Injection device and method for particles

Abstract

The invention relates to an injection device and method for particles, and the device comprises a liquid environment which is used for placing a carrier, and the particles are attached to the carrier; the special ultrasonic device is located in a liquid environment so as to generate a sound beam current in the liquid environment; the liquid supply flow channel is used for conveying an injection substance into a sound beam of the special ultrasonic device; the special ultrasonic device and the particles can be in a relatively fixed state, and the particles are located in an operable range of a sound beam of the special ultrasonic device, so that the injection substance is injected into the particles through the sound beam. According to the invention, injection aiming at single cells is realized, and particularly quantitative injection can be realized based on pulse type sound beam current.

Description

Technical Field

[0001] The present application relates to the fields of micro-electromechanical technology, acoustofluidics technology, and cell and other biological processing technologies, and in particular to a device and method for injecting microparticles. Background Art

[0002] Microparticle injection, whereby a target substance is injected into the interior of a microparticle through its surface, is widely used in treatment, research, or production. Examples include cell transfection (injecting DNA, RNA, protein, or other molecules into cells), cell labeling (introducing markers such as fluorescent proteins and reporter genes into cells), and cell culture (injecting nutrients into cells).

[0003] For microscopic particles (such as cells, bacteria, liposomes, etc.), the current method is batch injection followed by screening of particles that are effectively injected and maintain activity. The injection of a single particle is relatively difficult to achieve.

[0004] Based on this, the purpose of this application is to provide an implementation solution that can be used to inject single particles. Summary of the Invention

[0005] In view of the above problems in the prior art, the present application provides an injection device and method for microparticles, which can achieve quantitative injection processing or operation of single microparticles.

[0006] In a first aspect, the present application provides an injection device for microparticles, comprising:

[0007] a liquid environment for placing a carrier with microparticles attached thereto;

[0008] A super-ultrasonic device, wherein the super-ultrasonic device is in a liquid environment to generate an acoustic beam flow in the liquid environment;

[0009] A liquid supply channel, used for delivering the injection substance into the acoustic beam flow of the ultra-ultrasonic device;

[0010] The ultra-ultrasonic device and the microparticles can be in a relatively fixed position, and the microparticles are located within the effective range of the acoustic beam of the ultra-ultrasonic device, so that the injection substance can be injected into the microparticles through the acoustic beam.

[0011] As a possible implementation of the first aspect, the liquid outlet of the liquid supply channel is adjacent to the ultra-ultrasonic device; or the liquid outlet of the liquid supply channel passes through the ultra-ultrasonic device.

[0012] As a possible implementation of the first aspect, the ultra-ultrasonic device is located at the liquid outlet of the liquid supply channel.

[0013] As a possible implementation of the first aspect, the ultra-ultrasonic device is driven to generate a pulsed acoustic beam flow; the microparticles are injected with the injection substance under the action of at least two pulses of the pulsed acoustic beam flow.

[0014] As a possible implementation of the first aspect, the method further includes: an image acquisition device configured to acquire images of the particles; and adjusting the number of pulses of the generated pulsed acoustic beam flow according to morphological changes of the particles in the image.

[0015] As a possible implementation of the first aspect, the injection substance includes: a liquid containing second particles; the size of the second particles is smaller than the size of the particles attached to the carrier.

[0016] A second aspect of the present application provides a method for injecting microparticles, using any one of the microparticle injection devices described in the first aspect, the method comprising:

[0017] placing the carrier with the microparticles in a liquid environment;

[0018] The ultra-ultrasonic device is placed in a liquid environment so that the ultra-ultrasonic device and the particles are in a relatively fixed position;

[0019] The ultra-ultrasonic device is driven to generate an acoustic beam in the liquid environment to act on the particles, and an injection substance is transported into the acoustic beam of the ultra-ultrasonic device through a liquid supply channel, so that the injection substance is injected into the particles through the acoustic beam.

[0020] As a possible implementation of the second aspect, the acoustic beam generated by driving the ultra-ultrasonic device includes a pulsed acoustic beam; and the microparticles are injected with the injection substance under the action of at least two pulses of the pulsed acoustic beam.

[0021] As a possible implementation of the second aspect, the method further includes: acquiring an image of the particle by an image acquisition device; and adjusting the number of pulses of the generated pulsed acoustic beam flow according to morphological changes of the particle in the image.

[0022] As a possible implementation of the second aspect, the injection substance includes: a liquid containing second particles; the size of the second particles is smaller than the size of the particles attached to the carrier; and the second particles are carried by the injection substance and injected into the particles attached to the carrier.

[0023] From the above, it can be seen that the present application uses the acoustic beam generated by the super ultrasonic device to inject the target substance into a single target particle (such as a cell). In addition, the super ultrasonic device is a method for contactless cell injection / transfection of cells, which causes less damage to cells, can achieve single-cell precision operation, can control the amount of injected substance, improve transfection efficiency, and achieve transfection of difficult-to-transfect cells. In addition, the amount of injected target substance is regulated by the number of times (number of pulses) of the pulsed acoustic beam of the super ultrasonic device. In addition, the state of the injected target cells can be observed by an image acquisition device such as a microscope, and the number of pulses of the super ultrasonic device can be adjusted by feedback to realize the automation process of injection. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is an image and schematic diagram of jet flow phenomenon and secondary flow phenomenon;

[0025] Figure 2a and Figure 2b is a diagram of an acoustic beam flow generated in a liquid environment according to an embodiment of the present application;

[0026] Figure 3 This is a timing diagram of the output signal of the driving device of the ultra-ultrasonic device provided in an embodiment of the present application:

[0027] Figure 4 Schematic diagram of a driving device of an ultra-ultrasonic device provided in an embodiment of the present application;

[0028] Figure 5a-5e is a schematic diagram of a cell processing device provided in a second embodiment of the present application;

[0029] Figure 6 is a schematic diagram of a replaceable probe of a super-ultrasonic device with different characteristics in the present application;

[0030] Figure 7 It is a schematic diagram of the ultra-ultrasonic device and the liquid supply channel;

[0031] Figure 8 It is a schematic diagram of the dimensions of the ultra-ultrasonic device;

[0032] Figure 9a-9c This is an experimental diagram of the process of cell injection experiment using the scheme provided in the examples of this application.

[0033] The figures are marked as follows: 1-stage, 2-robotic arm, 21-first rotating part, 22-probe, 23-ultrasonic device, 3-second robotic arm, 31-linear motor, 32-second rotating part, 33-pipette, 4-image acquisition device, 41-lens, 42 eyepiece, liquid supply channel 5, liquid outlet 51 of the liquid supply channel.

[0034] It should be understood that the sizes and shapes of the blocks in the above structural diagrams are for reference only and should not constitute an exclusive interpretation of the embodiments of the present invention. The relative positions and inclusion relationships between the blocks presented in the structural diagrams are merely schematic representations of the structural relationships between the blocks and do not limit the physical connection methods of the embodiments of the present invention. DETAILED DESCRIPTION

[0035] The technical solution provided by this application is further described below with reference to the accompanying drawings and examples. It should be understood that the system structure and business scenarios provided in the examples of this application are mainly for illustrating possible implementation methods of the technical solution of this application and should not be interpreted as the sole limitation of the technical solution of this application. It is known to those skilled in the art that with the evolution of the system structure and the emergence of new business scenarios, the technical solution provided by this application is also applicable to similar technical problems.

[0036] It should be understood that the microparticle injection solutions provided in the embodiments of this application include microparticle injection devices and methods, as well as their applications. Because these technical solutions address the same or similar principles, some repetitions may not be repeated in the following descriptions of the specific embodiments. However, these specific embodiments should be considered as cross-references and can be used in conjunction with each other.

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art to which this application belongs. In the event of any inconsistency, the meanings described in this specification or the meanings derived from the contents recorded in this specification shall prevail. In addition, the terms used herein are only for the purpose of describing the embodiments of this application and are not intended to limit this application. In order to accurately describe the technical content in this application and to accurately understand the present invention, the following explanations or definitions are given for the terms used in this specification before describing the specific embodiments:

[0038] 1) Ultrasonic device: A high-frequency resonator can be a device that generates mechanical vibrations by applying voltage based on the piezoelectric effect. In this application, a piezoelectric resonator that generates ultrasonic waves of not less than 0.5 gigahertz (GHz) when in operation is used. Preferably, a piezoelectric resonator that generates ultrasonic waves of not less than 1 GHz and not more than 30 GHz when in operation is used. For example, it can be 2G-2.5 GHz. Such piezoelectric resonators can be, for example, surface acoustic wave (SAW) devices, bulk acoustic wave (BAW) devices, etc. For example, when it is a BAW, it can be a film bulk acoustic wave resonator (FBAR), a solid-state mounted resonator (SMR), or a Lamb wave resonator (LWR). For the sake of convenience, the piezoelectric resonator that can generate ultrasonic waves of not less than 0.5 GHz will be referred to as an ultrasonic device in the following.

[0039] 2) Jet phenomenon: This phenomenon occurs when the sound waves of a super-ultrasonic device act on a liquid. The regional vibrations generated by the working interface of the super-ultrasonic device can form traveling waves in the liquid, exerting a continuous thrust (i.e., body force) on the localized liquid in the liquid environment, causing at least a portion of the liquid to move linearly along the direction of sound wave propagation. This linear motion phenomenon is called the jet phenomenon.

[0040] Secondary flow phenomenon: including eddy currents and heat reflux, is another phenomenon produced when ultra-ultrasonic devices act on liquids. It includes eddy currents (or micro-vortices) caused by the local circulation of liquids driven by the jet, and heat reflux caused by the heat generated by the ultra-ultrasonic devices.

[0041] The jet phenomenon and secondary flow phenomenon can be found in Figure 1 The images and schematics are shown. Figure 1 Schematic diagram of particle capture using vortex flow is shown in FIG.

[0042] 3) Acoustic beam flow: In this application, acoustic beam flow is generated based on ultra-ultrasonic devices. In this application, acoustic beam flow is a unique type of jet phenomenon, characterized by the high-speed movement of the fluid along the direction of sound wave transmission and the jet presenting a thin cylindrical shape within its travel. Before the acoustic beam flow is significantly attenuated, it is basically in a laminar state with the surrounding liquid and has a low degree of mixing with the surrounding liquid. Figure 2a and Figure 2b An image of the generated acoustic beam flow captured by a high-speed camera is shown. Figure 2a and Figure 2b It can be seen that the acoustic beam is focused and no longer looks like Figure 1 The jets are clustered and Figure 2a and Figure 2b It is obviously not visible Figure 1 Secondary flow phenomenon in .

[0043] The conditions for generating the acoustic beam flow in the embodiment of the present application are: driving the ultra-ultrasonic device to work in at least one driving cycle at a first power, each driving cycle including a first stage and a second stage (see Figure 3 The device is configured to drive the ultra-ultrasonic device to generate an ultra-ultrasonic liquid environment with a frequency of 0.5-30 GHz in the first phase, and to stop driving the ultra-ultrasonic device in the second phase. The first power is sufficient to cause the ultra-ultrasonic device to generate a focused columnar acoustic beam perpendicular to the solid-liquid interface in the first phase. The duration of the first phase at the first power is less than a threshold value, or the duty cycle of the first phase within a driving cycle is less than a threshold value, so that the temperature of the ultra-ultrasonic device is suppressed within a controllable temperature threshold, and the ultra-ultrasonic device is prevented from substantially generating secondary flows (including eddies and backflows) in the liquid environment.

[0044] The intensity of the acoustic beam is related to the duration of the first phase (or the duty cycle within a drive cycle) and the intensity of the first power. The duration of the first phase and the first power constitute factors that determine the amount of energy input into the ultra-ultrasonic device within a drive cycle. For example, the shorter the duration of the first phase, the more effectively it can suppress temperature and secondary flow, but the acoustic beam intensity is also lower (a shorter first phase means a shorter time for energy accumulation). The higher the first power, the higher the acoustic beam intensity, which leads to a higher temperature rise in the ultra-ultrasonic device. Therefore, applying a higher first power and a shorter first phase can not only produce a higher-intensity acoustic beam, but also effectively suppress the temperature and secondary flow. Therefore, when real-time control of the acoustic beam intensity is required, the preferred solution is to use a shorter, fixed first phase (such as a fixed duty cycle within the cycle) and control the intensity of the generated acoustic beam by applying different first power levels. The second option is to apply the same first power and adjust different first stage values ​​(such as the duty cycle of the first stage value within the cycle) to control the intensity of the generated sound beam flow, or the second option is to control the intensity of the generated sound beam flow by adjusting the first stage value and the first power at the same time.

[0045] Once the liquid environment is determined and the ultrasonic device is selected, the relationship between the acoustic beam intensity and the control parameters (the first power and / or the periodic signal consisting of the first and second phases) can be calibrated. In one case, if the periodic signal is a constant (i.e., the first and second phases are constant), calibrating the acoustic beam intensity and the first power can yield the first power and the acoustic beam intensity.

[0046] 4) Regarding the first stage of the driving cycle: In the first stage of the driving cycle, the driving unit continuously outputs a number of signals. For example, when the signal generator outputs a 1GHz signal, assuming that the duration of the first stage is 1 microsecond, the driving unit will output 1000 signals during the first stage. The power loaded during the first stage will be loaded onto the 1000 signals and output to drive the ultra-ultrasonic device. In the second stage of the driving cycle (corresponding to Figure 3 Switch signal low level) no drive signal output.

[0047] In a specific case, for example, the first stage is completed after only one execution, which is equivalent to or regarded as executing only one driving cycle in the present application, and therefore this case also falls within the protection scope of the present application.

[0048] 5) Driving device of ultra-ultrasonic device: such as Figure 4 An embodiment is shown, including a control unit and a driving unit, wherein the driving unit includes a signal generator and a power amplifier. The signal output principle can be found in Figure 3 shown.

[0049] The signal generator is used to generate a high-frequency signal. The frequency of the original high-frequency signal generated by the signal generator is the same as or similar to the operating frequency of the ultra-ultrasonic device as the load (or the natural frequency of the ultra-ultrasonic device). The waveform of the signal generator can be a rectangular wave (such as Figure 3 The output signal of the corresponding driving unit is modulated into a sawtooth wave, a sharp pulse (or a triangle wave), a step wave, a sine wave, or a half-wave signal.

[0050] The power amplifier is used to amplify the signal to be output so as to drive the ultra-sonic device.

[0051] The control unit can be a switching power supply, which can output a controllable switching signal. The switching signal can be a periodic signal. The switch-on phase (such as the switching power supply switch tube conduction phase) corresponds to a high level, and the corresponding Figure 3 In the first stage, the switch off stage corresponds to the low level, corresponding to Figure 3 The control unit can also control the operating voltage or amplification factor input to the power amplifier to achieve different amplified output powers, and the output power is the first power mentioned above. The first power can also be understood as the average energy density input to the super-ultrasonic device during a drive cycle (a drive cycle consists of a first stage and a second stage). Since the amount of energy is related to power and time, the amount of energy input to the super-ultrasonic device during a drive cycle is related to the first power level, the duration of the first stage or its duty cycle.

[0052] 6) Pulsed acoustic beam flow. When the driving device of the ultra-ultrasonic device outputs the signal of each driving cycle, due to the viscosity characteristics of the liquid itself, when the second stage is short, below a certain time, the acoustic beam flow may still show a continuous characteristic. When the second stage exceeds a certain time (for example, the second stage accounts for 50%, 70%, etc.), the acoustic beam flow will show a clear interval characteristic. In the embodiment of the present application, the acoustic beam flow with a clear interval characteristic is called a pulsed acoustic beam flow. Pulsed acoustic beam flow is easier to quantitatively control or apply to measurement, etc.

[0053] Among them, the above-mentioned acoustic beam flow and related technologies for driving ultra-ultrasonic devices to generate acoustic beam flow can also be referred to the relevant introduction in Chinese patent application number CN20108322079.

[0054] 7) Penetration force: In the embodiments of the present application, it refers to the unit in which the acoustic beam can break through the membrane on the surface of the particle and enter the interior of the particle. This unit can refer to the number of pulses of the pulsed acoustic beam used when the ultra-ultrasonic device is facing the target particle, at a set distance, and under set driving signal parameters (including set power, set driving period, and set signal duty cycle). This number of pulses is just enough to enable the pulsed acoustic beam to break through the membrane on the surface of the particle and enter the interior of the particle.

[0055] In this application, the above-mentioned representation of injection force is based on the fact that membrane-bearing cells (such as single cells and bacteria) have pores in their membranes that, in certain circumstances, are associated with the sustained application of force. When cells are subjected to sustained stimulation, they respond to these stimuli through specific mechanisms, a process also known as mechanical signal transduction. For example, the following AC are several possible cellular responses associated with injection:

[0056] A. Activation of Mechanosensitive Channels: A special class of proteins called mechanosensitive channels exist on the cell membrane. These channels can directly sense and respond to changes in mechanical forces. When the cell membrane is squeezed, stretched, or subjected to other forms of mechanical stimulation, these channels can open or close, allowing ions such as sodium (Na+), calcium (Ca2+), or chloride (Cl-) to enter and exit the cell, changing the cell membrane potential, triggering electrical signal transmission, and further affecting physiological activities within the cell.

[0057] B. Activation of signal transduction pathways: Mechanical stimulation can also activate multiple signal transduction pathways within cells, such as those regulated by the Rho family of GTPases, affecting gene expression, cell proliferation, differentiation, and apoptosis. For example, when osteoblasts sense mechanical force, they promote bone formation and repair through these pathways.

[0058] C. Direct force perforation: When a sufficiently strong acoustic beam is used to act on the cell, it may also be that a pulse of a pulsed acoustic beam directly perforates the cell membrane. Compared with the above two cases A and B, the power of the acoustic beam used for direct perforation is higher, but the relative destructiveness to the cell (affecting activity) may also be greater. Therefore, in most cases, multiple pulses of a relatively low-power pulsed acoustic beam are used to achieve injection. The injection can be achieved based on the above method A or B to maintain more active cells. Therefore, unless otherwise stated, the use of a pulsed acoustic beam for injection mentioned in the following embodiments mostly refers to the use of multiple pulses of a relatively low-power pulsed acoustic beam to achieve membrane perforation injection.

[0059] In some embodiments, the injection force corresponding to cell perforation under different drive signal parameters (e.g., set power, set signal duty cycle) can be adjusted to observe the corresponding cell activity. The injection force corresponding to the higher activity (including drive signal parameters and pulse number) can be selected as the injection force for calibrating the cell type and used in the quantitative injection of that cell type.

[0060] The injection scheme for microparticles provided in the present application can control the ultra-ultrasonic device to generate a highly concentrated acoustic beam in the liquid, and transport the injection substance into the acoustic beam through the liquid supply channel, thereby injecting the injection substance into the target microparticles through the acoustic beam. In particular, a pulsed acoustic beam can be used to achieve injection. The injection scheme for microparticles provided in the present application can be applied to the injection of cells, bacteria, molecules, liposomes and other substances with membranes or surfaces, especially quantitative injection. The injected substance can include DNA, RNA, proteins or other molecules, which are injected into the cells to achieve cell transfection, labeling, culture, organoid drug delivery experiments, stem cell induced differentiation, etc.

[0061] The solution provided in this application is described in detail below with reference to the accompanying drawings and embodiments.

[0062] A first embodiment of the present application provides an injection device for microparticles, comprising:

[0063] a liquid environment for placing a carrier with microparticles attached thereto;

[0064] A super-ultrasonic device, wherein the super-ultrasonic device is in a liquid environment to generate an acoustic beam flow in the liquid environment;

[0065] A liquid supply channel, used for delivering the injection substance into the acoustic beam flow of the ultra-ultrasonic device;

[0066] The ultra-ultrasonic device and the microparticles can be in a relatively fixed position, and the microparticles are located within the effective range of the acoustic beam of the ultra-ultrasonic device, so that the injection substance can be injected into the microparticles through the acoustic beam.

[0067] In some embodiments, the liquid environment can be a container such as a cell culture dish containing liquid. In some embodiments, the carrier of the microparticles can be the inner wall (including the sidewall or bottom) of a container such as a cell culture dish. In other embodiments, the carrier can be a carrier such as a glass slide that can be placed in a container such as a cell culture dish containing liquid.

[0068] In some embodiments, microparticles refer to microparticles having a surface or membrane, for example, including proteins, cells, bacteria, molecules, molecular polymers, long-chain molecules, liposomes, and the like.

[0069] In some embodiments, the injection substance can be a liquid, or a liquid containing another substance, such as DNA, RNA, protein, molecule, molecular polymer, nanoparticles of medicine, etc. The size of the particles in these injection substances is smaller than the size of the particles attached to the carrier, and the particles in the injection substance will be injected into the target particles along with the substance.

[0070] In some embodiments, the liquid outlet of the liquid supply channel is adjacent to the ultra-ultrasonic device. For example, the liquid supply channel is a microchannel or a thin tube, and the liquid outlet is adjacent to the ultra-ultrasonic device. Figure 7 As shown in a.

[0071] In some embodiments, the liquid outlet of the liquid supply channel passes through the ultra-ultrasonic device. For example, the liquid supply channel is a microchannel or a thin tube, and the ultra-ultrasonic device has a through hole, which serves as the liquid outlet of the liquid supply channel. In some embodiments, the through hole can be located at the center of the ultra-ultrasonic device. Figure 7 As shown in b.

[0072] In some embodiments, the super ultrasonic device is located at the liquid outlet of the liquid supply channel. For example, the liquid supply channel is a microchannel, the super ultrasonic device is located in the microchannel or at the bottom of the microchannel (the bottom of the microchannel is the side opposite to the opening of the microchannel), and the direction of the working surface of the super ultrasonic device (i.e., the direction of the sound beam flow) corresponds to the opening of the microchannel, and the opening is the liquid outlet of the microchannel. Figure 7 As shown in c.

[0073] In some embodiments, the ultra-sonic device is driven to generate a pulsed acoustic beam; the microparticles are injected into the injection substance under the action of at least two pulses of the pulsed acoustic beam. Thus, injection is achieved using multiple pulses of a relatively low-power pulsed acoustic beam, which can maintain more active cells and facilitate quantitative injection control.

[0074] In some embodiments, the system further comprises: an image acquisition device for acquiring images of the microparticles; and adjusting the number of pulses of the generated pulsed acoustic beam (and the injection force as described herein) based on the morphological changes of the microparticles in the images. This allows for automatic control of the injection.

[0075] In some embodiments, after determining the injection force for a cell type or class based on the aforementioned method, this injection force can be used as a calibrated injection force for quantitative injection of the same cell type. The effective (effective: refers to the injection of the substance into the microparticle) calculation start time for the quantitative injection can be the moment the cell is pore-opened (this is because cells respond to continuous stimulation, and the pulsed acoustic beam flow before reaching the stimulation level can be considered as a pulse that has not yet been injected). Each pulse corresponds to one injection.

[0076] The second embodiment of the present application provides a processing device that can realize the injection of microparticles as described above. Figure 5a-5e As shown, the processing device may include:

[0077] A stage 1 on which a container such as a cell culture dish can be placed. The container contains a target object in a liquid environment, the target object including target particles to be injected, such as cells;

[0078] a first robotic arm 2, wherein a probe 22 is mounted on a movable end of the first robotic arm 2, and a super-ultrasonic device 23 is mounted on an end of the probe 22. The posture of the probe 22 above the stage 1 can be adjusted by the first robotic arm 2, thereby adjusting the posture of the super-ultrasonic device 23;

[0079] The ultra-ultrasonic device 23 can be extended into the liquid environment, and any one of the embodiments described in the first embodiment can be used to inject the microparticles on the carrier in the liquid environment.

[0080] In some embodiments, the first robotic arm 2 can be a three-axis robotic arm that can be positioned beside the stage 1. In one embodiment, the three-axis movement can be manually adjusted. In another embodiment, each axis of the three-axis robotic arm is equipped with a motor, and the motor drives the movement of the movable end of the first robotic arm 2. In some embodiments, the movable end of the first robotic arm 2 is also equipped with a first rotating portion 21, and the probe 22 is mounted on the first rotating portion 21. In some embodiments, the first rotating portion 21 can rotate along a horizontal axis. In some embodiments, the first rotating portion 21 has a clamping portion that clamps the probe 22. The rotation angle of the first rotating portion 21 can be manually adjusted, or the angle can be adjusted by being configured with a motor to drive the first rotating portion 21 to rotate. The combination of the three-axis robotic arm and the first rotating portion 21 enables the movement and rotation of the probe 22, thereby adjusting the ultra-ultrasonic device 23 at the end of the probe 22 to a desired position and desired posture, so that the ultra-ultrasonic device 23 contacts or immerses in the aforementioned liquid environment and faces the target particles.

[0081] In some embodiments, the ultra-ultrasonic device 23 can also be controlled to generate a jet or acoustic beam, or to adjust its posture to generate an acoustic beam or vortex to peel off the injected particles, and the peeled particles are suspended in the liquid.

[0082] In some embodiments, a second robotic arm 3 may also be included, and the movable end of the second robotic arm 3 is equipped with a pipette 33. The pipette 33 includes a pipette and a tip located at the end of the pipette. The posture of the pipette 33 can be adjusted by the second robotic arm 3, thereby adjusting the posture of the tip of the pipette 33. The tip of the pipette 33 can be extended into the liquid environment to absorb the particles in the liquid after peeling.

[0083] In some embodiments, the second robotic arm 3 can be a three-axis robotic arm. In one embodiment, the movement of the three axes can be manually adjusted. In another embodiment, each axis of the three-axis robotic arm is equipped with a motor for each axis, and the movement of the movable end of the second robotic arm 3 is driven by the motor. In some embodiments, the movable end of the second robotic arm 3 is also equipped with a linear motor 31 (or T-axis motor), and the movable end of the linear motor 31 is equipped with a clamping portion to clamp the main body of the pipette 33, wherein the linear motor 31 can be used to extend or retract the pipette tip toward the target cell. In some embodiments, the movable end of the second robotic arm 3 is also equipped with a second rotating portion 32, and the linear motor 31 is installed on the second rotating portion 32. The movement of the pipette 33 is achieved by the combination of the three-axis robotic arm, the second rotating portion 32 and the linear motor 31, thereby adjusting the pipette tip at the end of the pipette 33 to the desired position.

[0084] In some embodiments, the stage 1 is horizontally movable. The stage 1 can be mounted on a horizontal guide rail and driven by a motor to move along the rail. For example, the stage 1 can be mounted on a horizontal two-axis translation stage. The two-axis translation stage can be manually adjusted, or each axis can be equipped with a corresponding motor for motor-driven movement. This allows a container, such as a cell culture dish containing target particles, to be adjusted to a desired position on the stage 1.

[0085] In some embodiments, as Figure 6 The probe 22 end shown in FIG. 2 may be composed of two detachable parts, one part for being assembled on the rotating part 23 (for example, assembled on the clamping part of the rotating part 23), and the other part having the probe 22 end and equipped with the ultra-ultrasonic device 23, so that the probe 22 or the part having the probe 22 end can be replaced as needed. Figure 6 The size of the ultrasonic device 23 in the right figure is smaller than that in the left figure. Under the same conditions, the diameter of the sound beam flow generated by the ultrasonic device 23 in the right figure is much smaller than the diameter of the sound beam flow generated by the ultrasonic device 23 in the left figure, that is, it is more focused and the unit area that can be processed is smaller.

[0086] In some embodiments, as Figure 7 The end of the probe 22 is shown, wherein Figure 7 Figures a, b, and c in the figure respectively show the relative positions of the ultra-ultrasonic device 23, the liquid supply channel 5, and the liquid outlet 51 of the liquid supply channel.

[0087] In some embodiments, the special ultrasonic device 23 assembled at the end of the probe 22 can be located on both sides of the end of the probe 22, so that the probe 22 can be rotated 180 degrees along its axis and then clamped and fixed at the first rotating part 21, thereby realizing quick selection of the special ultrasonic device 23 for work.

[0088] In some embodiments, as Figure 5c As shown, at least a portion of the stage 1 is made of transparent material or hollowed out. Figure 5d As shown, an image acquisition device 4 is provided below the stage 1. The image acquisition device 4 includes at least one lens 41, which can be adjusted to face the stage 1. Thus, the image acquisition device 4 below can capture images (since video is composed of frames of images, images here include video) of the injection process and results of the pulsed acoustic beam on the target particles, for analysis, or to control the operating state of the ultra-ultrasonic device 23 (such as the number of pulses in the pulsed acoustic beam) and the position of the mobile end of the robotic arm based on the obtained image data or analyzed data.

[0089] In some embodiments, image acquisition device 4 may be a microscope having multiple lenses 41 , wherein the multiple lenses 41 are mounted on a rotating component to switch between the working lenses, so that the working lenses face upward. Image acquisition device 4 may also include an eyepiece 42 or a display to observe the images captured by the lenses 41 .

[0090] In some embodiments, the image capture device 4 may be positioned beside or above (e.g., diagonally above) the stage 1. The lens 41 of the image capture device 4 may be adjusted to face the target particles on the stage 1 for image capture. In some embodiments, the image capture device 4 may include multiple lenses 41 positioned at different locations (e.g., below, beside, or diagonally above the stage 1).

[0091] In some embodiments, as Figure 5e As shown, the incubator further includes a housing that houses at least the aforementioned stage 1, the first robotic arm 2 holding the probe 22, and the second robotic arm 3 holding the pipette 33, thereby forming a relatively isolated space. The housing can be used as an incubator. The housing has an openable door for users to perform operations (e.g., placing or removing containers on the stage 1). The housing or door can have a transparent window for users to observe the interior of the housing.

[0092] In some embodiments, the space of the box can also accommodate at least the lens 41 of the image acquisition device 4, so that the lens 41 is located in a relatively clean box.

[0093] In some embodiments, the interior of the box also includes partitions to accommodate necessary electrical equipment and other experimental equipment. These electrical equipment may include at least one of the following: a power supply, a drive device (such as a signal generator and power amplifier) ​​for driving the ultra-ultrasonic device 23, a pump and reservoir for use with the pipette 33, a drive device for driving the motors in the robotic arm or translation stage, a control device (such as an industrial computer or a computer), and lighting equipment. The control device signals connect to other equipment, such as the drive device, to acquire images captured by the image acquisition device 4, analyze and process them, and control the execution of corresponding components.

[0094] In some embodiments, the control device is also connected to an external display device and a human-computer interaction interface (such as a keyboard and touch screen, etc.). The control device receives the user's operating instructions through the human-computer interaction interface, controls the actions of other devices based on the instructions, or reads a preset program based on the user's operating instructions, and automatically controls other devices to cooperate with each other to achieve a series of operations (i.e., achieve automation). In some embodiments, the display device and the human-computer interaction interface can be located on the box, or exist independently of the box. In some embodiments, a conspicuous position on the box (such as Figure 5eAn emergency stop button can be set in the upper right corner of the box to trigger shutdown and can be used in emergencies.

[0095] In some embodiments, the robotic arm or the translation stage may also be connected to a manipulation device for direct operation, and the manipulation device may be mounted on the surface of the box or outside the box.

[0096] In some embodiments, the liquid environment, for example, is liquid contained in a vessel placed on stage 1, with the target particles located within the vessel. The vessel may also be made of a transparent material to facilitate image acquisition by the image acquisition device 4 below stage 1. In other embodiments, the liquid environment refers to liquid on the surface of the target particles. This liquid may be liquid carried on the surface of the target particles themselves, or may be liquid continuously sprayed or drained to the target location of the target particles during operation of the ultra-ultrasonic device 23.

[0097] In some embodiments, the target particles are fixed relative to the probe 22. For example, the target particles can be attached to the surface of a device (such as a glass slide) or on the surface of other tissues (such as organic tissues). The device or organic tissue can be fixedly placed in the aforementioned vessel.

[0098] In some embodiments, the ultra-sonic device 23 may be a polygon, particularly a polygon with an odd number of sides. For example, in one embodiment, it may be a regular pentagon or a scalene pentagon. In other embodiments, it may be an olive-shaped, triangular, elliptical, diamond-shaped, semicircular, or any combination of shapes or sizes.

[0099] The area of ​​the super ultrasonic device 23 (here refers to the radial dimension, not the thickness dimension, that is, the device interface dimension) is about 10-1000000 μm2, preferably about 100-40000 μm2, and more preferably 1000-10000 μm2. In order to intuitively understand the size of the super ultrasonic device 23, Figure 8 A dimensional diagram of a super-ultrasonic device 23, which is an integrated acoustic wave resonator, is shown. The size of the super-ultrasonic device 23 is negatively correlated with the achievable resonant frequency; therefore, reducing the size can increase the resonant frequency. In other embodiments, the resonant frequency of the super-ultrasonic device 23 can also be altered by varying the device's shape, such as the number of polygonal elements, the angles between adjacent sides, or the lengths of each side.

[0100] The diameter of the generated acoustic beam is related to the parameters of the ultra-ultrasonic device 23. The smaller the area (radial dimension, or interface dimension) of the ultra-ultrasonic device 23, the smaller the acoustic beam diameter. A smaller microscale also results in better acoustic wave focusing. Under the same first power, a smaller microscale ultra-ultrasonic device 23 produces a stronger acoustic beam, a smaller acoustic beam diameter, and a faster response speed (the time it takes to generate the acoustic beam).

[0101] A third embodiment of the present application provides a method for injecting microparticles. The method uses the first embodiment or the second embodiment and any optional embodiment to implement injection of microparticles. The method includes:

[0102] S1: placing the carrier with microparticles in a liquid environment.

[0103] For example, a glass slide with microparticles attached is placed in a cell culture dish containing liquid and can be placed on the stage described in the second embodiment.

[0104] S2: placing the ultra-ultrasonic device in a liquid environment, with the ultra-ultrasonic device and the particles in a relatively fixed position.

[0105] For example, adjust the first robotic arm in the second embodiment and the horizontal two-axis translation stage where the stage is located so that the ultra-ultrasonic device reaches the predetermined position and the glass slide is located at the observation position of the lens of the image acquisition device below the stage.

[0106] S3: driving the ultra-ultrasonic device to generate an acoustic beam in the liquid environment to act on the microparticles, and transporting the injection substance into the acoustic beam of the ultra-ultrasonic device through the liquid supply channel, so as to inject the injection substance into the interior of the microparticles through the acoustic beam.

[0107] For example, the ultra-ultrasonic device is driven to generate a pulsed acoustic beam flow, and the liquid supply device is driven to deliver the injection substance to the acoustic beam flow, so that the injection substance is injected into the interior of the microparticle through the acoustic beam flow.

[0108] During this process, an image acquisition device can be used to capture images of the particles to determine whether the injection has been successful, and the number of pulses in the pulsed acoustic beam can be adjusted accordingly.

[0109] The specific methods that can be adopted in each step can be found in the first embodiment, the second embodiment and the optional embodiments, and will not be described in detail.

[0110] The results of cell injection experiments using the above method are introduced below to illustrate the feasibility of this application.

[0111] like Figure 9aThese are several frames of images acquired by an image acquisition device (electron microscope) during the cell injection experiment using the above method. Figure 9a As can be seen, the application of a pulsed acoustic beam can induce subtle cell deformation, thereby altering the instantaneous permeability of the cell membrane and completing the process of injecting extracellular drugs into the cell. The vertical lines adjacent to the cells in the three images show the subtle physical deformation of the cells to the left after the pulsed acoustic beam is applied. After the pulsed acoustic beam is turned off, the target cells return to their normal morphology. Furthermore, MTT cell proliferation activity assays and calcein-AM staining have verified that this cell injection method using a pulsed acoustic beam generated by a super-ultrasonic device does not affect cell viability.

[0112] like Figure 9b During the cell injection experiment using the above method, Figures 9 (I) through (VI) show the changes in the cells as the ultra-sonic device power or the duty cycle of the first-stage signal increases. As can be seen from Figures 9 (I) through (VI), the cells gradually deform due to compression. Therefore, visual recognition using the image capture device's feedback can be used to determine the progress of cell injection. This visual recognition can be further combined with deep learning and big data recognition methods to improve recognition accuracy.

[0113] like Figure 9c In the cell injection experiment using the above method, the pulsed acoustic beam of the ultra-ultrasonic device is turned on for a period of time (which can also be converted into the number of pulses) from 0s to 80s, the target cells are gradually injected with the target substance (including fluorescence), and the change in fluorescence intensity is observed. Figure 9c It is also shown that non-target cells do not send changes, and the present application can achieve injection of single cells.

[0114] In the several embodiments provided in this application, it should be understood that the disclosed devices and methods are not limited to the above embodiments, and can also be implemented in other ways. For example, the device embodiments described above are only schematic. For example, the division of the units is only a logical function division. There may be other division methods in actual implementation, such as multiple units or components can be combined or integrated into another system, or some features can be ignored or not executed. Another point is that the mutual coupling or direct coupling or communication connection shown or discussed can be through some interfaces, indirect coupling or communication connection of devices or units, which can be electrical, mechanical or other forms.

[0115] The units described as separate components may or may not be physically separate, and the components shown as units may or may not be physical units, that is, they may be located in one place or distributed across multiple network units. Some or all of these units may be selected to achieve the purpose of this embodiment according to actual needs.

[0116] In addition, the words "first, second, third, etc." or module A, module B, module C and other similar terms in the specification and claims are only used to distinguish similar objects and do not represent a specific ordering of the objects. It can be understood that the specific order or sequence can be interchanged where permitted so that the embodiments of the present application described herein can be implemented in an order other than that illustrated or described herein.

[0117] In the above description, the numbers representing the steps, such as S10, S20, etc., do not necessarily mean that the steps must be executed in this manner. If permitted, the order of the steps can be interchanged or they can be executed simultaneously.

[0118] The term "comprising" as used in the specification and claims should not be construed as limiting to what is listed thereafter; it does not exclude other elements or steps. Thus, it should be interpreted as specifying the presence of the features, integers, steps, or components mentioned, but not excluding the presence or addition of one or more other features, integers, steps, or components, or groups thereof. Thus, the expression "a device comprising means A and B" should not be limited to a device consisting solely of components A and B.

[0119] References in this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present application. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment, but may refer to the same embodiment. Furthermore, in one or more embodiments, the particular features, structures, or characteristics can be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure.

[0120] Note that the above are only preferred embodiments of the present application and the technical principles employed. Those skilled in the art will understand that the present application is not limited to the specific embodiments described herein, and that various obvious changes, readjustments, and substitutions can be made by those skilled in the art without departing from the scope of protection of the present application. Therefore, although the present application has been described in more detail through the above embodiments, the present application is not limited to the above embodiments and may include many other equivalent embodiments without departing from the scope of protection of the present application, all of which fall within the scope of protection of the present application.

Claims

1. A device for injecting microparticles, characterized in that: include: a liquid environment for placing a carrier with microparticles attached thereto; A super-ultrasonic device, wherein the super-ultrasonic device is in a liquid environment to generate an acoustic beam flow in the liquid environment; A liquid supply channel, used for delivering the injection substance into the acoustic beam flow of the ultra-ultrasonic device; The ultra-ultrasonic device and the microparticles can be in a relatively fixed position, and the microparticles are located within the effective range of the acoustic beam of the ultra-ultrasonic device, so that the injection substance can be injected into the microparticles through the acoustic beam.

2. The device according to claim 1, characterized in that The liquid outlet of the liquid supply channel is adjacent to the ultra-ultrasonic device; or The liquid outlet of the liquid supply channel passes through the ultra-ultrasonic device.

3. The device according to claim 1, characterized in that The ultra-ultrasonic device is located at the liquid outlet of the liquid supply channel.

4. The device according to claim 1, characterized in that The ultra-ultrasonic device is driven to generate a pulsed acoustic beam flow; The microparticles achieve the injection of the injection substance under the action of at least two pulses of the pulsed acoustic beam.

5. The device according to claim 1 or 4, characterized in that Also includes: an image acquisition device for acquiring images of the particles; The number of pulses of the generated pulsed acoustic beam is adjusted according to the morphological changes of the particles in the image.

6. The device according to claim 1, characterized in that The injection substance comprises: a liquid containing second particles; the size of the second particles is smaller than the size of the particles attached to the carrier.

7. A method for injecting microparticles, characterized in that: Using the microparticle injection device according to any one of claims 1 to 6, the method comprises: placing the carrier with the microparticles in a liquid environment; The ultra-ultrasonic device is placed in a liquid environment so that the ultra-ultrasonic device and the particles are in a relatively fixed position; The ultra-ultrasonic device is driven to generate an acoustic beam in the liquid environment to act on the particles, and an injection substance is transported into the acoustic beam of the ultra-ultrasonic device through a liquid supply channel, so that the injection substance is injected into the particles through the acoustic beam.

8. The method according to claim 7, wherein: The acoustic beam generated by driving the ultra-ultrasonic device includes a pulsed acoustic beam; The microparticles achieve the injection of the injection substance under the action of at least two pulses of the pulsed acoustic beam.

9. The method according to claim 8, characterized in that Also includes: capturing images of the particles using an image capture device; The number of pulses of the generated pulsed acoustic beam is adjusted according to the morphological changes of the particles in the image.

10. The method according to claim 7, characterized in that The injection substance comprises: a liquid containing second particles; the second particles are smaller in size than the particles attached to the carrier; The second microparticles are carried by the injection substance and injected into the microparticles attached to the carrier.

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