CN209555218U

CN209555218U - Separating chips

Info

Publication number: CN209555218U
Application number: CN201821790069.9U
Authority: CN
Inventors: 陈欲超
Original assignee: Shenzhen Hui Core Bio Medical Technology Co Ltd
Current assignee: Shenzhen Hui Core Bio Medical Technology Co Ltd
Priority date: 2017-11-30
Filing date: 2018-10-31
Publication date: 2019-10-29
Anticipated expiration: 2028-10-31
Also published as: CN109439533A; CN209636238U; CN109439525B; CN109439525A; CN109439533B

Abstract

A kind of separating chips, comprising: sample pool；First filter membrane；Second filter membrane；First chamber, the first chamber are connected with the sample pool by first filter membrane, and the first chamber is provided with the first opening, and first opening is for making the first chamber be in communication with the outside；Second chamber, the second chamber are connected with the sample pool by second filter membrane, and the second chamber is provided with the second opening, and second opening is for making the second chamber be in communication with the outside.Wherein, the first chamber and the second chamber two sides that be located at the sample pool opposite.

Description

Separating chips

Technical field

The utility model relates to field of biotechnology, and in particular, to one kind is for separating target particles in liquid sample Separating chips.

Background technique

Liquid biopsy (liquid biopsy or fluid biopsy) is that one kind can reflect that tumour is thin comprehensively, in real time The sampling and analysis method of born of the same parents or tissue biological's information.Liquid biopsy has the advantages that Noninvasive, by separating, analyzing blood Or specific research object carries out dynamic observation to tumour in other body fluid (urine, saliva, pleural effusion, cerebrospinal fluid etc.), it can To instruct medical staff to carry out screening, diagnosis, judging prognosis, selection therapeutic scheme and monitoring recurrence etc. to tumour.Liquid biopsy In particular studies object include Circulating tumor DNA (circulating tumor DNA, ctDNA), circulating tumor cell (circulating tumor cell, CTC), microvesicle (also known as excretion body, exosome) etc..

It is general using following in the methods of centrifugation, immunocapture or filtering separation, purifying blood or body fluid in the prior art Ring tumour cell and/or excretion body.The separation method of centrifugation will cause one to the membrane structure of circulating tumor cell (or excretion body) The mechanical damage for determining degree influences subsequent analysis and research, and is centrifuged the affected cumbersome flux to liquid biopsy and causes to limit.Exempt from The separation method of epidemic disease capture needs to increase substantially sample process cost, and the elution requirement after immunocapture can using antibody The activity of circulating tumor cell (or excretion body) can be had an impact.It can be effectively using the separation method that filter membrane filters Various sizes of component in body fluid is separated, has the characteristics that low cost, high throughput, the target components obtained after filtering are able to maintain very High bioactivity.But in the actual operation process, the constituent part being easy on filter membrane in enriched biological sample, wherein Group branch greater than membrane pore size blocks fenestra.Pore Blocking can prevent the component less than membrane pore size from effectively through filtering Film influences the purity of target components after separation.It is excessive that Pore Blocking will also result in local pressure, even results in filter membrane rupture.

Utility model content

In view of the foregoing, it is necessary to provide it is a kind of can reduce be separated by filtration during filter membrane plug-hole phenomenon occurs Separating chips.

The utility model embodiment provides a kind of separating chips, for from liquid sample separating-purifying go out target particles, The separating chips include:

Sample pool；

First filter membrane, the aperture of first filter membrane are less than the partial size of target particles；

Second filter membrane, the aperture of second filter membrane are less than the partial size of target particles；

First chamber, the first chamber are connected with the sample pool by first filter membrane, first chamber Room is provided with the first opening, and first opening is for making the first chamber be in communication with the outside；

Second chamber, the second chamber are connected with the sample pool by second filter membrane, second chamber Room is provided with the second opening, and second opening is for making the second chamber be in communication with the outside, wherein the first chamber with The second chamber is located at the opposite two sides of the sample pool.

Compared to the prior art, separating chips provided by the utility model are using simply, and cost of manufacture is lower, when subsequent When connecting vacuum system, liquid sample to be separated can more effectively penetrate filter membrane, can be to following in liquid biopsy procedure The separation and Extraction of the progress such as ring tumour cell, excretion body quickly, high-throughput, mitigates the workload of experimenter, and it is living to reduce liquid The testing cost of inspection.

Detailed description of the invention

Fig. 1 is the structural schematic diagram of separating chips provided by the utility model embodiment.

Fig. 2 is the structure dismantling schematic diagram of separating chips shown in FIG. 1.

Fig. 3 is the structural schematic diagram of separating chips provided by another embodiment of the utility model.

Fig. 4 is the functional block diagram of separator provided by the utility model embodiment.

Fig. 5 a is the fluid path schematic diagram of separator shown in Fig. 4.

Fig. 5 b is the Program modual graph of the separation control system of separator shown in Fig. 4.

Fig. 6 is that liquid when carrying out separating-purifying using separating chips shown in FIG. 1 in sample pool flows to schematic diagram.

Fig. 7 a is the schematic diagram that the negative pressure of separating chips is applied in an embodiment of the present invention.

Fig. 7 b is the schematic diagram that the negative pressure of separating chips is applied in another embodiment of the utility model.

Fig. 7 c is the schematic diagram that the negative pressure of separating chips is applied in another embodiment of the utility model.

Fig. 8 a is the structural schematic diagram of the chip pad of one embodiment of separator shown in Fig. 3.

Fig. 8 b is the structural schematic diagram in the chip pad shown in Fig. 8 a after fixedly separated chip and gas conduit.

Fig. 9 a is the testing graininess figure of the component in original urine specimen.

Fig. 9 b is outer after carrying out separating-purifying to original urine specimen using the separating chips of the utility model embodiment Secrete the testing graininess figure of body.

Fig. 9 c is the testing graininess figure for carrying out the excretion body after separating-purifying to original urine specimen using qEV splitter.

Fig. 9 d is to carry out the excretion body after separating-purifying to original urine specimen using ExoQuick-TC excretion body kit Testing graininess figure.

Fig. 9 e is outer after carrying out separating-purifying to original urine specimen using Magcapture excretion body extracts kit Secrete the testing graininess figure of body.

Fig. 9 f is to carry out the excretion body after separating-purifying to original urine specimen using Exo-Spin excretion body purifying tubing string Testing graininess figure.

Figure 10 a is outer after carrying out separating-purifying to original urine specimen using the separating chips of the utility model embodiment Secrete the scanning electron microscope (SEM) photograph of body.

Figure 10 b is outer after carrying out separating-purifying to original urine specimen using the separating chips of the utility model embodiment Secrete the transmission electron microscope picture of body.

Figure 11 is to separating chips, the qEV splitter, ExoQuick-TC excretion for using the utility model embodiment respectively Body kit, Magcapture excretion body extracts kit and Exo-Spin excretion body purifying tubing string to original urine specimen into The excretion body obtained after row separating-purifying carries out the band map that proteins gel electrophoresis and Silver stain obtain.

Figure 12 is to using the separating chips of the utility model embodiment to divide the urine specimen of 11 parts of cancer patients The map that western blot analysis obtains is carried out from the excretion body obtained after purification.

Symbol description

The following detailed description will be further explained with reference to the above drawings the utility model.

Specific embodiment

The technical solution of the utility model is carried out below in conjunction with preferred embodiments of the present invention and embodiment Description.It should be noted that it can be directly to another when a unit is described as " connecting " in another unit A unit may be simultaneously present unit placed in the middle.When a unit is described as " being set to " another unit, it can be with It is to be set up directly on another unit or may be simultaneously present unit placed in the middle.Unless otherwise defined, institute used herein Some technical and scientific terms have the same meaning as commonly understood by one of ordinary skill in the art to which the utility model belongs.At this The title of the element or equipment used in the description of utility model is only for the purpose of describing specific embodiments It is intended to limitation the utility model.

The utility model embodiment provides a kind of separating chips, which is used for various sizes of in liquid sample Particle carries out separating-purifying, to obtain the target particles of specific dimensions.The liquid sample can for human plasma, serum, Cerebrospinal fluid, Saliva, urine and gastric juice etc..Fig. 1 is the structural schematic diagram of separating chips 10 provided by an embodiment of the present invention.Such as Shown in Fig. 1, separating chips 10 include sample pool 13, first chamber 15 and second chamber 17.The first chamber 15 and this second Chamber 17 is located at the opposite two sides of the sample pool 13.

The sample pool 13 includes sample pool substrate 136, the first inner coversheet 132 and the second inner coversheet 134, first inner coversheet 132 with second inner coversheet 134 two sides that be covered on the sample pool substrate 136 opposite, thus make the sample pool substrate 136, should First inner coversheet 132 and second inner coversheet 134 enclose the accommodating chamber set out for accommodating liquid sample jointly (figure is not marked).It should The first filter membrane 14 is provided in first inner coversheet 132, which is provided with the second filter membrane 16.First chamber Room 15 is connected with the sample pool 13 by first filter membrane 14.The first chamber 15 is provided with the first opening 152, this first Opening 152 is for making the first chamber 15 be in communication with the outside.The second chamber 17 and the sample pool 13 pass through second filter membrane 16 are connected, which is provided with the second opening 172, and second opening 172 is for making the second chamber 17 and the external world Connection.

When using the separating chips 10, sample pool 13 is added in liquid sample, by first opening 152 and second opening 172 are connected with vacuum system 30 (ginseng Fig. 3) respectively.When vacuum system 30 makes the first chamber 15 by first opening 152 When being aspirated, negative pressure is generated in the first chamber 15.Liquid under the suction function of the first chamber 15, in sample pool 13 Size flows into the first chamber 15 via the first filter membrane 14 less than the component of the pore size filter of the first filter membrane 14 in sample.When When vacuum system 30 aspirates the second chamber 17 by second opening 172, negative pressure is generated in the second chamber 17.In Under the suction function of the second chamber 17, pore size filter of the size less than the second filter membrane 16 in the liquid sample in sample pool 13 Component via the second filter membrane 16 flow into the second chamber 17.

Alternately and repeatedly make to generate negative pressure in the first chamber 15 and the second chamber 17, can effectively make liquid sample anti- It reestablishes diplomatic relations and alternately flows through the first filter membrane 14 and the second filter membrane 16, size in liquid sample is made to be greater than the first filter membrane 14 and second The component in 16 aperture of filter membrane stays in sample pool 13.The structure design of the separating chips 10 makes to be adsorbed in 14 He of the first filter membrane The component on 16 surface of the second filter membrane is easy to fall off from filter membrane surface in negative pressure variation alternately and repeatedly, can be effectively prevented The fenestra of filter membrane is blocked.

The sample pool 13 of the separating chips 10, first chamber 15, the main part of second chamber 17 can be by plastics, glass Glass, metal or composite material are made.In one embodiment, the sample pool 13 of the separating chips 10, first chamber 15, second chamber 17 main part is made of the transparent materials such as polyethyleneimine (PEI) or polymethyl methacrylate (PMMA).

First filter membrane 14 and second filter membrane 16 can be made of identical membrane material, can also be by different films Material is made.First filter membrane 14 and second filter membrane 16 can have identical average filtration membrane aperture and/or aperture Distribution, it is possible to have different average filtration membrane apertures and/or pore-size distribution.First filter membrane 14 (or second filtering Film 16) it can be made of a kind of membrane material, it is also possible to as made of multiple film Material cladding.First filter membrane, 14 He Second filter membrane 16 can be porous material, include but are not limited to porous ceramic film material, porous plastic materials and porous gold Belong to material.Specifically, first filter membrane 14 and second filter membrane 16 can be respectively selected from anodic alumina films (AAO), gather One or more of carbonic ester film, cellulose acetate film, polyethylene film, polypropylene screen and polystyrene film.More specifically, in view of Anodic alumina films porosity with higher and more uniform aperture, first filter membrane 14 and second filter membrane 16 use Anodic alumina films.

The aperture of first filter membrane 14 and second filter membrane 16 can be according to the class of the liquid sample and target particles Type is designed.In one embodiment, the aperture of first filter membrane 14 and second filter membrane 16 is between 2-20 microns； Preferably, between 5-10 microns.More specifically, the aperture of first filter membrane 14 and second filter membrane 16 is 8 microns, it can For the circulating tumor cell in separating-purifying plasma sample.

In another embodiment, the aperture of first filter membrane 14 and second filter membrane 16 is between 5-200 nanometers； Preferably, between 10-100 nanometers.In one embodiment, the aperture of first filter membrane 14 and second filter membrane 16 is 20 Nanometer can be used for separating-purifying and pass through excretion body in the plasma sample of 200 nano-filtration membranes.

Wherein, when first filter membrane 14 and 16 surface of the second filter membrane are not further embellished, the separating chips 10 according only to the various components in the aperture screening liquid sample of filter membrane, mainly include target in resulting sample after separating-purifying Particle (such as circulating tumor cell, excretion body), it is also possible to (e.g., highly dense with close or larger size particle containing other It spends lipoprotein (HDLs), low-density lipoprotein (LDLs), medium density lipoprotein (IDLs), very low density lipoprotein (VLDL), with And non-excretion body protein similar in the common granularity such as chylomicron (chylomicrons) and excretion body).To reduce porous material Expect absorption to albumen or gene in liquid sample, the surface of first filter membrane 14 and second filter membrane 16 can be by Chemical modification；For specifically separating-purifying target particles, the surface of first filter membrane 14 and second filter membrane 16 can Being modified by specific biological macromolecular, which can be specific one or more of antibody, resists Former, polypeptide or base sequence.It should be pointed out that target particles described herein can be the cell with biological significance Or component, it is also possible to other kinds of particle, such as liposome, the nanosphere, nanoparticle of synthesis.

It is understood that the volume of the sample pool 13 can be designed according to practical application scene.For biological biopsy The volume of application scenarios, the sample pool 13 can be between 0.1-10 milliliters, optionally, between 0.5-2 milliliters.Implement one In example, the volume of the sample pool 13 is 1 milliliter.The top of the sample pool 13 may include sample pool opening 138, for being added And/or take out liquid sample.

In the embodiment shown in figure 1, which includes first side cover opposite with first filter membrane 14 Piece 156, the first side cover piece 156 and first inner coversheet 132 with first filter membrane 14 enclose jointly set to be formed this One chamber 15, first opening 152 are set on the first side cover piece 156；The second chamber 17 includes and described second filters The opposite second side cover plate 176 of film 16, second side cover plate 176 and second inner coversheet with second filter membrane 16 134 enclose jointly and set to form the second chamber 17, which is set on second side cover plate 176.In present embodiment In, it is respectively fixed with an opening link block 18 on the first side cover piece 156 and second side cover plate 176 of the separating chips 10, it should It is offered in opening link block 18 channel (figure is not marked), which is aligned with the first opening 152 and the second opening 172 respectively And it is connected to.Further, which may also include a chip base 19, the chip base 19 for close this first Chamber 15 and the second chamber 17, and it is used to support other elements of the separating chips 10.The separating chips 10 have pair Claim structure.It should be noted that separating chips 10 are also possible to dissymmetrical structure or other any can be realized the utility model The structure of design.

Fig. 2 shows the decomposition diagrams of an embodiment of separating chips 10 provided by the utility model.Such as Fig. 2 institute Show, through-hole (figure is not marked) is offered in first inner coversheet 132, which is fixed on first inner coversheet 132 In through-hole；Through-hole (figure is not marked) is offered in second inner coversheet 134, which is fixed on second inner coversheet In 134 through-hole.The sample pool substrate 136 be with certain thickness U-shaped substrate, first inner coversheet 132 and this in second Cover plate 134 is covered on the opposite two sides of the sample pool substrate 136 along the thickness direction of the sample pool substrate 136.

The utility model embodiment also provides a kind of production method of above-mentioned separating chips 10 comprising following steps:

Step 1 provides above-mentioned sample pool substrate 136, the first inner coversheet 132, the second inner coversheet 134, the first side cover piece 156, second side cover plate 176, the first filter membrane 14 and the second filter membrane 16.

The first side cover piece 156 is assembled to first inner coversheet 132 to form first chamber 15 by step 2.

First filter membrane 14 is assembled to first inner coversheet 132 by step 3.

Step 4, with first filter membrane 14 the first inner coversheet 132 far from the side of the first side cover piece 156 according to Secondary assembling sample pool substrate 136 and second inner coversheet 134.

Second filter membrane 16 is assembled to second inner coversheet 134 by step 5.

Second side cover plate 176 is assembled to the second inner coversheet 134 with second filter membrane 16 far from this by step 6 The side of sample pool substrate 136 is to form second chamber 17.

In one embodiment, the sample pool substrate 136, the first inner coversheet 132, the second inner coversheet 134, the first side cover piece 156, second side cover plate 176, the first filter membrane 14 and the second filter membrane 16 are fixed by adhesive.Described adhesive can be with For ultraviolet cured adhesive or transparent silicone waterproof gasket cement.Based on above method, it can be made with lower cost and meet this The separating chips 10 of utility model spirit.

Referring to Fig. 3, another embodiment of the utility model also provides a kind of separating chips 10 '.With above-mentioned separating chips 10 Unlike, sample pool substrate 136, the first inner coversheet 132 and the second inner coversheet 134 are omitted in the separating chips 10 '.Due to The sample pool substrate 136, the first inner coversheet 132 and the second inner coversheet 134 are omitted, production can be reduced to a certain extent Cost.Specifically, which is equipped with the first convex block 154, which draws the first side cover piece 156 It is divided into the first cover plate portion 1561 positioned at 154 side of the first convex block and the second lid positioned at 154 other side of the first convex block Piece portion 1562.Second side cover plate 176 is equipped with second convex block 174 opposite with first convex block 154, second convex block 174 By second side cover plate 176 be divided into positioned at 174 side of the second convex block third cover plate portion 1761 and to be located at this second convex 4th cover plate portion 1762 of 174 other side of block.The first cover plate portion 1561, the third cover plate portion 1761, first convex block 154 with And second convex block 174 encloses jointly and sets to form the sample pool 13.

The bottom of the first side cover piece 156 position opposite with first convex block 154 is equipped with a chip base 19.This first Filter membrane 14 is set between first convex block 154 and the chip base 19 and opposite with the second cover plate portion 1562, this second Cover plate portion 1562, first filter membrane 14 and the chip base 19 are enclosed jointly to be set to form the first chamber 15.Second side cover The bottom of piece 176 and the position opposite with second convex block 174 are equipped with another chip base 19.Second filter membrane 16 is set to It is between second convex block 174 and the chip base 19 and opposite with the 4th cover plate portion 1762.4th cover plate portion 1762, this Two filter membranes 16 and chip base 19 are enclosed jointly to be set to form the second chamber 17.In the present embodiment, first convex block 154 Gap (figure is not marked) is equipped between second convex block 174, the gap is for enabling the liquid sample in sample pool 13 to flow out The sample pool 13, and the first chamber 15 or the second chamber are respectively enterd through first filter membrane 14 or second filter membrane 16 17.More specifically, the side of the first convex block 154 towards the chip base 19 offers one first card slot 1540, the chip base The corresponding position in bottom 19 offers one second card slot (figure is not marked), and first filter membrane 14 card sets and is fixed on first card slot Between 1540 and second card slot.Similarly, the side of the second convex block 174 towards the chip base 19 offers a third card slot 1740, which offers one the 4th card slot, and second filter membrane 16 card sets and is fixed on the third Between card slot 1740 and the 4th card slot.

The utility model embodiment also provides a kind of production method of above-mentioned separating chips 10 ' comprising following steps:

Step 1 provides above-mentioned first side cover piece 156, second side cover plate 176, the first filter membrane 14, the second filter membrane 16 And chip base 19.

First filter membrane 14 is assembled to the first convex block 154 of the first side cover piece 156 and corresponding chip by step 2 Between substrate 19.

Second filter membrane 16 is assembled to the second convex block 174 of second side cover plate 176 and corresponding chip by step 3 Between substrate 19.

Step 4 is assembled to second side cover plate 176 with by the first side cover piece 156, make first convex block 154 and this Two convex blocks 174 are opposite, and keep two chip bases 19 opposite.To, the first cover plate portion 1561, the third cover plate portion 1761, First convex block 154 and second convex block 174 enclose jointly to be set to form the sample pool 13；The second cover plate portion 1562, this first Filter membrane 14 and the chip base 19 are enclosed jointly to be set to form the first chamber 15；4th cover plate portion 1762, second filtering Film 16 and chip base 19 are enclosed jointly to be set to form the second chamber 17.

The utility model embodiment further provides a kind of separator.Fig. 4 shows the program of the separator 100 Module diagram.The separator 100 includes main body module 101, supplementary module 102 and human-computer interaction module 103.

The main body module 101 is used to carry out separating-purifying to liquid sample.The main body module 101 includes as described above Separating chips 10, liquid supplying unit 20, vacuum system 30 and frequency-variable module 40.

The liquid supplying unit 20 is used to inject liquid sample and cleaning into the sample pool 13 of separating chips 10,10 ' Liquid.As shown in figure 4, the liquid supplying unit 20 includes sample to be tested room 210, cleaning liquid chamber 230 and the first control valve 220.It should First control valve 220 can be connected to one of in the sample to be tested room 210 and the cleaning liquid chamber 230 respectively.This first Control valve 220 can be fluid path converter, include but are not limited to solenoid valve, rotary valve.First control valve 220 is connected to be measured Liquid sample in sample to be tested room 210 can be provided to the sample pool 13 of separating chips 10,10 ' for dividing by sample room 210 From target particles；First control valve 220 is switched to and is connected to the cleaning liquid chamber 230, can will be cleaned clear in liquid chamber 230 Washing lotion is provided to the sample pool 13 of separating chips 10,10 ' for cleaning separating chips 10.It may include above-mentioned in the cleaning solution Surfactant, for removing the protein molecular of separating chips 10,10 ' each Adsorption on Surface.The liquid supplying unit 20 is also It may include a power part, such as kinetic pump or aspiration pump, provide power for liquid stream.In other embodiments, the liquid Supply unit 20 or liquid-transfering gun or syringe, operator liquid sample can be added either manually or by liquid-transfering gun or syringe Sample pool 13.

It is negative that the vacuum system 30 is used to make respectively the first chamber 15 of the separating chips 10,10 ' and second chamber 17 to generate Pressure.The vacuum system 30 can be two independent vacuum systems, be also possible to a vacuum system by design.The vacuum System 30 also may include the equipment such as minipump or micro-suction pump.It is understood that the vacuum system 30 and this point It can be connected by the preferable pipeline of air-tightness between off-chip piece 10.In one embodiment, which includes first Vacuum pump 310 and the second vacuum pump 320, first vacuum pump 310 are connected with the first opening 152 of the separating chips 10,10 ' It connects, which is connected with the second opening 172 of the separating chips 10,10 '.

The frequency-variable module 40 is electrically connected with the vacuum system 30, which, which can control, is supplied to the vacuum system 30 supply voltage, to make to be alternately produced negative pressure in first chamber 15 and second chamber 17.In one embodiment, the frequency conversion Module 40 includes frequency converter 410 and the second control valve 420 connecting with the frequency converter 410.Second control valve 420 can be Fluid path converter includes but are not limited to solenoid valve, rotary valve.Second control valve 420 respectively with first vacuum pump 310 with And one of connection in second vacuum pump 320, to make the first vacuum pump 310 and the second vacuum pump 320 alternately and repeatedly Work.For example, second control valve 420 is connected to first vacuum pump 310, so that the frequency converter 410 controls first vacuum 310 operation of pump makes to generate negative pressure in first chamber 15 (left chamber as shown in FIG. 6), such as scheme by 152 pumping of the first opening Direction shown in 6 arrows, liquid in liquid sample and size in sample pool 13 exist less than the component in 14 aperture of the first filter membrane By the first filter membrane 14 under suction function, into first chamber 15, at the same time, the liquid sample in sample pool 13 is second Reflux (back flow) phenomenon can be generated at filter membrane 16, to reduce or remove the component for being adhered to the second filter membrane 16, kept away Exempt from the situation that filter membrane is blocked during being separated by filtration to occur；Then, which controls first vacuum pump 310 and stops Only run；Later, which is switched to and is connected to second vacuum pump 320, so that the frequency converter 410 controls Second vacuum pump 320 operation makes to produce in second chamber 17 (right chamber as shown in FIG. 6) by 172 pumping of the second opening Negative pressure is given birth to, as indicated by arrows in fig. 6 direction, so that the liquid and size in the liquid sample in sample pool 13 are less than the second filter membrane The component in 16 apertures passes through the second filter membrane 16 under the action of negative pressure, into second chamber 17, at the same time, in sample pool 13 Liquid sample can generate backflow phenomenon at the first filter membrane 14, to reduce or remove the group for being adhered to the first filter membrane 14 Point, avoid the situation that filter membrane is blocked during being separated by filtration from occurring；Followed by, which controls second vacuum It is out of service to pump 320；Above-mentioned steps are multiple repeatedly.Fig. 7 a is please referred to, in one embodiment, the vacuum system 30 is alternately at this The negative pressure generated in first chamber 15 and the second chamber 17 forms the rectangular pulse signal in period.The rectangular pulse signal Intensity be -70kpa, period 1min, alt time of the negative pressure between the first chamber 15 and the second chamber 17 be 0.5min.As shown in Fig. 7 b and 7c, in another embodiment, to prevent negative pressure direction sudden change to the first filter membrane 14 and Second filter membrane 16 damages, which may be replaced by periodic sinusoidal signal or filtered output.In It is more in view of protein content in plasma sample in other embodiments, it, can be at it in order to further avoid filter membrane clogging In positive pressure is generated in another chamber while generate negative pressure in a chamber, reinforce the backflow phenomenon at filter membrane.It, can in work Intensity, alt time, period and the total operating time of the negative pressure of alternating variation are accordingly adjusted according to the type of liquid sample Deng so that reflowing result is best at filter membrane.

Fig. 8 a and Fig. 8 b is please referred to, in one embodiment, which may also include a chip pad 50, the core Piece pedestal 50 is for accommodating and fixing the separating chips 10.The chip pad 50 is including a bottom plate 51 and is fixed on the bottom plate 51 On chip pedestal 52 and two fixed plates 53.It offers in the chip pedestal 52 and is matched with the separating chips 10,10 ' shapes Container 520, the separating chips 10,10 ' for being contained in wherein by the container 520.The chip pedestal 52 further includes phase To the two side walls 521 of setting, every one side wall 521 offers one from the top far from the bottom plate 51 to the direction of the bottom plate 51 and inserts Mouth 522.Therefore, when the separating chips 10,10 ' are inserted into the container 520 by operator, so that the separating chips 10,10 ' Opening link block 18 be inserted into the socket 522.As shown in Figure 8 b, the thickness of the opening link block 18 is greater than the side wall 521 thickness, thus, when the opening link block 18 is inserted into the socket 522, each opening link block 18 is far from the separating core The end of piece 10,10 ' protrudes out the socket 522.

Two fixed plates 53 are located at the opposite two sides of the chip pedestal 52.First vacuum pump 310 and this Two vacuum pumps 320 respectively include a gas conduit 330 and are sheathed on the gas conduit 330 far from first vacuum pump 310 or are somebody's turn to do One conduit of one end of the second vacuum pump 320 connects block 340.The gas conduit 330 connects in the conduit and forms a third on block 340 and open Mouth 341.Each gas conduit 330 passes through a fixed plate 53, so that the conduit on the gas conduit 330 is connect block 340 and is located at this Between fixed plate 53 and the chip pedestal 52.The gas conduit 330 be located at the fixed plate 53 and the conduit connect block 340 it Between part be arranged with a helical spring 331.When the separating chips 10,10 ' are inserted into the container 520, first opening 152 and second opening 172 connect the opening of the third on block 340 with the conduit and 341 be aligned and be connected to.At this point, due to this point Each opening link block 18 of off-chip piece 10,10 ' protrudes out the socket 522 far from the end of the separating chips 10,10 ', this is opened Mouth link block 18 pushes the conduit to connect block 340 and moves towards the fixed plate 53, to compress the helical spring 331.The spiral bullet The elastic-restoring force of spring 331 makes the separating chips 10,10 ' and the conduit connect the close contact of block 340, to prevent aspiration procedure Middle generation gas leakage.In one embodiment, which connects block 340 and is equipped with an annular in the position around third opening 341 Sealing ring 342, which can further increase the separating chips 10,10 ' and the conduit connects between block 340 Air-tightness.

As shown in Fig. 4 and Fig. 5 a, in one embodiment, which can also include a liquid collecting unit 60, The liquid collecting unit 60 is used for after liquid sample has completed separating-purifying, is received from the sample pool 13 of separating chips 10,10 ' Liquid sample after collection separation.The liquid collecting unit 60 may include sampling needle, this can be protruded into the sample pool 13 using needle inhales Liquid sample after taking separation.

Further, as shown in Figure 5 a, which can also include the first liquid locker room 350 and the second liquid Body locker room 360.The first liquid locker room 350 is set to first vacuum pump 310 and opens with the first of separating chips 10,10 ' Between mouthfuls 152, and the first liquid locker room 350 first chamber with the first vacuum pump 310 and separating chips 10,10 ' respectively 15 are connected.The second liquid locker room 360 is set to the second opening of second vacuum pump 320 and separating chips 10,10 ' Between 172, and the second liquid locker room 360 second chamber 17 with the second vacuum pump 320 and separating chips 10,10 ' respectively It is connected.First liquid locker room 350 and second liquid locker room 360 can be used as safety flack, avoid separating chips 10,10 ' In liquid enter vacuum pump, can also be used as waste liquid bottle collect every time separation after remain in the liquid in separating chips 10,10 ' Or cleaning solution.

The supplementary module 102 is for ensuring 100 stable operation of separator and improving separating-purifying effect.The auxiliary mould Block 102 includes a detector 70 and a controller 80.

The detector 70 is used to detect the liquid level in the sample pool 13 of the separating chips 10,10 '.

The controller 80 is electrically connected with the detector 70 and the frequency-variable module 40.The controller 80 is for obtaining the detection The liquid level that device 70 detects, and combine whether the default additional amount of the liquid level and liquid sample judges liquid sample Separating-purifying is completed.When judging that liquid sample has completed separating-purifying, which controls the frequency-variable module 40 It stops operation, generates negative pressure in the first chamber 15 and second chamber 17 of the separating chips 10,10 ' to stop at.Wherein, should Controller 80 can be the logical relation set being embedded on hardware or firmware (firmware), be also possible to programming language institute A series of programs being stored in memory or other firmwares write.In one embodiment, which is also used to basis The preset negative pressure state modulator frequency-variable module 40 is alternately produced corresponding negative in the first chamber 15 and second chamber 17 Pressure.The controller 80 is also used to control first control valve 220 and sample to be tested room 210 according to the default additional amount of liquid sample Lifetime, so that the liquid sample for making to meet the default additional amount flows into the sample pool 13.The controller 80 is also according to clear The default additional amount of washing lotion controls first control valve 220 and cleans the Lifetime of liquid chamber 230, thus make to meet this it is default plus The cleaning solution for entering amount flows into the sample pool 13.

The human-computer interaction module 103 is used to meet the use demand in actual separation purification process, makes the separator 100 With operability.The human-computer interaction module 103 includes a human-computer interaction interface 90.The human-computer interaction interface 90 is used for for operation Person inputs separating-purifying parameter by the input unit (such as: touch screen, keyboard, mouse) of the separator 100, that is, operation Person can preset separating-purifying parameter needed for separating-purifying process by the human-computer interaction interface 90.In an embodiment In, which includes the default additional amount of liquid sample, the default additional amount of cleaning solution and negative pressure parameter.This is negative Pressing parameter includes at least one of intensity, alt time, period and total operating time of the negative pressure etc..The controller 80 is also It is electrically connected with the human-computer interaction interface 90.To which the controller 80 can obtain the separation through the human-computer interaction interface 90 input and mention Pure parameter, and according to the separating-purifying state modulator frequency-variable module 40 or the corresponding operation of liquid supplying unit 20.

In one embodiment, which can also include a coffret 92, which is used for An external equipment (e.g., the USB flash disk, mobile phone etc. of operator) is connected, to transmit the separating-purifying parameter to the external equipment, makes to grasp Author can look back relevant separating-purifying data after the completion of each liquid sample separating-purifying.Wherein, which can be with It is USB interface or wireless interface.

Automatically the target particles in liquid sample can be carried out using separator provided by the utility model Separation, will can not be separated in sample pool by the component of filter membrane, while be become by the negative pressure in the cavity of sample pool two sides Change the Liquid Flow direction changed in sample pool, reduces the component for being adhered to filter membrane surface, avoid mistake during being separated by filtration The blocked situation of filter membrane occurs.Cost is relatively low for the separator, easy to use, significantly reduces the work of experimenter Amount.

The utility model embodiment further provides for a kind of separation control system 200, is applied to the separator 100 In.The supplementary module 102 of the separator 100 may also include a memory 82, which is stored in the storage In device 82.The separation control system 200 includes one or more program modules being made of program code.The controller 80 is used for Each program module of the separation control system 200 is loaded and executes, to realize the separating-purifying function of the separator 100 Energy.Wherein, as shown in Figure 4 and 5 b, which includes fluid path and mechanical module 202 and main control module 203.

The sample pool 13 that the fluid path and mechanical module 202 are used to control the liquid supplying unit 20 to separating chips 10,10 ' Middle injection liquid sample and cleaning solution.In one embodiment, the liquid supplying unit 20 include sample to be tested room 210, it is clear Washing lotion room 230 and the first control valve 220.The fluid path and mechanical module 202 are for controlling first control valve 220 and being somebody's turn to do to test sample This room 210 connection, so that the liquid sample in sample to be tested room 210 is provided to the sample pool 13.

The main control module 203 be used for by the frequency-variable module 40 control the vacuum system 30 respectively the separating chips 10, Negative pressure is alternately produced in 10 ' first chamber 15 and second chamber 17.In one embodiment, which includes first Vacuum pump 310 and the second vacuum pump 320, first vacuum pump 310 are connected with the first opening 152 of the separating chips 10,10 ' It connects, which is connected with the second opening 172 of the separating chips 10,10 '.The frequency-variable module 40 includes frequency conversion Device 410 and the second control valve 420 being connect with the frequency converter 410.The main control module 203 is for controlling second control valve 420 are connected to first vacuum pump 310, so that the frequency converter 410 controls first vacuum pump 310 operation, pass through the first opening 152 Pumping makes to generate negative pressure in first chamber 15.The main control module 203 be also used to control second control valve 420 switch to this The connection of two vacuum pumps 320 is made so that the frequency converter 410 controls second vacuum pump 320 operation by 172 pumping of the second opening Negative pressure is generated in second chamber 17.

In one embodiment, which further includes liquid collecting unit 60.The fluid path and mechanical module 202 are also Sample pool for after liquid sample has completed separating-purifying, controlling the liquid collecting unit 60 from separating chips 10,10 ' 13 collect the liquid sample after separation.

In one embodiment, which further includes detector 70.The detector 70 is for detecting the separating chips 10, the liquid level in 10 ' sample pool 13.The separation control system 200 further includes a drive control module 201, the driving Control module 201 combines the liquid level and liquid sample for obtaining the liquid level that the detector 70 detects Default additional amount judges whether liquid sample has completed separating-purifying.When judging that liquid sample has completed separating-purifying, The drive control module 201 sends a halt instruction to the main control module 203.The main control module 203 responds the halt instruction, and It controls the frequency-variable module 40 to stop operation, to stop in the first chamber 15 and second chamber 17 of the separating chips 10,10 ' Generate negative pressure.

In one embodiment, which is also used to obtain preset negative pressure parameter, and to the main control module 203 send one first control instruction including the preset negative pressure parameter.The main control module 203 for respond this first Control instruction, and according to described preset negative pressure state modulator frequency-variable module 40 in the first chamber 15 and second chamber 17 Inside it is alternately produced corresponding negative pressure.The drive control module 201 is also used to obtain the default additional amount of liquid sample, and to the liquid Road and mechanical module 202 send one second control instruction.The fluid path and mechanical module 202 are used to respond second control instruction, And the Lifetime of first control valve 220 and sample to be tested room 210 is controlled according to the default additional amount of the liquid sample, thus The liquid sample for meeting the default additional amount is set to flow into the sample pool 13.The drive control module 201 is also used to obtain cleaning solution Default additional amount, and send a third control instruction to the fluid path and mechanical module 202.The fluid path and mechanical module 202 are also First control valve 220 and cleaning solution are controlled for responding the third control instruction, and according to the default additional amount of the cleaning solution The Lifetime of room 230, so that the cleaning solution for meeting the default additional amount be made to flow into the sample pool 13.

The utility model embodiment further provides for a kind of method for separating target particles in liquid sample comprising as follows Step:

Step 1 provides separating chips 10,10 ' described in the utility model.

Step 2 provides liquid sample into the sample pool 13 of the separating chips 10,10 '.

In one embodiment, the liquid supplying unit 20 of separator 100 can be used that liquid-like is added to the sample pool 13 This, the sample pool 13 can be added by sample pool opening 138 in liquid sample.Protein molecular during separating-purifying in order to prevent It is adsorbed at the first filter membrane 14 and the second filter membrane 16 of the separating chips 10,10 ', it can be further into the sample pool 13 Surfactant and PBS buffer solution is added.The surfactant can be Polysorbate 20 (alias: tween 20) or polyoxyethylene polyoxypropylene (Pluronic F68), the mass concentration of the surfactant can be 5%.

Step 3 is made in first chamber 15 by the 152 suction first chamber 15 of the first opening of the separating chips 10,10 ' Generate negative pressure.

Wherein, before being aspirated, by the first of separating chips 10,10 ' the 152, second opening 172 of opening respectively with point Vacuum system 30 from device 100 is connected.In this way, vacuum system 30 makes first by 152 suction first chamber 15 of the first opening Negative pressure is generated in chamber 15.The component of liquid in liquid sample and size in sample pool 13 less than 14 aperture of the first filter membrane Under the action of negative pressure by the first filter membrane 14, into first chamber 15.In some cases, such as the volume phase of first chamber 15 To smaller, also or, the negative pressure variation in first chamber 15 is too fast, the component of liquid and size less than 14 aperture of the first filter membrane It may also be further by 152 outflow of the first opening, into the first liquid locker room 350.

More specifically, before being aspirated, if the sample pool 13 is added by sample pool opening 138 in liquid sample, It can be further by 138 closing of sample pool opening.When sample pool opening 138 is closed, in aspiration procedure, sample pool 13 In between first filter membrane 14 and second filter membrane 16 liquid flow velocity enhancing, to enhance first mistake The backflow phenomenon of filter membrane 14 or second filter membrane 16 reduces the component for being adhered to filter membrane surface, avoids being separated by filtration process The blocked situation of middle filter membrane occurs.

In other embodiments, more in view of protein content in plasma sample, it is existing in order to further avoid filter membrane blocking As step 3 can further comprise generating positive pressure in second chamber 17, reinforce the backflow phenomenon at filter membrane.

Step 4 stops suction first chamber 15.

Step 5 is made in second chamber 17 by the 172 suction second chamber 17 of the second opening of the separating chips 10,10 ' Generate negative pressure.

Wherein, vacuum system 30 makes to generate negative pressure in second chamber 17 by 172 suction second chamber 17 of the second opening. The component for being adhered to 14 surface of the first filter membrane can be with the liquid in air-flow and/or liquid stream sample pool 13, in sample pool 13 Liquid and size in sample pass through the second filter membrane 16 less than the component in 16 aperture of the second filter membrane under the action of negative pressure, enter Second chamber 17.In some cases, the volume such as second chamber 17 is relatively small, also or, the negative pressure in second chamber 17 Change it is too fast, liquid and size less than 16 aperture of the second filter membrane component may also further by 172 outflow of the second opening, Into second liquid locker room 360.It is to be appreciated that step 4 and step 5 successively can be executed successively, can also hold simultaneously Row.

In other embodiments, more in view of protein content in plasma sample, it is existing in order to further avoid filter membrane blocking As step 5 can further comprise generating positive pressure in first chamber 15, reinforce the backflow phenomenon at filter membrane.

Step 6 stops suction second chamber 17.

Then, step 3 is recyclable repeatedly to step 6, is removed the component in liquid sample less than filter membrane aperture, Component greater than filtering membrane aperture is trapped in sample pool 13, to realize better separating-purifying effect.Pass through first chamber 15, the alternating variation of negative pressure can improve the permeability of filter membrane in filter process in second chamber 17, reduce filter membrane plug-hole, Improve filter effect.

In one embodiment, after the step 6, this method can further comprise following steps:

Step 7: cleaning solution is provided into the sample pool 13 of the separating chips 10,10 '.Then, by executing step repeatedly Three clean separating chips 10,10 ' to step 6.The mass concentration of the surfactant can be 0.1%.

Separating-purifying is carried out using urine specimen of the separating chips 10 provided by the embodiment of the utility model to 10mL, The excretion body of high yield can be isolated in 30min.

In addition, also using commercially available qEV splitter (iZON Science company), Exoquick-TC excretion body kit (SBI company), MagCapture excretion body extracts kit (Wako company) and Exo-Spin excretion body purifying tubing string difference Excretion body is separated from same urine specimen, and using Particle Size Analyzer (Malvern company) to original urine specimen and The granularity for the excretion body that each separating-purifying means obtain is tested, and test result is as shown in Fig. 9 a to 9f.Wherein, it including uses The particle size range for the excretion body that various separating-purifying means including the separating chips 10,10 ' obtain is 30-150nm, is met The theoretical particle size range of excretion body.

Electronic Speculum and transmissioning electric mirror test, test result are scanned to the excretion body for using the separating chips 10,10 ' to obtain As shown in Figure 10 a and 10b.Wherein, isolated excretion body integrality with higher and purity.

Further, due to high-density lipoprotein (HDLs), low-density lipoprotein (LDLs), medium density lipoprotein (IDLs), very low density lipoprotein (VLDL) and chylomicron (chylomicrons) have and granularity similar in excretion body And density, to test the purity of excretion body obtained through various separating-purifying means, to original urine specimen and through various points The excretion body obtained from method of purification carries out proteins gel electrophoresis, after electrophoresis dying, is dyed using silver staining to albumen, Obtained band map is as shown in figure 11.Wherein, the corresponding Band signal of original urine specimen is stronger, shows original urine specimen In the albumen containing high level.After qEV splitter or Exo-Spin excretion body purifying tubing string separating-purifying, what is obtained is outer Secreting the corresponding band of body still has stronger signal, shows to adsorb in the excretion body after both separation means separating-purifyings There are a large amount of albumen.After MagCapture excretion body extracts kit or ExoQuick-TC excretion body kit separating-purifying, obtain The corresponding band intensity of the excretion body arrived weakens, and shows that a large amount of albumen have been removed in excretion body, and purification precision is higher.And it uses After separating chips 10 provided by the utility model embodiment carry out separating-purifying, the signal of the corresponding band of obtained body secretion Intensity is corresponding compared to the excretion body obtained after qEV splitter and Exo-Spin excretion body purifying tubing string separating-purifying Band signal intensity is greatly reduced, and compared to through MagCapture excretion body extracts kit and ExoQuick-TC excretion The corresponding Band signal intensity of the excretion body obtained after body kit separating-purifying is only a little to be improved.Therefore, practical using this It is unadsorbed in the isolated body secretion of separating chips 10 provided by new embodiment to have a large amount of albumen, purification precision compared with Height further demonstrates that the separating chips 10 have stronger competitiveness compared to commercially available various excretion body separation means.

Since the liquid sample of cancer patient usually has differences compared to ordinary person, for the separating chips for proving the present embodiment 10 can be equally used for the separating-purifying of the liquid sample of cancer patient, and the urine specimen for collecting 11 prostate cancer patients is each 10mL, and separating-purifying is carried out to every a urine specimen respectively using the separating chips 10 of the utility model embodiment, and adopt It carries out protein content to the excretion body after separation with micro ultraviolet-visible spectrophotometer to test, test result such as table 1 It is shown.

Table 1

Urine specimen	Protein content (mg/ml)
		1	0.275
2	0.731
		3	3.099
4	0.826
		5	0.321
6	0.165
		7	0.998
8	1.112
		9	2.21
10	0.624
		11	0.944

As known from Table 1, protein content is respectively less than 1mg/mL in the excretion body of 8 urine specimens, shows to use the separating core After piece 10 carries out separating-purifying to the liquid sample of cancer patient, obtained body secretion is equally unadsorbed a large amount of albumen, mentions Quality is higher.

Further, using immunoblotting in the excretion body obtained after 11 parts of urine specimen separating-purifyings CD81, CD9 protein marker are tested, and test result is as shown in figure 12.Wherein, at least after 7 parts of urine specimen separating-purifyings Two kinds of protein markers, the excretion body obtained after 2 parts of urine specimen separating-purifyings can be detected simultaneously by obtained excretion body In be able to detect that a kind of protein marker.It can be seen that the separating chips 10 of the utility model embodiment can be used in cancer The separating-purifying of the liquid sample of patient.

Further, it repeats to carry out 11 parts of urine specimens using the same separating chips 10 and in the same way Separating-purifying, and protein content survey is carried out to excretion body isolated every time using micro ultraviolet-visible spectrophotometer Examination finds coefficient of variation of the test result compared to table 1 less than 5%, shows that the separating chips 10 of the utility model embodiment have There is higher structural stability, and carries out separating-purifying repeatability with higher using the separating chips 10.Furthermore it uses Multiple separating chips 10 simultaneously repeat to carry out separating-purifyings to 11 parts of urine specimens in the same way, failure rate less than 5%, It further demonstrates that and carries out separating-purifying repeatability with higher using the separating chips 10.

Above-described embodiment is the preferable embodiment of the utility model, but the embodiments of the present invention is not by above-mentioned The limitation of embodiment, embodiment of above are only for interpreting the claims.The right protection scope of the utility model not office It is limited to specification.Anyone skilled in the art can think easily in the technical scope that the utility model discloses The variation or replacement arrived, are included within the protection scope of the utility model.

Claims

1. a kind of separating chips, for from liquid sample separating-purifying go out target particles, which is characterized in that the separating chips Include:

Sample pool；

First chamber, the first chamber are connected with the sample pool by first filter membrane, and the first chamber is set It is equipped with the first opening, first opening is for making the first chamber be in communication with the outside；And

Second chamber, the second chamber are connected with the sample pool by second filter membrane, and the second chamber is set Be equipped with the second opening, second opening is for making the second chamber be in communication with the outside, wherein the first chamber with it is described Second chamber is located at the opposite two sides of the sample pool.

2. separating chips as described in claim 1, which is characterized in that the sample pool includes sample pool substrate, the first inner cover Piece and the second inner coversheet, first inner coversheet and second inner coversheet two sides that be covered on the sample pool substrate respectively opposite To form the accommodating chamber for accommodating the liquid sample, first filter membrane is set in first inner coversheet, and described Two filter membranes are set in second inner coversheet, which further includes chip base, and the first chamber includes and institute State the first opposite side cover piece of the first filter membrane, the first side cover piece, first inner cover with first filter membrane Piece and the chip base are enclosed jointly to be set to form the first chamber, and first opening is set to the first side cover piece On, the second chamber includes second side cover plate opposite with second filter membrane, and second side cover plate has described the Second inner coversheet of two filter membranes and the chip base are enclosed jointly to be set to form the second chamber, second opening It is set on second side cover plate.

3. separating chips as described in claim 1, which is characterized in that the first chamber includes the first side cover piece, and described the Side cover plate is equipped with the first convex block, and the first side cover piece is divided into positioned at first convex block side by first convex block The first cover plate portion and the second cover plate portion positioned at first convex block other side, the second chamber include the second side cover Piece, second side cover plate are equipped with second convex block opposite with first convex block, and second convex block is by described second side Cover plate is divided into the third cover plate portion positioned at second convex block side and the 4th lid positioned at second convex block other side Piece portion, first cover plate portion, second cover plate portion, first convex block and second convex block enclose jointly and set to form institute State sample pool, the bottom of the first side cover piece and the position opposite with first convex block are equipped with chip base, and described first Filter membrane is set between first convex block and the chip base and opposite with second cover plate portion, second side cover The bottom of piece and the position opposite with second convex block are equipped with another chip base, and second filter membrane is set to described the It is between two convex blocks and the chip base and opposite with the 4th cover plate portion, second cover plate portion and first filter membrane It encloses jointly and sets to form the first chamber, the 4th cover plate portion and second filter membrane enclose jointly and set to form second chamber Room.

4. separating chips as claimed in claim 2 or claim 3, which is characterized in that the first side cover piece and second side cover On piece is respectively fixed with an opening link block, offers a channel in the opening link block, and the channel is respectively with described One opening and second register and connection.

5. separating chips as described in claim 1, which is characterized in that first filter membrane is with second filter membrane Anodic alumina films.

6. separating chips as described in claim 1, which is characterized in that the top of the sample pool is open including a sample pool.