CN207276609U - A kind of device for being used to capture biomolecule in cell or solution - Google Patents

A kind of device for being used to capture biomolecule in cell or solution Download PDF

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Publication number
CN207276609U
CN207276609U CN201720954998.8U CN201720954998U CN207276609U CN 207276609 U CN207276609 U CN 207276609U CN 201720954998 U CN201720954998 U CN 201720954998U CN 207276609 U CN207276609 U CN 207276609U
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capture
cell
sieve
cavity
outlet
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查梅特·杰罗姆
颜菁
鲍尔·沃尔夫冈安德烈斯·克里斯蒂安
余子夷
杨洋
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Jiangsu Huixian Pharmaceutical Technology Co.,Ltd.
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Suzhou Bofu Biological Medicine Technology Co Ltd
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Abstract

The utility model discloses a kind of device for being used to capture biomolecule in cell or solution.Above device, including capture mechanism, the capture mechanism includes the capture sieve of one or more stackings, the trapping layer that the capture sieve includes reticulated matrix and is formed on the reticulated matrix, and the trapping layer includes the catches that can be specifically bound with target cell or biomolecule.The device of the utility model has high specific and high throughput, and the biomolecule being suitable in the molecule trapping cell by being expressed by cell or capture solution, particularly suitable for capturing and sorting circulating tumor cell.

Description

A kind of device for being used to capture biomolecule in cell or solution
Technical field
It the utility model is related to for capturing biomolecule in cell or capture solution by the molecule expressed by cell Device, it is more particularly to a kind of to be used to by the biomolecule expressed by target cell capture target cell(Such as, circulating tumor Cell)Or the capture sieve of target biological molecules and the device with this capture sieve in capture solution.
Background technology
, there are great pass in circulating tumor cell, that is, sanguimotor tumour cell, the problems such as being considered the far-end transfer with tumour System.Only 1-10 circulating tumor cell in the 10ml blood of general cancer patient, its acquisition speed is slow or poor specificity is fast Speed detection patient's blood sample problem in the urgent need to address.
For example traditional immunological magnetic bead sorting method of present method for separating is reproducible, high sensitivity, the good, Ke Yiding of specificity Amount analysis circulating tumor cell, but service speed is slow, flux is small.Although membrane micropore filtering technique and Percoll gradient centrifugation behaviour Make simply, cytoactive is preferable after separation, but specificity is low and false positive rate is high.Microflow control technique is easy to operate, required antibody Amount is few, but cost is higher, false negative rate is high.And microflow control technique company primarily now for scientific research client, it is necessary to mix in advance Plurality of reagents is closed, excessively relies on old-fashioned chip, industrialization is difficult, therefore it is difficult to enter in-vitro diagnosis field.Other such as U.S. Cell Search system sensitivities are high, specificity is high, but hematozemia amount is big, required amount of antibody is big, of high cost, can not realize work Cell capture, can not be collected circulating tumor cell and be further cultured for, therefore can only do DNA sequencing and cannot but do RNA sequencings, no Medication guide can be used as.
Utility model content
The purpose of this utility model is to provide a kind of device for being used to capture biomolecule in cell or solution, it has height Specificity and high throughput, and suitable for by the biomolecule in the molecule trapping cell expressed by cell or capture solution, especially fitting In capture and sorting circulating tumor cell.
To reach above-mentioned purpose, the technical solution adopted in the utility model is:
It is a kind of to be used to capturing the device of biomolecule in cell or solution, described device include capture mechanism and have into The main body of mouth, first outlet and the cavity between the entrance and the first outlet, the capture mechanism are fixed on institute State in cavity, the entrance, the first outlet are connected with the cavity respectively, and the capture mechanism includes at least one capture Sieve, the capture sieve trapping layer that includes reticulated matrix and be formed on the reticulated matrix, the trapping layer is including can be with Target cell or the catches of biomolecule specific binding.The main body preferably uses existing manufacturing technology such as injection molded Technology manufactures.The material of main body should compatible solvent.For example, polyether-ether-ketone meets above-mentioned condition.
Preferably, the biomolecule is protein that is in solution or being expressed by cell, oligonucleotides(DNA and/or RNA), enzyme or combinations thereof.
Preferably, the catches is antibody(Including nano antibody), oligonucleotides(Including aptamer(aptamer))Or Molecularly imprinted polymer(molecularly imprinted polymers).
Preferably, the catches physical absorption and/or chemical bonded refractory are bonded to the reticulated matrix.
It is highly preferred that the catches is antibody, the antibody is using traut ' s reagents or with biotin-avidin Mercaptan molecules of salt be connected to the reticulated matrix.
Further, the biomolecule is signal transduction factor, and the target cell is circulating tumor cell, institute State antibody for can with by circulating tumor cell surface expression signal transduction factor specific binding anti-epithelium it is thin Born of the same parents' adhesion molecule antibodies.
Preferably, the size of the reticulated matrix is 1-100 mm × 1-100 mm, and the hole of the sieve is 2 μm of -800 μ m.It is highly preferred that the size of the reticulated matrix is 2-10 mm × 2-10 mm, the hole of the sieve is 20 μm -100 μm.
Preferably, the material of the reticulated matrix is golden or other noble metals.
Preferably, the reticulated matrix includes:
Body of stainless steel;And
It is formed at the protective layer on the body of stainless steel surface;
Wherein, the protective layer is by noble metal or its alloy(Such as AuPd)It is made, the catches is connected to the protection Layer.
It is highly preferred that the protective layer passes through physical coating(Such as, magnetron sputtering)Or chemical deposition forms.
Preferably, the capture mechanism includes the capture sieve of multiple stackings.
It is highly preferred that the cavity is divided into two parts of the first cavity and the second cavity by the capture mechanism.
Further, the capture sieve is horizontally disposed, and the entrance is located at the top of the capture mechanism and with described the One cavity communicates, and the first outlet is located at the lower section of the capture mechanism and is communicated with second cavity.
Further, the entrance and the central axis of the first outlet are in the residing plane of the capture sieve.
Preferably, the main body further has a second outlet, in the main body formed with the cavity and described The microfluidic channel for being used to collect captured cell or biomolecule that second outlet communicates.
Preferably, described device further comprises being arranged on is used for captured cell or life in the microfluidic channel The counting mechanism that thing molecule is counted.
It is highly preferred that the counting mechanism includes impedance measurement electrode.
It is highly preferred that the cavity is divided into two parts of the first cavity and the second cavity by the capture mechanism, it is described Entrance is communicated with first cavity, and the microfluidic channel is communicated with second cavity..
The utility model uses above scheme, has the following advantages that compared with prior art:
It is easy to functionalization and there is higher flux;The risk of blocking is low;Higher specificity;Higher efficiency;Device It is designed to that conventional fabrication processes can be used(Such as injection molded)Manufacture;Existing counting technology can be combined;Can after experiment by Cell is collected.
Brief description of the drawings
, below will be to needed in embodiment description in order to illustrate more clearly of the technical solution of the utility model Attached drawing is briefly described, it should be apparent that, drawings in the following description are only some embodiments of the utility model, for For those of ordinary skill in the art, without creative efforts, other can also be obtained according to these attached drawings Attached drawing.
Fig. 1 is according to the present utility model a kind of for capturing circulation cancer cell(Abbreviation CTCs)Device signal Figure;
Fig. 2 is a kind of signal of the net of functionalization with captured circulating tumor cell according to the present utility model Figure;
Fig. 3 is a kind of sectional view of the net of functionalization according to the present utility model;
Fig. 4 a are the flow charts for capturing circulating tumor cell;
Fig. 4 b show a kind of typical process of the device capture circulating tumor cell using shown in Fig. 1;
Fig. 5 a and 5b are respectively that the circulating tumor for two kinds of expression target acquistion molecules for showing that captured sieve is captured is thin The photo of born of the same parents.
In above-mentioned attached drawing,
1st, main body;10th, cavity;101st, upper cavity;102nd, lower chamber;11st, entrance;12nd, first outlet;13rd, microfluid leads to Road;14th, second outlet;
2nd, capture mechanism;20th, sieve;201st, body of stainless steel;202nd, protective layer;21st, antibody;
3rd, counting mechanism;30th, electrode;
4th, circulating tumor cell;5th, blood cell.
Embodiment
The preferred embodiment to the utility model is described in detail below in conjunction with the accompanying drawings, so that the advantages of the utility model It can be easier to be understood by the person skilled in the art with feature.It should be noted that the explanation for these embodiments It is used to help understand the utility model, but does not form the restriction to the utility model.In addition, this practicality disclosed below is new As long as involved technical characteristic does not form conflict and is combined with each other each other in each embodiment of type.
Device disclosed in the utility model is combining macroscopic view/small-fluid dress dependent on the capture sieve of one or more stackings Put(Shown in Fig. 1)Middle capture circulating tumor cell(CTCs)." macroscopic view " is partly used to reduce examination at the same time by increasing capture area The time is tested to optimize capture." small " is partly used to detecting and collecting circulating tumor cell.
Fig. 1 shows one embodiment of the fluid means for capturing circulating tumor cell.The device includes:
Inside has a main body 1 of cavity 10, and with an entrance 11 and a first outlet 12 and one the Two outlets 14;
Capture mechanism 2, including at least one capture sieve 20;And
Counting mechanism;
Wherein, main body 1 is manufactured using existing manufacturing technology such as injection molded technology.The material of main body also should be compatible The material of solvent.For example, PEEK(Polyether-ether-ketone, polyetheretherketone)It can meet all conditions.Preferably, main body 1 is manufactured using injection molding and forms the cavity 10.Capture mechanism 2 is arranged in cavity 10.Entrance 11 and first outlet 12 It is located at the opposite end of cavity respectively, capture mechanism 2 is arranged in cavity 10 and between entrance 11 and first outlet 12.More Body, entrance 11 is opened in 1 upper surface of main body, and first outlet is opened in the lower surface of main body 1, that is to say, that entrance 11 is high In capture mechanism 2, first outlet 12 is less than capture mechanism 2.The mechanism 2 that is captured of cavity 10 is divided into two parts, i.e. upper cavity 101(First cavity)With lower chamber 102(Second cavity).Upper cavity 101 and lower chamber 102 all substantially cuboids, and on The section in the horizontal direction of cavity 101(Refer specifically to area)More than the section in the horizontal direction of lower chamber 102.Capture mechanism 2 include the capture sieve 20 of one or more stackings, and capture sieve 20 includes reticulated matrix and the trapping layer being formed on reticulated matrix, Trapping layer contains the catches that can be specifically bound with target cell or biomolecule.The capture sieve 20 of capture mechanism 2 is made It is standby be with catches, the catches can use any technology based on compatibility specifically with target cell or solution By the molecule of cell expression or it is expressed in molecule on cell membrane surface.In medium, especially solution(Such as undressed blood Liquid)In target cell or biomolecule upper cavity 101 can be conducted to via entrance, flow through afterwards arresting structure 2 reach cavity of resorption In body 102, and finally main body 1 is flowed out via first outlet 12.In this process, target cell or biomolecule are captured Mechanism 2 captures, and the other parts of blood by capture mechanism 2 without captured.The advantages of capture sieve is high surface area-body Product ratio, it is allowed to which the processing of a large amount of samples reduces blocking risk again at the same time.Binding specificity antibody technique have devised antibody and catch Sieve is obtained, using epitope screening, ensure that hypersensitivity, specificity, cytoactive.Once target cell or biomolecule quilt Capture, they can be released(Such as, chemistry, thermodynamics, electrical method), for further analysis.Typically, the cell quilt Count, and be collected for further analyzing afterwards.Sieve can capture any target cell or biomolecule, and these molecules Can float over solution(Biofluid is other)In or be attached on cell(In this case, it will be that capture is thin Born of the same parents).Capture mechanism 2 may include the capture sieve 20 of multiple stackings, can have between direct mutually stacked together or adjacent two layers There is interval.
In one embodiment, as shown in Fig. 2, the antibody 21 that can capture the molecule on circulating tumor cell surface is connected To each sieve 20.Preferably, antibody 21 is anti-epithelial cell adhesion molecule antibody(anti - epithelial cell Adhesion molecule antibodies, abbreviation anti-EpCAM).Anti- epithelial cell adhesion molecule antibody can with molecule, Particularly epithelial cell adhesion molecule (EpCAM) specific binding of circulating tumor cell, the circulating tumor cell so expressed It can be captured.From such as 2 as can be seen that circulating tumor cell 4 is incorporated in on the antibody 21 on sieve 20, and other blood cells 5 Then influenced from sieve 20.
The material of sieve 20 can select golden or other noble metals and be coated with the stainless steel of golden or other noble metals.It is preferred that Ground, as shown in figure 3, capture sieve 20 includes:Body of stainless steel 201 and the protective layer 202 on body of stainless steel 201.
The material of protective layer 202 is gold or billon(Such as, AuPd), and antibody 21 is connected on protective layer 202.Protection Layer 202 is the AuPd layers being deposited on using magnetron sputtering or chemical method on body of stainless steel 201.Anti- epithelial cell adhesion molecule Antibody passes through traut ' s reagents(The reagent of Te Laote)It is connected on sieve 20, the mercaptan molecules of salt with biotin-avidin can Replace traut ' s reagents.
Microfluidic channel 13 is also arranged in main body 1, it is connected with the lower chamber of cavity 10, by the circulating tumor of the capture of sieve 20 Cell can be collected after being separated by cell dissociating buffer from sieve 20 by microfluidic channel 13.The counting mechanism 3 is set Counted with the circulating tumor cell to capture in the microfluidic channel 13.Counting mechanism 3 includes impedance measurement electrode 30.The Two outlets 14 are opened in the upper surface of main body 1, are connected with microfluidic channel 13, flow out master for the circulating tumor cell for capture Body 1.
A kind of method for capturing circulating tumor cell, as shown in fig. 4 a, including step:
(I)Undressed blood is injected in described device;
(II)Undressed blood is set to flow through capture mechanism, so that CTCs is incorporated on capture mechanism;
(III)Remove it is unwanted it is uncombined may not specifically interact with the capture mechanism it is miscellaneous Thing, cell or biomolecule;
(IV)Cell dissociating buffer is injected so that CTCs is separated from capture mechanism;
(V)Collect CTCs and CTCs is counted.
As shown in Figure 4 b, specifically, step(I)In, undressed blood is injected by cavity via entrance 11 using pump In 10;Step(II)In, undressed blood pump is gone down to sieve 20 by capturing, so that CTCs is incorporated in capture sieve 20 On, the operation direction of conversion pump makes blood multipass capture sieve 20, and to improve Percentage bound, while useless blood is via first outlet 12 Outflow;Step(III)In, it is all uncombined to be removed via first outlet 12 with water or other gentle solvent washing capture sieves 20 Debris, cell or molecule;Step(IV)In, close first outlet 12 and inject cell dissociating buffer(Such as contain tryptose The buffer solution of enzyme), so as to be departed from by the CTCs of the capture of sieve 20 from sieving 20;And in step(V)In, received by microfluidic channel 13 Collect CTCs, and the CTCs flowed through is counted by the impedance measurement electrode in microfluidic channel.It is described after counting CTCs is flowed out via second outlet 14.
Example 1
Here, give a kind of preparation method for the capture sieve for being used to sort CTCs.The preparation method includes:
(I)The selection of sieve;
Gold sieve:The hole of selection gold sieve(Such as, 64 μm or 40 μm)To maximize the time of contact with tumour cell, while in advance Anti-clogging risk.The size of gold sieve used is 2 × 2mm in once testing2.According to the size that concrete operations are preferably larger.
Stainless steel and gold sieve:Select 51 μm of holes and using magnetron sputtering cladding AuPd.This sieve is less expensive and than gold High mechanical strength, therefore this sieve is more suitable for integrating in a device.
(II)Pre- functionalization;
Various cleaning method is employed before functionalization and is sieved to prepare, including autoclaving, oxygen plasma are clear Wash and be cleaned by ultrasonic in a variety of solution, including Piranha solution(piranha solutions).For example, for 64 μm The best result of gold sieve, which is included in detergent, to be ultrasonically treated 15 minutes, rinses, 15 points are ultrasonically treated in 70% ethanol solution Clock, is rinsed, high purity water 5 minutes.Using more other volumes(Such as, Piranha solution)Processing time can be shortened.
(III)Antibody;
Typically, 10 μ l antibody are taken and are freezed, prepare reaction mixture with it thereafter(That is, antibody+with EDTA PBS), enough 2 × 50 μ l(50 μ l are 2 mm of submergence2Gold sieve minimum volume).
(IV)Traut ' s reagents;
Quick freeze after purchase.The ratio of Traut ' s reagents and antibody is 4:1.Other methods, including with biotin- Alternative Traut ' the s reagents of mercaptan molecules of salt of Avidin are sieved to be connected to sieve with forming the capture.
(V)The incubation time of Traut ' s reagents with antibody;
When optimum reacting time is 1 small.The time can be shortened under suitable conditions.
(VI)The incubation of above-mentioned solution with sieve;
Test different incubation times(10 minutes to 12 it is small when between)And different condition(4 DEG C, room temperature and 37 DEG C).Most It is good at room temperature 1 it is small when(It is that small improvement can be observed in longer incubation time but is not highly significant).
Example 2
Experiment has been carried out to confirm that sieve is captured by the efficiency of the EpCAM of tumor cells expression.In this case, sieve aperture 51 μm。
Cell selects --- there is the cell of high expression level to EpCAM albumen(Such as CaCo2 and MCF7 cells)Used.
Cell growth --- grown in 37 DEG C of DMEM buffer solutions.
Cell prepares and the incubation of sieve --- CaCo2 with MCF7 cell differentiations are into 1:2.Experiment is at one rotating 37 DEG C Carried out in heating dish.After separation, cell dilutes 1 in DMEM buffer solutions:10,0.5ml are used to be incubated the capture sieve, during incubation Between for 1 it is small when.
The flushing of the cell of non-specific binding, the sieve use pure water rinsing, are incubated 2 minutes in pure water solution, so Rinsed again before microexamination afterwards.
The cell being captured using microscopic evaluation:The photo of microscope photographing sieve(Fig. 5 a and 5b).Fig. 5 a are shown Be incubated 1 it is small when after be trapped in capture sieve on expression target acquistion molecule CaCo2 cells, Fig. 5 b be incubated 1 it is small when after quilt Capture the MCF7 cells of the expression target acquistion molecule on capture sieve.It can be seen that the EpCAM due to cell from Fig. 5 a and 5b With the combination of the anti-EpCAM antibody on sieve, multiple CaCo2 and MCF7 cells by it is described sieve capture, at the same time, sieve not by Block.It should be noted that described device according to the present utility model and the method can be used in capture various concentrations solution Target biological molecules.
The capture sieve according to the present utility model allows to contact with the time on the cell in blood while reduce blocking wind Danger.By using the sieve with bigger serface, it is possible to handle a large amount of samples in a few minutes.
Above-described embodiment is only to illustrate the technical concepts and features of the utility model, is a kind of preferred embodiment, its mesh Be that person skilled in the art can understand the content of the utility model and implement according to this, this reality can not be limited with this With new protection domain.The equivalent transformation or modification that all principles according to the present utility model are made, should all cover in this practicality Within new protection domain.

Claims (10)

  1. A kind of 1. device for being used to capture biomolecule in cell or solution, it is characterised in that:Described device includes capture mechanism And the main body with entrance, first outlet and the cavity between the entrance and the first outlet, the catching machine Structure is fixed in the cavity, and the entrance, the first outlet are connected with the cavity respectively, and the capture mechanism includes one The capture sieve of a or multiple stackings, the trapping layer that the capture sieve includes reticulated matrix and is formed on the reticulated matrix, institute State the catches that trapping layer includes to specifically bind with target cell or biomolecule.
  2. 2. device according to claim 1, it is characterised in that:The catches is antibody, oligonucleotides or molecular engram Polymer.
  3. 3. the apparatus of claim 2, it is characterised in that:The biomolecule is signal transduction factor, described Target cell is circulating tumor cell, and the antibody is can be with being sticked by the epithelial cell in circulating tumor cell surface expression The anti-signal transduction factor antibody that molecular specificity combines.
  4. 4. device according to claim 1, it is characterised in that:The size of the reticulated matrix is 1-100 mm × 1-100 Mm, the hole of the sieve is 2 μm -800 μm.
  5. 5. device according to claim 4, it is characterised in that:The size of the reticulated matrix is 2-10 mm × 2-10 Mm, the hole of the sieve is 20 μm -100 μm.
  6. 6. device according to claim 1, it is characterised in that:The material of the reticulated matrix is noble metal;Or
    The reticulated matrix includes:
    Body of stainless steel;And
    The protective layer on the body of stainless steel surface is formed at, the protective layer is made of noble metal or its alloy, the catches It is connected to the protective layer.
  7. 7. according to claim 1-6 any one of them devices, it is characterised in that:The cavity is divided into by the capture mechanism The central axis of two parts of first cavity and the second cavity, the entrance and the first outlet is in residing for the capture sieve Plane.
  8. 8. device according to claim 7, it is characterised in that:The capture sieve is horizontally disposed, and the entrance is positioned at described The top of capture mechanism is simultaneously communicated with first cavity, the first outlet be located at the lower section of the capture mechanism and with it is described Second cavity communicates.
  9. 9. according to claim 1-6 any one of them devices, it is characterised in that:The main body further has second outlet, Formed with being used to collect captured cell or biology point with what is communicated with the cavity and the second outlet in the main body The microfluidic channel of son.
  10. 10. device according to claim 9, it is characterised in that:Described device further comprises leading to arranged on the microfluid The counting mechanism for being used to count captured cell or biomolecule in road.
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707577A (en) * 2018-05-25 2018-10-26 苏州博福生物医药科技有限公司 The elution process and catching method of a kind of cell or biomolecule
CN109609336A (en) * 2018-11-19 2019-04-12 昆山汇先医药技术有限公司 It is a kind of to be sieved for capturing biomolecule, cell or the capture of bacterium
CN109874316A (en) * 2018-05-25 2019-06-11 昆山汇先医药技术有限公司 For enrichment isolation the object such as cell, bacterium or the device of biomolecule from sample
WO2019223214A1 (en) * 2018-05-25 2019-11-28 昆山汇先医药技术有限公司 Apparatus for enriching and screening target object such as cell, bacteria or biomolecule from sample
CN113124650A (en) * 2019-12-31 2021-07-16 苏州博福生物医药科技有限公司 Freeze-drying method of capture screen

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108707577A (en) * 2018-05-25 2018-10-26 苏州博福生物医药科技有限公司 The elution process and catching method of a kind of cell or biomolecule
CN109874316A (en) * 2018-05-25 2019-06-11 昆山汇先医药技术有限公司 For enrichment isolation the object such as cell, bacterium or the device of biomolecule from sample
WO2019223214A1 (en) * 2018-05-25 2019-11-28 昆山汇先医药技术有限公司 Apparatus for enriching and screening target object such as cell, bacteria or biomolecule from sample
CN108707577B (en) * 2018-05-25 2023-11-10 江苏汇先医药技术有限公司 Elution method and capture method of cells or biomolecules
CN109609336A (en) * 2018-11-19 2019-04-12 昆山汇先医药技术有限公司 It is a kind of to be sieved for capturing biomolecule, cell or the capture of bacterium
CN109609336B (en) * 2018-11-19 2021-05-18 江苏汇先医药技术有限公司 A catch sieve for catching biomolecule, cell or bacterium
CN113124650A (en) * 2019-12-31 2021-07-16 苏州博福生物医药科技有限公司 Freeze-drying method of capture screen
CN113124650B (en) * 2019-12-31 2022-05-06 江苏汇先医药技术有限公司 Freeze-drying method of capture screen

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