CN109470672A - The active apparatus and method of more single microalgae cells of light intensity excitation-detection - Google Patents
The active apparatus and method of more single microalgae cells of light intensity excitation-detection Download PDFInfo
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- CN109470672A CN109470672A CN201811488823.8A CN201811488823A CN109470672A CN 109470672 A CN109470672 A CN 109470672A CN 201811488823 A CN201811488823 A CN 201811488823A CN 109470672 A CN109470672 A CN 109470672A
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- G—PHYSICS
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
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Abstract
The present invention provides a kind of more single microalgae cells of light intensity excitation-detection active apparatus and method, which includes light source module, micro-fluidic chip, fluorescent collecting module, data processing module and power module.Microfluidic platform of the micro-fluidic chip as single microalgae cell Activity determination is made of substrate and the coating being fixed on substrate.Intaglio inlet opening, channel, detection zone and fluid hole on chip；It is all module integrated in a rectangular body device of cm × cm × cm in the present invention.The light intensity of the measurement light of the light source module excitation does not need the very weak horizontal fluorescence quantum yield to monitor dark adaptation, does not need to reach saturation light level to assess maximum fluorescence yield yet；Detectable microalgae cell is not limited by type, individual morphology, size dimension, cell structure.Technical solution of the present invention solves the problems such as active bring universality and error that single microalgae cell can not be quantitatively evaluated in existing microalgae cell activity test method.
Description
Technical field
The present invention relates to the Activity determination technical fields of single microalgae cell, specifically, more particularly to a kind of more light intensity
The active apparatus and method of the single microalgae cell of excitation-detection.
Background technique
3.2 ten thousand km of China's seashore wire length, seas under its jurisdiction area with sovereign right is up to 4,730,000 sq-kms, across 5 climate zones,
Ecosystem-type is numerous.This physical feature makes China be subject to the invasions of marine organisms.With China's sea transport industry
With the rise of mariculture industry, it is faced with growing number of biotic intrusion situation.Wherein ballast water for ship brought and ten several had
Cause the algae of red tide risk, once causing red tide, structure and the function of local marine ecosystems will almost entirely collapse, right
The stability of the original biocoene in sea area and the ecosystem constitutes great threat.
Traditional single microalgae cell device for detecting activity can only detect the activity of a microalgae group.However, when harm compared with
When high microalgae group shows lower activated state, traditional detection method can not exclude to have there are single microalgae cell higher
Activated state, this is unfavorable for the invasion for taking precautions against harmful microalgae.In addition, the activity that traditional approach detects single microalgae needs to use
The measurement procedure of conventional field sampling, lab analysis, but in practical applications it is necessary to carry out scene to the microalgae in water,
In real time, online and continuous detection.Moreover, the apparatus structure that laboratory uses is huge, expensive, complicated for operation, profession is needed
Personnel operate, cannot integrate it is portable, can not on-site test.
Summary of the invention
According to can not be quantitatively evaluated in existing microalgae cell activity test method set forth above single microalgae cell activity,
Can not on-site test, detection device is expensive, sample treatment is cumbersome, the technology of the more bring universalities of amount of samples and error is asked
Topic, and provide a kind of more single microalgae cells of light intensity excitation-detection active apparatus and method.Fundamentally solve single microalgae
The Activity determination problem of cell has important scientific meaning and realistic price for environmental science.
The technological means that the present invention uses is as follows:
The active device of a kind of more single microalgae cells of light intensity excitation-detection, comprising: light source module, micro-fluidic chip, glimmering
Light collection module, data processing module and power module；The power module respectively with light source module and fluorescent collecting module
And the input terminal of data processing module is connected；Micro-fluidic chip respectively with the output end of light source module and fluorescent collecting module
Input terminal connection；Data processing module is connect with the output end of fluorescent collecting module；It is the light source module, micro-fluidic chip, glimmering
Light collection module, data processing module and power module are incorporated into the rectangular body device of cm × cm × cm；
The micro-fluidic chip includes substrate and the coating that is fixed on substrate, the coating intaglio inlet opening, channel I,
Detection zone I, channel II, detection zone II, channel III and fluid hole；In use, being added dropwise in inlet opening thin containing single microalgae
The fluid sample of born of the same parents, liquid flow into detection zone I along channel I with fixed flow rate under the driving of Micropump, pass through extremely short channel
II flows into detection zone II, flows into fluid hole finally by channel III；
The light source module includes voltage regulator circuit, light source fixing structure and the laser fitted closely with light passing aperture member
I and laser II；
The fluorescent collecting module includes slit sheet, Red lightscreening plate and photoelectric sensor.
Further, the length in the channel II can be estimated it is known that when single microalgae cell appears in detection zone I
The time point that single microalgae cell occurs in detection zone II.
Further, the detection zone I in the I face micro-fluidic chip of laser issues weaker blue light, laser II
What the detection zone II in face micro-fluidic chip issued is stronger blue light.
Further, the substrate is sheet glass, and the coating is dimethyl silicone polymer coating.
The present invention also provides a kind of active methods of more single microalgae cells of light intensity excitation-detection, include the following steps:
Step 1: sample is added dropwise, the sample solution containing single microalgae cell is added drop-wise to the feed liquor of micro-fluidic chip
Hole；
Step 2: starter successively opens power module, light source module, fluorescent collecting module and data processing module,
Sample solution containing single microalgae cell flows into detection zone I along channel I with fixed flow rate under the driving of Micropump, leads to
It crosses extremely short channel II and flows into detection zone II, flow into fluid hole finally by channel III；
Step 3: multistage light intensity excites single microalgae cell to generate fluorescence, and the laser I in light source module issues weaker
Blue laser is radiated on the detection zone I in micro-fluidic chip, laser II issue stronger blue laser be radiated at it is micro-fluidic
On detection zone II in chip；When the single microalgae cell in sample solution successively area I and when detection zone II after testing, it is excited
Generate different fluorescence quantum yields；
Step 4: the acquisition of single microalgae cell fluorescence quantum yield, the fluorescence for the varying strength that single microalgae cell successively generates
It is filtered out by slit sheet, the Red lightscreening plate in fluorescent collecting module and is received by photoelectric sensor after veiling glare respectively；
Step 5: data analysis is carried out to above-mentioned collected single microalgae cell fluorescence quantum yield, it will be collected single micro-
Frustule fluorescence quantum yield is transmitted in data processing module through data line and carries out processing analysis；
Step 6: Activity Assessment being carried out to single microalgae cell, uses F0Indicate single microalgae cell by swashing in light source module
Light device I issues the fluorescence quantum yield that weaker blue laser excitation generates, and uses F1Indicate single microalgae cell by light source module
Laser II issues the fluorescence quantum yield that stronger blue laser excitation generates, and data processing module is according to formula Fr=(F1-F0)/
F1Obtain effective fluorescence quantum yield Fr, according to FrJudge the activity of single microalgae cell.
Further, according to effective fluorescence quantum yield F in the step 6rJudge the active process of single microalgae cell, including
Following steps:
Step 61: working as FrWhen > 0.6, indicate that the microalgae cell is in high activity state；
Step 62: as 0.3≤FrWhen≤0.6, indicate that the microalgae cell is in low activity state or the single microalgae
Cell is damaged to a certain extent but is not fatal；
Step 63: working as FrWhen < 0.3, indicates the microalgae cell dead or be at death's door.
Compared with the prior art, the invention has the following advantages that
1, the present invention solves the work that single microalgae cell can not be quantitatively evaluated in existing microalgae cell activity test method
Property, can not on-site test, detection device is expensive, sample treatment is cumbersome, more bring universalities of amount of samples and the problems such as error,
The Activity determination for fundamentally solving the problems, such as single microalgae cell, for environmental science with important scientific meaning and now
Net price value.
2, the present invention uses microfluidic platform of the micro-fluidic chip as single microalgae cell Activity determination, and relevant module also may be used
It is all module integrated in a rectangular body device of cm × cm × cm using the structure type of small volume.Relative to existing
Large-scale detection device, the present invention have it is small in size, light weight and cost is low, amount of samples is few, it is easy to carry, be able to carry out it is hand-held
The advantages that for on-site test.
3, for the present invention after sample is added in sample cell, subsequent step is intelligent completion, does not need any specialization and knows
To know, does not need the operation of specialized personnel, step is few, and it is easy to operate, solve the problems, such as that sample treatment is cumbersome.
4, the light intensity of the measurement light of light source module excitation of the present invention does not need very weak level to monitor dark adaptation
Fluorescence quantum yield, meanwhile, the light intensity for measuring light does not need to reach saturation light level to assess maximum fluorescence yield.It not only reduces
Technical difficulty, and due to not by different microalgae cell dark adaptation it is horizontal it is different limited, applicable surface is wider, detectable
Microalgae cell is not limited by type, individual morphology, size dimension, cell structure.
The present invention can be widely popularized in fields such as the Activity determinations of single microalgae cell based on the above reasons.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to do simply to introduce, it should be apparent that, the accompanying drawings in the following description is this hair
Bright some embodiments for those of ordinary skill in the art without any creative labor, can be with
It obtains other drawings based on these drawings.
Fig. 1 is structure of the detecting device schematic diagram of the present invention.
Fig. 2 is microfluidic chip structure schematic diagram of the present invention.
Fig. 3 is fluorescent collecting modular structure schematic diagram of the invention.
Fig. 4 is light source module structure schematic diagram of the invention.
In figure: 1, power module；2, light source module；3, micro-fluidic chip；4, fluorescent collecting module；5, data processing mould
Block；6, laser I；7, laser II；8, inlet opening；9, channel I；10, detection zone I；11, channel II；12, detection zone II；13,
Channel III；14, fluid hole；15, Micropump；16, slit sheet；17, Red lightscreening plate；18, photoelectric sensor.
Specific embodiment
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.The present invention will be described in detail below with reference to the accompanying drawings and embodiments.
In order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below in conjunction with the embodiment of the present invention
In attached drawing, technical scheme in the embodiment of the invention is clearly and completely described, it is clear that described embodiment is only
It is only a part of the embodiment of the present invention, instead of all the embodiments.It is real to the description of at least one exemplary embodiment below
It is merely illustrative on border, never as to the present invention and its application or any restrictions used.Based on the reality in the present invention
Example is applied, every other embodiment obtained by those of ordinary skill in the art without making creative efforts all belongs to
In the scope of protection of the invention.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to exemplary embodiments of the present invention.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
Unless specifically stated otherwise, positioned opposite, the digital table of the component and step that otherwise illustrate in these embodiments
It is not limited the scope of the invention up to formula and numerical value.Simultaneously, it should be clear that for ease of description, each portion shown in attached drawing
The size divided not is to draw according to actual proportionate relationship.Technology known for person of ordinary skill in the relevant, side
Method and equipment may be not discussed in detail, but in the appropriate case, and the technology, method and apparatus should be considered as authorizing explanation
A part of book.In shown here and discussion all examples, appointing should be construed as merely illustratively to occurrence, and
Not by way of limitation.Therefore, the other examples of exemplary embodiment can have different values.It should also be noted that similar label
Similar terms are indicated in following attached drawing with letter, therefore, once it is defined in a certain Xiang Yi attached drawing, then subsequent attached
It does not need that it is further discussed in figure.
In the description of the present invention, it is to be understood that, the noun of locality such as " front, rear, top, and bottom, left and right ", " it is laterally, vertical,
Vertically, orientation or positional relationship indicated by level " and " top, bottom " etc. is normally based on orientation or position shown in the drawings and closes
System, is merely for convenience of description of the present invention and simplification of the description, in the absence of explanation to the contrary, these nouns of locality do not indicate that
It must have a particular orientation or be constructed and operated in a specific orientation with the device or element for implying signified, therefore cannot manage
Solution is limiting the scope of the invention: the noun of locality " inside and outside " refers to inside and outside the profile relative to each component itself.
For ease of description, spatially relative term can be used herein, as " ... on ", " ... top ",
" ... upper surface ", " above " etc., for describing such as a device shown in the figure or feature and other devices or spy
The spatial relation of sign.It should be understood that spatially relative term is intended to comprising the orientation in addition to device described in figure
Except different direction in use or operation.For example, being described as if the device in attached drawing is squeezed " in other devices
It will be positioned as " under other devices or construction after part or construction top " or the device of " on other devices or construction "
Side " or " under its device or construction ".Thus, exemplary term " ... top " may include " ... top " and
" in ... lower section " two kinds of orientation.The device can also be positioned with other different modes and (is rotated by 90 ° or in other orientation), and
And respective explanations are made to the opposite description in space used herein above.
In addition, it should be noted that, limiting components using the words such as " first ", " second ", it is only for be convenient for
Corresponding components are distinguished, do not have Stated otherwise such as, there is no particular meanings for above-mentioned word, therefore should not be understood as to this
The limitation of invention protection scope.
Embodiment 1
As shown in Figure 1, the present invention provides a kind of active devices of more single microalgae cells of light intensity excitation-detection, comprising:
Light source module, micro-fluidic chip, fluorescent collecting module, data processing module and power module；The power module respectively with
Light source module is connected with the input terminal of fluorescent collecting module and data processing module；Micro-fluidic chip respectively with light source module
Output end is connected with the input terminal of fluorescent collecting module；Data processing module is connect with the output end of fluorescent collecting module；It is described
Light source module, micro-fluidic chip, fluorescent collecting module, data processing module and power module are incorporated into a 79mm × 49mm
In the rectangular body device of × 43 mm (length×width×height)；
As shown in Fig. 2, micro-fluidic chip includes substrate and the coating that is fixed on substrate, substrate is sheet glass, and coating is
Dimethyl silicone polymer coating, the coating intaglio inlet opening, channel I, detection zone I, channel II, detection zone II, III and of channel
Fluid hole；In use, be added dropwise the fluid sample containing single single microalgae cell in inlet opening, liquid under the driving of Micropump with
Fixed flow rate flows into detection zone I along channel I, flows into detection zone II by extremely short channel II, flows into finally by channel III
Fluid hole；The length in channel II can estimate single microalgae cell and exist it is known that when single microalgae cell appears in detection zone I
The time point that detection zone II occurs.Light source module includes voltage regulator circuit, light source fixing structure and closely pastes with light passing aperture member
The laser I and laser II of conjunction；Detection zone I in I face micro-fluidic chip of laser issues weaker blue light, laser II
What the detection zone II in face micro-fluidic chip issued is stronger blue light.Fluorescent collecting module includes slit sheet, red filter
Piece and photoelectric sensor.
Embodiment 2
On the basis of embodiment 1, the present invention also provides a kind of more single microalgae cells of light intensity excitation-detection are active
Method includes the following steps:
Step 1: sample is added dropwise, the sample solution containing single microalgae cell is added drop-wise to the feed liquor of micro-fluidic chip
Hole；
Step 2: starter successively opens power module, light source module, fluorescent collecting module and data processing module,
Sample solution containing single microalgae cell flows into detection zone I along channel I with fixed flow rate under the driving of Micropump, leads to
It crosses extremely short channel II and flows into detection zone II, flow into fluid hole finally by channel III；
Step 3: multistage light intensity excites single microalgae cell to generate fluorescence, and the laser I in light source module issues weaker
Blue laser is radiated on the detection zone I in micro-fluidic chip, laser II issue stronger blue laser be radiated at it is micro-fluidic
On detection zone II in chip；When the single microalgae cell in sample solution successively area I and when detection zone II after testing, it is excited
Generate different fluorescence quantum yields；
Step 4: the acquisition of single microalgae cell fluorescence quantum yield, the fluorescence for the varying strength that single microalgae cell successively generates
It is filtered out by slit sheet, the Red lightscreening plate in fluorescent collecting module and is received by photoelectric sensor after veiling glare respectively；
Step 5: data analysis is carried out to above-mentioned collected single microalgae cell fluorescence quantum yield, it will be collected single micro-
Frustule fluorescence quantum yield is transmitted in data processing module through data line and carries out processing analysis；
Step 6: Activity Assessment being carried out to single microalgae cell, uses F0Indicate single microalgae cell by swashing in light source module
Light device I issues the fluorescence quantum yield that weaker blue laser excitation generates, and uses F1Indicate single microalgae cell by light source module
Laser II issues the fluorescence quantum yield that stronger blue laser excitation generates, and data processing module is according to formula Fr=(F1-F0)/
F1Obtain effective fluorescence quantum yield Fr, according to FrJudge the activity of single microalgae cell.
Step 61: working as FrWhen > 0.6, indicate that the single microalgae cell is in high activity state；
Step 62: as 0.3≤FrWhen≤0.6, indicate that the single microalgae cell is in low activity state or this is single
Microalgae cell is damaged to a certain extent but is not fatal；
Step 63: working as FrWhen < 0.3, indicates the single microalgae cell dead or be at death's door.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Pipe present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that: its according to
So be possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features into
Row equivalent replacement；And these are modified or replaceed, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
Claims (6)
1. a kind of active device of more single microalgae cells of light intensity excitation-detection characterized by comprising light source module, miniflow
Control chip, fluorescent collecting module, data processing module and power module；The power module respectively with light source module and fluorescence
The input terminal of acquisition module and data processing module is connected；Micro-fluidic chip is adopted with the output end of light source module and fluorescence respectively
Collect the input terminal connection of module；Data processing module is connect with the output end of fluorescent collecting module；It is the light source module, micro-fluidic
Chip, fluorescent collecting module, data processing module and power module are incorporated into a length 79mm × width 49mm × height
In the rectangular body device of 43mm；
The micro-fluidic chip includes substrate and the coating that is fixed on substrate, the coating intaglio inlet opening, channel I, detection
Area I, channel II, detection zone II, channel III and fluid hole；In use, being added dropwise in inlet opening containing single microalgae cell
Fluid sample, liquid flow into detection zone I along channel I with fixed flow rate under the driving of Micropump, are flowed by extremely short channel II
Enter detection zone II, flows into fluid hole finally by channel III；
The light source module include voltage regulator circuit, light source fixing structure and the laser I fitted closely with light passing aperture member and
Laser II；
The fluorescent collecting module includes slit sheet, Red lightscreening plate and photoelectric sensor.
2. the active device of more single microalgae cells of light intensity excitation-detection according to claim 1, which is characterized in that described
The length in channel II can estimate single microalgae cell in detection zone II it is known that when single microalgae cell appears in detection zone I
The time point of appearance.
3. the active device of more single microalgae cells of light intensity excitation-detection according to claim 1, which is characterized in that described
Detection zone I in I face micro-fluidic chip of laser issues weaker blue light, the detection in II face micro-fluidic chip of laser
What area II issued is stronger blue light.
4. the active device of more single microalgae cells of light intensity excitation-detection according to claim 1, which is characterized in that described
Substrate is sheet glass, and the coating is dimethyl silicone polymer coating.
5. a kind of active method of more single microalgae cells of light intensity excitation-detection, which comprises the steps of:
Step 1: sample is added dropwise, the sample solution containing single microalgae cell is added drop-wise to the inlet opening of micro-fluidic chip；
Step 2: starter is successively opened power module, light source module, fluorescent collecting module and data processing module, is contained
The sample solution of single microalgae cell flows into detection zone I along channel I with fixed flow rate under the driving of Micropump, passes through pole
Short channel II flows into detection zone II, flows into fluid hole finally by channel III；
Step 3: multistage light intensity excites single microalgae cell to generate fluorescence, and the laser I in light source module issues weaker blue
On detection zone I in micro-fluidic chip, laser II issues stronger blue laser and is radiated at micro-fluidic chip laser irradiation
In detection zone II on；When the single microalgae cell in sample solution successively area I and when detection zone II after testing, it is excited to generate
Different fluorescence quantum yields；
Step 4: the acquisition of single microalgae cell fluorescence quantum yield, the fluorescence difference for the varying strength that single microalgae cell successively generates
It is filtered out by slit sheet, the Red lightscreening plate in fluorescent collecting module and is received by photoelectric sensor after veiling glare；
Step 5: data analysis is carried out to above-mentioned collected single microalgae cell fluorescence quantum yield, collected single microalgae is thin
Born of the same parents' fluorescence quantum yield is transmitted in data processing module through data line and carries out processing analysis；
Step 6: Activity Assessment being carried out to single microalgae cell, uses F0Indicate single microalgae cell by the laser I in light source module
The fluorescence quantum yield that weaker blue laser excitation generates is issued, F is used1Indicate single microalgae cell by the laser in light source module
II issues the fluorescence quantum yield that stronger blue laser excitation generates, and data processing module is according to formula Fr=(F1-F0)/F1It obtains
Ratio Fr, according to ratio FrJudge the activity of single microalgae cell.
6. the active method of more single microalgae cells of light intensity excitation-detection according to claim 5, which is characterized in that described
According to ratio F in step 6rJudge the active process of single microalgae cell, includes the following steps:
Step 61: working as FrWhen > 0.6, indicate that the single microalgae cell is in high activity state；
Step 62: as 0.3≤FrWhen≤0.6, indicate that the single microalgae cell is in low activity state or the single microalgae is thin
Born of the same parents are damaged to a certain extent but are not fatal；
Step 63: working as FrWhen < 0.3, indicates the single microalgae cell dead or be at death's door.
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| 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 |
| JP2021528970A 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 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020114327A1 (en) * | 2018-12-06 | 2020-06-11 | 大连海事大学 | Multi-light intensity excited apparatus and method for detecting activity of single microalgae cell |
| CN112461751A (en) * | 2020-10-16 | 2021-03-09 | 江苏大学 | Cancer cell activity detection and evaluation device and method based on multi-adhesion strength fusion |
| CN114717101A (en) * | 2022-03-10 | 2022-07-08 | 大连海事大学 | Double-field-of-view microalgae activity analysis device and method based on microfluidic light filtering chip |
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| CN114717101B (en) * | 2022-03-10 | 2024-09-27 | 大连海事大学 | Double-view-field microalgae activity analysis device and method based on microfluidic filter chip |
Also Published As
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| WO2020114327A1 (en) | 2020-06-11 |
| JP2022509803A (en) | 2022-01-24 |
| JP7266324B2 (en) | 2023-04-28 |
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