Observation device and method for controlling biological sample by using ultrasonic oscillation bubble array

Abstract

The invention discloses an observation device for controlling a biological sample by using an ultrasonic oscillation bubble array. The observation device comprises an observation device body, and an objective table is arranged on the observation device body; the objective table is connected with a bottom plate, the bottom plate is connected with a flow channel layer, and the flow channel layer is provided with a fluid chamber; a plurality of horseshoe-shaped protrusions are arranged in the fluid cavity, and the upper surface of the flow channel layer is connected with a cover plate. A feeding pipe and a discharging pipe are arranged on the cover plate, and the feeding pipe and the discharging pipe are communicated with the fluid chamber; the bottom plate is connected with an ultrasonic excitation unit, and the ultrasonic excitation unit is electrically connected with the drive circuit. Compared with the prior art, when the device works, the ultrasonic excitation unit is utilized to excite ultrasound, generated sound waves can be transmitted to the horseshoe-shaped protrusion array on the front portion of the bottom plate through the bottom plate, oscillating microbubbles can be generated in the horseshoe-shaped protrusion structure, and under the combined action of acoustic radiation force of the oscillating microbubbles and acoustic streaming induced resistance, the sound waves can be transmitted to the bottom plate through the acoustic streaming induced resistance. A biological sample can be captured in a sample solution.

Description

Technical Field

[0001] The present invention relates to the technical field of biological experimental devices, and in particular to an observation device and method for manipulating biological samples by using an ultrasonic oscillation bubble array. Background Art

[0002] At present, with the development of science and technology and the continuous progress of human civilization, people pay more and more attention to their own safety and the integrity of biological samples when observing and recording biological samples. Biological sample observation is an important means in life science research, involving in-depth analysis of the morphology, structure and function of samples such as cells, tissues, and microorganisms. The technology of how to accurately and quickly manipulate biological samples is particularly important.

[0003] At present, most of the means of manipulating biological samples use mechanical manipulation, electromagnetic manipulation, atomic force manipulation and other methods. Mechanical manipulation is likely to cause certain damage to the samples. Electromagnetic manipulation is difficult to accurately manipulate due to electromagnetic instability and susceptibility to external interference. Atomic force manipulation of biological samples is relatively expensive and difficult to promote widely. Therefore, a manipulation device for manipulating biological samples using an acoustic oscillation bubble array is proposed. Compared with mechanical, electromagnetic and atomic force methods, the low-cost acoustic flow platform is suitable for single-cell manipulation and analysis, and has the advantages of small physiological damage, non-contact operation, and low dependence on the biophysical properties of the sample.

[0004] Therefore, it is necessary to improve the existing technology to solve the shortcomings of the existing technology. Summary of the invention

[0005] The purpose of the present invention is to provide an observation device and method for manipulating biological samples using an ultrasonic oscillation bubble array. Ultrasound is excited by an ultrasonic excitation unit, and the generated sound waves can be transmitted to the horseshoe-shaped protrusion array on the front of the bottom plate through the bottom plate. Oscillating microbubbles will be generated in the horseshoe-shaped protrusion structure. Under the joint action of the acoustic radiation force of the oscillating microbubbles and the acoustic flow induced resistance, the biological sample can be captured in the sample solution. The sound waves will generate acoustic vortices in the biological sample solution. The continuous flow of the acoustic vortex can adjust the rotational angular velocity of the sample. The oscillation intensity can be controlled by changing the voltage applied to the ultrasonic excitation unit, thereby adjusting the rotation speed of the sample. When observing biological samples, the situation of the biological sample during rotation can be monitored in real time, and the obtained biological sample rotation video can be sliced ​​frame by frame through editing software, so that the obtained two-dimensional contour image frames are superimposed to reconstruct the three-dimensional morphology, and the surface area, volume, ellipticity, roughness and other geometric parameters of the biological sample can be collected to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions: An observation device for manipulating biological samples using an ultrasonic oscillation bubble array comprises an observation device body, on which a stage is provided; the stage is connected to a bottom plate, on which a flow channel layer is connected, on which a fluid chamber is provided; in the fluid chamber, a plurality of horseshoe-shaped protrusions are provided, and on the upper surface of the flow channel layer, a cover plate is connected; on the cover plate, a feed pipe and a discharge pipe are provided, and the feed pipe and the discharge pipe are connected to the fluid chamber; on the bottom plate, an ultrasonic excitation unit is connected, and the ultrasonic excitation unit is electrically connected to a drive circuit.

[0007] A further solution of the present invention is that the observation device body includes but is not limited to an optical microscope, an electron microscope, and the base plate is bonded or connected to the stage of the observation device body by screws; the material of the flow channel layer includes but is not limited to glass and acrylic, and the flow channel layer is bonded to the base plate.

[0008] A further solution of the present invention is that the horseshoe-shaped protrusions are arranged in an array on the substrate, the horseshoe-shaped protrusions are integrally formed with the substrate, the substrate is bonded to a bottom plate in the fluid chamber, the size of the substrate matches the fluid chamber, and the material of the horseshoe-shaped protrusions includes but is not limited to synthetic polymers, bio-based elastomers, and silicone rubber.

[0009] A further solution of the present invention is that the ultrasonic excitation unit includes but is not limited to a ceramic piezoelectric sheet, an ultrasonic transducer or an interdigital electrode.

[0010] A further solution of the present invention is that when the ultrasonic excitation unit is a ceramic piezoelectric piece, the vibration frequency of the ceramic piezoelectric piece is at an ultrasonic level, and the operating frequency is adjusted by sweeping the frequency from 20 kHz to 200 kHz, and the operating voltage used is 1-10vpp.

[0011] A further solution of the present invention is that the ceramic piezoelectric sheet is bonded to the bottom plate, and the ceramic piezoelectric sheet is connected to the driving circuit via a wire.

[0012] A further solution of the present invention is that the material of the bottom plate includes but is not limited to glass, ceramic, and alloy steel, and the material of the cover plate includes but is not limited to transparent glass and acrylic.

[0013] A further solution of the present invention is that the cover plate is bonded to the flow channel layer, through holes are opened at both ends of the cover plate, and the feed pipe and the discharge pipe are bonded to the two through holes respectively.

[0014] A further solution of the present invention is that the inner diameters of the feed pipe and the discharge pipe are in the range of 0.1-0.3 mm.

[0015] A method for observing biological samples by using an ultrasonic oscillation bubble array to manipulate biological samples, using the above-mentioned observation device for observing biological samples by using an ultrasonic oscillation bubble array, comprises the following steps: Step 1: The biological sample solution is introduced into the fluid chamber through the feeding tube.

[0016] Step 2: The ultrasonic excitation unit works, and the sound waves generated can be transmitted to the horseshoe-shaped protrusion array on the front of the bottom plate through the bottom plate. Oscillating microbubbles will be generated in the horseshoe-shaped protrusions. Under the combined effect of the acoustic radiation force of the oscillating microbubbles and the acoustic flow induced resistance, the biological sample solution will diffuse around the horseshoe-shaped protrusion array, and eventually 1-2 cells will be adsorbed around each horseshoe-shaped structure; and the sound waves will generate acoustic vortices in the biological sample solution. The continuous flow of the acoustic vortex can adjust the rotational angular velocity of the sample, and the oscillation intensity can be controlled by changing the voltage applied to the ultrasonic excitation unit, thereby adjusting the rotational speed of the sample.

[0017] Step 3: When observing biological samples, the rotation of the biological samples can be monitored in real time, and the obtained biological sample rotation video can be sliced ​​frame by frame through editing software, so that the obtained two-dimensional contour image frames are superimposed to reconstruct the three-dimensional shape, and the geometric parameters of the biological samples such as surface area, volume, ellipticity, roughness, etc. can be collected.

[0018] Step 4: After the experiment is completed, the feed tube is pressurized and the biological sample solution is discharged from the discharge tube.

[0019] Beneficial effects of the present invention: The observation device and method of the present invention for manipulating biological samples using an ultrasonic oscillation bubble array have the advantages of low cost, safety, reliability, and environmental friendliness, which give it a significant advantage in the field of micro-nano manipulation.

[0020] The observation device and method of the present invention for controlling biological samples using an ultrasonic oscillation bubble array uses ultrasound to control biological samples, which is a non-contact control method that can avoid direct contact with the biological samples and will not cause damage to the biological samples that need to be observed.

[0021] The observation device and method for controlling biological samples using an ultrasonic oscillation bubble array of the present invention can control tiny biological samples using ultrasound, and the control accuracy can be improved to the micrometer or even nanometer level compared with traditional control methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the overall structure of the present invention.

[0023] Figure 2 It is a schematic diagram of the local structure of the present invention.

[0024] Figure 3 It is a schematic diagram of the local structure of the present invention.

[0025] Figure 4 Experimental diagram of the vortex generated around a single horseshoe-shaped protrusion.

[0026] Figure 5Experimental diagram of the horseshoe-shaped protrusion array structure manipulating biological samples.

[0027] Figure 6 This is a simulation diagram of particle trajectories for a single horseshoe-shaped protrusion.

[0028] In the figure: 1-observation device body, 101-stage, 2-bottom plate, 3-channel layer, 301-fluid chamber, 4-horseshoe-shaped protrusion, 5-cover plate, 6-feed pipe, 7-discharge pipe, 8-ultrasonic excitation unit, 9-driving circuit. DETAILED DESCRIPTION

[0029] The present invention is further explained below in conjunction with the accompanying drawings and specific embodiments.

[0030] like Figures 1 to 3 As shown, an observation device for manipulating biological samples using an ultrasonic oscillation bubble array includes an observation device body, on which a stage 101 is provided; the stage 101 is connected to a base plate 2, the base plate 2 is connected to a flow channel layer 3, and the flow channel layer 3 is provided with a fluid chamber 301; a plurality of horseshoe-shaped protrusions 4 are provided in the fluid chamber 301, and a cover plate 5 is connected to the upper surface of the flow channel layer 3; a feed pipe 6 and a discharge pipe 7 are provided on the cover plate 5, and the feed pipe 6 and the discharge pipe 7 are connected to the fluid chamber 301; an ultrasonic excitation unit 8 is connected to the base plate 2, and the ultrasonic excitation unit 8 is electrically connected to a driving circuit 9.

[0031] The observation device body is an electron microscope, and the bottom plate 2 is connected to the stage 101 of the electron microscope by epoxy resin glue or screws; the material of the flow channel layer 3 is glass, and the flow channel layer 3 is bonded to the bottom plate 2 by epoxy resin glue.

[0032] The horseshoe-shaped protrusions 4 are arranged in an array on the substrate, which is bonded to the bottom plate 2 in the fluid chamber 301 through photoresist. The size of the substrate matches the fluid chamber 301, and the material of the horseshoe-shaped protrusions 4 is a PDMS film.

[0033] The horseshoe-shaped protrusion 4 is prepared by standard soft lithography and replication molding technology to form a base with a length of 19.6 mm and a width of 9 mm. The horseshoe-shaped protrusion 4 on the base is 60 μm high and is used to generate microbubbles to capture biological samples.

[0034] The ultrasonic excitation unit 8 is a ceramic piezoelectric piece, and the vibration frequency of the ceramic piezoelectric piece is at the ultrasonic level. The frequency is swept from 20 kHz to 200 kHz to adjust the working frequency, and the working voltage used is 1-10vpp.

[0035] The ceramic piezoelectric sheet is bonded to the bottom plate 2 by epoxy resin glue, and the ceramic piezoelectric sheet is connected to the driving circuit 9 by a wire.

[0036] The driving circuit 9 is composed of a signal generator (Tektronix AFG 3022C, 250MS / s, 25MHz) that sends an electrical signal to a power amplifier (Aigtek ATA-4052) for amplifying the power of the electrical signal to drive the ceramic piezoelectric sheet, and an oscilloscope (Tektronix TDS 2024, 200MHz, 2GS / s) displays the working voltage value and working current value of the ceramic piezoelectric sheet.

[0037] The base plate 2 is made of glass, and the cover plate 5 is made of glass.

[0038] The cover plate 5 is bonded to the flow channel layer 3. Through holes are opened at both ends of the cover plate 5. The feed pipe 6 and the discharge pipe 7 are bonded to the two through holes respectively, and the joints are sealed with epoxy resin glue.

[0039] The inner diameters of the feed pipe 6 and the discharge pipe 7 are in the range of 0.1-0.3 mm.

[0040] The specific working steps of the present invention are as follows: The selected HeLa cell suspension is introduced into the fluid chamber 301 through the feed tube 6. The suspension will diffuse around the horseshoe-shaped protrusion 4, and eventually 1-2 cells will be adsorbed around each horseshoe-shaped structure. After the ceramic piezoelectric piece is working, a microbubble will be generated in the middle of the horseshoe-shaped protrusion 4 structure and will vibrate continuously. Under the combined effect of the acoustic radiation force of the vibrating microbubble and the acoustic flow induced resistance, the experimental HeLa cells are attracted to the center of the acoustic flow vortex and self-rotate under the action of the acoustic flow. The angular velocity of the cell body rotation caused by the acoustic flow vortex depends on the oscillation intensity of the acoustic field on the propagation surface, and the oscillation intensity is controlled by changing the voltage applied to the piezoelectric buzzer. The schematic diagram of the vortex generated by the horseshoe-shaped protrusion 4 array structure is shown in FIG. Figure 4 , 5 As shown, the particle trajectory simulation diagram of the vibration of a single horseshoe-shaped protrusion 4 structure is as follows Figure 6 shown.

[0041] When observing biological samples, the rotation of the biological samples can be monitored in real time, and the obtained biological sample rotation video can be sliced ​​frame by frame through editing software, so that the obtained two-dimensional contour image frames can be superimposed to reconstruct the three-dimensional shape, and the surface area, volume, ellipticity, roughness and other geometric parameters of the biological samples can be collected.

[0042] After the experiment is finished, the feed pipe 6 is pressurized and the biological sample solution is discharged from the discharge pipe 7 .

[0043] The above embodiments are only for illustrating the technical concept and features of the present invention, and their purpose is to enable people familiar with the technology to understand the content of the present invention and implement it accordingly, and they cannot be used to limit the protection scope of the present invention. Any equivalent transformation or modification made according to the spirit of the present invention should be included in the protection scope of the present invention.

Claims

1. An observation device for manipulating biological samples using an ultrasonic oscillation bubble array, characterized in that: The observation device comprises an observation device body (1), wherein the observation device body (1) is provided with a stage (101); the stage (101) is connected to a bottom plate (2), the bottom plate (2) is connected to a flow channel layer (3), and the flow channel layer (3) is provided with a fluid chamber (301); a plurality of horseshoe-shaped protrusions (4) are provided in the fluid chamber (301), and a cover plate (5) is connected to the upper surface of the flow channel layer (3); a feed pipe (6) and a discharge pipe (7) are provided on the cover plate (5), and the feed pipe (6) and the discharge pipe (7) are in communication with the fluid chamber (301); an ultrasonic excitation unit (8) is connected to the bottom plate (2), and the ultrasonic excitation unit (8) is electrically connected to a drive circuit (9).

2. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 1, characterized in that: The observation device body (1) is an optical microscope or an electron microscope, and the bottom plate (2) is bonded or connected to the stage (101) of the observation device body (1) by screws; the flow channel layer (3) is made of glass or acrylic material, and the flow channel layer (3) is bonded to the bottom plate (2).

3. An observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 1 or 2, characterized in that: The horseshoe-shaped protrusions (4) are arranged in an array on the substrate. The horseshoe-shaped protrusions (4) are integrally formed with the substrate. The substrate is bonded to the bottom plate (2) in the fluid chamber (301). The size of the substrate matches the fluid chamber (301). The material of the horseshoe-shaped protrusions (4) is a synthetic polymer, a bio-based elastomer or silicone rubber.

4. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 1, characterized in that: The ultrasonic excitation unit (8) is a ceramic piezoelectric sheet, an ultrasonic transducer or an interdigital electrode.

5. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 4, characterized in that: When the ultrasonic excitation unit (8) is a ceramic piezoelectric piece, the vibration frequency of the ceramic piezoelectric piece is at an ultrasonic level, and the operating frequency is adjusted by sweeping the frequency from 20 kHz to 200 kHz. The operating voltage used is 1-10 Vpp.

6. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 5, characterized in that: The ceramic piezoelectric sheet is bonded to the base plate (2), and the ceramic piezoelectric sheet is connected to the drive circuit (9) via a wire.

7. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 1, characterized in that: The material of the bottom plate (2) is glass, ceramic or alloy steel, and the material of the cover plate (5) is transparent glass or acrylic.

8. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 1, characterized in that: The cover plate (5) is bonded to the flow channel layer (3), and through holes are provided at both ends of the cover plate (5), and the feed pipe (6) and the discharge pipe (7) are bonded to the two through holes respectively.

9. The observation device for manipulating biological samples using an ultrasonic oscillation bubble array as claimed in claim 8, characterized in that: The inner diameters of the feed pipe (6) and the discharge pipe (7) are in the range of 0.1-0.3 mm.

10. A method for observing biological samples by using an ultrasonic oscillating bubble array, using the observation device for observing biological samples by using an ultrasonic oscillating bubble array as described in any one of claims 1 to 9, characterized in that The steps include: Step 1: The biological sample solution is introduced into the fluid chamber (301) through the feeding tube (6); Step 2: The ultrasonic excitation unit (8) works, and the sound waves generated can be transmitted to the horseshoe-shaped protrusion (4) array at the front of the bottom plate (2) through the bottom plate (2). Oscillating microbubbles are generated in the horseshoe-shaped protrusions (4). Under the combined effect of the acoustic radiation force of the oscillating microbubbles and the acoustic flow induced resistance, the biological sample solution will diffuse around the horseshoe-shaped protrusion (4) array, and eventually 1-2 cells will be adsorbed around each horseshoe-shaped structure; and the sound waves will generate acoustic vortices in the biological sample solution. The continuous flow of the acoustic vortices can adjust the rotational angular velocity of the sample. The oscillation intensity can be controlled by changing the voltage applied to the ultrasonic excitation unit (8), thereby adjusting the rotational speed of the sample; Step 3: When observing biological samples, the rotation of the biological samples can be monitored in real time, and the obtained biological sample rotation video can be sliced ​​frame by frame through editing software, so that the obtained two-dimensional contour image frames are superimposed to reconstruct the three-dimensional shape, and the geometric parameters such as surface area, volume, ellipticity, roughness, etc. of the biological samples can be collected; Step 4: After the experiment is completed, the feed pipe (6) is pressurized and the biological sample solution is discharged from the discharge pipe (7).

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