CN103822959A - Device and method for detecting biomacromolecules by capillary electrophoresis - Google Patents
Device and method for detecting biomacromolecules by capillary electrophoresis Download PDFInfo
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- CN103822959A CN103822959A CN201410042598.0A CN201410042598A CN103822959A CN 103822959 A CN103822959 A CN 103822959A CN 201410042598 A CN201410042598 A CN 201410042598A CN 103822959 A CN103822959 A CN 103822959A
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Abstract
The invention provides a method and device for detecting biomacromolecules by capillary electrophoresis, and belongs to the technical field of biochemical equipment. The method comprises the following steps of pumping a fresh separation glue solution into a capillary by using an automatic high-pressure pump machine, placing the separation glue solution in the capillary in treated macromolecular biosample sample, to be separated, on a sample tray (96-pore) for electrophoresis sample loading, allowing the macromolecular biosample to be analyzed to be at one end of the capillary, connecting the two ends of the capillary to a high-voltage power supply to separate the biomacromolecules to be separated in the capillary, performing back-and-forth scanning detection by using the automatic macromolecule detection device mounted at one end of the capillary after separation, stopping electrophoresis after the biomacromolecules are detected, transmitting a signal to a processor, and analyzing the components and content of the macromolecular biosample in the capillary by using a system. The device is mounted at the end of the capillary, and is used for continuously detecting biomacromolecules on line; electrophoresis is stopped after biomacromolecules are detected, and data is transmitted to the system.
Description
Technical field
One utilizes capillary electrophoresis technique to biomacromolecule detection device, belongs to biochemical equipment technical field.
Background technology
In DC electric field, the phenomenon that charged particle moves to the contrary electrode of tape symbol is called electrophoresis (electropho-resis).Within 1809, first the physicist Pe н ce of Russia has found electrophoresis, but until nineteen thirty-seven Sweden Tiselius set up isolated protein moving boundary electrophoresis (boundary electrophoresis) afterwards, electrophoretic techniques just starts application.In the 60-70 age in this century, since the medium such as filter paper, polyacrylamide gel is introduced electrophoresis in succession, electrophoretic techniques is developed rapidly.Colourful electrophoresis form makes its application very extensive, except the compartment analysis for small-molecule substance, main for protein, nucleic acid, enzyme, the even research of virus and cell, because electrophoresis equipment is simple, easy to operate, there is high resolving power and selectivity feature, become technology conventional in medical test.
Biological ployose is as peptide, protein, nucleic acid, oligosaccharides etc., or its potpourri passes through gel electrophoresis analysis, conventionally object sample is loaded in a medium, as polyacrylamide gel, according to the difference of molecular size, electric charge and other physical propertys, in electric field, migration obtains different bands, and after electrophoresis, band is combined rear detected or these biological ployoses are transferred to the analysis of carrying out next step on NC, PVDF or nylon membrane and test with staining reagent by biomacromolecule.Wherein, aspect biological ployose dyeing, a large amount of colouring methods and reagent are developed the biomacromolecule for the medium as gel that dyes, these reagent can be divided into four kinds: the first staining reagent comprises the organic dyestuff being attached on polymkeric substance, as coomassie brilliant blue staining reagent, can be observed by naked eyes thereby make protein band become blueness; The second staining reagent comprises the fluorescent dye being attached on polymkeric substance, if EB is in conjunction with DNA or RNA, in the time that ultraviolet ray is irradiated, DNA or RNA is taken on a red color; The third staining reagent comprises silver-colored transfection reagent; The 4th kind is the reagent of dyeing background, is called as negative staining.These dyestuffs, mostly also with electric charge, can move in DC electric field.Because the factor that affects electrophoretic mobility mainly contains electric field intensity, pH value and the ionic strength of solution etc. of solution are relevant, therefore biomacromolecule is not identical with the mobility of dyestuff in the medium that applies electric field, biomacromolecule molecule is larger, slowly mobile in electric field, within the time limiting, can from medium, not shift out, it is much smaller that the relative biomacromolecule molecule of organic dye molecule comes, the dye molecule of not being combined with biomacromolecule molecule can shift out rapidly from medium, therefore mode that can biomacromolecule (combining dyestuff) is retained in medium by dyestuff is shifted out from medium in same electric field realizes the colour developing of biomacromolecule, thereby can obtain at short notice high resolving power, background influence is little even without the analysis image of the biomacromolecule of background, if but dyestuff and biomacromolecule are only through the combination of meeting once, some sensitivity are required to higher experiment, be difficult to meet the demands, even in detection, easily there is undetected situation.
Summary of the invention
The object of the invention is to solve the problem that the large molecule manipulation complex steps of electrophoretic techniques detection of biological in the middle of prior art, biomacromolecule move and are restricted, cannot fast detecting separate, provide a kind of simple in structure, with low cost, easy and simple to handle, what be easy to realize has a large molecular method of robotization electrophoretic techniques detection of biological, has adopted following technical scheme:
A kind of capillary electrophoresis technique detects the disposal route of mcroorganism molecule, comprise the steps: to pump into new separation glue in kapillary, put into damping fluid prerunning, afterwards the separation glue after prerunning is put into the to be separated large molecular biosciences sample of handling well and carried out loading, make large molecular biosciences sample to be analyzed as for kapillary one end; Then kapillary two ends are applied to voltage, carry out electrophoresis, when stop electrophoresis after the other end capillaceous detects biomacromolecule, pick-up unit mobile line scanning of going forward side by side on kapillary detects, and detection information is analyzed.
As the improvement to said method, before each detection, will automatically inject new separation glue to kapillary by discharge pump, used scrap rubber liquid is drained into waste liquid tank.
As the improvement to said method, auto injection termination receives after new separation glue, will newly separate glue and put into tank and carry out rinse, and the separation glue after rinse is put into damping fluid and carried out prerunning, by such improvement means, can before application of sample, remove impurity, polymkeric substance etc.
As the improvement to said method, entered rinse, in the kapillary that prerunning is crossed, separate glue and put into large molecular biosciences sample tray, in pallet, carry out electrophoresis, make separation glue to be analyzed as for kapillary one end, saddle moves afterwards, make capillary sample inlet end be arranged in tank rinse, the capillary sample inlet end of rinse moves to and moves in buffering liquid groove, kapillary two ends are by the voltage of setting, electrophoresis, at kapillary two ends loading gradient voltage, separation glue to be analyzed large molecule under gradient voltage is separated, damping fluid can be selected the mixed solution (as HAc and NaAc) of weak acid and salt thereof, the mixed solution (as NH3H2O and NH4Cl) of weak base and salt thereof etc.
Pick-up unit can be installed on kapillary interlude, also can be installed on exit capillaceous, so just can realize the continuity of biomacromolecule thing is detected online, without steps such as traditional dyeing-decolorzings, when biomacromolecule detected, stop electrophoresis, and data are passed to system, systematic analysis data and provide data exhibiting form.
As the improvement to said method,
As the improvement to said method, shift out buffering liquid groove through separating glue in the kapillary loading end detecting, insert waste liquid tank, meanwhile, fresh glue is injected kapillary by the high-pressure pump of the kapillary other end, replaces used glue.Used glue is pressed in waste liquid tank.
Another object of the present invention is to provide a kind of biomacromolecule detection device based on Capillary Electrophoresis, include kapillary and detecting device, one end capillaceous is provided with capillary sample inlet end, the other end is provided with high-pressure pump, be provided with the optical scanning parts that can slide along kapillary in kapillary outside, also include pallet, the top of saddle is provided with sample pallet, tank, buffering liquid groove, waste liquid tank, is also provided with saddle drive motor below pallet.
Further improve, described buffering liquid groove, waste liquid tank, tank and sample pallet are arranged successively on saddle.
Further improve, described sample pallet is 96 orifice plates.
Further improve, described pick-up unit comprises all band spectrum generator, photodiode battle array.
As the improvement to said apparatus, described material capillaceous can be selected from
in glass, stainless steel, copperone, caliber is 0.5 mm～5mm preferably.
technique effect
Capillary Electrophoresis biomacromolecule detection method and apparatus provided by the invention has avoided traditional electrophoretic separation mode to need a large amount of samples, the problem of dyeing-decolorzing, and adopt autoscan to collect data mode to have improved work efficiency.
Accompanying drawing explanation
Fig. 1 is the complete machine structure view of device.
Fig. 2 is the structural representation of a kind of embodiment of device.
Fig. 3 is the optical scanning modular construction schematic diagram of device.
Wherein, 1, upper cover; 2, pump state observation window; 3, sample tray inlet port; 4, high-pressure pump motor; 5, high-pressure pump; 6, optical scanning parts; 7, optical scanning parts drive motor; 8, saddle drive motor; 9, capillary sample inlet end; 10, large molecular biosciences sample pallet (96 hole); 11, kapillary; 12, tank; 13, waste liquid tank; 14, buffering liquid groove.
Embodiment
Above-mentioned device can be mounted to all-in-one as Fig. 1 and Fig. 2, comprising upper cover 1, and pump state observation window 2, sample tray inlet port 3 parts such as grade;
The inner structure of said apparatus as shown in Figure 2, by high-pressure pump motor 4, high-pressure pump 5, optical scanning parts 6, optical scanning parts drive motor 7, saddle drive motor 8, capillary sample inlet end 9, large molecular biosciences sample pallet (96 hole) 10, kapillary 11, tank 12, waste liquid tank 13, buffering liquid groove 14 forms.
Frame in the bottom of main frame, in frame, be provided with transportable saddle, saddle is connected with drive motor 8, drive motor can make saddle move around in frame, be disposed with buffering liquid groove 14, waste liquid tank 13, tank 12, large molecular biosciences sample pallet (96 hole) 10 on saddle top, kapillary 11 one end connect high-pressure pump 5 and pump into new glue, and the other end is placed on saddle and puts as sample introduction end 9; Optical scanning parts 6 are connected with optics drive motor 7, and high-pressure pump motor 4, high-pressure pump 5 are placed in frame and are connected with saddle.
In the inner structure of said apparatus optical scanning modular construction as shown in Figure 3, all band spectrum generator 15, photodiode battle array 16.
Before instrument work, by capillary sample inlet end 9, as in capillary sample inlet end 14, when work, saddle drive motor 8 drives saddle to move, by capillary sample inlet end 9 as in waste liquid tank 13.
When work, pump into fresh separation glue in kapillary 11 by high-pressure pump motor 4, high-pressure pump 5, replace the used glue staying before in kapillary 11, the glue waste liquid after use is to waste liquid tank 13.After this process completes, saddle moves capillary sample inlet end 9 is moved in tank 12, 5 seconds of rinse, again capillary sample inlet end 9 is moved in buffering liquid groove 14, prerunning 5 minutes, saddle moves afterwards, capillary sample inlet end 9 is inserted and be placed with in advance (a wherein row) in large molecular biosciences sample pallet (96 hole) 10, electrophoresis loading, make sample to be analyzed as for kapillary 11 one end, saddle moves afterwards, make capillary sample inlet end 9 be arranged in 12 5 seconds of rinse of tank, again capillary sample inlet end 9 is moved in damping fluid 14, kapillary 11 two ends are by the voltage of setting, electrophoresis.In above-mentioned method, adopt Capillary Electrophoresis mode.In Capillary Electrophoresis capillary column used, the in the situation that of specific pH-value, its inner surface belt negative electricity, while contact with damping fluid, form electrostatic double layer, under high-voltage electric field effect, the damping fluid that forms electrostatic double layer one side moves to negative pole direction due to positively charged, thereby forms electroosmotic flow.Meanwhile, in damping fluid, charged particle, under electric field action, moves to its electrically charged polarity reverse direction with friction speed separately, forms electrophoresis.The migration velocity of charged particle in kapillary damping fluid equals the vector of electrophoresis and electroosmotic flow.Various particles are because the equal factor of electrically charged how much, the quality of institute, volume and shape causes that migration velocity difference realizes separation.
On capillary sample inlet end, be also provided with pick-up unit, sweep unit carrys out flyback retrace from one end of kapillary 11 to the other end, and (during electrophoresis, this sweep unit does not move, and only stays the place of outlet and detects; In the time that it finds that there is sample and occurs, just stop electrophoresis, and then carry out flyback retrace, obtaining the sample message in kapillary length range) while having biomacromolecule to arrive sweep unit in kapillary 11, electrophoresis finishes; The light wave of launching according to demand corresponding wavelength in all band spectrum generator 15, light wave is received by photodiode battle array.In scanning process, biological specimen can absorb light wave, in receiver photodiode battle array 16, can produce corresponding signal, and software carries out collection analysis to it.
Claims (9)
1. a biomacromolecule detection method, it is characterized in that, comprise the steps: to pump into new separation glue in kapillary, put into damping fluid prerunning, afterwards the separation glue after prerunning is put into the to be separated large molecular biosciences sample of handling well and carried out loading, make large molecular biosciences sample to be analyzed as for kapillary one end; Then kapillary two ends are applied to voltage, carry out electrophoresis, when stop electrophoresis after the other end capillaceous detects biomacromolecule, pick-up unit mobile line scanning of going forward side by side on kapillary detects, and detection information is analyzed.
2. biomacromolecule detection method according to claim 1, is characterized in that: the new separation glue loading in described kapillary is to capillary sample inlet end, through water rinse, separates glue and puts into damping fluid and carry out pre-electricity and remove out impurity polymkeric substance.
3. biomacromolecule detection method according to claim 1, is characterized in that: described large molecule divine and mighty sample electrophoresis loading to be analyzed is as for kapillary one end, and trapeziodal voltage is connected at kapillary two ends, and described sample is separated in kapillary.
4. a biomacromolecule detection device, is characterized in that: described device is installed on kapillary one end, realizes the continuity of biomacromolecule thing is detected online, when biomacromolecule detected, stops electrophoresis, and data are passed to system.
5. the biomacromolecule detection device based on Capillary Electrophoresis, it is characterized in that: include kapillary and detecting device, one end capillaceous is provided with capillary sample inlet end, the other end is provided with high-pressure pump, be provided with the optical scanning parts that can slide along kapillary in kapillary outside, also include pallet, the top of saddle is provided with sample pallet, tank, buffering liquid groove, waste liquid tank, is also provided with saddle drive motor below pallet.
6. the biomacromolecule detection device based on Capillary Electrophoresis according to claim 5, is characterized in that: described buffering liquid groove, waste liquid tank, tank and sample pallet are arranged successively on saddle.
7. the biomacromolecule detection device based on Capillary Electrophoresis according to claim 5, is characterized in that: described sample pallet is 96 orifice plates.
8. the biomacromolecule detection device based on Capillary Electrophoresis according to claim 5, is characterized in that: described pick-up unit comprises all band spectrum generator photodiode battle array.
9. the biomacromolecule detection device based on Capillary Electrophoresis according to claim 5, is characterized in that: described material capillaceous can be selected from the one in glass, stainless steel, copper, and caliber is 0.5 mm～5mm.
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| CN201410042598.0A CN103822959A (en) | 2014-01-29 | 2014-01-29 | Device and method for detecting biomacromolecules by capillary electrophoresis |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593748A (en) * | 2018-01-26 | 2018-09-28 | 南京溯远基因科技有限公司 | capillary and DNA sequencer |
| CN108624493A (en) * | 2018-08-01 | 2018-10-09 | 德诺杰亿（北京）生物科技有限公司 | Integrated DNA analysis instrument |
-
2014
- 2014-01-29 CN CN201410042598.0A patent/CN103822959A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108593748A (en) * | 2018-01-26 | 2018-09-28 | 南京溯远基因科技有限公司 | capillary and DNA sequencer |
| CN108593748B (en) * | 2018-01-26 | 2024-04-30 | 南京溯远基因科技有限公司 | Capillary and DNA sequencer |
| CN108624493A (en) * | 2018-08-01 | 2018-10-09 | 德诺杰亿（北京）生物科技有限公司 | Integrated DNA analysis instrument |
| CN108624493B (en) * | 2018-08-01 | 2024-05-28 | 德诺杰亿（北京）生物科技有限公司 | Integrated DNA Analyzer |
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Application publication date: 20140528 |
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