CN108531455A - Polyphenol coating for circulating tumor cell capture - Google Patents
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Abstract
The present invention provides a kind of polyphenol coatings for circulating tumor cell capture.The present invention is modified polyphenol compound to material surface using the method that surface is modified, and polyphenol coating being capable of fast and effeciently enrichment cycles tumour cell.Circulating tumor cell capture technique provided by the present invention, it can be achieved that the specific capture of different cancer cell-types, and without introducing micro-nano matrix, has advantage of low cost, easy to operate without immobilized specific cancer cell identification antibody or aptamer.Prepared polyphenol coating can be used for capturing the circulating tumor cell in peripheral blood, be expected to be used for the early diagnosis of cancer patient.
Description
Technical field
The invention belongs to Biofunctional materials and technical field of analysis and detection, more particularly to swollen in material surface capture cycle
The technology of oncocyte.
Background technology
Cancer is current one of the disease for threatening human health most serious, and the invasion and transfer of tumour are cancer patient's clinics
The main reason for lethal, finds early with treatment to be to improve the most effective means of cancer patient's survival rate.Circulating tumor cell
(Circulating Tumor Cells, CTCs) be in the formation of tumour or growth course from situ tumor lesion be detached from into
Enter the cell of human peripheral circulatory system.Studies have shown that in blood the generation of the quantity of CTCs and Change of types and cancer and
Develop closely related, is one of most potential tumour non-invasive diagnosis and real-time curative effect monitoring means.Therefore development high sensitivity,
The CTCs catching methods of high specific, to realizing that the individualized treatment of cancer is of great significance.
With the development of micro-fluidic chip and micro-nano technology of preparing, CTCs capture techniques obtained in recent years it is larger into
Step, this is based primarily upon tumour cell and the difference in normal cell physical property and biochemical property in blood.Wherein, it is based on
The separation method of physical property mainly utilizes cancer cell and normal plasma cell thin to cancer in the difference of size, density etc.
Born of the same parents detach；Separating trap method based on biochemical property is based primarily upon the principle of Immune discrimination separation, can specific aim separation
The cancer cell of specific antigen is expressed, therefore there is higher specificity.At present mainly with epithelial cell adhesion molecule
(Epithelial cell adhesion molecule, EpCAM) antibody or aptamer are identification molecule, magnetic Nano
Particle, micro-nano matrix or micro-fluidic chip are capture of the carrier realization to CTCs.Document Small 2015,11,3850,
Angew.Chem.Int.Ed.2016,55,1252 and Chem.Soc.Rev., 2017,46,4245, which review CTCs in recent years, catches
The methods and techniques obtained.Although these methods all have certain CTCs capture effects, various deficiencies, such as material system are also deposited
Standby process is complex, and some capture rates are not high or enrichment time is longer etc..Further, since CTCs can occur between epithelial cell
Qualitative change, and be not that each tumour cell all expresses epithelium mark, therefore uses single EpCAM antibody captures CTCs often
Cause false negative result.In addition the production prices of antibody are expensive, can be led again using antibody combined use of a variety of Specific markers
Cause the increase of cost；And the stability of aptamer is insufficient, is easily degraded by nuclease.Chinese patent
Also distinguish in CN201210382382.X, CN201410153844.X, CN201510263669.4 and CN201710014061.7
The method or technique of capture CTCs is disclosed, but there is also above-mentioned similar defects.
Polyphenol compound refers to that one group of chemical substance is referred to as in plant, gains the name, is present in because containing multiple phenolic hydroxyl groups
In many common fruits and vegetables, derive from a wealth of sources, it is of low cost.Polyphenol compound is had not yet to see for CTCs captures
Similar report.
Invention content
The purpose of the present invention is to provide a kind of materials and preparation method thereof for circulating tumor cell capture.The material
Including polyphenol compound, polyphenol compound can be modified to base material, polyphenol coating be prepared, prepared is more
Phenol coating can be used for capturing the circulating tumor cell in peripheral blood, be expected to be used for the early diagnosis of cancer patient.
Technical scheme is as follows：
A kind of material for circulating tumor cell capture, which includes polyphenol compound.
Further, in the above-mentioned technical solutions, the polyphenol compound is tannic acid, epicatechin gallate
One kind in ester or Epigallo-catechin gallate (EGCG).
The present invention also provides a kind of polyphenol coating for circulating tumor cell capture, the method being modified using surface will
Polyphenol compound modification forms polyphenol coating to substrate material surface.
Further, in the above-mentioned technical solutions, the polyphenol compound is tannic acid, epicatechin gallate
One kind in ester or Epigallo-catechin gallate (EGCG).
Further, in the above-mentioned technical solutions, the base material is inorganic non-metallic material, metal material or has
Machine high molecular material.One kind in the preferred silica of the inorganic non-metallic material, aluminium oxide, iron oxide, the metal material
One kind in the preferred gold of material, copper, in the preferred dimethyl silicone polymer of the high-molecular organic material, polystyrene, polyurethane
One kind.
Further, in the above-mentioned technical solutions, the thickness of the polyphenol coating is no less than 80nm, coating surface phenol hydroxyl
The ratio of base content is not less than 20%.
In the present invention, the method being modified using surface, polyphenol compound is modified formed to substrate material surface it is more
Phenol coating.The surface modifying method is not specially limited, those skilled in the art can be modified based on conventional surface
Method modifies polyphenol compound to substrate material surface, it is preferred to use polyphenolic substance and metal ion chelated surface from
The method of assembling.
The present invention also provides a kind of methods of capture circulating tumor cell, include applying peripheral blood sample and above-mentioned polyphenol
The step of layer contact.It will be added dropwise to the polyphenol coating for the peripheral blood sample that examination person provides, applies educate in the incubator, polyphenol applies
Layer can specifically capture the tumour cell in sample, to achieve the purpose that capture tumour cell.In addition, polyphenol compound
Bioactivity containing anti-inflammatory, so as to further decrease adherency of the leucocyte in polyphenol coating, therefore polyphenol coating energy
Enough quickly and effectively enrichment cycles tumour cells from peripheral blood.
In the above-mentioned technical solutions, the peripheral blood sample is that peripheral blood is removed blood plasma, red blood cell and blood platelet
Obtained from.
The present invention has the advantages that compared with existing circulating tumor cell capture technique：
1. the present invention is using deriving from a wealth of sources, cheap, nontoxic polyphenol compound is not necessarily to immobilized specific cancer
Cell recognition antibody or aptamer can effectively reduce use cost.
2. the polyphenol coating of the present invention does not need complexity without introducing micro-nano matrix in entire coating preparation process
Surface etch technology, method is simple and quick.
3. the material provided by the present invention comprising Polyphenols and prepared polyphenol coating can be realized thin to different carcinoma
The specific capture of born of the same parents' type has preferable broad spectrum activity, can be applied to the early diagnosis of cancer patient.
Description of the drawings
Fig. 1 is the stereoscan photograph of polyphenol coating prepared by the embodiment of the present invention 1, and engineer's scale is 10 microns.
Fig. 2 is that the polyphenol coating of the embodiment of the present invention 1 captures the stereoscan photograph after cancer cell, and engineer's scale is 10 micro-
Rice.
Fig. 3 is capture rate block diagram before and after the modification polyphenol coating of the embodiment of the present invention 1.
Fig. 4 is capture rate block diagram of the polyphenol coating to different cancerous cell lines of the embodiment of the present invention 2.
Fig. 5 be the embodiment of the present invention 3 polyphenol coating human peripheral blood in different number cancer cell capture rate scattergram.
Specific implementation mode
Following non-limiting embodiments can make those skilled in the art be more fully understood the present invention, but not with
Any mode limits the present invention.In following embodiments, unless otherwise specified, used experimental method is conventional method, institute
It can be bought from biological or chemical company with material, reagent etc..
Embodiment 1
The polyphenol compound that the present embodiment is selected is tannic acid, and base material is plaine single crystal silicon chip；Utilize polyphenol
The chelating self assembly effect for closing object and metal ion is chemically modified base material；It is to be captured with cancer cell MCF-7
For circulating tumor cell, the capture system of the present invention is further elaborated and is verified, is included the following steps：
1) method of modifying for using the chelating self assembly of polyphenolic substance and metal ion, prepares more in substrate silicon chip surface
Phenol coating：
The tannin aqueous acid of a concentration of 3.2mg/mL and the ferric chloride aqueous solutions of 0.8mg/mL are respectively configured；To 1cm
The tannic acid solution of 1mL and the liquor ferri trichloridi of 1mL are sequentially added in the substrate silicon chip of × 1cm, are reacted 3 minutes, have been reacted
At rear taking-up silicon chip, deionized water washing, nitrogen drying；It repeats above-mentioned reaction step 5 times, obtains more after tannic acid modification
The thickness of phenol coating (Fig. 1), coating is 80nm, and the ratio of coating surface content of phenolic hydroxyl groups is 20%.
2) prepare circulating tumor cell sample to be measured：
100 μ L of MCF-7 cell suspending liquids are taken, is counted with cell counter and calculates its concentration；It draws a certain amount of above-mentioned
Cell suspending liquid, with DMEM cell culture mediums to 1 × 105A cell/mL；
3) circulating tumor cell in sample to be tested is captured：
It will be modified with the acid coated silicon chip of tannin in step 1) to be put into tissue culture plate, be then uniformly mixed step 2)
Cell suspending liquid 0.5mL be added drop-wise to coating surface, be subsequently placed in cell incubator, capture time be 90 minutes；As right
It according to the facts tests, the unmodified circulating tumor cell capture for having the silicon chip surface of polyphenol coating also to carry out in identical sample to be tested is real
It tests；
4) evaluation of capture effect：
After capture time, there is the polyphenol coating of circulating tumor cell to be cleaned with phosphate buffer (PBS) capture
Three times；With the cancer cell 30 minutes for the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%；The cancer cell of capture is impregnated
30 minutes in the DAPI aqueous solutions of a concentration of 10 μ g/mL, achieve the purpose that dyeing；Finally fluorescence microscopy is inverted in Olympus
It takes pictures respectively under 10 times of mirror, chooses the fluorescence photo under the different visuals field, using ImageJ softwares to the circulating tumor cell of capture
It is counted, calculates capture rate；Also above-mentioned steps carry out control experiment group, and calculate capture rate.
The experimental results showed that MCF-7 cancer cells can preferably spread over the silicon chip surface (figure for being modified with polyphenol coating
2), the capture rate of the MCF-7 cancer cells of polyphenol coating surface reaches 76%, and in control experiment, unmodified polyphenol coating table
The capture rate of the MCF-7 cancer cells in face is only 2% (Fig. 3), shows that efficiently catching for circulating tumor cell may be implemented in this method
It obtains.
Embodiment 2
The polyphenol compound that the present embodiment is selected is L-Epicatechin gallate, and base material is plane gold plaque；Profit
Base material is chemically modified with the chelating self assembly of polyphenolic substance and metal ion effect；It is with cancer cell MCF-7
For circulating tumor cell to be captured, the capture system of the present invention is further elaborated and is verified, is included the following steps：
1) method of modifying for using the chelating self assembly of polyphenolic substance and metal ion, prepares more on substrate gold plaque surface
Phenol coating：
The L-Epicatechin gallate aqueous solution of a concentration of 3.2mg/mL and the ferric trichloride of 0.8mg/mL is respectively configured
Aqueous solution；The L-Epicatechin gallate solution of 1mL and the tri-chlorination of 1mL are sequentially added into the substrate gold plaque of 1cm × 1cm
Ferrous solution reacts 3 minutes, and gold plaque, deionized water washing, nitrogen drying are taken out after the completion of reaction；Repeat above-mentioned reaction step
10 times, the polyphenol coating (Fig. 1) after L-Epicatechin gallate modification is obtained, the thickness of coating is 82nm, coating surface phenol
The ratio of hydroxy radical content is 25%.
2) prepare circulating tumor cell sample to be measured：
100 μ L of MCF-7 cell suspending liquids are taken, is counted with cell counter and calculates its concentration；It draws a certain amount of above-mentioned
Cell suspending liquid, with DMEM cell culture mediums to 1 × 105A cell/mL；
3) circulating tumor cell in sample to be tested is captured：
The gold plaque that L-Epicatechin gallate coating is modified in step 1) is put into tissue culture plate, it then will step
The rapid cell suspending liquid 0.5mL 2) being uniformly mixed is added drop-wise to coating surface, is subsequently placed in cell incubator, capture time 90
Minute；It tests as a contrast, the unmodified circulating tumor for thering is the gold plaque surface of polyphenol coating also to carry out in identical sample to be tested
Cell capture is tested；
4) evaluation of capture effect：
After capture time, there is the polyphenol coating of circulating tumor cell to be cleaned with phosphate buffer (PBS) capture
Three times；With the cancer cell 30 minutes for the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%；The cancer cell of capture is impregnated
30 minutes in the DAPI aqueous solutions of a concentration of 10 μ g/mL, achieve the purpose that dyeing；Finally fluorescence microscopy is inverted in Olympus
It takes pictures respectively under 10 times of mirror, chooses the fluorescence photo under the different visuals field, using ImageJ softwares to the circulating tumor cell of capture
It is counted, calculates capture rate；Also above-mentioned steps carry out control experiment group, and calculate capture rate.
The experimental results showed that the capture rate of the MCF-7 cancer cells of polyphenol coating surface reaches 80%, and control experiment
In, the capture rate of the MCF-7 cancer cells of unmodified polyphenol coating surface is 5%, shows that circulating tumor may be implemented in this method
The efficient capture of cell.
Embodiment 3
The polyphenol compound that the present embodiment is selected is tannic acid, and base material is dimethyl silicone polymer (PDMS)；Profit
Base material is chemically modified with the chelating self assembly of polyphenolic substance and metal ion effect；With cancer cell MCF-7,
A431, Hela and A549 be circulating tumor cell to be captured for, to the present invention capture system be further elaborated and
Verification, includes the following steps：
1) method of modifying for using the chelating self assembly of polyphenolic substance and metal ion, prepares more on the surfaces substrate PDMS
Phenol coating：
The tannin aqueous acid of a concentration of 3.2mg/mL and the ferric chloride aqueous solutions of 0.8mg/mL are respectively configured；To 1cm
The tannic acid solution of 1mL and the liquor ferri trichloridi of 1mL are sequentially added in the PDMS substrates of × 1cm, are reacted 3 minutes, have been reacted
At rear taking-up PDMS, deionized water washing, nitrogen drying；It repeats above-mentioned reaction step 5 times, obtains more after tannic acid modification
The thickness of phenol coating, coating is 90nm, and the ratio of coating surface content of phenolic hydroxyl groups is 22%.
2) prepare circulating tumor cell sample to be measured：
Each 100 μ L of MCF-7, A431, Hela and A549 cell suspending liquid are taken respectively, are counted with cell counter and are calculated it
Respective concentration；Draw a certain amount of above-mentioned cell suspending liquid respectively, with DMEM cell culture mediums respectively suspension to 1 ×
105A cell/mL；
3) circulating tumor cell in sample to be tested is captured：
The acid coated PDMS of tannin will be modified in step 1) to be put into tissue culture plate, then by the respective thin of step 2)
Born of the same parents' suspension 0.5mL is added drop-wise to coating surface, is subsequently placed in cell incubator, and capture time is 90 minutes；It is real as a contrast
It tests, the unmodified circulating tumor cell capture experiment for thering is the PDMS material of polyphenol coating also to carry out in identical sample to be tested；
4) evaluation of capture effect：
After capture time, the polyphenol coating for capturing circulating tumor cell is cleaned with phosphate buffer (PBS)
Three times；With the cancer cell 30 minutes for the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%；The cancer cell of capture is impregnated
30 minutes in the DAPI aqueous solutions of a concentration of 10 μ g/mL, achieve the purpose that dyeing；Finally fluorescence microscopy is inverted in Olympus
It takes pictures respectively under 10 times of mirror, chooses the fluorescence photo under the different visuals field, using ImageJ softwares to the circulating tumor cell of capture
It is counted, calculates capture rate；Also above-mentioned steps carry out control experiment group, and calculate capture rate.
The experimental results showed that polyphenol coating surface distinguishes the capture rate of MCF-7, A431, Hela and A549 cancer cell
Reach 76%, 76%, 78% and 83% (Fig. 4), and in the control experiment of unmodified polyphenol coating surface, these cancer cells are caught
It obtains efficiency and is below 2%, show that the efficient capture to different cancerous cell lines may be implemented in this method, there is preferable broad spectrum activity.
Embodiment 4
The polyphenol compound that the present embodiment is selected is tannic acid, and base material is plaine single crystal silicon chip；Utilize polyphenol
The chelating self assembly effect for closing object and metal ion is chemically modified base material；Cancer is added into peripheral blood mononuclear cells
Cell MCF-7 is circulating tumor cell model to be captured, and the capture system of the present invention is further elaborated and is verified, and is wrapped
Include following steps：
1) method of modifying for using the chelating self assembly of polyphenolic substance and metal ion, prepares more in substrate silicon chip surface
Phenol coating：
The tannin aqueous acid of a concentration of 3.2mg/mL and the ferric chloride aqueous solutions of 0.8mg/mL are respectively configured；To 1cm
The tannic acid solution of 1mL and the liquor ferri trichloridi of 1mL are sequentially added in the substrate silicon chip of × 1cm, are reacted 3 minutes, have been reacted
At rear taking-up silicon chip, deionized water washing, nitrogen drying；It repeats above-mentioned reaction step 5 times, obtains more after tannic acid modification
The thickness of phenol coating, coating is 85nm, and the ratio of coating surface content of phenolic hydroxyl groups is 20%.
2) prepare circulating tumor cell sample to be measured：
Take the fresh anticoagulations of 5mL, be added into blood peripheral blood mononuclear cells separating liquid (Beijing Suo Laibao biotechnologies,
P8610), using density-gradient centrifugation method separating periphery blood monocytic cell, peripheral blood mononuclear cells suspension, cell concentration are obtained
It is 0.8 × 106A cell/mL；100 μ L of MCF-7 cell suspending liquids are taken, is counted with cell counter and calculates its concentration；Outward
It is thin that the MCF-7 that cancer cell number is 50,100,200,400,600,800 and 1000 is separately added into all blood monocyte suspension
Born of the same parents are uniformly mixed；
3) circulating tumor cell in sample to be tested is captured：
It will be modified with the acid coated silicon chip of tannin in step 1) to be put into tissue culture plate, be then uniformly mixed step 2)
Cell suspending liquid 0.5mL be added drop-wise to coating surface, be subsequently placed in cell incubator, capture time be 90 minutes；
4) evaluation of capture effect：
After capture time, the polyphenol coating for capturing circulating tumor cell is cleaned with phosphate buffer (PBS)
Three times；With the cancer cell 30 minutes for the paraformaldehyde aqueous solution fixed trapped that mass concentration is 4%；The cancer cell of capture is impregnated
30 minutes in the DAPI aqueous solutions of a concentration of 10 μ g/mL, 60 in the Cytokeratin-FITC phosphate buffers of 10 μ g/mL
Minute, achieve the purpose that dyeing；It finally takes pictures, chooses under the different visuals field respectively under 10 times of laser confocal fluorescence microscope
Fluorescence photo counts the circulating tumor cell of capture using ImageJ softwares, calculates capture rate.
The experimental results showed that the capture of the MCF-7 cancer cells of different number is imitated in polyphenol coating surface human peripheral blood sample
Rate can reach 75% or more (Fig. 5).In addition, peripheral blood mononuclear cells polyphenol coating adherency quantity be (14 cells/
mm2), significantly lower than unmodified surface (the 73 cell/mm for having polyphenol coating2), show that this method may be implemented from peripheral blood
In efficiently specifically capture circulating tumor cell.
Claims (9)
1. a kind of material for circulating tumor cell capture, which is characterized in that the material includes polyphenol compound.
2. material according to claim 1, which is characterized in that the polyphenol compound is tannic acid, epicatechin
One kind in gallate or Epigallo-catechin gallate (EGCG).
3. a kind of polyphenol coating for circulating tumor cell capture, which is characterized in that the method being modified using surface, by polyphenol
Class is compound-modified to form polyphenol coating to substrate material surface.
4. polyphenol coating according to claim 3, which is characterized in that the polyphenol compound is tannic acid, table
One kind in catechin gallate or Epigallo-catechin gallate (EGCG).
5. polyphenol coating according to claim 3, which is characterized in that the base material be inorganic non-metallic material,
Metal material or high-molecular organic material.
6. polyphenol coating according to claim 3, which is characterized in that the polyphenol compound is through chelating self assembly
Method is modified in the substrate material surface.
7. polyphenol coating according to claim 3, which is characterized in that the thickness of the polyphenol coating is no less than 80nm,
The ratio of coating surface content of phenolic hydroxyl groups is not less than 20%.
8. a kind of method of capture circulating tumor cell, which is characterized in that include by peripheral blood sample and claim 3~7
The step of any one of them polyphenol coating layer touch.
9. according to the method described in claim 8, it is characterized in that, the peripheral blood sample is that peripheral blood is removed blood
Obtained from slurry, red blood cell and blood platelet.
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| WO2021088902A1 (en) * | 2019-11-06 | 2021-05-14 | 精拓生技股份有限公司 | Method, kit, and composite membrane for ex vivo expansion of circulating tumor cells, preparation method for composite membrane, medication test method, and cryopreservation solution |
| CN113332951A (en) * | 2021-06-11 | 2021-09-03 | 西南交通大学 | Magnetic nano material for efficiently enriching circulating tumor cells and preparation method thereof |
| CN113943653A (en) * | 2020-07-16 | 2022-01-18 | 中国科学院苏州纳米技术与纳米仿生研究所 | Tannin-based broad-spectrum CTC (CTC) capturing and separating substrate as well as preparation method and application thereof |
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| CN113332951A (en) * | 2021-06-11 | 2021-09-03 | 西南交通大学 | Magnetic nano material for efficiently enriching circulating tumor cells and preparation method thereof |
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