CN111423971A - Polymer microsphere for capturing circulating tumor cells and preparation method thereof - Google Patents
Polymer microsphere for capturing circulating tumor cells and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a polymer microsphere for capturing circulating tumor cells and a preparation method thereof. The rough structure of the surface-wrinkled polymer microsphere prepared by the invention is formed based on an interface instability mechanism of emulsion droplets, and accurate adjustment of the wrinkling degree can be realized by changing the preparation conditions (the concentration of a cosurfactant in an inner phase or the height of a collected liquid in a fixed container), the preparation method is simple and easy to implement, the requirement on reaction conditions is low, and the form of surface-wrinkled can be easily and accurately adjusted; the surface-wrinkled polymer microsphere can efficiently capture Circulating Tumor Cells (CTC) in peripheral blood.
Description
Technical Field
The invention relates to a method for capturing Circulating Tumor Cells (CTC), in particular to a polymer microsphere for capturing the circulating tumor cells and a preparation method thereof.
Background
Circulating Tumor Cells (CTCs), which are Tumor cells that spread from a primary Tumor into the peripheral Circulating blood system, appear to indicate that the Tumor cells have begun to spread from focal tissue to the surrounding area, possibly resulting in lesions in more organ tissues. CTCs can appear early in tumorigenesis and studies indicate that about 90% of cancer deaths are associated with CTC spread. Therefore, CTC detection has important significance in medical applications such as early prevention of major diseases, treatment effect evaluation, drug sensitivity test, tumor recurrence monitoring and the like.
Although the traditional macroscopic method can also realize cell separation, the traditional macroscopic method has the defects of long time consumption, large sample demand, serious target cell loss, high dependence on hardware equipment and the like. The micro-fluidic technology which has emerged in recent years can integrate physical, chemical and biological means in a micron scale range, is easy to realize the miniaturization, low-cost and portable development of the whole device, and provides an important potential technical means for the high-sensitivity and high-efficiency sorting of rare CTC.
Microfluidic technology refers to a technology that integrates different fluids into a system in a specific manner at a microscopic scale, and systematically controls and operates the behavior of the fluids. Through the development of recent decades, the microfluidic technology provides a new method for many scientific and technological fields, including chemical synthesis, biochemical analysis, instant diagnosis, drug development, environmental detection, and the like, and particularly in the aspect of material preparation, the microfluidic technology can precisely control the generated material morphology and structure.
In recent years, polymeric microspheres having different compositions and morphologies have gained increasing attention. The optical, electrical, chemical and other characteristics of the particles can be adjusted according to the change of the structure, size and components of the particles, so that the particles are widely applied to the biomedical and chemical industries. The morphology of the particles greatly affects their physicochemical properties, such as catalytic efficiency of the catalyst, platelet adhesion, etc., wherein the particle surface roughness plays an important role in regulating the interaction between biomaterials and biological systems, and the rough surface facilitates cell adhesion, propagation and growth, accelerating tissue healing. Therefore, artificial manipulation of surface microstructure will provide opportunities for designing new biomaterials.
Generally, microparticles polymerized in a two-phase or multi-phase system form a sphere due to surface tension minimization. Currently, a variety of methods have been created that complicate the surface of microparticles, but the generation of polymeric microspheres with controlled surface roughness remains a technical challenge.
Therefore, in the invention, a preparation method of a novel polymer microsphere is designed and invented by starting with the construction of the polymer microsphere with controllable surface wrinkles and relying on a microfluidic technology, and the novel microsphere can efficiently capture circulating tumor cells in peripheral blood by utilizing the rough surface characteristic of the novel microsphere.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a polymer microsphere for capturing circulating tumor cells and a preparation method thereof, aiming at the defects of the prior art.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows:
the invention provides a preparation method of polymer microspheres for capturing circulating tumor cells, which comprises the following steps:
(1) formation of monodisperse emulsion droplets: building a micro-fluidic chip, selecting a water phase solution of a surfactant as an external phase, selecting a mixed solution of a hydrophobic polymer and a cosurfactant in a volatile organic solvent as an internal phase, and preparing monodisperse emulsion droplets with uniform sizes through the micro-fluidic chip;
(2) preparing polymer microspheres with controllable surface wrinkles: collecting the droplets of the monodisperse emulsion prepared in the step 1) in a fixed container filled with a collecting liquid, forming polymer microspheres with wrinkled surfaces for capturing circulating tumor cells by diffusion and volatilization of an organic solvent in a water phase, and adjusting the surface wrinkling degree of the polymer microspheres by changing the concentration of a cosurfactant in an internal phase or the height of the collecting liquid.
Further, in the step (1), the microfluidic chip is assembled by a glass capillary tube subjected to hydrophilic and hydrophobic treatment, a glass slide, a cover glass, a sample application needle and quick-drying glue, wherein the glass capillary tube is assembled by coaxially nesting an outer phase capillary tube and an inner phase capillary tube.
Furthermore, the diameter of the outer phase capillary tube is 300-400 μm, the diameter of the inner phase capillary tube is 30-70 μm, and the particle size of the monodisperse emulsion droplet and the polymer microsphere with controllable surface wrinkles can be adjusted by changing the flow rate of the inner phase and the outer phase or the diameters of the inner phase and the outer phase capillary tubes.
Further, in the step (1), the hydrophobic polymer is selected from Polystyrene (PS), the cosurfactant is selected from n-cetyl alcohol (HD), the volatile organic solvent is selected from chloroform, the inner phase is composed of Polystyrene (PS) with a certain concentration and n-cetyl alcohol (HD) with different concentrations dissolved in chloroform, wherein the specific concentration of the Polystyrene (PS) dissolved in the chloroform is 10mg/m L, the concentration of the n-cetyl alcohol (HD) dissolved in the chloroform is 0-3 mg/m L, and the surface wrinkle degree of the polymer microspheres can be adjusted by changing the concentration of the n-cetyl alcohol (HD) in the inner phase within the range of 0-3 mg/m L.
Further, in the step (1), the aqueous solution of the surfactant is selected from a Sodium Dodecyl Sulfate (SDS)/glycerin solution, the concentration of the Sodium Dodecyl Sulfate (SDS)/glycerin solution is 3mg/m L, and the surfactant is prepared by dissolving SDS solid powder in a 30vol% glycerin/water solution.
Further, in the step (2), the collection liquid is an SDS/glycerol solution, droplets of the monodisperse emulsion are collected in a fixed container filled with the collection liquid and exposed to the air, and the surface morphology is changed by changing the height of the collection liquid, so that the volatilization rate of chloroform which is an organic solvent is influenced, and the surface wrinkle degree of the polymer microspheres is adjusted.
Further, the fixed container is a circular glass culture dish, and the inner diameter of the circular glass culture dish is 2.2 cm.
Further, in step (2), the prepared polymeric microspheres suspended in the collection liquid are dialyzed in deionized water for 7 days to remove surface glycerol, SDS and residual chloroform, and then dialyzed in ethanol for 7 days to remove HD, thereby obtaining purified polymeric microspheres for capturing circulating tumor cells.
The invention also provides the polymer microsphere for capturing the circulating tumor cells, which is prepared by the preparation method.
Compared with the prior art, the invention has the beneficial effects that:
1) compared with the traditional complex method for roughening the surface of the microsphere, the preparation method disclosed by the invention has the advantages that the surface wrinkles are directly formed by volatilization of the organic solvent and an emulsion droplet instability mechanism, the preparation process is simple and easy to implement, and the requirement on reaction conditions is low;
2) the invention relies on the micro-fluidic technology, adopts the micro-fluidic chip to prepare the liquid drop, has simple channel, less building steps, does not need complex machining process, has simple process, can adjust the size of the monodisperse emulsion liquid drop and the size of the polymer microsphere after corresponding solidification through the flow rate of the internal phase and the external phase or the pipe diameter of the internal phase and the external phase capillary, and has convenient operation;
3) according to the invention, the accurate adjustment of the surface wrinkle degree of the polymer microsphere for capturing the circulating tumor cells can be easily realized by changing the concentration of the cosurfactant or the height of the collection liquid in the internal phase, the rough wrinkle structure of the microsphere is favorable for increasing the adhesion effect with cells, the capability of capturing enriched cells can be greatly increased, the circulating tumor cells in peripheral blood can be efficiently captured, and the cell capturing capability can be enhanced by increasing the wrinkle degree, so that the invention can artificially and accurately adjust the surface microstructure of the microsphere and has good application prospects in the aspects of cell culture, enrichment analysis and the like;
4) the rough polymer microspheres prepared by the invention can also adsorb magnetic nanoparticles and the like, and are further functionalized so as to adapt to more various detection requirements.
Drawings
FIG. 1 is a flow chart of the preparation process of the polymeric microspheres for capturing circulating tumor cells according to the present invention;
fig. 2 is a real-time generated image of a monodisperse emulsion droplet and a surface-wrinkled polystyrene microsphere, wherein a is a real-time generated image of a droplet in a microfluidic chip channel observed by a high-speed camera, and b and c are optical mirror images of the monodisperse emulsion droplet and a corresponding PS microsphere thereof, respectively;
FIG. 3 is a graph showing the relationship between the flow rate of the internal phase and the flow rate of the external phase and the particle size of a monodisperse emulsion droplet in a microfluidic chip, wherein (a) is a graph showing the relationship between the flow rate of the external phase and the particle size of the droplet, and (b) is a graph showing the relationship between the flow rate of the internal phase and the particle size of the droplet;
FIG. 4 is a real-time diagram of the generation process of surface-wrinkled polystyrene microspheres observed under a body microscope, wherein (a) is an image taken at a certain time after collecting droplets, and (b) - (i) are images taken at time points 1s later, 3s later, 7s later, 13s later, 21s later, 30s later, 45s later and 60s later, respectively, in the diagram (a);
FIG. 5 is a graph of the optical and electron micrographs of polystyrene microspheres of example 1 having different degrees of surface wrinkling resulting from varying the HD concentration in the internal phase, corresponding to HD concentrations of (a, d) 0mg/m L, (b, e) 0.5mg/m L, (c, f) 1mg/m L, (g, j) 1.5mg/m L, (h, k) 2mg/m L, (i, l) 3mg/m L;
fig. 6 is a magnetic response state diagram of the polystyrene folded microsphere coated with ferroferric oxide nanoparticles in example 2, wherein (a) is a distribution diagram of the nanospheres in the aqueous dispersion when no magnetic field is initially applied, and (b) to (d) are distribution diagrams of the nanospheres in the aqueous dispersion with time after the magnetic field is applied;
FIG. 7 shows the results of experiments on capturing Circulating Tumor Cells (CTC) by surface-wrinkled polystyrene microspheres of example 3, wherein (a) - (c) are confocal laser scanning microscope images of circulating tumor cells captured on the surface of the surface-wrinkled polystyrene microspheres, (a) are cross-section fluorescence images, (b) are bright field images, and (c) are combined images of a and b; (d) the number of Circulating Tumor Cells (CTCs) captured versus HD concentration was plotted.
Detailed Description
In order to make those skilled in the art better understand the technical solution of the present invention, the following detailed description of the embodiments of the present invention is provided with reference to the accompanying drawings.
The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.
The invention provides a preparation method of polymer microspheres for capturing circulating tumor cells, which comprises the following steps:
(1) formation of monodisperse emulsion droplets: building a micro-fluidic chip, selecting a water phase solution of a surfactant as an external phase, selecting a mixed solution of a hydrophobic polymer and a cosurfactant in a volatile organic solvent as an internal phase, and preparing monodisperse emulsion droplets with uniform sizes through the micro-fluidic chip;
(2) preparing polymer microspheres with controllable surface wrinkles: collecting the droplets of the monodisperse emulsion prepared in the step 1) in a fixed container filled with a collecting liquid, forming polymer microspheres with wrinkled surfaces for capturing circulating tumor cells by diffusion and volatilization of an organic solvent in an aqueous phase, dialyzing the prepared polymer microspheres suspended in the collecting liquid in deionized water for 7 days to remove glycerol, SDS and residual chloroform on the surfaces, and then dialyzing in ethanol for 7 days to remove HD, so as to obtain purified polymer microspheres for capturing circulating tumor cells, wherein the surface wrinkling degree of the polymer microspheres is adjusted by changing the concentration of a cosurfactant in an internal phase or the height of the collecting liquid.
In the step (1), the microfluidic chip is assembled by a glass capillary tube subjected to hydrophilic and hydrophobic treatment, a glass slide, a cover glass, a sample application needle head and quick-drying glue, wherein the glass capillary tube is assembled by coaxially nesting an external phase capillary tube with the tube diameter of 300-400 microns and an internal phase capillary tube with the tube diameter of 30-70 microns, and the particle size adjustment of monodisperse emulsion droplets and polymer microspheres with controllable surface wrinkles can be realized by changing the flow velocity of the internal phase and the external phase or the tube diameters of the internal phase and the external phase capillaries.
In the step (1), Polystyrene (PS) is selected as a hydrophobic polymer, n-cetyl alcohol (HD) is selected as a cosurfactant, chloroform is selected as a volatile organic solvent, the inner phase is formed by dissolving Polystyrene (PS) with a certain concentration and n-cetyl alcohol (HD) with different concentrations in chloroform, the specific concentration of the Polystyrene (PS) dissolved in the chloroform is 10mg/m L, the concentration of the n-cetyl alcohol (HD) dissolved in the chloroform is 0-3 mg/m L, and the surface folding degree of the polymer microspheres can be adjusted by changing the concentration of the n-cetyl alcohol (HD) in the inner phase within the range of 0-3 mg/m L.
In the step (1), the aqueous solution of the surfactant is selected from a Sodium Dodecyl Sulfate (SDS)/glycerol solution, the concentration of the Sodium Dodecyl Sulfate (SDS)/glycerol solution is 3mg/m L, and the surfactant is prepared by dissolving SDS solid powder in 30vol% glycerol/water solution.
In the step (2), the collection liquid is an SDS/glycerol solution, droplets of the monodisperse emulsion are collected in a fixed container, preferably a round glass culture dish, filled with the SDS/glycerol solution, and exposed to the air, the inner diameter of the round glass culture dish is 2.2cm, and the surface wrinkle degree of the polymer microspheres is adjusted by changing the height of the SDS/glycerol solution in the culture dish, so that the volatilization rate of chloroform, which is an organic solvent, is influenced, the surface morphology is changed, and the surface wrinkle degree of the polymer microspheres is adjusted.
The following are examples:
example 1
A surface-wrinkled polystyrene microsphere for capturing circulating tumor cells is prepared by the following steps, wherein the preparation process is shown in figure 1:
(1) preparing an internal phase solution and an external phase solution, and collecting liquid:
1.1) inward solution which consists of Polystyrene (PS)/chloroform solution and n-Hexadecanol (HD)/chloroform solution, wherein the polystyrene is used as hydrophobic polymer, PS21k solid particles are adopted and dissolved in chloroform to form Polystyrene (PS)/chloroform solution of 10mg/m L, the n-Hexadecanol (HD) is used as cosurfactant, 1-hexadecanol solid particles are selected and dissolved in chloroform, and 1-Hexadecanol (HD)/chloroform solution (0 mg/m L, 0.5mg/m L, 1mg/m L, 1.5mg/m L, 2mg/m L and 3mg/m L) with different concentration gradients in the range of 0-3 mg/m L are respectively prepared;
1.2) external phase solution, which is prepared by dissolving 3mg/m L SDS/glycerol solution in 30vol% glycerol aqueous solution, and selecting sodium dodecyl sulfate solid powder.
1.3) collecting liquid, namely, adopting SDS/glycerol solution with the components of 3mg/m L as external phase solution;
(2) assembling an O/W single-emulsion microfluidic chip: drawing two glass capillary tubes with different sizes by using a microelectrode drawing instrument or an acetylene blowtorch, wherein the pipe diameter of the inner phase glass capillary tube is 30-70 mu m, and the pipe diameter of the outer phase glass capillary tube is 300-400 mu m; respectively carrying out hydrophilic and hydrophobic treatment on the inner and outer phase glass capillary tubes according to the requirements: the glass capillary into which the internal phase solution was injected was subjected to hydrophobic treatment using an acetone solution (OTS) containing 5% (v/v) octadecyltrimethoxysilane; the glass capillary tube injected into the external phase solution was subjected to hydrophilic treatment using an ethanol solution containing 5% (v/v) of 3-Aminopropyltriethoxysilane (APTES); the O/W single-emulsion microfluidic chip is formed by assembling internal and external phase glass capillary tubes subjected to hydrophilic and hydrophobic treatment, a glass slide, a cover glass, a sample application needle head and quick-drying glue, wherein the internal and external phase glass capillary tubes are assembled in a coaxial nested manner.
(3) Preparation of monodisperse emulsion droplets:
and pumping the internal and external phase solution into glass syringes with corresponding specifications, respectively placing the glass syringes on two peristaltic pumps, connecting the glass syringes with the O/W single-emulsion microfluidic chip through polyethylene pipes, setting the flow velocity of the internal and external phases, and starting the peristaltic pumps to work. In a microfluidic channel, when the internal and external phase fluids meet, the internal phase fluid is stretched and eventually breaks to form monodisperse emulsion droplets due to the combined action of viscous forces and interfacial tensions.
Fig. 2 is a real-time generated image of the monodisperse emulsion droplet and the surface-wrinkled polystyrene microsphere, wherein, a is a real-time generated image of the monodisperse emulsion droplet in the microfluidic chip channel observed by a high-speed camera, b and c are respectively optical lens pictures of the monodisperse emulsion droplet and the corresponding PS microsphere, as can be seen from fig. 2 (a) and 2 (b), the monodisperse emulsion droplet is generated in the O/W monodisperse microfluidic chip channel, and the size of the monodisperse emulsion droplet is uniform;
controlling the inner flow rate to be 0.1m L/h and other factors to be unchanged, and respectively setting the outer phase flow rates to be 5m L/h, 6m L/h, 7m L/h, 8m L/h, 9m L/h and 10m L/h, wherein the particle size of the monodisperse emulsion droplets is reduced along with the increase of the outer phase flow rate in the range of 5-10 m L/h as shown in FIG. 3 (a), and controlling the outer flow rate to be 5m L/h and other factors to be unchanged, wherein the particle size of the monodisperse emulsion droplets is increased along with the increase of the inner phase flow rate in the range of 0.1m L/h, 0.2m L/h, 0.3m 25/h, 0.4m L/h and 0.5m L/h as shown in FIG. 3 (b), and the particle size of the monodisperse emulsion droplets is adjusted along with the increase of the inner phase flow rate, and the results show that the inner phase flow rate and the outer phase flow rate can be changed to adjust the particle size of the monodisperse emulsion droplets.
The diameter of the outer phase and other factors are controlled to be unchanged, the diameter of the inner phase is reduced, and the particle size of the monodisperse emulsion droplets is reduced due to the reduction of the fluid volume passing through the single shearing; controlling the diameter of the inner phase pipe and other factors to be unchanged, increasing the diameter of the outer phase pipe, increasing the amount of the outer phase fluid to increase the shearing force of the interface of the emulsion droplets, and reducing the particle size of the monodisperse emulsion droplets; therefore, the diameter of the inner and outer phase capillary tubes is changed, and the particle size of the monodisperse emulsion liquid drop can be adjusted.
(4) Preparing polymer microspheres with controllable surface wrinkles:
collecting the monodisperse emulsion droplets with uniform size prepared in the step (3) in a circular glass culture dish with an inner diameter of 2.2cm, pouring a collecting solution (SDS/glycerol solution of 3mg/m L), observing a generation process real object diagram of the surface-wrinkled polystyrene microspheres by using a body-type microscope, wherein (a) is an image shot at a certain time (t) after the droplets are collected, (b) to (i) are images shot at a time (t + 1) after the image is shot at a time (a), at a time (t + 3) after 3s, at a time (t + 7) after 7s, at a time (t + 13) after 13s, at a time (t + 21) after 21s, at a time (t + 30) after 30s, at a time (t + 45) after 45s, and at a time (t + 60) after 60s, and thus an unstable phenomenon of an emulsion droplet interface is triggered, the droplets are gradually solidified and wrinkles are formed on a surface layer to form the surface-wrinkled polystyrene microspheres.
The size of the polymer microsphere prepared by the method is linearly related to the size of the monodisperse emulsion droplet, the size of the monodisperse emulsion droplet is increased, the size of the corresponding polymer microsphere is increased, and vice versa; therefore, the diameter of the inner and outer phase capillary tubes is changed, and the particle size of the polymer microspheres can be adjusted.
Firstly dialyzing the polystyrene microspheres suspended in the solution in deionized water for 7 days to remove glycerol, SDS and residual chloroform on the surface, and then dialyzing in ethanol for 7 days to remove 1-hexadecanol, so as to prepare purified surface-folded polystyrene microspheres for capturing circulating tumor cells; observing the morphology of the microspheres under a light mirror, as shown in FIG. 2 c;
the regulation and control of the surface wrinkle degree of the surface wrinkle polystyrene microsphere for capturing the circulating tumor cells can be mainly realized by regulating the HD concentration and the volume of a collecting solution:
the other factors in the preparation process are controlled to be unchanged, the HD concentrations in the internal phase are respectively 0mg/m L, 0.5mg/m L, 1mg/m L0, 1.5mg/m L1, 2mg/m L and 3mg/m L, and the polystyrene microspheres prepared by adopting different internal phase HD concentrations are subjected to optical microscope and electron microscope characterization by using a body type microscope and a field emission scanning electron microscope, and the results are shown in FIG. 5, wherein (a) - (c) and (g) - (i) are optical microscope characterization diagrams, and (d) - (f) and (j) - (l) are electron microscope characterization diagrams, and the corresponding HD concentrations are (a, d): 0mg/m L, (b, e): 0.5mg/m L, (c, f): 1mg/m L, (g, j): 1.5mg/m L, (h, k) 2mg/m L, (i, l): 3mg/m L, and the degree of surface wrinkle of the microspheres can be adjusted by adopting the method of gradually increasing the prepared HD concentration.
Other factor parameters in the preparation process are controlled to be unchanged, the height of a collecting liquid (SDS/glycerol solution) contained in a circular glass culture dish with the inner diameter of 2.2cm is changed, the increase of the height of the collecting liquid enables the distance between emulsion liquid drops at the bottom of the circular culture dish and the upper air to be increased, the volatilization rate of an organic solvent is reduced, the occurrence rate of an interface instability phenomenon is further reduced, the surface wrinkle degree of the microspheres prepared by the preparation method is further gradually reduced, and the accurate adjustment of the surface wrinkle degree of the microspheres can be realized by adjusting the height of the collecting liquid in the fixed culture dish.
Example 2: experiment for adsorbing magnetic nanoparticles by polystyrene microspheres with surface wrinkles
In order to adapt the microspheres to more widely applied functions, the surface-wrinkled polystyrene microspheres (abbreviated as surface-wrinkled polystyrene microspheres) for capturing circulating tumor cells, prepared in example 1, are soaked in a water-soluble ferroferric oxide nanoparticle dispersion liquid and kept for 3 hours to vibrate; because the polystyrene microspheres adsorbed with the magnetic nanoparticles have a high settling velocity in water, the microspheres are settled to the bottom by adopting a standing method, and then ultrapure water is added for multiple times for washing after supernatant liquid is removed; after the washing is completed, the polystyrene microspheres are quickly separated by the aid of the additional magnet, so that the folded microspheres are endowed with magnetic control characteristics to adapt to more various detection requirements; fig. 6 is a magnetic response state diagram of polystyrene folded microspheres (abbreviated as "nanospheres") coated with ferroferric oxide nanoparticles on the surface, wherein (a) is a distribution diagram of the nanospheres in an aqueous dispersion when a magnetic field is not initially applied, and (b) to (d) are distribution diagrams of the nanospheres in the aqueous dispersion with time after the magnetic field is applied, and it can be found that the nanospheres gradually approach to the magnet side after the magnetic field is added, which indicates that the polystyrene folded microspheres coated with ferroferric oxide nanoparticles on the surface are endowed with strong magnetic responsiveness.
Example 3: surface-wrinkled polystyrene microsphere used for capture experiment of Circulating Tumor Cells (CTC)
3.1) drying the surface-wrinkled polystyrene microspheres (abbreviated as surface-wrinkled polystyrene microspheres) for capturing circulating tumor cells, which are prepared in the embodiment 1, and then placing the dried microspheres into a reaction cavity of a vacuum plasma cleaning machine, and performing plasma treatment for 5 minutes to activate the inert surface of the microspheres so as to facilitate the grafting and specific fixation of subsequent antibodies;
3.2) mixing an Anti-Epithelial cell adhesion molecule (Epithelial cell adhesion molecule, EpCAM) specific antibody solution (Anti-EpCAM Antibody (APC), Nano Biological, Cat # 10694-R028-A) with the polystyrene microspheres treated in the step 3.1), shaking for 4 hours at 37 ℃ to allow the Anti-EpCAM antibody to stably and uniformly modify the surfaces of the polystyrene microspheres treated in the step 3.1), and washing with phosphate buffered saline solution to remove free excess Anti-EpCAM antibody;
3.3) adopting a human breast cancer cell MCF-7 tumor cell (purchased from Shanghai Guanzhou bioengineering Co., Ltd., product number C7105) as a target cell, adding the polystyrene microsphere with the modified antibody on the outer surface in the step 3.2) into the MCF-7 cell suspension, incubating for 30 minutes at 37 ℃, and washing with phosphate buffer saline solution to remove the excess MCF-7 cells which are not captured;
for convenient observation and counting, the cells in the step 3.3) are stained by Calcein, a 1mM Calcein (Calcein-AM) solution is prepared by dimethyl sulfoxide (DMSO), and the Calcein-AM solution is diluted by PBS to be 1-50 mu M; adding 1/10 Calcein-AM solution into cell culture medium, and culturing cells at 37 deg.C for 15-30 min; washing the surface fold polystyrene microspheres of the captured cells twice by phosphate buffered saline; then, a laser scanning confocal microscope is used for observing a fluorescence image of the surface-wrinkled polystyrene microsphere of the captured cell, as shown in fig. 7a-c, the MCF-7 cell dyed by calcein is attached to the surface of the surface-wrinkled polystyrene microsphere and emits bright green fluorescence (bright color part in the image);
in order to obtain higher capture efficiency, the relationship between the number of Circulating Tumor Cells (CTC) and the folding degree of the surface-folded polystyrene microspheres is continuously explored, namely, the Anti-EpCAM antibody in the step 3.2) with the same concentration is adopted to respectively modify the surface-folded polystyrene microspheres with different folding degrees, then the surface-folded polystyrene microspheres are respectively incubated with the human breast cancer cells MCF-7 tumor cells in the step 3.3) with the same concentration for 30 minutes at 37 ℃, excess MCF-7 cells which are not captured are washed by phosphate buffered saline solution, the surface-folded polystyrene microspheres with the same amount of captured cells are taken to calculate and analyze the captured cells through fluorescence images observed by a laser scanning confocal microscope, and the result is shown in figure 7 (d), wherein the transverse coordinate is HD concentration, the higher HD concentration is the corresponding higher folding degree of the surface-folded polystyrene microspheres, the longitudinal coordinate is the number of the peripheral blood MCF-7 tumor cells captured on the surface of the surface-folded polystyrene microspheres, the higher longitudinal coordinate is the peripheral blood folding degree of the surface-folded polystyrene microspheres, the peripheral blood cells captured by the MCF-7 tumor cells captured microspheres are prepared according to the higher folding degree, the obtained by the cycle tumor cell density regulating effect, the invention, the folding degree of the invention can be prepared by the invention, the higher the circulating tumor cell folding degree of the circulating tumor cells in the invention, the obtained by the invention, the invention.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
Claims (9)
1. A preparation method of polymer microspheres for capturing circulating tumor cells is characterized by comprising the following steps:
(1) formation of monodisperse emulsion droplets: building a micro-fluidic chip, selecting a water phase solution of a surfactant as an external phase, selecting a mixed solution of a hydrophobic polymer and a cosurfactant in a volatile organic solvent as an internal phase, and preparing monodisperse emulsion droplets with uniform sizes through the micro-fluidic chip;
(2) preparing polymer microspheres with controllable surface wrinkles: collecting the droplets of the monodisperse emulsion prepared in the step 1) in a fixed container filled with a collecting liquid, forming polymer microspheres with wrinkled surfaces for capturing circulating tumor cells by diffusion and volatilization of an organic solvent in a water phase, and adjusting the surface wrinkling degree of the polymer microspheres by changing the concentration of a cosurfactant in an internal phase or the height of the collecting liquid.
2. The method of claim 1, wherein: in the step (1), the microfluidic chip is assembled by a glass capillary tube, a glass slide, a cover glass, a sample application needle head and quick-drying glue, wherein the glass capillary tube is assembled by coaxially nesting an external phase capillary tube and an internal phase capillary tube.
3. The method of claim 2, wherein: the diameter of the external phase capillary is 300-400 mu m, the diameter of the internal phase capillary is 30-70 mu m, and the particle size of the monodisperse emulsion liquid drop and the polymer microsphere with controllable surface wrinkles can be adjusted by changing the flow rate of the internal phase and the external phase or the diameters of the internal phase and the external phase capillaries.
4. The preparation method according to claim 3, wherein in the step (1), the hydrophobic polymer is polystyrene, the cosurfactant is n-cetyl alcohol, the volatile organic solvent is chloroform, and the inner phase is composed of polystyrene with a certain concentration and n-cetyl alcohol with different concentrations dissolved in chloroform, wherein the specific concentration of polystyrene dissolved in chloroform is 10mg/m L, the specific concentration of n-cetyl alcohol dissolved in chloroform is 0-3 mg/m L, and the surface wrinkle degree of the polymer microspheres can be adjusted by changing the concentration of n-cetyl alcohol in the inner phase within the range of 0-3 mg/m L.
5. The method according to claim 4, wherein in the step (1), the aqueous solution of the surfactant is a sodium lauryl sulfate/glycerin solution having a concentration of 3mg/m L, and the surfactant is prepared by dissolving a solid powder of sodium lauryl sulfate in a 30vol% glycerin/water solution.
6. The method of claim 5, wherein: in the step (2), the collection liquid is SDS/glycerol solution, the monodisperse emulsion droplets are collected in a fixed container filled with the collection liquid and exposed in the air, and the surface wrinkle degree of the polymer microspheres can be adjusted by changing the height of the collection liquid.
7. The method of claim 6, wherein: the fixed container is a circular glass culture dish, and the inner diameter of the circular glass culture dish is 2.2 cm.
8. The method of claim 7, wherein: in the step (2), the prepared polymer microspheres suspended in the collecting liquid are subjected to deionized water and ethanol dialysis treatment in sequence to remove surface residual liquid, so that the purified polymer microspheres for capturing the circulating tumor cells are obtained.
9. A polymer microsphere for capturing circulating tumor cells, which is prepared by the preparation method of any one of claims 1 to 8.
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