Acoustic fluidic device for rapid tissue lysis and automated lysis and biopsy device

Abstract

The present invention discloses an acoustofluidic device for rapid tissue lysis and an automated lysis and biopsy device. The acoustofluidic device for rapid tissue lysis provided by the present invention includes: an acoustofluidic chip (100), a PCB board (200), a housing (300), and an RF connection line (400); wherein, the acoustofluidic chip (100) is disposed on the front surface of the PCB board (200), the signal terminal of the acoustofluidic chip (100) is connected to the signal terminal on the front surface of the PCB board (200), and the ground terminal of the acoustofluidic chip (100) is connected to the ground terminal of the PCB board (200); the housing (300) is sleeved on the RF connection line (400); two lines extend from one end of the RF connection line (400) close to the PCB board (200) as a signal line and a ground line respectively, and are respectively connected to the signal terminal and the ground terminal on the back surface of the PCB board (200) to form a power transmission path. The present invention also designs an automated device with simple operation, reduces manual operation, lowers the operation difficulty, shortens the time for preparing a single cell suspension, and can efficiently complete tissue lysis and liquid biopsy.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an acoustofluidic device for rapid tissue lysis and an automated lysis and biopsy device. Background Art

[0002] Rapid and effective lysis of biological tissues into single-cell suspensions is not only an essential part of biological research experiments but also a key issue in the field of medical clinical practice. In biological experiments, flow cytometers are often used to identify, count, and analyze parameters of cells in tissues. The key factor determining the accuracy of flow cytometer analysis lies in the quality of the input single-cell suspension. Therefore, during tissue lysis, researchers pursue lysis methods with high cell yield, fast lysis speed, small cell damage, and strong cell activity. In clinical practice, the demand for intraoperative rapid detection is increasing day by day, and related research is gradually advancing. Among single-cell-based detection methods, a convenient and fast sample pretreatment method can greatly shorten the overall detection time and improve the detection efficiency on the premise of ensuring high cell activity and high dispersion.

[0003] Currently, traditional methods mostly use enzymatic digestion and physical mechanical methods to process tissue specimens to prepare single-cell suspensions, which generally have problems such as too long digestion time, large mechanical damage to tissues, and poor cell dispersion effect. Among them, enzymatic digestion lyses tissues by destroying collagen fibers between tissues, connecting proteins between cells, and polysaccharide substances, greatly reducing physical damage to cells compared with physical mechanical methods, but there are problems such as slow digestion process and enzymes affecting cell activity. Therefore, a shaker is often used during the operation to achieve the purpose of shaking and mixing the tissues evenly, but the digestion process generally still requires 1h - 2h (different types and sizes of tissue blocks will affect the digestion time), and it has certain requirements for the operator's skills, consuming time and effort, and unable to meet the need for rapid lysis to prepare single cells.

[0004] The disadvantages of the shaker enzymatic digestion method are as follows: (1) The shaker plays a role in shaking and mixing the tissues evenly during digestion, but the overall digestion time is still too long, and cells are easily immersed in the enzyme solution for a long time, which can easily cause problems such as reduced cell activity or even cell death. (2) It can be seen from the operation steps that the shaker enzymatic digestion method has many operation steps and a large workload, is difficult to complete in a short time, and all steps need to be manually operated in a sterile environment, with high requirements for the operator's proficiency and operation experience. If not proficient, it is easy to cause problems such as bacterial contamination and over-digestion resulting in decreased cell activity.

[0005] The mechanical method cuts and disperses tissue blocks through the cutting force generated by mechanical contact or liquid, causing cells to shed and separate. The main methods include cutting, sieving extrusion, and pipetting with a syringe or pipette. The disadvantages of the mechanical method are as follows: Although the operation steps of the mechanical method are simpler than those of the enzymatic method, since its principle is to use cutting force to forcibly break the connections between cells in the tissue, cells are extremely likely to die and break during this process. The cell survival rate is much lower than that of the enzymatic method, and a large amount of tissue samples are required to obtain a sufficient number of live cells. Summary of the Invention

[0006] To make up for the deficiencies in the above fields, the main object of the present invention is to provide an acoustic fluidic device and an automated lysis and biopsy device for rapid tissue lysis, which helps to quickly and simply lyse tissue into a single-cell suspension, while not damaging cells, ensuring high cell viability and dispersibility, and supporting rapid cell biopsy and cancer cell screening.

[0007] The acoustic fluidic device for rapid tissue lysis provided by the present invention includes: an acoustic fluidic chip 100, a PCB board 200, a housing 300, and an RF connection line 400; wherein,

[0008] The acoustic fluidic chip 100 is disposed on the front surface of the PCB board 200. The signal terminal of the acoustic fluidic chip 100 is connected to the signal terminal on the front surface of the PCB board 200, and the ground terminal of the acoustic fluidic chip 100 is connected to the ground terminal of the PCB board 200; the housing 300 is sleeved on the RF connection line 400; two lines extend from one end of the RF connection line 400 close to the PCB board 200, which are respectively a signal line and a ground line, and are respectively connected to the signal terminal and the ground terminal on the back surface of the PCB board 200 to form a power transmission path.

[0009] Preferably, the acoustic fluidic chip 100 is a surface acoustic wave resonator chip, a bulk acoustic wave resonator chip, or a piezoelectric ceramic chip.

[0010] More preferably, the bulk acoustic wave resonator chip is a solidly mounted bulk acoustic wave resonator chip; the resonance frequency range of the solidly mounted bulk acoustic wave resonator is 1 GHz to 10 GHz, and the applied power range is 0.1 W to 10 W. The shape of the resonator piezoelectric layer can be circular, pentagonal, square, triangular, polygonal, etc. The length, width, and height dimensions of the solidly mounted bulk acoustic wave resonator chip are 0.1 mm * 0.1 mm * 0.5 mm to 3 cm * 3 cm * 0.5 mm.

[0011] Even more preferably, the resonance frequency of the solidly mounted bulk acoustic wave resonator is 2.49 GHz, the applied power is 2 W, and the shape of the resonator piezoelectric layer is pentagonal.

[0012] The present invention uses SMR chips, but it is not limited thereto. Piezoelectric resonators such as BAW (Bulk acoustic wave) resonators (SMR belongs to BAW resonators), SAW (Surface Acoustic Wave Resonator), and PZT (Piezoelectric) can all achieve this operation.

[0013] Under this packaging method, the length, width, and height dimensions of the SMR chip are 0.1 mm * 0.1 mm * 0.5 mm to 3 cm * 3 cm * 0.5 mm (array resonator, increasing the processing area). The number of resonators on one chip ranges from 1 to 100.

[0014] Under this packaging method, to adapt to the chip size, the length, width, and height dimensions of the pcb board are 1 mm * 1 mm * 0.3 mm to 4 cm * 4 cm * 0.3 mm. The shell (300) is a cylindrical hollow tube with an inner diameter of 1 to 3 mm, an outer diameter of 5 to 10 mm, and a tube length of 3 cm to 20 cm. The length of the RF connection line (400) is 3 cm to 20 cm.

[0015] Preferably, the acoustic fluid chip 100 is packaged and fixed on the front side of the PCB board 200.

[0016] Preferably, the signal terminal of the acoustic fluid chip 100 and the signal terminal on the front side of the PCB board 200 are connected by a gold wire in the middle, and the ground terminal of the acoustic fluid chip 100 and the ground terminal of the PCB board 200 are connected by two gold wires on the left and right.

[0017] The present invention also provides an automated lysis and biopsy device for rapid tissue lysis.

[0018] The automated lysis and biopsy device for rapid tissue lysis provided by the present invention includes a box body 11. Four vertical tubes are respectively arranged in the box body 11, which are in sequence the liquid inlet tube 12, the sample injection tube 13, the sample outlet tube 14, and the waste liquid tube 15. The injection tube 1 extends into the liquid inlet tube 12; a simple xy-axis displacement stage 3 is arranged at the upper end of the sample injection tube 13, and the xy-axis displacement stage is used to fix the upper part of the acoustic fluid device 4, and the lower part of the acoustic fluid device 4 extends into the sample injection tube 13; the liquid outlet tube 5 extends into the sample outlet tube 14; the waste liquid tube 6 extends into the waste liquid tube 15; the bottoms of the liquid inlet tube 12, the sample injection tube 13, the sample outlet tube 14, and the waste liquid tube 15 are connected by a transverse elbow 16. Filter membranes 7, 8, 9, and 10 are respectively arranged in the transverse elbow 16, wherein the filter membrane 7 is arranged between the liquid inlet tube 12 and the sample injection tube 13; the filter membranes 8 and 9 are arranged between the sample injection tube 13 and the sample outlet tube 14; the filter membrane 10 is arranged between the sample outlet tube 14 and the waste liquid tube 15; a lysis chamber 17 is arranged at the bottom in the box body 11 corresponding to the position of the acoustic fluid device 4, and an observation chamber 18 is arranged at the position corresponding to the sample outlet tube 14.

[0019] Preferably, the diameters of the liquid injection tube 1, the liquid outlet tube 5, and the waste liquid tube 6 are 1-10 mm for injecting enzyme solution and culture medium; the diameters of the liquid inlet tube 12, the sampling tube 13, the sample outlet tube 14, and the waste liquid tube 15 are 10 mm-30 mm; the lysis chamber 17 is an inverted conical chamber with an upper bottom diameter of 5-30 mm, a lower bottom diameter of 1-10 mm, and a depth of 5-30 mm; the observation chamber 18 is a cylindrical chamber with a tube diameter of 5-30 mm and a depth of 5-30 mm.

[0020] Preferably, the filter membranes 7 and 10 are 5-10 μm filter membranes, the filter membrane 8 is a 70-100 μm filter membrane, and the filter membrane 9 is a 30-50 μm filter membrane.

[0021] The present invention also provides a method for tissue lysis.

[0022] The method for tissue lysis provided by the present invention includes the following steps:

[0023] (1) Place the tissue to be lysed into the sampling tube of the automated lysis and biopsy device for rapid tissue lysis, and the displacement stage adjusts the position of the acoustic fluidic device to face the tissue block to be lysed;

[0024] (2) Inject the enzyme solution through the liquid injection tube 1 to immerse the tissue block to be lysed and the acoustic fluidic device in the solution;

[0025] (3) Turn on the acoustic fluidic device, and wait for the tissue block to rotate into the acoustic vortex to accelerate the enzymatic reaction process, and continuously act for 10-20 min;

[0026] (4) Filter and replace the enzyme solution. While injecting the culture medium through the liquid injection tube 1, suck out the solution from the waste liquid tube 6, and continuously circulate for 2-5 min. The original enzyme solution in the tube cavity will be replaced with cell culture medium;

[0027] (5) Open the liquid outlet tube 5 to suck out the single-cell suspension between the filter membranes 9 and 10.

[0028] The present invention also provides a method for tissue biopsy.

[0029] The method for tissue biopsy provided by the present invention includes the following steps:

[0030] (1) Place the tissue to be lysed into the sampling tube of the automated lysis and biopsy device for rapid tissue lysis, and the displacement stage adjusts the position of the acoustic fluidic device to face the tissue block to be lysed;

[0031] (2) Inject a mixed solution of enzyme solution and fluorescent specific antibody through the liquid injection tube 1 to immerse the tissue block to be lysed and the acoustic fluidic device in the solution;

[0032] (3) Activate the acoustofluidic device and wait for the tissue block to rotate into the acoustic vortex to accelerate the enzymatic reaction and antigen-antibody binding, and continuously act for 10 - 20 min;

[0033] (4) Filter and replace the mixed solution of the enzyme solution and the fluorescent specific antibody. While injecting the culture medium from the injection tube 1, suck out the solution from the waste liquid tube 6, and continuously circulate for 2 - 5 min. The mixed solution in the original lumen will be replaced with cell culture medium;

[0034] (5) Combine with an inverted fluorescence microscope to observe the single-cell morphology and fluorescence staining results through the observation chamber 18, and judge whether there are target type cells and the proportion of cells according to the presence or absence of fluorescence and the intensity of fluorescence.

[0035] The present invention has the following beneficial effects:

[0036] 1. The present invention utilizes the acoustofluidic device to accelerate the process of enzymatic reaction, shortens the tissue lysis time, ensures high cell activity and high dispersion, and solves the problems of long lysis time, poor cell activity, high cell mortality, and low cell acquisition rate in traditional methods.

[0037] 2. The present invention designs a simple and automated biopsy device, reduces manual operation, lowers the operation difficulty, and greatly simplifies the operation steps. It shortens the time for preparing single-cell suspension and reduces problems such as contamination caused by non-standard operation. It is suitable for applications that require rapid acquisition of single-cell suspension in scientific research experiments and rapid cell biopsy or cancer cell screening in clinics. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] For purposes of illustration and not limitation, the present invention will now be described in accordance with the preferred embodiments of the present invention, particularly with reference to the accompanying drawings, in which:

[0039] Figure 1 is a schematic diagram of the acoustofluidic device for rapid tissue lysis of the present invention; wherein, 100 - acoustofluidic chip, 200 - PCB board, 300 - housing, 400 - RF connection line.

[0040] Figure 2 is a schematic diagram of the stacked structure of the SMR chip.

[0041] Figure 3 is a schematic diagram of SMR digestion principle.

[0042] Figure 4Schematic diagram of the automated lysis and biopsy device for rapid tissue lysis of the present invention; wherein, 1 - liquid injection tube, 2 - tube mouth cap, 3 - xy-axis displacement stage, 4 - acoustic fluidic device, 5 - liquid outlet tube, 6 - waste liquid tube, 7 - filter membrane, 8 - filter membrane, 9 - filter membrane, 10 - filter membrane, 11 - box body, 12 - liquid inlet tube, 13 - sampling tube, 14 - sample outlet tube, 15 - waste liquid tube, 16 - horizontal elbow, 17 - lysis chamber, 18 - observation chamber.

[0043] Figure 5 3D diagram of the automated lysis and biopsy device for rapid tissue lysis of the present invention.

[0044] Figure 6 Primary cell culture conditions after two lysis methods, where a are the cells lysed by the shaker group and b are the cells lysed by the SMR group.

[0045] Figure 7 Cell numbers and cell growth rates after treatment with a shaker and a lysis device respectively.

[0046] Figure 8 Results of lysing 30 mg of renal tumor tissue under 5 different treatment conditions.

[0047] Figure 9 Fluorescent staining results at two different positions in the observation chamber 18 under an inverted fluorescence microscope. Detailed implementation mode

[0048] Reference Figure 1 shows the acoustic fluidic device for rapid tissue lysis of the present invention. Among them, 100 is an SMR chip with dimensions (length, width, height) of 0.8 mm * 0.5 mm * 0.5 mm. 200 is a thin PCB board with dimensions (length, width, height) of 3 mm * 3 mm * 0.3 mm. 100 and 200 are connected by 3 gold wires. The signal terminal of the SMR is connected to the signal terminal on the front of the PCB board (the middle gold wire), and the ground terminals are also connected (the left and right gold wires). 300 is a housing, a hollow cylinder made of copper with an inner diameter of 2 mm and an outer diameter of 3 mm. 400 is an RF connection wire with a wire diameter of 1.3 mm. Two wires extend from the end close to the PCB board 200, which are respectively soldered to two solder joints (signal terminal and ground terminal) on the back of the PCB board 200. This encapsulation forms the signal power transmission path of the SMR chip, and connecting the RF connection wire 400 to a signal generator can drive the SMR device to work.

[0049] The thin PCB board plays a connecting role in this acoustic fluid device. One side is connected to the SMR chip (connected by gold wire), and the other side is connected to the RF wire (connected by soldering). Since the SMR chip is small in size and the electrodes on it are also small, it is impossible to directly connect the RF wire to apply power.

[0050] The SMR chip is bonded to the specified position on the PCB board through AB glue, and the connection between the two is stable.

[0051] The SMR ground - PCB ground - RF wire ground are connected. The SMR signal - PCB signal - RF signal are connected.

[0052] The RF wire is relatively soft and easy to bend, which will cause problems such as unstable structure and difficult adjustment of the device position. Therefore, a rigid shell is processed and sleeved on the RF wire for stable connection.

[0053] First, the shell 300 is sleeved on the RF connecting wire 400, and AB glue is used for bonding at the connection end of the shell 300 and the RF connecting wire 400 (at the SMR joint).

[0054] The two wires at the end of the RF connecting wire 400 are the signal wire and the ground wire respectively. The two solder joints on the back of the thin PCB board 200 correspond to the signal end and the ground end of the SMR chip, and the purpose of soldering is to conduct this path. Figure 1 The hexagon and the subsequent thread on it are the SMR joint, which is part of the RF connecting wire.

[0055] This encapsulation forms the signal power transmission path of the SMR chip. Through the connection and conduction method of the PCB board and the RF connecting wire, the sine wave signal of the signal generator can be transmitted to the chip to drive its operation. The specific principle of the signal generator driving its operation is as follows: Based on the inverse piezoelectric effect, the piezoelectric material converts the sinusoidal voltage (the GHz sinusoidal signal provided by the signal generator) that changes at a specific frequency acting on the electrode into the mechanical deformation of the piezoelectric thin film AlN. This periodic deformation caused by the alternating current signal will generate a bulk acoustic wave propagating along the film thickness direction in the piezoelectric thin film AlN, that is, the special ultrasonic wave. Due to the use of the Bragg reflector structure, the special ultrasonic wave undergoes total reflection at the interface between the upper electrode and the air and at the interface between the lower electrode and the Bragg reflector respectively, thereby generating a sound wave in the opposite direction to the original propagation direction, and further superposing to form a standing wave and resonance. When the special ultrasonic wave propagates in the liquid, it will trigger the acoustic fluid effect, and the acoustic fluid effect is a non - linear fluid effect caused by the attenuation of the bulk acoustic wave propagating in the liquid. In terms of performance, it will form a forward - jetting jet and a vortex formed by swirling.

[0056] The purpose of this encapsulation is: (1) Designed as an insertable device, the package size is reduced, and it can work deep in the pipeline or cavity. (2) It is convenient to clamp and can be precisely controlled in cooperation with the displacement stage.

[0057] The RF connecting line is used to transmit power. One end of it is an SMA connector, which can be connected to the SMA connector transmission line extended from the signal generator.

[0058] The stacked structure of the SMR chip is as Figure 2 shown, and it is composed of a silicon substrate, a Bragg reflection layer formed by Mo and SiO2, a bottom electrode Mo, a piezoelectric layer AlN, a top electrode Mo, and a gold electrode.

[0059] As Figure 3 shown, the SMR chip (gigahertz bulk acoustic wave resonator) in the acoustic fluidic device of the present invention can generate strong jet force and strong shear force to lyse fresh tissue. The acoustic pressure field generated by gigahertz ultrasonic waves is used to drive the enzyme solution to form an acoustic fluidic vortex, and the tissue block is rotationally captured in the vortex for layer-by-layer interstitial digestion and cell stripping. The jet force impacts the tissue block, accelerating the diffusion of the enzyme between the solid-liquid media, increasing the surface area of the tissue, and accelerating the hydrolysis rate of the interstitium. The shear force quickly strips the hydrolyzed products from the tissue surface, exposing deeper interstitium and providing more binding sites for the enzyme. At the same time, the acoustic fluidic vortex can also increase the collision probability between the fluorescent antibody and the cell membrane surface antigen, promote antigen-antibody binding, and accelerate the process of immunofluorescence staining. Using the acoustic fluidic device of the present invention to process tissue specimens can greatly accelerate the lysis rate compared with traditional lysis methods, quickly lyse the tissue into single-cell suspension, and can maintain cell viability. Moreover, the acoustic fluidic device can simultaneously accelerate the tissue lysis and antigen-antibody staining processes, and it only takes 10 - 30 minutes to prepare the tissue block into an immunofluorescence-stained single-cell suspension, promoting the development of liquid biopsy and cancer cell detection for clinical applications.

[0060] Refer to Figure 4 and Figure 5, showing the automated lysis and biopsy device for rapid tissue lysis of the present invention. 1 is an injection tube with a pipe diameter of 1-10 mm, used for injecting enzyme solution and culture solution. 2 is a tube mouth cap, used to fix the injection tube 1 and prevent sundries from falling into the device. The lower end is a rubber stopper to seal the pipeline. 3 is a simple xy-axis displacement stage, used to adjust the position of 4 (SMR device) to facilitate alignment with the tissue block, and is made of transparent resin by 3D printing. 4 is the above-mentioned acoustic fluid device, that is, the SMR device. 5 is an outlet tube, used to extract the single-cell suspension after lysis. 6 is a waste liquid tube, used to suck out excess waste liquid and replace the enzyme solution. The injection tube 1, the outlet tube 5, and the waste liquid tube 6 are all connected to a peristaltic pump for injecting or extracting liquid. 7, 8, 9, and 10 are all filter membranes, used for filtering cells. The common cell size is generally 10-20 μm. The filter membrane 7 and the filter membrane 10 are 5-10 μm filter membranes, the filter membrane 8 is a 70-100 μm filter membrane, and the filter membrane 9 is a 30-50 μm filter membrane. The filter membrane 7 and the filter membrane 8 limit the tissue block within a certain range for lysis. The single cells after lysis cannot pass through the filter membrane 7 and the filter membrane 10, but can pass through the filter membrane 8 and the filter membrane 9. The filter membrane 8 and the filter membrane 9 also limit the passage of cell clusters. Therefore, the single cells will be between the filter membrane 9 and the filter membrane 10. 11 is the device box body, and the pipeline path therein is as shown, with a pipe diameter of 15 mm. The box body material is PMMA. There are 4 openings on the box body, and the pipeline path is all included in the box body. 17 is an inverted conical lysis chamber with an upper bottom diameter of 5-30 mm, a lower bottom diameter of 1-10 mm, and a depth of 5-30 mm., put the tissue block into the chamber, and the acoustic fluid device moves to align and extend into the chamber to rapidly lyse the tissue block. 18 is a cylindrical observation chamber with a pipe diameter of 5-30 mm and a depth of 5-30 mm. After the single-cell suspension is located behind the sampling tube 14, it can be combined with an inverted microscope to observe the cell morphology and fluorescence staining online from the bottom of the observation chamber.

[0061] The simple xy-axis displacement stage fixes the device by clamping. The displacement stage has a clamping hole. Pass the device through this hole and turn the screw to tighten and narrow the hole to clamp the end of the device (the end far from the SMR chip).

[0062] Such as Figure 4As shown in the figure, the automated lysis and biopsy device for rapid tissue lysis of the present invention includes a box body 11. Four vertical tubes are respectively arranged in the box body 11, which are the liquid inlet tube 12, the sample injection tube 13, the sample outlet tube 14, and the waste liquid tube 15 in sequence. The liquid injection tube 1 extends into the liquid inlet tube 12; a simple xy-axis displacement stage 3 is arranged at the upper end of the sample injection tube 13, and the xy-axis displacement stage is used to fix the upper part of the acoustic fluid device 4, and the lower part of the acoustic fluid device 4 extends into the sample injection tube 13; the liquid outlet tube 5 extends into the sample outlet tube 14; the waste liquid tube 6 extends into the waste liquid tube 15; the bottoms of the liquid inlet tube 12, the sample injection tube 13, the sample outlet tube 14, and the waste liquid tube 15 are connected by a horizontal elbow 16, and filter membranes 7, 8, 9, and 10 are respectively arranged in the horizontal elbow 16, wherein the filter membrane 7 is arranged between the liquid inlet tube 12 and the sample injection tube 13; the filter membranes 8 and 9 are arranged between the sample injection tube 13 and the sample outlet tube 14; the filter membrane 10 is arranged between the sample outlet tube 14 and the waste liquid tube 15. A lysis chamber 17 is arranged at the bottom in the box body 11 corresponding to the position of the acoustic fluid device 4, and an observation chamber 18 is arranged at the position corresponding to the sample outlet tube 14.

[0063] Example 1: Rapid tissue lysis

[0064] The operation process of the automated lysis and biopsy device for rapid tissue lysis of the present invention is as follows:

[0065] ① Cleaning: Soak the tissue in DMEM culture medium containing penicillin (concentration 400U / ml) and streptomycin (concentration 0.4mg / ml) for 15 minutes. Under the culture medium, use a scalpel to cut the fresh renal tumor tissue block into small pieces of 1mm*1mm, and soak and wash the tissue with D-Hanks 2-3 times. The fresh renal tumor tissue is provided by the Second Hospital of the Medical University, and is a small piece with a size of about 5*5*5mm and a mass of about 100mg.

[0066] ② Digestion:

[0067] 1. Put the tissue block into the lysis chamber 17 from the sample injection tube 13, and the displacement stage adjusts the position of the SMR device to face the tissue block, within a distance of 1 cm.

[0068] Specifically: Put the tissue block into the sample injection tube 13 through the opening above the sample injection tube 13. First put the tissue block in, and then clamp the acoustic fluid device on the displacement stage, extend it into the sample injection tube 13 to adjust the position for processing.

[0069] 2. Pass an appropriate amount of enzyme solution through the liquid injection tube 1 to immerse the tissue block and the SMR device in the solution.

[0070] 3. Turn on the SMR device, the signal generator outputs a sine signal of 2.49Ghz, and the power is adjusted to 2w. Wait for the tissue block to rotate into the acoustic vortex to accelerate the progress of the enzymatic hydrolysis reaction, and continuously act for 15 minutes.

[0071] 4. Filter and replace the enzyme solution. While injecting the culture medium from the injection tube 1, suck out the solution from the waste liquid tube 6. The cells will pass through the two filter membranes, namely filter membrane 8 and filter membrane 9, along the fluid direction (filtering out incompletely digested tissues and cell clusters). The cell debris will pass through filter membrane 10 along with the fluid and be sucked out as waste liquid. Continuously circulate for 3 minutes, and the original enzyme solution in the lumen will be replaced with cell culture medium.

[0072] 5. Open the liquid outlet tube 5 and suck out the single-cell suspension between filter membrane 9 and filter membrane 10.

[0073] Control experiment:

[0074] Shaker enzymatic digestion method, the specific method is as follows:

[0075] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 minutes.

[0076] ② Cutting: Under the culture medium, use a sharp scalpel to cut the fresh kidney tumor tissue into small pieces of 1 mm * 1 mm in size.

[0077] ③ Washing: Soak and wash the tissue 2 - 3 times with buffer solutions such as D-Hanks or PBS.

[0078] ④ Digestion: Add enzyme solution (type and concentration depend on the tissue type) to the centrifuge tube, put the tissue pieces into the centrifuge tube, and use a shaker to mix the tissue evenly at a constant temperature of 37°C. The digestion time on the shaker is carried out for 1 - 2 hours according to the standard operation (determine the digestion time according to the tissue digestion situation, and it is okay when the tissue shows a fluffy flocculent state).

[0079] ⑤ Centrifugation: After the treatment, perform rapid centrifugation with a centrifugal force of 200 g for 5 minutes. After centrifugation, suck out the enzyme solution above and replace it with culture medium. Repeat the entire centrifugation process three times to ensure complete removal of the enzyme solution.

[0080] ⑥ Filtration: Use an appropriate amount of culture medium to pipette repeatedly to fully disperse the cells, and filter the cells with a cell strainer to remove incompletely digested tissues and large impurities.

[0081] Control experiment with only enzyme, the specific method is as follows:

[0082] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 minutes.

[0083] ② Cutting: Under the culture medium, use a sharp scalpel to cut the fresh tumor tissue into small pieces of 1 mm * 1 mm in size.

[0084] ③ Washing: Soak and wash the tissue 2 - 3 times with buffer solutions such as D-Hanks or PBS.

[0085] ④ Digestion: Add enzyme solution (type and concentration depend on tissue type) into a centrifuge tube, put the tissue block into the centrifuge tube, and let it stand and digest at a constant temperature of 37°C for 1 - 2 hours.

[0086] Control experiment with no enzyme + shaker, the specific method is as follows:

[0087] ① Sterilization: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 min.

[0088] ② Cutting: Under the culture medium, use a sharp scalpel to cut the fresh tumor tissue block into small pieces of 1 mm * 1 mm.

[0089] ③ Washing: Soak and wash the tissue 2 - 3 times with buffer solutions such as D-Hanks or PBS.

[0090] ④ Digestion: Add cell culture medium (depending on tissue type) into a centrifuge tube, put the tissue block into the centrifuge tube, and use a shaker to mix the tissue evenly at a constant temperature of 37°C for 1 - 2 hours.

[0091] Control experiment with no enzyme + acoustic fluidic lysis device, the specific method is as follows:

[0092] ① Washing: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 min. Under the culture medium, use a scalpel to cut the fresh tumor tissue block into small pieces of 1 mm * 1 mm, and soak and wash the tissue 2 - 3 times with D-Hanks.

[0093] ② Digestion:

[0094] 1. Put the tissue block into the lysis chamber 17 from the injection tube 13, and the displacement stage adjusts the position of the SMR device to face the tissue block, with a distance within 1 cm.

[0095] Specifically: Put the tissue block into the injection tube 13 through the opening on the injection tube 13. First put the tissue block in, then clamp the acoustic fluidic device on the displacement stage, extend it into the injection tube 13 to adjust the position for processing.

[0096] 2. Inject an appropriate amount of cell culture medium from the injection tube 1 to immerse the tissue block and the SMR device in the solution.

[0097] 3. Turn on the SMR device, the signal generator outputs a sine signal of 2.49 GHz, adjust the power to 2 w, and act continuously for 15 min.

[0098] The fresh renal tumor tissues of the same source, with the same size and quality, were lysed by the traditional shaker enzymatic method and the automated lysis and biopsy device of the present invention respectively. The results are as follows: Figure 6 As shown, the experiment verified that the single-cell suspension lysed by the automated lysis and biopsy device of the present invention has successfully completed primary culture. The number of cells lysed in only 15 minutes has reached the amount that can be lysed by the shaker group in 1 hour and 15 minutes. Moreover, according to the processing results of the cell growth situation photos at the same site for 11 days (5 sites were randomly selected from both groups for photographing from day 1 to day 11), the total number of cells per day is slightly higher than that of the shaker group, and the cell division rate is basically the same. The automated lysis and biopsy device of the present invention has increased the lysis speed by 4 to 5 times compared with the traditional method on the premise of ensuring cell viability, demonstrating its feasibility in the application of rapid tissue lysis for preparing single-cell suspension.

[0099] Figure 7 The left figure shows the change results of the cell number over days after being treated with the shaker and the lysis device respectively. The statistical source is Figure 6 in the photographed results. The photographed field of view range is 2500*1900μm. 5 positions were randomly selected at the bottom of the culture flask, and the cell number of each group was recorded every day. The average value of the 5 points was taken as the final result. It can be seen that for tissues of the same quality, the cell number after being treated with the lysis device is more. Figure 7 The right figure shows the growth rate of cells in a cycle every two days. The growth rates of the lysis device group and the shaker group are almost the same in each cycle, proving that the lysis device does not reduce the cell viability or inhibit its normal division compared with the traditional dissociation method, but has an obvious lysis speed advantage.

[0100] Figure 8 For the lysis of 30mg renal tumor tissues under 5 different treatment conditions, 50ul of the solution was sampled at regular intervals during the treatment process. After calcein staining, photographs were taken for counting, and the number of single cells lysed at different digestion times was statistically analyzed. Then, the data was statistically analyzed and curve-fitted to draw the change curve of the cell number over the treatment time and the change curve of the lysis rate over the treatment time under different treatment conditions. As can be seen from Figure 8 it, the automated lysis device of the present invention can accelerate the digestion process of the enzyme on tissue blocks, and the lysis efficiency is much greater than other methods.

[0101] Example 2. Tissue biopsy

[0102] The operation process of the automated lysis and biopsy device for tissue biopsy of the present invention is as follows:

[0103] ① Washing: Soak the tissue in DMEM culture medium containing penicillin (concentration 400 U / ml) and streptomycin (concentration 0.4 mg / ml) for 15 min. Under the culture medium, use a scalpel to cut the fresh renal tumor tissue into small pieces of 1 mm * 1 mm in size, and soak and wash the tissue with D-Hanks 2 - 3 times. The fresh renal tumor tissue is provided by the Second Hospital of the Medical University, and it is a small piece with a size of about 5 * 5 * 5 mm and a mass of about 100 mg.

[0104] ② Digestion:

[0105] 1. Place the tissue block into the lysis chamber 17 from the injection tube 13, and the displacement stage adjusts the position of the SMR device to face the tissue block, with the distance within 1 cm.

[0106] 2. Inject an appropriate amount of the mixed solution of enzyme solution and fluorescent specific antibody from the injection tube 1, and immerse the tissue block and the SMR device in the solution. The fluorescent specific antibody is the CA9 antibody conjugated with the PE fluorescent group, and the antibody brand is Abcam.

[0107] 3. Turn on the SMR device, the signal generator outputs a sine signal of 2.49 GHz, and the power is adjusted to 2 w. Wait for the tissue block to rotate into the acoustic vortex to accelerate the enzymatic hydrolysis reaction and the process of antibody - antigen binding, and continuously act for 15 min.

[0108] 4. Filter and replace the enzyme solution. While injecting the culture medium from the injection tube 1, suck out the solution from the waste liquid tube 6. The cells will pass through the two - layer filter membranes of filter membrane 8 and filter membrane 9 along the fluid direction (filtering out incompletely digested tissue and cell clusters), and the cell debris will pass through filter membrane 10 along with the fluid and be sucked out with the waste liquid. Continuously circulate for 3 min, and the original enzyme solution in the lumen will be replaced with cell culture medium.

[0109] 5. Combine with an inverted fluorescence microscope to observe the single - cell morphology and fluorescence staining results through the observation chamber 18, and judge whether there are target - type cells and the approximate proportion of cells by the presence or absence of fluorescence and the intensity of fluorescence.

[0110] Figure 9To observe the fluorescence staining results at two different positions in chamber 18 under an inverted fluorescence microscope, calcein labels all living cells, and the CA9 antibody with PE fluorescence labels the CA9 protein highly expressed in clear renal carcinoma cells. As can be seen from the figure, the cells are fully dispersed into single cells, and the clear renal carcinoma cells with high CA9 expression are fully stained, with the fluorescence intensity far higher than that of other cells and can be easily distinguished, proving that the SMR device can simultaneously and fully accelerate the enzymatic digestion and antigen-antibody binding processes, and the single-cell preparation and full staining of fluorescent antibodies can be completed in 15 minutes. According to the imaging results at two positions in the figure, it can be preliminarily determined that the cancer cell type of this tissue is clear renal carcinoma cells. By counting the pictures, it is statistically found that the proportion of cancer cells is about 4%, and it can be inferred that the malignancy degree of this tissue is not high.

[0111] The specific operation of picture counting is to take cell images at 5 different positions in chamber 18 under the microscope and import the pictures into ImageJ. First, separate the RGB three-color channels, then perform binary processing on the images of different channels, and finally use the built-in counting plug-in in ImageJ to count the number of cells. The statistical results of the three different color channels of RGB represent the number of cells emitting red, green, and blue fluorescence respectively. For example, for the R-channel statistical result of this experiment, it is the number of all cells emitting red fluorescence, that is, the number of clear renal carcinoma cells showing PE red fluorescence due to high CA9 expression.

[0112] The above specific implementation manners do not constitute a limitation to the protection scope of the present invention. Those skilled in the art should understand that various modifications, combinations, sub-combinations, and substitutions can occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. An automated lysis and biopsy device for rapid lysis of tissues, characterized in that, It includes an acoustofluidic device for rapid tissue lysis, and a box body (11). Four vertical tubes are respectively arranged inside the box body (11), which are in sequence the liquid inlet tube (12), the sample injection tube (13), the sample outlet tube (14), and the first waste liquid tube (15). The liquid injection tube (1) extends into the liquid inlet tube (12); a simple xy-axis displacement stage (3) is arranged at the upper end of the sample injection tube (13), and the xy-axis displacement stage is used to fix the upper part of the acoustofluidic device (4), and the lower part of the acoustofluidic device (4) extends into the sample injection tube (13); the liquid outlet tube (5) extends into the sample outlet tube (14); the second waste liquid tube (6) extends into the first waste liquid tube (15); the bottoms of the liquid inlet tube (12), the sample injection tube (13), the sample outlet tube (14), and the first waste liquid tube (15) are connected through a transverse elbow (16), and a first filter membrane (7), a second filter membrane (8), a third filter membrane (9), and a fourth filter membrane (10) are respectively arranged inside the transverse elbow (16), wherein the first filter membrane (7) is arranged between the liquid inlet tube (12) and the sample injection tube (13); the second filter membrane (8) and the third filter membrane (9) are arranged between the sample injection tube (13) and the sample outlet tube (14); the fourth filter membrane (10) is arranged between the sample outlet tube (14) and the first waste liquid tube (15); a lysis chamber (17) is arranged at the bottom inside the box body (11) corresponding to the position of the acoustofluidic device (4), and an observation chamber (18) is arranged at the position corresponding to the sample outlet tube (14). The acoustofluidic device for rapid tissue lysis includes: an acoustofluidic chip (100), a PCB board (200), a tube shell (300), and an RF connecting wire (400); wherein, The acoustofluidic chip (100) is arranged on the front surface of the PCB board (200), the signal end of the acoustofluidic chip (100) is connected to the signal end on the front surface of the PCB board (200), and the grounding end of the acoustofluidic chip (100) is connected to the grounding end of the PCB board (200); the tube shell (300) is sleeved on the RF connecting wire (400); two wires extend from one end of the RF connecting wire (400) close to the PCB board (200), which are respectively the signal wire and the ground wire, and are respectively connected to the signal end and the grounding end on the back surface of the PCB board (200) to form a power transmission path. The acoustofluidic chip (100) is used to generate special ultrasonic waves. When the special ultrasonic waves propagate in a liquid, it will trigger an acoustofluidic effect, forming a forward jet and a vortex formed by the gyration to generate jet force, shear force, and acoustofluidic vortex; the acoustofluidic vortex is used to rotate and capture tissue blocks in the liquid in the vortex, the jet force is used to impact the tissue blocks, and the shear force is used to peel the hydrolyzed products from the tissue surface.

2. The automated lysis and biopsy device for rapid lysis of tissues according to claim 1, characterized in that: The diameters of the liquid injection tube (1), the liquid outlet tube (5), and the second waste liquid tube (6) are 1 - 10 mm, which are used to inject enzyme solution and culture medium; the diameters of the liquid inlet tube (12), the sample injection tube (13), the sample outlet tube (14), and the first waste liquid tube (15) are 10 mm - 30 mm.

3. The automated lysis and biopsy device for rapid lysis of tissues according to claim 1, characterized in that: The first filter membrane (7) and the fourth filter membrane (10) are 1-10 μm filter membranes, the second filter membrane (8) is a 70-200 μm filter membrane, and the third filter membrane (9) is a 30-70 μm filter membrane.

4. The automated lysis and biopsy device for rapid lysis of tissues according to claim 1, characterized in that: The acoustic fluidic chip (100) is a surface acoustic wave resonator chip, a bulk acoustic wave resonator chip, or a piezoelectric ceramic chip.

5. The automated lysis and biopsy device for rapid lysis of tissues according to claim 4, characterized in that: The bulk acoustic wave resonator chip is a solidly mounted bulk acoustic wave resonator chip; the resonance frequency range of the solidly mounted bulk acoustic wave resonator is 1 GHz to 10 GHz, and the applied power range is 0.1 W to 10 W.

6. The automated lysis and biopsy device for rapid lysis of tissues according to claim 1 or 4, characterized in that: The acoustic fluidic chip is packaged and fixed on the front side of the PCB board (200).

7. The automated lysis and biopsy device for rapid lysis of tissues according to claim 1 or 4, characterized in that: The signal terminal of the acoustic fluidic chip (100) and the signal terminal on the front side of the PCB board (200) are connected by a single gold wire in the middle, and the ground terminal of the acoustic fluidic chip (100) and the ground terminal of the PCB board (200) are connected by two gold wires on the left and right.

8. A method for tissue lysis, characterized in that, It includes the following steps: (1) Put the tissue to be lysed into the sampling tube of the automated lysis and biopsy device for rapid tissue lysis according to any one of claims 1 to 7, and the displacement stage adjusts the position of the acoustic fluidic device to face the tissue block to be lysed; (2) Inject the enzyme solution through the injection tube (1) to immerse the tissue block to be lysed and the acoustic fluidic device in the solution; (3) Turn on the acoustic fluidic device, and wait for the tissue block to rotate into the acoustic vortex to accelerate the enzymatic reaction process, and continuously act for 10-20 min; (4) Filter and replace the enzyme solution. While injecting the culture solution from the injection tube (1), suck out the solution from the second waste liquid tube (6), and continuously circulate for 2-5 min. The original enzyme solution in the lumen will be replaced by the cell culture solution; (5) Open the liquid outlet tube (5) to suck out the single cell suspension between the third filter membrane (9) and the fourth filter membrane (10).

9. A method for tissue detection for non-disease diagnosis purposes, characterized in that, It includes the following steps: (1) Put the tissue to be lysed into the sampling tube of the automated lysis and biopsy device for rapid tissue lysis according to any one of claims 1 to 7, and the displacement stage adjusts the position of the acoustic fluidic device to face the tissue block to be lysed; (2) Inject a mixed solution of an enzyme solution and a fluorescent specific antibody through the injection tube (1) to immerse the tissue block to be lysed and the acoustic fluidic device in the solution; (3) Turn on the acoustic fluidic device, and wait for the tissue block to rotate into the acoustic vortex to accelerate the enzymatic reaction and antigen-antibody binding, and continuously act for 10-20 min; (4) Filter and replace the mixed solution of the enzyme solution and the fluorescent specific antibody. While injecting the culture solution from the injection tube (1), suck out the solution from the second waste liquid tube (6), and continuously circulate for 2-5 min. The original mixed solution in the lumen will be replaced by the cell culture solution; (5) Combine with an inverted fluorescence microscope to observe the single cell morphology and fluorescence staining results through the observation chamber (18), and judge whether there are target type cells and the proportion of cells according to the presence or absence of fluorescence and the intensity of fluorescence.

Images