CN103461279B - For the micro flow chip of culture of nematodes and/or observation and equipment and application thereof - Google Patents
For the micro flow chip of culture of nematodes and/or observation and equipment and application thereof Download PDFInfo
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- CN103461279B CN103461279B CN201210189484.XA CN201210189484A CN103461279B CN 103461279 B CN103461279 B CN 103461279B CN 201210189484 A CN201210189484 A CN 201210189484A CN 103461279 B CN103461279 B CN 103461279B
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Abstract
The present invention relates to for the micro flow chip of culture of nematodes and/or observation and equipment and application thereof.Specifically, the invention provides a kind of micro flow chip for culture of nematodes, observation and/or research; Comprise the equipment of this micro flow chip; And the purposes of described micro flow chip and described equipment.Micro flow chip of the present invention and equipment can take into account high flux and high-resolution, the chemical reagent suppression larval growth avoiding use extra, manufacturing cost and use cost is low, operating automation degree is high, are with a wide range of applications.
Description
Technical field
The present invention relates to biotechnology and field of experiment equipment.Specifically, the present invention relates to a kind of cheapness, be easy to prepare, can automatic washing except larvae and eggs culture of nematodes micro flow chip, comprise the equipment of this micro flow chip and the application of this micro flow chip and equipment.
Background technology
Caenorhabditis elegans (Caenorhabditiselegans) 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, live in soil, the history of life is short, as long as become ripe adult two days more (need 52 hours when 25 DEG C, 48 hours can be reached the soonest) by a development of fertilized ova.In the embryonic development of wild type nematode, the formation of cell division and cell-line has the procedural of height, facilitates like this and carries out genetic analysis to its growth.
Obtain the important foundation that nematode life cycle data is accurately the aging of research nematode.The aging-related phenotype data (motion activity, body size, fat depot and various aging gene merge fluorescin signal etc.) simultaneously obtaining each period in senescence process more have 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, the growth that floxuridine (2'-Deoxy-5-fluorouridine, Floxuridine, FuDR) suppresses nematode offspring must be added in culture plate.Nearest research shows, FuDR can mutagenesis body tub-1 life-span significant prolongation, shows that FuDR can not be almost completely neglected the impact of adult, otherwise likely causes the erroneous judgement to aging gene.During liquid culture, because culture fluid and food cannot be changed, add easy pollution, be difficult to the to accomplish nematode only tracing observation of whole aging course of about 15 days.And these large scale experiments all need to add FuDR to suppress the growth of nematode offspring.
At present, sorting flow cytometer is the most common technology platform carrying 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.), carries out collection analysis because too greatly or too fragility can not apply this instrument.
The current equipment that uniquely can be applied to the small-sized biopsy sample sortings such as nematode and collection phenotypic data is on the market COPAS (ComplexObjectParametricAnalyzerandSorter) system of UnionBiometrica company of U.S. research and development, it utilizes the more wide-aperture flow chamber of comparison type cell instrument, analysis, the sample of sorting diameter in 20-1500 micrometer range.This equipment utilization principle of flow cytometer, form liquid stream by object in turn by analytical port, analytical port has laser to irradiate, can be obtained the size parameter of object by the measurement flight time of object in laser beam; To be blocked by object and the light intensity that decays can obtain the color depth of object and the parameter of transparency by measuring; Instrument, with the laser instrument of three different wave lengths and three groups of light filters, can obtain the information (parameter) of fluorescence labeling on body axis (fluorescin or fluorescence antibody) abundance situation by measuring fluorescence intensity.But there is obvious defect in COPAS system: it can only provide the integral fluorescence value of the cross section total fluorescent value of unit and 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, high flux and high-resolution can be taken into account in the urgent need to developing and avoid using extra chemical reagent to suppress the large scale experiment platform of larval growth in this area.
Summary of the invention
Main purpose of the present invention can take into account high flux and high-resolution to provide a kind of and avoid using extra chemical reagent to suppress the large scale experiment platform of larval growth.
In a first aspect of the present invention, provide a kind of micro flow chip 1 cultivated for Caenorhabditis elegans (Caenorhabditiselegans), observe and/or study, described micro flow chip 1 comprises microflow layer 2 and substrate 20, and described microflow layer 2 comprises:
At least one is for cultivating the culturing room 3 of nematode, and described culturing room 3 is the cavitys being enclosed in microflow layer, communicates with the external world by means of only microchannel, and described culturing room 3 is being greater than the diameter of nematode adult perpendicular to the size on micro flow chip direction;
Be connected with culturing room 3 at least one input microchannel 4 and be connected with culturing room 3 at least one screen pipeline 30, described input microchannel 4 is for infusion fluid in culturing room 3, described screening pipeline 30 exports microchannel 5 from least one of the larvae and eggs of the liquid of culturing room 3 through screening pipeline 30 and flowing out and nematode with for collecting be connected with discharging, and described screening pipeline 30 is less than or equal to 2/3 of the average traversal area of nematode adult and is more than or equal to the average traversal area 1/2 of nematode larval and ovum at the aperture area of culturing room's inwall.
In a preference, described screening pipeline 30 is less than or equal to 1/2 of the average traversal area of nematode adult and is more than or equal to the average traversal area 2/3 of nematode larval and ovum at the aperture area of culturing room's inwall.
In another preference, described screening pipeline 30 is less than or equal to 2/3 of nematode adult average diameter at the maximum inner diameter of culturing room's inwall and is more than or equal to 1/2 of nematode larval and ovum average diameter.
In another preference, described screening pipeline 30 is less than or equal to 1/2 of the average diameter of nematode adult and is more than or equal to the average diameter 2/3 of nematode larval and ovum at the maximum inner diameter of culturing room's inwall.
In certain embodiments of the present invention, described screening pipeline 30 is rectangle at the opening of culturing room 3 inwall, and described rectangular length is 15-25 μm, is preferably 18-22 μm, is more preferably 20 μm; Described rectangular wide be 8-12 μm, be preferably 9-11 μm, be more preferably 10 μm.
In other embodiments of the present invention, described screening pipeline 30 is circular at the opening of culturing room 3 inwall, and the diameter of described circle is 8-12 μm, is preferably 9-11 μm, is more preferably 10 μm.
In other embodiments of the present invention, described culturing room 3 is perpendicular to size >=100 μm on micro flow chip direction.
In other embodiments of the present invention, number >=1 of described input microchannel 4, number >=3 of described screening pipeline 30.
In other embodiments of the present invention, described culturing room 3 is strip, the opening of described input microchannel 4 in culturing room 3 is positioned at one end of the major axis of strip, and the opening of described screening pipeline 30 in culturing room 3 is positioned on the other end of the major axis of strip and two sides of strip.
In other embodiments of the present invention, one end that strip culturing room 3 is connected with described input microchannel 4 is circular arc, is preferably semicircle.
In other embodiments of the present invention, number >=12 of described screening pipeline 30, are wherein positioned at number >=4 of the screening pipeline 30 of one end of the major axis of described strip, are positioned at number >=4 of the screening pipeline 30 on arbitrary side of strip.
In other embodiments of the present invention, described micro flow chip 1 also comprises key-course 6 and articulamentum 7, is separated between described key-course 6 and described microflow layer 2 by articulamentum 7, and described key-course 6 comprises:
At least one controls pipeline 8, described control pipeline 8 and input microchannel 4 or export microchannel 5 and have at least one crosspoint 9, and the articulamentum 7 at described crosspoint 9 place is made up of elastomeric material,
Wherein, when controlling the pressure in pipeline 8 and being increased to certain value, the elastomer articulamentum 7 of position, described crosspoint 9 expands to microflow layer 2 direction, and makes the inner surface contact inputting microchannel 4 or export microchannel 5 to blocking miniflow completely, makes miniflow be in the state of disconnection; When controlling the pressure recover in pipeline 8, the elastomer articulamentum 7 of position, crosspoint 9 recovers ortho states, makes miniflow be in the state of circulation.
In other embodiments of the present invention, described microflow layer 2, key-course 6 and articulamentum 7 are made up of elastomeric material, and described control pipeline 8 forms cavity expanding with input microchannel 4 and crosspoint 9 place that exports microchannel 5.
In a preference, described cavity is circular or rectangle.
In other embodiments of the present invention, described elastomeric material be selected from lower group one or more: dimethyl silicone polymer, acrylonitrile-butadiene-styrene copolymer, Merlon, polymethyl methacrylate, polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), polyvinyl chloride, cyclic polyolefin co-polymer, polyvinylidene fluoride, polystyrene, polysulfones, nylon, Styrene-acrylic 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, provide a kind of device 100 cultivated for Caenorhabditis elegans, observe and/or study, described device comprises:
Micro flow chip 1 of the present invention;
The liquid input block 10 be connected with the input microchannel 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 microchannel 5.
In a preference, described control assembly comprises one or more that be selected from lower group: press device, the magnetic valve be arranged on external pipe 11, the calculator be connected with magnetic valve.
In another preference, described observation parts comprise one or more that be selected from lower group: microscopic system, preferred stereomicroscope, confocal microscope; Imaging system, preferred CCD imaging device.More preferably described observation parts are connected with recording-member, the calculator be such as connected with CCD imaging device.
In certain embodiments of the present invention, described liquid input block 10 comprises one or more closed container 15 that liquid is housed, described closed container 15 is connected with the external world by the pipeline below liquid level in two insertion containers, wherein a pipeline is connected with the input microchannel 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 of Caenorhabditis elegans, observation and/or research of micro flow chip 1 of the present invention or device of the present invention 100.
In certain embodiments of the present invention, the cultivation of described nematode, observation and/or research are used for Developmental Biology, genetics, genomics, pharmacy are observed and/or research.
In a preference, described cultivation, observation and/or research are used for the research of ontogeny and age, are preferred for the research of growing or the research of aging gene, the screening of anti-ageing material, old and feeble characteristic phenotypic and/or aging biology mark.
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Accompanying drawing explanation
Below in conjunction with accompanying drawing, the invention will be further described, and wherein these displays are only in order to illustrate embodiment of the present invention, instead of 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: every integrated 8 culturing room of chip block, are realized the parallel operation of 8 culturing room 3 by the opening and closing controlling pipeline 8; Wherein black part belongs to key-course 6, and grey parts belongs to microflow layer 2.
The schematic diagram of (b) single culturing room and accessory structure thereof: culture of nematodes room 3, its length, width and height can be such as 4000 μm × 1000 μm × 100 μm; Connect culturing room and the screening pipeline 30 exporting microchannel 5, its length, width and height can be such as 80 μm × 20 μm × 10 μm; Export microchannel 5 and can be rectangle with input microchannel 4, the wide height of its cross section can be 300 μm × 70 μm; Control pipeline 8 and export microchannel 5 and input microchannel 4 to form crosspoint 9 respectively, realize the access and exit control of miniflow.
The structure for amplifying at (c) crosspoint 9 place and miniflow access and exit control schematic diagram: substrate 20 bearer control layer 6, articulamentum 7 and microflow layer 2, separated by articulamentum 7 between key-course 6 and microflow layer 2; Key-course 6 comprises at least one control pipeline 8, this control pipeline 8 and input microchannel 4 or export microchannel 5 and have at least one crosspoint 9; The articulamentum 7 at crosspoint 9 place is made up of elastomeric material.
Wherein, when controlling the pressure in pipeline 8 and being increased to certain value, the elastomer articulamentum 7 of position, described crosspoint 9 expands to microflow layer 2 direction and makes the inner surface contact inputting microchannel 4 or export microchannel 5 to blocking miniflow completely, make it be in the state of disconnection, namely make micro-valve close; When controlling the pressure recover in pipeline 8, the elastomer articulamentum 7 of position, crosspoint 9 recovers ortho states, and miniflow is communicated with, and namely makes micro-valve open.
Fig. 2: chip operation flow chart:
A () modifies chip pipe surface with surfactant 2% pluronic (pluronic);
B () passes into nematode suspension;
C culture fluid is changed in () timing.
Fig. 3: energy automatic washing is except the culture of nematodes device schematic diagram of larva.
Liquid input block 10 comprises the closed container 15 that 8 are equipped with liquid, described closed container 15 is connected with the external world by the pipeline below liquid level in two insertion containers, wherein a pipeline is connected with the input microchannel 4 on micro flow chip 1, and an other pipeline is connected with compressed air pump.Control pipeline 8 to be connected with external pipe 11, control crosspoint 9 opening and closing by compressed air-driven.
Fig. 4: the process that nematode is old and feeble in time under different glucose environment and Life cycle curve figure:
A () adult stage is cultivated obviously fast than not containing the nematode aging rate grown in the culture fluid of glucose containing the nematode aging rate in the culture fluid of 2% and 4% glucose, the vigor rate of decay is obviously accelerated;
B () chip contains 2% with nematode in the culturing room of the glucose culture solution of 4% concentration compared with control group, the life-span significantly shortens;
C on the NGM solid culture plate of () glucose containing variable concentrations, nematode life cycle is different: 2% with the glucose of 4% concentration compared with control group, significantly shorten the nematode life-span.
Fig. 5: the process that nematode is old and feeble in time under different RNA i pressure environment and Life cycle curve figure:
(a) adult stage cultivate the culture fluid containing blank and respectively containing sptf-3, Y82E9BR.3 and age-1 gene RNAi clone culture fluid in nematode aging rate and the vigor rate of decay variant;
On (b) chip containing nematode in different RNA i pressure culturing room compared with control group, the life-span is variant;
C on () NGM solid culture plate containing different RNA i pressure, nematode life cycle is different.
Embodiment
The present inventor can not provide the problem of 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, develop a kind of cheapness, be easy to prepare and can automatic washing except the culture of nematodes micro flow chip of larva, and provide based on this chip, culture of nematodes and finder that image data can be provided in real time.In addition, the present inventor additionally provides the application of this micro flow chip and device.
term definition
In the present invention, term " chip " is identical with the chip concept that usual this area defines, namely outward appearance is the homogeneous smooth board of thickness, and modal shape is rectangle or square, but those of ordinary skill in the art also can adjust its shape as required.
In the present invention, term " miniflow " refers to that the material (comprising molecule and colloid) under micro-(receiving) metrical scale transmits, Momentum Transfer, heat trnasfer, and course of reaction in the transmission.
In the present invention, term " micro flow chip " refers to integrated micro-channels network and numerous analytic function element, can override receive and rise to the chip that skin rises volume fluid in micron scale construction, the resulting structure (comprising passage, reative cell and some other functional part) of its containing fluid be at least micron order yardstick in a dimension.As used herein, term " chip of the present invention " and " micro flow chip " are used interchangeably, and all refer to the chip for culture of nematodes and/or observation provided in the present invention.
" microchannel " in the present invention is with the opening of microchannel in culturing room for mark, a corresponding microchannel of opening.Accordingly, " N bar microchannel " refers to each microchannel corresponding with the N number of opening in culturing room, and the total quantity of described microchannel is N bar.
Term " input microchannel " refer to be communicated with culturing room, for one or more microchannel toward infusion fluid in culturing room (such as comprise the medium of food, comprise the medium etc. of drug candidate).As required, the mixture of same input microchannel input plurality of liquid can be adopted, many also can be adopted to input microchannel and input different liquid respectively.If needed, can at the other discharge duct arranging at least one and get rid of bubble in loading process of input microchannel.
In the present invention, term " screening pipeline " or " screening microchannel " are used interchangeably, and all refer to the sidewall of culturing room or away from inputting one end of microchannel and exporting microchannel and be connected, have and the nematode larval in culturing room and ovum can be allowed to enter through it export microchannel but the microchannel of the maximum inner diameter not allowing adult to pass through or cross-sectional area.
The shape of the cross section of screening pipeline can be rectangle, square, circle or oval usually.When the cross section screening pipeline is rectangle, " maximum inner diameter " refers to its size that is long and wide middle the greater; When the cross section screening pipeline is oval, " maximum inner diameter " refers to the diameter of its major axis.In a preference, screening pipeline is less than or equal to 2/3 of the average traversal area of nematode adult and is more than or equal to the average traversal area 1/2 of nematode larval and ovum at the aperture area of culturing room's inwall, do not discharge nematode adult for discharging nematode larval and ovum with liquid stream.In another preference, described screening pipeline (30) is less than or equal to 1/2 of the average diameter of nematode adult and is more than or equal to the average diameter 2/3 of nematode larval and ovum at the maximum inner diameter of culturing room's inwall.In another preference, screening pipeline is less than or equal to 2/3 of nematode adult average diameter at the maximum inner diameter of culturing room's inwall and is more than or equal to 1/2 of nematode larval and ovum average diameter, or be less than or equal to 1/2 of nematode adult average diameter and be more than or equal to 2/3 of nematode larval and ovum average diameter, thus can not discharge nematode adult with liquid stream discharge nematode larval and ovum.
Term " output microchannel " refers to and screens pipeline and be connected, for one or more microchannel of the liquid stream and nematode larval and ovum of collecting and eject self-sizing pipeline.
" crosspoint " in the present invention refers to microchannel and the joining controlling the projection of pipeline on micro flow chip.Being understood that, because microchannel and control pipeline belong to different layers, is disconnected between them.
micro flow chip
In certain embodiments of the present invention, the culture of nematodes micro flow chip of a kind of energy automatic washing except larva is provided.
Micro flow chip comprises a microflow layer, described microflow layer comprises at least one for cultivating the culturing room of nematode, described culturing room is a cavity being enclosed in microflow layer, communicate with the external world by means of only microchannel, the thickness of cavity (or claiming height) is preferably greater than the diameter of adult but is not more than the twice of adult diameter, stacked with what avoid between nematode, above-mentioned thickness refers to that cavity is perpendicular to the size on micro flow chip direction;
The number of the input microchannel be communicated with each culturing room is more than or equal to 1, and wherein at least one microchannel is used for toward infusion fluid in culturing room; The number of the screening pipeline be communicated with each culturing room is more than or equal to 1 (namely at least one), for discharging the larva of liquid and nematode; All screening pipelines are all less than the cross-sectional area of nematode adult at the area of the opening of culturing room's inwall and are greater than the cross-sectional area of nematode larval, and to ensure that nematode larval can discharge culturing room by screening pipeline, adult then can not.
The screening opening of pipeline in culturing room can be arranged on the other end contrary with inputting the opening of microchannel in culturing room and/or be arranged on the side (or claiming sidewall) of culturing room, preferably all arranges on described one end and sidewall and screens pipeline to guarantee unobstructed discharge nematode larval and ovum and other foreign material.
In other embodiments of the present invention, that this micro flow chip also can be included in culturing room's inner opening, that opening and closing is controlled large output channel (being namely greater than cross-sectional area or the diameter of adult), so that thoroughly cleaning culturing room or the content thoroughly changed in culturing room, thus make culturing room and micro flow chip reusable.
Adopt technique scheme can solve the most headachy technical problem in prior art dexterously: how to eliminate the FuDR that uses in conventional method to the impact of nematode.The impact of FuDR to be eliminated completely, just must not use FuDR.Adopt technique scheme of the present invention, all be less than the cross-sectional area of nematode adult due to screening pipeline at the area of the opening of culturing room's inwall and be greater than the cross-sectional area of nematode larval, thus nematode larval can discharge culturing room by screening pipeline through exporting microchannel, and adult then can not.In experimentation, by constantly injecting new liquid from input microchannel in culturing room, just can utilize and from exporting the liquid flowed out microchannel, the chalaza of nematode larval and nematode be gone out culturing room via screening pipeline, thus not use FuDR just can remove larvae and eggs.
In actual experiment operation, can experimentally in nematode (or other biology) adult that uses and larva body size difference determine to export and screen microchannel in the shape of the opening of culturing room's inwall and size.In fact, screening microchannel is not subject to special restriction in the shape of the opening of culturing room's inwall, and total principle ensures that nematode larval can discharge culturing room by screening microchannel through exporting microchannel, and adult then can not.For Caenorhabditis elegans, if screening pipeline is circular at the opening of culturing room's inwall, then the diameter of described circle can be 8-12 μm, is preferably 9-11 μm, is more preferably 10 μm; If screening pipeline is rectangle at the opening of culturing room's inwall, then can arrange described rectangular length is 15-25 μm, and be preferably 18-22 μm, be more preferably 20 μm, wide is 8-12 μm, is preferably 9-11 μm, is more preferably 10 μm.
Because culturing room is mainly used to provide living space to nematode adult, therefore the space size of culturing room should be suitable for the growth of nematode adult.For Caenorhabditis elegans, the diameter of its adult is generally at about 90 μm, and therefore the thickness of culturing room generally should be set at least be greater than this diameter, preferably >=100 μm.
Preferably, input microchannel number >=1, screening number of conduits >=3.The number of input microchannel can be determined according to the kind of infusion fluid; The number of usual screening pipeline is more, then discharge liquid, larva and ovum efficiency higher, generally dozens of is set.
Preferably, culturing room is strip, and the opening of input microchannel in culturing room is positioned at one end of the major axis of strip, and the opening of screening pipeline in culturing room is positioned on the other end of the major axis of strip and minor axis two sides of strip.Culturing room is set to strip, input microchannel and the opening of screening pipeline in culturing room are arranged on the two ends of the major axis of strip simultaneously, be conducive to liquid and enter culturing room from input microchannel glibly, then flow out culturing room by screening pipeline through exporting microchannel, the liquid flow simultaneously formed is also easy goes out culturing room by larva and chalaza.If liquid flow velocity is very fast, or adult quantity is more, then adult may be flushed to one end of strip and block the screening pipeline tapping of end face under the flowing of fluid, therefore the preferred opening also arranging screening pipeline on two sides of strip, still can eccysis larvae and eggs glibly when ensureing that said circumstances occurs.In the case, can arrange in the side of culturing room and one end accordingly and export microchannel accordingly.
Preferably, one end that strip culturing room is connected with input microchannel is set to circular arc, and more preferably, described circular arc is semicircle.So arrange and can prevent the retention of food culture fluid in the square dead angle of input microchannel one end.
Preferably, screening number of conduits >=6, more preferably >=12, more preferably >=20, the opening of screening pipeline in culturing room is positioned on two sides of the other end of the major axis of strip and the minor axis of strip, wherein be positioned at number of openings >=2 of the other end of described strip major axis, preferably >=4, more preferably >=6, are positioned at number of openings >=2 on arbitrary side of strip minor axis, preferably >=4, more preferably >=6.The screening pipeline arranging a greater number can ensure efficiently larvae and eggs to be flushed out culturing room.In a preferred embodiment, generally arrange 10 with upper shed at end face, the opening of more than 20 is all set in each side.
Preferably, all microchannel are all in microflow layer, and microchannel has opening and is in communication with the outside on micro flow chip, inject liquid or discharge liquid and larva from culturing room by described opening in culturing room, micro flow chip also comprises key-course, separated by articulamentum between key-course and microflow layer, described key-course has at least one control pipeline, described control pipeline and microchannel have at least one crosspoint, the articulamentum at described crosspoint place is made up of elastomeric material, when controlling ducted pressure and being increased to certain value, the elastomer articulamentum of at least one cross-point locations above-mentioned will expand to microflow layer direction, and make microchannel inner surface contact to blocking microchannel completely, it is made to be in the state of disconnection, when controlling ducted pressure recover, the elastomer articulamentum of cross-point locations recovers ortho states, microchannel is communicated with again.So just forming one can the micro flow chip of automatic control fluid flow, control pipeline is connected with providing the equipment of pressure (as air pump, peristaltic pump etc.), carry out the ducted pressure size of regulable control by press device, thus control the connected state of microchannel further.
In one preferred embodiment, some control pipelines are set, the connected state that pipeline can control arbitrarily each input microchannel and output microchannel is controlled by these, input microchannel is connected with providing the pressure source of steady pressure, thus in time inputting microchannel and output microchannel is in connected state, to continuously new liquid be had to enter culturing room from input microchannel, original liquid continuously leaves culturing room from output microchannel simultaneously, control pipeline to be connected with continuing to provide the press device of pressure by outside pipeline, external pipe arranges magnetic valve, magnetic valve is connected with calculator, opened or closed by the time that calculator Controlling solenoid valve is presetting, just can the connection of Controlling solenoid valve place external pipe or disconnection, thus control connection or the disconnection of the control pipeline be connected with this external pipe, the connection of the microchannel that this control pipeline of further control controls or disconnection, thus final realization injects liquid in the time preset toward culturing room, rinse larvae and eggs, reach full-automatic object of cultivating 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 managing the synchronization of multiple culturing room and controlling.
Certainly, those of ordinary skill in the art also can adopt structure and the method for other control liquid input and output as known in the art.Such as, the method controlling liquid input can use gravity, compressed-air actuated pressure, peristaltic pump pressure, magnetic force, electrical potential difference.Preferably, microflow layer, articulamentum and key-course (as shown in Figure 1 example) are formed by elastomeric material, and control pipeline expands at the crosspoint place with microchannel and forms cavity.Described cavity is preferably circular or rectangle.Microflow layer, key-course and articulamentum all use elastomeric material to be to prepare micro flow chip with easy laboratory method.Because many elastomeric materials have the performance of good machine-shaping, such as thermoplasticity, thermosetting, radiation-initiated crosslinking etc., without the need to the equipment of costliness and the method for complexity, adopt the simple common equipment in laboratory just can prepare the micro flow chip of various structure easily.Control pipeline expands at the crosspoint place with microchannel and forms cavity, because the articulamentum of empty cavity position is thinner, therefore when controlling ducted pressure and increasing, the articulamentum at empty cavity position place will expand at first, control suitable pressure and just can ensure to only have the articulamentum within the scope of cavity to expand, thus block microchannel in cavity position.
Preferably, elastomeric material is to be selected from lower group the mixture of one or any two kinds or more: 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 (CyclicOlefinCopolymers, COC), polyvinylidene fluoride, polystyrene, polysulfones, nylon, Styrene-acrylic 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.Use dimethyl silicone polymer to prepare micro flow chip in embodiment, this be manyly be conducive to micro-ly building because PDMS has, 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 the culture of nematodes of larva and/or finder, this device comprises aforesaid micro flow chip, the liquid input block be connected with the input microchannel of micro flow chip, the external pipe be connected with the control pipeline of micro flow chip, the control assembly (such as press device, the magnetic valve be arranged on said external pipeline, the calculator that is connected with magnetic valve) be connected with said external pipeline, optional observation parts (such as stereomicroscope and the CCD imaging device that is connected with stereomicroscope), optional recording-member.Those of ordinary skill in the art according to real needs, can adjust the parts in culture of nematodes and finder and do not depart from general plotting of the present invention and scope.
The not special restriction of liquid input block used in the present invention, as long as liquid can be driven to enter culturing room, such as, can be longer one section of pipeline being full of liquid, drive ducted liquid to enter culturing room by peristaltic pump.
Preferred a kind of liquid input block comprises the closed container that is equipped with liquid, described closed container is connected with the external world by the pipeline below liquid level in two insertion containers, wherein a pipeline is connected with the input microchannel 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 container bottom, the bubble of emerging from pipeline can make the Escherichia coli as nematode feeds be suspended in culture fluid, effectively prevent the cenobium blocking pipe deposited, in time inputting microchannel and output microchannel is 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 to input in microchannel and there is the power that one is injected liquid constantly in culturing room, the press device be connected with external pipe is opened, control all magnetic valves by calculator and be in open mode, make above-mentioned press device produce certain pressure in control pipeline, closed by all microchannel, nematode can grow in immobilising culture fluid, when needs are changing liquid to some culturing room sometime, when rinsing larvae and eggs, with computer programming, some magnetic valve is closed in this moment, so the distress resolves in the exterior line at above-mentioned magnetic valve place, the ducted pressure of the control be connected with said external pipeline also disappears, so the elastomer articulamentum at above-mentioned control pipeline and microchannel crosspoint place recovers ortho states, microchannel is communicated with again, the culturing room be connected with the above-mentioned microchannel be again communicated with starts to change liquid, new liquid enters culturing room from input microchannel, original liquid, larva and ovum leave culturing room from output microchannel, thus achieve and fully automatically cultivate nematode, rinse larva.
Those of ordinary skill in the art should know, and liquid input block as known in the art and control assembly can be adopted to realize liquid input and liquid current control and do not depart from general plotting of the present invention and scope.
Observation parts can be set according to the concrete needs observed, such as microscopic system (as stereomicroscope, confocal microscope), the imaging system (as CCD imaging device) that is connected with microscopic system.
In addition, also the optional recording-member that comprises is to record observation or measurement result and/or to analyze it for equipment of the present invention, and these parts can be connected with observation parts or integrate.
advantage of the present invention
1. high flux: energy automatic washing provided by the invention removes 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 in parallel or more culturing room of connecting, controlled by the grouping of different passage switching by-pass valve controls, after having carried 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: WormFarm system can be the food of nematode supply needs automatically, changing food does not need line of transference worm sample originally yet, automatically the T/A of food supply can be controlled as required, greatly reduce the step that classical culture protocols frequently shifts sample, save the time of experimenter, reduce human factor to the additional injury of experiment sample simultaneously.In normal experiment, in order to keep the nematode feeds in culture plate (being generally Escherichia coli) fresh, particularly in RNAi experiment, generally need experimenter to be transferred to by nematode every three days and be covered with on the culture plate of the new bacterial food cultivated, experimenter's a large amount of time and muscle power certainly will be consumed.When requiring to add up the life cycle of the nematode group of extensive different RNA i or different disposal or strain, workload will be heavier.In operation nematode body is damaged and sample size is caused damage and be difficult to avoid.Therefore in the old and feeble screening experiment of extensive high-throughout nematode, normal employing porous plate solid or liquid culture, by contrast control group, obtain maximum vital values, be difficult to obtain meticulous Life cycle curve, often miss and much nematode aging be correlated with and just affect the important gene of mean lifetime.
3. consume few: the working volume of each cell can be as small as 2.5 microlitres, save experiment material reagent.
4. chip can arbitrarily design, and makes simple: can arbitrarily design number of conduits and shape as required.
5. cost is low: magnetic valve and air compressor can meet the demands, cheap compared with large scale business instrument.
6. without the need to using floxuridine (2'-Deoxy-5-fluorouridine, Floxuridine, FuDR) larval growth is suppressed: WormFarm system can a large amount of offspring's larvae and eggs of producing of auto-flushing research nematode sample, avoid using FuDR to suppress larval growth, thus deduction outer chemical material is on the impact of nematode life cycle.Because nematode has very strong reproduction ability, an adult nematodes, can output about 300 ovum in 4 days.Be separated adult and larva in order to avoid frequently shifting in reproduction period, after avoiding larvae development, impact is female for old and feeble relevant data acquisition simultaneously, usually must add FuDR to suppress the growth of nematode offspring in solid culture plate.FuDR is that one can suppress DNA and RNA to synthesize, thus Profilin is synthesized and caused the medicine of Mitotic Cell Death.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 except 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 almost completely neglected the impact of adult, otherwise likely causes the erroneous judgement to aging gene.
7. the observation of pair experimental result is convenient, direct: the nematode sample cultivated in WormFarm system can directly at stereoscope or high power fluorescence microscopy Microscopic observation, and the nematode sample eliminating tradition cultivation needs the step making glass print.Making after glass print nematode sample just can not repeated application, and can carry out somatoscopy in WormFarm system, need not lose sample.
8. be with a wide range of applications: the application prospect in the research of WormFarm system nematode widely, such as be applied in old and feeble research, particularly based on needing more complicated food to control, such as erstricted diet (CaloricRestriction, CR), or the research of the Aging mechanism of the aspect such as intermittent feeding (IntermittentFasting, IF).Again such as, system of the present invention can also apply the screening that this platform carries out large-scale antiaging agent, delays senility to expect to find and prevents the drug target relevant with diseases associated with senescence.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.Those skilled in the art can make suitable amendment, variation to the present invention, and these amendments and variation are all within the scope of the present invention.Such as, below illustrate the preparation method of micro flow chip, easier photoresist SU-8, AZP4620, AZ50XT and PDMS preparation is used in example, but do not represent that micro flow chip can only be prepared by such method, 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, the conventional method in this area can be adopted, such as with reference to " Molecular Cloning: A Laboratory guide " (third edition, New York, CSH Press, NewYork:ColdSpringHarborLaboratoryPress, 1989) or the condition of advising according to supplier.The sequence measurement of DNA is the method for this area routine, also can provide test by commercial company.
Unless otherwise indicated, otherwise percentage and number calculate by weight.Unless otherwise defined, all specialties used in literary composition and scientific words and one skilled in the art the meaning be familiar with 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 (i.e. microchannel) and key-course pipeline (i.e. control valve
road) template
1. the making of key-course pipeline
Key-course pipeline makes of positive glue AZP4620 (purchased from AZElectronicMaterials).Silicon chip hexamethyldisilane (HMDS) is smoked 5 minutes, then positive glue AZP4620 is toppled on the silicon chip of hexamethyldisilane modified, 1 minute is rotated with the rotating speed of 1000rpm, 95 DEG C of heating plates toast 5 minutes, then toast 10 minutes on 115 DEG C of heating plates, and condition carries out exposing and developing routinely.
2. the making of fluid layer pipeline
The making of fluid layer pipeline is divided into 3 parts: controllable portion, cultivate nematode chamber portion and be used for tackling nematode and going out the narrow conduit of worm's ovum.
First, controllable portion is made: smoked 5 minutes by silicon chip hexamethyldisilane (HMDS) with positive glue AZ50XT (purchased from AZElectronicMaterials), then positive glue AZ50XT is toppled on the clean silicon chip smoked, 1 minute is rotated with the rotating speed of 1000rpm, 95 DEG C are heated 4 minutes, 115 DEG C are heated 8 minutes, exposure and development.Then 220 DEG C are progressively warmed up to the positive plastic pipe of sphering with the method for increase per hour 10 DEG C from 40 DEG C on hot plate.
Then, the chamber portion of nematode is cultivated: topple over negative glue SU-82050 on the masterplate etching positive glue with the making of negative glue SU-82050 (purchased from MicroChem), 1 minute is rotated with the rotating speed of 1000rpm, 65 DEG C of heating plates toast 5 minutes, then toast 10 minutes on 95 DEG C of heating plates, and expose.Repeat to toast 5 minutes on 65 DEG C of heating plates, 95 DEG C of heating plates toast 10 minutes, development.150 DEG C of heating plates heat 1 hour with reinforcing patterns.
Finally, the narrow conduit tackled nematode and go out worm's ovum is constructed for: topple over negative glue SU-82010 on the masterplate etching positive glue and negative glue with negative glue SU-82010 (purchased from MicroChem), 1 minute is rotated with the rotating speed of 1000rpm, 65 DEG C of heating plates toast 2 minutes, 95 DEG C of heating plates toast 3 minutes, exposure, repeat to toast 2 minutes on 65 DEG C of heating plates, 95 DEG C of heating plates toast 3 minutes, development, 150 DEG C of heater plate, 1 hour reinforcing patterns.
3. the making of micro flow chip
The method of Soft lithograph is adopted to make chip, material used is dimethyl silicone polymer (PDMS): before being used to make micro-fluidic chip (i.e. micro flow chip), and all templates all will by the smoked disengaging being beneficial to PDMS and the silicon chip be polymerized for 10 minutes of trim,ethylchlorosilane (TMCS).
First the component A of RTV615 (purchased from GEAdvancedMaterials) and B component (adhesion agent) are mixed with the ratio of 5:1, be poured on the silicon chip of fluid layer pipeline, vacuum suction de-bubble, heat 20 minutes in 80 DEG C of baking ovens, tear with the PDMS block of fluid layer pipeline from silicon chip, cut into 4 cm x, 2 centimetres of sizes, punching (diameter 500 microns).
The component A of RTV615 and B component (adhesion agent) are mixed with the ratio of 20:1 simultaneously, be poured on the silicon chip of key-course pipeline, with the speed of 1600rpm, mixing material is evenly thrown on silicon chip, heat 30 minutes in 80 DEG C of baking ovens, then the PDMS block alignment of the fluid layer pipeline had openning hole is fitted on the key-course silicon chip be baked, two-layer in 80 DEG C of baking ovens be polymerized 45 minutes, after tearing, punching (diameter 500 microns).
In addition, the component A of RTV615 and B component (adhesion agent) are mixed with the ratio of 10:1, be poured on slide, with the speed of 1000rpm, mixing material is evenly thrown on slide, heat 15 minutes in 80 DEG C of baking ovens, the two layers of polymer PDMS blocks had openning hole are attached on slide, heated overnight in 80 DEG C of baking ovens.
the making of preparation example 2. micro flow chip and device
Test in embodiment has the micro flow chip of following structure by using and device carries out.
1. the structure of micro flow chip
The structure of micro flow chip 1 is as shown in Figure 1:
Culture of nematodes room 3, its front (namely near input microchannel end) is for semi-circular, prevent the retention of food culture fluid in square dead angle, the height of culturing room is about 100 μm, and suitable adult nematodes is freely activity (diameter about 90 μm) in culturing room.
There is the screening pipeline 30 of 17 wide 20 μm high 10 μm at culturing room 3 rear (namely away from one end of input microchannel 4), this pipeline can stop adult nematodes (width is about 90 μm) to pass through, but (larvae development stage of nematode is divided into tetra-periods of L1, L2, L3 and L4 for ovum and L1 to L3, wherein the length of L1 to L3 nematode is 250-500 μm, the larva that width is about 20-40 μm of (L1-L3 nematode suitably can compress the pipeline by being slightly less than its width) size can be rinsed out pipeline, only leaves the adult nematodes needing to observe.For the screening pipeline preventing life cycle statistical experiment later death nematode from blocking below, respectively there are 28 screening pipelines 30 of same specification on the both sides of culturing room, effectively prevent the phenomenon of experiment later stage line clogging.
The other end of screening pipeline 30 is connected with output microchannel 5, thus by exporting microchannel 5 and collect and discharging from culturing room 3 through screening the liquid stream of pipeline 30 outflow, nematode larval and ovum and other impurity.
Input microchannel 4 or output microchannel 5 can be semicircular pipeline by the cross section of Valve controlling, highly about 70 μm.
This integrated chip 8 said structure unit (construction unit comprise culturing room 3,1 input microchannel 4 (otherly also having 1 pipeline getting rid of bubble in loading process at it), 73 30,1, screening pipelines export microchannel 5,4 control pipelines 8 and 4 the elastomer valves corresponding with 4 crosspoints 9), the structure of each construction unit as shown in Figure 1.
2. the overall structure of device
The structure of device as shown in Figure 3.
Adopt the 15ml centrifuge tube of transformation, utilize compressed air to provide pressure, designed and produced one group for providing the loading system of food to nematode.Being communicated with compressed-air actuated flexible pipe is inserted into bottom culture fluid, the pressure that compressed air produces after entering centrifuge tube is as the power ordering about culture fluid flowing, the bubble simultaneously produced continuously makes the Escherichia coli as food be suspended in culture fluid, effectively prevents the cenobium blocking pipe deposited.Other flexible pipe one end is immersed in the other end under liquid level and is connected to sample holes on chip, conveying culture fluid.
The opening and closing of each pipeline of Valve controlling on pneumatic braking chip is controlled with LabView software programming.Stereomicroscope (NikonSMZ1000) observation and Olympus CCD (DP72) is adopted to gather picture and view data, statistics nematode survival rate.
testing example 1. glucose is on the impact in nematode life-span
Select Wild-type C. elegans (Caenorhabditiselegans) N2 (purchased from CaenorhabditisGeneticsCenter) to be experimental subjects, culture of nematodes food selects OP50 (purchased from CaenorhabditisGeneticsCenter) and the HT115 Escherichia coli (purchased from SourceBioScienceLifeSciences) containing L4440 empty plasmid carrier.
The larva in synchronized L1 period, containing in the common NGM culture plate of OP50,20 DEG C, is cultivated 60 hours, with nematode M9 buffer solution (3gKH
2pO
4, 6gNa
2hPO
4, 5gNaCl, 1ml1MMgSO
4, add H
2o to 1 liter) rinse, the suspension containing nematode is passed into (first chip pipeline is modified with surfactant 2% pluronic) in miniflow hollow sheet, 30-40 bar nematode in each culturing room.
At S-Medium (5.85gNaCl, 1gK
2hPO
4, 6gKH
2pO
4, 1ml cholesterol (ethanolic solution of 5mg/ml), adds H
2o to 1 liter) in add respectively 0%, 2% or 4% glucose (Glucose), with the sterilizing of 0.22 μm of filter suction filtration.With the resuspended OP50 food of the culture fluid containing different glucose, (bacterium colony and other impurity assembled is removed with 5 μm of membrane filtrations, prevent from blocking chip pipeline) after add in corresponding centrifuge tube, and centrifuge tube is connected to corresponding pipeline on chip.
Chip is put into 25 DEG C of constant incubators to cultivate.Utilize valve control system to control the circulation of culture fluid, at interval of circulation in 2 minutes 30 seconds, flow 20 microliter per minute, was nematode and changes fresh food.Experimental implementation flow process as shown in Figure 3.With gathering stereomicroscope (NikonSMZ1000) and Olympus CCD (DP72) every day picture and view data once, statistics nematode survival rate.
As shown in Figure 4, from adult stage by culture of nematodes containing in the culture fluid of 2% and 4% glucose, the life cycle of nematode is not than significantly shortening containing the nematode life cycle grown in glucose culture solution, and the vigor rate of decay is obviously accelerated.This result with to report before known nematode containing 2% glucose NGM solid culture plate on the life-span obviously shorten (Leeetal., 2009, CellMetabolism10,379-391) and be consistent.A small amount of glucose just can suppress life-extending correlation factor, such as FOXO family member DAF-16, and heat shock factor HSF-1, makes nematode life cycle shorten (Leeetal., 2009, CellMetabolism10,379-391).Do not report in prior art and confirm that this phenomenon is effectively same in the nematode of liquid culture.
Above-mentioned result of the test confirms 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.
testing example 2.RNAi is on the impact in nematode life-span
According to prior art report, sptf-3 gene is longevity gene, makes the nematode life-span significantly shorten (Xueetal., 2007, MolSystBiol, 147) after its RNAi; Y82E9BR.3 gene code nematode mitochondria F1F0-ATP synthase, makes nematode life-span significant prolongation (Xueetal., 2007, MolSystBiol, 147) after its RNAi; Age-1 is known famous aging gene, and its RNAi also can cause nematode life-time dilatation (Johnsonetal., 1990, Science249,908-912).
By the HT115 coli strain of L4440 carrier respectively containing sptf-3, Y82E9BR.3 and age-1 genetic fragment as food, feed nematode, observe life cycle in PDMS chip culturing room of nematode after RNAi and senescent phenotypes.
Select wild type nematode N2 to be experimental subjects, culture of nematodes food selects the HT115 Escherichia coli and common HT115 Escherichia coli that contain sptf-3, Y82E9BR.3 and age-1 gene RNAi carrier (purchased from SourceBioScienceLifeSciences) respectively.
Synchronized L1 larva in period is added in the NGM culture plate containing each RNAi clone and plain edition escherichia coli cloning that (1ml cholesterol (ethanolic solution of 5mg/ml), adds H for 3gNaCl, 17g agar, 2.5g peptone
2after O to 1L. autoclaving, add 1ml1MCaCl
2, 1ml1MMgSO
4, 25ml phosphate buffer (pH6), 1mol/LIPTG, 1mg/LAmp), 20 DEG C, cultivate 60 hours, rinse with M9.
Suspension containing nematode is passed into (first chip pipeline is modified with surfactant 2% pluronic) in micro flow chip, 30-40 bar nematode in each culturing room.With containing 1mol/LIPTG, 1mg/LAmp, 250 μ g/L Fungizone Fungizone, the resuspended above-mentioned RNAi food of S-medium of 10mg/L quadracycline (Tet), (bacterium colony and other impurity assembled is removed with 5 μm of membrane filtrations, prevent from blocking chip pipeline) after add in corresponding centrifuge tube, be connected to corresponding pipeline on chip.
Chip is put into 25 DEG C of constant incubators to cultivate.Utilize valve control system to control the circulation of culture fluid, at interval of circulation in 2 minutes 30 seconds, flow was 20 microliter per minute, was nematode and changed fresh food.Experimental implementation flow process as shown in Figure 3.With gathering stereomicroscope (NikonSMZ1000) and Olympus CCD (DP72) every day picture and view data once, statistics nematode survival rate.
The nematode life-span as shown in Figure 5 after sptf-3 gene RNAi in culturing room compares remarkable shortening with control group, and growth rate is very fast; The nematode life-span after Y82E9BR.3 gene RNAi in culturing room compares significant prolongation with control group, and build is obviously tiny than contrast nematode; After RNAi falls age-1 gene, the nematode life-span meets the significant prolongation of expection.The nematode life cycle testing result that the solid NGM medium that the above results peace row carries out is cultivated is consistent.
Above-mentioned result of the test confirms 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 all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention, and these equivalent form of values fall within the application's appended claims limited range equally after having read above-mentioned instruction content of the present invention.
Claims (22)
1. the micro flow chip (1) cultivated for Caenorhabditis elegans (Caenorhabditiselegans), observe and/or study, described micro flow chip (1) comprises microflow layer (2) and substrate (20), and described microflow layer (2) comprising:
At least one is for cultivating the culturing room (3) of nematode, described culturing room (3) is a cavity being enclosed in microflow layer, communicate with the external world by means of only microchannel, described culturing room (3) is being greater than the diameter of nematode adult perpendicular to the size on micro flow chip direction;
Be connected with culturing room (3) at least one input microchannel (4) and be connected with culturing room (3) at least one screen pipeline (30), described input microchannel (4) is for infusion fluid in culturing room (3), described screening pipeline (30) exports microchannel (5) from least one of the larvae and eggs of the liquid of culturing room (3) through screening pipeline (30) and flowing out and nematode with for collecting be connected with discharging, described screening pipeline (30) is less than or equal to 2/3 of the average traversal area of nematode adult and is more than or equal to the average traversal area 1/2 of nematode larval and ovum at the aperture area of culturing room's inwall,
Wherein, described culturing room (3) is strip, the opening of described input microchannel (4) in culturing room (3) is positioned at one end of the major axis of strip, and the opening of described screening pipeline (30) in culturing room (3) is positioned on the other end of the major axis of strip and two sides of strip; And wherein, number >=6 of described screening pipeline (30), are wherein positioned at number of openings >=2 of the other end of described strip major axis, are positioned at number of openings >=2 on arbitrary side of strip minor axis.
2. micro flow chip according to claim 1 (1), 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 μm, and wide is 8-12 μm.
3. micro flow chip according to claim 2 (1), is characterized in that, described rectangular length is 18-22 μm, and wide is 9-11 μm.
4. micro flow chip according to claim 2 (1), is characterized in that, described rectangular length is 20 μm, and wide is 10 μm.
5. 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 μm.
6. micro flow chip according to claim 5 (1), is characterized in that, the diameter of described circle is 9-11 μm.
7. micro flow chip according to claim 5 (1), is characterized in that, the diameter of described circle is 10 μm.
8. the micro flow chip (1) according to any one of claim 1 ~ 7, is characterized in that, described culturing room (3) is perpendicular to size >=100 μm on micro flow chip direction.
9. micro flow chip according to claim 1 (1), is characterized in that, one end that strip culturing room (3) is connected with described input microchannel (4) is circular arc.
10. micro flow chip according to claim 9 (1), is characterized in that, one end that strip culturing room (3) is connected with described input microchannel (4) is for semicircle.
11. micro flow chips according to claim 1 (1), it is characterized in that, number >=12 of described screening pipeline (30), wherein be positioned at number >=4 of the screening pipeline (30) of one end of the major axis of described strip, be positioned at number >=4 of the screening pipeline (30) on arbitrary side of strip.
12. micro flow chips according to claim 1 (1), it is characterized in that, described micro flow chip (1) also comprises key-course (6) and articulamentum (7), separated by articulamentum (7) between described key-course (6) and described microflow layer (2), described key-course (6) comprising:
At least one controls pipeline (8), described control pipeline (8) is with input microchannel (4) or export microchannel (5) and have at least one crosspoint (9), the articulamentum (7) at described crosspoint (9) place is made up of elastomeric material
Wherein, when controlling the pressure in pipeline (8) and being increased to certain value, the elastomer articulamentum (7) of described crosspoint (9) position expands to microflow layer (2) direction, and make the inner surface contact inputting microchannel (4) or export microchannel (5) to blocking miniflow completely, make miniflow be in the state of disconnection; When controlling the pressure recover in pipeline (8), the elastomer articulamentum (7) of crosspoint (9) position recovers ortho states, makes miniflow be in the state of circulation.
13. micro flow chips according to claim 12 (1), it is characterized in that, described microflow layer (2), key-course (6) and articulamentum (7) are made up of elastomeric material, and described control pipeline (8) forms cavity expanding with input microchannel (4) and crosspoint (9) place that exports microchannel (5).
14. micro flow chips (1) according to claim 12 or 13, it is characterized in that, described elastomeric material be selected from lower group one or more: dimethyl silicone polymer, acrylonitrile-butadiene-styrene copolymer, Merlon, polymethyl methacrylate, polyurethane, polyethylene, polypropylene, polymethylpentene, polytetrafluoroethylene (PTFE), polyvinyl chloride, cyclic polyolefin co-polymer, polyvinylidene fluoride, polystyrene, polysulfones, nylon, Styrene-acrylic copolymer, polyisoprene, polybutadiene, ethane-acetic acid ethyenyl ester, polyimides or phenolic resins.
15. micro flow chips (1) according to claim 12 or 13, it is characterized in that, described elastomeric material be selected from lower group one or more: natural rubber, butyl rubber, butadiene-styrene rubber, acrylonitrile-butadiene rubber, chloroprene rubber, EP rubbers, epichlorohydrin rubber, lactoprene, silicone rubber, fluorosioloxane rubber or elastin laminin.
16. micro flow chips (1) according to claim 12 or 13, is characterized in that, described elastomeric material be selected from lower group one or more: halogenated butyl rubber or resilin.
17. micro flow chips (1) according to claim 12 or 13, it is characterized in that, described elastomeric material is fluoroelastomer.
18. micro flow chips (1) according to claim 12 or 13, it is characterized in that, described elastomeric material is Perfluoroelastomer.
19. 1 kinds of devices (100) cultivated for Caenorhabditis elegans, observe and/or study, it is characterized in that, described device comprises:
Micro flow chip (1) according to any one of claim 1 ~ 18;
The liquid input block (10) be connected with the input microchannel (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 microchannel (5).
20. devices according to claim 19 (100), it is characterized in that, described liquid input block (10) comprises one or more closed container (15) that liquid is housed, described closed container (15) is connected with the external world by the pipeline below liquid level in two insertion containers, wherein a pipeline is connected with the input microchannel (4) on micro flow chip (1), and an other pipeline is connected with compressed air pump.
The application in the cultivation of Caenorhabditis elegans, observation and/or research of 21. micro flow chips (1) according to any one of claim 1 ~ 18 or the device according to any one of claim 19 ~ 20 (100).
22. apply as claimed in claim 21, it is characterized in that, the cultivation of described nematode, observation and/or research are used for Developmental Biology, genetics, genomics, pharmacy are observed and/or research.
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| CN104920307B (en) * | 2015-06-19 | 2018-01-26 | 安徽省农业科学院农业工程研究所 | A kind of Caenorthaditis elegans ws123 fixing means suitable for single-particle microbeam device |
| CN106885807B (en) * | 2017-02-21 | 2020-04-24 | 澳门大学 | Large-scale living organism screening system based on micro-fluidic technology |
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