CN1695775A - Method and equipment for purifying and recovering biomacromolecules - Google Patents
Method and equipment for purifying and recovering biomacromolecules Download PDFInfo
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- CN1695775A CN1695775A CN 200510024257 CN200510024257A CN1695775A CN 1695775 A CN1695775 A CN 1695775A CN 200510024257 CN200510024257 CN 200510024257 CN 200510024257 A CN200510024257 A CN 200510024257A CN 1695775 A CN1695775 A CN 1695775A
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Abstract
A process for purifying and recovering the macromolecular biologic substances by electrophoresis method includes applying a voltage across two electrodes at both ends of electrophoretic channel for separating specimen, optically monitoring the position of biologic macromolecular band on the channel, switching between two electrodes when said band reaches proper position, and leading it in a branch tube and in turn in collecting pool. Its apparatus is also disclosed.
Description
Technical field
The present invention relates to method and device thereof that a kind of biological macromolecule purifying reclaims.The method and device can be applicable to molecular biological field.
Background technology
Biomacromolecules such as DNA, protein, DNA-albumen composition to cut that glue reclaims be a routine operation of biology laboratory, cut the purifying of product, marked product etc., the separation and purification of marking protein, DNA-albumen composition, RNA-albumen composition etc. etc. such as PCR product, enzyme.
At present, the method that the dna molecular purifying reclaims mainly contains two kinds, a kind of is conventional laboratory method: electrophoresis → cut glue → melt glue → phenol chloroform extracting → precipitation with alcohol → dissolving, this method complex operation step, technology and skill level to operating personnel have certain requirement, and include and poison reagent, and have the quantity of sample loss, thereby be unsuitable for the purifying recovery of micro-example.Another kind method is the commercially available reagent cassette method: electrophoresis → cut glue → melt glue → centrifugal is crossed post → wash-out dissolving, this method equally also has certain technical requirement to operating personnel, there is the quantity of sample loss equally, purifying recovery to micro-example is unfavorable, and existing kit price is expensive, be difficult for popularizing [Sheng Xiaoyu translates .DNA and RNA basic experiment technology. Beijing: the .2002.Frederick Ausubel et al.ShortProtocols in Molecular Biology.John Wiley of Science Press; Sons, Inc., Publishers, 2000].
At present, the method for purifying and recycling of protein molecule mainly contains several different methods [Richard J.Simpson.Purifyingproteins for proteomics:a Laboratory Manual.Cold Spring HarborLab (CSHL) Press such as the precipitation method, gel filtration, electrophoresis, ion exchange chromatography, affinity chromatography, 2004], the complicated operation that these methods have, consuming time longer, the organic efficiency that has is low, and causes albuminous degeneration easily, and what have needs cryogenic conditions.Lack a kind of easy and simple to handlely, albumen reclaims purification process fast and effectively.
Summary of the invention
The purpose of this invention is to provide a kind of method of biological macromolecule purifying recovery and the separating and the one step completed device of purifying biomacromolecule of Using such method design, simplify the operation steps that biological macromolecule purifying reclaims, the use of poisonous chemical reagent in avoiding operating, improve the recovery of sample simultaneously, reduce the sample cost recovery, and provide a kind of simple in structure, with low cost, easy and simple to handle, easily be automated, and can be used for the biological macromolecule purifying retracting device of microsystem.
The method that a kind of biological macromolecule purifying provided by the invention reclaims, it is characterized in that this method utilizes the selectivity of iontophoretic electrode to switch, at first biomacromolecule is separated by the electrophoresis mode, biomacromolecule with purifying to be recycled imports collecting pit then, thereby realizes the recovery purifying of biomacromolecule.
Particularly, when implementing the recovery of sample purifying, at first on-load voltage on the electrode that is positioned at the main track two ends makes sample be separated on main track.Monitor the position of swimming biomacromolecule band simultaneously by optical detecting method, after the biomacromolecule band of purifying to be recycled and other band are separated fully, by optionally switching electrode, the biomacromolecule of purifying to be recycled is introduced nearest lateral, and the final collecting pit that imports correspondence, the purifying of finishing sample reclaims.Whole electrophoresis process relates to the selectivity switching of plural electrode and electrode; The switching of electrode is by the electrophoretic band determining positions of the biomacromolecule of recovery to be purified.In the electrophoresis pipeline, fill solid-state sieving media, as Ago-Gel; Charge into electrophoresis buffer solution in the addition pool of duct end and the collecting pit, as TBE.
The present invention also provides a kind of device of implementing above-mentioned biological macromolecule purifying recovery method, this device comprises the electrophoresis parts that are processed with branch shape pipeline and some electrodes, the monitoring component of the big molecule electrophoretic band of monitoring bio position and control electrode ON-OFF control circuit parts.It is characterized in that: its electrophoresis parts comprise a main separating pipe and one or more lateral, and electrode is all arranged in the Xiao Chi of each duct end; Electrode can contact with damping fluid among the Xiao Chi by inserted mode, also can by sputter or electroplating processing process be fixed in the Xiao Chi bottom directly and the damping fluid among the Xiao Chi contact.Be positioned at the electrode of collecting pit or outside and coat one deck semi-permeable diaphragm, perhaps and isolate the material of one deck semi-permeable diaphragm between the lateral.
The present invention compares with biological macromolecule purifying recovery method commonly used at present, simplified the operation steps that biological macromolecule purifying reclaims, avoid the use of poisonous chemical reagent in the operation, improved the recovery of sample simultaneously, reduced the cost that the sample purifying reclaims.In addition, apparatus of the present invention are simple in structure, and easy operating is easy to realize integrated and robotization.
Description of drawings
Fig. 1 is apparatus of the present invention synoptic diagram.
Fig. 2 is the electrophoresis parts spatial structure view of the embodiment of the invention.
Fig. 3 is an electrophoresis parts bottom electrode substrate plane view shown in Figure 2.
Fig. 4 is the electrophoresis parts intermediate conduit shown in Figure 2 and the middle layer plan view of cavity.
Fig. 5 is an electrophoresis parts upper strata cover plate plan view shown in Figure 2.
Embodiment
Further specify substantive distinguishing features of the present invention and obvious improvement below in conjunction with accompanying drawing.
Fig. 1 is apparatus of the present invention synoptic diagram, the utilization of this device has the swimming that the electrophoresis parts 1 of branch shape electrophoresis pipeline and specific configuration electrode drive biomacromolecule in the sample and separates, position by electrophoretic band in the monitoring component 2 monitoring electrophoresis parts 1, and feed back to control assembly 3, utilize the switch and the alive size of each electrode on the control circuit control electrophoresis parts 1 in the control assembly 3.
Fig. 2 is the electrophoresis parts spatial structure view of the embodiment of the invention.These parts comprise three-decker, and the substrate 4 that is processed with the specific configuration electrode is positioned at bottom, and the middle layer is the pipe layers 5 that is processed with branch shape electrophoresis pipeline and cavity, and the superiors are the cover plate 6 that is processed with corresponding aperture.
Fig. 3 is the electrophoresis parts bottom electrode substrate plane view of the embodiment of the invention.The substrate zone line is placed with realizes separation and each electrode of collecting, is followed successively by keeper electrode 9, passive electrode 10 and the main separate mesh electrode 12 of application of sample electrode 7, band shaping electrode 8, recovery sample.The periphery is the connection electrode that is connected with control circuit 11 that each electrode is drawn.
Fig. 4 has the pipe layers plan view of branch shape electrophoresis pipeline and cavity for the electrophoresis component processing of the embodiment of the invention.According to the function difference, purifying reclaims electrophoresis pipeline 14 and has difformity with reference to electrophoresis pipeline 15.With reference to electrophoresis pipeline 15 is single straight pipeline, and two ends have addition pool 13 and waste liquid pool 17 respectively; Purifying reclaims electrophoresis pipeline 14 except the addition pool 13 and waste liquid pool 17 at main separating pipe two ends, then than reference electrophoresis pipeline 15 more laterals and collecting pit 16, main separating pipe has three laterals and corresponding collecting pit in the present embodiment as shown in the figure.
Fig. 5 is the electrophoresis parts upper strata cover plate plan view of the embodiment of the invention.The cover plate layer comprises the well 18 of being convenient to application of sample and sampling, collection hole 19 and waste liquid hole 20.The position of well 18 should be corresponding to the addition pool in the pipe layers 5 13, and collection hole 19 should reclaim lateral and collecting pit 16 on the electrophoresis pipeline 14 corresponding to purifying, and waste liquid hole 20 should be corresponding to the waste liquid pool on the pipe layers 3 17.
When implementing the recovery of sample purifying, at first in two wells 18 of electrophoresis parts 1, add sample respectively, reclaim the sample liquid that adds recovery to be purified in the well that electrophoresis pipeline 14 communicates with purifying, with add reference sample liquid in the well that reference electrophoresis pipeline 15 communicates, between application of sample electrode 7 and sample strip shaping electrode 8, load the voltage of suitable direction and intensity then, biomacromolecule in the sample liquid is enriched in rapidly on the sample strip shaping electrode 8, forms the sample strip that elongated and narrow is easy to differentiate.After finishing the sample strip shaping, switch electrode, between application of sample electrode 7 and main separate mesh electrode 12, load the voltage of suitable direction and intensity, make sample on main track, be separated.Monitor the position of the biomacromolecule band of swimming simultaneously by optical detecting method, after the biomacromolecule band of purifying to be recycled arrives suitable position, by optionally switching electrode, the biomacromolecule of purifying to be recycled is enriched on the nearest keeper electrode 9.The last voltage that loads suitable direction and intensity between the recovery electrode 10 of keeper electrode 9 and respective branches collection conduit terminal imports corresponding collecting pit with the biomacromolecule band to be recycled that is enriched on the keeper electrode 9, and the purifying of finishing sample reclaims.After this process is finished the separation of biomacromolecule, directly biomacromolecule to be recycled is imported in the damping fluid, saved and cut glue in the normal experiment method, melted glue, the step of the purifying that removes photoresist then.Collected biomacromolecule sample can be directly used in follow-up molecular biology operation, as order-checking, clone, mark, amplification, restriction enzyme digestion and in-vitro transcription translation etc.Obvious retracting device provided by the invention will separate and purifying is once finished, and be better than existing method and apparatus.
Claims (10)
1. the method that biological macromolecule purifying reclaims it is characterized in that described method is to utilize the selectivity of iontophoretic electrode to switch by the biomacromolecule importing collecting pit of electrophoresis mode with purifying to be recycled, thereby the purifying of realizing biomacromolecule reclaims.
2. the method that reclaims according to the described biological macromolecule purifying of claim 1 is characterized in that at first on-load voltage on the electrode that is positioned at the main track two ends of described recovery method, makes sample be separated on main track; Monitor the position of swimming biomacromolecule band simultaneously by optical detecting method, after the biomacromolecule band of purifying to be recycled and other band are separated fully, by optionally switching electrode, the biomacromolecule of purifying to be recycled is introduced nearest lateral, and the final collecting pit that imports correspondence, the purifying of finishing sample reclaims.
3. the method that reclaims according to claim 1 or 2 described biological macromolecule purifyings is characterized in that: fill solid-state sieving media in the electrophoresis pipeline, all charge into electrophoretic buffer in the addition pool of duct end and the collecting pit.
4. according to the method for the described biological macromolecule purifying recovery of claim 1, it is characterized in that: whole electrophoresis process relates to the selectivity switching of plural electrode and electrode; The switching of electrode is by the electrophoretic band determining positions of the biomacromolecule of recovery to be purified.
5. implement biological macromolecule purifying retracting device as claimed in claim 1, it is characterized in that: this device is by being processed with the branch shape pipeline and the electrophoresis parts (1) of the electrode of arranging, and the monitoring component (2) of the big molecule electrophoretic band of monitoring bio position and the control circuit parts (3) of control electrode switch constitute; Wherein the electrophoresis parts comprise a main separating pipe and one or more lateral, and electrode is all arranged in the Xiao Chi of each duct end; Electrode contacts with damping fluid among the Xiao Chi by inserted mode, or by sputter or electroplating processing process be fixed in the Xiao Chi bottom directly and the damping fluid among the Xiao Chi contact.
6. according to the described biological macromolecule purifying retracting device of claim 5, it is characterized in that described electrophoresis parts are three-decker: it is the upper strata cover plate (6) that is processed with the pipe layers (5) of the electrophoresis pipeline of branch shape and cavity and is processed with corresponding aperture that the substrate that is processed with the electrode of arranging is positioned at bottom (4), middle layer.
7. according to the described biological macromolecule purifying retracting device of claim 6, it is characterized in that the zone line of bottom substrate (4) arranges the keeper electrode (9) of application of sample electrode (7), band shaping electrode (8), recovery sample, passive electrode (10) and main separate mesh electrode (12) successively; The periphery is the connection electrode that is connected with control circuit (11) that each electrode is drawn.
8. according to the described biological macromolecule purifying retracting device of claim 6, it is characterized in that: in the pipe layers that is processed with branch shape electrophoresis pipeline and cavity, with reference to electrophoresis pipeline (15) is single straight pipeline, and two ends have addition pool (13) and waste liquid pool (17) respectively; Purifying reclaims electrophoresis pipeline (14) than with reference to how one or more lateral of electrophoresis pipeline (15) and collecting pit.
9. according to the described biological macromolecule purifying retracting device of claim 6, it is characterized in that described cover plate layer comprises the well of application of sample and sampling (18), collection hole (19) and waste liquid hole (20); Well (18) position is corresponding to the addition pool (13) in the pipe layers (5), and collection hole (19) is corresponding to lateral and collecting pit (16) on the purifying recovery electrophoresis pipeline (14), and waste liquid hole (20) are corresponding to the waste liquid pool (17) on the pipe layers (3).
10. according to the described biological macromolecule purifying retracting device of claim 6, it is characterized in that: be positioned at the electrode of collecting pit or outside and coat one deck semi-permeable diaphragm, perhaps and isolate the material of one deck semi-permeable diaphragm between the lateral; Described semi-permeable diaphragm only can penetrating small-molecule substance.
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| CNB2005100242571A CN1314476C (en) | 2005-03-09 | 2005-03-09 | Method and equipment for purifying and recovering biomacromolecules |
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| CNB2005100242571A CN1314476C (en) | 2005-03-09 | 2005-03-09 | Method and equipment for purifying and recovering biomacromolecules |
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| CN1695775A true CN1695775A (en) | 2005-11-16 |
| CN1314476C CN1314476C (en) | 2007-05-09 |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103827315A (en) * | 2011-06-16 | 2014-05-28 | 不列颠哥伦比亚癌症分社 | Automated size selection of nucleic acids |
| CN109456882A (en) * | 2019-01-16 | 2019-03-12 | 北京理工大学 | A kind of space biological sample micro process integrating device |
| CN112352157A (en) * | 2018-06-14 | 2021-02-09 | 海岸基因组学公司 | Devices for capturing macromolecules and methods of making and using the same |
| CN113939734A (en) * | 2019-06-20 | 2022-01-14 | 株式会社日立高新技术 | Method and apparatus for collecting biological material |
Family Cites Families (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP3920221B2 (en) * | 2001-05-02 | 2007-05-30 | アプレラ コーポレイション | Concentration and purification of analytes using electric fields |
| CN1172184C (en) * | 2001-07-11 | 2004-10-20 | 中国科学院大连化学物理研究所 | Stereo multi-dimensinal multi-mode capillary electrophoresis method and its special equipment |
| CN1223856C (en) * | 2002-07-18 | 2005-10-19 | 中国科学院大连化学物理研究所 | Microflow control chip for protein analysis and its application in protein analysis |
| JP2004184138A (en) * | 2002-11-29 | 2004-07-02 | Nec Corp | Separator, separation method, and mass spectrometric analysis system |
-
2005
- 2005-03-09 CN CNB2005100242571A patent/CN1314476C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103827315A (en) * | 2011-06-16 | 2014-05-28 | 不列颠哥伦比亚癌症分社 | Automated size selection of nucleic acids |
| US9952176B2 (en) | 2011-06-16 | 2018-04-24 | British Columbia Cancer Agency Branch | Automated size selection of nucleic acids |
| CN112352157A (en) * | 2018-06-14 | 2021-02-09 | 海岸基因组学公司 | Devices for capturing macromolecules and methods of making and using the same |
| CN109456882A (en) * | 2019-01-16 | 2019-03-12 | 北京理工大学 | A kind of space biological sample micro process integrating device |
| CN113939734A (en) * | 2019-06-20 | 2022-01-14 | 株式会社日立高新技术 | Method and apparatus for collecting biological material |
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| CN1314476C (en) | 2007-05-09 |
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Owner name: NINGBO RUIXIN BIOLOGY SCIENCE CO., LTD. Free format text: FORMER OWNER: SHANGHAI INST. OF MICROSYSTEM AND INFORMATION TECHNOLOGY, CHINESE ACADEMY OF SCI Effective date: 20080919 |
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Effective date of registration: 20080919 Address after: Room 301, Pioneer Building, academician Road, Ningbo science and Technology Park, Zhejiang Patentee after: Ningbo realchip Biotechnology Co. Ltd. Address before: No. 865, Changning Road, Shanghai, Changning District Patentee before: Shanghai Institute of Microsystem and Information Technology, Chinese Academy of Sciences |
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