JP6757527B2 - Measurement method and reaction vessel used for it - Google Patents
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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 measuring method and a reaction vessel used therein.
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, genes, etc. (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 such as an antigen-antibody reaction or an enzyme reaction is carried out according to the purpose, and light emission emitted from the reaction solution is carried out. Alternatively, a method of detecting or quantifying the target substance by measuring the fluorescence intensity can be mentioned. In recent years, microplates with a small well volume and a large number have been used to perform a plurality of analyzes at one time. With this microplate, a plurality of chemical reactions can be carried out at one time 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 detection sensitivity of light emission or fluorescence is lowered because the absolute amount of light emission or fluorescence from the reaction solution 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 problems, and an object of the present invention is to provide a measuring method in which the detection sensitivity of light emission or fluorescence does not decrease and a reaction vessel used for the measurement method.
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 aspects will be described as means for solving the problem. These aspects can be arbitrarily combined as needed.
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 measuring method of the present invention
At least two kinds of liquids are injected into the wells of the reaction vessel containing the microplate in which a plurality of wells are formed and the transparent mineral oil injected into the wells to react, and the transparent mineral oil generated from the reaction solution is formed. It is characterized in that it detects the light emission that has passed through the above and measures the intensity of the 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 this invention
At least one liquid is injected into the wells of the reaction vessel containing the microplate in which a plurality of wells are formed and the transparent mineral oil injected into the wells, and the liquid is irradiated with excitation light to form the liquid. It is characterized in that fluorescence is generated, fluorescence that has passed through a transparent mineral oil is detected, and the intensity of 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 this invention
At least two kinds of liquids are injected into the wells of the microplate in which a plurality of wells are formed to react, and a transparent mineral oil is injected immediately after the reaction to detect the luminescence generated from the reaction solution and passed through the transparent mineral oil. It is characterized by measuring 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 this invention
At least one liquid and transparent mineral oil are injected into the wells 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 is characterized by detecting and measuring the intensity 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。
Further, the reaction vessel of the present invention is
It is characterized by containing 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 measuring 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 to react. The light emission generated from the reaction solution and passed through the transparent mineral oil was detected, and the intensity of the 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, in the measuring method of the present invention, at least one liquid is injected 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 wells, and the liquid is liquid. Is irradiated with excitation light to generate fluorescence from the liquid, fluorescence that has passed through the 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ように構成した。 Further, in the measuring 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 to react, and immediately after the reaction, transparent mineral oil is injected to generate transparent mineral oil from the reaction solution. It was configured to detect the luminescence that passed through the mineral oil and measure the intensity of the luminescence.
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。 Further, in the measuring method of the present invention, at least one kind of liquid and transparent mineral oil are injected into the wells 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. , The fluorescence that passed 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 measuring method of the present invention, the detection sensitivity of light emission 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。 Further, the reaction vessel of the present invention was configured to contain 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 light emission 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 of the method for manufacturing the decorative product of the present invention will be described.
＜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 measuring method of the present invention, at least two kinds of liquids 2 are contained in the well 10a of the reaction vessel 1 including the microplate 10 in which a plurality of wells 10a are formed and the transparent mineral oil 11 injected into the wells 10a. 3 is injected and reacted, and the light emission generated from the reaction solution 4 and passed through the transparent mineral oil 11 is detected, and the intensity of the light emission 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, and a resin such as polyethylene, polystyrene, or polypropylene, or glass can be used as the material. A plurality of wells 10a are formed on the microplate 10. As the well 10a, a 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 4240000, 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 injected into the well 10a in advance (see FIGS. 1A and 3). The transparent mineral oil 11 is refined, has non-volatility, and has a specific gravity lower than that of the aqueous phase. For example, it is possible to use a carbon number of 6 or more hydrocarbon compounds such as liquid paraffin (straight chain saturated hydrocarbons, branched-chain saturated hydrocarbon, cyclic saturated hydrocarbon), ethyl ether, petroleum ether, a etc. diphenyl ether. These can be used alone or as a mixture. Among these, fluidized paraffin is preferred. 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. 1 (b)). For 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. 1 (c)). For injection, for example, a dispenser 8 can be used. As the liquid 3, for example, reagents, antigens, substrates and 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 liquids 2 and 3 into the well 10a, the chemical reaction is initiated (see FIG. 1D). Light is emitted 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)). Light is detected by the detection unit 9a, and the emission intensity is measured. 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, the light emission from the reaction solution is diffused (see FIG. 2B). On the other hand, in the present invention in which the transparent mineral oil 11 is present 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 downwardly convex meniscus. The transparent mineral oil 11 on the liquid surface also wets the inner wall of the well 10a, so that a downwardly convex meniscus is formed. Therefore, the luminescence generated from the reaction solution 4 is condensed by the transparent mineral oil 11 having a convex meniscus lens shape, and the luminescence detection sensitivity 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 reaction liquid 4 may be injected into the well 10a into which the transparent mineral oil 11 has been injected in advance, and immediately after that, a sample different from the liquids 2 and 3 may be further injected. Also in this form, the detection sensitivity of light emission can be increased by the convex meniscus lens effect of the 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 on 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。 The transparent mineral oil 11 is pre-injected into the well 10a. Therefore, when storing the reaction vessel 1, it is necessary to prevent the transparent mineral oil 11 from leaking from the well 10a. Examples of such an example include the forms shown in FIGS. 4 (a) to 4 (c). FIG. 4A shows a form in which the lid material 12 having the same size as the microplate 10 is used. The material of the lid material 12 can be the same as that of the microplate 10, for example. The lid material 12 can be fixed by using the lid material 12 having protrusions at the four corners and the microplate 10 having holes formed at the portions corresponding to the protrusions. Further, the lid material 12 can be fixed by being brought into close contact with the microplate 10 with an adhesive or the like. When the lid material 12 is fixed, it is possible to prevent the transparent mineral oil 11 from leaking from the well 10a. The lid material 12 does not have to be fixed to the microplate 10. When the lid material 12 is not fixed, the reaction vessel can be used immediately when it is desired to be used.
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 or the like can be used. The sealing material 13 is fixed to the microplate 10 with an adhesive. When the lid material 12 is fixed, it is possible to prevent the transparent mineral oil 11 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 the vacuum packaging material 14 and sealed with a degassing sealer or the like. By doing so, it is possible to more reliably prevent the transparent mineral oil 11 from leaking from the well 10a as compared with FIGS. 4A and 4B. It is also preferable to use the lid material 12 and the sealing material 13 described above and then seal them with the vacuum packaging material 14. By doing so, 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, the second embodiment of the present invention will be described. In the measuring method of the present invention, at least one liquid 5 is contained in the well 10a of the reaction vessel 1 including the microplate 10 in which the plurality of wells 10a are formed and the transparent mineral oil 11 injected into the wells 10a. Is injected, 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 differs from the first embodiment in that at least one liquid is injected into the well, and the liquid is irradiated with excitation light to generate fluorescence from the liquid. Hereinafter, the points different from those of the first embodiment will be mainly described.
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 in which the transparent mineral oil 11 is previously injected into the well 10a of the microplate 10 is prepared (FIG. 5A). 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 irradiates excitation light to emit fluorescence. As the fluorescent substance, for example, fluorescent dyes such as fluorescein, Cy3, and Cy5, fluorescent proteins such as GFP (Green Fluorescein Protein), and fluorescently labeled antibodies labeled with these can be used. A plurality of liquids of a type different from that of 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, the 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 substance injected into the well 10a. The excitation light may be irradiated from below the reaction vessel toward the liquid 5.
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 irradiating the liquid 5 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. 5E). Fluorescence is detected by the detection unit 9a, and the fluorescence intensity is measured. 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 those 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 measuring method of the present invention, at least two kinds of liquids 2 and 3 are injected into the wells 10a of the microplate 10 in which a plurality of wells 10a are formed to react, and immediately after the reaction, transparent mineral oil 11 is injected to react. It is characterized in that the light emission generated from the liquid 4 and passed through the transparent mineral oil 11 is detected and the intensity of the light emission 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 liquids 2 and 3 are first injected into the well and then transparent mineral oil is injected. Hereinafter, the points different from those of the first embodiment will be mainly described.
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 a dispenser 8 or the like (see FIG. 6A). The liquid 3 is then injected (see FIG. 6B). When the liquid 3 is injected, a chemical reaction starts and light emission occurs. Immediately after the reaction, the 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 for injecting liquid 3 and a dispenser for injecting transparent mineral oil 11 may be prepared, and when the dispenser for injecting liquid 3 moves, the dispenser for injecting transparent mineral oil 11 may track it. it can. In addition, the liquid 2 may be injected first, and then the transparent mineral oil 11 and the liquid 3 may be injected in this order. Further, the liquid 2 may be injected first, and the transparent mineral oil 11 and the liquid 3 may be injected at the same time.
In this way, since the transparent mineral oil 11 is injected immediately after the reaction or almost at the same time as the reaction, the luminescence generated from the reaction solution can be efficiently focused 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 measuring method of the present invention, at least one liquid and transparent mineral oil are injected into the wells of a microplate in which a plurality of wells are formed, and the liquid is irradiated with excitation light to cause fluorescence from the liquid to be transparent. It is characterized in that fluorescence that has passed through various mineral oils is detected and the intensity of fluorescence 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, then the transparent mineral oil 11 is injected, and the liquid 5 is irradiated with excitation light. Hereinafter, the points different from the second embodiment will be mainly described.
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。 Liquid 5 is injected into the well 10a using, for example, a dispenser 8 (see FIG. 7A). Then, the transparent mineral oil 11 is injected using a dispenser 8 or the like (see FIG. 7B). Irradiate with excitation light (see FIG. 7 (c)). When irradiated with excitation light, a chemical reaction begins 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 the excitation light and the chemical reaction starts, fluorescence is generated, and the time during which the fluorescence is generated is extremely short (for example, in nanosecond units). Therefore, the measuring instrument 9 measures the fluorescence intensity and the excitation light. Those capable of irradiation are preferable. Also in the fourth embodiment, the fluorescence detection sensitivity can be increased by the convex meniscus lens effect of the 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, but the liquid 5 and the transparent mineral oil 11 may be injected at the same time. Since the transparent mineral oil 11 has a smaller specific gravity than the liquid 5, even if it is injected at the same time, the transparent mineral oil 11 and the liquid 5 are separated into two layers after a lapse of a predetermined time, and the transparent mineral oil 11 is relative to the liquid 5. It becomes the upper layer. Therefore, the fluorescence generated from the reaction solution can be efficiently focused 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 flat. A reaction vessel was prepared by injecting 50 μL of clear mineral oil into the wells of the microplate in advance. Mineral oil was used as the transparent mineral oil. It is colorless and transparent, has a viscosity of 22.5 mPa · s at a specific gravity of 0.85 and 25 ° C., and is characterized by not containing DNase (deoxyribonuclease), RNase (ribonuclease), and Protease (protease).
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 and chemically reacted. Use 1 μM ATP solution (rATP 10 mM adjusted in concentration by adding Tris buffer) and Reagent solution (mixture of Kinase-Glo® Substrate and Kinase-Glo® Buffer) in an amount of 50 μL per well. The enzyme substrate reaction was carried out. The reaction time was 16 minutes. A pipette and a pipette tip were used to inject the sample.
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。 The luminescence generated from the reaction solution was detected by a measuring instrument to measure the luminescence intensity. The measuring instrument used was a multi-mode microplate reader (power scan HT manufactured by DS Pharma Biomedical Co., Ltd., compatible with absorption / fluorescence / chemiluminescence measurement). The measurement was performed three times, and the calculated average value 382631 RLU was used 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 above sample was injected into a well to cause a chemical reaction, and immediately after the reaction, 50 μL of clear mineral oil was injected. Other than that, the procedure was the same 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)
The above sample was injected into a well and chemically reacted without injecting clear mineral oil. Other than that, the procedure was the same as in Example 1. The average value was 33,859 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 is the amount of light emission (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 Tables 1 and 2, Examples 1 and 2 had a larger amount of light emission 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
1: Reaction vessel 10: Microplate 10a: Well 11: Clear mineral oil 12: Lid material 13: Sealing material 14: Vacuum packaging material 2, 3, 5: Liquid 4: Reaction liquid 8: Dispenser 9: Measuring instrument 9a: Detection Department
Claims (4)
前記少なくとも２種の液体を反応させて発光させるステップと、
前記発光による光を、反応液の液面と、前記透明な鉱油の液面とが下に凸のメニスカスになることで形成された、凸メニスカスレンズ状の前記透明な鉱油を通過させて、検出するステップと、
前記発光の強度を測定するステップとを備えた、測定方法。 A micro plate having a plurality of wells are formed, and a transparent mineral oil injected into the plurality of wells in the plurality of wells of including reaction vessel, a step of injecting at least two liquid,
The step of reacting at least two kinds of liquids to emit light,
The light generated by the light emission is detected by passing the convex meniscus lenticular transparent mineral oil formed by the liquid surface of the reaction solution and the liquid surface of the transparent mineral oil forming a downwardly convex meniscus. Steps to do and
A measuring method comprising the step of measuring the intensity of light emission.
前記少なくとも１種の液体に励起光を照射して、前記少なくとも１種の液体から蛍光を生じさせるステップと、
前記蛍光を、前記少なくとも１種の液体の液面と、前記透明な鉱油の液面とが下に凸のメニスカスになることで形成された、凸メニスカスレンズ状の前記透明な鉱油を通過させて、検出するステップと、
前記蛍光の強度を測定するステップとを備えた、測定方法。 A micro plate having a plurality of wells are formed, in said plurality of wells of the injected transparent mineral oil and a including reaction vessels in said plurality of wells, the step of injecting at least one liquid,
A step wherein by irradiating excitation light to at least one liquid, that cause fluorescence from the at least one liquid,
The fluorescence is passed through the transparent mineral oil having a convex meniscus lens shape, which is formed by forming the liquid surface of the at least one liquid and the liquid surface of the transparent mineral oil into a downwardly convex meniscus. , Steps to detect,
A measuring method comprising the step of measuring the intensity of fluorescence.
前記少なくとも２種の液体を反応させて発光させるステップと、
反応直後に透明な鉱油を注入するステップと、
前記発光による光を、反応液の液面と、前記透明な鉱油の液面とが下に凸のメニスカスになることで形成された、凸メニスカスレンズ状の前記透明な鉱油を通過させて、検出するステップと、
前記発光の強度を測定するステップとを備えた、測定方法。 In said plurality of wells of a microplate having a plurality of wells are formed, a step of implanting at least two liquids,
The step of reacting at least two kinds of liquids to emit light,
The step of injecting clear mineral oil immediately after the reaction ,
The light generated by the light emission is detected by passing the convex meniscus lenticular transparent mineral oil formed by the liquid surface of the reaction solution and the liquid surface of the transparent mineral oil forming a downwardly convex meniscus. Steps to do and
A measuring method comprising the step of measuring the intensity of light emission.
前記少なくとも１種の液体に励起光を照射して、前記少なくとも１種の液体から蛍光を生じさせるステップと、
前記蛍光を、前記少なくとも１種の液体の液面と、前記透明な鉱油の液面とが下に凸のメニスカスになることで形成された、凸メニスカスレンズ状の前記透明な鉱油を通過させて、検出するステップと、
前記蛍光の強度を測定するステップとを備えた、測定方法。
In said plurality of wells of a microplate having a plurality of wells are formed, and the step of injecting at least one liquid, a transparent mineral oil,
A step wherein by irradiating excitation light to at least one liquid, that cause fluorescence from the at least one liquid,
The fluorescence is passed through the transparent mineral oil having a convex meniscus lens shape, which is formed by forming the liquid surface of the at least one liquid and the liquid surface of the transparent mineral oil into a downwardly convex meniscus. , Steps to detect,
A measuring method comprising the step of measuring the intensity of fluorescence.
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