CN116426481A - Flower-shaped immunomagnetic beads for separating circulating tumor cells and preparation and application methods thereof - Google Patents
Flower-shaped immunomagnetic beads for separating circulating tumor cells and preparation and application methods thereof Download PDFInfo
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Abstract
The invention relates to a flower-shaped immunomagnetic bead for separating circulating tumor cells and a preparation and application method thereof, which belong to the technical field of nano material preparation, can selectively separate the circulating tumor cells and effectively enhance the separation efficiency of the circulating tumor cells; the preparation method comprises the steps of performing biomineralization by mixing a precursor, magnetic nano particles and SA solution, and performing antibody modification on the biomineralization magnetic particles to obtain flower-shaped immunomagnetic beads for separating circulating tumor cells; the magnetic nano particles are NH 2 ‑Fe 3 O 4 The precursor is CuSO 4 The method comprises the steps of carrying out a first treatment on the surface of the The antibody modification is realized by adopting a mode of incubating magnetic particles and biotin modified epithelial cell adhesion molecule antibodies together. The technical scheme provided by the invention is suitable for the process of separating the circulating tumor cells.
Description
Technical Field
The invention relates to the technical field of nano material preparation, in particular to a flower-shaped immunomagnetic bead for separating circulating tumor cells and a preparation method and an application method thereof.
Background
CTCs are tumor cells that escape from the primary tumor site to the peripheral blood circulation system, and CTCs are rarely abundant in human body fluids, so that a highly sensitive CTCs separation platform is required for monitoring them.
With the increasing demand for accurate, simple, economical, efficient and sensitive CTCs detection systems, many efforts have been made by related researchers to concentrate CTCs, such as density gradient centrifugation, flow cytometry, microfluidic, microarray, filtration, immunomagnetic separation, and the like. Among them, immunomagnetic separation is one of the most widely used techniques because of its simple operation, large specific surface area, rapid magnetic response and high recovery rate. In conventional immunomagnetic separation systems, the separation of CTCs relies solely on specific molecules, such as antibodies, aptamers and polypeptides, that are surface modified with magnetic particles. These immunomagnetic particles often encounter separation efficiency bottlenecks due to the inefficiency of antibody modification and neglecting the surface structure. First, the binding of these specific molecules always requires cumbersome reaction steps. For example, to modify CM-Fe with antibody (Ab) 3 O 4 The @ Au surface to achieve CTCs capture, steps include activation with 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to facilitate SA ligation and binding to biotin-modified epithelial cell adhesion molecule (EpCAM) antibodies. Fe in rapid magnetic reaction by complex layer-by-layer assembly (LBL) technique 3 O 4 Quantum dots and thiols are deposited on the nanoparticles, and CTCs are then further bio-friendly recovered using disulfide-bound antibodies. Multiple reaction steps may affect the modification efficiency of a particular molecule, resulting in poor CTCs separation efficiency. Second, existing immunomagnetic particles mostly have smooth surface structures. However, surface nanostructures can significantly increase the capture efficiency of CTCs due to the topological interactions between the surface and CTCs. Therefore, there is an increasing need to develop immunomagnetic particles with efficient specific molecular modifications and programmable surface nanostructures.
Accordingly, there is a need to develop a micro-nano hierarchical flower-like immunomagnetic beads for the isolation of Circulating Tumor Cells (CTCs) to address the deficiencies of the prior art to address or alleviate one or more of the problems described above.
Disclosure of Invention
In view of the above, the invention provides a micro-nano hierarchical flower-like immunomagnetic bead for separating Circulating Tumor Cells (CTCs), and a preparation method and an application method thereof, which can selectively separate the circulating tumor cells and effectively enhance the separation efficiency of the circulating tumor cells.
In one aspect, the present invention provides a method for preparing flower-shaped immunomagnetic beads for separating circulating tumor cells, wherein the method comprises the steps of biomineralization is performed in a mode of mixing a precursor, magnetic nanoparticles and SA (streptavidin), and antibody modification is performed on the magnetic particles after biomineralization, so that the flower-shaped immunomagnetic beads for separating circulating tumor cells are obtained.
In aspects and any one of the possible implementations described above, there is further provided an implementation, the steps of the preparation method include:
s1, preparing magnetic nano particles, and placing the magnetic nano particles in a buffer solution to form a magnetic nano particle suspension;
s2, mixing the obtained magnetic nanoparticle suspension with SA solution and precursor solution, standing for 24-72h at room temperature to carry out biomineralization, and then taking out the biomineralized magnetic particles;
s3, performing antibody modification on the magnetic particles obtained in the step S2 to obtain flower-shaped immunomagnetic beads for separating the circulating tumor cells.
In aspects and any one of the possible implementations as described above, there is further provided an implementation, the magnetic nanoparticle is NH 2 -Fe 3 O 4 。
Aspects and any one of the possible implementations as described above, further providing an implementation, the NH 2 -Fe 3 O 4 The preparation steps of (a) comprise:
s11, preparing Fe by using coprecipitation mode 3 O 4 A nanoparticle;
s12, fe to be prepared 3 O 4 Mixing the nano particles with anhydrous methanol and 3-aminopropyl triethoxysilane, performing ultrasonic treatment, and stirring at room temperature to obtain NH 2 -Fe 3 O 4 。
In aspects and any one of the possible implementations described above, there is further provided an implementation, in step S2, the concentration of the magnetic nanoparticle suspension is 5mg/mL, and the amount is 15 μl;
the concentration of SA solution was 5mg/mL and the amount was 20. Mu.L.
Aspects and any one of the possible implementations described above, further providing an implementation, the method comprisingThe precursor is CuSO 4 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the precursor solution was 120mM, in an amount of 17.5. Mu.L.
In the aspects and any possible implementation manner as described above, there is further provided an implementation manner, where the content of the antibody modification in step S3 includes: incubating the obtained biomineralization magnetic particles with biotin-modified epithelial cell adhesion molecule antibodies for 45-90 min; the precipitate was again adsorbed by magnet and washed repeatedly 3 times with PBS.
In the aspect and any possible implementation manner as described above, there is further provided an implementation manner, in which the method of removing the magnetic particles after biomineralization in step S2 is: and (3) washing the obtained magnetic precipitate with ultrapure water for 3-5 times through the magnetic precipitate generated by magnet adsorption to obtain the magnetic particles.
In another aspect, the invention provides a flower-shaped immunomagnetic bead for separating circulating tumor cells, wherein the flower-shaped immunomagnetic bead is prepared by adopting any one of the preparation methods.
In still another aspect, the present invention provides a method for applying the flower-shaped immunomagnetic beads for separating circulating tumor cells as described above, the content of the application method comprising:
adding a certain amount of the flower-shaped immunomagnetic beads into PC-3 cell solution, and placing at 37deg.C and 0.5% CO 2 Incubating for 45min in a gently shaking cell incubator; and then, adsorbing the precipitate by using a magnet and washing, wherein the obtained precipitate is the separated circulating tumor cells.
Compared with the prior art, one of the technical schemes has the following advantages or beneficial effects: the present invention employs Streptavidin (SA) to assist biomineralization and one-step antibody modification to prepare a flower-shaped immunomagnetic particle (FIMPs) which is highly efficient and has excellent ability to selectively isolate Circulating Tumor Cells (CTCs);
the other technical scheme has the following advantages or beneficial effects: according to the invention, the inorganic nano-sheets and the magnetic nano-particles are connected together through SA to obtain the hybrid magnetic particles with micro/nano hierarchical flower-like structures, and a huge binding site is provided for antibody post-modification;
the other technical scheme has the following advantages or beneficial effects: the unique surface nano structure and high-efficiency antibody modification of the magnetic beads enable FIMPs to separate CTCs with high selectivity and high efficiency;
the other technical scheme has the following advantages or beneficial effects: the scheme of the invention provides a promising platform for selectively separating trace biomolecules and particles from complex samples, and shows great potential for downstream detection and diagnosis.
Of course, it is not necessary for any of the products embodying the invention to achieve all of the technical effects described above at the same time.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and that other drawings can be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of micro/nano-sized flower-shaped immunomagnetic beads for enhancing separation of circulating tumor cells according to one embodiment of the present invention;
FIG. 2 is a scanning electron microscope image of flower-like immunomagnetic particles (FIMPs) of varying roughness provided by the present invention; wherein a, b and c are respectively the SEM images of nanoflower generated in the reactions for 24, 48 and 72 hours;
FIG. 3 is a transmission electron microscope image corresponding to flower-like immunomagnetic particles (FIMPs) produced 72 hours of reaction provided by the present invention; wherein a is a transmission electron microscope picture of the whole flower-shaped immunomagnetic particles, and b is a transmission electron microscope picture of part of the flower-shaped immunomagnetic particles;
FIG. 4 is a graph comparing capture efficiency of the present invention when PC-3 cells are captured by flower-like immunomagnetic particles (FIMPs) of varying roughness; wherein FIMPs-1, FIMPs-2 and FIMPs-3 are respectively the cancer cell capturing efficiency corresponding to nanoflower generated in 24 hours, 48 hours and 72 hours of reaction;
FIG. 5 is a scanning electron microscope image of PC-3 cells captured by flower-like immunomagnetic particles (FIMPs) of varying roughness in accordance with the present invention; wherein a, b and c are scanning electron microscope images corresponding to nanoflower capturing PC-3 generated in 24, 48 and 72 hours of reaction respectively;
FIG. 6 is a graph showing the comparison of the capture efficiency when different cells were captured by optimal flower-like immunomagnetic particles (FIMPs) according to examples 3-7 of the present invention.
Detailed Description
For a better understanding of the technical solution of the present invention, the following detailed description of the embodiments of the present invention refers to the accompanying drawings.
It should be understood that the described embodiments are merely some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
According to the simulation of biomineralization process in nature, a plurality of organic-inorganic hybrid materials with unique nano structures can be created, and FIMPs with high-efficiency antibody modification and programmable nano structures prepared by Streptavidin (SA) assisted biomineralization and one-step antibody modification can be prepared. The precursor, magnetic nanoparticles, and SA are mixed together for biomineralization, and the magnetic particles are modified with an epithelial cell adhesion molecule (EpCAM) antibody (Ab) by a one-step reaction. Among other things, SA can aid in the formation of micro/nano hierarchical flower-like magnetic bead structures and provide a large number of binding sites for post-antibody modification. The obtained FIMPs can separate CTCs from biological fluids with normal cells with high selectivity and high efficiency. FIMPs have good sensitivity and specificity under optimal detection conditions. This study may provide an important tool for rapid capture of CTCs.
The invention relates to a preparation method of micro-nano hierarchical flower-shaped immunomagnetic beads for separating Circulating Tumor Cells (CTCs), which comprises the following steps:
1.1. Preparation of Fe Using Co-precipitation method 3 O 4 Nanoparticles: 0.82g of FeCl 3 .6H 2 O and 0.6g FeCl 2 .4H 2 O was dissolved in 10mL of ultrapure water and rapidly stirred at a temperature of 30℃for 20min. Then, 2.5mL of 28% aqueous ammonia was added dropwise to adjust the pH to 11. And simultaneously stirring for 40min at 30 ℃, washing the precipitate with ultrapure water for 3-5 times after the reaction is finished, transferring the washed precipitate into a centrifuge tube, and drying in a vacuum drying oven at 60 ℃ for 48h for later use.
1.2. Preparation of NH 2 -Fe 3 O 4 Nanoparticles: weighing 20mg of Fe 3 O 4 Mixing with 20mL of anhydrous methanol and 1mL of 3-aminopropyl triethoxysilane (APTES), performing ultrasonic treatment for 5min, and stirring at room temperature for 7h to obtain amino modified magnetic beads. Washing the modified magnetic beads with ultrapure water and repeating for 3-5 times, and washing the washed NH 2 -Fe 3 O 4 Transferring into a centrifuge tube, adding 4mL of ultrapure water to prepare NH 2 -Fe 3 O 4 The suspension (5 mg/mL) was stored at 4℃until use.
the flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet attracted the magnetic particles and cells, the supernatant was removed, and the magnetic particles and cells were repeatedly washed 3 times with PBS. The following cell capture experiments were then performed.
Step 4, the functionalized flower-like immunomagnetic particles (FIMPs) are specifically combined with a target object to complete the efficient separation of the circulating tumor cells: functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 The solvent for FIMPs was water at a volume of one/mL) was added to PC-3 cell solution (1 mL,10 5 cell/mL), cell capturing and concentration are performed. The mixture was subjected to 37℃and 0.5% CO 2 Incubation was performed for 45min in a gentle shaking cell incubator, washed 3 times after magnet adsorption, and the supernatant was collected each time and centrifuged at 1000r/min for 3min. The pellet was then collected and mixed with 100. Mu.L of PBS, and 10. Mu.L of the mixture was placed in a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation.
The invention has the following advantages: the flower-like immunomagnetic particles (FIMPs) are simple to prepare, are readily available in materials, provide a large number of binding sites for post-antibody modification, and the unique surface nanostructure and efficient antibody modification enable FIMPs to selectively isolate CTCs from biological fluids containing normal cells with high selectivity and efficiency, as shown in fig. 1. FIMPs show good sensitivity and specificity under optimal detection conditions, with a capture efficiency of 83.3% + -2.1% for PC-3. This study provides an important tool for rapid capture of CTCs. The scheme of the invention provides a very promising platform for selectively separating trace biomolecules and particles from complex samples, and has great potential for subsequent detection and diagnosis.
Example 1:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 24h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed for 3 to 5 times by ultrapure water to remove unreacted CuSO 4 And SA. The precipitate is flower-like immunomagnetic particles (FIMPs), and is suspended in 100 μl PBS (10 mM) buffer solution, and stored at 4deg.CFor use and scanning electron microscopy as shown in figure 2, panel a.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Each/mL) was added to PC-3 cell solution (1 mL,10 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 (0.5％CO 2 Is 95% by volume of air and 5% by volume of CO 2 ) Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 4.
The captured cells were fixed overnight at room temperature with a 2.5% glutaraldehyde solution (where 2.5% means that the mass ratio of glutaraldehyde in the solution is 2.5%). Subsequently, the cells were dehydrated with ethanol at different concentrations (30%, 50%, 70%, 90%, 95% and 100%). The cells were then treated with 50% and 100% HMDS absolute ethanol solution for 15min, respectively. The cells were then dried in a vacuum freeze dryer for 12h and the morphology of the captured cells was observed with a scanning electron microscope as shown in fig. 5 a.
Example 2:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and left to stand at room temperature for 48h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed for 3 to 5 times by ultrapure water to remove unreacted CuSO 4 And SA. The precipitate is flower-like immunomagnetic particles (FIMPs), and is suspended in 100 μl PBS (10 mM) buffer solution, and then placedThe sample was kept at 4℃for further use and observed by a scanning electron microscope, and the observation result by the scanning electron microscope is shown in FIG. 2 b.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Each/mL) was added to PC-3 cell solution (1 mL,10 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 4.
The captured cells were fixed overnight with 2.5% glutaraldehyde solution at room temperature. Subsequently, the cells were dehydrated with ethanol at different concentrations (30%, 50%, 70%, 90%, 95% and 100%). The cells were then treated with 50% and 100% HMDS absolute ethanol solution for 15min, respectively. The cells were then dried in a vacuum freeze dryer for 12h and the morphology of the captured cells was observed with a scanning electron microscope as shown in figure 5 b.
Example 3:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 72h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed for 3 to 5 times by ultrapure water to remove unreacted CuSO 4 And SA. The precipitate is flower-like immunomagnetic particles (FIMPs), and is suspended in 100 μl PBS buffer (10 mM), stored at 4deg.C, and subjected to scanning electron microscopy as shown in figure 2 c and perspectiveThe observation by electron microscopy is shown in FIG. 3. As can be seen from the a, b, c diagrams of fig. 2, the structural complexity of the obtained flower-like immunomagnetic particles increases with increasing standing reaction time, and is optimal at 72h, and the structural complexity of the obtained flower-like immunomagnetic particles is also higher than that of the flower-like immunomagnetic particles prepared by reaction time of more than 72h. In fig. 3, a is a transmission electron microscope image showing that FIMPs have petal-like fine structures; panel b shows that the higher resolution transmission electron microscope image shows that the nanoparticles are uniformly embedded in FIMPs petals.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Each/mL) was added to PC-3 cell solution (1 mL,10 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation as shown in fig. 6.
The captured cells were fixed overnight with 2.5% glutaraldehyde solution at room temperature. Subsequently, the cells were dehydrated with ethanol at different concentrations (30%, 50%, 70%, 90%, 95% and 100%). The cells were then treated with 50% and 100% HMDS absolute ethanol solution for 15min, respectively. The cells were then dried in a vacuum freeze dryer for 12h and the morphology of the captured cells was observed by scanning electron microscopy as shown in figure 5, panel c.
The schematic of FIMPs capture PC-3 cells in FIG. 5 is the leftmost plot. The intermediate pseudo-low magnification shows the topographical interactions between cells and the different FIMPs surfaces. The pseudo-color high magnification on the right shows the interaction of cellular filament groups with different FIMPs surfaces, where (a) FIMPs nanostructures (FIMPs-1), (B) FIMPs nanostructures (FIMPs-2), and (C) FIMPs nanostructures (FIMPs-3).
Example 4:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 72h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed with ultrapure water for 3-5 times to remove unreacted CuSO 4 And SA. The precipitate was obtained as flower-like immunomagnetic particles (FIMPs), and the obtained precipitate was suspended in 100. Mu.L of PBS buffer (10 mM), stored at 4℃for use, and observed with a scanning electron microscope as shown in FIG. 2 c and a perspective electron microscope as shown in FIG. 3.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Each/mL) was added to MCF-7 cell solution (1 mL,10 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 6.
Example 5:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 72h. After the reaction, the resulting precipitate was repeatedly washed with ultrapure water by magnetic precipitation by magnet adsorptionWashing 3-5 times to remove unreacted CuSO 4 And SA. The precipitate was obtained as flower-like immunomagnetic particles (FIMPs), and the obtained precipitate was suspended in 100. Mu.L of PBS buffer (10 mM), stored at 4℃for use, and observed with a scanning electron microscope as shown in FIG. 2 c and a perspective electron microscope as shown in FIG. 3.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Individual/mL) was added to HepG2 cell solution (1 mL, 10) 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 6.
Example 6:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 72h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed with ultrapure water for 3-5 times to remove unreacted CuSO 4 And SA. The precipitate was obtained as flower-like immunomagnetic particles (FIMPs), and the obtained precipitate was suspended in 100. Mu.L of PBS buffer (10 mM), stored at 4℃for use, and observed with a scanning electron microscope as shown in FIG. 2 c and a perspective electron microscope as shown in FIG. 3.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Add Daudi cell solution (1 mL, 10) 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The pellet was then collected and mixed with 100 μl of PBS, and 10 μl of the mixture was placed into a counting plate to count the number of uncaptured cells. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 6.
Example 7:
SA solution (20. Mu.L, 5 mg/mL), NH 2 -Fe 3 O 4 Suspension (15. Mu.L, 5 mg/mL), cuSO 4 The solution (17.5 μl,120 mM) was added to 1mL of PBS buffer (100 mM, ph=7.4), mixed well and allowed to stand at room temperature for 72h. After the reaction is completed, the magnetic precipitate generated by the magnet adsorption is repeatedly washed for 3 to 5 times by ultrapure water to remove unreacted CuSO 4 And SA. The precipitate was obtained as flower-like immunomagnetic particles (FIMPs), and the obtained precipitate was suspended in 100. Mu.L of PBS buffer (10 mM), stored at 4℃for use, and observed with a scanning electron microscope as shown in FIG. 2 c and a perspective electron microscope as shown in FIG. 3.
The flower-like immunomagnetic particles (FIMPs) are incubated with biotin-modified epithelial cell adhesion molecule antibodies for 45min to 90min. After the magnet adsorption, the supernatant was removed, and the washing was repeated 3 times with PBS. The following cell capture experiments were then performed.
Functionalized flower-like immunomagnetic particles (FIMPs) (1 mL,10 7 Add Jurkat cell solution (1 mL, 10) 5 cell/mL), cell capturing and concentration are performed. The mixture was exposed to 0.5% CO at 37deg.C 2 Incubation for 45min in a gentle shaking cell incubator, washing 3 times after magnetic adsorption, collecting supernatant each time, and centrifuging at 1000r/min for 3min. The precipitate was then collected and mixed with 100. Mu.L of PBS, and 10. Mu.L of the mixture was put into a meterIn the plate, the number of cells not captured was counted. The capture rate of the captured cells was calculated by subtracting the number of cells in the supernatant after magnetic separation from the initial number of cells before separation, as shown in fig. 6.
The flower-shaped immunomagnetic beads for separating the circulating tumor cells, and the preparation and application methods thereof provided by the embodiment of the application are described in detail. The above description of embodiments is only for aiding in understanding the method of the present application and its core ideas; meanwhile, as those skilled in the art will have modifications in the specific embodiments and application scope in accordance with the ideas of the present application, the present description should not be construed as limiting the present application in view of the above.
It should also be noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a product or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such product or system. Without further limitation, an element defined by the phrase "comprising one … …" does not exclude the presence of other like elements in a commodity or system comprising such elements. By "substantially" is meant that within an acceptable error range, a person skilled in the art is able to solve the technical problem within a certain error range, substantially achieving the technical effect.
The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise. The term "and/or" as used herein is merely one association relationship describing the associated object, meaning that there may be three relationships, e.g., a and/or B, may represent: a exists alone, A and B exist together, and B exists alone. In addition, the character "/" herein generally indicates that the front and rear associated objects are an "or" relationship.
Claims (10)
1. The preparation method is characterized in that the preparation method carries out biomineralization by mixing a precursor, magnetic nano particles and SA, and then carries out antibody modification on the biomineralization magnetic particles, thereby obtaining the flower-shaped immunomagnetic beads for separating the circulating tumor cells.
2. The method for preparing flower-like immunomagnetic beads for isolating circulating tumor cells according to claim 1, wherein the steps of the preparation method comprise:
s1, preparing magnetic nano particles, and placing the magnetic nano particles in a buffer solution to form a magnetic nano particle suspension;
s2, mixing the obtained magnetic nanoparticle suspension with SA solution and precursor solution, standing for 24-72h at room temperature to carry out biomineralization, and then taking out the biomineralized magnetic particles;
s3, performing antibody modification on the magnetic particles obtained in the step S2 to obtain flower-shaped immunomagnetic beads for separating the circulating tumor cells.
3. The method for preparing flower-like immunomagnetic beads for separating tumor cells according to claim 1, wherein the magnetic nanoparticles are NH 2 -Fe 3 O 4 。
4. The method for preparing flower-like immunomagnetic beads for separating circulating tumor cells according to claim 3, wherein said NH 2 -Fe 3 O 4 The preparation steps of (a) comprise:
s11, preparing Fe by using coprecipitation mode 3 O 4 A nanoparticle;
s12, fe to be prepared 3 O 4 Mixing the nano particles with anhydrous methanol and 3-aminopropyl triethoxysilane, performing ultrasonic treatment, and stirring at room temperature to obtain NH 2 -Fe 3 O 4 。
5. The method for preparing flower-like immunomagnetic beads for circulating tumor cell separation according to claim 2, wherein in step S2, the concentration of the magnetic nanoparticle suspension is 5mg/mL, and the amount is 15 μl;
the concentration of SA solution was 5mg/mL and the amount was 20. Mu.L.
6. The method for preparing flower-like immunomagnetic beads for separating circulating tumor cells according to claim 2, wherein the precursor is CuSO 4 The method comprises the steps of carrying out a first treatment on the surface of the The concentration of the precursor solution was 120mM, in an amount of 17.5. Mu.L.
7. The method for preparing flower-like immunomagnetic beads for isolating circulating tumor cells according to claim 2, wherein the content of the antibody modification in step S3 comprises: incubating the obtained biomineralization magnetic particles with biotin-modified epithelial cell adhesion molecule antibodies for 45-90 min; the precipitate was again adsorbed by magnet and washed repeatedly 3 times with PBS.
8. The method for preparing flower-like immunomagnetic beads for separating tumor cells according to claim 2, wherein the method for removing the biomineralized magnetic particles in step S2 is as follows: and (3) washing the obtained magnetic precipitate with ultrapure water for 3-5 times through the magnetic precipitate generated by magnet adsorption to obtain the magnetic particles.
9. The flower-shaped immunomagnetic beads for separating the circulating tumor cells are characterized in that the flower-shaped immunomagnetic beads are prepared by the preparation method according to any one of claims 1-8.
10. A method of using the flower-shaped immunomagnetic beads for separating circulating tumor cells of claim 9, wherein the content of the method of using comprises:
adding a certain amount of the flower-shaped immunomagnetic beads into PC-3 cell solution, and placing at 37deg.C and 0.5% CO 2 Incubating for 45min in a gently shaking cell incubator; then the precipitate is adsorbed by a magnet and washed, and the obtained precipitate is separatedCirculating tumor cells obtained.
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