CN103461279A - Micro-fluidic chip and device for elegans cultivation and/or observation and application of micro-fluidic chip and device - Google Patents
Micro-fluidic chip and device for elegans cultivation and/or observation and application of micro-fluidic chip and device Download PDFInfo
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Abstract
The invention relates to a micro-fluidic chip and a device for elegans cultivation and/or observation and application of the micro-fluidic chip and the device, and specifically provides the micro-fluidic chip for elegans cultivation, observation and/or research, the device comprising the micro-fluidic chip and the application of the micro-fluidic chip and the device. According to the micro-fluidic chip and the device, which are provided by the invention, high throughput and high resolution can be both considered, the growth of larvas is prevented from being restrained by the use of an additional chemical reagent, the manufacturing and using cost is low, the operation automation degree is high, and the application prospect is wide.
Description
Technical field
The present invention relates to biotechnology and experimental facilities field.Particularly, the application that the present invention relates to a kind of cheapness, be easy to preparation, the energy automatic washing removes culture of nematodes micro flow chip, the equipment that comprises this micro flow chip and this micro flow chip and the equipment of larvae and eggs.
Background technology
Caenorhabditis elegans (Caenorhabditis elegans) is the important model material of modern Developmental Biology, genetics and genomics research.It is a kind of very little worm, the long only 1mm of its adult, and whole body is transparent, take bacterium as food, lives in soil, and the history of life is short, by a development of fertilized ova, becomes ripe adult two days as long as more (25 ℃ time need 52 hours, can reach the soonest 48 hours).In the embryonic development of wild type nematode, the formation of cell division and cell-line has the procedural of height, facilitates like this its growth is carried out to genetic analysis.
Obtaining nematode life cycle data accurately is important foundations of research nematode aging.The relevant phenotypic data of aging (motion activity, body size, fat depot and various aging gene are merged fluorescin signal etc.) that simultaneously obtains each period in senescence process more has important meaning to verifying old and feeble mechanism.
Traditional nematode durability analysis experiment is generally carried out in solid culture plate or liquid environment.These two kinds of methods all have some limitations.During solid culture, must in culture plate, add floxuridine (2'-Deoxy-5-fluorouridine, Floxuridine, FuDR) to suppress nematode offspring's growth.Recently research shows, FuDR can mutagenesis body tub-1 life-span significant prolongation, shows that FuDR can not be ignored fully on the impact of adult, otherwise likely causes the erroneous judgement to aging gene.During liquid culture, because can't change culture fluid and food, add easy pollution, be difficult to accomplish the only tracing observation of the whole aging course about 15 days of nematode.And these large scale experiments all need to add the growth that FuDR suppresses the nematode offspring.
At present, the sorting flow cytometer is the most common technology platform that carries out high flux, the sorting of automation sample and data acquisition, but its limitation is larger.The interested sample of Many researchers (such as nematode etc.), because fragility too greatly or too can not be applied this instrument and carries out collection analysis.
Current unique equipment that can be applied to the small-sized biopsy sample sorting such as nematode and gather phenotypic data on the market is COPAS (the Complex Object Parametric Analyzer and Sorter) system of U.S. Union Biometrica company research and development, it utilizes the more wide-aperture flow chamber of comparison type cell instrument, analysis, the sample of sorting diameter in the 20-1500 micrometer range.This equipment utilization the principle of flow cytometer, form liquid stream and allow object in turn by analytical port, analytical port has Ear Mucosa Treated by He Ne Laser Irradiation, can obtain the size parameter of object by measuring the flight time of object in laser beam; Blocked by object the light intensity decayed by measurement and can obtain the color depth of object and the parameter of transparency; Instrument, with laser instrument and three groups of light filters of three different wave lengths, can obtain by measuring fluorescence intensity the information (parameter) of fluorescence labeling on body axis (fluorescin or fluorescence antibody) abundance situation.But there is obvious defect in the COPAS system: it can only the cross section total fluorescent value of the unit of providing and the integral fluorescence value of sample, can not reach the resolution of cell or subcellsular level; The more important thing is that this system can not provide real-time visible image data; In addition, this system equipment is expensive, and use cost is relatively high.
In sum, can take into account high flux and high-resolution and avoid using extra chemical reagent to suppress the large scale experiment platform of larval growth in the urgent need to developing in this area.
Summary of the invention
Main purpose of the present invention is can take into account high flux and high-resolution and avoid using extra chemical reagent to suppress the large scale experiment platform of larval growth in order to provide a kind of.
In a first aspect of the present invention, a kind of micro flow chip 1 of cultivating, observing and/or study for Caenorhabditis elegans (Caenorhabditis elegans) is provided, described micro flow chip 1 comprises microflow layer 2 and substrate 20, described microflow layer 2 comprises:
At least one culturing room 3 for cultivating nematode, described culturing room 3 is cavitys that are enclosed in microflow layer, only by the miniflow pipeline, with the external world, communicates, described culturing room 3 is greater than the diameter of nematode adult in the size perpendicular on the micro flow chip direction;
At least one input miniflow pipeline 4 be connected with culturing room 3 and at least one screening pipeline 30 be connected with culturing room 3, described input miniflow pipeline 4 is for to culturing room's 3 infusion fluids, described screening pipeline 30 with for collecting with at least one output miniflow pipeline 5 of the larvae and eggs of discharging the liquid that flows out through screening pipeline 30 from culturing room 3 and nematode, be connected, described screening pipeline 30 is less than or equal to the average cross-section of nematode adult long-pending 2/3 and the average cross-section that is more than or equal to nematode larval and ovum at the aperture area of culturing room's inwall and amasss 1/2.
In a preference, described screening pipeline 30 the aperture area of culturing room's inwall be less than or equal to the average cross-section of nematode adult long-pending 1/2 and be more than or equal to the average cross-section long-pending 2/3 of nematode larval and ovum.
In another preference, described screening pipeline 30 the maximum inner diameter of culturing room's inwall be less than or equal to nematode adult average diameter 2/3 and be more than or equal to 1/2 of nematode larval and ovum average diameter.
In another preference, described screening pipeline 30 at the maximum inner diameter of culturing room's inwall, be less than or equal to the nematode adult average diameter 1/2 and be more than or equal to the average diameter 2/3 of nematode larval and ovum.
In some embodiments of the present invention, described screening pipeline 30 is rectangle at the opening of culturing room's 3 inwalls, and described rectangular length is 15-25 μ m, is preferably 18-22 μ m, more preferably 20 μ m; Described rectangular wide be 8-12 μ m, be preferably 9-11 μ m, more preferably 10 μ m.
In other embodiments of the present invention, described screening pipeline 30 is circular at the opening of culturing room's 3 inwalls, and the diameter of described circle is 8-12 μ m, is preferably 9-11 μ m, more preferably 10 μ m.
In other embodiments of the present invention, described culturing room 3 is at the size perpendicular on the micro flow chip direction >=100 μ m.
In other embodiments of the present invention, the number of described input miniflow pipeline 4 >=1, the number of described screening pipeline 30 >=3.
In other embodiments of the present invention, described culturing room 3 is strip, the opening of described input miniflow pipeline 4 in culturing room 3 is positioned at an end of the major axis of strip, and the opening of described screening pipeline 30 in culturing room 3 is positioned on two sides of the other end of major axis of strip and strip.
In other embodiments of the present invention, strip culturing room 3 is circular arc with the end that described input miniflow pipeline 4 is connected, and is preferably semicircle.
In other embodiments of the present invention, the number of described screening pipeline 30 >=12, wherein be positioned at the number >=4 of screening pipeline 30 of an end of the major axis of described strip, is positioned at the number of the screening pipeline 30 on arbitrary side of strip >=4.
In other embodiments of the present invention, described micro flow chip 1 also comprises key-course 6 and articulamentum 7, between described key-course 6 and described microflow layer 2, by articulamentum 7, separates, and described key-course 6 comprises:
At least one controls pipeline 8, and described control pipeline 8 has at least one crosspoint 9 with input miniflow pipeline 4 or output miniflow pipeline 5, and the articulamentum 7 at 9 places, described crosspoint consists of elastomeric material,
Wherein, when the pressure in controlling pipeline 8 is increased to certain value, the elastomer articulamentum 7 of 9 positions, described crosspoint expands to microflow layer 2 directions, and the inner surface contact that makes input miniflow pipeline 4 or output miniflow pipeline 5 makes the state of miniflow in disconnecting to blocking miniflow fully; When the pressure in controlling pipeline 8 recovers, the elastomer articulamentum 7 of 9 positions, crosspoint recovers ortho states, makes the state of miniflow in circulation.
In other embodiments of the present invention, described microflow layer 2, key-course 6 and articulamentum 7 consist of elastomeric material, and described control pipeline 8 enlarges and forms cavity at 9 places, crosspoint with input miniflow pipeline 4 and output miniflow pipeline 5.
In a preference, described cavity is circle or rectangle.
In other embodiments of the present invention, described elastomeric material is to be selected from one or more in lower group: dimethyl silicone polymer, acrylonitrile-butadiene-styrene copolymer, Merlon, polymethyl methacrylate, polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), polyvinyl chloride, the cyclic polyolefin co-polymer, polyvinylidene fluoride, polystyrene, polysulfones, nylon, the styrene-propene acid copolymer, natural rubber, polyisoprene, butyl rubber, halogenated butyl rubber, polybutadiene, butadiene-styrene rubber, acrylonitrile-butadiene rubber, chloroprene rubber, EP rubbers, epichlorohydrin rubber, lactoprene, silicone rubber, fluorosioloxane rubber, fluoroelastomer, Perfluoroelastomer, ethane-acetic acid ethyenyl ester, resilin (resilin), elastin laminin (elastin), polyimides, phenolic resins, or its combination.
In a second aspect of the present invention, a kind of device 100 of cultivating, observing and/or study for Caenorhabditis elegans is provided, described device comprises:
The liquid input block 10 be connected with the input miniflow pipeline 4 of micro flow chip 1;
The external pipe 11 be connected with the control pipeline 8 of micro flow chip 1;
The control assembly be connected with external pipe 11;
Optional observation parts;
Optional recording-member; And
The optional collecting part be connected with output miniflow pipeline 5.
In a preference, described control assembly comprises one or more that are selected from lower group: press device, be arranged on the magnetic valve on external pipe 11, the calculator be connected with magnetic valve.
In another preference, described observation parts comprise one or more that are selected from lower group: microscopic system, preferred body stereomicroscope, confocal microscope; Imaging system, preferably CCD imaging device.More preferably described observation parts are connected with recording-member, the calculator for example be connected with the CCD imaging device.
In some embodiments of the present invention, described liquid input block 10 comprises one or more closed containers 15 that liquid is housed, described closed container 15 is connected with the external world by the following pipeline of liquid level in two insertion containers, wherein a pipeline is connected with the input miniflow pipeline 4 on micro flow chip 1, and an other pipeline is connected with compressed air pump.
In a preference, described press device is air pressure pump, peristaltic pump or syringe pump.
In a third aspect of the present invention, provide the application in the cultivation at Caenorhabditis elegans of micro flow chip 1 of the present invention or device of the present invention 100, observation and/or research.
In some embodiments of the present invention, the cultivation of described nematode, observation and/or research are observed and/or research for Developmental Biology, genetics, genomics, pharmacy.
In a preference, described cultivation, observation and/or research, for the research of ontogeny and age, are preferred for the research of growth or the research of aging gene, the screening of anti-ageing material, old and feeble feature phenotype and/or aging biology mark.
Other side of the present invention, due to the disclosure of this paper, is apparent to those skilled in the art.
The accompanying drawing explanation
Below in conjunction with accompanying drawing, the invention will be further described, and wherein these show only in order to illustrate embodiment of the present invention, rather than in order to limit to scope of the present invention.
Fig. 1: culture of nematodes micro flow chip structural representation:
(a) culture of nematodes micro flow chip Structure of cross section schematic diagram: integrated 8 culturing room of every chip block, realize the parallel operation of 8 culturing room 3 by the switching of controlling pipeline 8; Wherein black part belongs to key-course 6, and grey color part belongs to microflow layer 2.
(b) schematic diagram of single culturing room and accessory structure thereof: culture of nematodes chamber 3, the high for example 4000 μ m * 1000 μ m * 100 μ m that can be of its length and width; Connect culturing room and the screening pipeline 30 of exporting miniflow pipeline 5, the high for example 80 μ m * 20 μ m * 10 μ m that can be of its length and width; Output miniflow pipeline 5 can be rectangle with input miniflow pipeline 4, and the wide height of its cross section can be 300 μ m * 70 μ m; Control pipeline 8 and form respectively crosspoint 9 with output miniflow pipeline 5 and input miniflow pipeline 4, realize the access and exit control of miniflow.
(c) structure for amplifying at 9 places, crosspoint and miniflow access and exit control schematic diagram: substrate 20 bearer control layers 6, articulamentum 7 and microflow layer 2 separate by articulamentum 7 between key-course 6 and microflow layer 2; Key-course 6 comprises at least one control pipeline 8, and this control pipeline 8 has at least one crosspoint 9 with input miniflow pipeline 4 or output miniflow pipeline 5; The articulamentum 7 at 9 places, crosspoint consists of elastomeric material.
Wherein, when the pressure in controlling pipeline 8 is increased to certain value, the elastomer articulamentum 7 of 9 positions, described crosspoint expands and makes the inner surface contact of input miniflow pipeline 4 or output miniflow pipeline 5 extremely block miniflow fully to microflow layer 2 directions, make its state in disconnecting, make micro-valve close; When the pressure in controlling pipeline 8 recovers, the elastomer articulamentum 7 of 9 positions, crosspoint recovers ortho states, makes miniflow be communicated with, and makes micro-valve open.
Fig. 2: chip operation flow chart:
(a) modify the chip pipe surface with surfactant 2% pluronic (pluronic);
(b) pass into the nematode suspension;
(c) regularly change culture fluid.
Fig. 3: the energy automatic washing is except the culture of nematodes device schematic diagram of larva.
Fig. 4: nematode old and feeble process and Life cycle curve figure in time under different glucose environment:
(a) the nematode aging rate of adult stage cultivation in the culture fluid that contains 2% and 4% glucose obviously, than not fast containing the nematode aging rate of growing in the culture fluid of glucose, obviously accelerate by the vigor rate of decay;
(b) contain 2% on chip and compare with control group with nematode in the culturing room of the glucose culture solution of 4% concentration, the life-span significantly shortens;
(c) nematode life cycle difference on the NGM solid culture plate of the glucose that contains variable concentrations: 2% compares with control group with the glucose of 4% concentration, significantly shortens the nematode life-span.
Fig. 5: nematode old and feeble process and Life cycle curve figure in time under different RNA i pressure environment:
(a) the adult stage cultivate the culture fluid that contains blank and contain respectively sptf-3, Y82E9BR.3 and age-1 gene RNAi clone's culture fluid in nematode aging rate and the vigor rate of decay variant;
(b) contain nematode in different RNA i pressure culturing room on chip and compare with control group, the life-span is variant;
(c) nematode life cycle difference on the NGM solid culture plate that contains different RNA i pressure.
Embodiment
The problem that the inventor can not provide real-time visible image data in order to solve adverse effect that in culture of nematodes in prior art and/or observation process, FuDR brings and art methods, developed a kind of cheapness, be easy to preparation and can remove the culture of nematodes micro flow chip of larva by automatic washing, and provide based on this chip, culture of nematodes and finder that image data can be provided in real time.In addition, the inventor also provides the application of this micro flow chip and device.
term definition
In the present invention, term " chip " is with the defined chip concept in this area is identical usually, be the smooth board that outward appearance is the thickness homogeneous, modally be shaped as rectangle or square, but those of ordinary skills also can be adjusted its shape as required.
In the present invention, term " miniflow " refers to material (comprising molecule and colloid) transmission, momentum transfer, the heat transmission under micro-(receiving) metrical scale, and the course of reaction in transmission.
In the present invention, term " micro flow chip " refers to integrated micro-channels network and numerous analytic function element, can be in the micron order structure override receive and rise to the chip that skin rises the volume fluid, the resulting structure of its containing fluid (comprising passage, reative cell and some other functional part) is at least the micron order yardstick on a dimension.As used herein, term " chip of the present invention " and " micro flow chip " are used interchangeably, and the chip for culture of nematodes and/or observation provided in the present invention all is provided.
" a miniflow pipeline " in the present invention is that to take the opening of miniflow pipeline in culturing room be sign, a corresponding miniflow pipeline of opening.Accordingly, " N bar miniflow pipeline " refers to each miniflow pipeline corresponding with N opening in culturing room, and the total quantity of described miniflow pipeline is the N bar.
Term " input miniflow pipeline " refer to culturing room, is communicated with, for one or more miniflow pipeline of past culturing room infusion fluid (such as the medium that comprises food, the medium etc. that comprises drug candidate).Can adopt the mixture of same input miniflow pipeline input plurality of liquid as required, also can adopt many input miniflow pipelines to input respectively different liquid.If necessary, the discharge duct of bubble at least one eliminating loading process can be set on input miniflow pipeline side.
In the present invention, term " screening pipeline " or " screening miniflow pipeline " are used interchangeably, and all refer to the sidewall of culturing room or be connected, have the nematode larval that can allow in culturing room and ovum away from an end of input miniflow pipeline and output miniflow pipeline to enter output miniflow pipeline but the maximum inner diameter that do not allow adult to pass through or the miniflow pipeline of cross-sectional area through it.
The shape of the cross section of screening pipeline can be rectangle, square, circle or oval usually.When the cross section of screening pipeline is rectangle, " maximum inner diameter " refers to its size long and wide middle the greater; When the cross section of screening pipeline is ellipse, " maximum inner diameter " refers to the diameter of its major axis.In a preference, the screening pipeline the aperture area of culturing room's inwall be less than or equal to the average cross-section of nematode adult long-pending 2/3 and to be more than or equal to the average cross-section of nematode larval and ovum long-pending 1/2, do not discharge the nematode adult for liquid stream, discharging nematode larval and ovum.In another preference, described screening pipeline (30) at the maximum inner diameter of culturing room's inwall, be less than or equal to the nematode adult average diameter 1/2 and be more than or equal to the average diameter 2/3 of nematode larval and ovum.In another preference, the screening pipeline the maximum inner diameter of culturing room's inwall be less than or equal to nematode adult average diameter 2/3 and be more than or equal to 1/2 of nematode larval and ovum average diameter, or be less than or equal to nematode adult average diameter 1/2 and be more than or equal to 2/3 of nematode larval and ovum average diameter, thereby can discharge nematode larval and ovum and not discharge the nematode adult with liquid stream.
Term " output miniflow pipeline " refer to screen pipeline be connected, for collection with eject the liquid stream of self-sizing pipeline and one or more miniflow pipeline of nematode larval and ovum.
" crosspoint " in the present invention refers to miniflow pipeline and the joining of controlling the projection of pipeline on micro flow chip.Being understood that, because miniflow pipeline and control pipeline belong to different layers, is disconnected between them.
micro flow chip
In some embodiments of the present invention, provide the culture of nematodes micro flow chip of a kind of energy automatic washing except larva.
Micro flow chip comprises a microflow layer, described microflow layer comprises that at least one is for cultivating the culturing room of nematode, described culturing room is a cavity be enclosed in microflow layer, only by the miniflow pipeline, with the external world, communicate, the thickness of cavity (or claim height) is preferably greater than the diameter of adult but is not more than the twice of adult diameter, to avoid stacked between nematode, above-mentioned thickness refers to that cavity is in the size perpendicular on the micro flow chip direction;
The number of the input miniflow pipeline be communicated with each culturing room is more than or equal to 1, and wherein at least one miniflow pipeline is for past culturing room infusion fluid; The number of the screening pipeline be communicated with each culturing room is more than or equal to 1 (at least one), for discharging the larva of liquid and nematode; All screening pipelines all are less than the cross-sectional area of nematode adult and are greater than the cross-sectional area of nematode larval at the area of the opening of culturing room's inwall, to guarantee nematode larval, can discharge culturing room by the screening pipeline, and adult can not.
The opening of screening pipeline in culturing room can be arranged on the other end contrary with inputting the opening of miniflow pipeline in culturing room and/or be arranged on the side of culturing room (or claiming sidewall) above, the screening pipeline preferably all is set on a described end and sidewall to guarantee unobstructed discharge nematode larval and ovum and other foreign material.
In other embodiment of the present invention, this micro flow chip also can be included in culturing room's inner opening, open and close controlled large output channel (being greater than cross-sectional area or the diameter of adult), so that thoroughly clean culturing room or thoroughly change the content in culturing room, thus make culturing room and micro flow chip reusable.
Adopt technique scheme can solve dexterously the most headachy technical problem in prior art: how to eliminate the FuDR that uses in the conventional method impact on nematode.The impact of FuDR to be eliminated fully, just FuDR must not be used.Adopt technique scheme of the present invention, because the screening pipeline all is less than the cross-sectional area of nematode adult and is greater than the cross-sectional area of nematode larval at the area of the opening of culturing room's inwall, thereby nematode larval can discharge culturing room through output miniflow pipeline by the screening pipeline, adult can not.In experimentation, constantly inject new liquid by from input miniflow pipeline toward culturing room, just can utilize the liquid flowed out from output miniflow pipeline via the screening pipeline that the chalaza of nematode larval and nematode is gone out to culturing room, thereby not use FuDR just can remove larvae and eggs.
In the actual experiment operation, can determine shape and the size of output screening miniflow pipeline at the opening of culturing room's inwall according to nematode (or other biology) adult of using in experiment and larva body size difference.In fact, screening miniflow pipeline is not subject to special restriction in the shape of the opening of culturing room's inwall, and total principle is to guarantee that nematode larval can pass through screening miniflow pipeline and discharge culturing room through output miniflow pipeline, and adult can not.Take Caenorhabditis elegans as example, if the screening pipeline is circular at the opening of culturing room's inwall, the diameter of described circle can be 8-12 μ m, is preferably 9-11 μ m, more preferably 10 μ m; If the screening pipeline is rectangle at the opening of culturing room's inwall, it is 15-25 μ m that described rectangular length can be set, and is preferably 18-22 μ m, 20 μ m more preferably, and wide is 8-12 μ m, is preferably 9-11 μ m, more preferably 10 μ m.
Because culturing room is mainly used to provide living space to the nematode adult, so the growth that the space size of culturing room should suitable nematode adult.Take Caenorhabditis elegans as example, and the diameter of its adult is generally in 90 μ m left and right, so the thickness of culturing room generally should be set at least be greater than this diameter, preferably >=100 μ m.
Preferably, input miniflow pipeline number >=1, screening pipeline number >=3.The number of input miniflow pipeline can be determined according to the kind of infusion fluid; Usually the number of screening pipeline is more, and the efficiency of discharging liquid, larva and ovum is higher, the operated by rotary motion dozens of.
Preferably, culturing room is strip, and the input opening of miniflow pipeline in culturing room is positioned at an end of the major axis of strip, and the opening of screening pipeline in culturing room is positioned on two sides of minor axis of the other end of major axis of strip and strip.Culturing room is set to strip, to input the two ends that miniflow pipeline and the opening of screening pipeline in culturing room are arranged on the major axis of strip simultaneously, be conducive to liquid and enter culturing room from input miniflow pipeline glibly, then flow out culturing room by the screening pipeline through output miniflow pipeline, the liquid flow simultaneously formed also easily goes out culturing room by larva and chalaza.If liquid flow velocity is very fast, or adult quantity is more, adult may be flushed to an end of strip and stop up the screening pipeline tapping of end face under the flowing of fluid, therefore preferably the opening of screening pipeline also is set on two sides of strip, eccysis larvae and eggs glibly still when guaranteeing that said circumstances occurs.In the case, can be accordingly at side and an end of culturing room, corresponding output miniflow pipeline be set.
Preferably, strip culturing room is set to circular arc with the end that input miniflow pipeline is connected, and more preferably, described circular arc is semicircle.So arrange and can prevent that the retention of food culture fluid is in the square dead angle of input miniflow pipeline one end.
Preferably, screening pipeline number >=6, more preferably >=12, more preferably >=20, the screening opening of pipeline in culturing room is positioned on two sides of minor axis of the other end of major axis of strip and strip, wherein be positioned at the number of openings of the other end of described strip major axis >=2, preferably >=4, more preferably >=6, be positioned at number of openings on arbitrary side of strip minor axis >=2, preferably >=4, more preferably >=6.The screening pipeline that a greater number is set can guarantee efficiently larvae and eggs to be flushed out to culturing room.In a preferred embodiment, generally at end face, arrange 10 with upper shed, in each side, the opening more than 20 all is set.
Preferably, all miniflow pipelines are all in microflow layer, and the miniflow pipeline has opening and is in communication with the outside on micro flow chip, discharge liquid and larva toward filling liquid in culturing room or from culturing room by described opening, micro flow chip also comprises key-course, between key-course and microflow layer, by articulamentum, separate, described key-course has at least one control pipeline, described control pipeline and microflow channels road have at least one crosspoint, the articulamentum at place, described crosspoint consists of elastomeric material, when the ducted pressure of control is increased to certain value, the elastomer articulamentum of above-mentioned at least one position, crosspoint will expand to the microflow layer direction, and make the contact of miniflow inner surface of pipeline to blocking the miniflow pipeline fully, make its state in disconnecting, when controlling ducted pressure recovery, the elastomer articulamentum of position, crosspoint recovers ortho states, the miniflow pipeline is communicated with again.But so just form the micro flow chip of an automatic control fluid flow, by controlling pipeline, with the equipment that pressure is provided (as air pump, peristaltic pump etc.), be connected, regulate the ducted pressure size of control by press device, thereby further control the connected state of miniflow pipeline.
One preferred embodiment in, some control pipelines are set, control by these connected state that pipeline can be controlled arbitrarily each input miniflow pipeline and output miniflow pipeline, input miniflow pipeline is connected with the pressure source that steady pressure is provided, thereby when inputting miniflow pipeline and output miniflow pipeline in connected state, to have continuously new liquid to enter culturing room from input miniflow pipeline, original liquid continuously leaves culturing room from output miniflow pipeline simultaneously, control pipeline by outside pipeline with continue to provide the press device of pressure to be connected, externally on pipeline, magnetic valve is set, magnetic valve is connected with calculator, by the computer control magnetic valve, in the predefined time, open or close, just can control connection or the disconnection of magnetic valve place external pipe, thereby control connection or the disconnection of the control pipeline be connected with this external pipe, connection or the disconnection of the miniflow pipeline that further this control pipeline of control is controlled, thereby final the realization in the predefined time toward culturing room's filling liquid, rinse larvae and eggs, reach the purpose of full-automatic cultivation nematode.
In some embodiments, for each culturing room arranges independent control pipeline, to realize the independent control to each culturing room.In other embodiments, for culturing room provides the control be interconnected pipeline, to realize synchronization management and the control to a plurality of culturing room.
Certainly, those of ordinary skills also can adopt as known in the art other to control structure and the method for liquid input and output.For example, the method for controlling the liquid input can be used gravity, compressed-air actuated pressure, peristaltic pump pressure, magnetic force, electrical potential difference.Preferably, microflow layer, articulamentum and key-course (example as shown in Figure 1) form by elastomeric material, control pipeline and enlarge and form cavity at place, the crosspoint with the miniflow pipeline.Described cavity is preferably circle or rectangle.It is to prepare micro flow chip for the laboratory method with easy that microflow layer, key-course and articulamentum are all used elastomeric material.Because many elastomeric materials have the performance of good machine-shaping, such as thermoplasticity, thermosetting, radiation-initiated crosslinking etc., without expensive equipment and complicated method, adopt the simple common equipment in laboratory just can prepare easily the micro flow chip of various structures.Controlling pipeline enlarges and forms cavity at place, the crosspoint with the miniflow pipeline, because the articulamentum of empty cavity position is thinner, therefore when controlling ducted pressure increase, the articulamentum at empty cavity position place will expand at first, control suitable pressure and just can guarantee to only have the articulamentum in the cavity scope to expand, thereby at cavity position blocking-up miniflow pipeline.
Preferably, elastomeric material is to be selected from one or any two kinds or above mixture in lower group: dimethyl silicone polymer (PDMS), acrylonitrile-butadiene-styrene copolymer, Merlon (PC), polymethyl methacrylate (PMMA), polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE) (PTFE), polyvinyl chloride (PVC), cyclic polyolefin co-polymer (Cyclic Olefin Copolymers, COC), polyvinylidene fluoride, polystyrene, polysulfones, nylon, the styrene-propene acid copolymer, natural rubber, polyisoprene, butyl rubber, halogenated butyl rubber, polybutadiene, butadiene-styrene rubber, acrylonitrile-butadiene rubber, chloroprene rubber, EP rubbers, epichlorohydrin rubber, lactoprene, silicone rubber, fluorosioloxane rubber, fluoroelastomer (FKM), Perfluoroelastomer (FFKM), ethane-acetic acid ethyenyl ester, resilin (resilin), elastin laminin (elastin), polyimides, phenolic resins.In embodiment, use dimethyl silicone polymer to prepare micro flow chip, this be because PDMS has, manyly be conducive to micro-ly to build, the material behavior of micro-molding and micro-patterning.Those skilled in the art can understand, and the existing elastomeric material in this area can be applied in micro flow chip of the present invention, and is not limited to above-mentioned preferred example.
culture of nematodes and finder
The present invention also provide a kind of can automatic washing except culture of nematodes and/or the finder of larva, the control assembly that this device comprises aforesaid micro flow chip, the liquid input block be connected with the input miniflow pipeline of micro flow chip, the external pipe be connected with the control pipeline of micro flow chip, be connected with the said external pipeline (for example press device, be arranged on the magnetic valve on the said external pipeline, the calculator be connected with magnetic valve), optional observation parts (for example stereomicroscope and the CCD imaging device that is connected with stereomicroscope), optional recording-member.Those of ordinary skills can, according to real needs, be adjusted and do not broken away from general plotting of the present invention and scope the parts in culture of nematodes and finder.
Can be used for the not special restriction of liquid input block of the present invention, as long as can drive liquid to enter culturing room, can be for example one section pipeline that is full of liquid growing, by peristaltic pump, drives ducted liquid to enter culturing room.
Preferred a kind of liquid input block comprises a closed container that liquid is housed, described closed container is connected with the external world by the following pipeline of liquid level in two insertion containers, wherein a pipeline is connected with the input miniflow pipeline on micro flow chip, and an other pipeline is connected with compressed air pump.Closed container can be test tube or the centrifuge tube of a band plug, compressed air pump passes into gas by coupled pipeline in closed container, this pipeline is inserted to the container bottom, the bubble of emerging from pipeline can make to be suspended in culture fluid as the Escherichia coli of nematode food, effectively prevent the cenobium blocking pipe of deposition, when inputting miniflow pipeline and output miniflow pipeline all in connected state, the liquid in closed container can be admitted in culturing room continuously.
The above-mentioned press device be connected with external pipe can be air pressure pump, peristaltic pump or injection pump etc.In practical operation, compressed air pump in liquid input device is opened, make in input miniflow pipeline and have one constantly toward the power of filling liquid in culturing room, the press device that will be connected with external pipe is opened, the magnetic valves all by computer control are in open mode, make above-mentioned press device produce certain pressure in controlling pipeline, by all miniflow closedown of pipeline, nematode can grow in immobilising culture fluid, when needs are being changed liquid to some culturing room sometime, when rinsing larvae and eggs, use computer programming, constantly close some magnetic valve at this, so the pressure in the exterior line at above-mentioned magnetic valve place disappears, the ducted pressure of control be connected with the said external pipeline also disappears, so the elastomer articulamentum at above-mentioned control pipeline and place, miniflow pipeline crosspoint recovers ortho states, the miniflow pipeline is communicated with again, the culturing room be connected with the above-mentioned miniflow pipeline again be communicated with starts to change liquid, new liquid enters culturing room from input miniflow pipeline, original liquid, larva and ovum leave culturing room from output miniflow pipeline, thereby realized cultivating nematode full-automaticly, rinse larva.
Those of ordinary skills should know, and can adopt liquid input block as known in the art and control assembly to realize liquid input and liquid current control and not break away from general plotting of the present invention and scope.
The observation parts can be set according to the needs of concrete observation, for example microscopic system (as stereomicroscope, confocal microscope), the imaging system (as the CCD imaging device) that is connected with microscopic system.
In addition, equipment of the present invention is also optional comprises that recording-member is to record observation or measurement result and/or it is analyzed, and these parts can be connected or integrate with the observation parts.
advantage of the present invention
1. high flux: provided by the invention can automatic washing except culture of nematodes and the finder (can be called " WormFarm system ") of larva, it is very little that complete equipment takes up an area space, can the in parallel or more culturing room of connecting, by different passage switching by-pass valve control groupings, control, after having carried the auto-translating platform, be easy to realize high-throughout observational study.And same function will utilize traditional cultural method to realize, it takes up an area space will be very large, and more difficultly realize high flux and automation.
2. automation, low infringement, high accuracy: the WormFarm system can supply with for nematode the food needed automatically, changing food does not need the line of transference worm sample originally yet, can automatically control as required the T/A of food supply, greatly reduce the step that traditional cultural method frequently shifts sample, save experimenter's time, reduce the additional injury of human factor to experiment sample simultaneously.In normal experiment, in order to keep the nematode food (being generally Escherichia coli) in culture plate fresh, particularly, in the RNAi experiment, generally need the experimenter nematode to be transferred on the culture plate of the bacterium food that is covered with new cultivation every three days, certainly will consume a large amount of time of experimenter and muscle power.When the life cycle of the nematode group that requires to add up extensive different RNA i or different disposal or strain, workload will be heavier.In operating process, the nematode body is damaged and sample size is caused damage and is difficult to avoid.Therefore in the old and feeble screening experiment of extensive high-throughout nematode, normal porous plate solid or the liquid culture of adopting, by the contrast control group, obtain maximum vital values, be difficult to obtain meticulous Life cycle curve, tend to miss the important gene that much old and feeble being correlated with of nematode is just affected mean lifetime.
3. consume few: the working volume of each cell can be as small as 2.5 microlitres, saves experiment material reagent.
4. chip can arbitrarily design, and makes simple: can arbitrarily design as required pipeline number and shape.
5. cost is low: magnetic valve and air compressor can meet the demands, and with the large scale business instrument, compare cheap.
6. without using floxuridine (2'-Deoxy-5-fluorouridine, Floxuridine, FuDR) suppress larval growth: a large amount of offspring's larvae and eggs that the WormFarm system can auto-flushing research nematode Sample producing, avoid using FuDR to suppress larval growth, thereby deduction outer chemical material is on the impact of nematode life cycle.Because nematode has very strong reproduction ability, an adult nematode, can output in 4 days about 300 ovum.For fear of in reproduction period, frequent transfer separates adult and larva, avoid affects old and feeble relevant data acquisition of female generation after larvae development simultaneously, usually must in the solid culture plate, add FuDR to suppress nematode offspring's growth.FuDR is that a kind of can to suppress DNA and RNA synthetic, thereby the medicine of Mitotic Cell Death is synthesized and caused to Profilin.The nematode adult cells overwhelming majority is all the cell of mitosis anaphase, so it is generally acknowledged that FuDR can suppress the growth of ovum and larva, except IDR increases 7%, do not find that the aging course of wild type (N2) nematode control group and FuDR processed group has significant difference.But recently research shows, FuDR can mutagenesis body tub-1 life-span significant prolongation, shows that FuDR can not be ignored fully on the impact of adult, otherwise likely causes the erroneous judgement to aging gene.
7. convenient, direct to the observation of experimental result: the nematode sample of cultivating in the WormFarm system can be directly at stereoscope or high power fluorescence microscopy Microscopic observation, has omitted the step that the nematode sample of traditional cultivation need to be made the glass print.Making after the glass print nematode sample just can not repeated application, and can carry out somatoscopy in the WormFarm system, needn't lose sample.
8. be with a wide range of applications: the application prospect in the research of WormFarm system nematode is very extensive, for example be applied in old and feeble research, particularly based on needs, more complicated food is controlled, erstricted diet (Caloric Restriction for example, CR), or the research of the Aging mechanism of the aspect such as intermittent feeding (Intermittent Fasting, IF).Again for example, system of the present invention can also be applied the screening that this platform carries out large-scale antiaging agent, with expectation, finds the drug target relevant with diseases associated with senescence with prevention that delay senility.
Embodiment
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only are not used in and limit the scope of the invention for the present invention is described.Those skilled in the art can make suitable modification, change to the present invention, and these modifications and change are all within the scope of the present invention.For example, below illustrate the preparation method of micro flow chip, use easier photoresist SU-8, AZP 4620, AZ50XT and PDMS preparation in example, but do not mean that micro flow chip can only be by such method preparation, those skilled in the art can understand, and the existing technology in any this area can be used for preparing the micro flow chip with structure of the present invention.
The experimental technique of unreceipted actual conditions in the following example, can adopt the conventional method in this area, for example, with reference to " molecular cloning experiment guide " (third edition, New York, publishing house of cold spring harbor laboratory, New York:Cold Spring Harbor Laboratory Press, 1989) or the condition of advising according to supplier.The method that the sequence measurement of DNA is this area routine, also can provide test by commercial company.
Unless otherwise indicated, otherwise percentage and umber calculate by weight.Unless otherwise defined, the familiar meaning of all specialties of using in literary composition and scientific words and one skilled in the art is identical.In addition, any method similar or impartial to described content and material all can be applicable in the inventive method.The use that better implementation method described in literary composition and material only present a demonstration.
preparation example 1. adopts photoetch method to make fluid layer pipeline (being the miniflow pipeline) and the key-course pipeline (is control valve
road) template
1. the making of key-course pipeline
Positive glue AZP 4620 for the key-course pipeline (purchased from AZ Electronic Materials) makes.Hexamethyldisilane for silicon chip (HMDS) is smoked 5 minutes, then topple over positive glue AZP 4620 to the silicon chip of hexamethyldisilane modified, rotating speed with 1000rpm rotates 1 minute, on 95 ℃ of heating plates, baking is 5 minutes, then on 115 ℃ of heating plates, toast 10 minutes, and condition is exposed and is developed routinely.
2. the making of fluid layer pipeline
The making of fluid layer pipeline is divided into 3 parts: controllable portion, the chamber portion of cultivating nematode and the narrow pipeline that is used for tackling nematode and goes out worm's ovum.
At first, make controllable portion with positive glue AZ50XT (purchased from AZ Electronic Materials): hexamethyldisilane for silicon chip (HMDS) is smoked 5 minutes, then topple over positive glue AZ50XT to the clean silicon chip of having smoked, rotating speed with 1000rpm rotates 1 minute, 95 ℃ are heated 4 minutes, 115 ℃ are heated 8 minutes, exposure and development.Then progressively be warmed up to 220 ℃ with the positive plastic pipe of sphering with the method that per hour increases by 10 ℃ from 40 ℃ on heating plate.
Then, make the chamber portion of cultivating nematode of negative glue SU-82050 (purchased from MicroChem): topple over negative glue SU-82050 to etching well on the masterplate of positive glue, rotating speed with 1000rpm rotates 1 minute, on 65 ℃ of heating plates, baking is 5 minutes, then toast 10 minutes on 95 ℃ of heating plates, and exposed.Repetition is toasted 5 minutes on 65 ℃ of heating plates, and on 95 ℃ of heating plates, baking is 10 minutes, develops.Heat 1 hour with reinforcing patterns on 150 ℃ of heating plates.
Finally, the narrow pipeline that is constructed for tackling nematode and goes out worm's ovum with negative glue SU-82010 (purchased from MicroChem): topple over negative glue SU-82010 to etching well on the masterplate of positive glue and negative glue, rotating speed with 1000rpm rotates 1 minute, on 65 ℃ of heating plates, baking is 2 minutes, and on 95 ℃ of heating plates, baking is 3 minutes, exposure, repetition is toasted 2 minutes on 65 ℃ of heating plates, on 95 ℃ of heating plates, baking is 3 minutes, develop, and 1 hour reinforcing patterns of 150 ℃ of heating plate heating.
3. the making of micro flow chip
Adopt the method for soft etching to make chip, material used is dimethyl silicone polymer (PDMS): be used to make micro-fluidic chip (being micro flow chip) before, all templates all will be by the smoked disengaging that is beneficial to PDMS and the silicon chip of polymerization in 10 minutes of trim,ethylchlorosilane (TMCS).
First the A component of RTV 615 (purchased from GE Advanced Materials) and B component (adhesion agent) are mixed with the ratio of 5:1, be poured on the silicon chip of fluid layer pipeline, the vacuum suction de-bubble, in 80 ℃ of baking ovens, heating is 20 minutes, from silicon chip, tear with the PDMS piece of fluid layer pipeline, cut into 2 centimetres of sizes of 4 cm x, punching (500 microns of diameters).
The A component of RTV 615 and B component (adhesion agent) are mixed with the ratio of 20:1 simultaneously, be poured on the silicon chip of key-course pipeline, speed with 1600rpm evenly is thrown on silicon chip by mixing material, in 80 ℃ of baking ovens, heating is 30 minutes, then the PDMS block alignment of the fluid layer pipeline that had openning hole is fitted on the key-course silicon chip dried, two-layer polymerization 45 minutes in 80 ℃ of baking ovens, after tearing, punching (500 microns of diameters).
In addition, the A component of RTV 615 and B component (adhesion agent) are mixed with the ratio of 10:1, be poured on slide, speed with 1000rpm evenly is thrown on slide by mixing material, in 80 ℃ of baking ovens, heating is 15 minutes, the two layers of polymer PDMS pieces that have openning hole are attached on slide to heated overnight in 80 ℃ of baking ovens.
the making of preparation example 2. micro flow chips and device
Micro flow chip and device that test in embodiment has following structure by use carry out.
1. the structure of micro flow chip
The structure of micro flow chip 1 is as shown in Figure 1:
Culture of nematodes chamber 3, its the place ahead (near input miniflow pipeline end) is semi-circular, prevent that the retention of food culture fluid from being 100 μ m left and right at the height of square dead angle Zhong， culturing room, suitable adult nematode freely movable (diameter 90 μ m left and right) in culturing room.
There is the screening pipeline 30 of 17 high 10 μ m of wide 20 μ m at culturing room 3 rears (away from an end of inputting miniflow pipeline 4), this pipeline can stop the nematode (width is about 90 μ m) that grows up to pass through, but (the larvae development stage of nematode is divided into L1, L2, L3 and tetra-periods of L4 for ovum and L1 to L3, wherein the length of L1 to L3 nematode is 250-500 μ m, the larva that width is about 20-40 μ m (the L1-L3 nematode can suitably compress by being slightly less than the pipeline of its width) size can be rinsed out pipeline, only stays and needs the adult nematode of observing.For preventing the screening pipeline of life cycle statistical experiment later death nematode obstruction below, respectively there are 28 screening pipelines 30 of same specification on the both sides of culturing room, have effectively avoided the phenomenon of experiment later stage line clogging.
The other end of screening pipeline 30 is connected with output miniflow pipeline 5, thereby collects and discharge by output miniflow pipeline 5 liquid stream, nematode larval and ovum and other impurity flowed out through screening pipeline 30 from culturing room 3.
The cross section that input miniflow pipeline 4 or output miniflow pipeline 5 can be controlled by valve is semicircular pipeline, highly approximately 70 μ m.
This integrated chip 8 said structure unit (construction unit comprise 3,1 input miniflow pipeline 4 of culturing room (its other also have 1 get rid of the loading process in the pipeline of bubble), 30,1 output 5,4 control pipeline 8 of miniflow pipeline of 73 screening pipelines and with 44 elastomer valves that crosspoint 9 is corresponding), the structure of each construction unit is as shown in Figure 1.
2. the overall structure of installing
The structure of device as shown in Figure 3.
Adopt the 15ml centrifuge tube of transformation, utilize compressed air that pressure is provided, designed and produced one group for the loading system of food is provided to nematode.Be communicated with compressed-air actuated flexible pipe and be inserted into the culture fluid bottom, compressed air enters the pressure that produces after centrifuge tube as ordering about the mobile power of culture fluid, the bubble simultaneously produced continuously makes to be suspended in culture fluid as the Escherichia coli of food, effectively prevents the cenobium blocking pipe of deposition.Other flexible pipe one end is immersed in the other end under liquid level and is connected to the sample holes on chip, carries culture fluid.
Control the switching of each pipeline with the valve on LabView software programming control pneumatic braking chip.Adopt stereomicroscope (NikonSMZ1000) to observe and Olympus CCD (DP72) collection picture and view data, statistics nematode survival rate.
the impact of test implementation example 1. glucose on the nematode life-span
Select wild type Caenorhabditis elegans (Caenorhabditis elegans) N2 (purchased from Caenorhabditis Genetics Center) for experimental subjects, the HT115 Escherichia coli (purchased from Source BioScience LifeSciences) that culture of nematodes food is selected OP50 (purchased from Caenorhabditis Genetics Center) and contained the blank plasmid vector of L4440.
The larva in synchronized L1 period, in containing the common NGM culture plate of OP50,20 ℃, is cultivated 60 hours, with nematode M9 buffer solution (3g KH
2pO
4, 6gNa
2hPO
4, 5g NaCl, 1ml 1M MgSO
4, add H
2o to 1 liter) rinse, the suspension that will contain nematode passes into (at first the chip pipeline is modified with surfactant 2% pluronic) in miniflow hollow sheet, 30-40 bar nematode in each culturing room.
At S-Medium (5.85g NaCl, 1gK
2hPO
4, 6g KH
2pO
4, 1ml cholesterol (ethanolic solution of 5mg/ml), add H
2o to 1 liter) in add respectively 0%, 2% or 4% glucose (Glucose), with 0.22 μ m filter suction filtration sterilizing.With the resuspended OP50 food of the culture fluid that contains different glucose, (remove bacterium colony and other impurity assembled with 5 μ m membrane filtrations, prevent from stopping up the chip pipeline) after add in corresponding centrifuge tube, and centrifuge tube is connected on chip to corresponding pipeline.
Chip is put into to 25 ℃ of constant incubators to be cultivated.Utilize valve control system to control the circulation of culture fluid, at interval of circulation in 2 minutes 30 seconds, flow 20 microlitres were per minute, were nematode and changed fresh food.The experimental implementation flow process as shown in Figure 3.Gather picture and view data every day once with stereomicroscope (Nikon SMZ1000) and Olympus CCD (DP72), statistics nematode survival rate.
As shown in Figure 4, start culture of nematodes the culture fluid that contains 2% and 4% glucose from the adult stage, the life cycle of nematode is not than significantly shortening containing the nematode life cycle of growing in glucose culture solution, and the vigor rate of decay is obviously accelerated.This result obviously shortens that (Lee et al., 2009, Cell Metabolism 10,379-391) be consistent with reporting before known nematode containing on the NGM solid culture plate of 2% glucose the life-span.A small amount of glucose just can suppress life-extending correlation factor, for example FOXO family member DAF-16, and heat shock factor HSF-1, make the nematode life cycle shorten (Lee et al., 2009, Cell Metabolism 10,379-391).In prior art, this phenomenon of report confirmation is effectively not same in the nematode of liquid culture.
Above-mentioned result of the test has confirmed that micro flow chip of the present invention provides the effective tool of culture of nematodes and observation, its visual result and have high confidence level.
the impact of test implementation example 2.RNAi on the nematode life-span
According to the prior art report, the sptf-3 gene is longevity gene, after its RNAi, makes the nematode life-span significantly shorten (Xue et al., 2007, Mol Syst Biol, 147); Y82E9BR.3 gene code nematode mitochondria F1F0-ATP synthase, make nematode life-span significant prolongation (Xue et al., 2007, Mol Syst Biol, 147) after its RNAi; Age-1 is known famous aging gene, and its RNAi also can cause the nematode life-time dilatation, and (Johnson et al., 1990, Science 249,908-912).
Using the HT115 coli strain of L4440 carrier that contains respectively sptf-3, Y82E9BR.3 and age-1 genetic fragment as food, feed nematode, observe life cycle and the old and feeble phenotype of nematode in PDMS chip culturing room after RNAi.
Selecting wild type nematode N2 is experimental subjects, and culture of nematodes food is selected HT115 Escherichia coli and the common HT115 Escherichia coli that contain respectively sptf-3, Y82E9BR.3 and age-1 gene RNAi carrier (purchased from Source BioScience LifeSciences).
Synchronized L1 larva in period is added in the NGM culture plate that contains each RNAi clone and plain edition escherichia coli cloning (3g NaCl, 17g agar, the 2.5g peptone, 1ml cholesterol (ethanolic solution of 5mg/ml), add H
2after O to 1L. autoclaving, add 1ml1M CaCl
2, 1ml 1M MgSO
4, 25ml phosphate buffer (pH 6), 1mol/L IPTG, 1mg/L Amp), 20 ℃, cultivate 60 hours, with M9, rinse.
The suspension that will contain nematode passes into (at first the chip pipeline is modified with surfactant 2% pluronic) in micro flow chip, 30-40 bar nematode in each culturing room.With containing 1mol/L IPTG, 1mg/L Amp, 250 μ g/L Fungizone Fungizone, the resuspended above-mentioned RNAi food of the S-medium of 10mg/L quadracycline (Tet), (remove bacterium colony and other impurity assembled with 5 μ m membrane filtrations, prevent from stopping up the chip pipeline) after add in corresponding centrifuge tube, be connected on chip corresponding pipeline.
Chip is put into to 25 ℃ of constant incubators to be cultivated.Utilize valve control system to control the circulation of culture fluid, at interval of circulation in 2 minutes 30 seconds, flow was that 20 microlitres are per minute, was nematode and changed fresh food.The experimental implementation flow process as shown in Figure 3.Gather picture and view data every day once with stereomicroscope (Nikon SMZ1000) and Olympus CCD (DP72), statistics nematode survival rate.
After the sptf-3 gene RNAi, the nematode life-span in culturing room is compared remarkable shortening with control group as shown in Figure 5, and growth rate is very fast; The nematode life-span after the Y82E9BR.3 gene RNAi in culturing room is compared significant prolongation with control group, and build is obviously tiny than the contrast nematode; After RNAi falls the age-1 gene, the nematode life-span meets the significant prolongation of expection.The nematode life cycle testing result of cultivating on the solid NGM medium that the above results peace row carries out is consistent.
Above-mentioned result of the test has confirmed that micro flow chip of the present invention provides the effective tool of culture of nematodes and observation, its visual result and have high confidence level.
All documents of mentioning in the present invention are all quoted as a reference in this application, just as each piece of document quoted separately as a reference.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (15)
1. cultivate, observe for Caenorhabditis elegans (Caenorhabditis elegans) and/or the micro flow chip (1) of research for one kind, described micro flow chip (1) comprises microflow layer (2) and substrate (20), and described microflow layer (2) comprising:
At least one culturing room (3) for cultivating nematode, described culturing room (3) is a cavity be enclosed in microflow layer, only by the miniflow pipeline, with the external world, communicate, described culturing room (3) is greater than the diameter of nematode adult in the size perpendicular on the micro flow chip direction;
The at least one input miniflow pipeline (4) be connected with culturing room (3) and at least one screening pipeline (30) be connected with culturing room (3), described input miniflow pipeline (4) is for to culturing room (3) infusion fluid, described screening pipeline (30) is connected with at least one output miniflow pipeline (5) of the larvae and eggs of the liquid flowed out through screening pipeline (30) from culturing room (3) with discharge for collection and nematode, described screening pipeline (30) the aperture area of culturing room's inwall be less than or equal to the average cross-section of nematode adult long-pending 2/3 and be more than or equal to the average cross-section long-pending 1/2 of nematode larval and ovum.
2. micro flow chip according to claim 1 (1), it is characterized in that, described screening pipeline (30) is rectangle at the opening of culturing room (3) inwall, and described rectangular length is 15-25 μ m, be preferably 18-22 μ m, more preferably 20 μ m; Described rectangular wide be 8-12 μ m, be preferably 9-11 μ m, more preferably 10 μ m.
3. micro flow chip according to claim 1 (1), is characterized in that, described screening pipeline (30) is circular at the opening of culturing room (3) inwall, and the diameter of described circle is 8-12 μ m, is preferably 9-11 μ m, more preferably 10 μ m.
4. according to the described micro flow chip of any one in claim 1～3 (1), it is characterized in that, described culturing room (3) is at the size perpendicular on the micro flow chip direction >=100 μ m.
5. according to the described micro flow chip of any one in claim 1～3 (1), it is characterized in that the number of described input miniflow pipeline (4) >=1, the number of described screening pipeline (30) >=3.
6. according to the described micro flow chip of any one in claim 1～3 (1), it is characterized in that, described culturing room (3) is strip, the opening of described input miniflow pipeline (4) in culturing room (3) is positioned at an end of the major axis of strip, and the opening of described screening pipeline (30) in culturing room (3) is positioned on two sides of the other end of major axis of strip and strip.
7. micro flow chip according to claim 6 (1), is characterized in that, strip culturing room (3) is circular arc with the end that described input miniflow pipeline (4) is connected, and is preferably semicircle.
8. micro flow chip according to claim 6 (1), it is characterized in that, the number of described screening pipeline (30) >=12, wherein be positioned at the number >=4 of screening pipeline (30) of an end of the major axis of described strip, be positioned at the number of the screening pipeline (30) on arbitrary side of strip >=4.
9. according to the described micro flow chip of any one in claim 1～3 (1), it is characterized in that, described micro flow chip (1) also comprises key-course (6) and articulamentum (7), between described key-course (6) and described microflow layer (2), by articulamentum (7), separate, described key-course (6) comprising:
At least one controls pipeline (8), described control pipeline (8) has at least one crosspoint (9) with input miniflow pipeline (4) or output miniflow pipeline (5), the articulamentum (7) that described crosspoint (9) is located consists of elastomeric material
Wherein, when the pressure in controlling pipeline (8) is increased to certain value, the elastomer articulamentum (7) of position, described crosspoint (9) expands to microflow layer (2) direction, and the inner surface contact that makes input miniflow pipeline (4) or output miniflow pipeline (5) makes the state of miniflow in disconnecting to blocking miniflow fully; When the pressure in controlling pipeline (8) recovers, the elastomer articulamentum (7) of position, crosspoint (9) recovers ortho states, makes the state of miniflow in circulation.
10. micro flow chip according to claim 9 (1), it is characterized in that, described microflow layer (2), key-course (6) and articulamentum (7) consist of elastomeric material, and described control pipeline (8) locates to enlarge the formation cavity in the crosspoint (9) with input miniflow pipeline (4) and output miniflow pipeline (5).
11. according to the described micro flow chip of claim 9 or 10 (1), it is characterized in that, described elastomeric material is to be selected from one or more in lower group: dimethyl silicone polymer, acrylonitrile-butadiene-styrene copolymer, Merlon, polymethyl methacrylate, polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), polyvinyl chloride, the cyclic polyolefin co-polymer, polyvinylidene fluoride, polystyrene, polysulfones, nylon, the styrene-propene acid copolymer, natural rubber, polyisoprene, butyl rubber, halogenated butyl rubber, polybutadiene, butadiene-styrene rubber, acrylonitrile-butadiene rubber, chloroprene rubber, EP rubbers, epichlorohydrin rubber, lactoprene, silicone rubber, fluorosioloxane rubber, fluoroelastomer, Perfluoroelastomer, ethane-acetic acid ethyenyl ester, resilin (resilin), elastin laminin (elastin), polyimides, phenolic resins, or its combination.
12. a device (100) of cultivating, observing and/or study for Caenorhabditis elegans, is characterized in that, described device comprises:
The described micro flow chip of any one (1) in claim 1～11;
The liquid input block (10) be connected with the input miniflow pipeline (4) of micro flow chip (1);
The external pipe (11) be connected with the control pipeline (8) of micro flow chip (1);
The control assembly be connected with external pipe (11);
Optional observation parts;
Optional recording-member; And
The optional collecting part be connected with output miniflow pipeline (5).
13. device according to claim 12 (100), it is characterized in that, described liquid input block (10) comprises one or more closed containers (15) that liquid is housed, described closed container (15) is connected with the external world by the following pipeline of liquid level in two insertion containers, wherein a pipeline is connected with the input miniflow pipeline (4) on micro flow chip (1), and an other pipeline is connected with compressed air pump.
14. micro flow chip as described as any one in claim 1～11 (1) or device as described as any one in claim 12～13 (100) application in cultivation, observation and/or the research of Caenorhabditis elegans.
15. application as claimed in claim 14, is characterized in that, the cultivation of described nematode, observation and/or research are observed and/or research for Developmental Biology, genetics, genomics, pharmacy.
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| CN111316957A (en) * | 2018-12-14 | 2020-06-23 | 深圳先进技术研究院 | Microfluidic chip, preparation method thereof, nematode culture device and application |
| WO2020181028A1 (en) * | 2019-03-05 | 2020-09-10 | Texas Tech University System | Automated microfluidic system for lifespan and healthspan analysis in nematodes |
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