JP2008096154A - Fine particle detector - Google Patents

Fine particle detector Download PDF

Info

Publication number
JP2008096154A
JP2008096154A JP2006275462A JP2006275462A JP2008096154A JP 2008096154 A JP2008096154 A JP 2008096154A JP 2006275462 A JP2006275462 A JP 2006275462A JP 2006275462 A JP2006275462 A JP 2006275462A JP 2008096154 A JP2008096154 A JP 2008096154A
Authority
JP
Japan
Prior art keywords
excitation light
light emitter
fluorescence detector
fine particles
amount
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
JP2006275462A
Other languages
Japanese (ja)
Inventor
Masamoto Torimura
政基 鳥村
Hiroaki Tao
博明 田尾
Hiroto Nishijima
裕人 西島
Kunihiko Nishibe
邦彦 西部
Satoru Sugitani
悟 杉谷
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sumika Chemical Analysis Service Ltd
National Institute of Advanced Industrial Science and Technology AIST
Hitachi Plant Technologies Ltd
Original Assignee
Sumika Chemical Analysis Service Ltd
National Institute of Advanced Industrial Science and Technology AIST
Hitachi Plant Technologies Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sumika Chemical Analysis Service Ltd, National Institute of Advanced Industrial Science and Technology AIST, Hitachi Plant Technologies Ltd filed Critical Sumika Chemical Analysis Service Ltd
Priority to JP2006275462A priority Critical patent/JP2008096154A/en
Publication of JP2008096154A publication Critical patent/JP2008096154A/en
Pending legal-status Critical Current

Links

Images

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fine particle detector capable of stably detecting desired fine particles, without being affected by changes in the utilization environment or the deterioration in the exciting light source, or the like. <P>SOLUTION: The fine particle detector is constituted so as to detect fine particles by the fluorescent dyeing of a sample, containing fine particles and performing capillary electrophoresis by using a migration liquid and equipped with an exciting light emitter 2, such as LED or the like, for emitting a pulsed light as the exciting light to the fine particles subjected to fluorescent dyeing; a fluorescence detector 5 for measuring fluorescent emission, synchronized to the pulse emission; and a luminous flux quantity adjusting means for adjusting the quantity of the luminous flux of the exciting light emitter 2, on the basis of the quantity of fluorescent luminous flux measured by the fluorescence detector 5. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

本発明は、微細管電気泳動を用いて、検体試料中に存在する細胞等の各種微粒子を検出する微粒子検出装置に関し、特に人体に有害なレジオネラ属菌等の微生物を迅速かつ高感度に分離検出することができる微粒子検出装置に関するものである。   The present invention relates to a microparticle detection apparatus for detecting various microparticles such as cells existing in a specimen sample by using microtubule electrophoresis, and particularly, for rapidly and highly sensitively detecting and detecting microorganisms such as Legionella spp. Harmful to the human body. The present invention relates to a fine particle detection apparatus capable of performing the above.

近年、試料中に細胞等の各種微粒子が存在するか否かを調べたり、また試料中に含まれる微生物の種類や量を迅速に調べるための分離・検出手法として、微細管内で予め蛍光染色した微生物を電気泳動させ、励起光を照射してその時発生する蛍光から検出する電気泳動技術が提案されている(例えば、特許文献1参照)。   In recent years, as a separation / detection method for examining the presence or absence of various microparticles such as cells in a sample, and for quickly examining the type and amount of microorganisms contained in a sample, fluorescent staining has been performed in advance in a microtubule. There has been proposed an electrophoresis technique in which microorganisms are electrophoresed and irradiated with excitation light and detected from fluorescence generated at that time (see, for example, Patent Document 1).

一方、微細管内で微生物を泳動分離する際には、微生物細胞と細管内壁、あるいは細胞と細胞との相互作用が大きいため、迅速かつ高感度に微細管内で微生物を分離するために、泳動液にアルギン酸塩を含有させることが有効であることが知られている(例えば、特許文献2参照)。
この方法において、微生物の分離効率を向上させる添加剤として用いられるアルギン酸塩は、すべての微生物に対して同様に機能するものではなく、多種多様な微生物種に対応できるより多くのタイプの分離効率向上剤の導入が期待されている。
しかしながら、上記従来の微粒子検出装置では、励起光を常時点灯としているため、励起光や蛍光発光以外の光が蛍光検出装置に入らないように、計測部分を周囲の光から隔離する必要があった。
On the other hand, when migrating and separating microorganisms in a microtubule, the interaction between the microorganism cell and the inner wall of the microtubule or between the cells and the cell is large. It is known that it is effective to contain an alginate (see, for example, Patent Document 2).
In this method, the alginate used as an additive to improve the separation efficiency of microorganisms does not function in the same way for all microorganisms, and more types of separation efficiency can be improved to cope with a wide variety of microorganism species. The introduction of agents is expected.
However, in the conventional fine particle detection device, since the excitation light is always turned on, it is necessary to isolate the measurement portion from the ambient light so that light other than the excitation light and the fluorescence emission does not enter the fluorescence detection device. .

また、蛍光発光は励起光に対し一般的に少なく、周囲の光に対し十分な蛍光発光の光束が得られるように、励起光源を高輝度(高発光)のものを使用する必要があった。
例えば、励起光源として安価なLED等を使用した場合、高輝度で常時点灯させると発熱が大きくなり、また寿命が短くなる等の問題があった。
したがって、従来の微粒子検出装置では、励起光源の劣化による光束量の低下等があるため、励起光源の光束量調整や、励起光源の光束量変動にともなう蛍光検出器における受光感度調整等が必要であった。
特開2002−345451号公報 特開2002−181781号公報
In addition, fluorescence emission is generally small with respect to excitation light, and it is necessary to use an excitation light source with high luminance (high emission) so that a sufficient luminous flux of fluorescence emission can be obtained with respect to ambient light.
For example, when an inexpensive LED or the like is used as an excitation light source, there is a problem that heat is increased and the life is shortened when the LED is constantly lit with high luminance.
Therefore, in the conventional particle detector, there is a decrease in the amount of light due to deterioration of the excitation light source, so adjustment of the amount of light of the excitation light source and adjustment of light receiving sensitivity in the fluorescence detector due to fluctuations in the amount of light of the excitation light source are necessary. there were.
JP 2002-345451 A JP 2002-181781 A

本発明は、上記従来の微粒子検出装置が有する問題点に鑑み、使用環境の変化や励起光源の劣化等の影響を受けることなく、所望微粒子の検出を安定的に実施することができる微粒子検出装置を提供することを目的とする。   In view of the problems of the conventional fine particle detection apparatus, the present invention is capable of stably detecting desired fine particles without being affected by changes in the use environment or deterioration of the excitation light source. The purpose is to provide.

上記目的を達成するため、本発明の微粒子検出装置は、微粒子を含む試料を蛍光染色し、泳動液を用いて微細管電気泳動することにより微粒子を検出する微粒子検出装置において、蛍光染色の励起光として間欠的に発光する励起光発光器と、励起光発光に同期して蛍光を計測する蛍光検出器とを備えたことを特徴とする。   In order to achieve the above object, the particle detection apparatus of the present invention is an excitation light for fluorescent staining in a particle detection apparatus that detects a particle by fluorescently staining a sample containing the particle and performing microtubule electrophoresis using an electrophoresis solution. And an excitation light emitter that emits light intermittently and a fluorescence detector that measures fluorescence in synchronization with excitation light emission.

この場合において、蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器の光束量を調整する光束量調整手段を備えることができる。   In this case, a light beam amount adjusting means for adjusting the light beam amount of the excitation light emitter can be provided based on the fluorescent light beam amount measured by the fluorescence detector.

また、光束量調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて、励起光発光器の光束量を調整するようにすることができる。   Further, the luminous flux amount adjusting means can adjust the luminous flux amount of the excitation light emitter based on the difference between when the excitation light emitter is turned on and when it is not lit, measured by the fluorescence detector.

また、蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器のON、OFF時間比率を調整するパルス幅調整手段を備えることができる。   Further, it is possible to provide a pulse width adjusting means for adjusting the ON / OFF time ratio of the excitation light emitter based on the amount of fluorescent light flux measured by the fluorescence detector.

また、パルス幅調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて、励起光発光器のON、OFF時間比率を調整することができる。   The pulse width adjusting means can adjust the ON / OFF time ratio of the excitation light emitter based on the difference between when the excitation light emitter is turned on and when it is not turned on, measured by the fluorescence detector.

さらに、励起光発光器における励起光源として、間欠的に発光するLEDを使用することができる。   Furthermore, LEDs that emit light intermittently can be used as the excitation light source in the excitation light emitter.

また、検出対象物である微粒子が微生物、例えば、レジオネラ属菌等の人体に有害な微生物とすることができる。   Moreover, the microparticles | fine-particles which are detection objects can be used as microorganisms harmful | toxic to human bodies, such as microorganisms, for example, Legionella genus bacteria.

本発明の微粒子検出装置によれば、微粒子を含む試料を蛍光染色し、泳動液を用いて微細管電気泳動することにより微粒子を検出する微粒子検出装置において、蛍光染色の励起光として間欠的に発光する励起光発光器と、励起光発光に同期して蛍光を計測する蛍光検出器とを備えることにより、励起光や蛍光発光以外の周囲の光及びパルス発光する励起光発光器の劣化等の影響を受けることなく、安定的に所望微粒子の検出を実施することができる。   According to the microparticle detection apparatus of the present invention, in a microparticle detection apparatus that detects microparticles by fluorescently staining a sample containing microparticles and performing microtubule electrophoresis using an electrophoresis solution, light is emitted intermittently as excitation light for fluorescent staining. By providing an excitation light emitter that emits light and a fluorescence detector that measures fluorescence in synchronization with the excitation light emission, influences such as deterioration of excitation light, ambient light other than fluorescence emission, and excitation light emitter that emits pulses The desired fine particles can be detected stably without being subjected to the above.

また、蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器の光束量を調整する光束量調整手段を備えることにより、パルス発光する励起光発光器の光束量を常時発光時の10〜300%程度に可変調整し、分析に適当な励起光の光束量とすることができ、これにより、一層安定的に所望微粒子の検出を実施することができる。   In addition, by providing a luminous flux amount adjusting means for adjusting the luminous flux amount of the excitation light emitter based on the fluorescent luminous flux amount measured by the fluorescence detector, the luminous flux amount of the excitation light emitter that emits pulses is constantly emitted. It is possible to variably adjust to about 10 to 300% to obtain a light flux amount of excitation light suitable for analysis, and thereby it is possible to detect desired fine particles more stably.

また、光束量調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて励起光発光器の光束量を調整することにより、光束を前もって調整する光束調整期間を不要にすることができる。   In addition, the luminous flux amount adjusting means adjusts the luminous flux amount of the excitation light emitter in advance based on the difference between when the excitation light emitter is turned on and when it is not lit, measured by the fluorescence detector, so that the light flux is adjusted in advance. The adjustment period can be eliminated.

また、蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器のON、OFF時間比率を調整するパルス幅調整手段を備えることにより、パルス発光する励起光発光器のパルス幅を可変調整し、分析に適当な励起光のパルス幅とすることができ、これにより、一層安定的に所望微粒子の検出を実施することができる。   The pulse width of the excitation light emitter that emits pulses can be varied by providing pulse width adjustment means that adjusts the ON / OFF time ratio of the excitation light emitter based on the amount of fluorescent light flux measured by the fluorescence detector. The pulse width of the excitation light can be adjusted to an appropriate value for the analysis, whereby the desired fine particles can be detected more stably.

また、パルス幅調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて、励起光発光器のON、OFF時間比率を調整することにより、光束を前もって調整する光束調整期間を不要にすることができる。   Further, the pulse width adjusting means adjusts the ON / OFF time ratio of the excitation light emitter based on the difference between the time when the excitation light emitter is turned on and the time when the excitation light emitter is turned off, which is measured by the fluorescence detector. The luminous flux adjustment period to be adjusted in advance can be eliminated.

また、励起光発光器における励起光源として、間欠的に発光するLEDを使用することにより、安価な装置を実現することができる。   Moreover, an inexpensive apparatus can be realized by using an LED that emits light intermittently as the excitation light source in the excitation light emitter.

また、検出対象物である微粒子が微生物、例えば、レジオネラ属菌等の人体に有害な微生物とすることにより、微生物、例えば、温泉やプール等で生息し易いレジオネラ属菌等の人体に有害な微生物の検出を行うことができる。   In addition, microparticles that are detection targets are microorganisms, for example, microorganisms that are harmful to the human body such as Legionella, so microorganisms that are harmful to the human body such as Legionella that are likely to live in hot springs or pools Can be detected.

以下、本発明の微粒子検出装置の実施の形態を、図面に基づいて説明する。
なお、本発明でいう分析とは、試料中に含まれる所望微粒子の濃縮、分離、検出(定性的分析)、同定、定量、スクリーニング、生死判別を包含するものである。
Hereinafter, embodiments of the particulate detection device of the present invention will be described with reference to the drawings.
The analysis referred to in the present invention includes concentration, separation, detection (qualitative analysis), identification, quantification, screening, and life / death discrimination of desired fine particles contained in a sample.

図1に、本発明の微粒子検出装置の基本的構成を一例として示す。
図において、1は電気泳動装置本体であり、LEDやレーザー等の励起光発光器2、対物レンズ4、CCDカメラ等の蛍光検出器5等から構成されている。
FIG. 1 shows an example of the basic configuration of the particle detector of the present invention.
In the figure, reference numeral 1 denotes an electrophoretic apparatus main body, which includes an excitation light emitter 2 such as an LED or a laser, an objective lens 4, a fluorescence detector 5 such as a CCD camera, and the like.

検出対象物である有害菌等の微粒子は、予め当該微粒子とのみ選択的に結合する蛍光抗体等を用いて蛍光染色しておき、例えば、キャピラリー等の微細管6の内部に、その両端に設置した溶液9a又は溶液9bより注入するようにする。
溶液9a又は溶液9bを検出の段階に応じて変更し、電極7a、7bにより微細管6の両端に高圧電源を印加する。高電圧は高圧電源7より供給し、その電圧の大きさ、印加時間等は、例えば、パソコン等の微粒子検出制御装置8より制御する。
なお、蛍光検出器5としては、上記のようなCCDカメラ等の画像入力装置のほかに、光電子倍増管(フォトマル)等の光束量センサを用いることができる。
Fine particles such as harmful bacteria, which are detection objects, are fluorescently stained in advance using a fluorescent antibody or the like that selectively binds only to the fine particles, for example, inside a microtube 6 such as a capillary, at both ends thereof. The solution 9a or the solution 9b is injected.
The solution 9a or the solution 9b is changed according to the detection stage, and a high voltage power source is applied to both ends of the microtube 6 by the electrodes 7a and 7b. The high voltage is supplied from the high voltage power source 7, and the magnitude of the voltage, the application time, and the like are controlled by the particulate detection control device 8 such as a personal computer, for example.
As the fluorescence detector 5, in addition to the above-described image input device such as a CCD camera, a light flux amount sensor such as a photomultiplier tube (photomultiplier) can be used.

励起光2aは、励起光発光器2から発した光であり、微細管6の検出窓6aより、蛍光染色された微粒子に照射される。
ここで、励起光発光器2の発光電力は、励起光発光器2用のパルス発生器2cより供給し、その電流やパルス間隔等は、微粒子検出制御装置8により制御する。
蛍光染色された微粒子は、蛍光2bを発光し、対物レンズ4より装置本体内に入射し、例えば、CCDカメラ等で構成される蛍光検出器5によりパルス発生器2cと同期して検出される。
蛍光検出器5により検出した検出画像信号は、画像信号記録装置5aに記録されるととともに、微粒子検出制御装置8に接続される。微粒子検出制御装置8では、この画像信号から微粒子の検出を行い、その結果を記録し表示する。
The excitation light 2 a is light emitted from the excitation light emitter 2 and is irradiated to the fluorescently stained fine particles from the detection window 6 a of the microtube 6.
Here, the emission power of the excitation light emitter 2 is supplied from the pulse generator 2 c for the excitation light emitter 2, and the current, pulse interval, and the like are controlled by the particle detection control device 8.
The fluorescently stained fine particles emit fluorescence 2b, enter the apparatus main body through the objective lens 4, and are detected in synchronization with the pulse generator 2c by a fluorescence detector 5 constituted by, for example, a CCD camera or the like.
The detected image signal detected by the fluorescence detector 5 is recorded in the image signal recording device 5a and connected to the particulate detection control device 8. The particulate detection control device 8 detects particulates from the image signal, and records and displays the result.

検出対象としての微粒子を含む試料である検水溶液を、例えば、溶液9aとして準備し、溶液9bの側から吸引することにより、微細管6内に対象とする検水溶液20として注入する。
次に、電極7a、7bはそれぞれ高圧電源7に接続されており、微粒子検出制御装置8により、例えば、電極7aには陽極、電極7bには陰極の電圧を印加するように接続する。
次いで、高圧電源7を作動させることにより、既知の電気泳動技術により、微粒子を微粒子塊25として濃縮し、さらには陰極の方向22に泳動させることができる。
A test solution, which is a sample containing fine particles as a detection target, is prepared as, for example, a solution 9a, and is sucked from the solution 9b side to be injected into the microtube 6 as a target test solution 20.
Next, the electrodes 7a and 7b are connected to the high-voltage power source 7, and are connected by the particulate detection control device 8 so as to apply, for example, a voltage of the anode to the electrode 7a and a voltage of the cathode to the electrode 7b.
Next, by operating the high-voltage power supply 7, the fine particles can be concentrated as a fine particle lump 25 by a known electrophoresis technique, and further migrate in the cathode direction 22.

このとき、微粒子塊25は、検出窓6aを通過する。
この微粒子塊25は、蛍光抗体等を用いて蛍光染色されており、図1で示したように、励起光2aが照射されると、蛍光2bを発光するため、CCDカメラ等で構成される蛍光検出器5により微粒子を検出することができる。
At this time, the fine particle lump 25 passes through the detection window 6a.
The fine particle lump 25 is fluorescently stained with a fluorescent antibody or the like, and as shown in FIG. 1, when irradiated with the excitation light 2a, the fluorescent light 2b is emitted, so that the fluorescent light constituted by a CCD camera or the like is used. Fine particles can be detected by the detector 5.

微細管6内への検体試料の注入方法は、特に制限されず、従来使用される重力法、加圧法及び減圧法のいずれをも使用することができる。
注入量も特に制限されないが、通常用いる微細管全域に試料を満たすため、その微細管のサイズに依存し、0.1〜100μL、好ましくは0.5〜100μL、より好ましくは0.5〜10μLを例示することができる。
The method of injecting the specimen sample into the microtube 6 is not particularly limited, and any of the conventionally used gravity method, pressurization method, and decompression method can be used.
Although the injection amount is not particularly limited, it depends on the size of the microtubule in order to fill the sample in the entire microtubule to be used, and it is 0.1 to 100 μL, preferably 0.5 to 100 μL, more preferably 0.5 to 10 μL. Can be illustrated.

また、上記で例示した一対の電極7a、7b及び高圧電源7は、微細管内に注入された微粒子が微細管内の泳動液中を泳動するのに必要な強さの電位勾配を、微細管中の泳動液に対して印加するための手段であり、かかる目的が達成できるものであれば、これらの電源及び一対の電極は何ら限定されることなく、その他の手段として任意の手段を使用することができる。   In addition, the pair of electrodes 7a and 7b and the high-voltage power source 7 exemplified above have a potential gradient in the intensity required for the fine particles injected into the microtube to migrate in the electrophoresis solution in the microtube. As long as it is a means for applying to the electrophoretic solution and the object can be achieved, the power source and the pair of electrodes are not limited at all, and any means can be used as other means. it can.

電極間にかけられる電圧としては、微細管長さに対して、一般に約1kV/m〜約500kV/m、好ましくは約2kV/m〜約100kV/m、より好ましくは約5kV/m〜約20kV/mを例示することができる。
微細管としては、内径1〜150μm、長さ0.1〜100cm、好ましくは内径20〜100μm、長さ1〜50cmの中空管を例示することができる。
その材質としては特に制限されず、ガラス(フューズドシリカ)やフッ素樹脂等を任意に例示することができる。
The voltage applied between the electrodes is generally about 1 kV / m to about 500 kV / m, preferably about 2 kV / m to about 100 kV / m, more preferably about 5 kV / m to about 20 kV / m, relative to the length of the microtube. Can be illustrated.
Examples of the fine tube include a hollow tube having an inner diameter of 1 to 150 μm and a length of 0.1 to 100 cm, preferably an inner diameter of 20 to 100 μm and a length of 1 to 50 cm.
It does not restrict | limit especially as the material, Glass (fused silica), a fluororesin, etc. can be illustrated arbitrarily.

図2は、検出対象とする微粒子21としての微生物を拡大して示したもので、その外壁21aに選択的に結合する抗体30、抗体30を蛍光標識した蛍光色素31等を示している。
なお、図示しないが、検出対象である微粒子21に結合している抗体及び蛍光色素31以外に、微粒子に結合しないままに溶液中を浮遊している抗体及び蛍光色素からも蛍光2bが発せられるが、泳動時間が検出対象微粒子とは異なるので、時間経過を見ることにより分離することが可能である。
FIG. 2 is an enlarged view of microorganisms as the fine particles 21 to be detected, and shows an antibody 30 that selectively binds to the outer wall 21a, a fluorescent dye 31 that is fluorescently labeled with the antibody 30, and the like.
Although not shown, fluorescence 2b is also emitted from antibodies and fluorescent dyes floating in the solution without being bound to the fine particles in addition to the antibodies and fluorescent dyes 31 bound to the fine particles 21 to be detected. Since the migration time is different from that of the detection target fine particles, it is possible to separate them by observing the passage of time.

図3に、図1に示す励起光発光器2の光束量とパルス幅、及び蛍光検出器5の検出フローの一例を示す。
励起光発光器2がON時の蛍光検出器5の蛍光光束量を測定する。
測定した蛍光光束量が予め決められた下限値以下であった場合、微粒子検出制御装置8が光束量調整手段及びパルス幅調整手段として、励起光束量を増大し、必要に応じてパルス幅を減少させる制御を行い、また、蛍光光束量が予め決められた上限値以上であった場合、励起光束量を減少し、必要に応じてパルス幅を増加させる制御を行い、適切な励起光発光量とする。
また、励起光にLED等の半導体を利用した場合、常時点灯に比較して発熱量が押さえられるので、パルス点灯により励起光束量を増大することにより、常時点灯時の300%程度までの光束出力が可能となることが一般的に知られており、パルス点灯を利用することにより、励起光の光束量を10〜300%と可変調整することができる。
FIG. 3 shows an example of the light flux amount and pulse width of the excitation light emitter 2 shown in FIG. 1 and the detection flow of the fluorescence detector 5.
The amount of fluorescent light flux of the fluorescence detector 5 when the excitation light emitter 2 is ON is measured.
When the measured fluorescent light flux amount is less than or equal to a predetermined lower limit value, the particulate detection control device 8 increases the excitation light flux amount as the light flux amount adjusting means and the pulse width adjusting means, and decreases the pulse width as necessary. If the amount of fluorescent light flux is greater than or equal to a predetermined upper limit, the amount of excitation light flux is decreased, and the pulse width is increased as necessary. To do.
In addition, when a semiconductor such as an LED is used for the excitation light, the amount of generated heat can be suppressed as compared with the case of constant lighting. Therefore, by increasing the amount of excitation light by pulse lighting, the luminous flux can be output up to about 300% during normal lighting. It is generally known that the amount of excitation light can be variably adjusted to 10 to 300% by using pulse lighting.

図4に、図1に示す励起光発光器2の光束量とパルス幅、及び蛍光検出器5の検出フローの他の例を示す。
励起光発光器2がOFF時の蛍光検出器5の蛍光光束量を測定する。
また、励起光発光器2より適当な光束量とパルス幅を持つ励起光のON時に合わせて蛍光検出器5の蛍光光束量を測定し、前述の励起光ON、OFF時の蛍光光束量の差から、次の励起光の光束量の大きさ、及び/又はパルス幅を制御し適切な励起光発光量とする。すなわち、励起光ON、OFF時の蛍光光束量の差がいつも有意差となり、かつ励起光ON時の蛍光光束量の値が下限値から上限値の間の値となるように励起光束量の大きさ、及び/又はパルス幅を制御する。
このフローを用いれば、図3のフローと比べ、一定時間内に微粒子検出サイクルが長くなるが、図3に示した光束調整期間が不要となる。
FIG. 4 shows another example of the light flux amount and pulse width of the excitation light emitter 2 shown in FIG. 1 and the detection flow of the fluorescence detector 5.
The amount of fluorescent light flux of the fluorescence detector 5 when the excitation light emitter 2 is OFF is measured.
Further, the amount of fluorescent light flux of the fluorescence detector 5 is measured when the excitation light having an appropriate light amount and pulse width from the excitation light emitter 2 is turned on, and the difference in the amount of fluorescent light flux when the excitation light is turned on and off. From the above, the magnitude of the luminous flux of the next excitation light and / or the pulse width are controlled to obtain an appropriate excitation light emission amount. That is, the excitation light beam amount is large so that the difference in the amount of fluorescent light beam when the excitation light is on and off is always a significant difference, and the value of the fluorescence light beam amount when the excitation light is on is between the lower limit value and the upper limit value. And / or control the pulse width.
If this flow is used, the particle detection cycle becomes longer within a fixed time than the flow of FIG. 3, but the light flux adjustment period shown in FIG. 3 is not necessary.

図5は、前記した対象微粒子の検出過程をフロー図で示したものである。
まず、検体溶液を微細管に注入し(ステップ32)、励起光の光束量調整(図3の光量調整期間)である光量調整を実施し(ステップ33)、微粒子を電気泳動し、微粒子塊(濃縮)する(ステップ34)。
次に、検出窓まで微粒子塊を電気泳動(微粒子塊の泳動)し(ステップ35)、CCDカメラ等で微粒子検出する(ステップ36)。
以上の過程で対象微粒子を検出する。
なお、図4の励起光調整方法の場合、ステップ33の光量調整の工程は行わない。
FIG. 5 is a flowchart showing the detection process of the target fine particles.
First, the sample solution is injected into the microtube (step 32), the light amount adjustment that is the adjustment of the luminous flux amount of the excitation light (the light amount adjustment period in FIG. 3) is performed (step 33), the fine particles are electrophoresed, and the fine particle lump ( Concentrated) (step 34).
Next, the fine particle lump is electrophoresed (migration of the fine particle lump) to the detection window (step 35), and the fine particle is detected with a CCD camera or the like (step 36).
The target fine particles are detected in the above process.
In the case of the excitation light adjustment method of FIG. 4, the light amount adjustment process in step 33 is not performed.

かくして、本実施例の微粒子検出装置によれば、微粒子を含む試料を蛍光染色し、泳動液を用いて微細管電気泳動することにより微粒子を検出する微粒子検出装置において、蛍光染色の励起光として間欠的に発光する励起光発光器2と、励起光発光に同期して蛍光を計測する蛍光検出器5とを備えることにより、励起光や蛍光発光以外の周囲の光及びパルス発光する励起光発光器の劣化等の影響を受けることなく、安定的に所望微粒子の検出を実施することができる。   Thus, according to the microparticle detection apparatus of the present embodiment, the sample containing microparticles is fluorescently stained, and the microparticles are detected by performing microtubule electrophoresis using an electrophoresis solution. Excitation light emitter 2 that emits light in a pulsed manner and excitation light emitter 2 that emits pulsed light and ambient light other than fluorescence light emission by including fluorescence detector 5 that measures fluorescence in synchronization with excitation light emission The desired fine particles can be detected stably without being affected by degradation of the liquid.

また、蛍光検出器5にて測定した蛍光光束量に基づいて、励起光発光器2の光束量を調整する光束量調整手段を備えることにより、パルス発光する励起光発光器の光束量を常時発光時の10〜300%程度に可変調整し、分析に適当な励起光の光束量とすることができ、これにより、一層安定的に所望微粒子の検出を実施することができる。   Further, by providing a luminous flux amount adjusting means for adjusting the luminous flux amount of the excitation light emitter 2 based on the fluorescent luminous flux amount measured by the fluorescence detector 5, the luminous flux amount of the excitation light emitter that emits pulses is always emitted. It is possible to variably adjust to about 10 to 300% of the hour to obtain a light flux amount of excitation light suitable for analysis, and thereby it is possible to more stably detect desired fine particles.

また、光束量調整手段が、蛍光検出器5にて測定した励起光発光器の点灯時と不点灯時の差に基づいて励起光発光器2の光束量を調整することにより、光束を前もって調整する光束調整期間を不要にすることができる。   Further, the luminous flux amount adjusting means adjusts the luminous flux in advance by adjusting the luminous flux amount of the excitation light emitter 2 based on the difference between when the excitation light emitter is turned on and when it is not lit, measured by the fluorescence detector 5. The light beam adjustment period to be performed can be eliminated.

また、蛍光検出器5にて測定した蛍光光束量に基づいて、励起光発光器のON、OFF時間比率を調整するパルス幅調整手段を備えることにより、パルス発光する励起光発光器のパルス幅を可変調整し、分析に適当な励起光のパルス幅とすることができ、これにより、一層安定的に所望微粒子の検出を実施することができる。   Further, by providing a pulse width adjusting means for adjusting the ON / OFF time ratio of the excitation light emitter on the basis of the amount of fluorescent light flux measured by the fluorescence detector 5, the pulse width of the excitation light emitter that emits a pulse is reduced. It is possible to variably adjust the pulse width of the excitation light suitable for the analysis, and thereby it is possible to more stably detect the desired fine particles.

また、パルス幅調整手段が、蛍光検出器5にて測定した励起光発光器2の点灯時と不点灯時の差に基づいて、励起光発光器2のON、OFF時間比率を調整することにより、光束を前もって調整する光束調整期間を不要にすることができる。   Further, the pulse width adjusting means adjusts the ON / OFF time ratio of the excitation light emitter 2 based on the difference between when the excitation light emitter 2 is turned on and when the excitation light emitter 2 is turned off, measured by the fluorescence detector 5. The light beam adjustment period for adjusting the light beam in advance can be eliminated.

また、励起光発光器2における励起光源として、間欠的に発光するLEDを使用することにより、安価な装置を実現することができる。   Moreover, an inexpensive apparatus can be realized by using an intermittently emitting LED as the excitation light source in the excitation light emitter 2.

また、励起光発光器2における励起光源として、安価なLED又はレーザー発光器等を利用することにより、所望の微粒子を高感度かつ特異的に検出することができ、また、試料を培養する等の増殖処理を施すことなく、微量の微粒子を精度よく分離検出することができ、試料中に存在する菌体数を判別測定(定量検出)することも可能である。
さらに、本実施例の装置によれば、試料中の微粒子の検出が可能であるだけでなく、種々の微粒子に特有の特異的検出試薬(例えば、抗体等)を使用することにより菌種の同定も可能である。
In addition, by using an inexpensive LED or laser light emitter as an excitation light source in the excitation light emitter 2, desired fine particles can be detected with high sensitivity and specificity, and a sample can be cultured. A small amount of microparticles can be separated and detected with high accuracy without performing a proliferation treatment, and the number of bacterial cells present in a sample can be discriminated and measured (quantitative detection).
Furthermore, according to the apparatus of the present embodiment, not only microparticles in a sample can be detected, but also the identification of bacterial species by using specific detection reagents (for example, antibodies) specific to various microparticles. Is also possible.

そして、この微粒子検出装置は、検出対象物である微粒子として、微生物、例えば、レジオネラ属菌等の人体に有害な微生物とすることにより、微生物、例えば、温泉やプール等で生息し易いレジオネラ属菌等の人体に有害な微生物の検出(定性検出、定量検出)を行うのに適しているため、病原性微生物によって汚染された環境水や食品が早期に排除でき、病気の発生の防止に有用である。
また、病原性微生物による患者の早期診断が可能になることから、有害微生物による被害の蔓延防止並びに早期治療の一助となる。さらには、食品の品質管理期間が短縮でき、また衛生管理の厳格性から賞味期間も延長可能であり、食品流通の経済性に大きく貢献することができる効果がある。
And this particulate detection device makes microorganisms, for example, Legionella spp. Easy to inhabit in microorganisms, for example, hot springs, pools, etc., by using microbes such as microorganisms, for example, Legionella spp. It is suitable for the detection of microorganisms harmful to the human body (qualitative detection, quantitative detection), so that environmental water and food contaminated with pathogenic microorganisms can be eliminated at an early stage, which is useful for preventing the occurrence of diseases. is there.
In addition, since early diagnosis of patients with pathogenic microorganisms becomes possible, it helps prevent the spread of damage caused by harmful microorganisms and helps early treatment. Furthermore, the quality control period of the food can be shortened, and the shelf life period can be extended due to the strictness of hygiene management, which has the effect of greatly contributing to the economics of food distribution.

以上、本発明の微粒子検出装置に関して、その実施例に基づいて説明したが、本発明は上記実施例に記載した構成に限定されるものではなく、その趣旨を逸脱しない範囲において適宜その構成を変更することができる。 As mentioned above, although the particulate detection device of the present invention has been described based on the embodiments thereof, the present invention is not limited to the configurations described in the above embodiments, and the configuration is appropriately changed without departing from the gist thereof. can do.

本発明の微粒子検出装置は、各種微粒子を迅速かつ高感度に分離検出するという特性を有していることから、例えば、環境、食品及び医療分野等において、検体試料中の微粒子量を精度よく又は一斉分析する用途に好適に用いることができる。   The fine particle detection apparatus of the present invention has the property of separating and detecting various fine particles quickly and with high sensitivity, so that, for example, in the environment, food and medical fields, etc. It can be suitably used for the purpose of simultaneous analysis.

本発明の微粒子検出装置の一実施例を示す概略構成図である。It is a schematic block diagram which shows one Example of the microparticle detection apparatus of this invention. 微粒子の詳細を示す拡大図である。It is an enlarged view showing details of fine particles. 励起光の光束調整動作の実施例を示すフロー図である。It is a flowchart which shows the Example of the light beam adjustment operation | movement of excitation light. 励起光の光束調整動作の他の実施例を示すフロー図である。It is a flowchart which shows the other Example of the light beam adjustment operation | movement of excitation light. 微粒子を検出する動作フロー図である。It is an operation | movement flowchart which detects fine particles.

符号の説明Explanation of symbols

1 電気泳動装置本体
2 励起光発光器
2a 励起光線
2b 蛍光
2c パルス発生器
4 対物レンズ
5 蛍光検出器
5a 画像信号記録装置
6 微細管
6a 検出窓
7 高圧電源
7a 電極
7b 電極
8 微粒子検出制御装置
9a 溶液
9b 溶液
20 検水溶液
21 微粒子
21a 微粒子の外壁
25 微粒子塊
30 抗体
31 蛍光色素
32 検体溶液の注入
33 光束調整(光束調整期間)
34 電気泳動(濃縮)
35 電気泳動(微粒子塊の移動)
36 微粒子検出
DESCRIPTION OF SYMBOLS 1 Electrophoresis apparatus main body 2 Excitation light emitter 2a Excitation light 2b Fluorescence 2c Pulse generator 4 Objective lens 5 Fluorescence detector 5a Image signal recording device 6 Fine tube 6a Detection window 7 High voltage power supply 7a Electrode 7b Electrode 8 Fine particle detection control device 9a Solution 9b Solution 20 Test solution 21 Fine particle 21a Outer wall of fine particle 25 Fine particle mass 30 Antibody 31 Fluorescent dye 32 Injection of sample solution 33 Light flux adjustment (light flux adjustment period)
34 Electrophoresis (concentration)
35 Electrophoresis (movement of fine particle mass)
36 Particle detection

Claims (8)

微粒子を含む試料を蛍光染色し、泳動液を用いて微細管電気泳動することにより微粒子を検出する微粒子検出装置において、蛍光染色の励起光として間欠的に発光する励起光発光器と、励起光発光に同期して蛍光を計測する蛍光検出器とを備えたことを特徴とする微粒子検出装置。   An excitation light emitter that emits light intermittently as excitation light for fluorescence staining, and an excitation light emission in a particle detection device that detects particles by fluorescently staining a sample containing the particles and performing microtubule electrophoresis using an electrophoresis solution A fine particle detection device comprising a fluorescence detector that measures fluorescence in synchronization with 蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器の光束量を調整する光束量調整手段を備えたことを特徴とする請求項1記載の微粒子検出装置。   2. The particle detecting apparatus according to claim 1, further comprising a light beam amount adjusting means for adjusting a light beam amount of the excitation light emitter based on a fluorescent light beam amount measured by the fluorescence detector. 光束量調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて、励起光発光器の光束量を調整することを特徴とする請求項2記載の微粒子検出装置。   The light beam amount adjusting means adjusts the light beam amount of the excitation light emitter based on a difference between when the excitation light emitter is turned on and when it is not turned on, which is measured by a fluorescence detector. Fine particle detector. 蛍光検出器にて測定した蛍光光束量に基づいて、励起光発光器のON、OFF時間比率を調整するパルス幅調整手段を備えたことを特徴とする請求項1、2又は3記載の微粒子検出装置。   4. The particle detection according to claim 1, further comprising pulse width adjusting means for adjusting an ON / OFF time ratio of the excitation light emitter based on the amount of fluorescent light flux measured by the fluorescence detector. apparatus. パルス幅調整手段が、蛍光検出器にて測定した励起光発光器の点灯時と不点灯時の差に基づいて、励起光発光器のON、OFF時間比率を調整することを特徴とする請求項4記載の微粒子検出装置。   The pulse width adjusting means adjusts the ON / OFF time ratio of the excitation light emitter based on a difference between when the excitation light emitter is turned on and when not turned on, which is measured by a fluorescence detector. 4. The fine particle detection apparatus according to 4. 励起光発光器における励起光源として、間欠的に発光するLEDを使用することを特徴とする請求項1、2、3、4又は5記載の微粒子検出装置。   6. The particulate detection device according to claim 1, wherein an LED that emits light intermittently is used as an excitation light source in the excitation light emitter. 微粒子が微生物であることを特徴とする請求項1、2、3、4、5又は6記載の微粒子検出装置。   The fine particle detection apparatus according to claim 1, 2, 3, 4, 5, or 6, wherein the fine particles are microorganisms. 微生物がレジオネラ属菌等の人体に有害な微生物であることを特徴とする請求項7記載の微粒子検出装置。   The microparticle detection apparatus according to claim 7, wherein the microbe is a microbe harmful to the human body such as Legionella spp.
JP2006275462A 2006-10-06 2006-10-06 Fine particle detector Pending JP2008096154A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2006275462A JP2008096154A (en) 2006-10-06 2006-10-06 Fine particle detector

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2006275462A JP2008096154A (en) 2006-10-06 2006-10-06 Fine particle detector

Publications (1)

Publication Number Publication Date
JP2008096154A true JP2008096154A (en) 2008-04-24

Family

ID=39379163

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2006275462A Pending JP2008096154A (en) 2006-10-06 2006-10-06 Fine particle detector

Country Status (1)

Country Link
JP (1) JP2008096154A (en)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012230000A (en) * 2011-04-26 2012-11-22 Sumika Chemical Analysis Service Ltd Method for inspecting fine particle
CN106770079A (en) * 2016-11-17 2017-05-31 无锡艾科瑞思产品设计与研究有限公司 A kind of fluorescent microorganism detector based on light emitting diode
CN112119339A (en) * 2018-04-23 2020-12-22 生德奈股份有限公司 Method for examining a liquid containing at least one cell and/or at least one particle
WO2021070218A1 (en) * 2019-10-07 2021-04-15 ナノティス株式会社 Method for detecting substance to be detected in sample

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2012230000A (en) * 2011-04-26 2012-11-22 Sumika Chemical Analysis Service Ltd Method for inspecting fine particle
CN106770079A (en) * 2016-11-17 2017-05-31 无锡艾科瑞思产品设计与研究有限公司 A kind of fluorescent microorganism detector based on light emitting diode
CN112119339A (en) * 2018-04-23 2020-12-22 生德奈股份有限公司 Method for examining a liquid containing at least one cell and/or at least one particle
WO2021070218A1 (en) * 2019-10-07 2021-04-15 ナノティス株式会社 Method for detecting substance to be detected in sample

Similar Documents

Publication Publication Date Title
St-Pierre et al. High-fidelity optical reporting of neuronal electrical activity with an ultrafast fluorescent voltage sensor
ES2659535T3 (en) Device and method for the determination and monitoring of water toxicity
US7307721B2 (en) Particle imaging system with a varying flow rate
EP2724160A2 (en) Acoustic cytometry methods and protocols
EP1478912A2 (en) Methods and apparatus for assays of bacterial spores
TWI622650B (en) Method of inspecting microorganisms
Werner et al. High speed and high resolution chemical imaging based on a new type of OLED-LAPS set-up
JP2008096154A (en) Fine particle detector
JP2008032440A (en) Device and method for measuring emission life
Strack et al. Individual mitochondrion characterization: a comparison of classical assays to capillary electrophoresis with laser-induced fluorescence detection
Skilitsi et al. Towards sensitive, high-throughput, biomolecular assays based on fluorescence lifetime
JP2004533853A (en) Apparatus and method for detecting photosynthesis inhibition
Li et al. Shifts in the fluorescence lifetime of EGFP during bacterial phagocytosis measured by phase-sensitive flow cytometry
JP2008096155A (en) Fine particle detector
KR20150050265A (en) bio-detecting apparatus and method of bio-detecting microoranisms using the same
WO2022185592A1 (en) Method for measuring viral particle, and device for measuring viral particle
Horká et al. Dynamic modification of microorganisms by pyrenebutanoate for fluorometric detection in capillary zone electrophoresis
Lacoste et al. Live-cell migration and adhesion turnover assays
Wang et al. Study of antagonism between some intestinal bacteria with high‐speed micellar electrokinetic chromatography
JP2008096157A (en) Fine particle detector
WO2021070884A1 (en) Method for detecting substance to be detected within sample
JP4990791B2 (en) Method and apparatus for analyzing dynamic samples
CN110907411A (en) Steady-state luminous magnetic field effect tester
JP2008096156A (en) Fine particle detector
US20210172002A1 (en) Detecting cell vitality