WO2020114327A1 - 多光强激发检测单个微藻细胞活性的装置与方法 - Google Patents

多光强激发检测单个微藻细胞活性的装置与方法 Download PDF

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WO2020114327A1
WO2020114327A1 PCT/CN2019/121979 CN2019121979W WO2020114327A1 WO 2020114327 A1 WO2020114327 A1 WO 2020114327A1 CN 2019121979 W CN2019121979 W CN 2019121979W WO 2020114327 A1 WO2020114327 A1 WO 2020114327A1
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module
fluorescence
activity
microalgae cell
cell
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PCT/CN2019/121979
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French (fr)
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王俊生
丁格格
王兰兰
潘新祥
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大连海事大学
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    • 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

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  • the invention relates to the technical field of activity detection of a single microalgae cell, in particular to a device and method for detecting the activity of a single microalgae cell with multi-light intensity excitation.
  • the traditional microalgae cell activity detection device can only detect the activity of a microalgae group.
  • the traditional detection method cannot exclude the existence of a single microalgae cell with a higher activity state, which is not conducive to preventing the invasion of harmful microalgae.
  • the traditional method of detecting the activity of a single microalgae requires the use of conventional on-site sampling, laboratory analysis and measurement process, but in practical applications, it is necessary to conduct on-site, real-time, online and continuous detection of microalgae in water.
  • the instruments used in the laboratory are huge in structure, expensive, and complicated in operation, requiring professional operation, and cannot be integrated and portable, and cannot be inspected on site.
  • a device and method for detecting the activity of a single microalgae cell with multi-light intensity excitation are provided. Fundamentally solving the problem of detecting the activity of single microalgae cells has important scientific significance and practical value for the field of environmental science.
  • a device for detecting the activity of a single microalgae cell with multi-intensity excitation includes: a light source module, a microfluidic chip, a fluorescence collection module, a data processing module and a power supply module; the power supply module is separately connected to the light source module and the fluorescence collection module and data The input end of the processing module is connected; the microfluidic chip is connected to the output end of the light source module and the input end of the fluorescence collection module; the data processing module is connected to the output end of the fluorescence collection module; the light source module, the microfluidic chip, and the fluorescence
  • the acquisition module, data processing module and power supply module are integrated in a 79cm ⁇ 49cm ⁇ 43cm cuboid device;
  • the microfluidic chip includes a substrate and a coating fixed on the substrate, the coating is recessed into a liquid hole, a channel I, a detection area I, a channel II, a detection area II, a channel III and a liquid outlet; use At the same time, the liquid sample solution of microalgae cells was added dropwise into the inlet hole, and the single microalgae cells in the sample solution were driven into the detection area I along the channel I at a fixed flow rate through the very short channel II and flowed in through the very short channel II. Detection area II, and finally flow into the outlet through channel III;
  • the light source module includes a voltage stabilizing circuit, a light source fixing structure, and a laser I and a laser II that are closely attached to the through hole assembly;
  • the fluorescence collection module includes a slit sheet, a red filter and a photoelectric sensor.
  • the length of the channel II is known.
  • the laser I is emitting weak blue light to the detection area I in the microfluidic chip
  • the laser II is emitting strong blue light to the detection area II in the microfluidic chip.
  • the substrate is a glass sheet
  • the coating is a polydimethylsiloxane coating.
  • the invention also provides a method for detecting the activity of a single microalgae cell with multiple light intensity excitations, including the following steps:
  • Step 1 Drop the sample, add the sample solution containing microalgae cells to the inlet of the microfluidic chip;
  • Step 2 Start the device, turn on the power module, light source module, fluorescence collection module and data processing module in sequence, and the single microalgae cells in the sample solution under the drive of the micropump flow into the detection area I along the channel I at a fixed flow rate in sequence, through The very short channel II flows into the detection zone II, and finally flows into the outlet through the channel III;
  • Step 3 Multi-level light intensity excites a single microalgae cell to produce fluorescence.
  • Laser I in the light source module emits a weak blue laser to illuminate the detection area I in the microfluidic chip, and Laser II emits a strong blue laser Irradiated on the detection zone II in the microfluidic chip; when a single microalgae cell in the sample solution passes through the detection zone I and the detection zone II successively, it is stimulated to produce different fluorescence yields;
  • Step 4 Collect the fluorescence output of a single microalgae cell.
  • the fluorescence of different intensity produced by a single microalgae cell is filtered by the slit sheet and red filter in the fluorescence collection module to filter out the stray light and then received by the photoelectric sensor;
  • Step 5 Perform data analysis on the fluorescence output of the single microalgal cells collected above, and transmit the collected fluorescence output of the single microalgal cells to the data processing module for processing and analysis through the data line;
  • Step 6 Evaluate the activity of individual microalgae cells, F w represents the fluorescence output of single microalgae cells excited by the weak blue laser emitted by laser I in the light source module, and F s represents the single microalgae cells are light source
  • the laser II in the module emits a strong blue laser excitation fluorescence output
  • the single process step 6 is determined based on the effective activity of microalgal cells fluorescence yield F r, comprising the steps of:
  • Step 61 When F r >0.6, it means that the microalgal cell is in a highly active state
  • Step 63 When F r ⁇ 0.3, it means that the microalgae cell has died or is about to die.
  • the present invention has the following advantages:
  • the present invention solves the problems of inability to quantitatively evaluate the activity of a single microalgae cell in the existing microalgae cell activity detection method, inability to perform on-site detection, expensive detection equipment, cumbersome sample processing, and the universality and error brought by the large sample amount.
  • To fundamentally solve the problem of detecting the activity of single microalgal cells it has important scientific significance and practical value for the field of environmental science.
  • the invention uses a microfluidic chip as a micro-platform for detecting the activity of a single microalgae cell.
  • the related modules can also adopt a structure with a smaller volume. All the modules are integrated into a 79cm ⁇ 49cm ⁇ 43cm cuboid device.
  • the present invention has the advantages of small size, light weight, low cost, low sample consumption, easy portability, and can be handheld for on-site detection.
  • the light intensity of the measurement light excited by the light source module of the present invention does not need a very weak level to monitor the fluorescence output of dark adaptation, and at the same time, the light intensity of the measurement light does not need to reach the saturation light level to evaluate the maximum fluorescence output. It not only reduces the technical difficulty, but also is not limited by the different dark adaptation levels of different microalgae cells, and has a wider application range.
  • the detectable microalgae cells are not restricted by the type, individual morphology, size, and cell structure.
  • the present invention can be widely promoted in the field of activity detection of single microalgal cells.
  • FIG. 1 is a schematic structural diagram of a detection device of the present invention.
  • FIG. 2 is a schematic structural diagram of a microfluidic chip of the present invention.
  • FIG. 3 is a schematic structural diagram of a fluorescence collection module of the present invention.
  • FIG. 4 is a schematic structural view of a light source module of the present invention.
  • orientation words such as “front, back, up, down, left, right", “horizontal, vertical, vertical, horizontal” and “top and bottom” indicate the orientation Or the positional relationship is usually based on the orientation or positional relationship shown in the drawings, just to facilitate the description of the present invention and simplify the description, in the case of no contrary description, these orientation words do not indicate and imply that the device or element It must have a specific orientation or be constructed and operated in a specific orientation, so it cannot be understood as a limitation of the scope of protection of the present invention: the orientation words “inside and outside” refer to inside and outside relative to the contour of each component itself.
  • spatially relative terms such as “above”, “above”, “above”, “above”, etc. can be used here to describe as shown in the figure The spatial relationship between a device or feature shown and other devices or features. It should be understood that spatially relative terms are intended to encompass different orientations in use or operation in addition to the orientation of the device described in the figures. For example, if the device in the drawings is turned upside down, a device described as “above another device or configuration” or “above another device or configuration” will then be positioned as “below other device or configuration” or “in Under its device or structure.” Thus, the exemplary term “above” may include both “above” and “below” orientations. The device can also be positioned in other different ways (rotated 90 degrees or at other orientations), and the relative description of the space used here is explained accordingly.
  • the present invention provides a device for detecting the activity of a single microalgae cell with multi-intensity excitation, including: a light source module, a microfluidic chip, a fluorescence collection module, a data processing module, and a power module; the power module It is connected to the input end of the light source module, the fluorescence collection module and the 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; the data processing module is connected to the output end of the fluorescence collection module;
  • the light source module, microfluidic chip, fluorescence collection module, data processing module, and power supply module are integrated into a 79mm ⁇ 49mm ⁇ 43mm (length ⁇ width ⁇ height) cuboid device;
  • the microfluidic chip includes a substrate and a coating fixed on the substrate, the substrate is a glass sheet, the coating is a polydimethylsiloxane coating, and the coating is recessed into the liquid hole , Channel I, detection zone I, channel II, detection zone II, channel III and outlet hole; when in use, the sample solution is added dropwise into the inlet hole, and a single microalgae cell in the sample solution is fixed under the drive of the micropump
  • the flow rate flows into the detection zone I along the channel I in sequence, flows into the detection zone II through the extremely short channel II, and finally flows into the liquid outlet through the channel III; the length of the channel II is known.
  • the light source module includes a voltage stabilizing circuit, a light source fixing structure, and a laser I and a laser II that are closely attached to the through hole assembly; the laser I is facing the detection area I in the microfluidic chip to emit weak blue light, and the laser II is facing the micro The detection area II in the flow control chip emits strong blue light.
  • the fluorescence collection module includes a slit sheet, a red filter and a photoelectric sensor.
  • the present invention also provides a method for detecting the activity of a single microalgae cell with multiple light intensity excitations, including the following steps:
  • Step 1 Drop the sample, add the sample solution containing microalgae cells to the inlet of the microfluidic chip;
  • Step 2 Start the device, turn on the power module, light source module, fluorescence collection module and data processing module in sequence, and the single microalgae cells in the sample solution under the drive of the micropump flow into the detection area I along the channel I at a fixed flow rate in sequence, through The very short channel II flows into the detection zone II, and finally flows into the outlet through the channel III;
  • Step 3 Multi-level light intensity excites a single microalgae cell to produce fluorescence.
  • Laser I in the light source module emits a weak blue laser to illuminate the detection area I in the microfluidic chip, and Laser II emits a strong blue laser Irradiated on the detection zone II in the microfluidic chip; when a single microalgae cell in the sample solution passes through the detection zone I and the detection zone II successively, it is stimulated to produce different fluorescence yields;
  • Step 4 Collect the fluorescence output of a single microalgae cell.
  • the fluorescence of different intensity produced by a single microalgae cell passes through the slit and red filter in the fluorescence collection module to filter out the stray light and then received by the photoelectric sensor;
  • Step 5 Perform data analysis on the fluorescence output of the single microalgal cells collected above, and transmit the collected fluorescence output of the single microalgae cells to the data processing module through a data line for processing and analysis;
  • Step 6 Evaluate the activity of single microalgae cells, F w represents the fluorescence output of single microalgae cells excited by the weak blue laser emitted by the laser I in the light source module, and F s represents the single microalgae cells are light source
  • the laser II in the module emits a strong blue laser excitation fluorescence output.
  • Step 61 When F r >0.6, it indicates that the single microalgae cell is in a highly active state
  • Step 63 When F r ⁇ 0.3, it indicates that the single microalgae cell has died or is about to die.

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Abstract

一种多光强激发检测单个微藻细胞活性的装置与方法，该装置包括光源模块（2）、微流控芯片（3）、荧光采集模块（4）、数据处理模块（5）以及电源模块（1）。微流控芯片（3）作为单个微藻细胞活性检测的微平台，由基片和固定在基片上的涂层组成。微流控芯片（3）上凹刻进液孔（8）、通道（9，11，13）、检测区（10，12）和出液孔（14）；所有模块整合在一个79cm×49cm×43cm的长方体装置中。光源模块（2）激发的测量光的光强不需要非常弱的水平以监测暗适应的荧光产量，也不需要达到饱和光水平来评估最大荧光产量；可检测的微藻细胞不受种类、个体形态、大小尺寸、细胞构造的限制。该检测方法解决了现有微藻细胞活性检测方法中无法定量评估单个微藻细胞的活性带来的普适性和误差等问题。

Description

多光强激发检测单个微藻细胞活性的装置与方法 技术领域
本发明涉及单个微藻细胞的活性检测技术领域，具体而言，尤其涉及一种多光强激发检测单个微藻细胞活性的装置与方法。
背景技术
我国海岸线长3.2万千米，主权管辖海域面积达473万平方千米，跨越5个气候带，生态系统类型众多。这种自然特征使我国容易遭受海洋生物的入侵。随着我国海洋运输业和海水养殖业的兴起，面临着不断增多的生物入侵形势。其中船舶压载水带来过十几种有引发赤潮风险的藻类，一旦引发赤潮，当地海洋生态系统的结构与功能将几乎彻底崩溃，对海域原有生物群落和生态系统的稳定性构成极大威胁。
传统微藻细胞活性检测装置只能检测一个微藻群体的活性。然而，当危害较高的微藻群体显示较低的活性状态时，传统检测方法并不能排除存在单个微藻细胞有较高的活性状态，这不利于防范有害微藻的入侵。另外，传统方式检测单个微藻的活性需要采用常规的现场取样、实验室分析的测量流程，但在实际应用中有必要对水中的微藻进行现场，实时，在线和连续检测。而且，实验室使用的仪器结构庞大、价格昂贵、操作复杂，需要专业人员进行操作，不能集成便携、无法现场检测。
发明内容
根据上述提出现有微藻细胞活性检测方法中无法定量评估单个微藻细胞的活性、无法现场检测、检测设备昂贵、样品处理繁琐、样品用量多带来的普适性和误差的技术问题，而提供一种多光强激发检测单个微藻细胞活性的装置与方法。从根本上解决单个微藻细胞的活性检测问题，对于环境科学领域具有重要的科学意义和现实价值。
本发明采用的技术手段如下：
一种多光强激发检测单个微藻细胞活性的装置，包括：光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块；所述电源模块分别与光源模块和荧光采集模块以及数据处理模块的输入端相连；微流控芯片分别与光源模块的输出端和荧光采集模块的输入端连接；数据处理模块与荧光采集模块的输出端连接；所述光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块整合在一个79cm×49cm×43cm的长方体装置中；
所述微流控芯片包括基片和固定在基片上的涂层，所述涂层凹刻进液孔、通道Ⅰ、检测区Ⅰ、通道Ⅱ、检测区Ⅱ、通道Ⅲ和出液孔；使用时，在进液孔滴加微藻细胞的液体样品溶液，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；
所述光源模块包括稳压电路、光源固定结构以及与通光孔组件紧密贴合的激光器Ⅰ和激光器Ⅱ；
所述荧光采集模块包括狭缝片、红色滤光片和光电传感器。
进一步地，所述通道Ⅱ的长度已知，当单个微藻细胞出现在检测区Ⅰ时，能够预估单个微藻细胞在检测区Ⅱ出现的时间点。
进一步地，所述激光器Ⅰ正对微流控芯片中的检测区Ⅰ发出较弱的蓝光，激光器Ⅱ正对微流控芯片中的检测区Ⅱ发出的是较强的蓝光。
进一步地，所述基片为玻璃片，所述涂层为聚二甲基硅氧烷涂层。
本发明还提供了一种多光强激发检测单个微藻细胞活性的方法，包括如下步骤：
步骤1：滴加样品，将含有微藻细胞的样品溶液滴加到微流控芯片的进液孔；
步骤2：启动装置，依次开启电源模块、光源模块、荧光采集模块和数据处理模块，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；
步骤3：多级光强激发单个微藻细胞产生荧光，光源模块中的激光器 Ⅰ发出较弱的蓝色激光照射在微流控芯片中的检测区Ⅰ上，激光器Ⅱ发出较强的蓝色激光照射在微流控芯片中的检测区Ⅱ上；当样品溶液中的单个微藻细胞先后经过检测区Ⅰ和检测区Ⅱ时，受激产生不同的荧光产量；
步骤4：单个微藻细胞荧光产量的采集，单个微藻细胞先后产生的不同强度的荧光分别通过荧光采集模块中的狭缝片、红色滤光片过滤掉杂光后被光电传感器接收；
步骤5：对上述采集到的单个微藻细胞荧光产量进行数据分析，将采集到的单个微藻细胞荧光产量经数据线传输至数据处理模块中进行处理分析；
步骤6：对单个微藻细胞进行活性评估，用F w表示单个微藻细胞被光源模块中的激光器Ⅰ发出较弱的蓝色激光激发产生的荧光产量，用F s表示单个微藻细胞被光源模块中的激光器Ⅱ发出较强的蓝色激光激发产生的荧光产量，数据处理模块根据公式F r＝(F s-F w)/F s得到有效荧光产量F r，根据F r判断单个微藻细胞的活性。
进一步地，所述步骤6中根据有效荧光产量F r判断单个微藻细胞活性的过程，包括如下步骤：
步骤61：当F r>0.6时，表示该微藻细胞处于高活性状态；
步骤62：当0.3<＝F r<＝0.6时，表示该微藻细胞处于低活性状态或者该单个微藻细胞在一定程度上受损但不致命；
步骤63：当F r<0.3时，表示该微藻细胞已死亡或濒临死亡。
较现有技术相比，本发明具有以下优点：
1、本发明解决了现有微藻细胞活性检测方法中无法定量评估单个微藻细胞的活性、无法现场检测、检测设备昂贵、样品处理繁琐、样品用量多带来的普适性和误差等问题，从根本上解决单个微藻细胞的活性检测问题，对于环境科学领域具有重要的科学意义和现实价值。
2、本发明采用微流控芯片作为单个微藻细胞活性检测的微平台，相关的模块亦可采用体积较小的结构形式，所有模块整合在一个79cm×49cm×43cm的长方体装置中。相对于现有大型检测设备，本发明具有体积小、重量轻、成本低、样品用量少、便于携带、能够进行手持用于现场检测等优点。
3、本发明在样品池加入样品后，后续步骤均为智能化完成，不需要任何专业化知识，不需要专业化人员的操作，步骤少，操作简单，解决了样品处理繁琐的问题。
4、本发明所述光源模块激发的测量光的光强不需要非常弱的水平以监测暗适应的荧光产量，同时，测量光的光强不需要达到饱和光水平来评估最大荧光产量。不仅降低了技术上的难度，而且由于不受不同微藻细胞暗适应水平不同的限制，适用面更广，可检测的微藻细胞不受种类、个体形态、大小尺寸、细胞构造的限制。
基于上述理由本发明可在单个微藻细胞的活性检测等领域广泛推广。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图做以简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动性的前提下，还可以根据这些附图获得其他的附图。
图1为本发明检测装置结构示意图。
图2为本发明微流控芯片结构示意图。
图3为本发明的荧光采集模块结构示意图。
图4为本发明的光源模块结构示意图。
图中：1、电源模块；2、光源模块；3、微流控芯片；4、荧光采集模块；5、数据处理模块；6、激光器Ⅰ；7、激光器Ⅱ；8、进液孔；9、通道Ⅰ；10、检测区Ⅰ；11、通道Ⅱ；12、检测区Ⅱ；13、通道Ⅲ；14、出液孔；15、微泵；16、狭缝片；17、红色滤光片；18、光电传感器。
具体实施方式
需要说明的是，在不冲突的情况下，本发明中的实施例及实施例中的特征可以相互组合。下面将参考附图并结合实施例来详细说明本发明。
为使本发明实施例的目的、技术方案和优点更加清楚，下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。以下对至少一个示例性实施例的描述实际上仅仅是说明性的，决不作为对本发明及其应用或使用的任何限制。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本发明的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
除非另外具体说明，否则在这些实施例中阐述的部件和步骤的相对布置、数字表达式和数值不限制本发明的范围。同时，应当清楚，为了便于描述，附图中所示出的各个部分的尺寸并不是按照实际的比例关系绘制的。对于相关领域普通技术人员己知的技术、方法和设备可能不作详细讨论，但在适当情况下，所述技术、方法和设备应当被视为授权说明书的一部分。在这里示出和讨论的所有示例中，任向具体值应被解释为仅仅是示例性的，而不是作为限制。因此，示例性实施例的其它示例可以具有不同的值。应注意到：相似的标号和字母在下面的附图中表示类似项，因此，一旦某一项在一个附图中被定义，则在随后的附图中不需要对其进行进一步讨论。
在本发明的描述中，需要理解的是，方位词如“前、后、上、下、左、右”、“横向、竖向、垂直、水平”和“顶、底”等所指示的方位或位置关系通常是基于附图所示的方位或位置关系，仅是为了便于描述本发明和简化描述，在未作相反说明的情况下，这些方位词并不指示和暗示所指的装置或元件必须具有特定的方位或者以特定的方位构造和操作，因此不能理解为对本发明保护范围的限制：方位词“内、外”是指相对于各部件本身的轮廓的内外。
为了便于描述，在这里可以使用空间相对术语，如“在……之上”、“在…… 上方”、“在……上表面”、“上面的”等，用来描述如在图中所示的一个器件或特征与其他器件或特征的空间位置关系。应当理解的是，空间相对术语旨在包含除了器件在图中所描述的方位之外的在使用或操作中的不同方位。例如，如果附图中的器件被倒置，则描述为“在其他器件或构造上方”或“在其他器件或构造之上”的器件之后将被定位为“在其他器件或构造下方”或“在其位器件或构造之下”。因而，示例性术语“在……上方”可以包括“在……上方”和“在……下方”两种方位。该器件也可以其他不同方式定位(旋转90度或处于其他方位)，并且对这里所使用的空间相对描述作出相应解释。
此外，需要说明的是，使用“第一”、“第二”等词语来限定零部件，仅仅是为了便于对相应零部件进行区别，如没有另行声明，上述词语并没有特殊含义，因此不能理解为对本发明保护范围的限制。
实施例1
如图1所示，本发明提供了一种多光强激发检测单个微藻细胞活性的装置，包括：光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块；所述电源模块分别与光源模块和荧光采集模块以及数据处理模块的输入端相连；微流控芯片分别与光源模块的输出端和荧光采集模块的输入端连接；数据处理模块与荧光采集模块的输出端连接；所述光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块整合在一个79mm×49mm×43mm(长度×宽度×高度)的长方体装置中；
如图2所示，微流控芯片包括基片和固定在基片上的涂层，基片为玻璃片，涂层为聚二甲基硅氧烷涂层，所述涂层凹刻进液孔、通道Ⅰ、检测区Ⅰ、通道Ⅱ、检测区Ⅱ、通道Ⅲ和出液孔；使用时，在进液孔滴加样品溶液，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；通道Ⅱ的长度已知，当单个微藻细胞出现在检测区Ⅰ时，能够预估单个微藻细胞在检测区Ⅱ出现的时间点。光源模块包括稳压电路、光源固定结构以及与通光孔组件紧密贴合的激光器Ⅰ和激光器Ⅱ；激光器Ⅰ正对微流控芯片中的检测区Ⅰ发出较弱的蓝光，激光器Ⅱ正对微流控芯片中的检测区Ⅱ发出的是较强的蓝光。荧光采集模块包括狭缝片、红色滤光片和光电传感器。
实施例2
在实施例1的基础上，本发明还提供了一种多光强激发检测单个微藻细胞活性的方法，包括如下步骤：
步骤1：滴加样品，将含有微藻细胞的样品溶液滴加到微流控芯片的进液孔；
步骤2：启动装置，依次开启电源模块、光源模块、荧光采集模块和数据处理模块，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；
步骤3：多级光强激发单个微藻细胞产生荧光，光源模块中的激光器Ⅰ发出较弱的蓝色激光照射在微流控芯片中的检测区Ⅰ上，激光器Ⅱ发出较强的蓝色激光照射在微流控芯片中的检测区Ⅱ上；当样品溶液中的单个微藻细胞先后经过检测区Ⅰ和检测区Ⅱ时，受激产生不同的荧光产量；
步骤4：单个微藻细胞荧光产量的采集，单个微藻细胞先后产生的不同强度的荧光分别通过荧光采集模块中的狭缝片、红色滤光片过滤掉杂光后被光电传感器接收；
步骤5：对上述采集到的单个微藻细胞荧光产量进行数据分析，将采集到的单个微藻细胞荧光产量经数据线传输至数据处理模块中进行处理分析；
步骤6：对单个微藻细胞进行活性评估，用F w表示单个微藻细胞被光源模块中的激光器Ⅰ发出较弱的蓝色激光激发产生的荧光产量，用F s表示单个微藻细胞被光源模块中的激光器Ⅱ发出较强的蓝色激光激发产生的荧光产量，数据处理模块根据公式F r＝(F s-F w)/F s得到有效荧光产量F r，根据F r判断单个微藻细胞的活性。
步骤61：当F r>0.6时，表示该单个微藻细胞处于高活性状态；
步骤62：当0.3<＝F r<＝0.6时，表示该单个微藻细胞处于低活性状态或者该单个微藻细胞在一定程度上受损但不致命；
步骤63：当F r<0.3时，表示该单个微藻细胞已死亡或濒临死亡。
最后应说明的是：以上各实施例仅用以说明本发明的技术方案，而非对其限制；尽管参照前述各实施例对本发明进行了详细的说明，本领域的普通 技术人员应当理解：其依然可以对前述各实施例所记载的技术方案进行修改，或者对其中部分或者全部技术特征进行等同替换；而这些修改或者替换，并不使相应技术方案的本质脱离本发明各实施例技术方案的范围。

Claims (6)

  1. 一种多光强激发检测单个微藻细胞活性的装置，其特征在于，包括：光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块；所述电源模块分别与光源模块和荧光采集模块以及数据处理模块的输入端相连；微流控芯片分别与光源模块的输出端和荧光采集模块的输入端连接；数据处理模块与荧光采集模块的输出端连接；所述光源模块、微流控芯片、荧光采集模块、数据处理模块以及电源模块整合在一个长度79mm×宽度49mm×高度43mm的长方体装置中；
    所述微流控芯片包括基片和固定在基片上的涂层，所述涂层凹刻进液孔、通道Ⅰ、检测区Ⅰ、通道Ⅱ、检测区Ⅱ、通道Ⅲ和出液孔；使用时，在进液孔滴加含有单个微藻细胞的样品溶液，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；
    所述光源模块包括稳压电路、光源固定结构以及与通光孔组件紧密贴合的激光器Ⅰ和激光器Ⅱ；
    所述荧光采集模块包括狭缝片、红色滤光片和光电传感器。
  2. 根据权利要求1所述的多光强激发检测单个微藻细胞活性的装置，其特征在于，所述通道Ⅱ的长度已知，当单个微藻细胞出现在检测区Ⅰ时，能够预估单个微藻细胞在检测区Ⅱ出现的时间点。
  3. 根据权利要求1所述的多光强激发检测单个微藻细胞活性的装置，其特征在于，所述激光器Ⅰ正对微流控芯片中的检测区Ⅰ发出较弱的蓝光，激光器Ⅱ正对微流控芯片中的检测区Ⅱ发出的是较强的蓝光。
  4. 根据权利要求1所述的多光强激发检测单个微藻细胞活性的装置，其特征在于，所述基片为玻璃片，所述涂层为聚二甲基硅氧烷涂层。
  5. 一种多光强激发检测单个微藻细胞活性的方法，其特征在于，包括如下步骤：
    步骤1：滴加样品，将含有单个微藻细胞的样品溶液滴加到微流控芯片的进液孔；
    步骤2：启动装置，依次开启电源模块、光源模块、荧光采集模块和数据处理模块，在微泵的驱动下样品溶液中的单个微藻细胞以固定流速依 次沿着通道Ⅰ流入检测区Ⅰ，通过极短的通道Ⅱ流入检测区Ⅱ，最后通过通道Ⅲ流入出液孔；
    步骤3：多级光强激发单个微藻细胞产生荧光，光源模块中的激光器Ⅰ发出较弱的蓝色激光照射在微流控芯片中的检测区Ⅰ上，激光器Ⅱ发出较强的蓝色激光照射在微流控芯片中的检测区Ⅱ上；当样品溶液中的单个微藻细胞先后经过检测区Ⅰ和检测区Ⅱ时，受激产生不同的荧光产量；
    步骤4：单个微藻细胞荧光产量的采集，单个微藻细胞先后产生的不同强度的荧光分别通过荧光采集模块中的狭缝片、红色滤光片过滤掉杂光后被光电传感器接收；
    步骤5：对上述采集到的单个微藻细胞荧光产量进行数据分析，将采集到的单个微藻细胞荧光产量经数据线传输至数据处理模块中进行处理分析；
    步骤6：对单个微藻细胞进行活性评估，用F w表示单个微藻细胞被光源模块中的激光器Ⅰ发出较弱的蓝色激光激发产生的荧光产量，用F s表示单个微藻细胞被光源模块中的激光器Ⅱ发出较强的蓝色激光激发产生的荧光产量，数据处理模块根据公式F r＝(F s-F w)/F s得到比值F r，根据比值F r判断单个微藻细胞的活性。
  6. 根据权利要求5所述的多光强激发检测单个微藻细胞活性的方法，其特征在于，所述步骤6中根据比值F r判断单个微藻细胞活性的过程，包括如下步骤：
    步骤61：当F r>0.6时，表示该单个微藻细胞处于高活性状态；
    步骤62：当0.3<＝F r<＝0.6时，表示该单个微藻细胞处于低活性状态或者该单个微藻细胞在一定程度上受损但不致命；
    步骤63：当F r<0.3时，表示该单个微藻细胞已死亡或濒临死亡。
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