CN106676004B - The method of micro-fluidic culture apparatus and its culture cell of application or microorganism - Google Patents
The method of micro-fluidic culture apparatus and its culture cell of application or microorganism Download PDFInfo
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- CN106676004B CN106676004B CN201510756962.4A CN201510756962A CN106676004B CN 106676004 B CN106676004 B CN 106676004B CN 201510756962 A CN201510756962 A CN 201510756962A CN 106676004 B CN106676004 B CN 106676004B
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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
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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
- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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- 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
- C12N1/20—Bacteria; Culture media therefor
Abstract
This provides a kind of micro-fluidic culture apparatus, including substrate and cover plate, and cover plate opens up fluted on one side, and cover plate sets a reeded cover and is located on substrate；Liquid supplying passage, gas delivery channels and gas chamber are formed between the upper surface of substrate and the groove of cover plate, gas chamber is arranged around liquid supplying passage, liquid supplying passage is connected with gas delivery channels by gas chamber, and the cofferdam for limiting liquid outflow liquid supplying passage is equipped between liquid supplying passage and gas chamber；Gas delivery channels offer the venthole and gas vent being connected with the external world；Liquid supplying passage offers the infusion hole and drainage hole being connected with the external world；Liquid supplying passage offers culture microchamber.The present invention can be used for cultivating cell or microorganism in gas-liquid interface differential growth and observable its growing state.
Description
Technical field
The present invention relates to micro-fluidic and biological cell culture fields, it particularly relates to micro-fluidic culture apparatus and application
It cultivates the method for cell or microorganism.
Background technique
In nature, there are the alternatively distributed microenvironments of many gas-liquid interfaces, such as soil gap, lake and ocean, people
The bronchus etc. of class.Some microorganisms and cell such as filamentous fungi, actinomyces, algae, bronchial epithelial cell etc. are in gas-liquid circle
Face shows the special differentiation form being adapted with environment.Streptomyces coelicolor (Streptomyces coelicolor) is
The trichobacteria being present in soil has complicated life cycle, becomes and studies multicellular differentiated mode prokaryotes (K.et al.Streptomyces morphogenetics:dissecting differentiation in a
Filamentous bacterium.Nature Rev.Microbiol.2009.7 (1): p.36-49), genome sequencing is
Through completing.Streptomycete can generate the secondary metabolite of a series of complex, such as antibiotic, anti-tumor agent, immunosuppressor
Deng (S.D.Bentley, et al.Complete genome sequence of the model actinomycete
Streptomyces coelicolor A3 (2) .Nature.2002.417 (6885): p.141-7.), secondary metabolite point
The starting secreted is often consistent with the differentiation of mycelia.Therefore, study streptomycete generation of the Growth and Differentiation to secondary metabolite,
Using with important directive significance.
The culture observation method of filamentous fungi used in traditional experiment room and actinomyces is mainly plate inserted sheet method, main
Defect is real-time dynamicly observe the process of biology growing development, and complicated operation, can not grow item to it
Part is accurately controlled.
Micro-fluidic (Microfluidics) is that skin nanoliter volumes fluid is manipulated using the channel network of micro-meter scale
Science and technology.Micro-fluidic chip can be handled and be analyzed to denier sample and reagent, realize high-resolution, highly sensitive
The separation and detection of degree, it is at low cost, shorten analysis time (G.M.Whitesides, et al.The origins and the
future of microfluidics.Nature.2006.442(7101):p.368-373).Function is carried out on micro-fluidic chip
Integrated chip lab (Lab-On-a-Chip) technology of energy unit, which is applied to biological study, can make complicated experiment flow
Simplify, sample volume is greatly reduced, reduces the consumption of reagent, obtain information as much as possible from precious sample, advises
The processing of modelling and detection gross sample provide bigger controlling and foresight to the change in time and space of cell micro-environment
(E.K.Sackmann,et al.The present and future role of microfluidics in
biomedical research.Nature.2014.507(7491):p.181-189)。
Using the control that micro-fluidic chip carries out micro-meter scale gas-liquid interface need to gas-liquid interface tension and micro-structure into
The accurate design of row and control.Difficult point is the location control for liquid level.There is gas-liquid interface differentiation for actinomyces etc.
RESEARCH ON CELL-BIOLOGY once had been reported that the actinomyces of culture and screening from soil using the drop high throughput of picoliters volume
(E.Zang,et al.Real-time image processing for label-free enrichment of
Actinobacteria cultivated in picolitre droplets.Lab Chip.2013.13(18):p.3707)。
It yet there are no the report of the micro fluidic device for realizing gas-liquid interface differential growth and spore collection.
Summary of the invention
The present invention provides a kind of micro-fluidic culture apparatus and its culture cell of application or the methods of microorganism, can be used for training
Cell or microorganism are supported in gas-liquid interface differential growth and observable its growing state.
One aspect of the present invention, provides a kind of micro-fluidic culture apparatus, including substrate and cover plate, and the cover plate is opened on one side
Equipped with groove, the cover plate sets a reeded cover and is located on the substrate；
Liquid supplying passage, gas delivery channels and gas are formed between the upper surface of the substrate and the groove of the cover plate
Fluid chamber, the gas chamber are arranged around the liquid supplying passage, and the liquid supplying passage and gas conveying are logical
Road is connected by the gas chamber, is equipped between the liquid supplying passage and the gas chamber for limiting liquid flow
The cofferdam of the liquid supplying passage out；
The gas delivery channels offer the venthole and gas vent being connected with the external world；The liquid supplying passage is opened
Equipped with the infusion hole and drainage hole being connected with the external world；
The liquid supplying passage offers culture microchamber.
Above-described micro-fluidic culture apparatus, the cofferdam structure are conveyed around the culture microchamber and the liquid
Channel is arranged, 0.5 μm to 5 μm of substrate upper surface described in the distance from top in the cofferdam.
Above-described micro-fluidic culture apparatus, the height of the gas chamber are 0.5 μm of -1mm.
Above-described micro-fluidic culture apparatus, the culture microchamber is in the two sides of the liquid supplying passage or side battle array
Column distribution.
Above-described micro-fluidic culture apparatus, the mouth for the side that the culture microchamber is connected to the liquid supplying passage
Diameter is gradually reduced.
Above-described micro-fluidic culture apparatus, open ended liquor capacity is the μ of 0.01nL~100 in the culture microchamber
L。
Above-described micro-fluidic culture apparatus, the length of the liquid supplying passage are 5mm~200mm, the width in channel
Degree is 50 μm of -10mm, and depth is 10 μm of -1mm.
Above-described micro-fluidic culture apparatus, the width of the gas delivery channels are 50-10mm, depth is 10 μm~
2mm。
Above-described micro fluidic device, equipped with can be used for observing the culture microchamber and the gas chamber coupling part
Structure.
Above-described micro fluidic device, the cover plate are to be detachably connected with the substrate.
Another aspect of the present invention provides a kind of observable cell or microorganism in the culture of gas-liquid interface Growth and Differentiation
Method includes the following steps:
A) cell or microorganism suspension are inoculated in liquid supplying passage or culture microchamber: by cell or microorganism suspension from
The infusion hole of upper any device injects in the liquid supplying passage and the culture microchamber, and cell or microorganism are being trained
Support that microchamber inner core on piece is adherent or suspension growth；
B) it is passed through culture medium: fluid nutrient medium needed for cell or microculture is passed through described in infusion hole injection
Liquid supplying passage；
C) it is passed through gas: gas needed for cell or microculture is gentle by venthole injection gas delivery channels
Fluid chamber；Ventilation treatment is not done for the cell or microorganism that can grow by air；
D) cell or microorganism growth and development process are observed and record in real time: introducing the cell or micro- of the culture microchamber
Biology is grown in the culture microchamber, is grown to after certain phase in the gas-liquid interface for cultivating microchamber and the gas chamber
Broken up, the gas first portion of cell or microorganism can be grown into the gas chamber, observe and record growing state.
Micro fluidic device provided by the invention can accommodate the liquid supplying passage and training of cell or microorganism in device
It supports in microchamber, persistently supplies fluid nutrient medium by liquid supplying passage, make cell or microorganism can be in sufficient nutrition supply
Under, continued propagation and Development And Differentiation.Air or culture are full of in the gas chamber around liquid supplying passage and culture microchamber
Required special proportion gas, cell can pass through gas-liquid interface by the gap between the cofferdam and substrate at culture microchamber edge,
It is grown in gas chamber, completes its complete gas hair tonic and educate atomization.Transparent glass, organic glass can be used in micro fluidic device
The materials such as glass, polycarbonate, are facilitated and are imaged using optical microscopy, realize the sight of cell or microorganism complete lifecycle
It examines；It can be by carrying out accurate regulation and control to culture medium, by changing different carbon source, nitrogen source, pH value or test
The condition of liquid and gas with various studies the influence of liquid and gas to cell or microorganism Growth and Differentiation.
The device of the invention can also be passed through glutaraldehyde by liquid input channel after cell or microculture are mature
Equal fixers, cell is fixed into form, opens cover plate, carries out high-resolution shape to cell or microorganism using electron microscope
State credit analysis and Nanoscale Surface structural analysis.
Detailed description of the invention
Fig. 1 is the micro-fluidic culture apparatus structural schematic diagram that the embodiment of the present invention 1 provides；
Fig. 2 is the sectional side elevation of micro-fluidic culture apparatus of the invention along the direction A-A of Fig. 1；
Fig. 3 is cell or microorganism growing state schematic diagram in the present invention micro-fluidic culture apparatus shown in Fig. 2；
Fig. 4 is the experiment flow schematic diagram of the embodiment of the present invention 1；
Fig. 5 is that pigment is full of in the microchamber and liquid supplying passage of the micro-fluidic culture apparatus material object of the embodiment of the present invention 1
The micro- enlarged photograph of solution；
Fig. 6 A is the bright of growth in streptomyces coelicolor 0 hour in the microchamber of the micro-fluidic culture apparatus of the embodiment of the present invention 1
Field micro-imaging observation；
Fig. 6 B is to grow for streptomyces coelicolor culture 20 hours in the microchamber of the micro-fluidic culture apparatus of the embodiment of the present invention 1
Light field micro-imaging observation；
Fig. 6 C is to grow for streptomyces coelicolor culture 45 hours in the microchamber of the micro-fluidic culture apparatus of the embodiment of the present invention 1
Light field micro-imaging observation；
Fig. 7 is the structural schematic diagram of the micro-fluidic culture apparatus of the embodiment of the present invention 2；
Fig. 8 is the partial enlarged view of the micro-fluidic culture apparatus of the embodiment of the present invention 2；
Fig. 9 is the pictorial diagram of the micro-fluidic culture apparatus of the embodiment of the present invention 2.
In the figures above, 1 is substrate, and 2 be cover plate；
21 be liquid supplying passage, and 211 be infusion hole, and 212 be drainage hole, and 213 be culture microchamber；
22 be gas delivery channels, and 221 be venthole, and 222 be gas vent；
23 be gas chamber；
24 be cofferdam；
25 be second liquid transfer passage.
Specific embodiment
Below in conjunction with drawings and examples, a specific embodiment of the invention is described in more details, so as to energy
The advantages of enough more fully understanding the solution of the present invention and its various aspects.
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Embodiment 1
Shown in Fig. 1, the structural schematic diagram of the micro fluidic device of the embodiment of the present invention 1, including substrate 1 and cover plate 2, cover plate 2
Open up on one side fluted, cover plate 2 sets a reeded cover and is located on substrate 1.
Liquid supplying passage 21, gas delivery channels 22 and gas are formed between the upper surface of substrate 1 and the groove of cover plate 2
Chamber 23, gas delivery channels 22 are arranged around liquid supplying passage 21, and liquid supplying passage 21 and gas delivery channels 22 are logical
Gas chamber 23 is crossed to be connected.Liquid supplying passage width is 500 μm, length 20mm, and depth is 40 μm.
It is 50 μm that array in the present embodiment equipped with microtrabeculae composition between substrate 1 and cover plate 2, which is many a length and width, a height of
1.5 μm of microtrabeculae, can play the role of supporting cover plate 2, one 1.5 μm of slit be formed between substrate 1 and cover plate 2, i.e.,
Form gas chamber 23.
Gas delivery channels 22 offer the venthole 221 and gas vent 222 being connected with the external world；Liquid supplying passage 21
The infusion hole 211 and drainage hole 212 being connected with the external world are offered, the diameter of infusion hole 211 is 0.8mm.Solution interface diameter
For 0.6mm or so, can directly be connect with the syringe of the Teflon pump line of connecting external diameter 0.8mm by pump line.
Liquid supplying passage 21 offers culture microchamber 213, and the length of culture microchamber 213 is 150 μm, and width is 100 μm,
Maximum height is 15 μm, and the side bore that culture microchamber 213 is connected with liquid supplying passage 21 is gradually reduced.
The substrate 1 and cover plate 2 of micro fluidic device are glass (perfluor silanization) material, whole clearing, convenient for observation note
Record.
Substrate 1 and 2 surface of cover plate in the present embodiment have done silanization treatment, surface hydrophobicity, solution surface tension compared with
Greatly, the bore that surface-hydrophobicized processing and culture microchamber 213 reduce guarantees that solution can be retained in the micro- indoor cofferdam area of culture
Within domain, there is no leakages.The fixed requirement of specification for cultivating microchamber 213, can be adjusted according to the cell to be cultivated.Training
Supporting the bore that microchamber 213 reduces can also guarantee when cell is grown in microchamber not by caused by liquid flowing in transfer passage
The influence of shearing force.
The brief step that micro fluidic device of the invention uses is as shown in figure 4, specifically include that
Cell or microorganism suspension are inoculated in microchamber: choosing streptomyces coelicolor A3 (2) as type strain.Sky blue chain
Mould has complicated life cycle, including machine substrate mycelium, aerial hyphae and fibrillae of spores stage, is that research is multicellular differentiated
Model organism.A3 (2) is scraped from solid plate, is placed in MM (Minimal Medium) culture medium, is filtered out with cotton
Aerial hyphae, only spore suspension is made in remaining spore, measurement OD=0.1 or so；Spore suspension is drawn from defeated with 10 μ L pipettors
Fluid apertures 211 is injected into liquid supplying passage 21 and culture microchamber 213, is then sucked out from drainage hole 212 by the solution in channel,
Microchamber 213 is cultivated since with closing in configuration, the liquid stream in liquid supplying passage is faint to micro- indoor impulse effect, can be retained
The streptomycete spore of introducing.
It is passed through culture medium: fluid nutrient medium is loaded into the syringe of 1mL, and syringe is fixed to PLC technology
Syringe pump on, with the speed of 100nL/min by fluid nutrient medium inject liquid supplying passage 21 in, continue 72 hours.
Be passed through gas: gas in the present embodiment is air, due to air diffusion velocity quickly, need to only keep air logical
The entrance for entering channel opens wide, that is, can guarantee the growth of aerial hyphae in gas microchamber.
Streptomyces coelicolor growth and development process is observed and record in real time: micro fluidic device is placed in the training of microscope constant temperature
It supports in cover, the growing state of streptomycete is observed by inverted fluorescence microscope, and continuously photograph to record.Fig. 6 A is 0 hour: being trained
Feeding microchamber is successfully inoculated with the spore of single streptomyces coelicolor (white arrow is signified).Fig. 6 B is 20 hours: spore is small by 20
When growth, grown substrate mycelium (white arrow signified)；Fig. 6 C is 45 hours: solution mycelia penetrates cofferdam structure, to sky
Aerial hyphae and fibrillae of spores are grown in gas microchamber (white arrow is signified).Using this micro-fluidic chip, can be clearly viewed
To streptomyces coelicolor different phase complete growth and development situation.
It should be noted that glutaraldehyde can be squeezed by solution input channel 21 when cell or microorganism growth and maturity,
Mycelia is fixed, then the cover plate of micro fluidic device is opened, utilizes the high score of electron microscope observation cell and microorganism
Resolution structure or mesostructure.
In order to further illustrate the operation of the above micro fluidic device, Fig. 5 illustrates the micro fluidic device of the embodiment of the present invention 1
Pictorial diagram.Haematochrome solution is passed through in channel.
It should be noted that can according to need the culture medium for injecting heterogeneity into liquid supplying passage, research is not
Influence of same carbon source, nitrogen source, inorganic ion and the pH to protokaryon or eukaryotic Growth and Differentiation.
Also the gas to gas delivery channels injection heterogeneity, the gas such as research oxygen, carbon dioxide be can according to need
Influence of the body to cell or microorganism Growth and Differentiation.
Embodiment 2
The control at multiple gas-liquid alternating interface also may be implemented in the present invention, to realize to complexity such as soil in natural environment
The simulation of microorganism and cell growth state in phase interface.Fig. 7 is a schematic structural view of Embodiment 2 of the present invention.
The channel design of device is similar to Example 1, is passed through unwrapping wire from intermediate wider 21 inlet of liquid supplying passage
Bacterium spore suspension, and continue to flow into culture medium, culture medium can be passed through in second liquid transfer passage 25 or contains other not
With the solution of component, it should be noted that second liquid transfer passage 25 can according to need equipped with multiple, and each liquid conveying is logical
It is provided with cofferdam identical with 1 structure of embodiment around road, is leaked out to avoid the liquid in channel, second liquid transfer passage 25
Outside is also provided with gas delivery channels 22, gas chamber 23 is formed between each liquid supplying passage, and pass through gas chamber 23
It separates, forms being alternately distributed for multiple gas-liquid interfaces.Spore growth and development in culture microchamber 213, grows substrate mycelium, connects
It is divided into aerial hyphae after contacting air, can be grown into wherein after encountering culture medium again, and reversible differentiation occurs.The width in channel
Spending range is 200 μm -1000 μm.The fluid passage width of surrounding is 100 μm, and culture 213 length of microchamber is 350 μm, and width is
200 μm, maximum height is 15 μm.
Using second liquid transfer passage 25, can after aerial hyphae Development And Differentiation to certain phase, be passed through culture medium,
Water, heavy metal solution, organic oil-based liquid or other reagent solutions observe the aerial hyphae of actinomyces when encountering different liquids
Growth and Differentiation behavior.Present method be advantageous in that multiple gas-liquid interfaces can be created continuously, and essence can be carried out to solution composition
The growth behavior of actinomyces is observed in really control in real time.
Fig. 9 is the pictorial diagram of the micro fluidic device of the embodiment of the present invention 2, and for demo function, wherein liquid conveying is logical
Microchamber 213 is passed through red pigments solution, second liquid transfer passage 25 has been passed through green pigment solution for road 21 and culture.Each
It is gas chamber between liquid supplying passage.
Finally, it should be noted that obviously, the above embodiment is merely an example for clearly illustrating the present invention, and simultaneously
The non-restriction to embodiment.For those of ordinary skill in the art, it can also do on the basis of the above description
Other various forms of variations or variation out.There is no necessity and possibility to exhaust all the enbodiments.And thus drawn
The obvious changes or variations that Shen goes out are still in the protection scope of this invention.
Claims (11)
1. a kind of micro-fluidic culture apparatus, including substrate (1) and cover plate (2), which is characterized in that
The cover plate (2) opens up fluted on one side, and the cover plate (2) sets a reeded cover and is located on the substrate (1)；
Liquid supplying passage (21) are formed between the upper surface of the substrate (1) and the groove of the cover plate (2), gas conveying is led to
Road (22) and gas chamber (23), the gas chamber (23) are arranged around the liquid supplying passage (21), and the liquid is defeated
Channel (21) is sent to be connected with the gas delivery channels (22) by the gas chamber (23), the liquid supplying passage
(21) cofferdam (24) that the liquid supplying passage (21) is flowed out for limiting liquid is equipped between the gas chamber (23)；
The gas delivery channels (22) offer the venthole (221) and gas vent (222) being connected with the external world；The liquid
Transfer passage (21) offers the infusion hole (211) and drainage hole (212) being connected with the external world；
The liquid supplying passage (21) offers culture microchamber (213).
2. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the cofferdam (24) is around the culture microchamber
(213) and the liquid supplying passage (21) is arranged, 0.5 μ of substrate (1) upper surface described in the distance from top of the cofferdam (24)
M to 5 μm.
3. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the height of the gas chamber (23) is 0.5 μ
m-1mm。
4. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the culture microchamber (213) is in the liquid
The two sides of transfer passage (21) or side array distribution.
5. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the culture microchamber (213) and the liquid
The bore of the side of transfer passage (21) connection is gradually reduced.
6. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the culture microchamber (213) is open ended molten
Liquid product is 0.01nL-100 μ L.
7. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the length of the liquid supplying passage (21) is
5mm-200mm, the width in channel are 50 μm of -10mm, and depth is 10 μm of -1mm.
8. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the width of the gas delivery channels (22) is
50-10mm, depth are 10 μm of -2mm.
9. micro-fluidic culture apparatus as described in claim 1, which is characterized in that equipped with can be used for observing the culture microchamber
(213) with the structure of the gas chamber (23) coupling part.
10. micro-fluidic culture apparatus as described in claim 1, which is characterized in that the cover plate (2) is with the substrate (1)
It is detachably connected.
11. a kind of observable cell or microorganism are in the cultural method of gas-liquid interface Growth and Differentiation, which is characterized in that including as follows
Step:
A) cell or microorganism suspension are inoculated in liquid supplying passage or culture microchamber: by cell or microorganism suspension (3) from power
Benefit requires infusion hole (211) the injection liquid supplying passage (21) of any device in 1 to 10 and the culture micro-
In room (213), cell or the microorganism adherent or suspension growth in culture microchamber (213)；
B) it is passed through culture medium: fluid nutrient medium needed for cell or microculture is injected into institute by the infusion hole (211)
State liquid supplying passage (21)；
C) it is passed through gas: gas needed for cell or microculture is injected into gas delivery channels by venthole (221)
(22) and gas chamber (23)；For the cell that can be grown by air or microorganism, ventilation treatment is not done；
D) cell or microorganism growth and development process observe in real time with record: introduce it is described culture microchamber (213) cell or
Microorganism growth in the culture microchamber (213), grows to after certain phase in the culture microchamber (213) and the air chamber
The gas-liquid interface of room (23) is broken up, and the gas first portion of cell or microorganism can be grown into the gas chamber (23), observation
Record growing state.
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| CN108753596B (en) * | 2018-04-09 | 2022-05-17 | 暨南大学 | Microchamber microfluidic system for detecting microorganism growth images |
| CN112403542B (en) * | 2020-11-05 | 2021-09-28 | 清华大学 | Micro-fluidic chip for quantitatively researching biofilm and manufacturing method thereof |
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