CN114208780A - Micro-fluidic chip device for nematode culture and instant fixation - Google Patents
Micro-fluidic chip device for nematode culture and instant fixation Download PDFInfo
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- CN114208780A CN114208780A CN202111646562.XA CN202111646562A CN114208780A CN 114208780 A CN114208780 A CN 114208780A CN 202111646562 A CN202111646562 A CN 202111646562A CN 114208780 A CN114208780 A CN 114208780A
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- 241000244206 Nematoda Species 0.000 title claims abstract description 123
- 239000004205 dimethyl polysiloxane Substances 0.000 claims abstract description 24
- 235000013870 dimethyl polysiloxane Nutrition 0.000 claims abstract description 24
- CXQXSVUQTKDNFP-UHFFFAOYSA-N octamethyltrisiloxane Chemical compound C[Si](C)(C)O[Si](C)(C)O[Si](C)(C)C CXQXSVUQTKDNFP-UHFFFAOYSA-N 0.000 claims abstract description 24
- 238000004987 plasma desorption mass spectroscopy Methods 0.000 claims abstract description 24
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 claims abstract description 24
- 239000001963 growth medium Substances 0.000 claims abstract description 16
- 238000009630 liquid culture Methods 0.000 claims abstract description 10
- 239000011521 glass Substances 0.000 claims abstract description 8
- 239000000758 substrate Substances 0.000 claims abstract description 8
- 239000002609 medium Substances 0.000 claims description 17
- 239000007788 liquid Substances 0.000 claims description 14
- 238000004458 analytical method Methods 0.000 claims description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
- 238000012634 optical imaging Methods 0.000 claims description 5
- 241000588724 Escherichia coli Species 0.000 claims description 4
- 230000001105 regulatory effect Effects 0.000 claims description 3
- 238000007906 compression Methods 0.000 claims description 2
- 230000035790 physiological processes and functions Effects 0.000 abstract description 5
- 238000011084 recovery Methods 0.000 abstract description 3
- 238000007599 discharging Methods 0.000 abstract 2
- BHPQYMZQTOCNFJ-UHFFFAOYSA-N Calcium cation Chemical compound [Ca+2] BHPQYMZQTOCNFJ-UHFFFAOYSA-N 0.000 description 9
- 229910001424 calcium ion Inorganic materials 0.000 description 9
- 238000005516 engineering process Methods 0.000 description 9
- 230000000968 intestinal effect Effects 0.000 description 8
- 239000000523 sample Substances 0.000 description 8
- 230000008859 change Effects 0.000 description 7
- 238000005457 optimization Methods 0.000 description 7
- 238000010586 diagram Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 3
- 238000003384 imaging method Methods 0.000 description 3
- 238000012258 culturing Methods 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 1
- 241000244203 Caenorhabditis elegans Species 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005056 compaction Methods 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 238000003825 pressing Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000000384 rearing effect Effects 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Environmental Sciences (AREA)
- Health & Medical Sciences (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Zoology (AREA)
- Dispersion Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Analytical Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Hematology (AREA)
- Clinical Laboratory Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention discloses a micro-fluidic chip device for nematode culture and immediate fixation, which comprises a glass substrate, wherein a nematode culture chamber is arranged on the glass substrate, the nematode culture chamber is of a circular structure surrounded by fences, a liquid culture medium (CeMM) sample feeding and discharging channel and a nematode sample feeding and discharging channel are respectively arranged at the outer side of each circular fence, a circular air chamber is arranged above the nematode culture chamber, a PDMS film is arranged between the nematode culture chamber and the circular air chamber, air pressure is applied in the circular air chamber to force the PDMS film to be pressed downwards to push nematodes, the nematodes are pushed to the fences, and a slender nematode body is compressed parallel to the fences to realize fixation of the nematodes. The invention has simple structure principle and convenient operation, can realize the immediate harmless fixation in the nematode culture, and furthest reduces the influence of the fixation operation on the natural physiological state of the nematode. The circular fence is used, the nematode culture is convenient, zero loss of the nematode entering and exiting the fence can be realized, and the sample introduction and recovery rate is 100 percent.
Description
Technical Field
The invention belongs to the technical field of microfluidic chip analysis, relates to a nematode control technology, a nematode liquid culture medium culture technology and a nematode optical imaging analysis technology of a microfluidic chip, and particularly relates to a microfluidic chip device capable of culturing and instantly fixing nematodes.
Background
The model animal Caenorhabditis elegans has the advantages of rapid growth and reproduction, easy culture, small individual, simple structure, convenient molecular biological operation and the like. The nematodes are taken from soil environment, have strong vitality and are easy to culture, and the nematode culture can be divided into a solid culture medium and a liquid culture medium. Coli is the main food for nematodes cultured on a fixed Medium, and liquid media are divided into bacteria-containing and sterile media, wherein the sterile media do not contain nutrients necessary for the growth and development of nematodes, such as salts, amino acids, nucleotides, vitamins, energy substances, etc., such as C.elegans Maintenance Medium (CeMM) liquid media, available from Lu and Goetsch. The liquid culture medium is required to be domesticated for culturing the nematodes, the individual shape of the nematodes is slender, and the service life of the nematodes is prolonged by 1 time. The liquid medium culture method allows nematodes to be cultured in closed environments, for example, within microfluidic chip channels. The liquid culture medium is added with nematode food OP50 type Escherichia coli to accelerate the growth of nematodes.
The nematode body is tiny, the adult body is 1 mm long and about 80 microns wide, and the trapping operation, the fixed observation operation and the like of the nematode body are difficult to perform, and the vigorously developed micro-fluidic chip technology is widely applied to the control of the nematode due to the excellent control capability of the micro-fluidic chip technology in the micro-nano scale. The micro-fluidic chip is used for fixing the nematodes, the nematodes can be re-cultured after the experiment is finished, and the nematodes can keep better activity, so that the micro-fluidic chip is a harmless nematode fixing technology. The nematodes can no longer be fixed using conventional fixation techniques, such as the widely used glue adhesion method. Various physiological functions of the nematodes in the culture are in a natural state, and the nematodes are fixed in real time for optical imaging analysis, so that the influence of the fixing operation on the natural physiological state of the nematodes can be reduced to the greatest extent.
The invention discloses a method for immediately fixing nematodes in culture on a microfluidic chip by using a membrane down-pressure method in combination with a nematode culture technology on the chip.
Disclosure of Invention
The purpose of the invention is as follows: in order to solve the defects of the prior art, the invention provides a micro-fluidic chip device for nematode culture and instant fixation, which uses the micro-fluidic chip to realize the instant non-damage fixation of nematodes in culture.
The technical scheme is as follows: the utility model provides a can be used to nematode to cultivate and instant fixed micro-fluidic chip device, includes the glass substrate, the glass substrate on be equipped with the nematode and cultivate the room, the nematode cultivate the room for the circular space that fence structure encloses, the outside of circular fence is equipped with liquid medium (CeMM) respectively and advances the passageway of appearance and the passageway that the nematode advances appearance and goes out the appearance, the top of nematode cultivation room be equipped with a circular air cavity room, cultivate and be one deck PDMS film between room and the circular air cavity room, circular air cavity indoor apply atmospheric pressure and force PDMS film to push down, push away the nematode, extrude the nematode to circular fence, compress tightly long and thin polypide parallel to the fence, realize the fixed to the nematode.
As an optimization: the nematodes are housed in a circular fence with the diameter of 8 mm and the height of 100 microns, rectangular columns are adopted to enclose the circular fence, and the width of gaps between the circular fence is 20 microns.
As an optimization: liquid medium (CeMM) and water can freely pass through the circular fence, but nematodes can not pass through the circular fence and are restrained in the circular fence for feeding.
As an optimization: fresh liquid medium (CeMM) can enter the nematode culture chamber from the medium feeding channel at one end of the circular fence through the circular fence and flow out from the other end, and the feeding speed of the liquid medium (CeMM) is 50 microliters per minute.
As an optimization: the thickness of the PDMS film is 100 micrometers.
As an optimization: the specific mode of applying air pressure in the circular air cavity to press the PDMS film is as follows: the round air cavity chamber is filled with water, the PDMS film allows gas molecules to pass through, water molecules cannot pass through, after the round air cavity chamber is filled with water, the pressure is regulated and controlled to compress the PDMS film, the compression process needs to be carried out slowly, the nematode is pushed to the edge and then can be further compressed, and fixation of the nematode is completed.
As an optimization: after the nematodes are compacted and fixed, optical imaging analysis can be carried out; after the analysis is finished, the air pressure is removed, the nematode culture chamber can be recovered, and the nematodes can continue to move.
As an optimization: the nematode is obtained by acclimatizing liquid culture medium (CeMM) at 21 deg.C, and the CeMM culture medium contains OP50 type Escherichia coli (10)9Each per milliliter).
Has the advantages that: the device has simple structure principle and convenient operation, can realize the real-time harmless fixation in the nematode culture, and furthest reduces the influence of the fixation operation on the natural physiological state of the nematode. The circular fence is used, the nematode culture is convenient, zero loss of the nematode entering and exiting the fence can be realized, and the sample introduction and recovery rate is 100 percent.
The invention can realize the imaging detection analysis of the dynamic change of calcium ions of the intestinal cells of the nematodes, and can carry out on-chip culture on the nematodes with calcium ion fluorescent probes in the intestinal cells, and the change level of the calcium ions of the intestinal cells can be observed in real time after the nematodes are fixed.
Drawings
FIG. 1 is a schematic structural view of the present invention; wherein, the left picture is a chip structure schematic diagram: the gray structure of the upper graph is a round palisade nematode culture room, a culture medium access passage and a nematode access passage. In the lower diagram, the black circle is an air pressure cavity above the nematode culture chamber. Right panel: the round fence is a nematode culture room, a plurality of adult nematode worms are arranged in the round fence, and the picture is magnified by 20 times.
FIG. 2 is a schematic view of the immobilization of nematodes under a PDMS membrane on a microfluidic chip (cross-sectional view); the upper diagram is a sectional diagram of the chip structure, and the lower diagram is a schematic diagram of the PDMS film pressing down for fixing the nematodes.
FIG. 3 shows the imaging detection analysis experiment of the dynamic change of calcium ion in nematode intestinal cells by using the method of the present technology. The left graph is a fluorescence graph marked by the intestinal cell calcium ion fluorescence probe, and the right graph is the detected change trend of the fluorescence brightness in the square frame area of the left graph, namely the change of the calcium ion concentration.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below so that those skilled in the art can better understand the advantages and features of the present invention, and thus the scope of the present invention will be more clearly defined. The embodiments described herein are only a few embodiments of the present invention, rather than all embodiments, and all other embodiments that can be derived by one of ordinary skill in the art without inventive faculty based on the embodiments described herein are intended to fall within the scope of the present invention.
Examples
A micro-fluidic chip device for nematode culture and immediate fixation comprises a glass substrate, wherein a nematode culture chamber is arranged on the glass substrate, the nematode culture chamber is a circular space surrounded by a fence structure, a liquid culture medium (CeMM) sample outlet channel and a nematode sample inlet and sample outlet channel are respectively arranged on the outer side of the circular fence, a circular air chamber is arranged above the nematode culture chamber, a PDMS film is arranged between the culture chamber and the circular air chamber, air pressure is applied in the circular air chamber to force the PDMS film to press downwards to push nematodes, the nematodes are pushed to the fence, a slender nematode body is pressed in parallel to the fence, and fixation of the nematodes is realized, as shown in the lower graph of figure 2.
Rearing is a common way of raising animals. The micro-fluidic chip technology can realize the captive breeding of the micro-scale biological nematodes. As shown in figure 1, the nematodes are housed in a circular fence with the diameter of 8 mm and the height of 100 microns, rectangular columns are adopted to enclose the circular fence, and the width of gaps between the circular fence is 20 microns.
Liquid medium (CeMM) and water can freely pass through the circular fence, but nematodes can not pass through the circular fence and are restrained in the circular fence for feeding.
As shown in figure 1, a liquid culture medium (CeMM) sample inlet and outlet channel and a nematode sample inlet and outlet channel are arranged on the outer side of the circular fence. Fresh liquid medium (CeMM) can enter the nematode culture chamber from the medium feeding channel at one end of the circular fence through the circular fence and flow out from the other end, and the feeding speed of the liquid medium (CeMM) is 50 microliters per minute.
A layer of PDMS film is arranged above the circular fence, and the thickness of the PDMS film is 100 micrometers. A circular air chamber is arranged above the PDMS film, and is coaxial with the center of the circular fence structure and has an upper layer structure and a lower layer structure. The area of the circular air chamber is larger than that of the circular fence structure.
The specific mode of applying air pressure in the circular air cavity to press the PDMS film is as follows: the circular air cavity chamber is filled with water, the PDMS film allows gas molecules to pass through, water molecules cannot pass through, and the circular air cavity chamber is filled with water, and then the pressure is regulated and controlled to compress the PDMS film. The compaction process needs to be carried out slowly, and the nematode can be further compacted after being pushed to the edge, so that the fixation of the nematode is completed.
After the nematodes are compacted and fixed, optical imaging analysis can be carried out; after the analysis is finished, the air pressure is removed, the nematode culture chamber can be recovered, and the nematodes can continue to move.
The nematode is obtained by acclimatizing liquid culture medium (CeMM) at 21 deg.C, and the CeMM culture medium contains OP50 type Escherichia coli (10)9Each per milliliter).
The device has simple structure principle and convenient operation, can realize the real-time harmless fixation in the nematode culture, and furthest reduces the influence of the fixation operation on the natural physiological state of the nematode. The circular fence is used, the nematode culture is convenient, zero loss of the nematode entering and exiting the fence can be realized, and the sample introduction and recovery rate is 100 percent.
The invention can realize the imaging detection analysis of the dynamic change of calcium ions of the intestinal cells of the nematodes, the nematodes with calcium ion fluorescent probes in the intestinal cells are cultured on a chip, and the change level of the calcium ions of the intestinal cells can be observed in real time after the nematodes are fixed, as shown in figure 3.
Claims (8)
1. A can be used to the nematode to cultivate and the fixed micro-fluidic chip device in time, its characterized in that: the nematode feeding device comprises a glass substrate, the glass substrate on be equipped with nematode culture chamber, nematode culture chamber be the circular structure that the fence encloses, the outside of circular fence is equipped with liquid medium (CeMM) respectively and advances kind of passageway and nematode and advance kind and go out the passageway of appearance, the top of nematode culture chamber be equipped with a circular air cavity room, nematode culture chamber and circular air cavity room between be a layer PDMS film, circular air cavity room in apply atmospheric pressure and force PDMS film to push down, push away the nematode, crowd the fence with the nematode, compress tightly slender polypide parallel to the fence, realize the fixed to the nematode.
2. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: the nematodes are housed in a circular fence with the diameter of 8 mm and the height of 100 microns, rectangular columns are adopted to enclose the circular fence, and the width of gaps between the circular fence is 20 microns.
3. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: liquid medium (CeMM) and water can freely pass through the circular fence, but nematodes can not pass through the circular fence and are restrained in the circular fence for feeding.
4. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: fresh liquid medium (CeMM) can enter the nematode culture chamber from the medium feeding channel at one end of the circular fence through the circular fence and flow out from the other end, and the feeding speed of the liquid medium (CeMM) is 50 microliters per minute.
5. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: the thickness of the PDMS film is 100 micrometers.
6. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: the specific mode of applying air pressure in the circular air cavity to press the PDMS film is as follows: the round air cavity chamber is filled with water, the PDMS film allows gas molecules to pass through, water molecules cannot pass through, after the round air cavity chamber is filled with water, the pressure is regulated and controlled to compress the PDMS film, the compression process needs to be carried out slowly, the nematode is pushed to the edge and then can be further compressed, and fixation of the nematode is completed.
7. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: after the nematodes are compacted and fixed, optical imaging analysis can be carried out; after the analysis is finished, the air pressure is removed, the nematode culture chamber can be recovered, and the nematodes can continue to move.
8. The microfluidic chip device of claim 1, wherein the microfluidic chip device comprises: the nematode is obtained by acclimatizing liquid culture medium (CeMM) at 21 deg.C, and the CeMM culture medium contains OP50 type Escherichia coli (10)9Each per milliliter).
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| Application Number | Priority Date | Filing Date | Title |
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| CN202111646562.XA CN114208780A (en) | 2021-12-30 | 2021-12-30 | Micro-fluidic chip device for nematode culture and instant fixation |
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| CN202111646562.XA CN114208780A (en) | 2021-12-30 | 2021-12-30 | Micro-fluidic chip device for nematode culture and instant fixation |
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| CN114208780A true CN114208780A (en) | 2022-03-22 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115487887A (en) * | 2022-09-13 | 2022-12-20 | 中国石油大学(华东) | High-temperature high-pressure micro-nanofluidic chip holder device and temperature and pressure control method thereof |
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