CN111621400B - Device for carrying out cell thermal cracking by utilizing surface acoustic wave acousto-thermal effect - Google Patents
Device for carrying out cell thermal cracking by utilizing surface acoustic wave acousto-thermal effect Download PDFInfo
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- CN111621400B CN111621400B CN202010434007.XA CN202010434007A CN111621400B CN 111621400 B CN111621400 B CN 111621400B CN 202010434007 A CN202010434007 A CN 202010434007A CN 111621400 B CN111621400 B CN 111621400B
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M23/00—Constructional details, e.g. recesses, hinges
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- C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
- C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
- C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
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Abstract
A device for carrying out cell thermal cracking by utilizing surface acoustic wave acousto-thermal effect comprises a micro-fluidic chip, wherein a high-speed camera and a microscope are arranged above the micro-fluidic chip, the signal input of the micro-fluidic chip is connected with the output of a signal generator through a power amplifier, the signal generator generates an electric signal, and the electric signal is amplified by the power amplifier and transmitted to the micro-fluidic chip to realize the generation and control of surface acoustic waves; the flow channel is internally suspended with liquid drops, cells are wrapped in the liquid drops, and after the cracking is finished, nucleic acid is wrapped in the liquid drops; the invention uses the surface acoustic wave microfluidic device to realize the control of the temperature in the micro-channel, further realizes the rapid and effective cracking of cells, and has the advantages of small equipment size, simple operation, small required clean space and the like; meanwhile, the method has the advantages of high compatibility to various medicines or reagents, low requirements on experimental environment and personnel and the like.
Description
Technical Field
The invention belongs to the technical field of microfluidic chips, and particularly relates to a device for carrying out cell thermal cracking by using Surface Acoustic Wave (SAW) acoustooptical effect.
Background
Cell lysis is an important means for nucleic acid detection, organelle research and the like in biomedical experiments, and with the continuous development of microfluidic chip technology, it is gradually possible to provide a simple, fast and practical lysis mode for cell lysis by using a microfluidic chip.
Currently, cell lysis is mainly achieved by chemical or physical methods. Chemical lysis is the lysis of lipids and proteins in cell membranes using lysis buffers or biological enzymes, and although simple to perform, the remaining chemicals will affect subsequent extraction and amplification. Physical lysis is the tearing or puncturing of cell membranes by some physical action, including electroporation, laser and mechanical disruption, which is less harmful to cellular components but generally requires a complex system.
In contrast, thermal cracking by surface acoustic waves is a non-contact cell processing method that can be controlled by simple electrical signals, thus providing good conditions for high-quality cell cracking and having high dissolution efficiency and dissolution rate. However, in the existing research, the flow driving capability of the surface acoustic wave is utilized to accelerate the flow of the fluid medium in the surface acoustic wave cell lysis, so that the cell is continuously collided with the structure such as the microcolumn, the microsphere and the like in the flow channel to realize the cell lysis, and the surface acoustic wave thermal effect is not involved.
Disclosure of Invention
In order to overcome the disadvantages of the prior art, the present invention provides a device for thermal cracking of cells by using surface acoustic wave thermoacoustic effect, which uses a surface acoustic wave microfluidic device to control the temperature in a micro-channel and further achieve rapid and effective cell cracking.
In order to achieve the purpose, the invention adopts the technical scheme that:
a device for carrying out cell thermal cracking by utilizing a surface acoustic wave acousto-thermal effect comprises a micro-fluidic chip 1, wherein a high-speed camera 2 and a microscope 3 are arranged above the micro-fluidic chip 1, the signal input of the micro-fluidic chip 1 is connected with the output of a signal generator 4 through a power amplifier 5, the signal generator 4 generates an electric signal, and the electric signal is amplified through the power amplifier 5 and transmitted to the micro-fluidic chip 1, so that the generation and control of surface acoustic waves are realized.
The micro-fluidic chip 1 comprises a piezoelectric substrate 1-1, an interdigital transducer 1-2 and a flow channel 1-3 are manufactured on the piezoelectric substrate 1-1, the interdigital transducer 1-2 generates surface acoustic waves, and the surface acoustic waves generate a thermal effect to heat a fluid medium after entering the flow channel 1-3 and are cracked;
droplets 1-4 are suspended in the flow channels 1-3, cells 1-5 are wrapped in the droplets 1-4, and nucleic acids 1-6 are wrapped in the droplets 1-4 after the lysis is finished; the inside and the outside of the interface of the liquid drop 1-4 are biocompatible fluid media which are not mutually soluble, the inside of the liquid drop 1-4 is a physiological water environment suitable for the survival of cells, and the cells 1-5 are heated and cracked in the liquid drop 1-4 to release genetic material nucleic acid 1-6 and are continuously wrapped in the liquid drop 1-4.
The piezoelectric substrate 1-1 is a 128-degree Y-direction cut lithium niobate, the interdigital transducer 1-2 is a planar patterned conductive metal layer sputtered on the piezoelectric substrate 1-1, and the flow channel 1-3 is a PDMS micro flow channel bonded on the piezoelectric substrate 1-1.
The finger space 1-2-1 and the finger width 1-2-2 of the interdigital transducer 1-2 determine the frequency of the surface acoustic wave 1-7, the channel width 1-3-1 and the channel height 1-3-2 of the channel 1-3 influence the propagation of the surface acoustic wave 1-7 in the channel 1-3 and the positions of the node 1-7-1 and the antinode 1-7-2; the distance of the channel 1-3 from the channel-electrode distance 1-3-3 of the interdigital transducer 1-2 also affects the position of the node 1-7-1 and the antinode 1-7-2, and further affects the distribution of the droplets 1-4 and the cells 1-5 within the channel 1-3.
The liquid drop 1-4 wrapped with the cell 1-5 is generated by the T-shaped flow channel 1-3-4, and the discrete phase liquid 1-3-5 suspended with the cell 1-5 forms the liquid drop 1-4 after entering the flow channel 1-3.
The invention has the beneficial effects that:
the invention adopts the interdigital transducer 1-2 to generate the surface acoustic wave 1-7 to heat the fluid medium in the flow channel 1-3, and can realize the cell 1-5 lysis. Compared with a chemical method using cell lysate and the like, the method of the invention has no chemical reagent residue. Compared with the method using microsphere collision and the like, the method of the invention does not need to carry out microsphere screening and separation.
The invention adopts the method that the cells 1-5 are cracked in the liquid drops 1-4, the cracking operation of the cells 1-5 in the independent microenvironment can be realized, the classification screening and the detection of different samples are convenient, and the low-damage and residue-free cell cracking in the liquid drops is difficult to realize by other cell cracking methods: the method of cell destruction using microcolumn collision or fluid shear force cannot lyse cells in the droplets and provide independent microenvironment for the cells and the released genetic material; the method of cell disruption using collision of cell lysate and microspheres is suitable for cell lysis in droplets, but has a residue of chemical agent and microspheres. The method is suitable for most cells, has small harm to genetic materials and is convenient for the next step of genetic material detection.
The invention adopts the high-speed camera 2 and the microscope 3 to observe the experimental phenomenon in real time, the signal generator 4 generates an electric signal, the electric signal is amplified by the power amplifier 5 and is transmitted to the microfluidic chip 1, and the generation and control of the surface acoustic waves 1-7 are realized, so that the whole experimental process can be monitored in real time and can be controlled in real time.
The micro-fluidic chip 1 is adopted, so that the micro-fluidic chip has the advantages of small equipment size, simplicity in operation, small required clean space and the like; meanwhile, the method has the advantages of high compatibility to various medicines or reagents, low requirements on experimental environment and personnel and the like.
Drawings
Fig. 1 is a schematic diagram of an experimental platform according to an embodiment of the present invention.
Fig. 2 is a schematic thermal cracking diagram of the acousto-thermal microfluidic chip used in the present invention, which is a top view angle.
Fig. 3 is a schematic view of the action mechanism of sound waves according to the present invention, which is a front view.
FIG. 4 is a schematic diagram of the generation of a droplet encapsulated with cells according to the present invention, shown from a top view.
Detailed Description
The present invention will be described in detail below with reference to the accompanying drawings.
Referring to fig. 1, a device for performing thermal cracking of cells by using a surface acoustic wave thermoacoustic effect comprises a microfluidic chip 1, wherein a high-speed camera 2 and a microscope 3 are arranged above the microfluidic chip 1, and experimental phenomena generated in the microfluidic chip 1 are recorded by the high-speed camera 2 and the microscope 3; the signal input of the micro-fluidic chip 1 is connected with the output of the signal generator 4 through the power amplifier 5, the signal generator 4 generates an electric signal, and the electric signal is amplified by the power amplifier 5 and transmitted to the micro-fluidic chip 1, so that the generation and control of surface acoustic waves are realized.
Referring to fig. 2, the microfluidic chip 1 includes a piezoelectric substrate 1-1, an interdigital transducer 1-2 and a flow channel 1-3 are fabricated on the piezoelectric substrate 1-1, a surface acoustic wave is generated by the interdigital transducer 1-2, and the surface acoustic wave generates a thermal effect after entering the flow channel 1-3 to heat a fluid medium and perform cracking.
The piezoelectric substrate 1-1 is a 128-degree Y-direction cut lithium niobate, the interdigital transducer 1-2 is a planar patterned conductive metal layer sputtered on the piezoelectric substrate 1-1, and the flow channel 1-3 is a PDMS micro flow channel bonded on the piezoelectric substrate 1-1.
Referring to fig. 2, droplets 1-4 are suspended in the flow channels 1-3, cells 1-5 are wrapped in the droplets 1-4, and nucleic acids 1-6 are wrapped in the droplets 1-4 after lysis is completed; the inside and the outside of the interface of the liquid drop 1-4 are biocompatible fluid media which are not mutually soluble, the inside of the liquid drop 1-4 is a physiological water environment suitable for the survival of cells, and the cells 1-5 are heated and cracked in the liquid drop 1-4 to release genetic material nucleic acid 1-6 and are continuously wrapped in the liquid drop 1-4.
Referring to fig. 3, the interdigital transducer 1-2 has a finger pitch 1-2-1 and a finger width 1-2-2 which determine the frequency of the surface acoustic wave 1-7, and the channel width 1-3-1 and the channel height 1-3-2 of the channel 1-3 affect the propagation of the surface acoustic wave 1-7 in the channel 1-3 and the positions of the node 1-7-1 and the antinode 1-7-2; the distance of the channel 1-3 from the channel-electrode distance 1-3-3 of the interdigital transducer 1-2 also affects the position of the node 1-7-1 and the antinode 1-7-2, and further affects the distribution of the droplets 1-4 and the cells 1-5 within the channel 1-3.
Referring to fig. 4, a droplet 1-4 in which cells 1-5 are packed is generated from a T-shaped flow channel 1-3-4, and a discrete phase liquid 1-3-5 in which the cells 1-5 are suspended forms the droplet 1-4 after entering the flow channel 1-3.
The working principle of the invention is as follows:
the nominal input frequency of the device is the maximum amplitude corresponding frequency. The measurement shows that the temperature of the flow channel 1-3 changes with the square of the applied voltage and is in direct proportion to the applied power, and the heat effect and the power of the surface acoustic wave 1-7 are in a simple linear relation. In addition to this, the introduction of the effect of the fluid flow to remove heat establishes a mathematical model for analyzing the temperature rise of the droplets 1-4. Comparing the model with experimental data, determining that the temperature change is mainly determined by the power and action time of the surface acoustic wave 1-7 and is influenced by the specific heat capacity, the mass of the heated fluid, the area surface area of the heated fluid, the heat transfer coefficient and the ambient temperature.
The frequency and power of the input electrical signal determine the wavelength and amplitude of the surface acoustic waves 1-7, which has a direct effect on the dissolution efficiency of the lysis of the cells 1-5. Rapid mass lysis of cells 1-5 occurs only near the resonant frequency determined by the structure of interdigital transducer 1-2. Within these frequency neighborhoods, the lysis onset time is taken to a minimum when the resonant frequency is reached. The resonant frequency is required to satisfy the electrode finger width of the interdigital transducer 1-2 with the surface acoustic wave 1-7 four times of the wavelength. As power increases, the start-up time decreases and then stabilizes after the power exceeds a threshold. In addition, the starting time is short when the number of electrode pairs is large, and the number of electrode pairs has positive influence on the cracking efficiency.
When the surface acoustic waves 1-7 are turned on, the cells 1-5 form parallel lines in the channel under the action of the acoustic radiation force. The sound waves enter the channel in the form of longitudinal waves and are reflected onto the side walls of the flow channels 1-3. The interference of the reflected sound wave and the original sound wave forms a pressure node in the channel, and the pressure amplitude is minimum. The position of the pressure node is determined by the position of the node 1-7-1, namely the stagnation point formed by interference of the original sound wave and the reflected sound wave and offset of the amplitude. The reason for the cells 1-5 to accumulate at these pressure nodes is that the acoustic radiation forces are the weakest and the pressure is the lowest at these locations. That is, when the cells 1 to 5 are arranged in parallel, the influence of the acoustic radiation force on the cells 1 to 5 is small. Such an acoustic field is not harmful to the cells if the temperature is kept in a lower range (below the threshold for cell lysis). In the rectangular flow channel 1-3, the liquid flow at the pressure node is almost stationary, the influence of the acoustic radiation force and the fluid shear force on the cells 1-5 is low, and the cells 1-5 are only lysed by the temperature increase. After the droplets 1-4 are used to wrap the cells 1-5, the influence of the fluid shear force is more isolated.
The experiment shows that the highest temperature reached in the flow passages 1-3 can reach 90 ℃. The temperature rise in the flow channel 1-3 is detected under different powers, and under different powers, 1.6w, 2.1w, 2.8w, the starting time of the lysis of the lower cell 1-5 is 303s, 200s, 110s, which are all started when the temperature reaches about 85 ℃, and the temperature can be used as the threshold value of the thermal lysis. Thus, the overall lysis mechanism is that the thermal effect caused by SAW causes the liquid temperature to exceed the thermal lysis threshold, causing damage and lysis of cellular structures.
The dimensions of the interdigital transducer 1-2, finger spacing 1-2-1 and finger width 1-2-2 determine the frequency of the surface acoustic wave 1-7. The sound velocity on the piezoelectric substrate 1-1 is determined by the substrate material, and the sum of the finger distance 1-2-1 and the finger width 1-2-2 needs to be half wavelength of the surface acoustic wave 1-7. The channel width 1-3-1 and channel height 1-3-2 of the channel 1-3 affect the propagation of the surface acoustic waves 1-7 within the channel 1-3, as well as the location of the node 1-7-1 and the antinode 1-7-2. The distance of the channel 1-3 from the channel-electrode distance 1-3-3 of the interdigital transducer 1-2 also affects the position of the node 1-7-1 and the antinode 1-7-2, and further affects the distribution of the droplets 1-4 and the cells 1-5 within the channel 1-3. The width of the flow channel is 1-3-1, and the distance between the flow channel and the electrode is 1-3-3, so that the nearest node 1-7-1 to the side wall in the flow channel 1-3 is not the antinode 1-7-2, otherwise, the liquid drop 1-4 or the cell 1-5 is attached to the side wall to influence the experimental effect. The flow channel height 1-3-2 should be within four wavelengths of the surface acoustic wave 1-7, and an excessively high flow channel height 1-3-2 would result in an insignificant partial area node 1-7-1 and excessive turbulence.
A discrete phase liquid 1-3-5, such as physiological saline, in which cells 1-5 are suspended is injected into the T-shaped flow channel 1-3-4 region, and a continuous phase liquid, such as perfluorinated oil, incompatible with the discrete phase liquid is injected into the flow channel 1-3. After the discrete phase liquid 1-3-5 enters the main flow channel of the flow channel 1-3, individual liquid drops 1-4 are formed and are suspended in the flow channel 1-3 as a dispersed phase. The size of the droplets 1-4 can be controlled by various conditions, such as the size of the T-shaped flow channels 1-3-4, the flow rates of the continuous and discrete phase liquids 1-3-5, and the like. The flow velocity can be adjusted by adjusting the conditions of pressure, flow and the like at the inlet, so that the size of the liquid drops can be controlled.
Claims (2)
1. The utility model provides a device for utilize surface acoustic wave thermoacoustic effect to carry out cell thermal cracking, includes micro-fluidic chip (1), its characterized in that: a high-speed camera (2) and a microscope (3) are arranged above the micro-fluidic chip (1), the signal input of the micro-fluidic chip (1) is connected with the output of the signal generator (4) through a power amplifier (5), the signal generator (4) generates an electric signal, and the electric signal is amplified by the power amplifier (5) and transmitted to the micro-fluidic chip (1) to realize the generation and control of surface acoustic waves;
the micro-fluidic chip (1) comprises a piezoelectric substrate (1-1), wherein an interdigital transducer (1-2) and a flow channel (1-3) are manufactured on the piezoelectric substrate (1-1), a surface acoustic wave is generated by the interdigital transducer (1-2), and the surface acoustic wave generates a thermal effect after entering the flow channel (1-3) to heat a fluid medium and is cracked;
the flow channel (1-3) is internally suspended with liquid drops (1-4), the liquid drops (1-4) are internally wrapped with cells (1-5), and after the lysis is finished, the liquid drops (1-4) are internally wrapped with nucleic acids (1-6); the inside and the outside of the interface of the liquid drop (1-4) are biocompatible fluid media which are not mutually soluble, the inside of the liquid drop (1-4) is a physiological water environment suitable for the survival of cells, and the cells (1-5) are heated and cracked in the liquid drop (1-4) to release genetic material nucleic acid (1-6) and are continuously wrapped in the liquid drop (1-4);
the piezoelectric substrate (1-1) is a 128-degree Y-direction cut lithium niobate, the interdigital transducer (1-2) is a planar patterned conductive metal layer sputtered on the piezoelectric substrate (1-1), and the flow channel (1-3) is a PDMS micro flow channel bonded on the piezoelectric substrate (1-1);
the finger space (1-2-1) and the finger width (1-2-2) of the interdigital transducer (1-2) determine the frequency of the surface acoustic wave (1-7), and the channel width (1-3-1) and the channel height (1-3-2) of the channel (1-3) influence the propagation of the surface acoustic wave (1-7) in the channel (1-3) and the positions of the node (1-7-1) and the antinode (1-7-2); the distance (1-3-3) of the flow channel (1-3) from the flow channel-electrode of the interdigital transducer (1-2) also affects the positions of the nodes (1-7-1) and the antinodes (1-7-2), and further affects the distribution of the liquid droplets (1-4) and the cells (1-5) in the flow channel (1-3).
2. The apparatus for thermal cracking of cells using surface acoustic wave thermoacoustic effect as claimed in claim 1, wherein: the liquid drops (1-4) wrapped with the cells (1-5) are generated by the T-shaped flow channels (1-3-4), and the discrete phase liquid (1-3-5) in which the cells (1-5) are suspended forms the liquid drops (1-4) after entering the flow channels (1-3).
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102612405A (en) * | 2009-08-24 | 2012-07-25 | 格拉斯哥大学理事会 | Fluidics apparatus and fluipics substrate |
| CN106596487A (en) * | 2016-12-14 | 2017-04-26 | 中国科学院苏州生物医学工程技术研究所 | Intracellular protein detection method based on microdrop and nano-fluorescence probe |
| CN109868218A (en) * | 2019-03-26 | 2019-06-11 | 西安交通大学 | A kind of nanobacteria cracking reactor and bacteria lysis method based on surface acoustic wave |
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| EP1548702A1 (en) * | 2003-12-24 | 2005-06-29 | Interuniversitair Microelektronica Centrum Vzw | Method for ultra-fast controlling of a magnetic cell and related devices |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102612405A (en) * | 2009-08-24 | 2012-07-25 | 格拉斯哥大学理事会 | Fluidics apparatus and fluipics substrate |
| CN106596487A (en) * | 2016-12-14 | 2017-04-26 | 中国科学院苏州生物医学工程技术研究所 | Intracellular protein detection method based on microdrop and nano-fluorescence probe |
| CN109868218A (en) * | 2019-03-26 | 2019-06-11 | 西安交通大学 | A kind of nanobacteria cracking reactor and bacteria lysis method based on surface acoustic wave |
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