CN110628614A - Microfluidic whole blood cell multistage sorting chip and method based on surface acoustic waves - Google Patents
Microfluidic whole blood cell multistage sorting chip and method based on surface acoustic waves Download PDFInfo
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
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Abstract
A microfluidic whole blood cell multistage sorting chip based on surface acoustic waves and a method thereof are disclosed, wherein the chip comprises a piezoelectric substrate, two groups of interdigital transducers are sputtered on the piezoelectric substrate, and a PDMS micro-channel system is bonded on the piezoelectric substrate and positioned on one side of the two groups of interdigital transducers; the method utilizes two groups of interdigital transducers, combines two-stage sorting micro-channels, and realizes the sequential sorting of white blood cells, red blood cells and platelets by applying different frequencies and voltages.
Description
Technical Field
The invention relates to the technical field of biological sample processing, in particular to a microfluidic whole blood cell multistage sorting chip and a microfluidic whole blood cell multistage sorting method based on surface acoustic waves.
Background
The micro-fluidic technology is rapidly developed in the last two decades, and a micro-fluidic chip is also commonly called a chip laboratory, so that the processes of preparation, treatment, transmission, reaction and the like of a sample can be concentrated on a chip with the square centimeter level, the size of experimental equipment is reduced, and the consumption of reagents is reduced. Therefore, more and more microfluidic technologies are applied to the sorting of cells, including dielectrophoretic manipulation, fluid inertial force manipulation, microfluidic microfiltration membrane methods, magnetic manipulation, and acoustic field force manipulation. In the technologies, the surface acoustic wave manipulation particles are accurate in control, non-labeled, non-invasive, good in biocompatibility and good in application prospect in the aspect of biological particle manipulation. The acoustic surface wave is an acoustic wave which is transmitted along the surface of a piezoelectric substrate, and is characterized in that acoustic energy is converged on the surface of the substrate, so that the control on fluid on the surface of the substrate can be realized, and the acoustic surface wave has high-efficiency fluid-solid coupling characteristics, so that the acoustic surface wave is widely applied to the field of microfluidics.
The current studies of blood cell sorting related to surface acoustic waves have emerged: nam et al deflect leukocytes and erythrocytes in a microfluidic chip using a standing wave field of surface acoustic waves to realize sorting of platelets, and the collection purity is about 98% (J.Nam, H.Lim, D.Kim, S.shin, Separation of platelets from floor blood using stationary surfaces in a microchannel, Lab on a chip,2011,11, 3361.); TonyJun Huang et al use surface acoustic waves in microfluidic chips to achieve sorting and washing of leukocytes in red blood cell debris (s.li, x.ding, z.mao, y.chen, n.nama, f.guo, p.li, L Wang, c.e.cameronbc, t.j.huang, Stacking Surface Acid Wave (SSAW) -basedcell washing, Lab on a Chip 2015,15, 331.); tony Jun Huang et al invented a microfluidic chip for High speed sorting of platelets using sound waves, which completed sorting of platelets by eliminating white and red blood cells, with a fluid throughput that was 2500 times higher than previous methods for sorting platelets based on sound waves (Y.Chen, M.Wu, L.ren, J.Liu, Pamela H.Whitley, L.Wang, T.J.Huang, High-throughput classification of platelets from white blood, Lab on a chip,2016,16, 3346.).
Although the above methods are successfully applied to blood cell sorting, they are only limited to sorting of single kind of blood cells, and can not simultaneously sort white blood cells, red blood cells and platelets on one microfluidic chip.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a microfluidic whole blood cell multistage sorting chip and a method based on surface acoustic waves.
In order to achieve the purpose, the invention adopts the following technical scheme:
the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises a piezoelectric substrate 1, two groups of interdigital transducers are sputtered on the piezoelectric substrate 1, and a PDMS micro-channel system is bonded on the piezoelectric substrate 1 and is positioned on one side of the two groups of interdigital transducers.
The PDMS micro flow channel system comprises a first sheath flow liquid inflow channel 4, wherein the inlet end of the first sheath flow liquid inflow channel 4 is connected with a sheath flow liquid inflow connector, and the outlet end of the first sheath flow liquid inflow channel 4 is connected with the inlet end of a leukocyte sorting flow channel 7; the inlet end of the leukocyte sorting flow channel 7 is connected with the outlet end of the blood cell sample liquid inflow channel 5, and the inlet end of the blood cell sample liquid inflow channel 5 is connected with a blood cell sample liquid inflow connector; the inlet end of the leukocyte sorting flow channel 7 is connected with the outlet end of the second sheath flow liquid inflow channel 6, and the inlet end of the second sheath flow liquid inflow channel 6 is connected with a sheath flow liquid inflow connector; the outlet end of the leukocyte sorting flow channel 7 is connected with the inlet end of a leukocyte collecting channel 8, and the outlet end of the leukocyte collecting channel 8 is connected with a leukocyte outlet connector;
the outlet end of the leukocyte sorting flow channel 7 is connected with the inlet end of a red blood cell and platelet inflow channel 9, and the outlet end of the red blood cell and platelet inflow channel 9 is connected with the inlet end of a red blood cell sorting flow channel 11;
the inlet end of the erythrocyte sorting flow channel 11 is connected with the outlet end of a third sheath flow liquid inflow channel 10, and the inlet end of the third sheath flow liquid inflow channel 10 is connected with a sheath flow liquid inflow connector; the outlet end of the red blood cell sorting flow channel 11 is connected with the inlet end of a red blood cell collecting channel 12, and the outlet end of the red blood cell collecting channel 12 is connected with a red blood cell collecting connector; the outlet end of the red blood cell sorting flow channel 11 is connected with the inlet end of a platelet collecting channel 13, and the outlet end of the platelet collecting channel 13 is connected with a platelet collecting connector.
The two groups of interdigital transducers comprise a first group of interdigital transducer 2 and a second group of interdigital transducer 3, and the relative positions of the first group of interdigital transducer 2, the second group of interdigital transducer 3 and the PDMS micro-channel system are as follows: the interdigital of the first group of interdigital transducers 2 is parallel to the leukocyte sorting flow channel 7, the vertical distance between the first group of interdigital transducers 2 and the leukocyte sorting flow channel 7 is 1mm, and the acoustic aperture acts within the length range of the leukocyte sorting flow channel 7; the interdigital of the second group of interdigital transducers 3 is parallel to the red blood cell sorting flow channel 11, the vertical distance between the second group of interdigital transducers 3 and the red blood cell sorting flow channel 11 is 1mm, and the acoustic aperture acts within the length range of the red blood cell sorting flow channel 11.
The two sets of interdigital transducers comprise a plurality of pairs of interdigital transducers.
The first group of interdigital transducers 2 comprises 100 pairs of interdigital, the width of an interdigital strip is 20um, and the acoustic aperture is 2 mm; the second group of interdigital transducers 3 comprises 100 pairs of interdigital, the width of the finger strip is 10um, and the acoustic aperture is 2 mm.
The PDMS micro flow channel system is characterized in that all flow channels are 70um in height, and the width of each part of the flow channel is as follows: the width of the first sheath flow liquid inflow channel 4 is 150um, the width of the blood cell sample liquid inflow channel 5 is 300um, the width of the second sheath flow liquid inflow channel 6 is 250um, the width of the white blood cell sorting flow channel 7 is 500um, the width of the white blood cell collecting channel 8 is 300um, the width of the red blood cell and platelet inflow channel 9 is 240um, the width of the third sheath flow liquid inflow channel 10 is 300um, the width of the red blood cell sorting flow channel 11 is 500um, the width of the red blood cell collecting channel 12 is 300um, and the width of the platelet collecting channel 13 is 240 um; the first sheath flow liquid inflow channel 4, the second sheath flow liquid inflow channel 6, the white blood cell collecting channel 8, the third sheath flow liquid inflow channel 10 and the red blood cell collecting channel 12 are all circular arc-shaped channels, and the blood cell sample liquid inflow channel 5, the white blood cell sorting channel 7, the red blood cell and platelet inflow channel 9, the red blood cell sorting channel 11 and the platelet collecting channel 13 are all straight channels.
The piezoelectric substrate 1 is made of 128-degree Y-cut lithium niobate.
The two groups of interdigital transducers adopt a double-layer structure of 50 nm bottom layer chromium and 200 nm upper layer gold.
The sorting method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises the following steps:
1) fixing a microfluidic whole blood cell multistage sorting chip based on surface acoustic waves on an objective table of a microscope, and observing and determining a white blood cell sorting flow channel 7 and a red blood cell sorting flow channel 11 in a microscope field under an eyepiece;
2) a sheath flow liquid inflow joint connected with the first sheath flow liquid inflow channel 4, a sheath flow liquid inflow joint connected with the second sheath flow liquid inflow channel 6 and a sheath flow liquid inflow joint connected with the third sheath flow liquid inflow channel 10 are respectively connected with three PBS solution storage bottles on a nitrogen pressure injection pump through Teflon catheters, a blood cell sample liquid inflow joint connected with the blood cell sample liquid inflow channel 5 is connected with a blood cell sample liquid storage bottle to be separated on the nitrogen pressure injection pump through a Teflon catheter, and a leukocyte outlet joint, an erythrocyte collection joint and a platelet collection joint are respectively connected with a leukocyte collection container, an erythrocyte collection container and a platelet collection container through Teflon catheters;
3) connecting the positive and negative poles of the signal output ports of the two signal generators with the positive and negative poles of the first group of interdigital transducers 2 and the second group of interdigital transducers 3 respectively, adjusting the output signals of the two signal generators to be sine continuous output, wherein the output voltage of the signal generator connected with the first group of interdigital transducers 2 is 20-40Vpp, and the output frequency is 48.5 MHz; the output voltage of the signal generator connected with the second group of interdigital transducers 3 is 20-40Vpp, and the output frequency is 99 MHz;
4) and starting the injection pump, enabling the blood cell sample liquid and the sheath flow liquid to form stable laminar flow by adjusting the pressure of the inlet ends of the first sheath flow liquid inflow channel 4, the blood cell sample liquid inflow channel 5, the second sheath flow liquid inflow channel 6 and the third sheath flow liquid inflow channel 10, and then opening an output switch of the signal generator to perform whole blood cell sorting.
The preparation method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises the following steps:
1) manufacturing a layer of photoresist with two groups of interdigital transducer patterns on the surface of a clean lithium niobate substrate by utilizing a photoetching technology;
2) manufacturing two groups of interdigital transducers on the surface of the lithium niobate substrate by adopting a sputtering and stripping process;
3) manufacturing an SU8 mold on a silicon substrate by adopting a photoetching technology;
4) manufacturing a PDMS micro-channel system made of PDMS material by using an SU8 mold, and performing inlet and outlet joint treatment;
5) and aligning the PDMS micro-channel system cleaned by the plasma with the lithium niobate substrate, and then preserving the heat at 150 ℃ for 2 hours to complete bonding.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention utilizes the surface acoustic wave technology to sort the whole blood cells in the microfluidic whole blood cell multistage sorting chip, not only fully utilizes the characteristics of high energy density, non-labeling, non-invasion and good biocompatibility of the surface acoustic wave, but also concentrates the whole blood cell sorting process on a chip of a square centimeter grade, reduces the size of experimental equipment, reduces the reagent consumption and reduces the sorting cost.
(2) The invention realizes the sequential sorting of white blood cells, red blood cells and platelets by utilizing two groups of interdigital transducers on a microfluidic whole blood cell multistage sorting chip, and breaks the limitation of sorting single blood cells by utilizing surface acoustic waves before.
(3) The invention realizes the sorting of blood cells by utilizing the traveling wave field of the surface acoustic wave, the distance of the traveling wave deflected cells is several times of the wavelength of the acoustic wave, the sorting effect is obvious without being limited by the distance of the acoustic wave node, and the defect that the distance of the deflected target cells can only be 1/2 wavelengths by utilizing the standing wave field of the surface acoustic wave is overcome.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic whole blood cell multistage sorting chip based on surface acoustic waves.
FIG. 2 is a schematic diagram of the microfluidic whole blood cell multistage sorting chip based on surface acoustic waves.
FIG. 3a is an enlarged partial view of a flow channel for leukocyte sorting during sorting of whole blood cells; FIG. 3b is a close-up view of the red blood cell sorting flow path during sorting of whole blood cells.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings and examples.
Referring to fig. 1 and 2, the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises a piezoelectric substrate 1, two sets of interdigital transducers are sputtered on the piezoelectric substrate 1, and a PDMS micro-channel system is bonded on the piezoelectric substrate 1 and is positioned on one side of the two sets of interdigital transducers.
The PDMS micro flow channel system comprises a first sheath flow liquid inflow channel 4, wherein the inlet end of the first sheath flow liquid inflow channel 4 is connected with a sheath flow liquid inflow connector, and the outlet end of the first sheath flow liquid inflow channel 4 is connected with the inlet end of a leukocyte sorting flow channel 7; the inlet end of the leukocyte sorting flow channel 7 is connected with the outlet end of the blood cell sample liquid inflow channel 5, and the inlet end of the blood cell sample liquid inflow channel 5 is connected with a blood cell sample liquid inflow connector; the inlet end of the leukocyte sorting flow channel 7 is connected with the outlet end of the second sheath flow liquid inflow channel 6, and the inlet end of the second sheath flow liquid inflow channel 6 is connected with a sheath flow liquid inflow connector; the outlet end of the leukocyte sorting flow channel 7 is connected with the inlet end of a leukocyte collecting channel 8, and the outlet end of the leukocyte collecting channel 8 is connected with a leukocyte outlet connector;
the outlet end of the leukocyte sorting flow channel 7 is connected with the inlet end of a red blood cell and platelet inflow channel 9, and the outlet end of the red blood cell and platelet inflow channel 9 is connected with the inlet end of a red blood cell sorting flow channel 11;
the inlet end of the erythrocyte sorting flow channel 11 is connected with the outlet end of a third sheath flow liquid inflow channel 10, and the inlet end of the third sheath flow liquid inflow channel 10 is connected with a sheath flow liquid inflow connector; the outlet end of the red blood cell sorting flow channel 11 is connected with the inlet end of a red blood cell collecting channel 12, and the outlet end of the red blood cell collecting channel 12 is connected with a red blood cell collecting connector; the outlet end of the red blood cell sorting flow channel 11 is connected with the inlet end of a platelet collecting channel 13, and the outlet end of the platelet collecting channel 13 is connected with a platelet collecting connector;
the first sheath flow liquid inflow channel 4, the second sheath flow liquid inflow channel 6 and the third sheath flow liquid inflow channel 10 are respectively introduced with sheath flow liquid, the blood cell sample liquid inflow channel 5 is introduced with blood cell sample liquid, the three-phase fluid forms a first stable laminar flow I in the leukocyte sorting flow channel 7, and forms a second stable laminar flow II in the erythrocyte sorting flow channel 11; the outlet end of the white blood cell collecting channel 8 is used for collecting white blood cells, the outlet end of the red blood cell collecting channel 12 is used for collecting red blood cells, and the platelet collecting channel 13 is used for collecting platelets.
The two groups of interdigital transducers comprise a first group of interdigital transducer 2 and a second group of interdigital transducer 3, and the relative positions of the first group of interdigital transducer 2, the second group of interdigital transducer 3 and the PDMS micro-channel system are as follows: the interdigital of the first group of interdigital transducers 2 is parallel to the leukocyte sorting flow channel 7, the vertical distance between the first group of interdigital transducers 2 and the leukocyte sorting flow channel 7 is 1mm, and the acoustic aperture acts within the length range of the leukocyte sorting flow channel 7; the interdigital of the second group of interdigital transducers 3 is parallel to the red blood cell sorting flow channel 11, the vertical distance between the second group of interdigital transducers 3 and the red blood cell sorting flow channel 11 is 1mm, and the acoustic aperture acts within the length range of the red blood cell sorting flow channel 11; the first group of interdigital transducers 2 and the second group of interdigital transducers 3 are arranged on the same side of the PDMS micro-channel, the first group of interdigital transducers 2 generate acoustic radiation force in the white blood cell sorting channel to realize sorting of white blood cells, red blood cells and platelets, the second group of interdigital transducers 3 generate acoustic radiation force in the red blood cell sorting channel to realize sorting of red blood cells and platelets, and the two groups of interdigital transducers are as close to the PDMS micro-channel system as possible in the vertical direction mainly for reducing acoustic energy loss.
The two groups of interdigital transducers comprise a plurality of pairs of interdigital, generate surface acoustic waves on the piezoelectric substrate according to the inverse piezoelectric effect, and the surface acoustic waves realize the manipulation of cells in the micro-channel.
The first group of interdigital transducers 2 comprises 100 pairs of interdigital, the width of the interdigital is 20um, the acoustic aperture is 2mm, and acoustic surface waves with the frequency of 48.5MHz are generated on the piezoelectric substrate 1 under the action of sine alternating current; the second group of interdigital transducers 3 comprises 100 pairs of interdigital, the width of the interdigital is 10um, the acoustic aperture is 2mm, and the surface acoustic wave with the frequency of 99MHz is generated at the piezoelectric base 1 under the action of sine alternating current.
The PDMS micro flow channel system is characterized in that all flow channels are 70um in height, and the width of each part of the flow channel is as follows: the width of the first sheath flow liquid inflow channel 4 is 150um, the width of the blood cell sample liquid inflow channel 5 is 300um, the width of the second sheath flow liquid inflow channel 6 is 250um, the width of the white blood cell sorting flow channel 7 is 500um, the width of the white blood cell collecting channel 8 is 300um, the width of the red blood cell and platelet inflow channel 9 is 240um, the width of the third sheath flow liquid inflow channel 10 is 300um, the width of the red blood cell sorting flow channel 11 is 500um, the width of the red blood cell collecting channel 12 is 300um, and the width of the platelet collecting channel 13 is 240 um; the first sheath flow liquid inflow channel 4, the second sheath flow liquid inflow channel 6, the white blood cell collecting channel 8, the third sheath flow liquid inflow channel 10 and the red blood cell collecting channel 12 are all circular arc-shaped channels, and the blood cell sample liquid inflow channel 5, the white blood cell sorting channel 7, the red blood cell and platelet inflow channel 9, the red blood cell sorting channel 11 and the platelet collecting channel 13 are all straight channels.
The piezoelectric substrate 1 is made of 128-degree Y-cut lithium niobate (128-degree Y-cut LiNbO 3).
The two groups of interdigital transducers adopt a double-layer structure of 50 nm bottom layer chromium and 200 nm upper layer gold, wherein the chromium is used as an adhesion layer for enhancing the adhesion strength of the gold and the piezoelectric substrate 1, and the gold is used as a conductive layer.
The PDMS micro-channel system is made of Polydimethylsiloxane (PDMS) with good light transmittance and biocompatibility, and is convenient for optical detection and recording in the whole blood cell sorting process.
The sorting method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises the following steps:
1) fixing a microfluidic whole blood cell multistage sorting chip based on surface acoustic waves on an objective table of a microscope, and observing through an eyepiece to ensure that a leukocyte sorting flow channel 7 and an erythrocyte sorting flow channel 11 in a micro flow channel system are positioned in a microscope field of view and have no inclination;
2) a sheath flow liquid inflow joint connected with the first sheath flow liquid inflow channel 4, a sheath flow liquid inflow joint connected with the second sheath flow liquid inflow channel 6 and a sheath flow liquid inflow joint connected with the third sheath flow liquid inflow channel 10 are respectively connected with three PBS solution storage bottles on a nitrogen pressure injection pump through Teflon catheters, a blood cell sample liquid inflow joint connected with the blood cell sample liquid inflow channel 5 is connected with a blood cell sample liquid storage bottle to be separated on the nitrogen pressure injection pump through a Teflon catheter, and a leukocyte outlet joint, an erythrocyte collection joint and a platelet collection joint are respectively connected with a leukocyte collection container, an erythrocyte collection container and a platelet collection container through Teflon catheters; the pressure inputs of a sheath flow liquid inflow joint connected with the first sheath flow liquid inflow channel 4, a blood cell sample liquid inflow joint connected with the blood cell sample liquid inflow channel 5, a sheath flow liquid inflow joint connected with the second sheath flow liquid inflow channel 6 and a sheath flow liquid inflow joint connected with the third sheath flow liquid inflow channel 10 are respectively set to be 12mbar, 22mbar, 33mbar and 35 mbar;
3) connecting the positive and negative poles of the signal output ports of the two signal generators with the positive and negative poles of the first group of interdigital transducers 2 and the second group of interdigital transducers 3 respectively, adjusting the output signals of the two signal generators to be sine continuous output, wherein the output voltage of the signal generator connected with the first group of interdigital transducers 2 is 20-40Vpp, and the output frequency is 48.5 MHz; the output voltage of the signal generator connected with the second group of interdigital transducers 3 is 20-40Vpp, and the output frequency is 99 MHz; the size of the acoustic radiation force of the traveling wave field of the acoustic surface wave is in direct proportion to the frequency of the interdigital transducers and the particle size of cells, the acoustic surface wave with the frequency of 48.5MHz excited by the first group of interdigital transducers 2 is used for realizing the deflection of white blood cells, red blood cells and platelets keep the original motion state, the acoustic surface wave with the frequency of 99MHz excited by the second group of interdigital transducers 3 is used for realizing the deflection of the red blood cells, and the platelets keep the original motion state;
4) and starting the injection pump, enabling the blood cell sample liquid and the sheath flow liquid to form stable laminar flow by adjusting the pressure of the inlet ends of the first sheath flow liquid inflow channel 4, the blood cell sample liquid inflow channel 5, the second sheath flow liquid inflow channel 6 and the third sheath flow liquid inflow channel 10, and then opening an output switch of the signal generator to perform whole blood cell sorting.
The sorting process is shown in FIG. 2, FIG. 3a and FIG. 3b, wherein the large, middle and small circles in FIG. 2 represent white blood cells, red blood cells and platelets, respectively, the first sheath flow liquid inflow channel 4, the second sheath flow liquid inflow channel 6 and the third sheath flow liquid inflow channel 10 are respectively introduced with sheath flow liquid, the blood cell sample liquid inflow channel 5 is introduced with the whole blood cell sample liquid to be separated, the inlet end pressures of the first sheath flow liquid inflow channel 4, the second sheath flow liquid inflow channel 6, the third sheath flow liquid inflow channel 10 and the blood cell sample liquid inflow channel 5 are adjusted by a nitrogen pressure injection pump, forming a first stable laminar flow I shown by a dotted line in FIG. 3a and a second stable laminar flow II shown by a dotted line in FIG. 3b by the blood cell sample liquid and the sheath flow liquid on both sides in the leukocyte sorting flow channel 7 and the erythrocyte sorting flow channel 11, the blood cell sample liquid being squeezed by the sheath flow liquid within the dotted lines shown in FIGS. 3a and 3 b; the two groups of interdigital transducers apply alternating current, surface acoustic waves are generated on the piezoelectric substrate according to the inverse piezoelectric effect, blood cells mainly receive four forces of acoustic radiation force, fluid viscous force, gravity and buoyancy in the white blood cell sorting flow channel 7 and the red blood cell sorting flow channel 11, the gravity and the buoyancy borne by the blood cells can be basically counteracted, the acoustic radiation force is in direct proportion to the cell volume, the fluid viscous force is in direct proportion to the cell radius, and therefore the larger the cell particle size is, the more obvious the effect of the acoustic radiation force is; in the flow channel 7 for sorting white blood cells, the first group of interdigital transducers 2 excite the surface acoustic wave with the frequency of 48.5MHz, white blood cells are deflected to a white blood cell collecting channel 8 through a first stable laminar flow I under the action of larger acoustic radiation force, red blood cells and platelets with smaller particle sizes are not obviously acted by the acoustic radiation force, the original motion track can be kept to enter the flow channel 11 for sorting red blood cells, in the flow channel 11 for sorting red blood cells, the second group of interdigital transducers 3 excite the surface acoustic wave with the frequency of 99MHz, red blood cells are deflected to a red blood cell collecting channel 12 through a second stable laminar flow II under the action of larger acoustic radiation force, platelets are not obviously acted by the acoustic radiation force, and the original track can be kept to enter the platelet collecting channel 13.
The preparation method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic waves comprises the following steps:
1) manufacturing a layer of photoresist with two groups of interdigital transducer patterns on the surface of a clean lithium niobate substrate by utilizing a photoetching technology;
2) manufacturing two groups of interdigital transducers on the surface of the lithium niobate substrate by adopting a sputtering and stripping process;
3) manufacturing an SU8 mold on a silicon substrate by adopting a photoetching technology;
4) manufacturing a PDMS micro-channel system made of PDMS material by using an SU8 mold, and performing inlet and outlet joint treatment;
5) and aligning the PDMS micro-channel system cleaned by the plasma with the lithium niobate substrate, and then preserving the heat at 150 ℃ for 2 hours to complete bonding.
Claims (9)
1. The utility model provides a micro-fluidic whole blood cell multistage sorting chip based on surface acoustic wave, includes piezoelectric substrate (1), its characterized in that: two groups of interdigital transducers are sputtered on the piezoelectric substrate (1), and a PDMS micro-channel system is bonded on the piezoelectric substrate (1) and positioned on one side of the two groups of interdigital transducers;
the PDMS micro flow channel system comprises a first sheath flow liquid inflow channel (4), wherein the inlet end of the first sheath flow liquid inflow channel (4) is connected with a sheath flow liquid inflow connector, and the outlet end of the first sheath flow liquid inflow channel (4) is connected with the inlet end of a leukocyte sorting flow channel (7); the inlet end of the leukocyte sorting flow channel (7) is connected with the outlet end of the blood cell sample liquid inflow channel (5), and the inlet end of the blood cell sample liquid inflow channel (5) is connected with a blood cell sample liquid inflow connector; the inlet end of the leukocyte sorting flow channel (7) is connected with the outlet end of the second sheath flow liquid inflow channel (6), and the inlet end of the second sheath flow liquid inflow channel (6) is connected with a sheath flow liquid inflow connector; the outlet end of the leukocyte sorting flow channel (7) is connected with the inlet end of a leukocyte collecting channel (8), and the outlet end of the leukocyte collecting channel (8) is connected with a leukocyte outlet connector;
the outlet end of the leukocyte sorting flow channel (7) is connected with the inlet end of an erythrocyte and platelet inflow channel (9), and the outlet end of the erythrocyte and platelet inflow channel (9) is connected with the inlet end of an erythrocyte sorting flow channel (11);
the inlet end of the erythrocyte sorting flow channel (11) is connected with the outlet end of a third sheath flow liquid inflow channel (10), and the inlet end of the third sheath flow liquid inflow channel (10) is connected with a sheath flow liquid inflow connector; the outlet end of the red blood cell sorting flow channel (11) is connected with the inlet end of a red blood cell collecting channel (12), and the outlet end of the red blood cell collecting channel (12) is connected with a red blood cell collecting connector; the outlet end of the red blood cell sorting flow channel (11) is connected with the inlet end of a platelet collecting channel (13), and the outlet end of the platelet collecting channel (13) is connected with a platelet collecting joint.
2. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 1, wherein: the two groups of interdigital transducers comprise a first group of interdigital transducers (2) and a second group of interdigital transducers (3), wherein the relative positions of the first group of interdigital transducers (2) and the second group of interdigital transducers (3) and the PDMS micro-channel system are as follows: the interdigital of the first group of interdigital transducers (2) is parallel to the leukocyte sorting flow channel (7), the vertical distance between the first group of interdigital transducers (2) and the leukocyte sorting flow channel (7) is 1mm, and the acoustic aperture acts within the length range of the leukocyte sorting flow channel (7); the interdigital of the second group of interdigital transducers (3) is parallel to the red blood cell sorting flow channel (11), the vertical distance between the second group of interdigital transducers (3) and the red blood cell sorting flow channel (11) is 1mm, and the acoustic aperture acts within the length range of the red blood cell sorting flow channel (11).
3. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 1, wherein: the two sets of interdigital transducers comprise a plurality of pairs of interdigital transducers.
4. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 2, wherein: the first group of interdigital transducers (2) comprises 100 pairs of interdigital, the width of an interdigital strip is 20um, and the acoustic aperture is 2 mm; the second group of interdigital transducers (3) comprises 100 pairs of interdigital, the width of the finger strip is 10um, and the acoustic aperture is 2 mm.
5. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 1, wherein: the PDMS micro flow channel system is characterized in that all flow channels are 70um in height, and the width of each part of the flow channel is as follows: the width of the first sheath flow liquid inflow channel (4) is 150um, the width of the blood cell sample liquid inflow channel (5) is 300um, the width of the second sheath flow liquid inflow channel (6) is 250um, the width of the white blood cell sorting flow channel (7) is 500um, the width of the white blood cell collecting channel (8) is 300um, the width of the red blood cell and platelet inflow channel (9) is 240um, the width of the third sheath flow liquid inflow channel (10) is 300um, the width of the red blood cell sorting flow channel (11) is 500um, the width of the red blood cell collecting channel (12) is 300um, and the width of the platelet collecting channel (13) is 240 um; the first sheath flow liquid inflow channel (4), the second sheath flow liquid inflow channel (6), the white blood cell collecting channel (8), the third sheath flow liquid inflow channel (10) and the red blood cell collecting channel (12) are all arc-shaped flow channels, and the blood cell sample liquid inflow channel (5), the white blood cell sorting flow channel (7), the red blood cell and platelet inflow channel (9), the red blood cell sorting flow channel (11) and the platelet collecting channel (13) are all straight flow channels.
6. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 1, wherein: the piezoelectric substrate (1) is made of 128-degree Y-cut lithium niobate.
7. The acoustic surface wave based microfluidic whole blood cell multistage sorting chip of claim 1, wherein: the two groups of interdigital transducers adopt a double-layer structure of 50 nm bottom layer chromium and 200 nm upper layer gold.
8. The sorting method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic wave according to claim 2, comprising the following steps:
1) fixing a microfluidic whole blood cell multistage sorting chip based on surface acoustic waves on an objective table of a microscope, and observing and determining a white blood cell sorting flow channel (7) and a red blood cell sorting flow channel (11) in a microscope field under an eyepiece;
2) a sheath flow liquid inflow joint connected with the first sheath flow liquid inflow channel (4), a sheath flow liquid inflow joint connected with the second sheath flow liquid inflow channel (6), and a sheath flow liquid inflow joint connected with the third sheath flow liquid inflow channel (10) are respectively connected with three PBS solution storage bottles on the nitrogen pressure injection pump through Teflon catheters, a blood cell sample liquid inflow joint connected with the blood cell sample liquid inflow channel (5) is connected with a blood cell sample liquid storage bottle to be separated on the nitrogen pressure injection pump through the Teflon catheter, and a leukocyte outlet joint, an erythrocyte collection joint and a platelet collection joint are respectively connected with a leukocyte collection container, an erythrocyte collection container and a platelet collection container through the Teflon catheters;
3) connecting the positive and negative poles of the signal output ports of the two signal generators with the positive and negative poles of the first group of interdigital transducers (2) and the second group of interdigital transducers (3) respectively, adjusting the output signals of the two signal generators to be sine continuous output, and connecting the output voltage of the signal generator of the first group of interdigital transducers (2) to be 20-40Vpp and the output frequency to be 48.5 MHz; the output voltage of a signal generator connected with the second group of interdigital transducers (3) is 20-40Vpp, and the output frequency is 99 MHz;
4) and starting the injection pump, enabling the blood cell sample liquid and the sheath flow liquid to form stable laminar flow by adjusting the pressure of the inlet ends of the first sheath flow liquid inflow channel (4), the blood cell sample liquid inflow channel (5), the second sheath flow liquid inflow channel (6) and the third sheath flow liquid inflow channel (10), and then opening an output switch of the signal generator to perform whole blood cell sorting.
9. The preparation method of the microfluidic whole blood cell multistage sorting chip based on the surface acoustic wave according to claim 1, characterized by comprising the following steps:
1) manufacturing a layer of photoresist with two groups of interdigital transducer patterns on the surface of a clean lithium niobate substrate by utilizing a photoetching technology;
2) manufacturing two groups of interdigital transducers on the surface of the lithium niobate substrate by adopting a sputtering and stripping process;
3) manufacturing an SU8 mold on a silicon substrate by adopting a photoetching technology;
4) manufacturing a PDMS micro-channel system made of PDMS material by using an SU8 mold, and performing inlet and outlet joint treatment;
5) and aligning the PDMS micro-channel system cleaned by the plasma with the lithium niobate substrate, and then preserving the heat at 150 ℃ for 2 hours to complete bonding.
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