ウェル１０ａ内に注入された透明な鉱油１１とを含んでなる反応容器１のウェル１０ａ内に少なくとも１種の液体５を注入し、液体５に励起光を照射して液体５から蛍光を生じさせ、透明な鉱油１１を通過した蛍光を検出し、蛍光の強度を測定することを特徴とするものである（図５参照）。
Second Embodiment
Next, a second embodiment of the present invention will be described. In the measurement method of the present invention, at least one kind of liquid 5 is contained in a well 10a of a reaction vessel 1 including a microplate 10 in which a plurality of wells 10a are formed and a transparent mineral oil 11 injected into the well 10a. The liquid 5 is irradiated with excitation light to generate fluorescence from the liquid 5, the fluorescence that has passed through the transparent mineral oil 11 is detected, and the intensity of the fluorescence is measured (FIG. 5). reference).
第２実施形態は、ウェル内に注入する液体が少なくとも１種であり、液体に励起光を照射して液体から蛍光を生じさせる点において、第１実施形態とは異なる。以下、第１実施形態と異なる点を中心に説明する。 The second embodiment is different from the first embodiment in that at least one type of liquid is injected into the well, and the liquid is irradiated with excitation light to generate fluorescence from the liquid. Hereinafter, a description will be given focusing on differences from the first embodiment.
まず、マイクロプレート１０のウェル１０ａ内にあらかじめ透明な鉱油１１を注入した反応容器１を準備する（図５（ａ））。この反応容器１に、たとえばディスペンサ８を用いて液体５を注入する（図５（ｂ）参照）。液体５は、励起光を照射して蛍光を発するものであれば、特に制限されない。蛍光物質としては、たとえば、フルオレセイン、Ｃｙ３、Ｃｙ５などの蛍光色素、ＧＦＰ（Ｇｒｅｅｎ Ｆｌｕｏｒｅｓｃｅｎｔ Ｐｒｏｔｅｉｎ；緑色蛍光タンパク質）をはじめとする蛍光タンパク質、およびこれらが標識された蛍光標識抗体などを用いることができる。なお、液体５とは異なる種類の液体を複数注入してもよい。 First, a reaction vessel 1 is prepared in which a transparent mineral oil 11 is poured into a well 10a of a microplate 10 in advance (FIG. 5A). A liquid 5 is injected into the reaction vessel 1 using, for example, a dispenser 8 (see FIG. 5B). The liquid 5 is not particularly limited as long as it emits fluorescence when irradiated with excitation light. As the fluorescent substance, for example, fluorescent dyes such as fluorescein, Cy3, and Cy5, fluorescent proteins such as GFP (Green Fluorescent Protein), and fluorescently labeled antibodies labeled with these can be used. A plurality of types of liquid different from the liquid 5 may be injected.
次に、励起光を反応容器の上方から液体５に向けて照射する（図５（ｃ）参照）。励起光としては、たとえば紫外線、可視光線、Ｘ線、赤外線などを用いることができ、ウェル１０ａ内に注入する蛍光物質によって励起光の種類を変えてもよい。なお、励起光の照射は反応容器の下方から液体５に向けて照射してもよい。 Next, excitation light is irradiated from above the reaction vessel toward the liquid 5 (see FIG. 5C). As the excitation light, for example, ultraviolet rays, visible rays, X-rays, infrared rays, and the like can be used, and the type of excitation light may be changed depending on the fluorescent material injected into the well 10a. The excitation light may be irradiated toward the liquid 5 from below the reaction vessel.
液体５に励起光を照射した後、液体５から蛍光が発生する（図５（ｄ）参照）。その蛍光が透明な鉱油１１を通過して、測定器９の検出部９ａに届く（図５（ｅ）参照）。検出部９ａで蛍光を検出し、蛍光強度を測定する。測定器９としては、マイクロプレートリーダ、マイクロアレイスキャナ、ＣＣＤイメージャー、蛍光顕微鏡、共焦点顕微鏡などを用いることができる。 After the liquid 5 is irradiated with excitation light, fluorescence is generated from the liquid 5 (see FIG. 5D). The fluorescence passes through the transparent mineral oil 11 and reaches the detection unit 9a of the measuring instrument 9 (see FIG. 5 (e)). The detection unit 9a detects the fluorescence and measures the fluorescence intensity. As the measuring instrument 9, a microplate reader, a microarray scanner, a CCD imager, a fluorescence microscope, a confocal microscope, or the like can be used.
液体５から生じた蛍光の検出感度を高めることができる理由および透明な鉱油１１の好ましい注入量については、第１実施形態と同様である。 The reason why the detection sensitivity of the fluorescence generated from the liquid 5 can be increased and the preferable injection amount of the transparent mineral oil 11 are the same as in the first embodiment.
＜第３実施形態＞
次に、本発明の第３実施形態について説明する。本発明の測定方法は、複数のウェル１０ａが形成されたマイクロプレート１０のウェル１０ａ内に少なくとも２種の液体２、３を注入して反応させ、反応直後に透明な鉱油１１を注入し、反応液４から生じ透明な鉱油１１を通過した発光を検出し、発光の強度を測定することを特徴とするものである（図６参照）。
<Third Embodiment>
Next, a third embodiment of the present invention will be described. In the measurement method of the present invention, at least two kinds of liquids 2 and 3 are injected and reacted in the well 10a of the microplate 10 in which a plurality of wells 10a are formed, and transparent mineral oil 11 is injected immediately after the reaction. The luminescence emitted from the liquid 4 and passing through the transparent mineral oil 11 is detected, and the intensity of the luminescence is measured (see FIG. 6).
第３実施形態は、ウェル内にまず液体２、３を注入してから透明な鉱油を注入する点において、第１実施形態とは異なる。以下、第１実施形態と異なる点を中心に説明する。 The third embodiment is different from the first embodiment in that the liquids 2 and 3 are first injected into the well and then the transparent mineral oil is injected. Hereinafter, a description will be given focusing on differences from the first embodiment.
まず、マイクロプレート１０のウェル１０ａ内にディスペンサ８などを用いて液体２を注入する（図６（ａ）参照）。次いで液体３を注入する（図６（ｂ）参照）。液体３を注入すると化学反応が始まり、発光が生じる。反応直後に、ディスペンサ８などを用いて透明な鉱油１１を注入する（図６（ｃ）参照）。反応液４から生じた発光は、透明な鉱油１１を通過して測定器９の検出部９ａに届く（図６（ｄ）参照）。 First, the liquid 2 is injected into the well 10a of the microplate 10 using the dispenser 8 or the like (see FIG. 6A). Next, the liquid 3 is injected (see FIG. 6B). When the liquid 3 is injected, a chemical reaction starts and light emission occurs. Immediately after the reaction, transparent mineral oil 11 is injected using a dispenser 8 or the like (see FIG. 6C). The luminescence generated from the reaction solution 4 passes through the transparent mineral oil 11 and reaches the detection unit 9a of the measuring instrument 9 (see FIG. 6D).
液体２と液体３との化学反応が始まるとすぐに発光が生じるため、透明な鉱油１１はできるだけ早く注入することが好ましい。たとえば、液体３を注入するディスペンサと透明な鉱油１１を注入するディスペンサとを用意し、液体３を注入するディスペンサが移動すると、透明な鉱油１１を注入するディスペンサがそれを追跡する形態とすることができる。なお、始めに液体２を注入し、その後、透明な鉱油１１、液体３を順番に注入する形態であってもよい。また、始めに液体２を注入し、透明な鉱油１１と液体３を同時に注入する形態であってもよい。
このようにすると、反応直後もしくは反応とほぼ同時に透明な鉱油１１を注入することになるため、反応液から生じた発光を凸メニスカスレンズ効果により効率よく集光できる（図２（ａ）参照）。
Since light emission occurs as soon as the chemical reaction between the liquid 2 and the liquid 3 starts, it is preferable to inject the transparent mineral oil 11 as soon as possible. For example, a dispenser that injects the liquid 3 and a dispenser that injects the transparent mineral oil 11 are prepared, and when the dispenser that injects the liquid 3 moves, the dispenser that injects the transparent mineral oil 11 tracks the dispenser. it can. The liquid 2 may be injected first, and then the transparent mineral oil 11 and the liquid 3 may be sequentially injected. Moreover, the form which inject | pours the liquid 2 first and injects the transparent mineral oil 11 and the liquid 3 simultaneously may be sufficient.
In this way, since the transparent mineral oil 11 is injected immediately after the reaction or almost simultaneously with the reaction, the light emitted from the reaction solution can be efficiently collected by the convex meniscus lens effect (see FIG. 2A).
＜第４実施形態＞
次に、本発明の第４実施形態について説明する。本発明の測定方法は、複数のウェルが形成されたマイクロプレートのウェル内に、少なくとも１種の液体と透明な鉱油を注入し、液体に励起光を照射して液体から蛍光を生じさせ、透明な鉱油を通過した蛍光を検出し、蛍光の強度を測定することを特徴とするものである（図７参照）。
<Fourth embodiment>
Next, a fourth embodiment of the present invention will be described. In the measurement method of the present invention, at least one liquid and transparent mineral oil are injected into a well of a microplate having a plurality of wells, and the liquid is irradiated with excitation light to generate fluorescence from the liquid. The fluorescent light passing through the mineral oil is detected and the intensity of the fluorescent light is measured (see FIG. 7).
第４実施形態は、ウェル１０ａ内にまず液体５を注入し、その後に透明な鉱油１１を注入し、液体５に励起光を照射する点において、第２実施形態とは異なる。以下、第２実施形態と異なる点を中心に説明する。 The fourth embodiment is different from the second embodiment in that the liquid 5 is first injected into the well 10a, the transparent mineral oil 11 is then injected, and the liquid 5 is irradiated with excitation light. Hereinafter, a description will be given focusing on differences from the second embodiment.
ウェル１０ａ内に、たとえばディスペンサ８を用いて液体５を注入する（図７（ａ）参照）。その後、ディスペンサ８などを用いて透明な鉱油１１を注入する（図７（ｂ）参照）。励起光を照射する（図７（ｃ）参照）。励起光を照射すると化学反応が始まり、液体５から蛍光が発生する。生じた蛍光は、透明な鉱油１１を通過して測定器９の検出部９ａに届く（図７（ｄ）参照）。液体５に励起光を照射して化学反応が始まるとすぐに蛍光が生じ、蛍光が発生している時間はきわめて短い（たとえばナノ秒単位）ため、測定器９は蛍光強度の測定と励起光の照射とが可能なものが好ましい。第４実施形態においても、透明な鉱油の凸メニスカスレンズ効果によって、蛍光の検出感度を高めることができる。 The liquid 5 is injected into the well 10a using, for example, the dispenser 8 (see FIG. 7A). Thereafter, a transparent mineral oil 11 is injected using a dispenser 8 or the like (see FIG. 7B). Excitation light is irradiated (see FIG. 7C). When the excitation light is irradiated, a chemical reaction starts and fluorescence is generated from the liquid 5. The generated fluorescence passes through the transparent mineral oil 11 and reaches the detection unit 9a of the measuring instrument 9 (see FIG. 7D). As soon as the liquid 5 is irradiated with excitation light and a chemical reaction starts, fluorescence is generated, and the time during which the fluorescence is generated is extremely short (for example, in nanosecond units). What can be irradiated is preferable. Also in the fourth embodiment, the fluorescence detection sensitivity can be increased by the convex meniscus lens effect of transparent mineral oil.
上記では液体５を先に注入し、その後に透明な鉱油１１を注入しているが、液体５と透明な鉱油１１は同時に注入してもよい。透明な鉱油１１は液体５よりも比重が小さいため、同時に注入しても、所定の時間が経過すれば透明な鉱油１１と液体５は二層に分かれ、透明な鉱油１１が液体５に対して上の層となる。したがって、反応液から生じた蛍光を凸メニスカスレンズ効果により効率よく集光できる（図２（ａ）参照）。 In the above, the liquid 5 is injected first and then the transparent mineral oil 11 is injected. However, the liquid 5 and the transparent mineral oil 11 may be injected simultaneously. Since the transparent mineral oil 11 has a specific gravity smaller than that of the liquid 5, the transparent mineral oil 11 and the liquid 5 are separated into two layers when a predetermined time elapses even when injected simultaneously. It becomes the upper layer. Therefore, the fluorescence generated from the reaction solution can be efficiently collected by the convex meniscus lens effect (see FIG. 2A).
（実施例１）
マイクロプレートは白色であり、９６個のウェルを有するものを用いた。ウェルの各寸法は、直径６．３５ｍｍ、深さ１０．６７ｍｍ、容積３３８μＬである。ウェルの底面はフラットのものを用いた。このマイクロプレートのウェル内にあらかじめ透明な鉱油を５０μＬ注入した反応容器を準備した。透明な鉱油としてミネラルオイルを用いた。これは無色透明であり、比重０．８５、２５℃における粘度が２２．５ｍＰａ・s、ＤＮａｓｅ（デオキシリボヌクレアーゼ）、ＲＮａｓｅ（リボヌクレアーゼ）、Ｐｒｏｔｅａｓｅ（プロテアーゼ）は含まれないことが特徴である。
Example 1
The microplate was white and had 96 wells. Each dimension of the well has a diameter of 6.35 mm, a depth of 10.67 mm, and a volume of 338 μL. The bottom of the well was a flat one. A reaction vessel in which 50 μL of a transparent mineral oil was previously injected into the well of the microplate was prepared. Mineral oil was used as a transparent mineral oil. This is colorless and transparent, and has a specific gravity of 0.85, a viscosity at 25 ° C. of 22.5 mPa · s, DNase (deoxyribonuclease), RNase (ribonuclease), and protease (protease) are not included.
次に、反応容器に次の２種の試料を注入して化学反応させた。１μＭのＡＴＰ溶液（ｒＡＴＰ１０ｍＭをＴｒｉｓバッファーを加えて濃度調整したもの）とＲｅａｇｅｎｔ溶液（Ｋｉｎａｓｅ‐Ｇｌｏ（登録商標）ＳｕｂｓｔｒａｔｅとＫｉｎａｓｅ‐Ｇｌｏ（登録商標）Ｂｕｆｆｅｒの混合液）を１ウェルあたり５０μＬずつ使用して酵素基質反応を行った。なお、反応時間は１６分とした。試料の注入にはピペットとピペットチップを用いた。 Next, the following two types of samples were injected into the reaction vessel to cause a chemical reaction. Use 1 μM ATP solution (rATP 10 mM with concentration adjusted by adding Tris buffer) and Reagent solution (mixture of Kinase-Glo (registered trademark) Substrate and Kinase-Glo (registered trademark) Buffer) at 50 μL per well. Then, an enzyme substrate reaction was performed. The reaction time was 16 minutes. Pipettes and pipette tips were used for sample injection.
反応液から生じた発光を測定器で検出して発光強度を測定した。使用した測定器はマルチモードマイクロプレートリーダー（ＤＳファーマバイオメディカル社製 パワースキャンＨＴ、吸光・蛍光・化学発光測定に対応）である。３回測定を行い、算出した平均値３８２６３１ＲＬＵを測定結果とした。 Luminescence generated from the reaction solution was detected with a measuring instrument to measure luminescence intensity. The measuring instrument used was a multi-mode microplate reader (Power Scan HT, manufactured by DS Pharma Biomedical Co., Ltd., corresponding to absorbance / fluorescence / chemiluminescence measurement). The measurement was performed three times, and the calculated average value 382631 RLU was taken as the measurement result.
（実施例２）
上記の試料をウェル内に注入して化学反応させ、反応直後に透明な鉱油を５０μＬ注入した。それ以外は実施例１と同様にして行った。平均値は３９２９３５ＲＬＵであった。
(Example 2)
The sample was injected into the well for chemical reaction, and 50 μL of transparent mineral oil was injected immediately after the reaction. Other than that was carried out in the same manner as in Example 1. The average value was 392935 RLU.
（比較例１）
透明な鉱油を注入せず、上記の試料をウェル内に注入して化学反応させた。それ以外は、実施例１と同様にして行った。平均値は３３０８５９ＲＬＵであった。
(Comparative Example 1)
Without injecting clear mineral oil, the above sample was injected into the well for chemical reaction. Otherwise, the same procedure as in Example 1 was performed. The average value was 330859 RLU.
実施例１および２と比較例１の結果を、表１と表２に示す。表２に示すグラフ中の縦軸は発光量（ＲＬＵ）である。 The results of Examples 1 and 2 and Comparative Example 1 are shown in Tables 1 and 2. The vertical axis in the graph shown in Table 2 represents the light emission amount (RLU).
表１および表２より、実施例１と２は比較例１よりも発光量が大きいものであった。 From Table 1 and Table 2, Examples 1 and 2 had larger light emission amounts than Comparative Example 1.
１ ：反応容器
１０ ：マイクロプレート
１０ａ ：ウェル
１１ ：透明な鉱油
１２ ：蓋材
１３ ：シール材
１４ ：真空包装材
２、３、５：液体
４ ：反応液
８ ：ディスペンサ
９ ：測定器
９ａ ：検出部
DESCRIPTION OF SYMBOLS 1: Reaction container 10: Microplate 10a: Well 11: Transparent mineral oil 12: Cover material 13: Sealing material 14: Vacuum packaging material 2, 3, 5: Liquid 4: Reaction liquid 8: Dispenser 9: Measuring instrument 9a: Detection Part
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