CN207276608U - A kind of capture sieve for being used to capture biomolecule in cell or solution - Google Patents
A kind of capture sieve for being used to capture biomolecule in cell or solution Download PDFInfo
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- CN207276608U CN207276608U CN201720954857.6U CN201720954857U CN207276608U CN 207276608 U CN207276608 U CN 207276608U CN 201720954857 U CN201720954857 U CN 201720954857U CN 207276608 U CN207276608 U CN 207276608U
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Abstract
The utility model discloses a kind of capture sieve for being used to capture biomolecule in cell or solution.Above-mentioned capture sieve, including reticulated matrix and the trapping layer that is formed on the reticulated matrix, the trapping layer include the catches that can be specifically bound with target cell or biomolecule.The capture sifter 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
Technical field
It the utility model is related to and be used to capture target cell by the molecule expressed by target cell with a kind of（Such as circulation
Tumour cell）Or the capture of target biological molecules is sieved 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 capture sieve for being used to capture biomolecule in cell or solution, it has
High specific and high throughput, and the biomolecule being suitable in the molecule trapping cell by being expressed by cell or capture solution, especially
Suitable for capturing and sorting circulating tumor cell.
To reach above-mentioned purpose, the technical solution adopted in the utility model is：
A kind of capture sieve for being used to capture biomolecule in cell or solution, including reticulated matrix and be formed at described netted
Trapping layer on matrix, the trapping layer include the catches that can be specifically bound with target cell or biomolecule.
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 selects free antibody（Including nano antibody）, oligonucleotides（Including aptamer
（aptamer））And molecularly imprinted polymer（molecularly imprinted polymers）The group of composition.
Preferably, the catches is bonded to the reticulated matrix by physical absorption and/or chemical bonded refractory.It is highly preferred that
The catches is antibody, and the antibody is connected to using traut ' s reagents or the mercaptan molecules of salt with biotin-avidin
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 gold, stainless steel or the combination of the two.
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.The material of protective layer
Expect for AuPd.
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；It can tie
Close existing counting technology；Cell can be collected after experiment.
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 means dependent on one or more capture sieves（Fig. 1
It is shown）Middle capture circulating tumor cell（CTCs）." macroscopic view " is partly used to reduce test period at the same time by increasing capture area
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)
- A kind of 1. capture sieve for being used to capture biomolecule in cell or solution, it is characterised in that：Including reticulated matrix and formation Trapping layer on the reticulated matrix, the trapping layer include to catch with what target cell or biomolecule were specifically bound Obtain thing.
- 2. capture sieve according to claim 1, it is characterised in that：The biomolecule is in solution or is expressed by cell Protein, oligonucleotides, enzyme or combinations thereof.
- 3. capture sieve according to claim 1, it is characterised in that：The catches is by antibody, oligonucleotides and/or divides Sub- imprinted polymer.
- 4. capture sieve according to claim 3, it is characterised in that：The catches is antibody.
- 5. capture sieve according to claim 4, it is characterised in that：The biomolecule is signal transduction factor, institute It is circulating tumor cell to state target 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 of attached molecule specific binding.
- 6. capture sieve 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.
- 7. capture sieve according to claim 1, 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.
- 8. capture sieve according to claim 1, it is characterised in that：The material of the reticulated matrix is noble metal.
- 9. capture sieve according to claim 1, it is characterised in that：The reticulated matrix includes：Body of stainless steel；AndIt is formed at the protective layer on the body of stainless steel surface；Wherein, the protective layer is made of noble metal or its alloy, and the catches is connected to the protective layer.
- 10. capture sieve according to claim 9, it is characterised in that：The protective layer passes through physical coating or chemical deposition Form.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338184A (en) * | 2017-08-02 | 2017-11-10 | 苏州博福生物医药科技有限公司 | A kind of capture sieve and device for being used to capture biomolecule in cell or solution |
| 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 |
| CN111175503A (en) * | 2020-03-05 | 2020-05-19 | 上海邦先医疗科技有限公司 | Catch sieve and preparation method thereof |
| WO2022147196A3 (en) * | 2020-12-31 | 2022-08-04 | Iovance Biotherapeutics, Inc. | Devices and processes for automated production of tumor infiltrating lymphocytes |
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2017
- 2017-08-02 CN CN201720954857.6U patent/CN207276608U/en active Active
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107338184A (en) * | 2017-08-02 | 2017-11-10 | 苏州博福生物医药科技有限公司 | A kind of capture sieve and device for being used to capture biomolecule in cell or solution |
| 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 |
| EP3805354A4 (en) * | 2018-05-25 | 2022-04-13 | Hemosmart Medical Technology Ltd. | Apparatus for enriching and screening target object such as cell, bacteria or biomolecule from sample |
| CN111175503A (en) * | 2020-03-05 | 2020-05-19 | 上海邦先医疗科技有限公司 | Catch sieve and preparation method thereof |
| CN111175503B (en) * | 2020-03-05 | 2023-07-25 | 上海邦先医疗科技有限公司 | Capturing screen and preparation method thereof |
| WO2022147196A3 (en) * | 2020-12-31 | 2022-08-04 | Iovance Biotherapeutics, Inc. | Devices and processes for automated production of tumor infiltrating lymphocytes |
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Address after: Room 12, no.1798, West Zhonghua Garden Road, Yushan Town, Kunshan City, Suzhou City, Jiangsu Province Patentee after: Jiangsu Huixian Pharmaceutical Technology Co.,Ltd. Address before: 215300 Pilot Building 101, No. 168 Yuanfeng Road, Yushan Town, Kunshan City, Suzhou City, Jiangsu Province Patentee before: KUNSHAN HUIXIAN MEDICAL TECHNOLOGY Co.,Ltd. |
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