A flow cytometry magnetic labeling microdevice based on ultrasound

Abstract

The invention discloses a flow cell magnetic labeling microdevice based on ultrasound, comprising a microfluidic chip and an ultrasonic device, wherein a first channel, a second channel, a mixing channel and a labeling channel are arranged inside the microfluidic chip, the first channel is used to inject a cell suspension, the second channel is used to inject a magnetic nanoparticle solution, the first channel and the second channel are respectively provided with injection ports on the surface of the microfluidic chip, the first channel and the second channel are both connected to the inflow end of the mixing channel, the outflow end of the mixing channel is connected to the inflow end of the labeling channel, the outflow end of the labeling channel is provided with a mixed liquid outlet on the surface of the microfluidic chip, and the flow area of ​​the mixing channel is smaller than the flow area of ​​the labeling channel; the ultrasonic device comprises a container and an ultrasonic transducer arranged at the bottom of the container, and the ultrasonic transducer is connected to a matching circuit. This scheme can continuously, quickly and safely carry out sufficient magnetic labeling of cells in batches, reducing the influence on the activity and proliferation function of the cells.

Description

Technical Field

[0001] The invention relates to the technical field of medical devices, and in particular to an ultrasound-based flow cell magnetic labeling microdevice. Background Art

[0002] Cell therapy is a promising strategy for the precision treatment of tumors, but it still faces difficulties in clinical application because the in vivo distribution, migration and targeting mechanisms of infused cells are unclear, and the effectiveness and safety of infused cells are difficult to directly verify. In order to solve the above problems, researchers have proposed a method of labeling cells with magnetic nanoparticles (MNs) so that magnetically labeled cells can be tracked using magnetic resonance imaging (MRI). Therefore, cell magnetic labeling technology is an important technology used in fields such as cell therapy and cell tracing. However, due to the non-permeability of cell membranes to magnetic nanoparticles, it remains a major challenge to quickly, safely and efficiently load magnetic nanoparticles into cells.

[0003] At present, there are three methods for magnetic labeling of cells: incubation, electroporation and sonoporation. Among them, the incubation method is a conventional strategy for loading magnetic nanoparticles. Cells are incubated with magnetic nanoparticles, and positively charged transfection agents need to be added to promote the transport of magnetic nanoparticles across the cell membrane. The incubation method is simple and easy, but very time-consuming. In addition, the transfection agents used are usually toxic to cells and are therefore not recommended. Electroporation is a method that applies a high electric field to transiently increase the permeability of the cell membrane and then increase the entry of magnetic nanoparticles across the cell membrane into the cell. Compared with the incubation method, the labeling speed of electroporation is faster, so better magnetic labeling results can be obtained in a shorter time. Sonoporation is a method based on the ultrasonic cavitation effect, which can temporarily and reversibly increase the permeability of the cell membrane, thereby improving the efficiency of cell uptake of genes, drugs and magnetic nanoparticles. Compared with electroporation, sonoporation not only has the advantages of short cell labeling time and high cell labeling efficiency, but also the range of cell damage and death is usually less than electroporation. Therefore, sonoporation can achieve relatively higher cell activity.

[0004] In the current sonoporation method, ultrasound is mostly applied directly to a mixture of cells and magnetic nanoparticles in a test tube. Therefore, cells cannot be labeled continuously and in batches. In recent years, microfluidic technology has developed rapidly, which can not only load substances into cells by precisely manipulating microcell samples, but also process cell samples continuously, quickly and efficiently. Although ultrasound-based microfluidic technology has made great progress in drug release, due to the relatively large size and potential toxicity of magnetic nanoparticles, there has been no report on the application of microfluidic technology to cell magnetic labeling so far.

[0005] Therefore, how to continuously, quickly and safely load magnetic nanoparticles into cells is a technical problem that technicians in this field currently need to solve. Summary of the invention

[0006] In view of this, the purpose of the present invention is to provide an ultrasound-based flow cytometry magnetic labeling microdevice, which can be used for continuous, rapid, safe and efficient cell magnetic labeling, and can also be expanded for the loading of cells with drugs or genes.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A flow cell magnetic labeling microdevice based on ultrasound, comprising:

[0009] A microfluidic chip, wherein a fluid channel is provided inside the microfluidic chip, wherein the fluid channel comprises a first channel, a second channel, a mixing channel and a labeling channel, wherein the first channel is used to inject a cell suspension, and the second channel is used to inject a magnetic nanoparticle solution, wherein the first channel has a cell suspension injection port on the surface of the microfluidic chip, and the second channel has a magnetic nanoparticle solution injection port on the surface of the microfluidic chip, wherein the outflow end of the first channel and the outflow end of the second channel are both connected to the inflow end of the mixing channel, and the outflow end of the mixing channel is connected to the inflow end of the labeling channel, and the outflow end of the labeling channel has a mixed liquid outlet on the surface of the microfluidic chip, and the flow area of ​​the mixing channel is smaller than the flow area of ​​the labeling channel;

[0010] The ultrasonic device comprises a container for accommodating the microfluidic chip and the connecting coupling agent and an ultrasonic transducer arranged at the bottom of the container, wherein the ultrasonic transducer is connected to a matching circuit.

[0011] Preferably, the mixing channel is a Tesla mixer structure.

[0012] Preferably, the marking channel comprises a plurality of straight channels arranged in parallel and connected end to end.

[0013] Preferably, the microfluidic chip includes a chip body and a cover sheet, one side surface of the chip body is provided with a recessed groove structure, the cover sheet fixedly covers the groove structure to form the fluid channel, and the other side surface of the chip body or the cover sheet is provided with the cell suspension injection port, the magnetic nanoparticle solution injection port and the mixed liquid outlet connected to the fluid channel.

[0014] Preferably, the chip body is made of polydimethylsiloxane or organic glass, and the cover sheet is a glass sheet.

[0015] Preferably, the maximum flow area of ​​the mixing channel is 0.5 mm×0.5 mm, and the maximum flow area of ​​the marking channel is 1.5 mm×1.5 mm.

[0016] Preferably, the container is an open vessel, a transducer mounting hole is provided at the bottom of the container, and the ultrasonic transducer is sealed and connected to the transducer mounting hole.

[0017] Preferably, the open container is a plastic container.

[0018] Preferably, the number of the first channels is one, and the number of the second channels is two.

[0019] Preferably, the center frequency of the ultrasonic transducer is 1 MHz.

[0020] In the ultrasound-based flow cytometry magnetic labeling microdevice provided by the present invention, a cell suspension is subjected to flow cytometry on a microfluidic chip, merged with a magnetic nanoparticle solution on the microfluidic chip and rapidly and evenly mixed in a mixing channel, and cell magnetic labeling is performed in a labeling area and with an ultrasound device.

[0021] The present invention has the following beneficial effects:

[0022] 1) This scheme combines ultrasound technology and microfluidics technology. The design of the flow cell allows for rapid and effective cell labeling. The magnetic labeling of the flow cell can be achieved within two minutes. The cells can be magnetically labeled in sufficient quantities in batches continuously, and a high average magnetic load of the cells can be achieved.

[0023] 2) The online mixing design can safely label cells and improve the integration of the chip, greatly reducing the impact on cell activity and proliferation function, simplifying the operation of cell magnetic labeling, and has the advantages of simple operation, high efficiency and high safety;

[0024] 3) In addition, the microdevice of the present invention can also be used to load cells, drugs or genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0026] Figure 1 Schematic diagram of the channel structure of the microfluidic chip in a specific embodiment of the present invention;

[0027] Figure 2is a schematic structural diagram of an ultrasonic device in a specific embodiment of the present invention;

[0028] Figure 3 is a longitudinal cross-sectional view of an ultrasonic device in a specific embodiment of the present invention;

[0029] Figure 4 Schematic diagram of the overall structure of the ultrasound-based flow cytometry magnetic labeling microdevice in a specific embodiment of the present invention;

[0030] Figure 5 It is a longitudinal cross-sectional view of an ultrasound-based flow cytometry magnetic labeling microdevice in a specific embodiment of the present invention.

[0031] Figures 1 to 5 middle:

[0032] 1-first channel, 11-cell suspension injection port, 2-second channel, 21-magnetic nanoparticle solution injection port, 3-mixing channel, 4-labeling channel, 5-mixed solution outlet, 6-microfluidic chip, 7-container, 8-ultrasonic transducer, 9-connection coupling agent. DETAILED DESCRIPTION

[0033] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] Please refer to Figures 1 to 5 , Figure 1 Schematic diagram of the channel structure of the microfluidic chip in a specific embodiment of the present invention; Figure 2 and Figure 3 They are respectively a structural schematic diagram and a longitudinal cross-sectional view of an ultrasonic device in a specific embodiment of the present invention; Figure 4 Schematic diagram of the overall structure of the ultrasound-based flow cytometry magnetic labeling microdevice in a specific embodiment of the present invention; Figure 5 It is a longitudinal cross-sectional view of an ultrasound-based flow cytometry magnetic labeling microdevice in a specific embodiment of the present invention.

[0035] The present invention provides an ultrasound-based flow cell magnetic labeling microdevice, comprising: a microfluidic chip 6 and an ultrasonic device, wherein a fluid channel is arranged inside the microfluidic chip 6, the fluid channel comprising a first channel 1, a second channel 2, a mixing channel 3 and a labeling channel 4, the first channel 1 is used to inject a cell suspension, the second channel 2 is used to inject a magnetic nanoparticle solution, the first channel 1 is provided with a cell suspension injection port 11 on the surface of the microfluidic chip 6, the second channel 2 is provided with a magnetic nanoparticle solution injection port 21 on the surface of the microfluidic chip 6, the outflow end of the first channel 1 and the outflow end of the second channel 2 are both connected to the inflow end of the mixing channel 3, the outflow end of the mixing channel 3 is connected to the inflow end of the labeling channel 4, the outflow end of the labeling channel 4 is provided with a mixed liquid outlet 5 on the surface of the microfluidic chip 6, and the flow area of ​​the mixing channel 3 is smaller than the flow area of ​​the labeling channel 4; the ultrasonic device comprises a container 7 for accommodating the microfluidic chip 6 and a connecting coupling agent 9, and an ultrasonic transducer 8 arranged at the bottom of the container 7, and the ultrasonic transducer 8 is connected to a matching circuit. When in use, the microfluidic chip 6 is located above the ultrasonic device, and the marking area of ​​the microfluidic chip 6 is located above the ultrasonic transducer 8. The marking channel 4 of the microfluidic chip 6 and the ultrasonic transducer 8 together constitute the ultrasonic marking area. The connection coupling agent 9 is contained in the container 7 and covers the marking channel 4 of the microfluidic chip 6, that is, the connection between the microfluidic chip 6 and the ultrasonic device is completed by the connection coupling agent 9 in the container 7. Among them, the connection coupling agent 9 is preferably ice water, and can also be water or other coupling agents.

[0036] It should be noted that this solution has no special restrictions on the shape and material of the microfluidic chip 6, as long as the actual operating conditions are met. Specifically, the microfluidic chip 6 can be designed in the shape of a rectangular parallelepiped, a square disk, a round disk, etc. The material of the microfluidic chip 6 can be polydimethylsiloxane (PDMS), or organic glass (PMMA), or glass, etc. Preferably, the shape of the microfluidic chip 6 is a rectangular parallelepiped, the material is polydimethylsiloxane, and the length, width and height are preferably 2.5cm×7.5cm×0.5cm. This solution can adjust the size of the microfluidic chip 6 by changing the depth and width of the fluid channel. The area of ​​the microfluidic chip 6 can be 15 to 25cm 2 , the thickness can be 4 to 8 mm.

[0037] It should be noted that a fluid channel is provided inside the microfluidic chip 6, and the channel depth is less than the chip thickness. This solution can realize the opening of a fluid channel inside the microfluidic chip 6 through a variety of processing methods or structural designs, for example, by molding a fluid channel structure in an integral chip board by injection molding, or by processing a groove structure on the surface of a plate, and then covering another layer of plate on the groove surface of the plate, using the groove between the two layers of plate to form a fluid channel structure, and so on. Preferably, in this solution, the microfluidic chip 6 includes a chip body and a cover sheet, a recessed groove structure is provided on one side surface of the chip body, and the cover sheet is fixed to cover the groove structure to form a fluid channel, and a cell suspension injection port 11, a magnetic nanoparticle solution injection port 21 and a mixed liquid outlet 5 connected to the fluid channel are provided on the other side surface of the chip body or the cover sheet. When in use, the side of the chip body provided with the groove structure can be placed downward. The cover sheet and the chip body can be fixed as a whole by gluing, hot pressing or bonding to form an integral chip structure. Further preferably, the material of the chip body is polydimethylsiloxane or organic glass, and the cover sheet is a glass sheet.

[0038] The microfluidic chip 6 mainly provides the following three functions: one of the functions is to realize flow cytometry, in which the cell suspension is continuously injected into the microfluidic chip 6, so that the cells flow in the microfluidic channel to form a flow cell, which is conducive to the continuous batch operation and processing of cells. In this scheme, by engraving channels on the microfluidic chip 6, the cell suspension is driven externally to flow in the channel. The second function is to realize on-chip mixing operation, in which the cell suspension and the magnetic nanoparticle solution are fully and evenly mixed in the mixing channel 3 at a certain flow ratio, so as to realize on-chip online mixing, reduce the toxic damage to the cells caused by long-term incubation of high-concentration magnetic nanoparticles with cells, and prepare for the next step of labeling. In this scheme, the mixing channel 3 preferably adopts a Tesla mixer structure, specifically an improved Tesla mixer, with a width range of 0.2mm to 0.5mm and a depth of 0.5mm. The specific structural schematic diagram is shown as follows Figure 1 As shown. The third function is to realize on-chip ultrasonic labeling. When the mixture of cells and magnetic nanoparticles passes through the labeling channel 4, it receives the ultrasonic waves emitted by the ultrasonic device and induces the cavitation effect, which causes the cells to temporarily and reversibly open holes to allow the magnetic nanoparticles to enter the cells, thereby realizing the magnetic labeling of the cells. In this scheme, the labeling area adopts a circuitous straight line channel design, that is, the labeling channel 4 provided in the microfluidic chip 6 is a circuitous straight line channel. The labeling channel 4 includes a plurality of straight line channels arranged in parallel and connected end to end. The purpose of such a setting is to prolong the residence time of the mixture of cells and magnetic nanoparticles in the labeling channel 4. Its width is 1.5mm and its depth is 1.5mm. The specific structural diagram is shown in Figure 1 shown.

[0039] Among them, the cross-sectional sizes of the mixing channel 3 and the marking channel 4 in the microfluidic chip 6 are different, that is, the flow areas of the two are different, the maximum flow area of ​​the mixing channel 3 is 0.5mm×0.5mm, and the maximum flow area of ​​the marking channel 4 is 1.5mm×1.5mm. When the injection flow rate is constant, since the flow area of ​​the mixing channel 3 is smaller than the flow area of ​​the marking channel 4, the liquid can obtain a relatively high flow rate in the mixing channel 3, which is conducive to the mixing of the cell suspension and the magnetic nanoparticle solution in the mixing channel 3; the cross-sectional area of ​​the marking channel 4 is larger than the mixing channel 3, so that a relatively low flow rate can be obtained, so that the residence time of the cell and magnetic nanoparticle mixture in the marking area is prolonged, that is, the ultrasonic treatment time of the cell and magnetic nanoparticle mixture is prolonged, so that large-sized magnetic nanoparticles can enter the cell. In other embodiments, the implementation mode of the mixing channel 3 and the marking channel 4 is variable, and the width and depth are variable. For example, the mixing channel 3 can also adopt a circuitous straight channel end-to-end structure, and the marking channel 4 can adopt a spiral channel structure, etc.

[0040] It should be noted that the present solution can be designed with a specific number of channels 1 and second channels 2 according to specific mixing requirements. Preferably, Figure 1 As shown, in this solution, the number of the first channel 1 is one, the number of the second channels 2 is two, and accordingly, there is one cell suspension injection port 11 and two magnetic nanoparticle solution injection ports 21.

[0041] The ultrasound-based flow cell magnetic labeling microdevice provided by the present invention includes an ultrasound device with a matching circuit. Figure 2As shown, the ultrasonic device includes an ultrasonic transducer 8, a container 7 and a matching circuit. Among them, the container 7 is used to hold the microfluidic chip 6 and the connection coupling agent 9. The container 7 is preferably designed as an open vessel, and the ultrasonic transducer 8 can be fixed to the bottom of the container 7 by means of a connector, adhesive fixation, etc. The material of the container 7 can be plastic, glass, organic glass, etc., and there is no special requirement for its source. Preferably, the open vessel in this scheme is a plastic dish. In addition, in order to reduce the ultrasonic loss caused by the ultrasonic wave passing through the bottom surface of the container 7, preferably, the present scheme has a transducer mounting hole at the bottom of the container 7, and the ultrasonic transducer 8 is sealed and connected to the transducer mounting hole. In this scheme, the diameter of the plastic dish is 8.5cm and the height is 1.5cm. Of course, this scheme can also design the plastic dish to other sizes according to the size of the chip and the ultrasonic transducer. The function of the ultrasonic transducer 8 is to convert electrical energy into ultrasonic waves, providing an ultrasonic source for the marking channel 4 of the microfluidic chip 6. In this solution, the ultrasonic transducer 8 adopts a piezoelectric ceramic transducer with a center frequency of 1 MHz and a diameter of 2.2 cm. In other embodiments, except that the center frequency of the ultrasonic transducer 8 is 1 MHz and the output power meets the requirements, there are no special restrictions on its type and source, and it can be purchased from the market. The function of the matching circuit is to maximize the ultrasonic output of the ultrasonic transducer 8 by resonant matching and impedance matching the ultrasonic transducer 8 and the interface device. In this solution, the matching circuit is designed by capacitors, inductors and resistors according to the actual conditions of the center frequency of the ultrasonic transducer 8 and the load impedance. In other embodiments, the matching circuit should be designed according to parameters such as the frequency, static capacitance and actual load impedance of the ultrasonic transducer, which will not be repeated in this article.

[0042] The ultrasound-based flow cell magnetic labeling microdevice provided by the present invention includes ice water (i.e., a connecting coupling agent 9) arranged between the ultrasonic device and the microfluidic chip 6. The ice water is placed in a plastic dish and covers the labeling channel 4 portion of the microfluidic chip 6. The ice water in the plastic dish has two main functions. One is to serve as a connecting coupling agent between the ultrasonic transducer 8 and the microfluidic chip 6, so that the ultrasonic wave generated by the ultrasonic transducer 8 can directly act on the labeling channel 4 of the microfluidic chip 6, so that the two together constitute an ultrasonic labeling area; the second is to use the good specific heat capacity of water as a thermal inhibitor of the ultrasonic transducer 8 to prevent the heating of the ultrasonic transducer 8 from affecting the cell activity. The ice water in the present invention is made by adding ice cubes to deionized water, and there is no special restriction on the source.

[0043] The ultrasound-based flow cell magnetic labeling microdevice provided by the present invention comprises a microfluidic chip 6 and an ultrasound device, such as Figure 4 and Figure 5In the present invention, the microfluidic chip 6 is placed above the ultrasonic device, wherein the side of the microfluidic chip 6 with the channel faces downward, and the opening of the plastic dish of the ultrasonic device faces upward. The microfluidic chip 6 is arranged to be inserted into the plastic dish of the ultrasonic device at an angle of about 5°, as shown in FIG. Figure 5 As shown, one end of the microfluidic chip 6 provided with the mixed liquid outlet 5 extends into the water, which can effectively suppress the standing wave effect, thereby facilitating the maximization of ultrasonic energy transmission. The marking area of ​​the microfluidic chip 6 is placed directly above the ultrasonic transducer 8 of the ultrasonic device, and together they constitute the ultrasonic marking area. When the mixed liquid of cells and magnetic nanoparticles passes through the marking area, it is subjected to ultrasonic treatment emitted by the ultrasonic device, thereby achieving the purpose of magnetic marking. In other embodiments, the placement of the microfluidic chip 6 and the ultrasonic transducer 8 should enable the ultrasonic wave to effectively act on the marking area of ​​the microfluidic chip 6.

[0044] In the present invention, the inlet of the microfluidic chip 6 is composed of a cell suspension injection port 11 and two magnetic nanoparticle solution injection ports 21. Wherein, when in use, the injection speed of the two magnetic nanoparticle solution injection ports 21 is set to be equal, and its flow rate is half of the cell suspension flow rate. The excitation signal of the ultrasonic device is mainly provided by a signal generator and a power amplifier, wherein the signal generator can provide a sinusoidal signal or a pulse signal, which is amplified by the power amplifier and drives the ultrasonic transducer 8 through a matching circuit. Through the continuously injected cell suspension and magnetic nanoparticle solution, the two are merged in the microfluidic chip 6, and then fully mixed through the mixing channel 3 so that the magnetic nanoparticles are evenly distributed around the cells, and then through the labeling channel 4, the cell membrane is temporarily opened under the cavitation induced by ultrasound to allow the magnetic nanoparticles to enter, thereby completing the cell magnetic labeling, and finally the mixed solution containing the magnetically labeled cells is collected from the mixed solution outlet 5, and the magnetically labeled cells can be obtained after centrifugation and washing, so that the cells are magnetically labeled continuously, quickly, safely and efficiently.

[0045] The ultrasound-based flow cell magnetic labeling microdevice provided by the present invention can not only perform cell magnetic labeling continuously and in batches, but also be expanded to be used for loading cells with drugs or genes.

[0046] In one embodiment, the preparation method of the microdevice is as follows. The preparation of the microfluidic chip 6: First, a three-dimensional model of the microfluidic channel is drawn by drawing software, then a PMMA material is used to process a raised channel pattern by a precision CNC machine tool to obtain a PMMA mold, and then a PDMS prepolymer is cast on the PMMA mold. After curing, separation, punching, cutting and other operations, the patterned side of the PDMS is finally bonded to the glass using a plasma bonding method to obtain a microfluidic chip 6. In other embodiments, commonly used microfluidic production methods can be used. Production of ultrasonic device: First, the position of the bottom of the plastic dish corresponding to the marked channel 4 of the microfluidic chip 6 is determined by placing the microfluidic chip 6, and a hole is drilled, and the diameter of the hole is consistent with the diameter of the ultrasonic transducer 8; then, the ultrasonic transducer 8 is combined with the drilled hole of the bottom of the plastic dish, wherein the surface of the ultrasonic transducer 8 is consistent with the height of the bottom of the plastic dish, and glue is used for sealing; then, the center frequency, static capacitance and actual load impedance parameters of the ultrasonic transducer 8 are measured by an impedance analyzer, and matching circuits are made using electronic components such as inductors and capacitors based on these parameters; finally, a proper amount of water is added to the plastic dish, and then one end of the microfluidic chip 6 provided with the mixed liquid outlet 5 is tilted downward by about 5° and placed in the plastic dish. In other embodiments, the microfluidic chip 6 and the ultrasonic device can be fastened and connected by glue.

[0047] Example 1

[0048] With attached Figures 1 to 5 A microfluidic chip, an ultrasonic device and a schematic diagram of the overall structure of a flow cell magnetic labeling microdevice based on ultrasound, including:

[0049] The length, width and height of the microfluidic chip 6 are 2.5cm×7.5cm×0.5cm cuboid chips; the materials of the microfluidic chip 6 are polydimethylsiloxane (PDMS) and glass. The microfluidic chip 6 includes a first channel 1 (cell suspension channel), a second channel 2 (magnetic nanoparticle solution channel), a mixing channel 3 and a labeling channel 4, wherein the mixing channel 3 is composed of an improved Tesla structure (width range of 0.2mm to 0.5mm, depth of 0.5mm), the labeling area is composed of a circuitous straight channel (straight channel width of 0.5mm, depth of 0.5mm), the overall length of the labeling area is 2cm, and the width is 2cm. A cell suspension injection port 11 is set, two magnetic nanoparticle solution injection ports 21 are set, and there is also a mixed liquid outlet 5.

[0050] The ultrasonic device includes an ultrasonic transducer 8, a plastic dish and a matching circuit. The plastic dish is combined with the ultrasonic transducer 8 by punching holes (the hole diameter is consistent with the diameter of the ultrasonic transducer 8). The ultrasonic transducer 8 is connected to the matching circuit for resonance matching and impedance matching. The matching circuit is designed according to parameters such as the center frequency of the ultrasonic transducer 8 and the actual load impedance, and is composed of capacitors, inductors and resistors.

[0051] The microfluidic chip 6 and the ultrasonic device are placed up and down, wherein the glass side of the microfluidic chip 6 faces downward, and the opening of the plastic dish in the ultrasonic device faces upward. One end of the microfluidic chip 6 containing the mixed liquid outlet 5 is set to be tilted downward by 5° and placed in the plastic dish of the ultrasonic device. The marking channel 4 of the microfluidic chip 6 is set to be placed just above the ultrasonic transducer 8 of the ultrasonic device, and together constitutes an ultrasonic marking area. The microfluidic chip 6 and the ultrasonic device are connected by adding ice water to the plastic dish to cover the marking area of ​​the microfluidic chip 6. The microfluidic chip 6 and the ultrasonic device are fastened by glue.

[0052] Example 2

[0053] The superparamagnetic iron oxide nanoparticles (SPIONs) labeling of DC2.4 cells was performed using an ultrasound-based flow cytometry magnetic labeling microdevice provided in Example 1. The specific process is as follows:

[0054] The signal generator and the power amplifier provide the ultrasonic device described in Example 1 with a sinusoidal wave excitation with a center frequency of 1 MHz and a signal strength of 6 W, so that the ultrasonic device emits ultrasonic waves with a center frequency of 1 MHz at a certain intensity.

[0055] The density is 10 6 A DC2.4 cell suspension with a concentration of 1.5 cell / mL was injected from the cell suspension injection port 11 provided in Example 1 at a flow rate of 75 μL / min, and a SPIONs solution with a concentration of 800 μg / mL was injected from the two magnetic nanoparticle solution injection ports 21 provided in Example 1 at a flow rate of 37.5 μL / min.

[0056] The injected cell suspension and SPIONs solution merge in the channel in the microfluidic chip 6, and then pass through the mixing channel 3 at a certain flow rate to be fully mixed, so that the SPIONs particles are evenly distributed around the cells to obtain a mixed solution of cells and SPIONs.

[0057] The mixed solution passes through the circuitous straight labeling channel 4 at a certain flow rate. In the labeling channel 4, the mixed solution receives ultrasonic treatment, and the cavitation effect induced by ultrasound allows the cell membrane to open holes temporarily and reversibly, allowing the SPIONs around the cells to enter the cells, thereby completing the SPIONs labeling of the cells. Among them, the water temperature in the plastic dish is continuously monitored, and the water temperature is maintained below 10°C by adding ice cubes, providing a safe microenvironment for cell survival.

[0058] After the cell suspension and SPIONs solution are stably run in the ultrasonic flow cytometry magnetic labeling microdevice provided in Example 1 of the present invention, the sample is collected at the outlet 5 of the mixed solution with a centrifuge tube, the cell pellet is obtained by centrifugation with a cell centrifuge, and then washed three times with phosphate buffered saline (PBS) to obtain magnetically labeled cells. The average magnetic load of the magnetically labeled cells, the activity of the cells, and the proliferation characteristics of the cells are tested and calculated respectively. The test results show that the SPIONs labeling of DC2.4 cells using the ultrasonic flow cytometry magnetic labeling microdevice provided in Example 1 of the present invention can complete the cell magnetic labeling within 2 minutes, the average magnetic load of the cells is above 19pg / cell, the cell survival rate is above 94%, and the normal proliferation characteristics of the cells are maintained above 92%. This result shows that the ultrasonic flow cytometry magnetic labeling microdevice provided in this scheme can continuously, quickly, safely and efficiently label cells without affecting the activity and basic functions of the cells.

[0059] The above embodiments are only for explaining the basic principle and preferred embodiments of the present invention, but the present invention is not limited by the above embodiments. Any replacement, change and improvement made without departing from the spirit and scope of the present invention shall be included in the protection scope of the present invention.

[0060] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A flow cytometry magnetic labeling microdevice based on ultrasound, characterized in that: include: A microfluidic chip (6), wherein a fluid channel is provided inside the microfluidic chip (6), wherein the fluid channel comprises a first channel (1), a second channel (2), a mixing channel (3) and a labeling channel (4), wherein the first channel (1) is used to inject a cell suspension, and the second channel (2) is used to inject a magnetic nanoparticle solution, wherein the first channel (1) is provided with a cell suspension injection port (11) on the surface of the microfluidic chip (6), and the second channel (2) is provided with a magnetic nanoparticle solution injection port (21) on the surface of the microfluidic chip (6), wherein the outflow end of the first channel (1) and the outflow end of the second channel (2) are both connected to the inflow end of the mixing channel (3), wherein the outflow end of the mixing channel (3) is connected to the inflow end of the labeling channel (4), wherein the outflow end of the labeling channel (4) is provided with a mixed liquid outlet (5) on the surface of the microfluidic chip (6), and the flow area of ​​the mixing channel (3) is smaller than the flow area of ​​the labeling channel (4); An ultrasonic device, comprising a container (7) for accommodating the microfluidic chip (6) and the connecting coupling agent (9), and an ultrasonic transducer (8) arranged at the bottom of the container (7), wherein the ultrasonic transducer (8) is connected to a matching circuit; The mixing channel (3) is a Tesla mixer structure; the marking channel (4) comprises a plurality of straight channels arranged in parallel and connected end to end; the maximum flow area of ​​the mixing channel (3) is 0.5 mm×0.5 mm, and the maximum flow area of ​​the marking channel (4) is 1.5 mm×1.5 mm.

2. The ultrasound-based flow cytometry magnetic labeling microdevice according to claim 1, characterized in that: The microfluidic chip (6) comprises a chip body and a cover sheet, wherein a recessed groove structure is provided on one side surface of the chip body, and the cover sheet fixedly covers the groove structure to form the fluid channel, and the cell suspension injection port (11), the magnetic nanoparticle solution injection port (21) and the mixed solution outlet (5) which are connected to the fluid channel are provided on the other side surface of the chip body or the cover sheet.

3. The ultrasound-based flow cytometry magnetic labeling microdevice according to claim 2, characterized in that: The chip body is made of polydimethylsiloxane or organic glass, and the cover sheet is a glass sheet.

4. The ultrasound-based flow cytometry magnetic labeling microdevice according to claim 1, characterized in that: The container (7) is an open vessel, a transducer mounting hole is provided at the bottom of the container (7), and the ultrasonic transducer (8) is sealed and connected to the transducer mounting hole.

5. The ultrasound-based flow cytometry magnetic labeling microdevice according to claim 4, characterized in that: The open container is a plastic container.

6. The ultrasound-based flow cytometry magnetic labeling microdevice according to claim 1, characterized in that: The number of the first channel (1) is one, and the number of the second channel (2) is two.

7. The ultrasound-based flow cytometry magnetic labeling microdevice according to any one of claims 1 to 6, characterized in that: The central frequency of the ultrasonic transducer (8) is 1 MHz.

Images