CN101993472A - Miniature multifunctional large biological molecule separating and purifying device - Google Patents
Miniature multifunctional large biological molecule separating and purifying device Download PDFInfo
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- CN101993472A CN101993472A CN2009100566403A CN200910056640A CN101993472A CN 101993472 A CN101993472 A CN 101993472A CN 2009100566403 A CN2009100566403 A CN 2009100566403A CN 200910056640 A CN200910056640 A CN 200910056640A CN 101993472 A CN101993472 A CN 101993472A
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Abstract
The invention relates to a miniature multifunctional large biological molecule separating and purifying device which comprises a chromatographic column, a cover capable of being sleeved with an upper opening of the chromatographic column, a sleeve capable of being sleeved with a column body of the chromatographic column, and a sample injector used for injecting a sample in the chromatographic column. The device integrates multiple functions into a whole, can be used for separating large biological molecules by adopting a centrifugal manner or injection pressurizing manner, has the advantages of good separation effect and low cost, and is convenient and simple for operation.
Description
Technical field
The invention belongs to biological instrument field; More specifically, the present invention relates to a kind of multiduty biological macromolecule purifying device.
Background technology
In the basic and applied research of biomedicine field, there is a large amount of separation and purification operations, particularly in processes such as preparation protein, nucleic acid.
The chromatography complex operation complexity of peristaltic pump as the hydrodynamic force source manipulated in traditional separation and purification, and a large amount of funds of Technology Need input such as high performance liquid chromatography, and use complicated more.For some breadboard routine operations, adopt high performance liquid chromatography installation cost height and complicated operation.
Therefore, this area also needs to develop some easy devices that are used for separation and purification of biological macromolecule, to reach the purpose of separation and purification on economical and practical basis.
Summary of the invention
The object of the present invention is to provide multiduty separation and purification of biological macromolecule device, it is simple, convenient, and practicality is good.
The present invention also aims to provide the using method of described separation and purification of biological macromolecule device.
In a first aspect of the present invention, a kind of biological macromolecule purifying device is provided, this device comprises:
One chromatography column comprises: cylinder, suitable for reading and internal diameter be less than the end opening of cylinder, and the end opening end comprises the salable or sealing connection opened;
One lid comprises: loam cake end, lower cover end and internal cavities, and the suitable for reading of lower cover end and chromatography column detachably socket-connects, and the loam cake end comprises the salable or sealing connection opened;
One sleeve pipe, described telescopic internal diameter detachably is placed in outside the chromatography column greater than the external diameter of the cylinder of chromatography column.
In a preference, described biological macromolecule purifying device can place whizzer centrifugal, need not to change lid or removes lid.
In another preference, described biomacromolecule includes but not limited to: protein or nucleic acid.
In another preference, described sleeve pipe is a centrifuge shield, and the hole that the loading centrifuge shield is used in its external diameter and the centrifugal device is complementary.
In another preference, the outside surface of the cylinder of described chromatography column also is provided with a circle projection near place suitable for reading, and the external diameter of described projection is greater than the telescopic internal diameter, thus fixedly chromatography column and telescopic relative position.
In another preference, described sealing connection is and the whole integrally formed form of lid, is provided with one at the boundary of the loam cake end of sealing connection and lid and encloses groove.When needs are removed sealing connection, along groove excision sealing connection.
In another preference, in the described lid, a sealing-ring is set also, thus the socket sealably suitable for reading of lid and chromatography column.
In another preference, lid and chromatography column belled mode include but not limited to: the spiral socket.
In another preference, the bottom of the cylinder of described chromatography column also is provided with at least one and filters core.
In another preference, the bottom of the cylinder of described chromatography column also is provided with a porous support plate.
In another preference, described porous support plate is positioned at the below of filtering core.
In another preference, this device also comprises:
One sample injector is used for adding sample in chromatography column.
In another preference, described sample injector is a syringe.
In another preference, the external diameter of the application of sample end of described sample injector and the internal cavities of lid are complementary.
In another preference, the external diameter of the application of sample end of described sample injector and the sealing of the internal cavities of lid.
In another preference, chromatography media is housed also in the cylinder of described chromatography column.
In another preference, described chromatography media includes but not limited to: sieve chromatography resin, affinity chromatography resin.
In a second aspect of the present invention, the purposes of described device is provided, be used for the purifying biological macromole.
In a preference, described device is used for centrifugation separation and purification biomacromolecule (as protein or nucleic acid) or exchange buffering solution.
In another preference, described device is used to inject pressuring method separation and purification biomacromolecule (as protein or nucleic acid) or exchange buffering solution.
In another preference, described device is used for removing the used probe (as fluorescent probe) of the unnecessary mark biomacromolecule of biomacromolecule (as protein or nucleic acid) solution etc.
In a third aspect of the present invention, a kind of lid that is used for separation and purification of biological macromolecule is provided, described lid comprises: loam cake end, lower cover end and internal cavities, loam cake end comprise the salable or sealing connection opened.
In a preference, described sealing connection is and the whole integrally formed form of lid, is provided with one at the boundary of the loam cake end of sealing connection and lid and encloses groove.
In a preference, described lid can match with chromatography column and sleeve pipe, is directly used in whizzer.
In another preference, when needs are removed sealing connection, along groove excision sealing connection.
In another preference, described internal cavities comprises two parts (containing 231,232), and a part (231) is used for being complementary (can closely cooperate) with the application of sample end of sample injector, is used to inject pressuring method separation and purification sample; Another part (232) is used for suitable for reading the socket-connecting (salable socket) with chromatography column.
In another preference, in the described lid, a sealing-ring is set also, thus the socket sealably suitable for reading of lid and chromatography column.
In a fourth aspect of the present invention, a kind of method of utilizing described device purifying biological macromole sample (comprising albumen or nucleic acid etc.) is provided, comprising:
(a) chromatography media of in the cylinder of chromatography column, packing into, described chromatography media can be removed impurity in the biomacromolecule sample by the chromatography effect;
(b) sealing connection of the end opening end of unlatching chromatography column;
(c) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected; With
(d) sample is joined in the cylinder by the suitable for reading of chromatography column, sleeve pipe is placed in outside the chromatography column, centrifugal, thus the end opening of the sample of the intravital chromatography media of post of flowing through (sample that is purified) by chromatography column is collected in the sleeve pipe.
In a preference, in the step (d), before adding sample, also comprise: excess liquid in the centrifugal removal chromatography media.More preferably, chromatography column is put in the sleeve pipe into centrifugal removal excess liquid.
In another preference, in the step (d), before adding sample, also comprise: chromatography media is carried out at least once balance.
In another preference, after step (d) obtains sample, repeat step (d) 1-10 time, thereby further improve the purity of sample.
In a fifth aspect of the present invention, a kind of method of utilizing described device purifying biological macromole sample (comprising albumen or nucleic acid etc.) is provided, comprising:
(i) chromatography media of in the cylinder of chromatography column, packing into, the impurity in the adsorbable biomacromolecule sample of described chromatography media;
(ii) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected, the application of sample end that is added with the sample injector of sample is inserted the suitable for reading of chromatography column via internal cavities;
(iii) open the sealing connection of the end opening end of chromatography column, in chromatography column, add sample with sample injector; With
(iv) collect from the effusive sample of the end opening of chromatography column (sample that is purified).
In a preference, step (ii) in, before adding sample, also comprise: chromatography media is carried out at least once balance.
In another preference, after step (iv) obtains sample, repeat step (iii)-(iv) 1-10 time, thereby further improve the purity of sample.
In a sixth aspect of the present invention, a kind of method of utilizing described device to change the damping fluid of biomacromolecule sample (comprising albumen or nucleic acid etc.) is provided, the damping fluid of described sample is replaced by buffer B by buffer A, and described method comprises:
(a1) chromatography media of in the cylinder of chromatography column, packing into;
(b1) sealing connection of the end opening end of unlatching chromatography column;
(c1) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected;
(d1) buffer B is joined in the cylinder by the suitable for reading of chromatography column, sleeve pipe is placed in outside the chromatography column, centrifugal, thereby with buffer B balance chromatography media;
(e1) sample is joined in the cylinder by the suitable for reading of chromatography column, after treating that sample infilters chromatography media, cover one deck buffer B at the chromatography media upper surface, sleeve pipe is placed in outside the chromatography column, thereby the sample of the intravital chromatography media of post of flowing through is collected in the sleeve pipe by the end opening of chromatography column, and the damping fluid of sample is replaced by buffer B by buffer A.
In a preference, in the step (d1), before adding sample, also comprise: excess liquid in the centrifugal removal chromatography media.More preferably, chromatography column is put in the sleeve pipe into centrifugal removal excess liquid.
In a seventh aspect of the present invention, a kind of method of utilizing described device purifying biological macromole sample (comprising albumen or nucleic acid etc.) is provided, comprising:
(a2) chromatography media of in the cylinder of chromatography column, packing into, the adsorbable biomacromolecule sample of described chromatography media;
(b2) sealing connection of the end opening end of unlatching chromatography column;
(c2) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected; With
(d2) pending sample is joined in the cylinder by the suitable for reading of chromatography column, sleeve pipe is placed in outside the chromatography column, centrifugal, thus sample is trapped within on the chromatography media, and impure solution is collected in the sleeve pipe;
(e2) with elutriant from the chromatography media with the sample wash-out, obtain purified sample.
In a preference, in the step (d2), before adding sample, also comprise: excess liquid in the centrifugal removal chromatography media.More preferably, chromatography column is put in the sleeve pipe into centrifugal removal excess liquid.
In another preference, in the step (d2), before adding sample, also comprise: chromatography media is carried out at least once balance.
In another preference, in the step (d2), be collected in the centrifuge shield described impure solution repeatedly (as 2-10 time) join in the cylinder from the suitable for reading of chromatography column, sleeve pipe is placed in outside the chromatography column, centrifugal, be trapped in ratio on the chromatography media thereby further improve sample.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown the schematic representation of apparatus of embodiment 1.
Fig. 2 has shown the cross-sectional view of lid in the device of embodiment 1.
Fig. 3 has shown the synoptic diagram behind sealing connection that the removal of the device of embodiment 1 is connected with the chromatography column end opening and removal and the sealing connection that the loam cake end of lid is connected.
Fig. 4 has shown a kind of synoptic diagram of sample injector.
Fig. 5 has shown the connection state synoptic diagram of sample injector and lid.
Fig. 6 has shown a kind of preferable biological macromolecule purifying device.Wherein last figure is the synoptic diagram of lid, and figure below is the synoptic diagram after lid, chromatography column and the outer tube combination.
In the above-mentioned accompanying drawing, each description of reference numerals is as follows:
1, chromatography column; 11, cylinder; 12, suitable for reading; 13, end opening; 14, sealing connection; 15, filter core; 16, porous support plate; 17, chromatography media; 18, projection; 19, outside screw.
2, lid; 21, the loam cake end; 22, the lower cover end; 23 (containing 231,232), internal cavities; 24, sealing-ring; 25, sealing connection; 26, groove; 27, internal thread.
3, sleeve pipe.
4, sample injector; 41, the application of sample end.
Embodiment
In order to overcome defectives such as separation and purification of biological macromolecule device complicated operation, cost height in the prior art, the inventor has developed a kind of separation and purification of biological macromolecule device of Multifunction, described device integrates multiple use, can adopt centrifugation or injection pressuring method to come the separating bio macromole, and simple to operation, with low cost.More preferably, described biological macromolecule purifying device can place whizzer centrifugal, need not to change lid or removes lid.
Among the present invention, having no particular limits for described " biomacromolecule ", can be the various biomaterials that need isolated or purified.For example, described biomacromolecule includes but not limited to: protein, nucleic acid etc.
Among the present invention, unless otherwise indicated, described " sample " is generalized, and they can various biomacromolecules, washings, damping fluid, balance liquid, elutriant etc.
The biological macromolecule purifying device
Biological macromolecule purifying device of the present invention comprises: but but chromatography column, with the belled lid suitable for reading of chromatography column and with the cylinder belled sleeve pipe of chromatography column.More preferably, also comprise the sample injector that is used in chromatography column, adding sample.
Chromatography column
Described chromatography column comprises: cylinder, suitable for reading and end opening, and the end of end opening comprises the salable or sealing connection opened.The cylinder of described chromatography column can be used for loading chromatography media, is used for the purifying of biomacromolecule.The type of chromatography media has no particular limits, and usually decides according to the kind of the biomacromolecule of required purifying, and this is that those skilled in the art are easy to make one's options.For example, described chromatography media includes but not limited to: sieve chromatography resin, affinity chromatography resin etc.The ratio of the shared chromatography column volume of the volume of described chromatography media has no particular limits, and can decide according to those skilled in the art's experience.Preferably, also can on chromatography media, cover one deck core, thereby prevent the disturbance of chromatography process medium.
Diameter and length for chromatography column have no particular limits, and generally can decide according to the scale of the biomacromolecule of required purifying.
The described application of sample that is used for suitable for reading makes sample flow flow to end opening behind cylinder (comprising chromatography media usually).The internal diameter of described end opening (or cross-sectional area) is less than cylinder.
For the ease of the dress post of chromatography media or be convenient to application of sample under the end opening sealed state, described end opening end also is provided with sealing connection.As long as can play the effect of sealing, described sealing connection is had no particular limits, it can be integrally formed with chromatography column, or the lid that is complementary with the end opening end of chromatography column or piston etc.When sealing connection is the form integrally formed with chromatography column, usually be convenient to part that sealing connection is separated (as fracture or cut) from the chromatography column end opening in the sealing connection and the boundary setting of chromatography column end opening, for example the material thickness of this part or firmly degree be lower than other part (as groove is set).
As optimal way of the present invention, the bottom of the cylinder of described chromatography column also is provided with at least one and filters core, and biomacromolecule can be flow through the core aperture, and resin particle can't pass through.More preferably, the internal diameter of the diameter that filters core and cylinder is identical or be slightly less than the internal diameter of cylinder.The number of the filtration core that is provided with can be decided according to the actual requirements, and for example can be 1-10, preferably 1-5.The model or the specification of filtering core are well-known to those skilled in the art, and those skilled in the art are easy to make one's options according to the actual requirements, and the present invention has no particular limits this.
As optimal way of the present invention, the bottom of the cylinder of described chromatography column also is provided with a porous support plate, be used for directly supporting chromatography media, perhaps support and filter core (be provided with under the situation of filtering core, described porous support plate is positioned at the below of filtering core).Adopt porous support plate to be supported, can guarantee to filter the intensity of core or chromatography media, prevent they in purification process (as centrifugal or injection) process because of liquid pressure cause discrete, break or be out of shape.
As optimal way of the present invention, the outside of the cylinder of described chromatography column also is provided with a circle projection near place suitable for reading, and the external diameter of described projection is greater than the telescopic internal diameter, thereby after chromatography column enters sleeve pipe, fixedly chromatography column and telescopic relative position are convenient to operation.
Lid
Described lid comprises: loam cake end, lower cover end and internal cavities.
Described lower cover end can detachably socket-connect with the suitable for reading of chromatography column, thereby plays sealing chromatography column purpose suitable for reading.The present invention has no particular limits for lid and chromatography column belled mode, includes but not limited to: the spiral socket.Preferably, in order to realize sealing well, in lid, one sealing-ring also is set, thereby realizes socket hermetically with the chromatography column place that engages suitable for reading.
Described loam cake end comprises the salable or sealing connection opened.As long as can play the effect of sealing, described sealing connection is had no particular limits, it can be whole integrally formed with lid, or second lid, piston, sealed strip or the sealant tape etc. that are complementary with the loam cake end of layer lid.When sealing connection is and the whole integrally formed form of lid, usually be convenient to sealing connection is separated from the loam cake end part of (as fracture or cut) in the sealing connection and the boundary setting of the loam cake end of lid, for example the material thickness of this part or firm degree are lower than other part (as groove is set), when needs are removed sealing connection, along this part excision sealing connection.Described sealing connection can play the effect of sealing, convenient transportation, and it opens the convenient centrifugal and injecting type chromatography operation in back.
After being positioned at the sealing connection unlatching of described loam cake end, cavity is exposed, thereby sample can be added in the chromatography column by this cavity.When sample adds fashionablely by sample injector, described cavity can be arranged to the size that the external diameter with the application of sample end of sample injector is complementary, thereby after the application of sample end 41 of sample injector inserted cavitys, cavity can be sealed.
Described lid can directly place whizzer centrifugal after assembling with chromatography column and sleeve pipe, need not to remove extraly lid or changes lid, thereby be convenient to operation.
Sleeve pipe
Described sleeve pipe detachably is placed in outside the chromatography column, is used to collect from the effusive sample of the end opening of chromatography column.Described telescopic diameter and length have no particular limits, and need only the external diameter of its internal diameter greater than the cylinder of chromatography column, and are complementary with described chromatography column.
As optimal way of the present invention, centrifugal for the ease of being directly used in, described sleeve pipe is a centrifuge shield, and the hole that the loading centrifuge shield is used in its external diameter and the centrifugal device is complementary.
Sample injector
As optimal way of the present invention, described purification devices also comprises sample injector, is used for adding in chromatography column sample.
The present invention has no particular limits the size and the length of sample injector, according to size, the length of chromatography column or lid decides usually, or decide according to the scale of the sample for the treatment of application of sample.
For handled easily when the application of sample or form the effect of sealing, the external diameter of the application of sample end 41 of described sample injector and the internal cavities of lid are complementary, and preferably can seal between them.More preferably, described sample injector is a syringe.
Purposes
Device of the present invention can be used for the isolated or purified biomacromolecule.For example its purposes includes but not limited to: the free fluorescent probe behind protein and purification of nucleic acids, protein soln desalination, exchange buffering liquid, the removal fluorescent mark etc.
Adopt device of the present invention can realize centrifugal and injection pressurization dual mode is finished sepn process, easy to use, can be according to selecting one carry out the chromatography operation in the different needs.Device of the present invention also can be made into prepacked column.
Purification process
The invention still further relates to and utilize described device to come the method for isolated or purified biomacromolecule.
As optimal way of the present invention, a kind of to utilize the method for described device purifying biological macromole sample be centrifuging, comprising: the chromatography media of (a) packing in the cylinder of chromatography column; (b) sealing connection of the end opening end of unlatching chromatography column joins sample in the cylinder by the suitable for reading of chromatography column; (c) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected; (d) sleeve pipe is placed in outside the chromatography column, centrifugal, thus the end opening of the sample that is purified of the intravital chromatography media of post of flowing through by chromatography column is collected in the sleeve pipe.
As optimal way of the present invention, it is injection that another kind utilizes the method for described device purifying biological macromole sample, comprising: the chromatography media of (i) packing in the cylinder of chromatography column; (ii) open the sealing connection of the loam cake end of lid, the lower cover end and the suitable for reading of chromatography column of lid socket-connected, the application of sample end that is added with the sample injector of sample is inserted the suitable for reading of chromatography column via internal cavities; (iii) open the sealing connection of the end opening end of chromatography column, in chromatography column, add sample with sample injector; (iv) collect from the effusive sample that is purified of the end opening of chromatography column.
When carrying out the isolated or purified of sample or before, also need the chromatography media in the chromatography column is carried out suitable pre-treatment (as column equilibration, washing etc.) usually, these methods are well-known to those skilled in the art, all may be implemented in the method for the present invention.For example, in abovementioned steps (b), before adding sample, also comprise: chromatography media is carried out at least once balance.
As one embodiment of the present invention, the chromatography process comprises as follows: pretreated chromatography media is joined in the chromatography column natural subsidence; Open chromatography column lower end sealing connection, with suitable damping fluid balance chromatography media; Add the sample solution that needs processing; Open the sealing connection of lid, build lid; Chromatography column is put in the sleeve pipe centrifugal, or draws required damping fluid with syringe and put the interface of lid into and operate.
Should be understood that after having obtained device of the present invention those skilled in the art can design the method for isolated or purified biomacromolecule easily, and described method is carried out suitable accommodation, these methods all are included in the present invention.
Assembling and fractionation
Before use, the chromatography column of described biological macromolecule purifying device, lid, sleeve pipe and sample injector can be to split to place, and are easy to like this pack and deposit.
When using, can assemble up by above-mentioned each parts, for example: lid is sheathed on chromatography column suitable for reading, casing pipe sleeve is located at outside the cylinder of chromatography column and/or the application of sample end of sample injector sample injector is inserted in the cavity of lid.
Device of the present invention can be made disposable form (deserted), and is easy to use and be not easy to pollute; Also can make the form of using repeatedly, save cost.
Material
The present invention has no particular limits for the material of making described biological macromolecule purifying device, and any material that can be made into described chromatography column, lid, sleeve pipe and sample injector and can make above-mentioned each parts be assembled into complete biological macromolecule purifying device all is an available.Described material includes but not limited to: polypropylene (PP), polyethylene (PE), polyamide (PA), vinyl cyanide, divinyl, cinnamic synthetics (ABS), rubber (as polyisoprene), glass or metal.
In addition, also can adopt two or more materials to make each parts of described biological macromolecule purifying device, such as adopting rubber to make described lid; Adopt glass, polypropylene (PP) or polyethylene (PE) to make described chromatography column, sleeve pipe or sample injector.
Major advantage of the present invention
(1) device of the present invention both can be used for whizzer, also can be used for injection, need not change lid, and was simple to operation, with low cost, overcome defectives such as separation and purification of biological macromolecule device complicated operation, cost height in the prior art.
(2) device of the present invention has also carried out multiple optimization design, particularly in the bottom of chromatography column back up pad is set, and can guarantee the intensity of core or chromatography media, prevent centrifugal or when injection liquid pressure cause distortion or break.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The basic structure of embodiment 1, biological macromolecule purifying device
Shown in seeing figures.1.and.2, described apparatus structure is as follows:
Chromatography column 1 comprises: cylinder 11, suitable for reading 12 and internal diameter less than the end opening 13 of cylinder, end opening 13 ends comprise the salable or sealing connection 14 opened;
One lid 2 comprises: loam cake end 21, lower cover end 22 and internal cavities 23, and lower cover end 22 detachably socket-connects with suitable for reading 12 of chromatography column 1, and loam cake end 21 comprises the salable or sealing connection 25 opened;
One sleeve pipe 3, the internal diameter of described sleeve pipe 3 detachably is placed in outside the chromatography column 1 greater than the external diameter of the cylinder 11 of chromatography column 1.Described sleeve pipe is used for centrifugal method and finishes the chromatography process.
In addition, also be provided with one in the bottom of the cylinder 11 of described chromatography column 1 and filter a core 15 and a porous support plate 16.Described porous support plate 16 is positioned at the below of filtering core 15.
In addition, in the described lid 2, a sealing-ring 24 is set also, thus suitable for reading 12 sockets sealably of lid and chromatography column 1.
In addition, the outside surface of the cylinder 11 of described chromatography column 1 also is provided with a circle projection 18 near 12 places suitable for reading, and described protruding 18 external diameter is greater than the internal diameter of sleeve pipe 3, thus the fixing relative position of chromatography column 1 and sleeve pipe 3.
Lid and chromatography column are by the spiral way socket.Promptly internal thread is set, the outside screw that is complementary with described internal thread is set at suitable for reading 12 of chromatography column in lower cover end 22 inboards of lid.
Described sealing connection 14 can be by the intersection excision from itself and end opening 13, and the end opening 13 after the excision as shown in Figure 3.
Described sealing connection 25 can be by loam cake end 21 excisions from lid, and the loam cake end 21 after the excision as shown in Figure 3.
Preferably, can pack in use chromatography media and with reference to shown in the right figure of Fig. 1 of described chromatography column is mounted with chromatography media 17 in the cylinder of chromatography column.
In the present embodiment, on the device basic of embodiment 1, described device also is furnished with sample injector 4, is used for adding in chromatography column 1 sample.Described sample injector is a syringe, as described in Figure 4.The external diameter of the application of sample end 41 of described syringe and the internal cavities 23 of lid 2 are complementary and can seal.
Behind the loam cake tip cut-off of described lid, the application of sample end 41 of syringe is inserted in the internal cavities 23 of lid 2, as shown in Figure 5.Thereby the method that can use injection is finished the chromatography process.
In the present embodiment, a kind of preferable biological macromolecule purifying device is provided, and it is suitable for Beckman JEJSE09B28 whizzer (rotary head is 24 * 1.5ml angle rotor) or loads the whizzer of hole that centrifuge shield uses other the various models identical with this whizzer.Described purification devices does not need to change lid, and can be used for whizzer under the situation of lid centrifugal carrying.
The lid 2 of described purification devices is referring to the last figure of Fig. 6, and the diameter of loam cake end 21 is 8mm, and the external diameter of lower cover end 22 is 12mm, and internal diameter is 10mm.Loam cake end 21 comprises the salable or sealing connection 25 opened, and for the ease of the excision of sealing connection 25, about 0.6mm place is provided with groove 26 below sealing connection 25 end faces, and the degree of depth of described groove is 0.5mm, and width is 0.2mm.
The internal cavities 23 of described lid 2 is divided into cavity 231 and 232, the upper diameter of cavity 231 is about 4.5mm, lower diameter is about 4.0mm, the cross section that also is cavity is for trapezoidal, the side constitutes the suitable gradient (shown in oblique arrow among the figure), and the application of sample end 41 of this gradient and syringe can closely cooperate.Cavity 232 is used for socket-connecting with suitable for reading 12 of chromatography column 1.
The chromatography column 1 of described purification devices and sleeve pipe 3 are referring to Fig. 6 figure below, and the internal diameter of the cylinder 11 of described chromatography column 1 is 8mm, the thickness of pipe 0.5mm of chromatography column 1.End opening 13 length are 3mm, and internal diameter is 4mm.Sealing connection 14 long 4mm, diameter 6mm.
The bottom of the cylinder 11 of chromatography column 1 also is provided with one and filters core 15, and its thickness is 1mm.
The bottom of the cylinder 11 of chromatography column 1 also is provided with a porous support plate 16, and its thickness is 1mm.
The outside surface distance suitable for reading 12 of the cylinder 11 of chromatography column 1 topmost about 20mm place also is provided with a circle projection 18, and projection 18 protrudes from outside surface 1.5mm.
Suitable for reading 12 arranged outside outside screw 19 of chromatography column 1, but internal thread 27 driving fits of this outside screw 19 and lid 2 lower cover ends 22 inwalls.
Above-mentioned lid 2 is with after chromatography column 1 and outer tube 3 are assembled mutually, can directly put into to carry out centrifugally in the whizzer, do not need to remove lid or change lid.In use, can excise the sealing connection 25 of lid 2 by groove 26 places, thereby the application of sample end 41 of syringe is inserted in the internal cavities 23, and the application of sample end 41 and internal cavities 23 driving fits of syringe.Also promptly, can realize multiple function, comprise centrifugal and injection by one group of device (with a lid).
In the present embodiment, adopt the small-molecule substance in the centrifugation removal protein soln.
Pending sample is: the protein B solution that contains fluorescent probe molecule A (molecular weight is lower than 1000).Also promptly: the purpose of present embodiment is for removing the fluorescent probe molecule A in the protein B solution (being dissolved in the PBS damping fluid).
Operation steps is as follows:
1) the internal diameter 8mm of the cylinder 11 of chromatography column 1, volume 800 μ L;
2) swelling is good Sephadex-G25 (chromatography media 17) joins in the cylinder 11 of chromatography column 1, and the column volume after the sedimentation is at 200～300 μ L;
3) twist into two parts the sealing connection 14 that is connected with the end opening 13 of chromatography column 1, cut off the sealing connection 25 of lid 2 loam cake ends or lid 2 is unscrewed, at 12 cover lids 2 suitable for reading of chromatography column 1, chromatography column 1 is put in the sleeve pipe 3, the centrifugal 1min of 1500g abandons sleeve pipe 3, the sleeve pipe 3 that more renews;
4) add pending sample (volume is no more than 100 μ L) in chromatography media 17 post bed upper surface mid-ways, treat that sample infilters resin after, cover the PBS solution of one deck 15 μ L at post bed upper surface; Sleeve pipe 3 is placed in outside the chromatography column 1;
5) the centrifugal 1min of 1500g, fluorescent probe molecule A is trapped within the post bed, and protein B flows in the sleeve pipe 3.
After measured, through above-mentioned purification step, the protein B purification effect is good, and wherein the fluorescent probe molecule A more than 95% is removed, and at least 90% protein B is recovered.
In the foregoing description, fluorescent probe molecule A is FITC, and protein B is BSA.
Embodiment 5, device make use-case 2--injection pressuring method purification of samples
In the present embodiment, adopt the small-molecule substance in the injection pressuring method removal protein soln.
Pending sample is: the protein B solution that contains fluorescent probe molecule A (molecular weight is lower than 1000).Also promptly: the purpose of present embodiment is for removing the fluorescent probe molecule A in the protein B solution (being dissolved in the PBS damping fluid).
Operation steps is as follows:
1) the internal diameter 8mm of the cylinder 11 of chromatography column 1, volume 800 μ L;
2) swelling is good Sephadex-G25 (chromatography media 17) joins in the cylinder 11 of chromatography column 1, and the column volume after the sedimentation is at 600 μ L;
3) cut off the sealing connection 25 is connected with the loam cake end 21 of lid 2, with lid 2 cover tight chromatography column 1 suitable for reading 12 on, in chromatography media 17, can not leave bubble;
4) balance: syringe 4 sucks the PBS solution of 3mL～3.5mL, the application of sample end 41 of syringe 4 passes lid 1 loam cake end 21 and enters internal cavities 23, and driving fit with it, twist into two parts the sealing connection 14 that is connected with chromatography column 1 end opening 13, at the uniform velocity slowly be injected into PBS solution in the chromatography column;
5) twist the lid off 2, when liquid layer drops near chromatography media 17 above the resin, on chromatography media 17, add pending sample (volume is no more than 100 μ L) in the liquid layer, cover tight lid 2, can not leave bubble in the chromatography media 17.
6) syringe 4 sucks the PBS solution of 1.5mL, and the application of sample end 41 of syringe 4 passes lid 1 loam cake end 21 and enters internal cavities 23, and driving fit with it, with PBS solution slowly at the uniform velocity (flow velocity is no more than 0.3ml/min) slowly be injected in the chromatography column; Collect and flow out solution, finish sepn process.
After measured, through above-mentioned purification step, the protein B purification effect is good, and wherein the free fluorescent probe molecule A more than 95% is removed, and at least 90% protein B is recovered.
In the foregoing description, fluorescent probe molecule A is FITC, and protein B is BSA.
Embodiment 6, device make use-case 3--centrifugation change proteinic buffered soln
In the present embodiment, adopt centrifugation to change proteinic buffered soln.
Pending sample is: the protein B solution that is dissolved in buffer A.Also promptly: the purpose of present embodiment is for to be replaced by damping fluid C with buffer A.
Operation steps is as follows:
1) the internal diameter 8mm of the cylinder 11 of chromatography column 1, volume 800 μ L;
2) swelling is good Sephadex-G25 (chromatography media 17) joins in the cylinder 11 of chromatography column 1, and the column volume after the sedimentation is at 200～300 μ L;
3) twist into two parts the sealing connection 14 that is connected with the end opening 13 of chromatography column 1, cut off the sealing connection 25 of lid 2 loam cake ends or lid 2 is unscrewed, at 12 cover lids 2 suitable for reading of chromatography column 1, chromatography column 1 is put in the sleeve pipe 3, the centrifugal 1min of 1500g abandons sleeve pipe 3, the sleeve pipe 3 that more renews;
4) with the damping fluid C balance of 1ml～1.5mL (can by centrifugal realization: the damping fluid C that each adding equates with column volume, the centrifugal 1min of 1500g, centrifugal 3-5 time);
5) add pending sample (volume is no more than 100 μ L) in chromatography media 17 post bed upper surface mid-ways, treat that sample infilters resin after, cover the damping fluid C of one deck 15 μ L at post bed upper surface;
6) the centrifugal 2min of 1500g, buffer A is replaced by damping fluid C.
After measured, through above-mentioned purification step, the buffer A component more than at least 95% is removed, and at least 95% protein B is recovered.
In the foregoing description, buffer A is TBS (Tris-Buffered Saline), and protein B is BSA, and damping fluid C is PBS (Phosphate Buffered Saline).
Embodiment 7, device make use-case 4--centrifugation antibody purification
In the present embodiment, adopt the antibody in the centrifugation purified blood serum.
Pending sample is: the human serum sample that contains IgG antibody.Also promptly: the purpose of present embodiment is the IgG antibody in the purified blood serum, removes all the other the biomacromolecule components in the serum.
Operation steps is as follows:
1) the internal diameter 8mm of the cylinder 11 of chromatography column 1, volume 800 μ L;
2) swelling is good Protein G (Protein G can combine with the IgG antibodies specific) affinity chromatography resin (chromatography media 17) joins in the cylinder 11 of chromatography column 1, and the column volume after the sedimentation is at 400～500 μ L;
3) twist into two parts the sealing connection 14 that is connected with the end opening 13 of chromatography column 1, cut off the sealing connection 25 of lid 2 loam cake ends or lid 2 is unscrewed,, chromatography column 1 is put in the sleeve pipe 3 at 12 cover lids 2 suitable for reading of chromatography column 1, the centrifugal 1min of 1500g abandons the waste liquid of collecting in the sleeve pipe 3;
4) carry out balance (can be by centrifugal realization: each add the balance liquid A that equates with column volume, the centrifugal 1min of 1500g, centrifugal 3-5 time) with the balance liquid A of 1ml～1.5mL;
5) add pending sample (volume is no more than 100 μ L, and IgG content is no more than 3mg) in chromatography media 17 post bed upper surface mid-ways, treat that sample infilters resin after, cover the balance liquid A of one deck 15 μ L at post bed upper surface;
6) repeating step 4) once, abandon the waste liquid of collecting in the sleeve pipe 3;
7) carry out wash-out (can be by centrifugal realization: each add the elutriant B that equates with column volume, the centrifugal 1min of 1500g, centrifugal 10-15 time) with the elutriant B of 4ml～5mL, the i.e. IgG antibody of purifying of collection liquid in the sleeve pipe 3;
After measured, through above-mentioned purification step, the IgG purification effect is good, and wherein the components such as foreign protein more than 95% are removed, and at least 90% IgG is recovered.
In the foregoing description, balance liquid A contains: 100mmol/L Tris-HCl, 100mmol/L NaCl, pH7.5.Elutriant B contains: 100mmol/L glycine-HCl, pH2.5.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (14)
1. a separation and purification of biological macromolecule device is characterized in that, this device comprises:
One chromatography column (1) comprising: cylinder (11), (12) suitable for reading and internal diameter be less than the end opening (13) of cylinder, and end opening (13) end comprises the salable or sealing connection (14) opened;
One lid (2) comprising: loam cake end (21), lower cover end (22) and internal cavities (23), and (12) suitable for reading of lower cover end (22) and chromatography column (1) detachably socket-connect, and loam cake end (21) comprises the salable or sealing connection (25) opened;
One sleeve pipe (3), the internal diameter of described sleeve pipe (3) detachably is placed in outside the chromatography column (1) greater than the external diameter of the cylinder (11) of chromatography column (1).
2. device as claimed in claim 1 is characterized in that, in the described lid (2), a sealing-ring (24) is set also, thus (12) suitable for reading socket sealably of lid and chromatography column (1).
3. device as claimed in claim 1 is characterized in that, the bottom of the cylinder (11) of described chromatography column (1) also is provided with at least one and filters core (15).
4. as claim 1 or 3 described devices, it is characterized in that the bottom of the cylinder (11) of described chromatography column (1) also is provided with a porous support plate (16).
5. device as claimed in claim 1 is characterized in that, this device also comprises:
One sample injector (4) is used for adding sample in chromatography column (1).
6. device as claimed in claim 5 is characterized in that, the external diameter of the application of sample end (41) of described sample injector (4) and the internal cavities (23) of lid (2) are complementary.
7. device as claimed in claim 1 is characterized in that, in the cylinder (11) of described chromatography column (1) chromatography media (17) is housed also.
8. the purposes of the arbitrary described device of claim 1-7 is used for the separation and purification biomacromolecule.
9. a lid that is used for separation and purification of biological macromolecule is characterized in that, described lid comprises: loam cake end (21), lower cover end (22) and internal cavities (23), loam cake end (21) comprise the salable or sealing connection (25) opened.
10. lid as claimed in claim 9 is characterized in that, described sealing connection (25) is and the whole integrally formed form of lid, at the boundary of sealing connection (25) and the loam cake end (21) of lid one circle groove (26) is set.
11. a method of utilizing the described device separation and purification of claim 1 biomacromolecule sample comprises:
(a) chromatography media (17) of in the cylinder (11) of chromatography column (1), packing into, the impurity in the adsorbable biomacromolecule sample of described chromatography media (17);
(b) the terminal sealing connection (14) of the end opening (13) of unlatching chromatography column (1);
(c) sealing connection (25) of the loam cake end (21) of unlatching lid (2) socket-connects the lower cover end (22) of lid (2) and (12) suitable for reading of chromatography column (1); With
(d) suitable for reading (12) of sample by chromatography column (1) are joined in the cylinder (11), sleeve pipe (3) is placed in outside the chromatography column (1), centrifugal, thus the end opening (13) of the sample of the chromatography media in the cylinder of flowing through (11) by chromatography column (1) is collected in the sleeve pipe (3).
12. a method of utilizing claim 1 or 5 described device separation and purification biomacromolecule samples comprises:
(i) chromatography media (17) of in the cylinder (11) of chromatography column (1), packing into, the impurity in the adsorbable biomacromolecule sample of described chromatography media (17);
(ii) open the sealing connection (25) of the loam cake end (21) of lid (2), the lower cover end (22) of lid (2) (12) suitable for reading with chromatography column (1) are socket-connected the application of sample end (41) that is added with the sample injector (4) of sample is inserted chromatography column (1) via internal cavities (23) (12) suitable for reading;
(iii) open the terminal sealing connection (14) of end opening (13) of chromatography column (1), in chromatography column (1), add sample with sample injector (4); With
(iv) collect the effusive sample of end opening (13) from chromatography column (1).
13. a method of utilizing the described device of claim 1 to change the damping fluid of biomacromolecule sample, the damping fluid of described sample is replaced by buffer B by buffer A, and described method comprises:
(a1) chromatography media (17) of in the cylinder (11) of chromatography column (1), packing into;
(b1) the terminal sealing connection (14) of the end opening (13) of unlatching chromatography column (1);
(c1) sealing connection (25) of the loam cake end (21) of unlatching lid (2) socket-connects the lower cover end (22) of lid (2) and (12) suitable for reading of chromatography column (1);
(d1) suitable for reading (12) of buffer B by chromatography column (1) are joined in the cylinder (11), sleeve pipe (3) is placed in outside the chromatography column (1), centrifugal, thereby with buffer B balance chromatography media (17);
(e1) suitable for reading (12) of sample by chromatography column (1) are joined in the cylinder (11), after treating that sample infilters chromatography media (17), cover one deck buffer B at chromatography media (17) upper surface, sleeve pipe (3) is placed in outside the chromatography column (1), thereby the sample of the chromatography media in the cylinder of flowing through (11) is collected in the sleeve pipe (3) by the end opening (13) of chromatography column (1), and the damping fluid of sample is replaced by buffer B by buffer A.
14. a method of utilizing the described device separation and purification of claim 1 biomacromolecule sample comprises:
(a2) chromatography media (17) of in the cylinder (11) of chromatography column (1), packing into, the adsorbable biomacromolecule sample of described chromatography media (17);
(b2) the terminal sealing connection (14) of the end opening (13) of unlatching chromatography column (1);
(c2) sealing connection (25) of the loam cake end (21) of unlatching lid (2) socket-connects the lower cover end (22) of lid (2) and (12) suitable for reading of chromatography column (1); With
(d2) suitable for reading (12) of pending sample by chromatography column (1) are joined in the cylinder (11), sleeve pipe (3) is placed in outside the chromatography column (1), centrifugal, thus sample is trapped within on the chromatography media (17), and impure solution is collected in the sleeve pipe (3);
(e2) go up the sample wash-out from chromatography media (17) with elutriant, obtain purified sample.
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009100566403A CN101993472A (en) | 2009-08-19 | 2009-08-19 | Miniature multifunctional large biological molecule separating and purifying device |
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| Application Number | Priority Date | Filing Date | Title |
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| CN2009100566403A CN101993472A (en) | 2009-08-19 | 2009-08-19 | Miniature multifunctional large biological molecule separating and purifying device |
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| CN105413233A (en) * | 2015-12-10 | 2016-03-23 | 中国医学科学院生物医学工程研究所 | Micro-column separation device |
| CN106124282A (en) * | 2016-07-26 | 2016-11-16 | 广州海力特生物科技有限公司 | A kind of method secreting body outside lamination centrifugal filtration separation and Extraction |
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Application publication date: 20110330 |