CN106148315B - A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof - Google Patents
A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof Download PDFInfo
- Publication number
- CN106148315B CN106148315B CN201510173357.4A CN201510173357A CN106148315B CN 106148315 B CN106148315 B CN 106148315B CN 201510173357 A CN201510173357 A CN 201510173357A CN 106148315 B CN106148315 B CN 106148315B
- Authority
- CN
- China
- Prior art keywords
- ctc
- cell
- capture
- substrate
- chitin nanometer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
CTC (circulating tumor cell) capture that the invention discloses a kind of based on chitin nanometer and purifying substrate, its preparation method and application.The present invention selects the good chitosan of cell compatibility to construct 3-D nano, structure " soft " substrate, introduce anti-adhesion molecule, such as polyethylene glycol (PEG) molecule reduces non-specific adherency of the cell on interface, utilize CTC affinity capture molecule, such as signal transduction factor (EpCAM) aptamers realize the special capture of the height of CTC cell, attribute difference is then proliferated based on CTC and blood cell, the purity of target cell is further increased by way of being captured cell in-situ culture.CTC capture of the invention has good cell compatibility with purifying substrate, is able to maintain the activity of captured cell, and preparation process is simple and convenient, low in cost, and is able to achieve the Culture in situ purifying of captured cell, is also easy to scale implementation.
Description
Technical field
The present invention relates to a kind of CTC isolation and purification method, it is specifically related to a kind of CTC based on chitin nanometer and catches
It obtains and purifies substrate and the preparation method and application thereof, belong to clinical medicine CTC separation technology field.
Background technique
Circulating tumor cell (CTC) refers to the tumour cell that human peripheral blood is shedded into from source hair knurl.Circulating tumor
Cell, which enters after blood circulation, moves to the transfer that body other tissue sites cause tumour, and the tumour also referred to as spread is thin
Born of the same parents.CTC and cancer metastasis, therapeutic effect, cancer return, medication guide and prognosis are closely related, thus by as cancer metastasis
The important biomolecule marker of early diagnosis and therapy.The research of CTC is expected to illustrate cancer metastasis, medicaments insensitive and drug resistance production
Raw inherent mechanism effectively treats the individuation of cancer patient to realize.Purity is high and bioactivity are good in order to obtain
Sample, the efficient capture and isolation technics for developing CTC be extremely important.
Research currently based on the nanostructure substrate of CTC separation has become a hot fields and direction.By close
Research and development in several years, by the nanostructures material such as silicon nano-pillar, carbon nanotube, graphene, nanoparticle and nanofiber
Material is applied in the research of CTC capture substrate.However current result of study shows that 3-D nano, structure is improving CTC capture effect
While rate, the absorption to haemocyte is also increased, this can actually reduce the capture purity of CTC.And the Molecular Identification of CTC and
Functional analysis needs the CTC sample of higher purity.Hsian-Rong Tseng seminar passes through the electrospinning on micro-fluidic chip
PLGA polymer nanofiber captures CTC, and cuts technology using laser microdissection and be collected to single CTC, and carry out molecule
Biological analysis.However, the method is very time-consuming, and is difficult to operate, also it is easy to damage cell.
In addition, realizing the holding of its bioactivity after CTC is trapped on interface, then the composition of substrate interface is required to have
There is better cell compatibility.However native biopolymer better for cell compatibility, including chitosan, do not have also at present
There is the research for being applied to the capture of the CTC based on nanoparticle interface.
Summary of the invention
The main purpose of the present invention is to provide a kind of good CTC based on chitin nanometer of biocompatibility to catch
It obtains and purifies substrate, which is designed Molecular Recognization on nano-interface, by anti-adhesion molecule and affinity capture
The effect of molecule is organically combined, and can effectively inhibit cell non-specificity to capture and the specificity for improving target cell simultaneously is known
Not, so realize CTC efficient capture and purifying.
Another object of the present invention is to provide a kind of method for preparing the CTC capture and purifying substrate, this method works
Skill is simple and convenient, at low cost, and has universality to material of different nature.
Another object of the present invention is to provide the purposes of the CTC capture and purifying substrate.
For realization aforementioned invention purpose, the technical solution adopted by the present invention includes:
A kind of CTC capture based on chitin nanometer and purifying substrate, including partial size 100-1000nm chitosan nano
Rice corpuscles, the CTC for being connected to the anti-adhesion molecule on chitin nanometer surface and connecting with the anti-adhesion molecule is affine
Capture molecule.
Further, CTC capture and purifying substrate include the main plural chitosan nano by being distributed in substrate surface
The molecular 3-D nano, structure of the grain of rice.
Wherein, the chitin nanometer surface of use has good cell compatibility.
Further, the anti-adhesion molecule is intended to reduce non-specific adherency of the cell on interface, may include various
Polyethylene glycol (PEG) molecule of functionalization, also that is, having the peg molecule of functional group, the functional group includes ammonia
Base and/or carboxyl, and it is without being limited thereto.
Among a preferred embodiment, NH is can be used in the anti-adhesion molecule2-PEG-COOH。
Further, the CTC affinity capture molecule may include to the special capture of height for realizing CTC cell
The various decorations products of EpCAM aptamers (DNA molecular) or EpCAM aptamers, such as DNA-NH2, and it is without being limited thereto.
A kind of CTC capture and the preparation method of purifying substrate based on chitin nanometer comprising:
(1) low-molecular weight chitoglycan that molecular weight is 40000~50000Da of Mw is dissolved in Organic Acid System and is formed
Mutually transparent chitosan solution；
(2) by electrostatic spinning apparatus by the chitosan solution by EFI in a manner of be applied to substrate surface, it is poly- to form shell
Sugared nanoparticle；
(3) in the chitin nanometer surface scion grafting anti-adhesion molecule；
(4) CTC affinity capture molecule is coupled with the anti-adhesion molecule for being connected to the chitin nanometer surface, is obtained
Obtain the CTC capture and purifying substrate.
Among one more preferred embodiment, step (1) includes: that Organic Acid System is added in low-molecular weight chitoglycan
In, after stirring 4-8h at room temperature, homogeneous phase transparent chitosan solution is obtained, the organic acid soln includes that concentration is 60v/v%-
The acetic acid or formic acid solution of 90v/v%.
Among one more preferred embodiment, step (2) includes: by step (1) obtained chitosan solution in electrostatic
Chitin nanometer is made in substrate surface in EFI 0.1-4h in device for spinning.
Wherein, the substrate can be sheet glass, monocrystalline silicon piece, metal aluminium flake, organic film, metal etc., and be not limited to
This.
Among one more preferred embodiment, step (3) includes:
By step (2) obtained chitin nanometer in the mixed solution of ammonium hydroxide and methanol (for example, the body of ammonium hydroxide and methanol
Product is than standing 1-4h for 4:96) is middle, then more than 40-60 DEG C of vacuum drying for 24 hours；
And the chitin nanometer is placed in the glutaraldehyde solution that concentration is 2.5v/v% and reacts 2-4h, so
12-24h is impregnated in the sodium cyanoborohydride solution that concentration is 0.1wt%-1wt% afterwards, is later 0.1-10mg/mL with concentration
NH2- PEG-COOH solution reaction 8-24h, thus in the chitin nanometer surface scion grafting PEG molecule.
Among one more preferred embodiment, step (4) includes: in neutral conditions, to make CTC affinity capture molecule
Under the action of the coupling reagent with activated carboxyl, there is the chitin nanometer of PEG molecule to be coupled with surface scion grafting.
Further, step (4) includes:
In neutral conditions, make EpCAM aptamers in coupling reagent EDC and NHS with activated carboxyl (for example, wherein
The concentration of EDC is 0.2mol/L, and the concentration of NHS is 0.05mol/L) under the action of, with NH2The chitosan of-PEG-COOH grafting
Nanoparticle coupling；
And the chitin nanometer is placed in the ethanolamine solutions that concentration is 0.1-1M and reacts 10-60min,
To close the NHS group not reacted completely, the CTC capture and purifying substrate are obtained.
Among one more specifically case study on implementation, a kind of CTC capture based on chitin nanometer and purifying substrate
Preparation method may include steps of:
A) using low-molecular weight chitoglycan in Organic Acid System, after stirring 4-8h at room temperature, homogeneous phase transparent shell is obtained
Glycan solution；
B) chitosan solution in step a is subjected in electrostatic spinning apparatus EFI 0.1-4h, shell is made in substrate surface
Glycan nanoparticle；
C) chitin nanometer for being distributed in substrate surface for being obtained step b is placed in the mixed solution of ammonium hydroxide and methanol
In static 1-4h, and be placed on 40-60 DEG C of drying of vacuum oven for 24 hours；
D) the chitin nanometer surface in step c is placed in the glutaraldehyde solution that concentration is 2.5v/v% and reacts 2-
4h is then immersed in 12-24h in the sodium cyanoborohydride solution that concentration is 0.1wt%-1wt%, is then 0.1- with concentration
The NH of 10mg/mL2PEG molecule is grafted to chitosan surface by-PEG-COOH solution reaction 8-24h；
E) in neutral conditions, make 0.1-10 μM of concentration of aptamers DNA-NH2In the coupling reagent example of activated carboxyl
Under the action of EDC and NHS, with NH2The chitosan of-PEG-COOH grafting is coupled；
F) the acquisition chitin nanometer surface in step e is placed in the ethanolamine solutions that concentration is 0.1-1M and is reacted
10-60min obtains the substrate to close the NHS group not reacted completely.
Any CTC capture based on chitin nanometer above-mentioned is with purifying substrate in CTC is captured and purified
Using.
For example, one such application scheme may is that a kind of device, it includes above-mentioned any based on chitosan nano
The CTC capture of rice corpuscles and purifying substrate.
In another example one such application scheme may is that a kind of CTC purification process, comprising: captured CTC exists
The CTC capture and Culture in situ in purifying substrate.
It wherein, can be into one by the cell captured progress Culture in situ because of the proliferation attribute difference of CTC and blood cell
Step improves the purity of target cell." Culture in situ " i.e.: the cell after capture is " winning during the substrate follow-up cultivation
It is bad to eliminate ", in other words: target cell (CTC) guarantees activity and continues to multiply, and haemocyte will gradually lose activity and be eliminated.
Compared with prior art, the present invention at least has the following beneficial effects:
1) the CTC capture based on chitin nanometer can provide cell compatibility good " soft " three with purifying substrate
Tie up nano-interface, can preferably with cell nano structure matching and effect；
2) with purifying substrate on the basis of modifying special capture molecule, introducing anti-adhesion molecule can be protected for CTC capture
The non-specific adherency of non-target cell is reduced under the premise of demonstrate,proving efficient capture target cell；
3) CTC capture has good biological activity with purifying the captured CTC of substrate, can carry out the original position of cell at interface
Being further purified for CTC is realized in culture.Culture in situ can improve the quantity of CTC with massive amplification CTC, this reflects to the molecule of CTC
It is fixed to wait researchs very helpful.
4) preparation method of the invention is utilized, the uniform nanoparticle surface of different-grain diameter can be prepared, it is simple and convenient,
It is at low cost, it is practical.
5) preparation method of the present invention, the preparation method of the electrostatic spinning nano particle is to material of different nature
There is universality.
6) preparation method of the present invention can be prepared on different substrate materials, including sheet glass, monocrystalline silicon piece, metal aluminium flake,
Organic film, metal surface etc..
Detailed description of the invention
Technical solution of the present invention is further elaborated below in conjunction with the drawings and specific embodiments.
Fig. 1 is the CTC capture and purifying base among one embodiment of this invention using a kind of based on chitin nanometer
Bottom carries out the schematic diagram of CTC capture and purifying；
Fig. 2 is in the embodiment of the present invention 1 with the chitin nanometer surface of the different nanometer particle sizes of electro-blowing processes preparation
SEM photograph；
Fig. 3 is the schematic diagram for carrying out modifying interface in the embodiment of the present invention 1 to chitin nanometer substrate；
Fig. 4 is that the fluorescence photo of tri- kinds of substrate A, B, C different interface capture cells is utilized in the embodiment of the present invention 1；
Fig. 5 a- Fig. 5 c is the capture rate comparison diagram of the substrate of different-grain diameter and modifying interface in the embodiment of the present invention 1；
Fig. 6 is investigation of the chitin nanometer P4 substrate to different cell strains capture specificity in the embodiment of the present invention 1
Result figure；
Fig. 7 be in the embodiment of the present invention 1 chitin nanometer P4 substrate to the capture row of different proportion cell mixing strain
For investigation result figure；
Fig. 8 a be in the embodiment of the present invention 1 chitin nanometer P4 substrate to having captured target cell/leucocyte by 104/
The investigation figure of Culture in situ purification effect after 106 ratio hybrid acquisitions；
Fig. 8 b be in the embodiment of the present invention 1 chitin nanometer P4 substrate to having captured target cell/leucocyte by 10/
The investigation figure of Culture in situ purification effect after 106 ratio hybrid acquisitions.
Specific embodiment
Above scheme is described further below in conjunction with specific embodiment.It should be understood that these embodiments are for illustrating
The present invention and be not limited to limit the scope of the invention.Implementation condition used in the examples can be done into one according to actual needs
Successive step, the implementation condition being not specified are usually the condition in routine experiment.
In view of the deficiencies in the prior art, inventor is studied for a long period of time and is practiced, and proposes technical solution of the present invention,
That is, providing a kind of circulating tumor cell (CTC) capture based on chitin nanometer and purifying substrate and preparation method thereof.
Specifically, one aspect of the present invention includes: by selecting the good chitosan of cell compatibility to construct three-dimensional
Nanostructure " soft " substrate, and non-specific adherency of the anti-adhesion molecule reduction cell on interface is introduced, and, utilize CTC parent
The special capture of height of CTC cell is realized with capture molecule；Attribute difference is then proliferated based on CTC and blood cell, by being caught
The mode for obtaining cell in-situ culture further increases the purity of target cell.
Another aspect of the present invention includes: uniform using the method construction structure of EFI low-molecular weight chitoglycan weak solution
Chitin nanometer shell is connected to by chemical reaction then by anti-adhesion molecule, such as polyethylene glycol (PEG) molecule
Glycan nanoparticle surface, then by affinity capture molecule, such as signal transduction factor (EpCAM) aptamers, with PEG points
Son combines, and obtains the substrate.
CTC capture provided by the invention has good cell compatibility with purifying substrate, is able to maintain surface attached cell
Activity, and prepare simple economy, and be able to achieve the Culture in situ purifying of captured CTC cell.
Embodiment 1 should be captured based on the CTC of chitin nanometer with the specific steps of the preparation method of purifying substrate such as
Under:
A) using low-molecular weight chitoglycan in Organic Acid System, after stirring 4-8h at room temperature, homogeneous phase transparent shell is obtained
Glycan solution.
B) chitosan solution in step a is subjected to EFI 0.1-4h in electrostatic spinning apparatus, in glass baseplate surface system
Obtain chitin nanometer.Different nanometer particle sizes and density is made by adjusting EFI fluidity matter, EFI parameter and EFI time
Chitin nanometer surface P1, P2, P3, P4, P5.Obtained different chitin nanometer surface texture patterns are shown in figure
2。
C) the chitin nanometer surface in step b is placed in ammonia hydroxide/methanol (volume ratio of ammonium hydroxide and methanol is
Static 1-4h in 4:96), and it is placed on 40-60 DEG C of drying of vacuum oven for 24 hours.Obtain unmodified chitin nanometer
Substrate A, i.e. bare chitin nanometer substrate.
D) the chitin nanometer surface in step c is placed in the glutaraldehyde solution of 2.5v/v% and reacts 2-4h, so
Afterwards immerse 0.1wt%-1wt% sodium cyanoborohydride solution in 12-24h, then with the NH of 0.1-10mg/mL2-PEG-COOH
Reaction solution acts on 8-24h, and PEG molecule is grafted to chitosan surface.The chitin nanometer substrate B of PEG modification is obtained, i.e.,
PEG chitin nanometer substrate.
E) in neutral conditions, make 0.1-10 μM of aptamers DNA-NH2In the coupling reagent EDC/NHS of activated carboxyl
Under the action of (wherein the concentration of EDC is 0.2mol/L, and the concentration of NHS is 0.05mol/L), with NH2The shell of-PEG-COOH grafting
Glycan is coupled.
F) the acquisition chitin nanometer surface in step e is placed in the ethanolamine solutions of 0.1-1M and reacts 10-60
Minute, to close the NHS group not reacted completely.Obtain chitin nanometer the substrate C, i.e. PEG- of aptamers modification
Aptamer (PEG-DNA) chitin nanometer substrate.It is as shown in Figure 3 that it modifies flow chart.
Embodiment 2 has investigated nanostructured surface P4 using the breast cancer cell line mcf-7 of the EpCAM positive as model cell
To the capturing behavior of cancer cell.Fig. 4 is the different capturing behaviors of the surface P4 to MCF-7 cell at 3 kinds of different modifying interfaces.Through
Surface (Fig. 4, a)) the cell capture amount of PEG-aptamer modification is maximum, while the surface (Fig. 4, c) of PEG modification) cell adherence
Amount is minimum.
Embodiment 3 has systematically investigated different micro-nanos using the breast cancer cell line mcf-7 of the EpCAM positive as model cell
Capture rate and specificity of the rice body structure surface to cancer cell.Meanwhile every kind of nanostructured surface has been constructed and has not been done any repair
The interface bare of decorations and the interface for only doing PEG modification are used as control.It will cultivate two days and the good MCF-7 of growth conditions thin
Born of the same parents digest removing using 0.25% pancreatin, then discard trypsin solution, and fresh culture solution is added and blows and beats cell uniformly,
Count cell, adjustment cell suspension to 105/ml.The nanometer substrate for having modified completion is placed in 24 orifice plates, is infused into each hole
Enter the configured cell suspension of 1ml, after being incubated for 10-60min in cell incubator, is cleaned 3-5 times using PBS, utilize fluorescence
The cell that micro- sem observation captures, and count.The experimental results showed that though cell adherence amount slightly has on the surface of PEG modification
Increase but obvious suppressed, i.e. the introducing of PEG can effectively reduce the non-specific adhesion (Fig. 5 a) of cell.In order to more
The cell capture behavior on each surface is accurately evaluated, Fig. 5 b, 5c summarize the surface different structure PEG-aptamer and PEG respectively
The ratio of the ratio of superficial cell capture rate and the surface different structure PEG and bare superficial cell capture rate, with more careful standard
The specific quantity of the catch and non-specific adhesion situation of true each superficial cell of analysis.P4 table is had finally chosen by being comprehensively compared
Structural model of the face as following cell capture substrate both ensure that the efficient capture of cell while inhibit as far as possible thin
The non-specific adhesion of born of the same parents.
It is above-mentioned the experimental results showed that, by regulate and control substrate surface nano-structure and interfacial property it is available have efficiently
The 3D nanostructure substrate of acquisition performance.
The specificity of the test aptamers of embodiment 4
Selected other 2 kinds of EpCAM- positive cell strain PC-3 and Sk-Br-3,2 kinds of EpCAM- negative cells strain 293T and
CCRM-CEM and freshly extd leucocyte have carried out cell capture experiment.The assessment result of the capture specificity of aptamers is converged
Always see Fig. 6) the experimental results showed that, aptamers modification chitin nanometer surface to EpCAM- positive cell strain all have compared with
High capture rate and it is lower to the capture rate of the strain of EpCAM- negative cells and human leucocyte.
Substrate described in embodiment 5 captures specificity to cell mixing strain and investigates
Prepare leucocyte solution first: acquisition venous blood 2ml, which injects in the test tube for filling 0.1ml heparin, to be mixed, be added etc.
The PBS equimultiple dilute blood of volume.It draws lymphocytes separating solution 2ml to be added in centrifuge tube, then by dilute blood carefully along pipe
Wall adds on layering liquid, it should be noted that keeps the two interface clear.Room temperature 2000r/min is centrifuged 20min.Four layers can be divided into pipe,
Linen mononuclearcell is gently sucked out with capillary syring, is added in another centrifuge tube containing 5ml PBS, mixes.
At room temperature, 10min is centrifuged with 1500rpm, can remove the blood platelet mixed.Supernatant is abandoned, repeated washing is primary.With complete RPMI-
16401ml constant volume counts cell, adjustment cell suspension to 106/ml。
It is thin that the MCF-7 that 5,10,100,1000,10000 have been dyed with DIO in advance is added into every milliliter of leucocyte solution
It is stand-by that born of the same parents are prepared into cell mixing sample.The slide modified is mounted in 4 well culture plates, 1ml is added in every hole and prepares
Cell suspension, be placed in 37 DEG C, 5%CO2Under the conditions of cultivate 30-45min after, cleaned 2-5 times with PBS.Utilize fluorescence microscope
The cell captured is observed, and is counted.
Chitin nanometer surface is fitted by the capturing behavior of the artificial mixing samples of different proportion target cell as the result is shown
Ligand modified chitin nanometer surface has high specificity to the capture of target cell.Especially when target cell content pole
When low, which is up to 90% to the capture rate of MCF-7 cell, and to the capture rate of leucocyte less than 2%.Experimental result is shown in
Fig. 7.
Embodiment 6 has captured the Culture in situ of cell mixing strain
It is 10 to target cell amount4(1mL contains 106A leucocyte) solution sample carried out capture and Culture in situ (Fig. 8 a).
The results show that the target cell purity after capture is about 35% ± 5%.Through Culture in situ two days later we can observe that,
The leucocyte on chitin nanometer surface gradually loses activity by " superseded ", while target cell starts to be proliferated.After 4 days, target
The purity of cell is up to 99% or more, realizes the proliferation purifying of target cell.
The capture and Culture in situ behavior of the cell mixing sample of (5-10) few to target cell population are investigated
(Fig. 8 b).When target cell injection rate is 7,7 cells are all captured and keep activity.After 2 days, leucocyte starts to lose work
Property is gradually eliminated, and target cell starts to be proliferated simultaneously, and target cell population reaches 200 or so after 7 days, and purity reaches 90%.
It is summarized, it is (that is, above-mentioned that the present invention has constructed the chitin nanometer surface with good cell compatibility
3-D nano, structure), surface cell capture specificity with higher and sensitivity, and by the original position for having captured cell
The proliferation purifying of target cell is realized in culture.
The foregoing examples are merely illustrative of the technical concept and features of the invention, its object is to allow the person skilled in the art to be
It cans understand the content of the present invention and implement it accordingly, it is not intended to limit the scope of the present invention.It is all smart according to the present invention
The equivalent transformation or modification that refreshing essence is done, should be covered by the protection scope of the present invention.
Claims (8)
1. a kind of product that the CTC capture based on chitin nanometer is captured and purified for CTC in preparation with purifying substrate
In purposes, which is characterized in that the substrate includes:
The 3-D nano, structure that plural chitin nanometer by being distributed in substrate surface forms, the chitin nanometer
Structure is uniform and partial size is 100-1000nm,
It is connected to the anti-adhesion molecule on chitin nanometer surface, and
The CTC affinity capture molecule being connect with the anti-adhesion molecule.
2. purposes according to claim 1, it is characterised in that: the anti-adhesion molecule includes the poly- second with functional group
Glycol molecules, the functional group include amino and/or carboxyl.
3. purposes according to claim 1, it is characterised in that: the CTC affinity capture molecule include EpCAM aptamers or
The modification product of EpCAM aptamers.
4. purposes according to claim 1, which is characterized in that it is described based on chitin nanometer CTC capture with it is pure
Change substrate preparation method include:
(1) low-molecular weight chitoglycan that molecular weight is 40000~50000Da of Mw is dissolved in Organic Acid System formed it is homogeneous saturating
Bright chitosan solution；
(2) by electrostatic spinning apparatus by the chitosan solution by EFI in a manner of be applied to substrate surface, form chitosan nano
Rice corpuscles；
(3) in the chitin nanometer surface scion grafting anti-adhesion molecule；
(4) CTC affinity capture molecule is coupled with the anti-adhesion molecule for being connected to the chitin nanometer surface, obtains institute
State CTC capture and purifying substrate.
5. purposes according to claim 4, which is characterized in that the step (1) includes: to add low-molecular weight chitoglycan
Enter in Organic Acid System, after stirring 4-8h at room temperature, obtains homogeneous phase transparent chitosan solution, the organic acid soln includes dense
Degree is the acetic acid or formic acid solution of 60v/v%-90v/v%.
6. purposes according to claim 4, which is characterized in that the step (3) includes:
Step (2) obtained chitin nanometer is stood into 1-4h in the mixed solution of ammonium hydroxide and methanol, then in 40-60 DEG C
It is more than vacuum drying for 24 hours；
And by the chitin nanometer be placed in concentration be 2.5v/v% glutaraldehyde solution in react 2-4h, then in
12-24h is impregnated in the sodium cyanoborohydride solution that concentration is 0.1wt%-1wt%, is later 0.1-10mg/mL's with concentration
NH2- PEG-COOH solution reaction 8-24h, thus in the chitin nanometer surface scion grafting PEG molecule.
7. purposes according to claim 4, which is characterized in that the step (4) includes: in neutral conditions, to make CTC
Affinity capture molecule has the chitosan nano of PEG molecule with surface scion grafting under the action of the coupling reagent with activated carboxyl
Particle is coupled.
8. purposes according to claim 4, which is characterized in that the step (4) includes:
In neutral conditions, make EpCAM aptamers under the action of the coupling reagent EDC and NHS with activated carboxyl, with NH2-
The chitin nanometer coupling of PEG-COOH grafting；
And the chitin nanometer is placed in the ethanolamine solutions that concentration is 0.1-1M and reacts 10-60min, with envelope
The NHS group not reacted completely is closed, the CTC capture and purifying substrate are obtained.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510173357.4A CN106148315B (en) | 2015-04-14 | 2015-04-14 | A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510173357.4A CN106148315B (en) | 2015-04-14 | 2015-04-14 | A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106148315A CN106148315A (en) | 2016-11-23 |
| CN106148315B true CN106148315B (en) | 2019-02-01 |
Family
ID=57335894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510173357.4A Active CN106148315B (en) | 2015-04-14 | 2015-04-14 | A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106148315B (en) |
Families Citing this family (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108330054B (en) * | 2017-01-20 | 2020-12-08 | 北京赛特超润界面科技有限公司 | Graphene chip for specific capture of circulating tumor cells in whole blood and preparation method and application thereof |
| CN111225608B (en) * | 2017-06-28 | 2023-09-15 | 魏秉庆 | Carbon nanotube sponge capturing circulating tumor cells |
| CN108102881A (en) * | 2018-02-05 | 2018-06-01 | 中国科学院苏州纳米技术与纳米仿生研究所 | The solid-phase purified extracting method of micro-fluidic chip and nucleic acid with micro structure array |
| CN108841774B (en) * | 2018-05-02 | 2021-11-26 | 南开大学 | Preparation of double-response type nano bionic interface and application of double-response type nano bionic interface in cell capture and on-demand lossless release |
| CN108866063A (en) * | 2018-06-28 | 2018-11-23 | 中国水产科学研究院珠江水产研究所 | A kind of aptamer and its preparation method and application of PEG modification |
| CN111334426A (en) * | 2018-12-18 | 2020-06-26 | 中国科学院苏州纳米技术与纳米仿生研究所 | PLGA nano-fiber CTCs capturing substrate, preparation method and application thereof |
| CN111424013B (en) * | 2019-01-10 | 2023-08-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | CTC capturing and separating substrate based on polystyrene nanospheres and preparation method thereof |
| CN112300994B (en) * | 2019-08-02 | 2024-01-12 | 中国科学院苏州纳米技术与纳米仿生研究所 | Nanometer magnetic bead for capturing circulating tumor cells and preparation method and application thereof |
| CN112300995B (en) * | 2019-08-02 | 2024-01-30 | 中国科学院苏州纳米技术与纳米仿生研究所 | Substrate material for capturing circulating tumor cells and preparation method and application thereof |
| CN112394167A (en) * | 2019-08-19 | 2021-02-23 | 中国科学院苏州纳米技术与纳米仿生研究所 | Fluorescent nano magnetic bead for capturing and identifying CTCs (biological chemical centers), and preparation method and application thereof |
| CN113730332B (en) * | 2021-08-02 | 2022-12-30 | 深圳市众循精准医学研究院 | Promoter targeting tumor surface collagen, and preparation method and application thereof |
| CN114478828B (en) * | 2021-12-08 | 2023-03-31 | 深圳先进技术研究院 | Detection material, detector and detection method for circulating tumor cells |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN103235124A (en) * | 2013-04-22 | 2013-08-07 | 复旦大学 | Nitrocellulose-membrane-based chip for conveniently capturing cancer cells |
| CN103998932A (en) * | 2011-06-29 | 2014-08-20 | 中央研究院 | Capture, purification and release of biological substance using a surface coating |
| CN104353082A (en) * | 2014-11-13 | 2015-02-18 | 福州大学 | Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells |
-
2015
- 2015-04-14 CN CN201510173357.4A patent/CN106148315B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102405411A (en) * | 2009-03-18 | 2012-04-04 | 加利福尼亚大学董事会 | Device for capturing circulating cells |
| CN103998932A (en) * | 2011-06-29 | 2014-08-20 | 中央研究院 | Capture, purification and release of biological substance using a surface coating |
| CN103235124A (en) * | 2013-04-22 | 2013-08-07 | 复旦大学 | Nitrocellulose-membrane-based chip for conveniently capturing cancer cells |
| CN104353082A (en) * | 2014-11-13 | 2015-02-18 | 福州大学 | Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells |
Non-Patent Citations (2)
| Title |
|---|
| Boran Cheng 等.Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture.《International Journal of Nanomedicine》.2014,(第9期),第2569-2580页. * |
| Transparent, biocompatible nanostructured surfaces for cancer cell capture and culture;Boran Cheng 等;《International Journal of Nanomedicine》;20140522(第9期);第2569-2580页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106148315A (en) | 2016-11-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106148315B (en) | A kind of CTC capture based on chitin nanometer and purifying substrate and preparation method thereof | |
| Wang et al. | A microfluidic chip integrated with hyaluronic acid-functionalized electrospun chitosan nanofibers for specific capture and nondestructive release of CD44-overexpressing circulating tumor cells | |
| CN107354134A (en) | Target cell capture substrate of nanometer stick array and its preparation method and application | |
| CN104651315A (en) | Method for sorting tumor cells in microfluidic chip by simultaneously utilizing specific antigen-antibody recognition and cell size difference | |
| CN105078890B (en) | A kind of preparation method of the multi-layer biological base vesica of releasable insulin | |
| CN109142712B (en) | The preparation method of dendritic nano-tube array, the method for tumor cell and for capturing and the microfluidic devices of regulation cancer cell in situ | |
| CN104353082A (en) | Functional nano material drug delivery system for identifying, capturing and restraining circulating tumor cells | |
| CN103275934A (en) | Separation method of micro circulating tumor cells | |
| CN103611192B (en) | Partially reduced graphene oxide-silk fibroin composite film, and preparation method and application thereof | |
| CN111569092B (en) | Preparation method of drug-loaded ultrasonic contrast agent with macrophages as carriers | |
| CN100560137C (en) | Bacillus nanometer magnetosome of a kind of carrier band primary amino medicine and preparation method thereof | |
| CN102532580A (en) | Method for preparing multifunctional nano-carrier | |
| CN1850058A (en) | Alginate nano capsule and its preparing method | |
| CN103741467A (en) | Preparation method for hyaluronic acid functionalized nano fibers applied to capturing cancer cells in targeted manner | |
| CN104857521A (en) | Preparation method of bio-based macromolecular vesicles with glucose response | |
| CN105259096A (en) | Zinc phosphate nano-substrate, preparation method thereof and application of zinc phosphate nano-substrate in capture and release of CTCs (circulating tumor cells) | |
| CN106511318B (en) | Preparation method with the nano-composite fiber film for carrying medicine and killing cancer cell | |
| CN103962112B (en) | Preparation method for the photoresponse intelligent gel microsphere of three-dimensional cell cultivation | |
| CN110907416A (en) | Circulating tumor cell detection device based on hollow nano needle tube electroporation system and detection method thereof | |
| CN105854966A (en) | Microfluidic chip embedding functional nanofiber membrane and application of microfluidic chip | |
| CN105999290A (en) | Phosphatidylserine-modified curcumin nanoparticles | |
| CN109045304A (en) | A kind of kernel targeted nano carrier and its preparation method and application carrying Polymerase I inhibitor | |
| CN104857502A (en) | Vascular endothelial growth factor loaded nano controlled-release compound and a preparation method thereof | |
| CN105497903B (en) | A kind of drug delivery carrier of double control and preparation method thereof based on inorganic nano silicon materials | |
| CN110403915A (en) | DNA and the hybridized nucleic acids pharmaceutical carrier of polymer and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |