JP2022509803A - Single microalgae cell activity detection device and method based on multi-level light intensity stimulation - Google Patents
Single microalgae cell activity detection device and method based on multi-level light intensity stimulation Download PDFInfo
- Publication number
- JP2022509803A JP2022509803A JP2021528970A JP2021528970A JP2022509803A JP 2022509803 A JP2022509803 A JP 2022509803A JP 2021528970 A JP2021528970 A JP 2021528970A JP 2021528970 A JP2021528970 A JP 2021528970A JP 2022509803 A JP2022509803 A JP 2022509803A
- Authority
- JP
- Japan
- Prior art keywords
- single microalgae
- module
- fluorescence
- microalgae cell
- sample solution
- 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.)
- Granted
Links
- 238000001514 detection method Methods 0.000 title claims abstract description 73
- 230000000694 effects Effects 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000000638 stimulation Effects 0.000 title claims abstract description 17
- 239000012488 sample solution Substances 0.000 claims abstract description 47
- 238000012545 processing Methods 0.000 claims abstract description 37
- 239000011247 coating layer Substances 0.000 claims abstract description 17
- 238000004519 manufacturing process Methods 0.000 claims abstract description 15
- 239000000758 substrate Substances 0.000 claims abstract description 14
- 239000000523 sample Substances 0.000 claims description 13
- 230000008569 process Effects 0.000 claims description 9
- 206010011953 Decreased activity Diseases 0.000 claims description 4
- 238000004458 analytical method Methods 0.000 claims description 4
- 230000005540 biological transmission Effects 0.000 claims description 4
- 239000004205 dimethyl polysiloxane Substances 0.000 claims description 4
- 239000011521 glass Substances 0.000 claims description 4
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims description 4
- -1 polydimethylsiloxane Polymers 0.000 claims description 4
- 239000003381 stabilizer Substances 0.000 claims description 4
- 238000001914 filtration Methods 0.000 claims description 2
- 239000012530 fluid Substances 0.000 claims description 2
- 230000001678 irradiating effect Effects 0.000 claims description 2
- 230000004936 stimulating effect Effects 0.000 claims description 2
- 238000010586 diagram Methods 0.000 abstract description 5
- 238000007796 conventional method Methods 0.000 abstract description 4
- 230000004300 dark adaptation Effects 0.000 abstract description 3
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 230000004913 activation Effects 0.000 description 2
- 238000007405 data analysis Methods 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 241000195493 Cryptophyta Species 0.000 description 1
- 238000009360 aquaculture Methods 0.000 description 1
- 244000144974 aquaculture Species 0.000 description 1
- 230000000712 assembly Effects 0.000 description 1
- 238000000429 assembly Methods 0.000 description 1
- 238000013475 authorization Methods 0.000 description 1
- 230000014155 detection of activity Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000013535 sea water Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6486—Measuring fluorescence of biological material, e.g. DNA, RNA, cells
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N2035/00178—Special arrangements of analysers
- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
本発明は、光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールを含む、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置及び方法を提供する。マイクロ流体チップは単一微細藻類細胞の活性を検出するマイクロプラットフォームとして、基板と基板に固定されたコーティング層とからなる。チップには、試料溶液供給孔、通路、検出領域、及び試料溶液排出孔が凹んで形成され、本発明において全てのモジュールが７９ｃｍ×４９ｃｍ×４３ｃｍの直方体装置に集積されている。光源モジュールによって励起する検出光の光強度は暗順応の蛍光生成量を監視するための非常に弱いレベルにする必要がなく、また、最大蛍光生成量を評価するための飽和レベルにする必要がなく、検出可能な微細藻類細胞は種類、個体形態、サイズ、細胞構造に制限されない。本発明の技術的手段は従来の微細藻類細胞活性検出方法において単一微細藻類細胞の活性を定量評価できないことに起因した一般性や誤差等における問題点を解決した。
【選択図】図１
The present invention provides a single microalgae cell activity detection device and method based on multi-level light intensity stimulation, including a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and a power supply module. The microfluidic chip consists of a substrate and a coating layer fixed to the substrate as a microplatform for detecting the activity of a single microalgae cell. The chip is formed with a sample solution supply hole, a passage, a detection area, and a sample solution discharge hole recessed, and all the modules in the present invention are integrated in a 79 cm × 49 cm × 43 cm rectangular parallelepiped device. The light intensity of the detected light excited by the light source module does not need to be at a very weak level for monitoring dark adaptation fluorescence production, nor does it need to be at a saturation level for assessing maximum fluorescence production. , Detectable microalgae cells are not limited by type, individual morphology, size, or cell structure. The technical means of the present invention has solved problems in generality and error caused by the inability to quantitatively evaluate the activity of a single microalgae cell in the conventional method for detecting microalgae cell activity.
[Selection diagram] Fig. 1
Description
本発明は、単一微細藻類細胞の活性を検出する技術分野に関し、具体的に言えば、特にマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置及び方法に関する。 The present invention relates to a technical field for detecting the activity of a single microalgae cell, specifically, to a device and a method for detecting the activity of a single microalgae cell based on a multi-level light intensity stimulus.
中国の海岸線は３．２万キロメートルと長く、主権管轄海域面積が４７３万平方キロメートルに至り、５つの気候帯に跨がり、生態系種類が多い。このような自然特徴のため、中国は海洋生物の侵入に遭う可能性が高い。中国の海上輸送産業や海水養殖産業が興るにつれて、侵入生物が多くなってきている情勢に直面している。その中で、赤潮を引き起こすリスクのある十数種の藻類が船舶バラスト水により持ち込まれたことがあるが、赤潮が引き起こされると、地元の海洋生態系の構造や機能が殆ど完全に崩壊することになり、海域の元の生物群集や生態系の安定性をひどく脅かしてしまう。 China's coastline is as long as 32,000 kilometers, and the area of sovereign jurisdiction reaches 4.73 million square kilometers, straddling five climatic zones, and there are many types of ecosystems. Due to these natural characteristics, China is likely to be invaded by marine life. With the rise of China's sea shipping industry and seawater aquaculture industry, we are facing a situation where the number of invading organisms is increasing. Among them, more than a dozen species of algae at risk of causing red tide have been brought in by ship ballast water, but when red tide is caused, the structure and function of the local marine ecosystem is almost completely destroyed. And seriously threatens the stability of the original marine communities and ecosystems.
従来の微細藻類細胞活性検出装置では、活性検出は１つの微細藻類群集にしか実施できない。しかしながら、危害性が高い微細藻類群集が低い活性状態を示す場合でも、従来の検出方法では、高い活性状態を有する単一微細藻類細胞の存在を排除できず、有害微細藻類の侵入を防ぐことに不利である。なお、従来の方式によって単一の微細藻類の活性を検出するには、現場で試料を採取し実験室で分析するという通常の検出プロセスを採用することを必要とされるが、実際の応用では水中の微細藻類を現場でリアルタイムでオンラインで連続して検出する必要がある。また、実験室で使用される機器は大型で、高価で、操作が複雑で、操作に専門知識を持つ者が必要とされ、集積して便利に携帯することができないので、現場での検出が不可能である。 With conventional microalgae cell activity detection devices, activity detection can only be performed on one microalgae community. However, even when a highly harmful microalgae community shows a low active state, the conventional detection method cannot eliminate the presence of a single microalgae cell having a high active state, and prevents the invasion of harmful microalgae. It is disadvantageous. In order to detect the activity of a single microalgae by the conventional method, it is necessary to adopt a normal detection process of collecting a sample in the field and analyzing it in the laboratory, but in actual application. It is necessary to continuously detect microalgae in water in real time online. In addition, the equipment used in the laboratory is large, expensive, complicated to operate, requires specialized knowledge in operation, and cannot be integrated and conveniently carried, so it can be detected in the field. It is impossible.
本発明は、従来の微細藻類細胞活性検出方法における、単一微細藻類細胞の活性を定量評価できず、現場での検出が不可能で、検出機器が高価で、試料処理が煩わしく、試料使用量が多い等のことによる一般性や誤差に関する上記技術的問題点に鑑みて、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置及び方法を提供する。本発明により、単一微細藻類細胞の活性検出における問題点を根本的に解決でき、環境科学分野に対し重要な科学的意義と現実的な価値を有する。 INDUSTRIAL APPLICABILITY The present invention cannot quantitatively evaluate the activity of a single microalgae cell in the conventional method for detecting microalgae cell activity, cannot detect it in the field, the detection device is expensive, the sample processing is troublesome, and the amount of sample used. In view of the above technical problems regarding generality and error due to the large number of cells, etc., an apparatus and method for detecting the activity of single microalgae cells based on multi-level light intensity stimulation are provided. INDUSTRIAL APPLICABILITY According to the present invention, problems in detecting the activity of single microalgae cells can be fundamentally solved, and the present invention has important scientific significance and practical value in the field of environmental science.
本発明の技術的手段は以下の通りである。 The technical means of the present invention are as follows.
本発明の一態様は、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置であって、光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールを含み、電源モジュールは、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールの入力端にそれぞれ接続され、マイクロ流体チップは、光源モジュールの出力端と蛍光採集モジュールの入力端とにそれぞれ接続され、データ処理モジュールは、蛍光採集モジュールの出力端に接続され、光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールは、長さ７９ｍｍ×幅４９ｍｍ×高さ４３ｍｍの直方体装置に集積され、
マイクロ流体チップは、基板と基板に固定されたコーティング層とを含み、コーティング層には、試料溶液供給孔、通路Ｉ、検出領域Ｉ、通路ＩＩ、検出領域ＩＩ、通路ＩＩＩ、及び試料溶液排出孔が凹んで形成され、使用する際に、試料溶液供給孔に単一微細藻類細胞を含有する試料溶液を滴下し、マイクロポンプの駆動により、試料溶液中の単一微細藻類細胞は所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入し、
光源モジュールは、電圧安定回路、光源固定構造、及び光透過孔アセンブリに緊密に貼り合わせられるレーザー発生装置Ｉとレーザー発生装置ＩＩとを含み、
蛍光採集モジュールは、スリットシート、赤色光フィルター、及び光電センサーを含む。
One aspect of the present invention is a single microalgae cell activity detection device based on multi-level light intensity stimulation, which includes a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and a power supply module, and is a power supply module. Is connected to the input ends of the light source module, fluorescence collection module, and data processing module, respectively, the microfluidic chip is connected to the output end of the light source module and the input end of the fluorescence collection module, respectively, and the data processing module is fluorescent. Connected to the output end of the collection module, the light source module, microfluidic chip, fluorescent collection module, data processing module, and power supply module are integrated into a square device measuring 79 mm long x 49 mm wide x 43 mm high.
The microfluidic chip comprises a substrate and a coating layer fixed to the substrate, in which the coating layer includes a sample solution supply hole, a passage I, a detection region I, a passage II, a detection region II, a passage III, and a sample solution discharge hole. Is formed as a dent, and when used, a sample solution containing a single microalgae cell is dropped into the sample solution supply hole, and by driving a micropump, the single microalgae cell in the sample solution is at a predetermined flow rate. In turn, it flows into the detection area I along the passage I, flows into the detection area II through the extremely short passage II, and finally flows into the sample solution discharge hole through the passage III.
The light source module includes a voltage stabilizer, a light source fixed structure, and a laser generator I and a laser generator II that are tightly coupled to the light transmission hole assembly.
The fluorescence collection module includes a slit sheet, a red light filter, and a photoelectric sensor.
さらに、通路ＩＩの長さは既知であり、単一微細藻類細胞が検出領域Ｉに現れた時、単一微細藻類細胞が検出領域ＩＩに現れる時点を推定できる。 Furthermore, the length of the passage II is known, and when a single microalgae cell appears in the detection area I, the time point at which the single microalgae cell appears in the detection area II can be estimated.
さらに、レーザー発生装置Ｉはマイクロ流体チップ中の検出領域Ｉに対向して比較的弱い青色光を発し、レーザー発生装置ＩＩはマイクロ流体チップ中の検出領域ＩＩに対向して比較的強い青色光を発する。 Further, the laser generator I emits relatively weak blue light toward the detection region I in the microfluidic chip, and the laser generator II emits relatively strong blue light toward the detection region II in the microfluidic chip. Emit.
さらに、基板はガラス板であり、コーティング層はポリジメチルシロキサンコーティング層である。 Further, the substrate is a glass plate and the coating layer is a polydimethylsiloxane coating layer.
本発明の他の一態様は、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法であって、
試料の滴下：単一微細藻類細胞を含有する試料溶液をマイクロ流体チップの試料溶液供給孔に滴下する工程１と、
装置の起動：順に電源モジュール、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールをオンにし、マイクロポンプの駆動により、試料溶液中の単一微細藻類細胞が所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入する工程２と、
マルチレベルの光強度刺激による単一微細藻類細胞の蛍光生成：光源モジュール中のレーザー発生装置Ｉが比較的弱い青色レーザーを発してマイクロ流体チップ中の検出領域Ｉに照射し、レーザー発生装置ＩＩが比較的強い青色レーザーを発してマイクロ流体チップ中の検出領域ＩＩに照射し、試料溶液中の単一微細藻類細胞が先後して検出領域Ｉと検出領域ＩＩを通る時、刺激されて異なる生成量の蛍光を生成する工程３と、
単一微細藻類細胞の蛍光生成量の採集：単一微細藻類細胞が先後して生成した異なる強度の蛍光が、それぞれ、蛍光採集モジュール中のスリットシート、赤色光フィルターを通ることにより迷光をフィルタアウトした後、光電センサーに採集される工程４と、
以上で採集した単一微細藻類細胞の蛍光生成量に対するデータ分析：採集した単一微細藻類細胞の蛍光生成量をデータ線によってデータ処理モジュールに伝送して処理分析を行う工程５と、
単一微細藻類細胞に対する活性評価：単一微細藻類細胞が光源モジュール中のレーザー発生装置Ｉが発した比較的弱い青色レーザーに刺激されて生成した蛍光生成量をＦｗで示し、単一微細藻類細胞が光源モジュール中のレーザー発生装置ＩＩが発した比較的強い青色レーザーに刺激されて生成した蛍光生成量をＦｓで示すと、データ処理モジュールは、式Ｆｒ＝（Ｆｓ－Ｆｗ）／Ｆｓによって有効蛍光生成量Ｆｒ得、Ｆｒに基づいて単一微細藻類細胞の活性を判断する工程６とを、
備える。
Another aspect of the present invention is a method for detecting the activity of a single microalgae cell based on multi-level light intensity stimulation.
Dropping the sample: Step 1 of dropping the sample solution containing a single microalgae cell into the sample solution supply hole of the microfluidic chip, and
Device activation: Turn on the power supply module, light source module, fluorescence collection module, and data processing module in order, and by driving the micropump, single microalga cells in the sample solution are detected along the passage I in order at a predetermined flow rate.
Fluorescence generation of single microalgae cells by multi-level light intensity stimulation: Laser generator I in the light source module emits a relatively weak blue laser to irradiate the detection area I in the microfluidic chip, and laser generator II When a relatively strong blue laser is emitted to irradiate the detection area II in the microfluidic chip and the single microalgae cells in the sample solution pass through the detection area I and the detection area II, they are stimulated to produce different amounts. Step 3 to generate the fluorescence of
Collection of fluorescence production of single microalgae cells: Different intensities of fluorescence generated earlier by single microalgae cells filter out stray light by passing through a slit sheet and a red light filter in the fluorescence collection module, respectively. After that,
Data analysis for the amount of fluorescence produced by the collected single microalgae cells: Step 5 of transmitting the amount of fluorescence produced by the collected single microalgae cells to the data processing module via a data line for processing analysis.
Evaluation of activity against single microalgae cells: The amount of fluorescence generated by single microalgae cells stimulated by a relatively weak blue laser emitted by the laser generator I in the light source module is indicated by Fw, and the single microalgae are shown. When the amount of fluorescence generated by the cells stimulated by the relatively strong blue laser emitted by the laser generator II in the light source module is indicated by F s , the data processing module has the formula F r = (F s − F w ).
Be prepared.
さらに、前記工程６において有効蛍光生成量Ｆｒに基づいて単一微細藻類細胞の活性を判断するプロセスは、
Ｆｒ＞０．６である場合、当該単一微細藻類細胞が高活性状態にあることを示す工程６１と、
０．３≦Ｆｒ≦０．６である場合、当該単一微細藻類細胞が低活性状態にあるか、又はこの単一微細藻類細胞がある程度で損傷を受けたが、致命的ではない状態にあるかを示す工程６２と、
Ｆｒ＜０．３である場合、当該単一微細藻類細胞が既に死亡したか、又は死亡に瀕しているかを示す工程６３とを、備える。
Further, in
When F r > 0.6, step 61 indicating that the single microalgae cell is in a highly active state, and
If 0.3 ≤ Fr ≤ 0.6, the single microalgae cell is in a hypoactive state, or the single microalgae cell is damaged to some extent but is not fatal. Step 62 to show if there is,
When F r <0.3, a step 63 indicating whether the single microalgae cell has already died or is on the verge of death is provided.
従来技術と比べると、本発明は以下のメリットを有する。
１．本発明は、従来の微細藻類細胞活性検出方法において単一微細藻類細胞の活性を定量評価できず、現場で検出できず、検出機器が高価で、試料処理が煩わしく、試料使用量が多いことによってもたらされる一般性や誤差等の問題を解決し、単一微細藻類細胞の活性検出問題を根本的に解決しており、環境科学分野に対し重要な科学的意義と現実的な価値を有する。
２．本発明は、単一微細藻類細胞の活性を検出するマイクロプラットフォームとしてマイクロ流体チップを採用し、関連モジュールに対して小体積の構造形態を採用してもよく、全てのモジュールを７９ｃｍ×４９ｃｍ×４３ｃｍの直方体装置に集積している。従来の大型検出機器と比べると、本発明は、小型で、軽量で、コストが低く、試料使用量が少なく、携帯しやすく、手持ち携帯型として現場検出に用いることができる等のメリットを有する。
３．本発明は、試料槽に試料を加えた後、後続の工程は全てインテリジェント化が完成しているので、いかなる専門知識も要らず、専門知識を持つ者の操作が要らず、工程が少なく、操作が簡単であり、試料処理が煩わしいという問題を解決した。
４．本発明に記載の光源モジュールによって励起する検出光の光強度は、暗順応の蛍光生成量を監視するための非常に弱いレベルにする必要がなく、また、検出光の光強度は、最大蛍光生成量を評価するための飽和レベルにする必要がない。技術上の難度を低減すると共に、異なる微細藻類細胞の暗順応レベルが異なることに制限されないため、適用範囲がより広くなり、検出可能な微細藻類細胞は種類、個体形態、サイズ、細胞構造に制限されない。
The present invention has the following merits as compared with the prior art.
1. 1. According to the present invention, the activity of a single microalgae cell cannot be quantitatively evaluated by the conventional method for detecting the activity of microalgae cells, cannot be detected in the field, the detection device is expensive, the sample processing is troublesome, and the amount of sample used is large. It solves problems such as generality and error that are brought about, and fundamentally solves the problem of detecting the activity of single microalgae cells, and has important scientific significance and practical value in the field of environmental science.
2. 2. The present invention employs a microfluidic chip as a microplatform for detecting the activity of single microalgae cells, may adopt a small volume structural form for related modules, and all modules are 79 cm x 49 cm x 43 cm. It is integrated in the rectangular parallelepiped device of. Compared with the conventional large-sized detection device, the present invention has advantages such as small size, light weight, low cost, low sample usage, easy portability, and can be used for on-site detection as a handheld portable type.
3. 3. In the present invention, after the sample is added to the sample tank, all subsequent processes have been made intelligent, so that no specialized knowledge is required, no operation by a person with specialized knowledge is required, there are few processes, and operations are performed. It solves the problem that it is easy and the sample processing is troublesome.
4. The light intensity of the detection light excited by the light source module according to the present invention does not need to be at a very weak level for monitoring the amount of fluorescence generation of dark adaptation, and the light intensity of the detection light is the maximum fluorescence generation. There is no need for a saturation level to evaluate the quantity. It has a wider range of applications because it reduces technical difficulty and is not limited to different levels of dark adaptation of different microalgae cells, limiting detectable microalgae cells by type, individual morphology, size, and cell structure. Not done.
上記理由から、本発明は単一微細藻類細胞の活性検出等の分野に広範に普及可能である。 For the above reasons, the present invention can be widely used in fields such as detection of activity of single microalgae cells.
本発明の実施例又は従来技術における技術的手段をより明確に説明するために、以下、実施例又は従来技術の記述に使用する必要がある図面を簡単に紹介するが、下記説明に用いられる図面は本発明の一部の実施例であり、当業者であれば、創造的労動を行わずにこれらの図面に基づいて他の図面を得るのができることはいうまでもない。 In order to more clearly explain the technical means in the embodiment or the prior art of the present invention, the drawings which need to be used for the description of the embodiment or the prior art will be briefly introduced below, but the drawings used in the following description will be briefly introduced. Is an embodiment of the present invention, and it goes without saying that those skilled in the art can obtain other drawings based on these drawings without any creative effort.
相互に違反しない限り、本発明における実施例及び実施例における特徴を相互に組み合わせることができるのを説明する必要がある。以下、図面を参照して実施例に基づいて本発明を詳細に説明する。 It is necessary to explain that the examples in the present invention and the features in the examples can be combined with each other as long as they do not violate each other. Hereinafter, the present invention will be described in detail with reference to the drawings.
本発明の実施例の目的、技術手段及びメリットをより明らかにするために、以下、本発明の実施例における図面を参照しながら、本発明の実施例における技術手段を明らかで完全に説明するが、説明される実施例が全ての実施例ではなく、本発明の一部の実施例に過ぎないことはいうまでもない。以下の少なくとも１つの例示的実施例に対する説明は実際に説明するためのものに過ぎなく、決して本発明及びその応用や使用に何の制限も加えない。当業者が本発明における実施例に基づいて、創造的労動を行うことなく得た他の実施例は、全て本発明が保護する範囲に含まれるものとする。 In order to further clarify the purpose, technical means and merits of the examples of the present invention, the technical means in the examples of the present invention will be clearly and completely described below with reference to the drawings in the examples of the present invention. It goes without saying that the examples described are not all examples, but only some examples of the present invention. The description for at least one exemplary embodiment below is for practical purposes only and does not impose any restrictions on the invention and its applications or uses. All other examples obtained by those skilled in the art based on the examples in the present invention without any creative effort shall be included in the scope of protection of the present invention.
本明細書で使用される技術用語は具体的な実施形態を記述するためのものに過ぎず、本発明による例示的実施形態を限定する意図がないことに注意されたい。例えば、文脈に別に明記しない限り、本明細書で使用される単数形態は複数形態も含む意図があり、なお、本明細書で技術用語の「含有する」及び／又は「含む」を使用する時に、特徴、工程、操作、デバイス、アセンブリ及び／又はそれらの組合せが存在するのを示すことも理解されたい。 It should be noted that the technical terms used herein are merely for describing specific embodiments and are not intended to limit the exemplary embodiments according to the invention. For example, unless otherwise specified in the context, the singular form used herein is intended to include multiple forms, and when the technical terms "contains" and / or "contains" are used herein. It should also be appreciated that there are features, processes, operations, devices, assemblies and / or combinations thereof.
別に具体的に説明しない限り、これらの実施例に記載の部材と工程の相対的配置、数式及び数値は本発明の範囲を限定するものではない。また、説明の便宜上、図面に示す各部分のサイズは実際の比例関係に基づいて描かれたものではないことを理解すべきである。当業者に既知の技術、方法及びデバイスについての詳細な検討を省略する場合があるが、適切な場合に、前記技術、方法及びデバイスを授権明細書の一部と見なすべきである。本明細書に示し、及び検討する全ての例において、全ての具体的な値は限定するものではなく、例示的なものと解釈すべきである。従って、例示的実施例の他の例は異なる値を有してもよい。類似する符号と文字は下記の図面において類似項目を示すため、ある項目が１つの図面で定義されると、その以降の図面でそれについて更に検討する必要がないことに注意されたい。 Unless specifically described separately, the relative arrangements, mathematical formulas and numerical values of the members and processes described in these examples do not limit the scope of the present invention. Also, for convenience of explanation, it should be understood that the size of each part shown in the drawings is not drawn based on the actual proportional relationship. Detailed discussion of techniques, methods and devices known to those of skill in the art may be omitted, but where appropriate, said techniques, methods and devices should be considered as part of the authorization specification. In all examples presented and considered herein, all specific values are not limited and should be construed as exemplary. Therefore, other examples of the exemplary embodiment may have different values. Note that similar signs and letters indicate similar items in the drawings below, so once an item is defined in one drawing, it does not need to be considered further in subsequent drawings.
本発明の記述では、「前、後、上、下、左、右」、「横方向、縱方向、垂直、水平」及び「頂、底」等の方位詞で示す方位又は位置関係は一般に図面に基づいて示す方位又は位置関係であり、その目的はただ本発明を容易に記述、及び記述を簡単化するためであり、反対に説明しない限り、これらの方位詞は示される装置又は素子が必ず特定の方位を有し又は特定の方位で構成され操作されることを明示も暗示もしないので、本発明の保護範囲を限定するものと理解してはならず、方位詞の「内、外」は各部材自身の輪郭に対する内外を指すことを理解されたい。 In the description of the present invention, the orientation or positional relationship indicated by the directional words such as "front, rear, top, bottom, left, right", "horizontal, horizontal, vertical, horizontal" and "top, bottom" is generally a drawing. It is an orientation or positional relationship shown based on the above, the purpose of which is merely to facilitate the description of the present invention and to simplify the description, and unless otherwise explained, these orientations are always expressed by the device or element shown. It should not be understood as limiting the scope of protection of the present invention, as it does not express or imply that it has a particular orientation or is composed and manipulated in a particular orientation, and the directional terms "inside, outside". It should be understood that refers to the inside and outside of the contour of each member itself.
説明の便宜上、本明細書では、例えば図に示すデバイス又は特徴と他のデバイス又は特徴との空間位置関係を記述するために、例えば、「…の上」、「…の上方」、「…の上面」、「上の」等の空間相対技術用語を用いてよい。空間相対技術用語は、図に記載の方位以外、デバイスの使用又は操作での異なる方位をも含むのを意図することを理解すべきである。例えば、図面におけるデバイスを反対に配置した場合に、「他のデバイス又は構造の上方」又は「他のデバイス又は構造の上」と記述したデバイスはその後で「他のデバイス又は構造の下方」又は「他のデバイス又は構造の下」と位置決めされる。従って、例示的技術用語の「…の上方」は「…の上方」及び「…の下方」といった２種の方位を含むことが可能である。このデバイスを他の異なる方式で位置決めすることもでき（９０度回転させ又は他の方位に位置させる）、そしてここで使用する空間相対記載を対応的に解釈する。 For convenience of explanation, in the present specification, for example, in order to describe the spatial positional relationship between the device or feature shown in the figure and another device or feature, for example, "above ...", "above ...", "..." Spatial relative technical terms such as "top" and "top" may be used. It should be understood that spatial relative technical terminology is intended to include different orientations in the use or operation of the device, in addition to the orientations shown in the figure. For example, when the devices in the drawings are placed in reverse, a device described as "above another device or structure" or "above another device or structure" is subsequently described as "below another device or structure" or "above another device or structure". Positioned "under other device or structure". Therefore, the exemplary technical term "above ..." can include two orientations, such as "above ..." and "below ...". The device can also be positioned in other different ways (rotated 90 degrees or positioned in other orientations), and the spatial relative description used herein is interpreted correspondingly.
なお、「第１」、「第２」等の用語を用いて部品を限定する目的は、ただ対応する部品を区別しやすくすることにあり、特に断らない限り、上記用語は特別な意味がないため、本発明の保護範囲を限定するものと理解してはならないことを説明すべきである。 The purpose of limiting parts by using terms such as "first" and "second" is simply to make it easier to distinguish the corresponding parts, and the above terms have no special meaning unless otherwise specified. Therefore, it should be explained that it should not be understood as limiting the scope of protection of the present invention.
（実施例１）
図１に示すように、本発明の一実施形態は、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置であって、光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールを含み、電源モジュールは、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールの入力端にそれぞれ接続され、マイクロ流体チップは、光源モジュールの出力端と蛍光採集モジュールの入力端とにそれぞれ接続され、データ処理モジュールは蛍光採集モジュールの出力端に接続され、光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールは、長さ７９ｍｍ×幅４９ｍｍ×高さ４３ｍｍの直方体装置に集積される。
(Example 1)
As shown in FIG. 1, one embodiment of the present invention is a single microalgae cell activity detection device based on multi-level light intensity stimulation, which comprises a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and the like. And the power supply module, the power supply module is connected to the input end of the light source module, the fluorescence collection module, and the data processing module, respectively, and the microfluidic chip is connected to the output end of the light source module and the input end of the fluorescence collection module, respectively. The data processing module is connected to the output end of the fluorescent collection module, and the light source module, microfluidic chip, fluorescent collection module, data processing module, and power supply module are connected to a rectangular device measuring 79 mm in length × 49 mm in width × 43 mm in height. Accumulate.
図２に示すように、マイクロ流体チップは、基板と基板に固定されたコーティング層とを含み、基板はガラス板であり、コーティング層はポリジメチルシロキサンコーティング層であり、コーティング層には、試料溶液供給孔、通路Ｉ、検出領域Ｉ、通路ＩＩ、検出領域ＩＩ、通路ＩＩＩ、及び試料溶液排出孔が凹んで形成されており、使用する際に、試料溶液供給孔に試料溶液を滴下し、マイクロポンプの駆動により、試料溶液中の単一微細藻類細胞が所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入する。通路ＩＩの長さは既知であり、単一微細藻類細胞が検出領域Ｉに現れた時、単一微細藻類細胞が検出領域ＩＩに現れる時点を推定できる。光源モジュールは、電圧安定回路、光源固定構造、及び光透過孔アセンブリに緊密に貼り合わせられるレーザー発生装置Ｉとレーザー発生装置ＩＩとを含み、レーザー発生装置Ｉはマイクロ流体チップ中の検出領域Ｉに対向して比較的弱い青色光を発し、レーザー発生装置ＩＩはマイクロ流体チップ中の検出領域ＩＩに対向して比較的強い青色光を発する。蛍光採集モジュールはスリットシート、赤色光フィルター及び光電センサーを含む。 As shown in FIG. 2, the microfluidic chip includes a substrate and a coating layer fixed to the substrate, the substrate is a glass plate, the coating layer is a polydimethylsiloxane coating layer, and the coating layer is a sample solution. The supply hole, passage I, detection area I, passage II, detection area II, passage III, and sample solution discharge hole are recessed, and when used, the sample solution is dropped into the sample solution supply hole to make a micro. By driving the pump, single microalgae cells in the sample solution flow into the detection region I in sequence along the passage I at a predetermined flow velocity, flow into the detection region II through the extremely short passage II, and finally in the passage III. It flows into the sample solution discharge hole through the sample solution. The length of the passage II is known, and when a single microalgae cell appears in the detection area I, the time point at which the single microalgae cell appears in the detection area II can be estimated. The light source module includes a voltage stabilizer, a light source fixation structure, and a laser generator I and a laser generator II that are tightly coupled to the light transmission hole assembly, the laser generator I in the detection area I in the microfluidic chip. The laser generator II emits a relatively strong blue light facing the detection region II in the microfluidic chip. The fluorescence collection module includes a slit sheet, a red light filter and a photoelectric sensor.
（実施例２）
実施例１を基に、更に、マルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法を提供する。該検出方法は、
試料の滴下：単一微細藻類細胞を含有する試料溶液をマイクロ流体チップの試料溶液供給孔に滴下する工程１と、
装置の起動：順に電源モジュール、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールをオンにし、マイクロポンプの駆動により、試料溶液中の単一微細藻類細胞が所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入する工程２と、
マルチレベルの光強度刺激による単一微細藻類細胞の蛍光生成：光源モジュール中のレーザー発生装置Ｉが比較的弱い青色レーザーを発してマイクロ流体チップ中の検出領域Ｉに照射し、レーザー発生装置ＩＩが比較的強い青色レーザーを発してマイクロ流体チップ中の検出領域ＩＩに照射し、試料溶液中の単一微細藻類細胞が先後して検出領域Ｉと検出領域ＩＩを通る時、刺激されて異なる生成量の蛍光を生成する工程３と、
単一微細藻類細胞の蛍光生成量の採集：単一微細藻類細胞が先後して生成した異なる強度の蛍光は、それぞれ、蛍光採集モジュール中のスリットシート、赤色光フィルターを通ることにより迷光をフィルタアウトした後、光電センサーに採集される工程４と、
以上で採集した単一微細藻類細胞の蛍光生成量に対するデータ分析：採集した単一微細藻類細胞の蛍光生成量をデータ線によってデータ処理モジュールに伝送して処理分析を行う工程５と、
単一微細藻類細胞に対する活性評価：単一微細藻類細胞が光源モジュール中のレーザー発生装置Ｉが発した比較的弱い青色レーザーに刺激されて生成した蛍光生成量をＦｗで示し、単一微細藻類細胞が光源モジュール中のレーザー発生装置ＩＩが発した比較的強い青色レーザーに刺激されて生成した蛍光生成量をＦｓで示すと、データ処理モジュールは、式Ｆｒ＝（Ｆｓ－Ｆｗ）／Ｆｓによって有効蛍光生成量Ｆｒを得、有効蛍光生成量Ｆｒに基づいて単一微細藻類細胞の活性を判断する工程６と、を備え、
さらに、工程６において有効蛍光生成量Ｆｒに基づいて単一微細藻類細胞の活性を判断するプロセスは、
Ｆｒ＞０．６である場合、当該単一微細藻類細胞が高活性状態にあることを示す工程６１と、
０．３≦Ｆｒ≦０．６である場合、当該単一微細藻類細胞が低活性状態にあるか、又はこの単一微細藻類細胞がある程度で損傷を受けたが、致命的ではない状態にあるかを示す工程６２と、
Ｆｒ＜０．３である場合、当該単一微細藻類細胞が既に死亡したか、又は死亡に瀕しているかを示す工程６３と、を備える。
(Example 2)
Based on Example 1, a method for detecting the activity of single microalgae cells based on multi-level light intensity stimulation is further provided. The detection method is
Dropping the sample: Step 1 of dropping the sample solution containing a single microalgae cell into the sample solution supply hole of the microfluidic chip, and
Device activation: Turn on the power supply module, light source module, fluorescence collection module, and data processing module in order, and by driving the micropump, single microalga cells in the sample solution are detected along the passage I in order at a predetermined flow rate.
Fluorescence generation of single microalgae cells by multi-level light intensity stimulation: Laser generator I in the light source module emits a relatively weak blue laser to irradiate the detection area I in the microfluidic chip, and laser generator II When a relatively strong blue laser is emitted to irradiate the detection area II in the microfluidic chip and the single microalgae cells in the sample solution pass through the detection area I and the detection area II, they are stimulated to produce different amounts. Step 3 to generate the fluorescence of
Collection of fluorescence production of single microalgae cells: The fluorescence of different intensities generated by the single microalgae cells is filtered out by passing through the slit sheet in the fluorescence collection module and the red light filter, respectively. After that,
Data analysis for the amount of fluorescence produced by the collected single microalgae cells: Step 5 of transmitting the amount of fluorescence produced by the collected single microalgae cells to the data processing module via a data line for processing analysis.
Evaluation of activity against single microalgae cells: The amount of fluorescence generated by single microalgae cells stimulated by a relatively weak blue laser emitted by the laser generator I in the light source module is indicated by Fw, and the single microalgae are shown. When the amount of fluorescence generated by the cells stimulated by the relatively strong blue laser emitted by the laser generator II in the light source module is indicated by F s , the data processing module has the formula F r = (F s − F w ). A
Further, in
When F r > 0.6, step 61 indicating that the single microalgae cell is in a highly active state, and
If 0.3 ≤ Fr ≤ 0.6, the single microalgae cell is in a hypoactive state, or the single microalgae cell is damaged to some extent but is not fatal. Step 62 to show if there is,
When F r <0.3, a step 63 indicating whether the single microalgae cell has already died or is on the verge of death is provided.
最後に以下のことを説明すべきである。以上の各実施例は本発明の技術的手段を説明するためのものに過ぎず、それを限定するものではなく、上述した各実施例を参照して本発明を詳細に説明したが、上述した各実施例に記載の技術的手段を修正するか、その術的特徴の一部又は全部に同等な取り替えを実施することも可能であり、これらの修正や取り替えによって、対応する技術的手段の本質が本発明の各実施例の技術的手段の範囲から逸脱しないことは当業者に自明である。 Finally, the following should be explained. Each of the above examples is merely for explaining the technical means of the present invention and is not limited thereto, and the present invention has been described in detail with reference to the above-mentioned Examples. It is also possible to modify the technical means described in each embodiment or to carry out equivalent replacements for some or all of its technical features, and by these modifications or replacements, the essence of the corresponding technical means. It is obvious to those skilled in the art that does not deviate from the scope of the technical means of each embodiment of the present invention.
［付記］
［付記１］
光源モジュール、マイクロ流体チップ、蛍光採集モジュール、データ処理モジュール、及び電源モジュールを含み、前記電源モジュールは、前記光源モジュール、前記蛍光採集モジュール、及び前記データ処理モジュールの入力端にそれぞれ接続され、前記マイクロ流体チップは、前記光源モジュールの出力端と前記蛍光採集モジュールの入力端とにそれぞれ接続され、前記データ処理モジュールは、前記蛍光採集モジュールの出力端に接続され、前記光源モジュール、前記マイクロ流体チップ、前記蛍光採集モジュール、前記データ処理モジュール、及び前記電源モジュールは、長さ７９ｍｍ×幅４９ｍｍ×高さ４３ｍｍの直方体装置に集積され、
前記マイクロ流体チップは、基板と前記基板に固定されたコーティング層とを含み、前記コーティング層には、試料溶液供給孔、通路Ｉ、検出領域Ｉ、通路ＩＩ、検出領域ＩＩ、通路ＩＩＩ、及び試料溶液排出孔が凹んで形成され、使用する際に、前記試料溶液供給孔に単一微細藻類細胞を含有する試料溶液を滴下し、マイクロポンプの駆動により、前記試料溶液中の前記単一微細藻類細胞は所定の流速で順に前記通路Ｉに沿って前記検出領域Ｉに流入し、極めて短い前記通路ＩＩを通って前記検出領域ＩＩに流入し、最後に前記通路ＩＩＩを通って前記試料溶液排出孔に流入し、
前記光源モジュールは、電圧安定回路、光源固定構造、及び光透過孔アセンブリに緊密に貼り合わせられるレーザー発生装置Ｉとレーザー発生装置ＩＩとを含み、
前記蛍光採集モジュールは、スリットシート、赤色光フィルター、及び光電センサーを含む、
ことを特徴とするマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置。
[Additional Notes]
[Appendix 1]
The power supply module includes a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and a power supply module, and the power supply module is connected to the input end of the light source module, the fluorescence collection module, and the data processing module, respectively, and the micro. The fluid chip is connected to the output end of the light source module and the input end of the fluorescent collection module, respectively, and the data processing module is connected to the output end of the fluorescent collection module, and the light source module, the microfluidic chip, and the like. The fluorescence collection module, the data processing module, and the power supply module are integrated in a rectangular device having a length of 79 mm, a width of 49 mm, and a height of 43 mm.
The microfluidic chip comprises a substrate and a coating layer immobilized on the substrate, the coating layer includes a sample solution supply hole, a passage I, a detection region I, a passage II, a detection region II, a passage III, and a sample. The solution discharge hole is formed as a recess, and when used, the sample solution containing a single microalgae cell is dropped into the sample solution supply hole, and the single microalgae in the sample solution is driven by a micropump. The cells sequentially flow into the detection region I along the passage I at a predetermined flow velocity, flow into the detection region II through the extremely short passage II, and finally through the passage III to the sample solution discharge hole. Inflow to
The light source module includes a voltage stabilizer, a light source fixation structure, and a laser generator I and a laser generator II that are tightly coupled to the light transmission hole assembly.
The fluorescence collection module includes a slit sheet, a red light filter, and a photoelectric sensor.
A single microalgae cell activity detection device based on multi-level light intensity stimulation.
［付記２］
前記通路ＩＩの長さは既知であり、前記単一微細藻類細胞が前記検出領域Ｉに現れた時、前記単一微細藻類細胞が前記検出領域ＩＩに現れる時点を推定できる、付記１に記載のマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置。
[Appendix 2]
The length of the passage II is known, and when the single microalgae cells appear in the detection area I, the time point at which the single microalgae cells appear in the detection area II can be estimated, according to Appendix 1. A device for detecting the activity of single microalgae cells based on multi-level light intensity stimulation.
［付記３］
前記レーザー発生装置Ｉは前記マイクロ流体チップ中の前記検出領域Ｉに対向して比較的弱い青色光を発し、前記レーザー発生装置ＩＩは前記マイクロ流体チップ中の前記検出領域ＩＩに対向して比較的強い青色光を発する、付記１に記載のマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置。
[Appendix 3]
The laser generator I emits relatively weak blue light toward the detection region I in the microfluidic chip, and the laser generator II relatively faces the detection region II in the microfluidic chip. The device for detecting the activity of a single microalgae cell based on the multi-level light intensity stimulus according to Appendix 1, which emits strong blue light.
［付記４］
前記基板はガラス板であり、前記コーティング層はポリジメチルシロキサンコーティング層である、付記１に記載のマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置。
[Appendix 4]
The device for detecting the activity of single microalgae cells based on the multi-level light intensity stimulus according to Appendix 1, wherein the substrate is a glass plate and the coating layer is a polydimethylsiloxane coating layer.
［付記５］
単一微細藻類細胞を含有する試料溶液をマイクロ流体チップの試料溶液供給孔に滴下する工程１と、
順に電源モジュール、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールをオンにし、マイクロポンプの駆動により、前記試料溶液中の前記単一微細藻類細胞が所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入する工程２と、
前記光源モジュール中のレーザー発生装置Ｉが比較的弱い青色レーザーを発して前記マイクロ流体チップ中の前記検出領域Ｉに照射し、レーザー発生装置ＩＩが比較的強い青色レーザーを発して前記マイクロ流体チップ中の前記検出領域ＩＩに照射し、前記試料溶液中の前記単一微細藻類細胞が先後して前記検出領域Ｉと前記検出領域ＩＩを通る時に刺激されて異なる生成量の蛍光を生成する工程３と、
前記単一微細藻類細胞が先後して生成した異なる強度の前記蛍光が、それぞれ、前記蛍光採集モジュール中のスリットシート、赤色光フィルターを通ることにより迷光をフィルタアウトした後、光電センサーに採集される工程４と、
採集した単一微細藻類細胞の蛍光生成量をデータ線によって前記データ処理モジュールに伝送して処理分析を行う工程５と、
前記単一微細藻類細胞が前記光源モジュール中の前記レーザー発生装置Ｉが発した比較的弱い青色レーザーに刺激されて生成した前記蛍光生成量をＦｗで示し、前記単一微細藻類細胞が前記光源モジュール中のレーザー発生装置ＩＩが発した比較的強い青色レーザーに刺激されて生成した前記蛍光生成量をＦｓで示すと、前記データ処理モジュールは、式Ｆｒ＝（Ｆｓ－Ｆｗ）／Ｆｓによって有効蛍光生成量Ｆｒを得、前記有効蛍光生成量Ｆｒに基づいて前記単一微細藻類細胞の活性を判断する工程６と、
を含む、
ことを特徴とするマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法。
[Appendix 5]
Step 1 of dropping the sample solution containing a single microalgae cell into the sample solution supply hole of the microfluidic chip, and
The power supply module, the light source module, the fluorescence collection module, and the data processing module are turned on in order, and by driving the micropump, the single microalga cells in the sample solution are sequentially detected along the passage I at a predetermined flow rate.
The laser generator I in the light source module emits a relatively weak blue laser to irradiate the detection region I in the microfluidic chip, and the laser generator II emits a relatively strong blue laser in the microfluidic chip. 3 and the step 3 of irradiating the detection region II of the above and stimulating the single microalgae cells in the sample solution as they pass through the detection region I and the detection region II to generate different amounts of fluorescence. ,
The fluorescence of different intensities generated by the single microalgae cells is collected by the photoelectric sensor after filtering out the stray light by passing through the slit sheet and the red light filter in the fluorescence collection module, respectively.
Step 5 of transmitting the amount of fluorescence generated from the collected single microalgae cells to the data processing module by a data line for processing analysis.
The amount of fluorescence generated by the single microalgae cell stimulated by the relatively weak blue laser emitted by the laser generator I in the light source module is indicated by Fw, and the single microalgae cell is the light source. When the amount of fluorescence generated by being stimulated by a relatively strong blue laser emitted by the laser generator II in the module is indicated by F s , the data processing module has the formula F r = (F s − F w ) /.
including,
A method for detecting the activity of single microalgae cells based on multi-level light intensity stimulation.
［付記６］
前記工程６において前記有効蛍光生成量Ｆｒに基づいて前記単一微細藻類細胞の活性を判断するプロセスは、
Ｆｒ＞０．６である場合、当該単一微細藻類細胞が高活性状態にあることを示す工程６１と、
０．３≦Ｆｒ≦０．６である場合、当該単一微細藻類細胞が低活性状態にあるか、又は当該単一微細藻類細胞がある程度で損傷を受けたが致命的ではない状態にあるかを示す工程６２と、
Ｆｒ＜０．３である場合、当該単一微細藻類細胞が既に死亡したか、又は死亡に瀕しているかを示す工程６３と、
を含む、付記５に記載のマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法。
[Appendix 6]
The process of determining the activity of the single microalgae cell based on the effective fluorescence production amount Fr in the
When F r > 0.6, step 61 indicating that the single microalgae cell is in a highly active state, and
If 0.3 ≤ Fr ≤ 0.6, the single microalgae cell is in a hypoactive state, or the single microalgae cell is damaged to some extent but is not fatal. Step 62 to indicate
When F r <0.3, step 63 indicating whether the single microalgae cell has already died or is on the verge of death, and
5. The method for detecting the activity of a single microalgae cell based on the multi-level light intensity stimulation according to Appendix 5.
１ 電源モジュール
２ 光源モジュール
３ マイクロ流体チップ
４ 蛍光採集モジュール
５ データ処理モジュール
６ レーザー発生装置Ｉ
７ レーザー発生装置ＩＩ
８ 試料溶液供給孔
９ 通路Ｉ
１０ 検出領域Ｉ
１１ 通路ＩＩ
１２ 検出領域ＩＩ
１３ 通路ＩＩＩ
１４ 試料溶液排出孔
１５ マイクロポンプ
１６ スリットシート
１７ 赤色光フィルター
１８ 光電センサー
1
7 Laser generator II
8 Sample
10 Detection area I
11 Passage II
12 Detection area II
13 Passage III
14 Sample
Claims (6)
前記マイクロ流体チップは、基板と前記基板に固定されたコーティング層とを含み、前記コーティング層には、試料溶液供給孔、通路Ｉ、検出領域Ｉ、通路ＩＩ、検出領域ＩＩ、通路ＩＩＩ、及び試料溶液排出孔が凹んで形成され、使用する際に、前記試料溶液供給孔に単一微細藻類細胞を含有する試料溶液を滴下し、マイクロポンプの駆動により、前記試料溶液中の前記単一微細藻類細胞は所定の流速で順に前記通路Ｉに沿って前記検出領域Ｉに流入し、極めて短い前記通路ＩＩを通って前記検出領域ＩＩに流入し、最後に前記通路ＩＩＩを通って前記試料溶液排出孔に流入し、
前記光源モジュールは、電圧安定回路、光源固定構造、及び光透過孔アセンブリに緊密に貼り合わせられるレーザー発生装置Ｉとレーザー発生装置ＩＩとを含み、
前記蛍光採集モジュールは、スリットシート、赤色光フィルター、及び光電センサーを含む、
ことを特徴とするマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出装置。 The power supply module includes a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and a power supply module, and the power supply module is connected to the input end of the light source module, the fluorescence collection module, and the data processing module, respectively, and the micro. The fluid chip is connected to the output end of the light source module and the input end of the fluorescent collection module, respectively, and the data processing module is connected to the output end of the fluorescent collection module, and the light source module, the microfluidic chip, and the like. The fluorescence collection module, the data processing module, and the power supply module are integrated in a rectangular device having a length of 79 mm, a width of 49 mm, and a height of 43 mm.
The microfluidic chip comprises a substrate and a coating layer immobilized on the substrate, the coating layer includes a sample solution supply hole, a passage I, a detection region I, a passage II, a detection region II, a passage III, and a sample. The solution discharge hole is formed as a recess, and when used, the sample solution containing a single microalgae cell is dropped into the sample solution supply hole, and the single microalgae in the sample solution is driven by a micropump. The cells sequentially flow into the detection region I along the passage I at a predetermined flow velocity, flow into the detection region II through the extremely short passage II, and finally through the passage III to the sample solution discharge hole. Inflow to
The light source module includes a voltage stabilizer, a light source fixation structure, and a laser generator I and a laser generator II that are tightly coupled to the light transmission hole assembly.
The fluorescence collection module includes a slit sheet, a red light filter, and a photoelectric sensor.
A single microalgae cell activity detection device based on multi-level light intensity stimulation.
順に電源モジュール、光源モジュール、蛍光採集モジュール、及びデータ処理モジュールをオンにし、マイクロポンプの駆動により、前記試料溶液中の前記単一微細藻類細胞が所定の流速で順に通路Ｉに沿って検出領域Ｉに流入し、極めて短い通路ＩＩを通って検出領域ＩＩに流入し、最後に通路ＩＩＩを通って試料溶液排出孔に流入する工程２と、
前記光源モジュール中のレーザー発生装置Ｉが比較的弱い青色レーザーを発して前記マイクロ流体チップ中の前記検出領域Ｉに照射し、レーザー発生装置ＩＩが比較的強い青色レーザーを発して前記マイクロ流体チップ中の前記検出領域ＩＩに照射し、前記試料溶液中の前記単一微細藻類細胞が先後して前記検出領域Ｉと前記検出領域ＩＩを通る時に刺激されて異なる生成量の蛍光を生成する工程３と、
前記単一微細藻類細胞が先後して生成した異なる強度の前記蛍光が、それぞれ、前記蛍光採集モジュール中のスリットシート、赤色光フィルターを通ることにより迷光をフィルタアウトした後、光電センサーに採集される工程４と、
採集した単一微細藻類細胞の蛍光生成量をデータ線によって前記データ処理モジュールに伝送して処理分析を行う工程５と、
前記単一微細藻類細胞が前記光源モジュール中の前記レーザー発生装置Ｉが発した比較的弱い青色レーザーに刺激されて生成した前記蛍光生成量をＦｗで示し、前記単一微細藻類細胞が前記光源モジュール中のレーザー発生装置ＩＩが発した比較的強い青色レーザーに刺激されて生成した前記蛍光生成量をＦｓで示すと、前記データ処理モジュールは、式Ｆｒ＝（Ｆｓ－Ｆｗ）／Ｆｓによって有効蛍光生成量Ｆｒを得、前記有効蛍光生成量Ｆｒに基づいて前記単一微細藻類細胞の活性を判断する工程６と、
を含む、
ことを特徴とするマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法。 Step 1 of dropping the sample solution containing a single microalgae cell into the sample solution supply hole of the microfluidic chip, and
The power supply module, the light source module, the fluorescence collection module, and the data processing module are turned on in order, and by driving the micropump, the single microalga cells in the sample solution are sequentially detected along the passage I at a predetermined flow rate. Step 2, which flows into the detection region II through the extremely short passage II, and finally flows into the sample solution discharge hole through the passage III.
The laser generator I in the light source module emits a relatively weak blue laser to irradiate the detection region I in the microfluidic chip, and the laser generator II emits a relatively strong blue laser in the microfluidic chip. 3 and the step 3 of irradiating the detection region II of the above and stimulating the single microalgae cells in the sample solution as they pass through the detection region I and the detection region II to generate different amounts of fluorescence. ,
The fluorescence of different intensities generated by the single microalgae cells is collected by the photoelectric sensor after filtering out the stray light by passing through the slit sheet and the red light filter in the fluorescence collection module, respectively. Step 4 and
Step 5 of transmitting the amount of fluorescence generated from the collected single microalgae cells to the data processing module by a data line for processing analysis.
The amount of fluorescence generated by the single microalgae cell stimulated by the relatively weak blue laser emitted by the laser generator I in the light source module is indicated by Fw, and the single microalgae cell is the light source. When the amount of fluorescence generated by being stimulated by a relatively strong blue laser emitted by the laser generator II in the module is indicated by F s , the data processing module has the formula F r = (F s − F w ) /. Step 6 of obtaining an effective fluorescence production amount Fr by F s and determining the activity of the single microalgae cell based on the effective fluorescence production amount Fr.
including,
A method for detecting the activity of single microalgae cells based on multi-level light intensity stimulation.
Ｆｒ＞０．６である場合、当該単一微細藻類細胞が高活性状態にあることを示す工程６１と、
０．３≦Ｆｒ≦０．６である場合、当該単一微細藻類細胞が低活性状態にあるか、又は当該単一微細藻類細胞がある程度で損傷を受けたが致命的ではない状態にあるかを示す工程６２と、
Ｆｒ＜０．３である場合、当該単一微細藻類細胞が既に死亡したか、又は死亡に瀕しているかを示す工程６３と、
を含む、請求項５に記載のマルチレベル光強度刺激に基づく単一微細藻類細胞の活性検出方法。 The process of determining the activity of the single microalgae cell based on the effective fluorescence production amount Fr in the step 6 is
When F r > 0.6, step 61 indicating that the single microalgae cell is in a highly active state, and
If 0.3 ≤ Fr ≤ 0.6, the single microalgae cell is in a hypoactive state, or the single microalgae cell is damaged to some extent but is not fatal. Step 62 to indicate
When F r <0.3, step 63 indicating whether the single microalgae cell has already died or is on the verge of death, and
5. The method for detecting the activity of a single microalgae cell based on the multi-level light intensity stimulation according to claim 5.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811488823.8 | 2018-12-06 | ||
| CN201811488823.8A CN109470672A (en) | 2018-12-06 | 2018-12-06 | The active apparatus and method of more single microalgae cells of light intensity excitation-detection |
| PCT/CN2019/121979 WO2020114327A1 (en) | 2018-12-06 | 2019-11-29 | Multi-light intensity excited apparatus and method for detecting activity of single microalgae cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2022509803A true JP2022509803A (en) | 2022-01-24 |
| JP7266324B2 JP7266324B2 (en) | 2023-04-28 |
Family
ID=65675804
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2021528970A Active JP7266324B2 (en) | 2018-12-06 | 2019-11-29 | Apparatus and method for detecting activity of single microalgae cells based on multi-level light intensity stimulation |
Country Status (3)
| Country | Link |
|---|---|
| JP (1) | JP7266324B2 (en) |
| CN (1) | CN109470672A (en) |
| WO (1) | WO2020114327A1 (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109470672A (en) * | 2018-12-06 | 2019-03-15 | 大连海事大学 | The active apparatus and method of more single microalgae cells of light intensity excitation-detection |
| CN112461751B (en) * | 2020-10-16 | 2022-04-26 | 江苏大学 | Cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61283848A (en) * | 1985-06-10 | 1986-12-13 | Japan Spectroscopic Co | Flow site meter |
| JPH1123467A (en) * | 1997-06-30 | 1999-01-29 | Suzuki Motor Corp | Equipment for measuring fluorescent quantity |
| JP2000246263A (en) * | 1999-02-26 | 2000-09-12 | Hitachi Ltd | Method and apparatus for purifying water |
| JP2006511825A (en) * | 2002-12-09 | 2006-04-06 | アドバンスド フルイディックス ラボラトリーズ， エルエルシー | Male fertility assay method and device |
| JP2006284335A (en) * | 2005-03-31 | 2006-10-19 | Univ Nagoya | Chlorophyll fluorescence measuring method and chlorophyll fluorescence measuring device |
| JP2010501851A (en) * | 2006-08-24 | 2010-01-21 | エージェンシー フォー サイエンス， テクノロジー アンド リサーチ | Compact optical detection system |
| JP2014503822A (en) * | 2010-12-21 | 2014-02-13 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Compact and wide-field fluorescence imaging in portable devices |
| JP2016503489A (en) * | 2012-10-15 | 2016-02-04 | ナノセレクト バイオメディカル， インコーポレイテッド | System, apparatus and method for sorting particles |
| CN107748138A (en) * | 2017-12-05 | 2018-03-02 | 山东交通学院 | Activity of microalgae detection means and method based on filter liquor principle |
| US20180284009A1 (en) * | 2017-03-31 | 2018-10-04 | Life Technologies Corporation | Apparatuses, systems and methods for imaging flow cytometry |
Family Cites Families (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1869662A (en) * | 2006-05-15 | 2006-11-29 | 清华大学 | Multi-channel column imaging fluorescent detector |
| CN102539394A (en) * | 2011-09-14 | 2012-07-04 | 中国科学院安徽光学精密机械研究所 | Device and method for carrying out in-situ detection on photosynthesis activity of algae in water body based on fluorescence method |
| JP2013113690A (en) * | 2011-11-28 | 2013-06-10 | Sharp Corp | Analysis element, analyzer and analysis method |
| CN103234949A (en) * | 2013-01-30 | 2013-08-07 | 大连海事大学 | Microalgae activity detection method and device in ship ballast water |
| CN103529006B (en) * | 2013-10-18 | 2016-08-17 | 大连海事大学 | A kind of Portable fluorescence based on micro-fluidic chip detection device |
| CN104849444B (en) * | 2015-05-20 | 2016-11-30 | 大连海事大学 | Fluorescence and block the cell counter and method simultaneously measured |
| CN105136763B (en) * | 2015-09-10 | 2017-10-13 | 大连海事大学 | The active dynamic monitoring new method of single microalgae cell and device characterized based on the unicellular capture of gas-liquid interface and chlorophyll fluorescence |
| CA3045796A1 (en) * | 2016-12-02 | 2018-06-07 | EMULATE, Inc. | In vitro epithelial models comprising lamina propria-derived cells |
| CN107543812A (en) * | 2017-10-09 | 2018-01-05 | 上海欧陆科仪有限公司 | Ballast water work algae device for fast detecting and its detection method |
| CN109470672A (en) * | 2018-12-06 | 2019-03-15 | 大连海事大学 | The active apparatus and method of more single microalgae cells of light intensity excitation-detection |
-
2018
- 2018-12-06 CN CN201811488823.8A patent/CN109470672A/en active Pending
-
2019
- 2019-11-29 JP JP2021528970A patent/JP7266324B2/en active Active
- 2019-11-29 WO PCT/CN2019/121979 patent/WO2020114327A1/en active Application Filing
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPS61283848A (en) * | 1985-06-10 | 1986-12-13 | Japan Spectroscopic Co | Flow site meter |
| JPH1123467A (en) * | 1997-06-30 | 1999-01-29 | Suzuki Motor Corp | Equipment for measuring fluorescent quantity |
| JP2000246263A (en) * | 1999-02-26 | 2000-09-12 | Hitachi Ltd | Method and apparatus for purifying water |
| JP2006511825A (en) * | 2002-12-09 | 2006-04-06 | アドバンスド フルイディックス ラボラトリーズ， エルエルシー | Male fertility assay method and device |
| JP2006284335A (en) * | 2005-03-31 | 2006-10-19 | Univ Nagoya | Chlorophyll fluorescence measuring method and chlorophyll fluorescence measuring device |
| JP2010501851A (en) * | 2006-08-24 | 2010-01-21 | エージェンシー フォー サイエンス， テクノロジー アンド リサーチ | Compact optical detection system |
| JP2014503822A (en) * | 2010-12-21 | 2014-02-13 | ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア | Compact and wide-field fluorescence imaging in portable devices |
| JP2016503489A (en) * | 2012-10-15 | 2016-02-04 | ナノセレクト バイオメディカル， インコーポレイテッド | System, apparatus and method for sorting particles |
| US20180284009A1 (en) * | 2017-03-31 | 2018-10-04 | Life Technologies Corporation | Apparatuses, systems and methods for imaging flow cytometry |
| CN107748138A (en) * | 2017-12-05 | 2018-03-02 | 山东交通学院 | Activity of microalgae detection means and method based on filter liquor principle |
Non-Patent Citations (5)
| Title |
|---|
| ERICKSON, R. A. AND JIMENEZ, R.: "Microfluidic cytometer for high-throughput measurement of photosynthetic characteristics and lipid a", LAB ON A CHIP, vol. 13, JPN6022032095, 10 May 2013 (2013-05-10), pages 2893 - 2901, ISSN: 0004842311 * |
| TAKAYAMA, K. AND NISHINA, H.: "Chlorophyll Fluorescence Imaging of the Chlorophyll Fluorescence Induction Phenomenon for Plant Moni", ENVIRON. CONTROL BIOL., vol. 47, no. 2, JPN6022032097, 20 November 2009 (2009-11-20), pages 101 - 109, XP055376286, ISSN: 0004842315, DOI: 10.2525/ecb.47.101 * |
| WANG, J. ET AL.: "A Label-Free Microfluidic Biosensor for Activity Detection of Single Microalgae Cells Based on Chlor", SENSORS, vol. 13, no. 12, JPN6022032093, 26 November 2013 (2013-11-26), pages 16075 - 16089, XP055429740, ISSN: 0005029448, DOI: 10.3390/s131216075 * |
| WANG, J. ET AL.: "Detection of activity of single microalgae cells in a new microfluidic cell capturing chip", MEASUREMENT SCIENCE AND TECHNOLOGY, vol. 27, no. 12, JPN6022032092, 26 October 2016 (2016-10-26), pages 125701, XP020310909, ISSN: 0005029447, DOI: 10.1088/0957-0233/27/12/125701 * |
| 園池 公毅: "クロロフィル蛍光と吸収による光合成測定", 低温科学, vol. 67, JPN7022003674, 25 May 2017 (2017-05-25), pages 507 - 524, ISSN: 0005029449 * |
Also Published As
| Publication number | Publication date |
|---|---|
| JP7266324B2 (en) | 2023-04-28 |
| WO2020114327A1 (en) | 2020-06-11 |
| CN109470672A (en) | 2019-03-15 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Colson et al. | Flow-through quantification of microplastics using impedance spectroscopy | |
| Kniggendorf et al. | Microplastics detection in streaming tap water with Raman spectroscopy | |
| Zamyadi et al. | A review of monitoring technologies for real-time management of cyanobacteria: Recent advances and future direction | |
| US9310334B2 (en) | Method and apparatus for characterizing and counting particles, in particular biological particles | |
| EP2389447B1 (en) | Chlorophyll and turbidity sensor system | |
| Babin et al. | Approachesand | |
| KR102390747B1 (en) | Microorganism test method and device therefor | |
| JP4990909B2 (en) | Method and apparatus for advanced detection of toxic substances | |
| JP2022509803A (en) | Single microalgae cell activity detection device and method based on multi-level light intensity stimulation | |
| Hou et al. | Smartphone based microfluidic lab-on-chip device for real-time detection, counting and sizing of living algae | |
| CN103234949A (en) | Microalgae activity detection method and device in ship ballast water | |
| US20130101468A1 (en) | Method and apparatus for controlling the propagation of cyanobacteria in a body of water | |
| TWI619809B (en) | Method and device for inspecting microorganisms | |
| CN106018688B (en) | A kind of evaluation method of metal nanoparticle ion and nano effect toxicity contribution rate | |
| JP6201285B2 (en) | Microorganism testing method and apparatus | |
| CN110554015A (en) | method for realizing visual detection of Cr (VI) by micro-fluidic sensor based on photoluminescence xylan carbon quantum dots | |
| KR20240019826A (en) | UV spectrophotometric detection module of polymer particles and phytoplankton for autonomous water analysis stations and detection processes | |
| JP7274605B2 (en) | Fluidic device for optical investigation of individual microorganisms | |
| Jagadhane et al. | Histotoxicity of AIEgen Based on Triphenylamine for the Simultaneous and Discriminatory Sensing of Hg2+ and Ag+ Directly in Aqueous Media for Environmental Applications | |
| Xu et al. | Electrokinetic motion and viability assessment of algae with a polyethylene glycol–dextran interface | |
| Li et al. | Ultra-sensitive detection using integrated waveguide technologies | |
| Fries et al. | Non-acoustic sensors | |
| Childers et al. | Portable On-Site Optical Detection and Quantification of Microplastics | |
| Zhang et al. | A fiber-based fluorometric system for in situ algal classification | |
| Mowlem et al. | Micro system technology for marine measurement |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20210715 |
|
| A977 | Report on retrieval |
Free format text: JAPANESE INTERMEDIATE CODE: A971007 Effective date: 20220607 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20220802 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20221102 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20221227 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20230123 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20230404 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20230411 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 7266324 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 |