CN116344189A - Microfluidic modification method of magnetic nanoparticles - Google Patents
Microfluidic modification method of magnetic nanoparticles Download PDFInfo
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Abstract
The invention discloses a microfluidic modification method of magnetic nanoparticles, which is based on a microfluidic system, prepares ferroferric oxide magnetic nanoparticles with uniform size and coated with a silicon dioxide shell layer, introduces monodisperse ferroferric oxide magnetic nanoparticles and a silicon precursor into a microfluidic device, and modifies the magnetic nanoparticles by combining passive diffusion mixing of the microfluidic system with active ultrasonic mixing. The core-shell structure of the magnetic nanoparticle is formed based on hydrolysis reaction, the precise adjustment of the thickness of the silicon dioxide shell layer is realized by changing the microfluidic modification conditions, the preparation method is simple and easy to implement, the requirements on the reaction conditions are low, and the morphology of the core-shell structure can be easily and precisely adjusted; the novel core-shell structure magnetic nanoparticle can be used for preparing materials for capturing circulating tumor cells due to the active surface, and the micro-fluidic system combined by active and passive mixing can be used for modifying the magnetic nanoparticle by various substances.
Description
Technical Field
The invention relates to the biomedical field, in particular to a microfluidic modification method of magnetic nanoparticles.
Background
Ferroferric oxide (Fe) 3 O 4 ) The magnetic nano particles have the advantages of stable property, high magnetic susceptibility, low biotoxicity, high responsiveness and the like, and are widely applied to targeted drug delivery, cell enrichment, biological imaging, biological sensing and the like, so that the magnetic nano particles become one of the most popular magnetic materials in the biomedical field. However, common Fe 3 O 4 The inert surface of magnetic nanoparticles is greatly limited in its application range due to the lack of functional groups or antibodies that have high affinity for the substance being loaded or the target cells or that can specifically bind. Thus, for common Fe 3 O 4 The magnetic nano particles are subjected to surface modification, and have important significance for improving the specific recognition capability, stability and biocompatibility.
Although the traditional macroscopic method can also finish Fe 3 O 4 Modification of magnetic nanoparticles has the defects of low yield, long time consumption, incapability of accurately regulating and controlling reaction, poor repeatability and the like. The micro-fluidic technology which is emerging in recent years is a technology which penetrates through the fields of biology, chemistry and material science, can carry out high integration and good control on fluid in the micrometer scale range, accords with the trend of the precise miniaturization of the current instrument, and can be Fe 3 O 4 The magnetic nanoparticles provide a good modification platform.
Microfluidics refers to the science and technology involved in systems that use microtubes to process or manipulate microscopic fluids, which are capable of continuously processing small-scale fluids in microtubes with internal diameters from tens to hundreds of microns, and which can perform high-throughput experiments requiring precise control. The existence of passive mixing behavior among laminar flows in a microfluidic system, high integration, automation and excellent control of the fluid can ensure uniformity of the morphology structure of the product, so that the microfluidic system has competitiveness in mass production and modification of materials.
In recent years, magnetic nanoparticles having different compositions and morphologies have been widely used in the biomedical field. The optical, magnetic, chemical and other properties of the magnetic nanoparticles can be changed along with the change of the components and the morphology, such as photo-thermal efficiency, drug loading rate and the like, wherein the surface activity of the magnetic nanoparticles has great influence on the adaptation degree of biological materials to biological systems, the active surface is favorable for drug loading and functional group grafting, and the drug delivery efficiency and the targeting property of the materials can be greatly improved. Therefore, great attention and research are paid to the modification of magnetic nano particles, and opportunities are provided for the development of novel multifunctional materials.
In general, modification of the shell layer on the surface of the magnetic nanoparticle requires a long loading and mechanical stirring process to prevent formation of a low-dispersion sample. At present, various macroscopic methods for modifying the surface of magnetic nanoparticles have been generated, but the generation of controllable and structural microspheres with precisely controllable morphology still faces technical challenges.
Disclosure of Invention
The invention provides a microfluidic modification method of magnetic nanoparticles, which aims at the defects in the prior art.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
a microfluidic modification method of magnetic nanoparticles, comprising the steps of:
step one, taking an organic solution containing a surfactant as a solvent, mixing the solvent with an iron source, and preparing monodisperse magnetic nano particles by a solvothermal method;
and step two, constructing a microfluidic chip, dispersing the monodisperse magnetic nanoparticles prepared in the step one in an alkaline ethanol solution to serve as an external phase, taking an ethanol solution of a silicon precursor as an internal phase, respectively introducing the external phase and the internal phase into a microfluidic pipeline, and actively mixing by ultrasound to prepare the magnetic nanoparticles with a core-shell structure. The silicon precursor is hydrolyzed in alkaline environment to form a silicon dioxide layer coating the magnetic ferroferric oxide nano particles, and the thickness of the shell layer of the magnetic nano particles is adjusted by changing the concentration of reactants in the internal phase and the external phase, the speed ratio of the internal phase and the external phase or the reaction time.
In order to optimize the technical scheme, the specific measures adopted further comprise:
further, in the first step, the solvent includes ethylene glycol (solvent), anhydrous sodium acetate (mineralizer), poly (4-styrenesulfonic acid-co-maleic acid) sodium salt (surfactant), ascorbic acid (antioxidant) and water (dispersant), and the iron source is anhydrous ferric trichloride.
Further, the preparation method of the monodisperse magnetic nanoparticle comprises the following steps: after mixing 16mL of ethylene glycol, 0.26g of anhydrous ferric trichloride, 1.20g of anhydrous sodium acetate, 0.40g of poly (4-styrenesulfonic acid-co-maleic acid) sodium salt, 45mg of ascorbic acid and 20 mu L of water uniformly, adding 0.24g of sodium hydroxide as an alkali source, mixing, adding into a reaction kettle, and heating at 190 ℃ for 9 hours to prepare the monodisperse magnetic nanoparticle.
In the second step, the microfluidic chip is assembled by a glass capillary, a glass slide, a cover slip, a sample application needle, quick-drying glue and a microfluidic pipeline, wherein the glass capillary is assembled by coaxially nesting an external phase capillary and an internal phase capillary; the diameter of the outer phase capillary tube is 2000 mu m, the diameter of the inner phase capillary tube is 800 mu m, and the diameter of the microfluidic pipeline is 2000 mu m and the length of the microfluidic pipeline is about 3.34m.
Further, in the second step, the alkaline ethanol solution comprises water, ammonia water and ethanol; the addition amount of the water is 0.5mL; the concentration range of the ammonia water is 0-1.5 mg/mL, and the thickness of the silica shell layer on the surface of the magnetic nano particle is adjusted, preferably 0.803mg/mL, by changing the concentration of the ammonia water in the external phase within the range of 0-1.5 mg/mL; the addition amount of the ethanol is 10mL; the addition amount of the monodisperse magnetic nano particles is 0-2.0 mg, and the thickness of the silicon dioxide shell layer on the surfaces of the magnetic nano particles is adjusted, preferably 2.0mg, by changing the addition amount of the magnetic nano particles in the external phase within the range of 0-2.0 mg.
Further, in the second step, the silicon precursor is tetraethyl orthosilicate, the concentration of the tetraethyl orthosilicate is 0-0.4 mg/mL, and the thickness of a silicon dioxide shell layer on the surface of the magnetic nano particle is adjusted, preferably 0.179mg/mL, by changing the adding amount of the tetraethyl orthosilicate in the internal phase within the range of 0-0.4 mg/mL; the addition amount of the ethanol is 5mL.
Further, in the second step, the external phase and the internal phase are respectively represented by 6:1 into a microfluidic chip, and immersing the microfluidic pipeline in a spiral water bath.
Further, the reaction time is 90min, and the thickness of the silica shell on the surface of the magnetic nanoparticle can be adjusted by changing the reaction time.
Further, the monodisperse magnetic nanoparticle prepared in the first step and the magnetic nanoparticle with the core-shell structure prepared in the second step are respectively washed and magnetically separated to remove surface residual liquid, so as to obtain the purified monodisperse magnetic nanoparticle and the purified magnetic nanoparticle with the core-shell structure.
The magnetic nano particle with the core-shell structure is obtained by the preparation method.
The beneficial effects of the invention are as follows:
1) Unlike traditional mechanical stirring and method of completing the silicon coating of magnetic nanometer particle by using micro-fluidic technology, the preparation method of the invention is based on micro-fluid passive diffusion mixing mode, and the active diffusion mixing is initiated by the power provided by sonic chemistry, thereby achieving the aim of high-efficiency and accurate modification of magnetic nanometer particle; firstly, the microfluidic technology is selected to accurately control the reaction process; secondly, since the fluid generally flows according to a laminar flow state under the microscale, the material exchange and the fluid mixing only depend on molecular diffusion, and the ultrasonic wave is added to realize the external energy input to realize the active mixing; microbubbles filled with gas in the liquid form cavitation microfluid under the ultrasonic action, and stirring and disturbance in the fluid are initiated to accelerate and enhance the mixing process; in addition, the ultrasonic treatment can also avoid the oxidation and agglomeration of the magnetic nano particles, and can improve the hydrolysis efficiency of TEOS, so that the monodisperse core-shell structure magnetic nano particles can be obtained rapidly and efficiently; the preparation method has the advantages of precise regulation and control of the micro-fluidic technology on the reaction programming, high efficiency, rapidness and uniformity of the sonic chemistry, simple and easy preparation process and low reaction condition requirements;
2) According to the invention, a microfluidic chip is adopted for coaxial preparation by means of a microfluidic technology, the channels are simple, the construction steps are few, a complex machining process is not needed, the process is simple, the size of the magnetic nano particles with the core-shell structure and the thickness of the silicon dioxide shell layer can be adjusted through the flow velocity of the inner phase and the outer phase, the concentration of reaction liquid in the inner phase and the outer phase or the reaction time, and the operation is convenient;
3) According to the invention, the precise adjustment of the thickness of the silicon dioxide shell layer of the magnetic nano particle with the core-shell structure can be easily realized by changing the flow rate of the inner phase and the outer phase and the concentration or the reaction time of the reaction liquid in the inner phase and the outer phase, the inorganic shell layer of the magnetic nano particle with the core-shell structure is beneficial to increasing the biocompatibility of the particle and improving the surface activity, the affinity and the specific recognition capability for medicines or cells can be greatly increased, and the target substance is efficiently combined, so that the novel magnetic nano particle with the core-shell structure can be artificially and precisely adjusted, and has good application prospects in the aspects of medicine delivery, cell enrichment analysis and the like;
4) The core-shell structure magnetic nanoparticle prepared by the invention can be further functionalized by ammoniation, folic acid grafting and the like so as to adapt to more various application requirements.
Drawings
FIG. 1 is a process flow diagram of a microfluidic modified magnetic nanoparticle of the present invention;
FIG. 2 is an image of magnetic nanoparticles before and after microfluidic modification, wherein FIG. (a) and FIG. (b) are Fe taken by a transmission electron microscope, respectively 3 O 4 Magnetic nanoparticle and core-shell structure Fe 3 O 4 @SiO 2 Images of magnetic nanoparticles, panels (c) and (d) are Fe photographed by a field emission scanning electron microscope, respectively 3 O 4 Magnetic nanoparticle and core-shell structure Fe 3 O 4 @SiO 2 An image of the magnetic nanoparticles;
FIG. 3 is a view of the generation of microfluidic modified core-shell magnetic nanoparticles observed under a field emission electron microscope, wherein the views (a) to (f) are images taken 5min after the reaction starts, 10min after, 15min after, 30min after, 60min after, 120min after, respectively;
FIG. 4 is a graph showing the relationship between the reaction time, the velocity ratio of internal and external phases, the TEOS concentration of external phases, and the concentration of internal phase ammonia water with the particle size of magnetic nanoparticles having a core-shell structure, wherein the graph (a) is a graph showing the relationship between the reaction time and the particle size, the graph (b) is a graph showing the relationship between the velocity ratio of internal and external phases and the particle size, the graph (c) is a graph showing the relationship between the TEOS concentration of external phases and the particle size, and the graph (d) is a graph showing the relationship between the concentration of internal phase ammonia water and the particle size;
FIG. 5 is an image of a magnetic nanoparticle coated with an erythrocyte membrane, wherein FIG. (a) is a view of Fe in a core-shell structure coated with an erythrocyte membrane, which is photographed by a field emission scanning electron microscope 3 O 4 Image of RMBC magnetic nanoparticles, plot (b) is Fe 3 O 4 Core-shell structure Fe wrapped by magnetic nano particles and erythrocyte membrane 3 O 4 Particle size and surface potential of RMBC magnetic nanoparticles;
FIG. 6 shows core-shell magnetic nanoparticles (Fe 3 O 4 @SiO 2 -FA) and Folic Acid (FA) and Fe 3 O 4 @SiO 2 Is an infrared contrast plot of (2);
FIG. 7 is a view showing the images before and after capturing Hela cells, wherein the images (a) to (b) are fluorescence microscope images (light parts in the figure) before and after capturing Hela cells pre-stained with calcein; FIGS. (c) - (d) show the cell surfaces before and after capture as observed by a field emission scanning electron microscope.
Detailed Description
Example 1: preparation of core-shell structure magnetic nanoparticle
The preparation process of the microfluidic modified core-shell structure magnetic nanoparticle is shown in figure 1, and specifically comprises the following steps:
1) Preparation of Fe 3 O 4 Monodisperse magnetic nanoparticles:
1.1 16mL of ethylene glycol, 0.26g of anhydrous ferric trichloride, 1.20g of anhydrous sodium acetate, 0.40g of sodium poly (4-styrenesulfonic acid-co-maleic acid) salt, 45mMixing ascorbic acid and 20 μl water uniformly, adding 0.24g sodium hydroxide as alkali source, thoroughly mixing, adding into a reaction kettle, heating at 190 deg.C for 9 hr to obtain Fe 3 O 4 Monodisperse magnetic nanoparticles;
1.2 The prepared monodisperse magnetic nano particles dispersed in the reaction liquid are washed by ethanol and deionized water and magnetically separated in sequence to remove surface residual liquid, and are dispersed in 12mL of deionized water, so that the purified monodisperse magnetic nano particle dispersion liquid is obtained.
2) Preparing an internal and external phase solution and a collecting solution:
2.1 Internal phase solution): consists of tetraethyl orthosilicate (TEOS)/ethanol solution; tetraethyl orthosilicate is used as a silicon precursor, a tetraethyl orthosilicate solution is adopted, the concentration of the tetraethyl orthosilicate solution dissolved in ethanol is 0.179mg/mL, and tetraethyl orthosilicate (TEOS)/ethanol solutions (0 mg/mL, 0.0448mg/mL, 0.0896mg/mL, 0.1341mg/mL, 0.1792mg/mL and 0.3576 mg/mL) with different concentration gradients within the range of 0-0.4 mg/mL are respectively prepared;
2.2 External phase solution): from ammonia (NH) 3 ) Magnetic nanoparticle/water/ethanol solution composition; ammonia water is used as alkaline hydrolysis agent, ammonia water solution is adopted, the concentration of the ammonia water dissolved in ethanol is 0.803mg/mL, and ammonia water (NH) with different concentration gradients within the range of 0-1.5 mg/mL is respectively prepared 3 ) Magnetic nanoparticle/water/ethanol solution (0.277 mg/mL, 0.533mg/mL, 0.803mg/mL, 1.070mg/mL, 1.338 mg/mL); the magnetic nano particles are used as cores of core-shell structures, and the concentration of the magnetic nano particles dissolved in ethanol is 0.2mg/mL; water was used as the hydrolyzer, ultrapure water was used, and the concentration dissolved in ethanol was 5%.
3) Assembling a two-phase microfluidic chip: two kinds of glass capillaries with different sizes are selected, the diameter of the inner phase glass capillary is 800 mu m, and the diameter of the outer phase glass capillary is 2000 mu m; the double-phase microfluidic chip is formed by assembling an inner-phase glass capillary tube, an outer-phase glass capillary tube, a glass slide, a sample application needle head and quick-drying glue, wherein the inner-phase glass capillary tube and the outer-phase glass capillary tube are coaxially nested and assembled.
4) Preparing core-shell structure magnetic nano particles: the internal and external phase solution is extracted into glass syringes of corresponding specifications and respectively placed on two microinjection pumps, the glass syringes are connected with a double-phase microfluidic chip through polyethylene pipes, an outlet of the microfluidic chip is connected with a section of polyethylene pipe with the length of 3.34m to serve as a reaction microchannel, and the reaction microchannel is placed in an ultrasonic water bath kettle. Setting the flow rate of the inner phase and the outer phase, starting the micro injection pump to work, and starting the ultrasonic. In the microfluidic channel, when the inner phase fluid and the outer phase fluid meet, the inner phase solution and the outer phase solution are fully mixed and hydrolysis reaction occurs to form the core-shell structure magnetic nano particles under the combined action of diffusion mixing caused by laminar flow and active mixing caused by ultrasound.
FIG. 2 is an electron microscope image of magnetic nanoparticles before and after microfluidic modification, wherein graphs (a) and (b) are Fe photographed by a transmission electron microscope, respectively 3 O 4 Magnetic nanoparticle and core-shell structure Fe 3 O 4 @SiO 2 Images of magnetic nanoparticles, figures (c) and (d) are Fe taken by field emission scanning electron microscopy, respectively 3 O 4 Magnetic nanoparticle and core-shell structure Fe 3 O 4 @SiO 2 As can be seen from fig. 2 (b) and fig. 2 (d), the image of the magnetic nanoparticle, core-shell structure Fe 3 O 4 @SiO 2 The magnetic nano particles are generated in the two-phase microfluidic chip channel, and the core-shell structure magnetic nano particles have uniform size and good dispersibility. Controlling other factors to be unchanged, setting sampling time points to be 5min, 10min, 15min, 30min, 60min, 90min and 120min respectively, and carrying out electron microscope characterization on the core-shell structure magnetic nano particles prepared by adopting the different time points by using a field emission scanning electron microscope, wherein the result is shown in a figure 3, and the reaction time corresponding to the figures (a) - (f) is (a): 5min, (b): 10mg/mL, (c): 15mg/mL, (d): 30mg/mL, (e) 60mg/mL, (f): 120mg/mL, as can be seen from FIG. 4 (a), the particle size of the core-shell magnetic nanoparticle increases with the increase of the reaction time in the range of 5 to 120 min; therefore, the adjustment of the particle size of the core-shell structure magnetic nano particles can be realized by changing the reaction time.
The flow rate of the external phase is controlled to be 6mL/h, other factors are unchanged, the flow rate of the internal phase is respectively set to be 0.1mL/h, 0.3mL/h, 0.6mL/h, 0.8mL/h, 1mL/h and 1.2mL/h, and as can be seen from FIG. 3 (b), the particle size of the core-shell structure magnetic nanoparticle is increased along with the increase of the flow rate of the internal phase within the range of 0.1-1.2 mL/h; the results show that the particle size of the core-shell structure magnetic nano particles can be adjusted by changing the velocity ratio of the internal phase flow and the external phase flow.
Control of external phase ammonia (NH) 3 ) The concentration and other factors were unchanged, and the internal phase tetraethyl orthosilicate (TEOS) concentration was set to 0mg/mL, 0.0448mg/mL, 0.0896mg/mL, 0.1341mg/mL, 0.1792mg/mL and 0.3576mg/mL, respectively, as the silicon precursor concentration was changed, so too was the particle size of the core-shell magnetic nanoparticles. As can be seen from fig. 3 (c), the particle size of the core-shell magnetic nanoparticle increases with increasing concentration of the inner phase tetraethyl orthosilicate (TEOS). Therefore, the concentration of the external phase silane is changed, and the particle size of the core-shell structure magnetic nano particle can be adjusted.
The concentration of tetraethyl orthosilicate (TEOS) in the internal phase and other factors are controlled to be unchanged, and the ammonia water (NH) in the external phase 3 ) The concentration of the external phase alkaline hydrolysis agent is respectively set to be 0.277mg/mL, 0.533mg/mL, 0.803mg/mL, 1.070mg/mL and 1.338mg/mL, so that the silicon shell generation speed is accelerated and the particle size of the magnetic nano particles with the core-shell structure is increased. As can be seen from fig. 3 (d), the particle size of the core-shell magnetic nanoparticle follows the external phase ammonia (NH 3 ) Increasing the concentration; therefore, the particle size of the core-shell magnetic nano particle can be adjusted by changing the concentration of the internal phase alkaline hydrolysis agent.
Example 2: erythrocyte membrane-coated magnetic nanoparticle experiment
1) Preparing an internal and external phase solution and a collecting solution:
1.1 Internal phase solution): consists of erythrocyte membrane (RMBC)/Phosphate Buffer (PBS); the erythrocyte membrane is taken as a biological organic material, rat erythrocytes are adopted, and erythrocyte membranes (RMBC)/Phosphate Buffer Solution (PBS) with different concentration gradients within the range of 0-0.519 mg/mL (0 mg/mL, 0.103mg/mL, 0.207mg/mL, 0.311mg/mL, 0.415mg/mL and 0.519 mg/mL) are respectively prepared;
1.2 External phase solution): consists of magnetic nanoparticle/Phosphate Buffer (PBS); magnetic nanoparticle/Phosphate Buffer (PBS) solutions (0.546 mg/mL, 1.323mg/mL, 2.906mg/mL, 4.463mg/mL, and 6.443 mg/mL) with different concentration gradients were prepared using the monodisperse magnetic nanoparticle prepared in step 1) of example 1.
2) Assembling a two-phase microfluidic chip: the dual-phase microfluidic chip was assembled as in step 3) of example 1 above.
3) Preparing magnetic nano particles wrapped by erythrocyte membranes: the internal and external phase solution is extracted into glass syringes of corresponding specifications and respectively placed on two microinjection pumps, the glass syringes are connected with a double-phase microfluidic chip through polyethylene pipes, an outlet of the microfluidic chip is connected with a section of polyethylene pipe with the length of 3.34m to serve as a reaction microchannel, and the reaction microchannel is placed in an ultrasonic water bath kettle. Setting the flow rate of the inner phase and the outer phase, starting the micro injection pump to work, and starting the ultrasonic. In the microfluidic channel, when the inner phase fluid and the outer phase fluid meet, the inner phase solution and the outer phase solution are fully mixed under the combined action of diffusion mixing caused by laminar flow and active mixing caused by ultrasound, so that the magnetic nano particles wrapped by the erythrocyte membrane are formed.
FIG. 5 is an image of a magnetic nanoparticle coated with an erythrocyte membrane, wherein FIG. (a) is a view of Fe in a core-shell structure coated with an erythrocyte membrane, which is photographed by a field emission scanning electron microscope 3 O 4 Image of RMBC magnetic nanoparticles, plot (b) is Fe 3 O 4 Core-shell structure Fe wrapped by magnetic nano particles and erythrocyte membrane 3 O 4 As can be seen from FIG. 5 (a) and FIG. 5 (b), the particle size and surface potential of the @ RMBC magnetic nanoparticle are that Fe of core-shell structure is encapsulated by erythrocyte membrane 3 O 4 The @ RMBC magnetic nano-particles are generated in the two-phase microfluidic chip channel, and the sizes of the magnetic nano-particles wrapped by the erythrocyte membranes are uniform.
Example 3: core-shell structure magnetic nanoparticle grafted folic acid experiment
To adapt the core-shell magnetic nanoparticles to a wider range of applications, the core-shell magnetic nanoparticles prepared in example 1 were dispersed in basic aminopropyl triethoxysilane (APTES) and subjected to mechanical stirring for 4 hours to complete amino modification; because the amino modified core-shell structure magnetic nano particles still have magnetism, the magnetic nano particles are separated by a magnetic separation method, and then ethanol and ultrapure water are added for washing for a plurality of times after the supernatant is removed; after washing completely, dispersing amino modified core-shell structure magnetic nano particles in the solution of NHydroxysuccinimide (NHS), 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC) and Folic Acid (FA) in dimethyl sulfoxide (DMSO) and stirred for 24 hours with time to graft folic acid. Because the core-shell structure magnetic nano particles grafted with folic acid still have magnetism, the magnetic nano particles are separated by a magnetic separation method, and then ethanol and ultrapure water are added for washing for a plurality of times after the supernatant is removed. The surface modification of the core-shell structure magnetic nano particle is realized by grafting folic acid, so that the folic acid specificity of the core-shell structure magnetic nano particle is endowed to adapt to more various application requirements; FIG. 6 shows core-shell magnetic nanoparticles (abbreviated as Fe) with folic acid grafted on the surface 3 O 4 @SiO 2 FA) IR image, fe after grafting folic acid 3 O 4 @SiO 2 FA has the same infrared absorption peak as folic acid, which shows that the core-shell magnetic nano particle grafted with folic acid on the surface is endowed with folic acid specificity.
Example 4: core-shell structured magnetic nanoparticles with folic acid grafted on surface for capturing experiment of Circulating Tumor Cells (CTC)
1) The core-shell structured magnetic nanoparticle for capturing circulating tumor cells prepared in example 2, which is prepared by taking human cervical cancer cells Hela tumor cells as target cells, is added into a Hela cell suspension, incubated for 30 minutes at 37 ℃, and then the uncaptured redundant Hela cells are removed by washing with a phosphate buffer solution.
2) For ease of observation and counting, cells in step (1) were stained with Calcein, 1mM Calcein (Calcetin-AM) solution was prepared with dimethyl sulfoxide (DMSO), and diluted with medium to 50. Mu.M Calcein-AM solution; adding 1/20 of the volume of the Calcein-AM solution to the cell culture medium, and culturing the cells at 37 ℃ for 20 minutes; washing of captured cells with phosphate buffered saline solution 3 O 4 @SiO 2 FA twice; then, fluorescence images before and after the addition of the magnetic field, i.e., before and after the capturing of the cells, are observed by a fluorescence microscope, as shown in fig. 7 (a) to (b), calcein-stained Hela cells can be attracted by the magnetic field and emit bright green fluorescence (bright part in the figure); observing the cell surface before and after capturing by using a field emission scanning electron microscopeAs shown in FIGS. 7 (c) to (d), the captured Hela cells had specifically bound Fe on the surface 3 O 4 @SiO 2 -FA。
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 examples, and all technical solutions belonging to the concept of the present invention belong to the protection scope of the present invention. It should be noted that modifications and adaptations to the invention without departing from the principles thereof are intended to be within the scope of the invention as set forth in the following claims.
Claims (10)
1. The microfluidic modification method of the magnetic nano particles is characterized by comprising the following steps of:
step one, taking an organic solution containing a surfactant as a solvent, mixing the solvent with an iron source, and preparing monodisperse magnetic nano particles by a solvothermal method;
and step two, constructing a microfluidic chip, dispersing the monodisperse magnetic nanoparticles prepared in the step one in an alkaline ethanol solution and taking the alkaline ethanol solution as an external phase, taking the ethanol solution of a silicon precursor as an internal phase, respectively introducing the external phase and the internal phase into a microfluidic pipeline, and actively mixing by ultrasound to prepare the magnetic nanoparticles with a core-shell structure.
2. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
in the first step, the solvent comprises ethylene glycol, anhydrous sodium acetate, sodium salt of poly (4-styrenesulfonic acid-co-maleic acid), ascorbic acid and water, and the iron source is anhydrous ferric trichloride.
3. The microfluidic modification method of magnetic nanoparticles according to claim 2, wherein the preparation method of the monodisperse magnetic nanoparticles comprises the following steps: after mixing 16mL of ethylene glycol, 0.26g of anhydrous ferric trichloride, 1.20g of anhydrous sodium acetate, 0.40g of poly (4-styrenesulfonic acid-co-maleic acid) sodium salt, 45mg of ascorbic acid and 20 mu L of water uniformly, adding 0.24g of sodium hydroxide as an alkali source, mixing, adding into a reaction kettle, and heating at 190 ℃ for 9 hours to prepare the monodisperse magnetic nanoparticle.
4. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
in the second step, the microfluidic chip is assembled by a glass capillary, a glass slide, a cover glass, a sample application needle, quick-drying adhesive and a microfluidic pipeline, wherein the glass capillary is assembled by coaxially nesting an outer phase capillary and an inner phase capillary; the diameter of the outer phase capillary tube is 2000 mu m, the diameter of the inner phase capillary tube is 800 mu m, and the diameter of the microfluidic pipeline is 2000 mu m and the length of the microfluidic pipeline is 3.34m.
5. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
in the second step, the alkaline ethanol solution comprises water, ammonia water and ethanol; the addition amount of the water is 0.5mL, the concentration range of the ammonia water is 0-1.5 mg/mL, the addition amount of the ethanol is 10mL, and the addition amount of the monodisperse magnetic nano particles is 0-2.0 mg.
6. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
in the second step, the silicon precursor is tetraethyl orthosilicate, the concentration of the tetraethyl orthosilicate is 0-0.4 mg/mL, and the addition amount of the ethanol is 5mL.
7. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
in the second step, the external phase and the internal phase are respectively 6:1 into a microfluidic chip, and immersing the microfluidic pipeline in a spiral water bath.
8. The microfluidic modification method of magnetic nanoparticles according to claim 7, wherein the reaction time is 90min.
9. A microfluidic modification method for magnetic nanoparticles according to claim 1, wherein,
and (3) respectively cleaning and magnetically separating the monodisperse magnetic nanoparticles prepared in the step (A) and the magnetic nanoparticles with the core-shell structure prepared in the step (B) to obtain purified monodisperse magnetic nanoparticles and purified magnetic nanoparticles with the core-shell structure.
10. A magnetic nanoparticle having a core-shell structure obtained by the method according to any one of claims 1 to 9.
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