CN205347420U - Full -automatic nucleic acid extraction and PCR increase micro -fluidic chip - Google Patents
Full -automatic nucleic acid extraction and PCR increase micro -fluidic chip Download PDFInfo
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- CN205347420U CN205347420U CN201521002869.6U CN201521002869U CN205347420U CN 205347420 U CN205347420 U CN 205347420U CN 201521002869 U CN201521002869 U CN 201521002869U CN 205347420 U CN205347420 U CN 205347420U
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Abstract
The utility model provides a full -automatic nucleic acid extraction and PCR increase micro -fluidic chip, including at least one main part, along the distribution of chip circumference equidistance, the main part includes: the nucleic acid extraction unit, it is located in the main part for extraction, washing, the elution of nucleic acid is carried out to the order under the drive of centrifugal force and capillary force, the PCR unit that amplifys for PCR reacts the increase, the waste liquid unit, it includes the waste liquid cavity for the storage waste liquid, and the exhaust unit, it is including exhaust hole and many gas -exhausting flow path, many gas -exhausting flow path communicates with nucleic acid extraction unit, PCR increase unit, waste liquid unit for discharge gaseously. The utility model provides a micro -fluidic chip who utilizes centrifugal force, capillary force and siphonage to realize the reaction of full -automatic nucleic acid extraction and PCR does not have any integrated pump, valve equipment on this micro -fluidic chip, greatly reduced the manufacturing degree of difficulty and the cost of chip to the reliability of chip has been improved.
Description
Technical field
This utility model relates to field of nucleic acid detection.More specifically, this utility model relates to a kind of full-automatic nucleic acid extraction and pcr amplification microfluidic chip structure.
Background technology
Fluidic chip has had a lot of application in nucleic acid extraction with pcr amplification, but most application also rests on laboratory stage.Reason is to realize the processes such as the cracking of blood sample or tissue, nucleic acid binding, cleaning, eluting, these nucleic acid extraction and pcr amplification micro-fluidic chip are all integrated with a lot of Micropumps and micro-valve, add the complexity of micro-fluidic chip, manufacture difficulty, cost, reduce reliability.Meanwhile, equipment and device required for these pumps of external drive, valve are also more complicated, limit the application to clinic of these chips.
Thus, this utility model abandons integrated pump and valve on existing detection of nucleic acids pcr chip, it is proposed to utilizes the centrifugal force rotating apparatus as driving, and assists the function controlling realizing whole fluid with capillary force and siphonage.Fluid operated in whole nucleic acid extraction, purification and PCR course of reaction has only to a rotary centrifuger and can complete.Without any integrated pump, valve equipment on whole micro-fluidic chip, greatly reducing manufacture difficulty and the cost of chip, and improve the reliability of chip, the clinical practice for nucleic acid extraction Yu pcr amplification has positive effect.
Utility model content
The purpose of this utility model is to propose a kind of to utilize centrifugal force, capillary force and siphonage to realize full-automatic nucleic acid extraction and the PCR micro-fluidic chip reacted, without any integrated pump, valve equipment on this micro-fluidic chip, greatly reduce manufacture difficulty and the cost of chip, and improve the reliability of chip.
In order to realize according to these purposes of the present utility model and further advantage, it is provided that a kind of full-automatic nucleic acid extraction and pcr amplification micro-fluidic chip, including at least one main body, being equally spaced along chip circumference, described main body includes:
Nucleic acid extraction unit, it is positioned in described main body, carries out the extraction of nucleic acid, cleaning, eluting for order under the driving of centrifugal force and capillary force；
Pcr amplification unit, it is positioned in described main body, and it is connected by trident runner with described nucleic acid extraction unit, for PCR reaction amplification；
Waste unit, it includes waste liquid cavity, is connected by trident runner with described nucleic acid extraction unit, is used for storing waste liquid；
And exhaust unit, it includes steam vent and a plurality of grate flow channel, and described a plurality of grate flow channel connects with nucleic acid extraction unit, pcr amplification unit, waste liquid cavity, is used for discharging gas.
Preferably, described nucleic acid extraction unit includes:
Sample adding device, it is positioned at the left upper end of main body, and described sample adding device includes application of sample cavity and is arranged on the well on application of sample cavity, and described sample adding device is for adding and the mixture of storing samples with lysate, binding liquid；
Rinser, it is arranged in the upper end of main body, and described rinser includes cleanout fluid well and cleanout fluid cavity, and described rinser is used for adding and depositing cleanout fluid；
Washing device, it is positioned at the upper right side of main body, and described washing device includes eluent well and eluent cavity, and described washing device is used for adding and depositing eluent；
And nucleic acid solid-phase extraction device, it includes nucleic acid Solid-Phase Extraction cavity, is provided with nucleic acid Solid-Phase Extraction material in described nucleic acid Solid-Phase Extraction cavity；
Wherein said application of sample cavity is connected with described nucleic acid Solid-Phase Extraction cavity by first flow, described cleanout fluid cavity is connected with described nucleic acid Solid-Phase Extraction cavity by the second runner, and described eluent cavity is connected with described in nucleic acid Solid-Phase Extraction cavity by the 3rd runner.
Preferably, described pcr amplification unit includes:
Nucleic acid quantification runner, itself and nucleic acid Solid-Phase Extraction unit are by trident flow passage；
Nucleic acid quantification cavity, it is with nucleic acid quantification flow passage, for quantitative nucleic acid；
PCR reaction cavity, itself and nucleic acid quantification cavity are by the 6th flow passage, and described PCR cavity is closed cavity, and inside is placed with the required primer of PCR reaction and reagent；
And unnecessary nucleic acid cavity, itself and nucleic acid quantification flow passage, for storing unnecessary nucleic acid.
Another purpose of the present utility model is to provide the application process of full-automatic nucleic acid extraction and pcr amplification micro-fluidic chip, comprises the steps；
Step 1): full-automatic nucleic acid extraction and pcr amplification micro-fluidic chip are arranged on centrifuge, in application of sample cavity, sample and lysate, the mixture binding liquid is added then through well, in cleanout fluid cavity, add cleanout fluid, in eluent cavity, add eluent.
Step 2): turn clockwise centrifugal, rotating speed 1200～1600rpm, 3～5 minutes time.Sample will enter Solid-Phase Extraction cavity with lysate, binding liquid mixture after first flow, and cleanout fluid will both enter Solid-Phase Extraction cavity after the 4th runner, cleanout fluid secondary cavity, the 3rd runner；And eluent will enter in eluent secondary cavity under centrifugal action, after solution reaches the siphon of the second runner, owing to powerful centrifugal force is more than capillary force, eluent cannot enter Solid-Phase Extraction cavity through the second runner；
Simultaneously, owing to first flow has less fluid resistance relative to the 3rd runner, can so that when the mixture of sample and lysate, binding liquid flows completely out Solid-Phase Extraction cavity, cleanout fluid still arrive Solid-Phase Extraction cavity, and then realization extracts and the order of cleaning performs；
Due to for high speed centrifugation clockwise, under the effect of Coriolis force so that sample and lysate, the mixture of binding liquid and cleanout fluid all will flow to waste liquid cavity through trident runner, without flowing to nucleic acid quantification runner.
Step 3): speed is to vulgar rotation counterclockwise after 0, rotating speed 20～80rpm, and time 20～60s, now due to centrifugal less so that eluent can be full of whole second runner by capillary force.
Step 4): high speed centrifugation counterclockwise, rotating speed 1200～1600rpm, time 60～120s, under the influence of centrifugal force, utilize siphonage that eluent flows to Solid-Phase Extraction cavity after the second runner, and under the effect of Coriolis force, nucleic acid complete for eluting is flowed to after trident runner quantitative runner, and be full of nucleic acid quantification cavity successively, unnecessary nucleic acid flows into waste liquid cavity, completes the eluting of nucleic acid；Now owing to the 6th runner has less cross sectional dimensions, and PCR reaction cavity is closed cavity so that centrifugal force is not enough to the interior pressure of capillary force and the sample chamber overcoming the 6th runner, and nucleic acid cannot be introduced into PCR reaction cavity.
Step 5): improve rotating speed further, 1800～2500rpm, time 20～40s, under more powerful centrifugal action, the complete nucleic acid of eluting enters PCR reaction cavity after quantitative cavity, and with the combination of PCRmix, primer, under the control of outside temperature control, complete pcr amplification, the preferred 2500rpm of described rotating speed.
Preferably, described PCR reaction is required reagent or primer comprise PCRmix, PCR primer, described reagent or primer and deposit in PCR reaction cavity with lyophilizing or air-dry form.
Preferably, described Solid-Phase Extraction cavity has slim-lined construction, and length direction points to chip center, to ensure that, under centrifugal action, fluid can have less residual；Described Solid-Phase Extraction cavity has rectangular cross section, its equivalent diameter 1mm～2mm, length 5mm～15mm；Described Solid-Phase Extraction material one in pellosil, silicagel column.
Preferably, described cleanout fluid cavity includes cleanout fluid one-level cavity and cleanout fluid secondary cavity, cleanout fluid one-level cavity and cleanout fluid secondary cavity are by the 4th flow passage, described eluent sap cavity body includes washing liquid and will not enter cleanout fluid secondary cavity, more will not enter the 3rd runner, and then enter Solid-Phase Extraction cavity in advance；Described scheme adds an eluent secondary cavity, and the advantage of this design is in that, due to the existence of the 5th runner, in capillary force and capillary existence, eluent will not enter eluent secondary cavity, more will not the second runner, and then in advance enter Solid-Phase Extraction cavity.
Preferably, described second runner is syphon runner, described eluent secondary cavity is connected to Solid-Phase Extraction cavity through syphon runner, the advantage of this design is in that, existence due to syphon runner, making under first time is centrifugal clockwise, eluent will not enter Solid-Phase Extraction cavity in advance through syphon runner；Only when second time is reversely centrifugal, eluent just can enter Solid-Phase Extraction cavity through syphon runner；The equivalent diameter of described syphon runner is 0.8mm～1.5mm, to ensure that it has less fluid resistance；The minimum range of described syphon runner distance chip center is less than the distance at eluent secondary cavity distance chip center minimum place；Described 3rd runner has local increases fluid resistance flow passage structure, and described flow passage structure has less equivalent diameter, and is " S " type；Described 4th runner and described 5th runner have equivalent diameter 0.4mm～0.8mm；To ensure that it has suitable capillary force and fluid resistance, when static, solution will not flow out, and when centrifugal, solution can quickly flow out.
Preferably, described 6th runner has equivalent diameter 0.1mm～0.2mm；Described application of sample cavity has volume 500～800 μ l；Described cleanout fluid cavity, cleanout fluid secondary cavity have volume 500～800 μ l；Described eluent cavity, eluent secondary cavity have volume 150～200 μ l；Described nucleic acid quantification cavity has volume 40 μ l；Described PCR reaction cavity has volume 35 μ l.
Preferably, main body described in this programme can also have multiple cleanout fluid one-level cavity and cleanout fluid secondary cavity, can be carried out with multiple different cleanout fluid to meet, or further amounts of same cleanout fluid cleans；Multiple nucleic acid quantification cavity and PCR reaction cavity can be comprised, each PCR cavity is placed with different nucleic acid reaction reagent, in order to detect multiple different target；This programme chip can comprise multiple such nucleic acid extraction and the PCR structure reacted, in order to detecting several different sample, it is equally spaced along chip circumference simultaneously.
Chip described in the utility model is applicable to can realize on the equipment of rotating centrifugal, utilize and rotate the centrifugal force and capillary force, siphonage produced, by adding sample and the lysate of sample chamber, binding liquid mixture, the cleanout fluid adding cleanout fluid cavity, the eluent that adds eluent cavity flow through Solid-Phase Extraction cavity successively, reach the purpose of nucleic acid cleavage, absorption, eluting.The complete nucleic acid of eluting through quantitative cavity quantitatively after flow into PCR reaction cavity, and here carry out combination with PCR reagent, primer, detection etc. and complete PCR and react.
This utility model at least includes following beneficial effect:
(1) micro-fluidic chip of the present utility model utilizes centrifugal force, capillary force and siphonage to realize what full-automatic nucleic acid extraction and PCR reacted, there is no integrated Micropump and micro-valve, its manufacture difficulty and cost are had relative to prior art significantly reduce, improve again the reliability of chip simultaneously, significant for improving the productivity of chip and reliability and accuracy of detection, reduction cost of manufacture；
(2) micro-fluidic chip of the present utility model integrates nucleic acid extraction, pcr amplification, facilitates experimental implementation, shortens experimental period.
Part is embodied by further advantage of the present utility model, target and feature by description below, and part is also by by being understood by those skilled in the art the research of this valve and practice.
Accompanying drawing explanation
Fig. 1 is this utility model nucleic acid detection chip structure chart；
Fig. 2 be this utility model nucleic acid detection chip clockwise with direction of rotation schematic diagram counterclockwise.
Detailed description of the invention
Below in conjunction with accompanying drawing, this utility model is described in further detail, to make those skilled in the art can implement according to this with reference to description word.
Should be appreciated that used herein such as " have ", existence or the interpolation of other elements one or more or its combination do not allotted in " comprising " and " including " term.
In conjunction with Fig. 1 and Fig. 2, present embodiment, full-automatic nucleic acid extraction and pcr amplification micro-fluidic chip are described, including installing hole 2, locating slot 3 and 4 main bodys 1, main body 1 is equally spaced along chip circumference, and each main body 1 includes: be positioned at the application of sample cavity 34 in main body 1, well 4, cleanout fluid well 8, cleanout fluid one-level cavity 10, cleanout fluid secondary cavity 12, eluent well 7, eluent one-level cavity 6, eluent secondary cavity 16, nucleic acid Solid-Phase Extraction cavity 29, nucleic acid Solid-Phase Extraction material 28, waste liquid cavity 19, nucleic acid quantification cavity 23, unnecessary nucleic acid deposits cavity 24, PCR reaction cavity 20, the required primer of PCR reaction and reagent 21, and for connecting the first flow 30 of application of sample cavity 34 and Solid-Phase Extraction cavity 29, for connecting the 4th runner 11 of cleanout fluid one-level cavity 10 and cleanout fluid secondary cavity 12, for connecting the 3rd runner 14 of cleanout fluid secondary cavity 12 and Solid-Phase Extraction cavity 29, for connecting the 5th runner 13 of eluent one-level cavity 6 and eluent secondary cavity 16, for connecting the second runner 31 between eluent secondary cavity 16 and Solid-Phase Extraction cavity 29, for connecting the trident runner 17 between Solid-Phase Extraction cavity 29 and waste liquid cavity 19 and nucleic acid quantification runner 25, for connecting the 6th runner 22 between nucleic acid quantification cavity 23 and PCR reaction cavity 20, a plurality of runner 26 for aerofluxus, runner 33, runner 5 and runner 18 and steam vent 35 and 9.
A kind of in present embodiment utilize centrifugal force, capillary force and siphonage to realize full-automatic nucleic acid extraction and the PCR micro-fluidic chip reacted, material is acrylic, chip diameter 80mm, thickness 3mm, directly being processed by CNC, its basic size structural parameters are:
Application of sample cavity 34 has volume 400 μ l；Cleanout fluid one-level cavity 10 and cleanout fluid secondary cavity volume are 800 μ l, eluent one-level cavity 6 and eluent secondary cavity 16 volume are 150 μ l, quantitative cavity 23 volume is 40 μ l, PCR reaction cavity volume is 35 μ l, unnecessary nucleic acid cavity 24 volume is 60 μ l, and waste liquid cavity 19 volume is 1500 μ l.Chip has a cleanout fluid one-level cavity 10 and cleanout fluid secondary cavity 12.
Second runner 31 is syphon runner, and syphon runner is square groove type, and cross section is the positive square of length of side 1mm, and what the 4th runner 14 had the local increase fluid main force is " S " type flow passage structure 15；Trident runner 17 is the width 0.1mm, degree of depth 0.1mm of width 1mm, degree of depth 0.5mm, the 6th runner 22.Chip has three nucleic acid quantification cavitys 23 and PCR reaction cavity 20, and inside is placed with the PCR reagent of lyophilizing, primer and probe.
Detailed description of the invention two, present embodiment is the application process of the full-automatic nucleic acid extraction described in detailed description of the invention one and pcr amplification micro-fluidic chip:
Step 1: the installing hole 2 of full-automatic nucleic acid extraction Yu pcr amplification micro-fluidic chip is arranged on centrifuge, and fixed by locating slot 3 and centrifuge rotating shaft, then through well 4 to adding sample and lysate, the mixture 300 μ l binding liquid in application of sample cavity 34, in cleanout fluid cavity 10, add cleanout fluid 700 μ l, in eluent cavity, add eluent 130 μ l.
Step 2: turn clockwise centrifugal, centrifugal speed 1500rpm, 5 minutes time.Sample will enter Solid-Phase Extraction cavity 29 with lysate, binding liquid mixture after first flow 30, it is achieved the absorption of nucleic acid, and flow into waste liquid cavity 19 through trident runner 17 under the effect of Coriolis force；Subsequently, cleanout fluid will initially enter Solid-Phase Extraction cavity 29 after the 4th runner 11, cleanout fluid secondary cavity the 12, the 3rd runner 14, rinse: complete waste liquid enters waste liquid cavity 19.
Step 3: rotate counterclockwise after being decelerated to 0, rotating speed 40rpm, time 20s, eluent is full of whole second runner 31 by capillary force.
Step 4: high speed centrifugation counterclockwise, rotating speed 1500rpm, time 60s, eluent flows to Solid-Phase Extraction cavity 29 and by Nucleic Acid Elution after the second runner 31, after trident runner 17, runner 25 is flowed under the effect of Coriolis force, and it being full of nucleic acid quantification cavity 23 successively, unnecessary nucleic acid flows into waste liquid cavity 19.
Step 5: improve further rotating speed to 2000rpm, the complete nucleic acid of eluting enters PCR reaction cavity after quantitative cavity 23, and with the combination of PCR reagent, primer, under the control of outside temperature control, complete pcr amplification.
The main body of said chip can be provided with multiple cleanout fluid one-level cavity 10 and cleanout fluid secondary cavity 12 according to actually used situation by those skilled in the art, can be carried out with multiple different cleanout fluid to meet, or further amounts of same cleanout fluid cleans.
Although embodiment of the present utility model is disclosed as above, but listed utilization that it is not restricted in description and embodiment, it can be applied to various applicable field of the present utility model completely, for those skilled in the art, it is easily achieved other amendment, therefore, under the general concept limited without departing substantially from claim and equivalency range, this utility model is not limited to specific details and shown here as the legend with description.
Claims (9)
1. full-automatic nucleic acid extraction and a pcr amplification micro-fluidic chip, including at least one main body, is equally spaced along chip circumference, it is characterised in that described main body includes:
Nucleic acid extraction unit, it is positioned in described main body, carries out the extraction of nucleic acid, cleaning, eluting for order under the driving of centrifugal force and capillary force；
Pcr amplification unit, it is positioned in described main body, and it is connected by trident runner with described nucleic acid extraction unit, for PCR reaction amplification；
Waste unit, it includes waste liquid cavity, is connected by trident runner with described nucleic acid extraction unit, is used for storing waste liquid；
And exhaust unit, it includes steam vent and a plurality of grate flow channel, and described a plurality of grate flow channel connects with nucleic acid extraction unit, pcr amplification unit, waste unit, is used for discharging gas.
2. full-automatic nucleic acid extraction as claimed in claim 1 and pcr amplification micro-fluidic chip, it is characterised in that described nucleic acid extraction unit includes:
Sample adding device, it is positioned at the left upper end of main body, and described sample adding device includes application of sample cavity and is arranged on the well on application of sample cavity, and described sample adding device is for adding and the mixture of storing samples with lysate, binding liquid；
Rinser, it is arranged in the upper end of main body, and described rinser includes cleanout fluid well and cleanout fluid cavity, and described rinser is used for adding and depositing cleanout fluid；
Washing device, it is positioned at the upper right side of main body, and described washing device includes eluent well and eluent cavity, and described washing device is used for adding and depositing eluent；
And nucleic acid solid-phase extraction device, it includes nucleic acid Solid-Phase Extraction cavity, is provided with nucleic acid Solid-Phase Extraction material in described nucleic acid Solid-Phase Extraction cavity；
Wherein said application of sample cavity is connected with described nucleic acid Solid-Phase Extraction cavity by first flow, described cleanout fluid cavity is connected with described nucleic acid Solid-Phase Extraction cavity by the second runner, and described eluent cavity is connected with described in nucleic acid Solid-Phase Extraction cavity by the 3rd runner.
3. full-automatic nucleic acid extraction as claimed in claim 2 and pcr amplification micro-fluidic chip, it is characterised in that described pcr amplification unit includes:
Nucleic acid quantification runner, itself and nucleic acid Solid-Phase Extraction unit are by trident flow passage；
Nucleic acid quantification cavity, it is with nucleic acid quantification flow passage, for quantitative nucleic acid；
PCR reaction cavity, itself and nucleic acid quantification cavity are by the 6th flow passage, and described PCR cavity is closed cavity, and inside is placed with the required primer of PCR reaction and reagent；
And unnecessary nucleic acid cavity, itself and nucleic acid quantification flow passage, for storing unnecessary nucleic acid.
4. full-automatic nucleic acid extraction as claimed in claim 3 and pcr amplification micro-fluidic chip, it is characterized in that, described cleanout fluid cavity includes cleanout fluid one-level cavity and cleanout fluid secondary cavity, cleanout fluid one-level cavity and cleanout fluid secondary cavity are by the 4th flow passage, described eluent cavity includes eluent one-level cavity and eluent secondary cavity, eluent one-level cavity and eluent secondary cavity by the 5th flow passage.
5. full-automatic nucleic acid extraction as claimed in claim 4 and pcr amplification micro-fluidic chip, it is characterised in that described Solid-Phase Extraction cavity has slim-lined construction, and length direction points to chip center, to ensure that, under centrifugal action, fluid can have less residual；Described Solid-Phase Extraction cavity has rectangular cross section, its equivalent diameter 1mm～2mm, length 5mm～15mm；Described Solid-Phase Extraction material is a kind of in pellosil, silicagel column.
6. full-automatic nucleic acid extraction as claimed in claim 5 and pcr amplification micro-fluidic chip, it is characterized in that, described second runner is syphon runner, the equivalent diameter of described syphon runner is 0.8mm～1.5mm, and the minimum range of described syphon runner distance chip center is less than the distance at eluent secondary cavity distance chip center minimum place；Described 3rd runner have local increase fluid resistance in " S " flow passage structure of type；Described 4th runner and described 5th runner have equivalent diameter 0.4mm～0.8mm, and described 6th runner has equivalent diameter 0.1mm～0.2mm.
7. full-automatic nucleic acid extraction as claimed in claim 6 and pcr amplification micro-fluidic chip, it is characterised in that described application of sample cavity has volume 500～800 μ l；Described cleanout fluid one-level cavity, cleanout fluid secondary cavity have volume 500～800 μ l；Described eluent one-level cavity, eluent secondary cavity have volume 150～200 μ l；Described nucleic acid quantification cavity has volume 40 μ l；Described PCR reaction cavity has volume 35 μ l.
8. full-automatic nucleic acid extraction as claimed in claim 7 and pcr amplification micro-fluidic chip, it is characterised in that described nucleic acid quantification cavity is multiple, and described PCR reaction cavity is identical with described nucleic acid quantification cavity number.
9. full-automatic nucleic acid extraction as claimed in claim 8 and pcr amplification micro-fluidic chip, it is characterised in that described cleanout fluid cavity is multiple and/or described rinser is multiple.
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| CN105316224A (en) * | 2015-12-07 | 2016-02-10 | 中国科学院苏州生物医学工程技术研究所 | Full-automatic nucleic acid extraction and PCR amplification micro-fluidic chip and application method thereof |
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| CN108732339B (en) * | 2017-04-19 | 2021-04-13 | 光宝电子(广州)有限公司 | Flow channel device for multiple reaction biological detection and detection method thereof |
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| CN108043478A (en) * | 2017-12-04 | 2018-05-18 | 国家纳米科学中心 | A kind of micro-fluidic chip, manual centrifugal device and nucleic acid detection method |
| CN108949507A (en) * | 2018-08-21 | 2018-12-07 | 苏州德思普生物科技有限公司 | The micro-fluidic chip of bacterium total nucleic acid in a kind of extraction human whole blood |
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| CN110777049A (en) * | 2019-11-12 | 2020-02-11 | 北京金豪制药股份有限公司 | Microfluidic chip, nucleic acid detector and nucleic acid detection method |
| CN110732357A (en) * | 2019-11-25 | 2020-01-31 | 前海奥斯韦尔生物科技(深圳)有限公司 | Sample shunting pipeline of microfluidic chips |
| CN111575176A (en) * | 2020-05-22 | 2020-08-25 | 中国科学院上海技术物理研究所 | Closed full-automatic nucleic acid extraction and detection system based on CRISPR technology |
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