CN108517284A - It is a kind of to be used to study algal grown and the micro-fluidic chip of reproduction - Google Patents
It is a kind of to be used to study algal grown and the micro-fluidic chip of reproduction Download PDFInfo
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- CN108517284A CN108517284A CN201810226044.4A CN201810226044A CN108517284A CN 108517284 A CN108517284 A CN 108517284A CN 201810226044 A CN201810226044 A CN 201810226044A CN 108517284 A CN108517284 A CN 108517284A
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
- C12M23/02—Form or structure of the vessel
- C12M23/16—Microfluidic devices; Capillary tubes
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
- C12M29/06—Nozzles; Sprayers; Spargers; Diffusers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Abstract
The invention belongs to algal grown reproduction technique fields, particularly for the micro-fluidic chip of research algal grown and breeding.The micro-fluidic chip of the present invention is made of sample channel layer, valve control layer and substrate layer；Sample channel layer includes four parts being sequentially connected in series:Sample inlet, frond observation on Growth chamber, reproduction cell capture chamber, sample export；The function that it realizes algae culture using continuous pouring technology, reproduction cell release, is automatically separated, can observe and record the long-time of alga cells and subcellular structure in real time；And influence of the manual intervention to frond growth conditions can be avoided with the culture medium of the replacement algae of fast accurate；The life cell for being automatically separated out can be cultivated individually again.Reproduction and increment study of the chip of the present invention especially suitable for all kinds of large-scale algaes.The cost of manufacture of chip is cheap, and method is simple, and easy to operate, convenient for universal.
Description
Technical field
The invention belongs to algal grown reproduction technique fields, and in particular to for studying the micro-fluidic of algal grown and breeding
Chip.
Background technology
In recent years after body eutrophication, the algae large-scale outbreak event such as green tide and red tide takes place frequently, and has been one complete
The problem of ball.Tangleweed excessive multiplication can caused by green tide, when green tide is serious entirely inshore sea all by frond
Covering consumes the oxygen in water, causes the death of water body zooplankter and plant.And it is likely to form marshland, generates H2S gas
Body all causes serious injury to the ecosystem of entire ocean.On the other hand, green tide can also influence the inshore activity of people,
Aquatic products industry is damaged, shipping traffic etc. is influenced.So studying the mechanism of alga eruption to solve the environment of green tide or red tide formation
Problem is particularly significant.
Existing research shows that some algae that can quickly breed include mainly since there are many modess of reproduction for it
It sexual reproduction, vegetative propagation and nourishes and generates.Green tide primarily forms algae Enteromorpha, and the vegetative propagation mainly by reproduction cell is able to greatly
Scale Growth.Almost all of Enteromorpha cell may be converted into reproduction cell capsule, and a sporangium about generates 8 spores, and one
A gametocyst about generates 16 gametes.1cm according to the study2Enteromorpha（Single side）10 can about be generated6-107A spore and gamete, 1g watersides
Tongue（Fresh weight）10 can theoretically be grown up to8-109A new frond.Two anisotropic gametes can be combined into zygote to grow up to new algae
Body can also grow up to new frond as spore by vegetative propagation, and spore or gamete under suitable conditions can also
Self-replacation is carried out, a large amount of individuals are divided out.Explosive it will increase in short, once possessing suitable growth conditions frond.And
And spore or gamete are more sensitive to the variation of the factor of environment relative to ripe frond, it is easier to which generation environment is coerced.Institute
It is particularly significant to the formation mechenism for opening green tide or red tide with the reproduction cell for studying algae.
There are some problems to need to solve optimization in the research done to algae reproduction and growth：It can not achieve training in situ
It supports, culture medium control is not accurate；Spatial resolution cannot meet frustule or subcellular structure metamorphosis（Spore or gamete
Differentiation, the cohesive process etc. of two gametes）Observation；The growth conditions of frond cannot be observed in real time；The reproduction cell of algae cannot
It is automatically separated.These problems are all hopeful through micro-fluidic solution.Microfluidic chip technology (Microfluidics) is can be life
The basic operation units such as object, chemistry, the sample preparation of medical analysis process, reaction, separation, detection are integrated into one piece of micro-meter scale
Chip on, be automatically performed analysis overall process.
The research method of algae is confined to macro -examination method at present, and the generation releasing mechanism of reproduction cell is not yet clear,
So Burst mechanism needs to be probed into.Micro-fluidic chip has been widely applied in the culture studies of zooblast, but is seldom answered
Use plant cell field.The present invention devises a kind of micro-fluidic chip and studies algal grown reproduction from micro-meter scale thus.
The chip of design meets algal grown, the needs that reproduction cell is diffused and captured and reproduction cell is individually selectively cultivated.
Invention content
Present invention aims at providing one kind can finely manipulating fluid, and production method is simple, versatile micro-
See the micro-fluidic chip of research algal grown and reproduction.
Provided by the present invention for studying the micro-fluidic chip of algal grown and reproduction, using optically transparent material as base material,
Using micro-fluidic continuous pouring system, being automatically separated for algae reproduction cell is realized from microcosmic point, monitors frustule in real time
A series of grow changes with reproduction, solves tradition research method and is asked what the uneven diffusion of nutrient solution and Toxic Metabolites accumulated
Topic.Specifically, being exactly to gate frond growing element and reproduction cell processed using the pneumatic control valve of micro-fluidic chip to capture list
Member makes free reproduction frustule enter reproduction cell capturing unit, to realize the capture and selection of single reproduction frustule
Property culture.Chip can be placed in by the present invention to be connected under the fluorescence inverted microscope of computer and high-speed CCD, to acquisition point
The optical signalling for analysing the specific binding generation of the variation of fluorescence intensity, reagent or antibody and reproduction cell all can be by photosensor
Part array acquisition is simultaneously transferred to microprocessor（Computer）The database for neutralizing standard items is made comparisons, to realize automation inspection
It surveys.
Provided by the present invention for studying the micro-fluidic chip of algal grown and reproduction, if can be set according to actual needs
Dry parallel modules (such as 6-10 etc.), each modular structure are three layers：Sample channel layer, valve control layer, substrate layer.Substrate
Layer is the substrate of entire micro-fluidic chip, with support valve control layer and sample channel layer；Sample channel layer is thin for accommodating algae
Born of the same parents and nutrient solution, valve control layer is for controlling the flowing of sample channel layer liquid and the movement of algae；Wherein, sample channel layer
Including four parts:Sample inlet, frond observation on Growth chamber, reproduction cell capture chamber, sample export, four parts are successively
Series connection；Frond observation on Growth chamber be height be 300-800 μm, the cuboid that width is 300-800 μm, for online observe algae
Body growing state, and enough spaces are provided for the growth of frustule, while being provided enough for the formation of reproduction cell diffusion mouth
Space length；Reproduction cell capture chamber be height be 10-30 μm, a diameter of 50-150 μm of cylindrical pipe, for automatic
Separation, capture reproduction cell；Sample inlet uses syringe, autosampler and dress for injecting culture medium, injection culture medium
There is the syringe of culture medium to be connected；Sample export is connected with waste liquid tank, for waste liquid to be discharged.
In the present invention, valve control layer includes two control valves, is respectively arranged at the front and back of reproduction cell capture chamber；
Two control valves have valve to control entrance respectively；By pulsometer or electronic component control valve, and then control frond life
The valve switch of long observation chamber and reproduction cell capture chamber sample channel layer.The valve is semi-transparent valve, the reproduction cell of algae
（Spore and gamete）It cannot pass through, culture liquid can pass freely through.
If the valve after reproduction cell capture chamber is the first valve, the valve before reproduction cell capture chamber is the second valve
Door；When pulsometer is filled with gas from valve control entrance, corresponding valve working, liquid can pass freely through at this time, but algae
Body cell cannot pass through.When the first valve 1 works, when the second valve 2 does not work, the reproduction spore of frond cell release can be by
Reproduction cell capture chamber captures.When the work of the second valve 2, when the first valve 1 does not work, culture solution is injected by sample export,
The independent culture of frond reproduction cell may be implemented.
In the present invention, the height and depth of frond observation on Growth chamber are all bigger, are provided enough for the growth of frustule
Space, while providing sufficient space distance for the formation of reproduction cell diffusion mouth.When sample introduction, fluid nutrient medium carrying releases
The free reproduction cell come enters reproduction cell capturing unit；Reproduction cell is in spot distribution in cell capture unit, is caught
The free reproduction cell received can be quickly in the adherent attachment of cell capture unit, therefore reproduction cell can be in cell capture unit
It is captured.
In the present invention, reproduction cell capture chamber can also be used as reproduction cell and individually cultivate unit.Reproduction cell is collected
After, culture medium is injected from former outlet, different types of culture medium makes frond show different states.The reproduction of algae is thin
Born of the same parents may develop into new filamentous, brood body or carry out clone type self-replacation.The present invention can also be to the algae of attached state
Body reproduction cell carries out the environment-stress such as polycyclic aromatic hydrocarbon or heavy metal, and then realizes frond antioxidase with fluorescence analysis
Automatic detection in situ.
In the present invention, the base material of micro-fluidic chip be with flexible optically transparent material, including：Polydimethylsiloxanes
Alkane polymer（PDMS）；Hard high molecular polymer：Makrolon, polyethylene terephthalate, polymethyl-benzene olefin(e) acid first
Ester, polystyrene, polypropylene；Inorganic material：Quartz, glass.Mold materials are silicon chip.
The present invention also provides the production methods of micro-fluidic chip, are as follows：
（1）Substrate prepares：Monocrystalline silicon piece cleans deoxidation with Piranha solution, after being dried up with nitrogen, just with AZ 50XT series
After the spin-coated machine whirl coating of glue, the soft baking on constant temperature heating plate makes photoetching adhesive curing；
（2）Exposure imaging：The mask of sample cell channel layer and valve control layer is placed on the substrate spun coating, by ultraviolet exposure
After ray machine exposure, it is placed in developer for positive photoresist and develops；
（3）Re-expose：It is soft on constant temperature heating plate after the 2150 spin-coated machine whirl coatings of series of SU-8 by the silicon chip after development
It dries, makes photoetching adhesive curing.Then re-expose is carried out to frond observation chamber, dries solidification after being placed in heating plate；
（4）Silicon chip after exposure is baked, which is placed in developer for negative photoresist, to develop, and examines development to finish with isopropanol, nitrogen drying；
（5）It is hard to dry：Silicon chip is placed in heat in heating plate and fix；
（6）Cast：Dimethyl silicone polymer monomer and curing agent are pressed 5:1 and 20:1 difference mixing, is poured on corresponding mold,
In oven for curing stripping；
（7）Bonding：Microchannel chamber is formed after valve layer and sample cell channel layer aligning key are closed, then is bonded with substrate layer.
The present invention also provides above-mentioned micro-fluidic filtrating chip application method, be as follows：
（1）The a small amount of frond of picking is as experiment sample from frond female parent, it is ensured that and frond cell is in identical growth conditions,
Sporangium and gametocyst are not formed in body cell；
（2）About 200 μm -500 μm of segment for cutting frond top, the algae of the frond segment implantation micro-fluidic chip after cutting
Viewing area；
（3）After frond sample implantation success, sample inlet is connected with the syringe for enclosing culture medium, sample export and waste liquid
Slot is connected；
（4）Chip is placed under the inverted microscope for being connected to computer and high-speed CCD, by equipped with culture medium syringe with from
Dynamic injector is connected, and sample introduction speed is 25 μ l/L-60 μ l/L, and the operated pneumatic valve at capture chamber both ends is controlled by pulsometer
Switch and closure, to capture frond reproduction cell.
When individually research reproduction cell, making original outlet into injection port, injection port originally makes outlet into,
Sample introduction speed is about 25 μ l/L-60 μ l/L, injects different culture mediums or carries out different environment-stress, to realize reproduction
The independent culture of cell individually carries out in situ detection with fluorescence detection after culture to frond reproduction cell antioxidase.
The present invention devises a reproduction cell capture chip, has frond observation on Growth chamber and reproduction cell catcher
Room, and it is automatic that the chip with autosampler, fluoroscopic imaging systems constituted a frond cell based on microflow control technique
Culture and monitoring system.The system uses micro-fluidic continuous pouring system, so that cell is in state full of nutrition, improves algae
The growth rate of body cell, and utmostly avoid the manual operation in frustule growth cycle, realize frond it is long when
Between automatic real-time monitoring record.Micro-fluidic chip provided by the invention has the advantage finely manipulated to fluid, and the side of making
Method is simple, versatile, can be used in combination with various designs.
Above-mentioned micro-fluidic filter core can be used for that frond is observed online, the separation of frond reproduction cell, frond reproduction cell individually trains
Support the fields of grade.
Description of the drawings
Fig. 1 is the three-layered node composition of the micro-fluidic chip for studying algal grown and reproduction.Pipe layers are by height difference
Micro chamber composition, frond observe chamber height be higher than reproduction cell capture chamber, frond cell is in reproduction cell catcher
It can only be arranged in the form of cell monolayer in room.In reproduction capture chamber both sides, respectively there are one operated pneumatic valves, are controlled by pulsometer
The capture and individually culture of reproduction cell may be implemented in valve.
Fig. 2 is the plan view of the micro-fluidic chip for studying algal grown and reproduction.When pulsometer from valve control into
When mouth is filled with gas, corresponding valve working, liquid can pass freely through at this time, but frond cell cannot pass through.When 1 work of valve
Make, when valve 2 does not work, the reproduction spore of frond cell release can be captured by reproduction cell capture chamber.When 2 work of valve
Make, when valve 1 does not work, culture solution is injected by sample export, the independent culture of frond reproduction cell may be implemented.
Specific implementation mode
（1）Substrate prepares：Monocrystalline silicon piece is placed on Piranha solution（98% concentrated sulfuric acid：30% hydrogen peroxide）Clean 15min deoxidations
Change, rinsing rear nitrogen well with deionized water dries up, and 30min is baked at 200 DEG C.
（2）Whirl coating：By ready substrate High Pressure Absorption spin coater（Spin-Coater KW-4A, Chemat
Technology, Inc.）Spin coating position is poured on substrate center with AZ 50XT series glue, and 2000r/min rotates 2min, stands
10min reduces the bubble of silicon chip surface.
（3）Front baking：Glue-coated silicon chip is sequentially placed into baking 2min, 7min in 65 DEG C, 90 DEG C and 65 DEG C of heating plate
And 2min.
（4）Exposure：The mask of sample cell channel layer and valve control layer is placed on the substrate spun coating, uv-exposure is used
Machine exposes.
（5）Development：It is developed in draught cupboard and carries out, silicon chip is placed in developer for positive photoresist, first in the developer solution of 1/3 concentration
Glue of the middle development to silicon chip periphery is shunk inward, and the developer solution for then placing 1/5 concentration finishes.It is rinsed well with deionized water,
Nitrogen dries up.
（6）Re-expose：Sample channel layer substrate is after first time exposes, with the 2150 spin-coated machine whirl coatings of series of SU-8
Afterwards, the soft baking on constant temperature heating plate, makes photoetching adhesive curing.With the mask of frond observation unit to its former pipeline, secondary exposure is carried out
Light.
（7）After dry：It is silicon chip is rear on 65 DEG C and 95 degrees Centigrade plates successively to dry 2min and 5min, it is cooled to room temperature.
（8）Developer for negative photoresist is equally used in draught cupboard（Main component is propylene glycol methyl ether acetate PGMEA）Development is used
Isopropanol examines development to finish, nitrogen drying.
（9）It is hard to dry：Sample channel layer silicon chip is placed in 120 DEG C of heating in heating plate and fixes.
（10）PDMS polymer is poured into a mould：Dimethyl silicone polymer monomer and curing agent are pressed 5:1 is uniformly mixed, and is poured on through three
On the silicon chip of the processed sample channel layer of chloro chlorosilane, after vacuum pump eliminates bubble, it is solid to be placed in baking 1h in 80 DEG C of baking ovens
Change.
（11）Valve layer and slide basal layer：Dimethyl silicone polymer monomer and curing agent are pressed 20:1 is uniformly mixed, point
Be not poured on valve silicon chip and slide, with 2000r/min on spin coater whirl coating 1min, it is solid that 1h is then toasted in 80 DEG C of baking ovens
Change.
Bonding and making：Sample channel layer and valve layer chip are removed with silicon chip, by valve layer and sample cell channel layer school
Microchannel chamber is formed after quasi- bonding, 1h is toasted in 80 DEG C of baking ovens and is allowed to closely be bonded together.After punching again with slide layer key
It closes, the solidification overnight of 80 DEG C of last baking oven.
The frond sample cut is filled into syringe pump in the frond observation chamber of chip, the injection port of chip connects
Different sample solutions is perfused in injector.The reproduction cell released enters reproduction cell capture chamber with liquid.Pass through
The switch of pressure control valve, by reproduction cell capture in chamber.After reproduction cell captures, stops sample introduction, go out from original
Specific sample solution is perfused in sample mouth, probes into the individual environment-stress that does of reproduction cell.And using fluorescence analysis point
Analyse the variation of reproduction cell activities of antioxidant enzymes.
Claims (4)
1. a kind of for studying algal grown and the micro-fluidic chip of reproduction, which is characterized in that include multiple parallel modules, each
Modular structure is three layers：Sample channel layer, valve control layer, substrate layer；Substrate layer is the substrate of entire micro-fluidic chip, with branch
Support valve control layer and sample channel layer；Sample channel layer is for accommodating frustule and nutrient solution, and valve control layer is for controlling
The flowing of sample channel layer liquid and the movement of algae；Wherein：
The sample channel layer includes four parts:Sample inlet, frond observation on Growth chamber, reproduction cell capture chamber, sample
Product export, and four parts are sequentially connected in series；Frond observation on Growth chamber be height be 300-800 μm, the length that width is 300-800 μm
Cube for observing frond growing state online, and provides enough spaces for the growth of frustule, while being put for reproduction cell
The formation of raw edge provides sufficient space distance；Reproduction cell capture chamber be height be 10-30 μm, a diameter of 50-150 μm of circle
Cylindrical tube, for being automatically separated, capturing reproduction cell；Sample inlet is for injecting culture medium, and injection culture medium is using injection
Device, autosampler are connected with the syringe equipped with culture medium；Sample export is connected with waste liquid tank, for waste liquid to be discharged.
The valve control layer includes two control valves, is respectively arranged at the front and back of reproduction cell capture chamber；Two controls
Valve has valve to control entrance respectively；By pulsometer or electronic component control valve, and then control frond observation on Growth chamber
The valve switch of room and reproduction cell capture chamber sample channel floor.
2. according to claim 1 for studying algal grown and the micro-fluidic chip of reproduction, which is characterized in that the valve
Door is semi-transparent valve, and the reproduction cell of algae cannot pass through, and culture liquid can pass freely through.
3. the application method of micro-fluidic chip as described in claim 1, which is characterized in that be as follows：
（1）The a small amount of frond of picking is as experiment sample from frond female parent, it is ensured that and frond cell is in identical growth conditions,
Sporangium and gametocyst are not formed in body cell；
（2）About 200 μm -500 μm of segment for cutting frond top, the algae of the frond segment implantation micro-fluidic chip after cutting
Viewing area；
（3）After frond sample implantation success, sample inlet is connected with the syringe for enclosing culture medium, sample export and waste liquid
Slot is connected；
（4）Chip is placed under the inverted microscope for being connected to computer and high-speed CCD, by equipped with culture medium syringe with from
Dynamic injector is connected, and sample introduction speed is 25 μ l/L-60 μ l/L, and the operated pneumatic valve at capture chamber both ends is controlled by pulsometer
Switch and closure, to capture frond reproduction cell.
4. the application method of micro-fluidic chip as described in claim 1, which is characterized in that when individually research reproduction cell,
Make original outlet into injection port, injection port originally makes outlet into, and sample introduction speed is about 25 μ l/L-60 μ l/L, note
Enter different culture mediums or carry out different environment-stress, to realize the independent culture of reproduction cell, is individually used after culture
Fluorescence detection carries out in situ detection to frond reproduction cell antioxidase.
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