CN117305102A - Acoustic flow control device for sorting outer vesicles in plasma sample and use method thereof - Google Patents
Acoustic flow control device for sorting outer vesicles in plasma sample and use method thereof Download PDFInfo
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Abstract
The invention discloses an acoustic flow control device for sorting outer vesicles in a plasma sample and a use method thereof, wherein the acoustic flow control device comprises an upper-layer micro-fluidic chip and a lower-layer piezoelectric transducer, and the piezoelectric transducer is reversibly connected with the micro-fluidic chip through a liquid film layer; the micro-fluidic chip is internally provided with a double-row micro-ridge structure, and the section of each row of micro-ridge is a congruent right triangle with two bevel edges opposite to each other; the micro-ridge structure adjusts the sound wave excited by the piezoelectric transducer, a standing wave sound field is formed above the interval of the two rows of micro-ridges, a symmetrical vortex field is formed above each row of micro-ridges through the sound induced micro-flow effect, and simultaneously, the separation of cells and outer vesicles in a plasma sample is realized by utilizing the sound radiation force and the sound flow drag force. The acoustic flow control sorting device has the advantages of simple structure, low control difficulty and small influence of temperature, and can meet the disposable requirement of biological samples. The invention also discloses a use method of the acoustic streaming control device, which can realize the efficient and stable separation of the outer vesicles in the plasma sample.
Description
Technical Field
The invention relates to the technical field of microfluidic technology and biological particle treatment, in particular to an acoustic flow control device for sorting outer vesicles in a plasma sample and a use method thereof.
Background
In recent years, biological particle separation methods and devices such as cells based on microfluidic chips are continuously emerging, and the method and the device have important significance for biochemical analysis processes such as specific cell detection, drug screening and tissue engineering. However, the microfluidic sorting devices such as centrifugal type, electrophoresis type and electromagnetic separation type commonly used at present generally have influence on the activity of biological particles, and have complex chip structure, difficult manufacture and high operation difficulty, so that the popularization and application of the microfluidic-based biological sorting technology are greatly limited.
The acoustic flow control is a micro-fluidic technology combining an acoustic field and a micro-fluid, and particles in the micro-fluid are captured, sorted and the like under the action of acoustic radiation force and acoustic flow drag force. Due to the advantages of non-contact and high efficiency of sound waves, the particle sorting device based on sound flow control has the advantages of simple equipment, high controllability, flexible operation and the like, and has great application prospect in the sorting field of micro-nano biological particles such as blood cells (5-25 mu m), outer vesicles (40-1000 nm) and the like. The current acoustic flow control sorting devices are mainly two types, one is based on piezoelectric single crystals such as lithium niobate (LiNbO) 3 ) Lithium tantalate (LiTaO) 3 ) The device is extremely easy to be influenced by temperature and the like, the sorting process is unstable, the adopted interdigital transducer is complex in manufacturing process and high in control difficulty, and the interdigital transducer is irreversibly connected with a sorting chip, so that the disposable processing requirement of biological samples is difficult to be met; the other is to use piezoelectric ceramics such as lead zirconate titanate piezoelectric ceramics (PZT) and the like to generate longitudinal waves, form a standing wave sound field through reflection of the chip on the acoustic waves, and realize separation by utilizing the difference of acoustic radiation forces borne by particles with different sizes. In addition, the current acoustic flow control sorting device mainly realizes the separation of particles with different sizes based on the difference of acoustic radiation force, and the influence of the acoustic micro-flow effect is not considered. Research shows that when the diameter of particles to be sorted is smaller than 2um, the influence of sound flow drag force is larger than sound radiation force, and when the particle sorting device is applied to sorting of submicron particles such as outer vesicles, most of the problems of low controllability, low stability, low efficiency and the like exist. Therefore, how to effectively regulate and control and utilize the sound induced micro-flow effect to realize the stable, rapid and convenient separation of submicron particles such as outer vesicles and the like is an important development direction of a micro-fluidic separation device.
Disclosure of Invention
In view of the defects existing at present, the invention provides an acoustic flow control device for sorting outer vesicles in a plasma sample and a use method thereof, which fully consider the influence of an acoustic micro-flow effect on the sorting of the outer vesicles and simultaneously realize the separation of the outer vesicles from blood cells and the like in the plasma sample by utilizing acoustic radiation force and acoustic flow drag force. The acoustic flow control sorting device has the advantages of small influence on cell activity, small influence on temperature, stable sorting process, high sorting efficiency, simple manufacturing process, small control difficulty, capability of meeting the disposable processing requirement of biological samples and the like.
In order to achieve the above purpose, the invention provides an acoustic flow control device for sorting outer vesicles in a plasma sample, which comprises an upper micro-fluidic chip and a lower acoustic wave excitation device, wherein the micro-fluidic chip comprises a sorting chip and a cover plate, and the acoustic wave excitation device comprises a piezoelectric transducer and a liquid film layer; the piezoelectric transducer is reversibly connected with the cover plate through a liquid film layer; the piezoelectric transducer is lead zirconate titanate piezoelectric ceramic, a sorting channel is arranged on the sorting chip, the sorting channel comprises a plasma sample sorting channel, a double-row micro-ridge structure corresponding to the plasma sample sorting channel is arranged on the cover plate, and the section of each row of micro-ridges is a congruent right triangle with two bevel edges opposite to each other; the micro-ridge structure adjusts the sound wave excited by the piezoelectric transducer, so that the sound wave is modulated in the space formed by the sorting chip and the cover plate to form the required sound and flow field; forming a symmetrical vortex field above each row of micro ridges by using an acoustic micro-flow effect, and forming a standing wave sound field above the interval between the two rows of micro ridges;
the invention aims at solving the defects that the existing acoustic flow control sorting device ignores the influence of acoustic flow effect, is easily influenced by temperature, has high requirement on chip manufacturing precision, is difficult to manufacture and is not suitable for one-time use.
It should be noted that the cover plate separates the sound field modulation module (double-row micro-ridge structure) from the piezoelectric transducer, thereby realizing the disposable use of the sorting channel.
The sorting channel is positioned on the inner surface of the sorting chip and comprises a sheath flow inlet, a sheath flow channel, a shunt channel, a first sheath flow channel, a plasma sample inlet, a plasma sample channel, a second sheath flow channel, a plasma sample sorting channel, a first outer vesicle outlet, a cell channel, a cell outlet, a second outer vesicle channel and a second outer vesicle outlet; the sheath flow inlet realizes sheath flow split through the sheath flow channel and the split channel; two ends of the shunt channel are respectively communicated with the first sheath flow channel and the second sheath flow channel; the plasma sample inlet is in communication with the plasma sample channel, and the plasma sample channel, the first sheath flow channel, and the second sheath flow channel are in communication at their outlet ends and in communication with the inlet end of the plasma sample sorting channel; the outlet end of the plasma sample separation channel is communicated with the cell outlet through the cell channel in the middle; the outlet end of the plasma sample sorting channel is respectively communicated with the first and the second vesicle outlets at one sides of the first and the second sheath flow channels through the first and the second outer vesicle channels;
in the separation chip, the plasma sample flow to be separated is fixed in the middle of the separation channel through the sheath flow focusing action at two sides, and the separation of cells and outer vesicles in the plasma sample is realized through the combined action of the acoustic radiation force and the acoustic flow drag force.
The sorting chip is also provided with a sheath flow sample inlet, a plasma sample inlet, a first outer vesicle collecting port, a second outer vesicle collecting port and a cell collecting port in a penetrating way; the sheath flow sample inlet, the plasma sample inlet, the first outer vesicle collecting port, the second outer vesicle collecting port and the cell collecting port are respectively communicated with a sheath flow inlet, a plasma sample inlet, a first outer vesicle outlet, a second outer vesicle outlet and a cell outlet on the sorting channel in sequence; the sheath flow sample inlet and the plasma sample inlet are respectively connected with an external sample injection device; the first outer vesicle collecting port, the second outer vesicle collecting port and the cell collecting port are respectively connected with an external collecting device.
According to one aspect of the invention, the side of the cover sheet containing the double-row micro-ridge structure is bonded with the side of the sorting chip containing the sorting channels; the double-row micro-ridge structure is distributed in the region where the plasma sample separation channel is located; cross-sectional width w of the single row of micro-ridges 1 Cross-sectional depth h=150-250 μm of the micro-ridges =400-500 μm, spacing w of two rows of micro-ridges 2 =100-200 μm. The extending directions of the plasma sample sorting channel and the double-row micro-ridge structure are parallel to the long side of the rectangular cover plate.
According to one aspect of the invention, the cross section of the plasma sample sorting channel is rectangular, the width is 1000-1200 μm, and the depth is 300-500 μm; the included angle between the primary sheath flow channel and the plasma sample channel is 30-60 degrees; the included angle between the second sheath flow channel and the plasma sample channel is 30-60 degrees; the included angle between the cell channel and the first outer vesicle channel is 30-60 degrees; the angle between the cell channel and the second outer vesicle channel is 30-60 degrees.
It should be noted that, by setting an included angle at the focusing position of each channel, the focusing sheath flow will form a certain extrusion to the plasma sample flow to be sorted, so that the plasma sample flow keeps moving in the middle of the channel, and the sheath flow will move along the channel to the separating opening. Through setting up the contained angle in each passageway separation mouth department, can make plasma sample passageway and separation passageway fluid streamline form certain angle, more do benefit to the separation.
According to one aspect of the invention, the cover sheet is made of polymethyl methacrylate or cycloolefin copolymer and is manufactured by injection molding; the sorting chip is made of polydimethylsiloxane through reverse molding and thermosetting molding; the sorting chip and the cover plate are cleaned by silanization treatment and oxygen plasma, and bonding is completed at normal temperature; the cover plate is provided with a first positioning block and a second positioning block; the sorting chip is provided with a first positioning hole and a second positioning hole; the first positioning block and the second positioning block are respectively matched with the first positioning hole and the second positioning hole, so that after the chip is bonded, the double-row micro-ridge structure is positioned in the plasma sample sorting channel.
The separation chip and the cover plate are connected irreversibly through silanization treatment and oxygen plasma bonding, and the PZT piezoelectric transducer and the cover plate are connected reversibly through a liquid film, so that the reuse of the PZT transducer and the disposable use of the acoustic flow control separation channel chip are realized.
According to one aspect of the invention, the liquid film layer is an aqueous layer having a thickness of 100-300 μm.
Based on the same inventive concept, the invention also provides a use method of the acoustic streaming control device for sorting the outer vesicles in the plasma sample, which comprises the following steps:
step 1: preparing sheath flow solution; preparing a plasma sample to be measured, properly diluting the plasma sample, and preparing a plasma sample solution; wherein the sheath fluid solution is a phosphate buffer solution;
step 2: sequentially cleaning a sorting channel of the acoustic flow control device by using absolute ethyl alcohol and phosphate buffer solution for 1-2min;
step 3: injecting the phosphoric acid buffer solution prepared in the step 1 from the sheath flow port by adopting a micro-injection pump, and injecting a plasma sample solution to be detected at the plasma sample injection port by adopting the micro-injection pump; controlling the flow rates of the sheath flow solution and the plasma sample solution in the plasma sample sorting channel such that the plasma sample solution is in the middle of the plasma sample sorting channel;
step 4: sinusoidal alternating current signals are applied to the piezoelectric transducer, a required standing wave sound field and a required vortex field are generated in the plasma sample sorting channel, and cells and outer vesicles are collected at a cell outlet and a first outer vesicle outlet and a second outer vesicle outlet respectively, so that sorting of the cells and the outer vesicles in the plasma sample is realized.
According to one aspect of the invention, the flow rate of the body fluid sample solution is 5-15 mu L/min, and the flow rate ratio of the sheath flow solution to the body fluid sample solution is greater than or equal to 3:1.
Sorting principle of outer vesicles in plasma samples according to the invention:
injecting sheath flow solution (phosphate buffer solution) into the sheath flow port through the polyethylene hose by adopting a micro-injection pump, and respectively entering the first through the diversion channel1. The two sheath flow channels are converged with the plasma sample flow to be sorted in the sorting channel (direct current channel), the plasma sample flow to be sorted forms an extrusion effect, the flow rate ratio of the sheath flow solution on two sides to the plasma sample to be sorted is controlled to be more than or equal to 3:1, the plasma sample to be sorted is ensured to be compressed in the middle of the channel, and the PZT piezoelectric transducer is kept to be electrified for sorting. The cells and outer vesicles in the plasma sample to be sorted are subjected to the action of acoustic radiation force and acoustic flow drag force in the focusing area, wherein the size of the acoustic radiation force and the acoustic flow drag force is positively related to the volume of the cells or the vesicles, namely F Radiation of ＝k 1 d 3 ，F Drag (drag) ＝k 2 d 2 . Wherein F is Radiation of Acoustic radiation force to cells or outer vesicles, F Drag (drag) Acoustic flow drag, k, to cells or outer vesicles 1 K is a proportionality coefficient related to the density, sound field intensity, etc. of cells or outer vesicles 2 D is the diameter of the cell or exosome, which is a scaling factor related to the sound field strength, flow field strength, etc. Cells with larger size in the plasma sample to be sorted are dominated by acoustic radiation force, captured at a sound pressure node in a focusing region, outer vesicles with smaller size are dominated by acoustic flow drag force, and deviate from the focusing region to vortex regions on two sides under the action of an acoustic micro-flow field, as shown in fig. 6 and 7. The influence of the sound induced micro-fluidic effect on submicron particles is fully considered and utilized, the adverse effect generated by the sound flow drag force when the separation is carried out by only adopting the sound radiation force is avoided, and the separation efficiency and effect are higher. Through sorting in a plasma sample sorting channel (direct current channel), outer vesicles moving out of a focusing area along with sound flow pass through a first outer vesicle channel, a second outer vesicle channel, a first outer vesicle outlet and a second outer vesicle outlet, are collected through a recovery bottle, and the rest cells captured in the focusing area flow to a cell collecting tube through a cell channel and a cell outlet to finish sorting.
The invention has the beneficial effects that:
(1) The acoustic flow control device for sorting the outer vesicles in the plasma sample has the advantages of simple structure, simple manufacture and operation, small influence of temperature and the like, simultaneously meets the disposable and low-cost requirements of biochemical sample treatment, has high sorting efficiency, and has wide application prospect in the field of sorting the outer vesicles.
(2) The acoustic flow control device fully considers the influence of the acoustic micro-flow effect on submicron particles, simultaneously utilizes the acoustic radiation force and the acoustic flow drag force to realize the capturing of cells and the separation of outer vesicles, greatly reduces the manufacturing requirements and the cost of a transducer and a channel chip by controlling the size, the position and the acoustic input parameters of the micro-ridges and regulating and controlling the sound field and the flow field parameters in a separation channel, can realize the flexible and efficient separation of the cells and the outer vesicles in a plasma sample to be separated, improves the separation flux and the separation efficiency, and has good biological application value.
(3) The sorting chip and the cover plate of the acoustic flow control device are manufactured through injection molding, a reverse molding process and a room temperature bonding process, and are used as consumable materials, the formed micro-flow control chip and the PZT piezoelectric transducer are connected in a reversible bonding mode through a water film layer, so that the acoustic flow control device is suitable for disposable use requirements of biological samples and the like, and the PZT piezoelectric transducer is used as an energy conversion device, stable in performance and reusable. The channel chip has simple integral structure, simple and convenient manufacturing process and lower cost, and is beneficial to popularization and application of the acoustic flow control sorting device.
Drawings
FIG. 1 is a schematic diagram of an acoustic streaming device for sorting outer vesicles in a plasma sample according to the invention;
FIG. 2 is a schematic diagram of the structure of the inner surface of a sorting chip of an acoustic streaming device for sorting outer vesicles in a plasma sample according to the invention;
FIG. 3 is a schematic diagram of the structure of the outer surface of the sorting chip of the acoustic streaming device for sorting outer vesicles in a plasma sample according to the invention;
FIG. 4 is a schematic illustration of the structure of the inner surface of the cover plate of the acoustic streaming device for sorting outer vesicles in a plasma sample according to the invention;
FIG. 5 is a schematic cross-sectional view of a plasma sample separation channel with a double-row micro-ridge structure according to the present invention;
FIG. 6 is a schematic diagram showing the stress and movement trend (sorting principle) of cells and outer vesicles in a sorting channel of a plasma sample containing a double-row micro-ridge structure according to the invention;
FIG. 7 is a graph showing the effect of sorting outer vesicles in a plasma sample according to the invention.
Reference numerals illustrate:
1. a piezoelectric transducer; 2. a liquid film layer; 3. a cover plate; 4. sorting chips; 31. the inner surface of the cover plate; 32. a first positioning block; 33. a double-row micro-ridge structure; 34; a second positioning block; 41. sorting the inner surface of the chip; 42. a sheath inflow port; 43. a sheath flow channel; 44. a shunt channel; 45. a primary sheath flow channel; 46. a plasma sample inlet; 47. a plasma sample channel; 48. a secondary sheath flow channel; 49. a first positioning hole; 410. a plasma sample sorting channel; 411. a second outer vesicle channel; 412. a second outer vesicle outlet; 413. a cell outlet; 414. a cell channel; 415. a first outer vesicle outlet; 416. a first outer vesicle channel; 417. a second positioning hole; 418. a sheath flow sample inlet; 419. a plasma sample inlet; 420. a first outer vesicle collection port; 421. a cell collection port; 422. a second outer vesicle collection port; 423. a first vortex region; 424. a focusing region; 425. a second vortex region.
Detailed Description
In order that the invention may be more readily understood, the invention will be further described with reference to the following examples. It should be understood that these examples are intended to illustrate the invention and not to limit the scope of the invention, and that the described embodiments are merely some, but not all, of the embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention. Unless defined otherwise, the terms of art used hereinafter are consistent with the meanings understood by those skilled in the art; unless otherwise indicated, all the materials and reagents referred to herein are commercially available or may be prepared by well-known methods.
Example 1
An acoustic flow control device for sorting outer vesicles in a plasma sample is used for separating the outer vesicles from blood cells in the plasma sample, and the structure schematic diagram is shown in figures 1-4. The micro-fluidic chip comprises a sorting chip 4 of Polydimethylsiloxane (PDMS) and a cover plate 3 of polymethyl methacrylate (PMMA) which are manufactured by reverse molding and injection molding; and the PDMS sorting chip and the PMMA cover plate are subjected to silanization treatment and oxygen plasma cleaning, and bonding is completed at normal temperature. The sound wave excitation device comprises a piezoelectric transducer 1 and a liquid film layer 2; the piezoelectric transducer 1 and the cover plate 3 are connected in a reversible bonding way through a liquid film layer 2, and the thickness of the liquid film layer is 200 mu m; the piezoelectric transducer 1 is lead zirconate titanate piezoelectric ceramic (PZT), a sorting channel is arranged on the sorting chip 4, the sorting channel comprises a plasma sample sorting channel 410, the cover plate 3 is provided with a double-row micro-ridge structure 33 corresponding to the plasma sample sorting channel 410, and the double-row micro-ridge structure 33 regulates sound waves excited by the piezoelectric transducer 1 to be modulated in a space formed by the sorting chip 4 and the cover plate 3 to form a required sound field and a required flow field; and forming a symmetrical vortex field above each row of micro ridges by using an acoustic micro-flow effect, and forming a standing wave sound field above the interval between the two rows of micro ridges.
The sorting channel is located on the inner surface 41 of the sorting chip, and comprises a sheath flow inlet 42, a sheath flow channel 43, a shunt channel 44, a first sheath flow channel 45, a plasma sample inlet 46, a plasma sample channel 47, a second sheath flow channel 48, a plasma sample sorting channel 410, a second outer vesicle channel 411, a second outer vesicle outlet 412, a cell channel 414, a cell outlet 413, a first outer vesicle channel 416 and a first outer vesicle outlet 415; the sheath inflow port 42 is connected with the shunt channel 44 through the sheath flow channel 43 to shunt sheath flow; both ends of the shunt channel 44 are respectively communicated with the first sheath flow channel 45 and the second sheath flow channel 48; the plasma sample inlet 46 communicates with the plasma sample channel 47, and the plasma sample channel 47, the primary sheath flow channel 45, and the secondary sheath flow channel 48 communicate at their outlet ends and with the inlet end of the plasma sample sorting channel 410; the outlet end of the plasma sample sorting channel 410 is centrally connected to a cell outlet 413 via a cell channel 414; the outlet end of the plasma sample sorting channel 410 is respectively communicated with the outlets of the first 415 and the second 412 through the first 416 and the second 411 outer vesicle channels at one side of the first 416 and the second 411 outer vesicle channels.
The sorting chip is also provided with a sheath flow sample inlet 418, a plasma sample inlet 419, a first outer vesicle collecting port 420, a second outer vesicle collecting port 422 and a cell collecting port 421 in a penetrating way; the sheath flow sample inlet 418, the plasma sample inlet 419, the first outer vesicle collection port 420, the second outer vesicle collection port 422, and the cell collection port 421 are respectively and sequentially communicated with the sheath flow inlet 42, the plasma sample inlet 46, the first outer vesicle outlet 415, the second outer vesicle outlet 412, and the cell outlet 413 of the sorting channel; the sheath flow sample inlet 418 and the plasma sample inlet 419 are respectively connected with an external sample injection device; the first outer vesicle collecting port 420, the second outer vesicle collecting port 422 and the cell collecting port 421 are respectively connected with an external collecting device.
One side of the cover plate 3 containing the double-row micro-ridge structure 33 is bonded with one side of the sorting chip 4 containing the sorting channel; the double-row micro-ridge structures 33 are distributed in the region where the plasma sample sorting channel 410 is located; cross-sectional width w of the single row of micro-ridges 1 Cross-sectional depth h=200μm of the micro-ridges =500 μm, spacing w of two rows of micro-ridges 2 =200 μm. The plasma sample sorting channel 410 and the double row micro-ridge structure 33 extend in directions parallel to the long sides of the rectangular cover plate.
The cross section of the plasma sample sorting channel 410 is rectangular, the width is 1200 μm, and the depth is 500 μm; the angle between the primary sheath flow channel 45 and the plasma sample channel 47 is 45 °; the angle between the secondary sheath flow channel 48 and the plasma sample channel 47 is 45 °; the angle between the cell channel 414 and the first outer vesicle channel 416 is 45 °; the cell channel 414 is at an angle of 45 ° to the second outer vesicle channel 411.
The cover plate 3 is provided with a first positioning block 32 and a second positioning block 34; the sorting chip 4 is provided with a first positioning hole 49 and a second positioning hole 417; the first positioning block 32 and the second positioning block 34 are respectively matched with the first positioning hole 49 and the second positioning hole 417, so that the double-row micro-ridge structure 33 is located inside the plasma sample sorting channel 410 after the chip is bonded.
The method for using the acoustic flow control device for sorting the outer vesicles in the plasma sample comprises the following steps:
step 1: preparing sheath flow solution; preparing a plasma sample to be measured, properly diluting the plasma sample, and preparing a plasma sample solution; wherein the sheath fluid solution is a phosphate buffer solution;
step 2: sequentially cleaning a sorting channel of the acoustic flow control device for 2min by using absolute ethyl alcohol and phosphoric acid buffer solution;
step 3: injecting the phosphoric acid buffer solution of the step 1 from the sheath fluid sample inlet 418 by a micro-injection pump, and injecting the plasma sample of the step 1 from the body fluid sample inlet 419 by another micro-injection pump; controlling the flow rate of the phosphoric acid buffer solution in the plasma sample separation channel to be 25 mu L/min, and controlling the flow rate of the plasma sample in the plasma sample separation channel to be 8 mu L/min, so that the plasma sample solution is in the middle of the plasma sample separation channel;
step 4: sinusoidal alternating current signals are applied to the piezoelectric transducer, a required standing wave sound field and a required vortex field are generated in the plasma sample sorting channel 410, and cells and outer vesicles are collected at the cell collecting port 421 and the outlets of the first 420 and the second 422 outer vesicles respectively, so that sorting of the cells and the outer vesicles in the plasma sample is realized. Cells with larger size in the plasma sample to be sorted are dominated by acoustic radiation force, captured at a sound pressure node in a focusing region, outer vesicles with smaller size are dominated by acoustic flow drag force, and deviate from a focusing region 424 to vortex regions 423 and 424 on two sides under the action of an acoustic micro-flow field. After sorting in the plasma sample sorting channel 410 (direct current channel), the outer vesicles moving out of the focusing region 424 with the acoustic flow pass through the first 416 and second 411 outer vesicle channels and the first 415 and second 412 outer vesicle outlets, are collected by the recovery bottle, and the remaining cells trapped in the focusing region 424 flow to the cell collection tube through the cell channel 414 and the cell outlet 413 to complete sorting.
Example 2
An acoustic flow control device for sorting outer vesicles in a plasma sample is used for separating the outer vesicles from blood cells in the plasma sample, and the structure schematic diagram is shown in figures 1-4. The micro-fluidic chip comprises a Polydimethylsiloxane (PDMS) sorting chip 4 and a cycloolefin copolymer (COC) cover plate 3, and is manufactured through reverse molding and injection molding; and the PDMS sorting chip and the COC cover plate are subjected to silanization treatment, oxygen plasma cleaning and bonding at normal temperature. The sound wave excitation device comprises a piezoelectric transducer 1 and a liquid film layer 2; the piezoelectric transducer 1 and the cover plate 3 are connected in a reversible bonding way through a liquid film layer 2, and the thickness of the liquid film layer is 200 mu m; the piezoelectric transducer 1 is lead zirconate titanate piezoelectric ceramic (PZT), a sorting channel is arranged on the sorting chip 4, the sorting channel comprises a plasma sample sorting channel 410, the cover plate 3 is provided with a double-row micro-ridge structure 33 corresponding to the plasma sample sorting channel 410, and the double-row micro-ridge structure 33 regulates sound waves excited by the piezoelectric transducer 1 to be modulated in a space formed by the sorting chip 4 and the cover plate 3 to form a required sound field and a required flow field; and forming a symmetrical vortex field above each row of micro ridges by using an acoustic micro-flow effect, and forming a standing wave sound field above the interval between the two rows of micro ridges.
The sorting channel is located on the inner surface 41 of the sorting chip, and comprises a sheath flow inlet 42, a sheath flow channel 43, a shunt channel 44, a first sheath flow channel 45, a plasma sample inlet 46, a plasma sample channel 47, a second sheath flow channel 48, a plasma sample sorting channel 410, a second outer vesicle channel 411, a second outer vesicle outlet 412, a cell channel 414, a cell outlet 413, a first outer vesicle channel 416 and a first outer vesicle outlet 415; the sheath inflow port 42 is connected with the shunt channel 44 through the sheath flow channel 43 to shunt sheath flow; both ends of the shunt channel 44 are respectively communicated with the first sheath flow channel 45 and the second sheath flow channel 48; the plasma sample inlet 46 communicates with the plasma sample channel 47, and the plasma sample channel 47, the primary sheath flow channel 45, and the secondary sheath flow channel 48 communicate at their outlet ends and with the inlet end of the plasma sample sorting channel 410; the outlet end of the plasma sample sorting channel 410 is centrally connected to a cell outlet 413 via a cell channel 414; the outlet end of the plasma sample sorting channel 410 is respectively communicated with the outlets of the first 415 and the second 412 through the first 416 and the second 411 outer vesicle channels at one side of the first 416 and the second 411 outer vesicle channels.
The sorting chip is also provided with a sheath flow sample inlet 418, a plasma sample inlet 419, a first outer vesicle collecting port 420, a second outer vesicle collecting port 422 and a cell collecting port 421 in a penetrating way; the sheath flow sample inlet 418, the plasma sample inlet 419, the first outer vesicle collection port 420, the second outer vesicle collection port 422, and the cell collection port 421 are respectively and sequentially communicated with the sheath flow inlet 42, the plasma sample inlet 46, the first outer vesicle outlet 415, the second outer vesicle outlet 412, and the cell outlet 413 of the sorting channel; the sheath flow sample inlet 418 and the plasma sample inlet 419 are respectively connected with an external sample injection device; the first outer vesicle collecting port 420, the second outer vesicle collecting port 422 and the cell collecting port 421 are respectively connected with an external collecting device.
One side of the cover plate 3 containing the double-row micro-ridge structure 33 is bonded with one side of the sorting chip 4 containing the sorting channel; the double-row micro-ridge structures 33 are distributed in the region where the plasma sample sorting channel 410 is located; cross-sectional width w of the single row of micro-ridges 1 Cross-sectional depth h=250μm of the micro-ridges =400 μm, spacing w of two rows of micro-ridges 2 =200 μm. The plasma sample sorting channel 410 and the double row micro-ridge structure 33 extend in directions parallel to the long sides of the rectangular cover plate.
The cross section of the plasma sample sorting channel 410 is rectangular, the width is 1000 μm, and the depth is 400 μm; the angle between the primary sheath flow channel 45 and the plasma sample channel 47 is 40 °; the angle between the secondary sheath flow channel 48 and the plasma sample channel 47 is 40 °; the angle between the cell channel 414 and the first outer vesicle channel 416 is 45 °; the cell channel 414 is at an angle of 45 ° to the second outer vesicle channel 411.
The cover plate 3 is provided with a first positioning block 32 and a second positioning block 34; the sorting chip 4 is provided with a first positioning hole 49 and a second positioning hole 417; the first positioning block 32 and the second positioning block 34 are respectively matched with the first positioning hole 49 and the second positioning hole 417, so that the double-row micro-ridge structure 33 is located inside the plasma sample sorting channel 410 after the chip is bonded.
The method for using the acoustic flow control device for sorting the outer vesicles in the plasma sample comprises the following steps:
step 1: preparing sheath flow solution; preparing a plasma sample to be measured, properly diluting the plasma sample, and preparing a plasma sample solution; wherein the sheath fluid solution is a phosphate buffer solution;
step 2: sequentially cleaning a sorting channel of the acoustic flow control device for 2min by using absolute ethyl alcohol and phosphoric acid buffer solution;
step 3: injecting the phosphoric acid buffer solution of the step 1 from the sheath fluid sample inlet 418 by a micro-injection pump, and injecting the plasma sample of the step 1 from the body fluid sample inlet 419 by another micro-injection pump; controlling the flow rate of the phosphoric acid buffer solution in the plasma sample separation channel to be 22 mu L/min, and controlling the flow rate of the plasma sample in the plasma sample separation channel to be 7 mu L/min, so that the plasma sample solution is in the middle of the plasma sample separation channel;
step 4: sinusoidal alternating current signals are applied to the piezoelectric transducer, a required standing wave sound field and a required vortex field are generated in the plasma sample sorting channel 410, and cells and outer vesicles are collected at the cell collecting port 421 and the outlets of the first 420 and the second 422 outer vesicles respectively, so that sorting of the cells and the outer vesicles in the plasma sample is realized. Cells with larger size in the plasma sample to be sorted are dominated by acoustic radiation force, captured at a sound pressure node in a focusing region, outer vesicles with smaller size are dominated by acoustic flow drag force, and deviate from a focusing region 424 to vortex regions 423 and 424 on two sides under the action of an acoustic micro-flow field. After sorting in the plasma sample sorting channel 410 (direct current channel), the outer vesicles moving out of the focusing region 424 with the acoustic flow pass through the first 416 and second 411 outer vesicle channels and the first 415 and second 412 outer vesicle outlets, are collected by the recovery bottle, and the remaining cells trapped in the focusing region 424 flow to the cell collection tube through the cell channel 414 and the cell outlet 413 to complete sorting.
The foregoing is merely illustrative of the present invention, and the present invention is not limited thereto, and any changes or substitutions easily contemplated by those skilled in the art within the technical scope of the present invention should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.
Claims (6)
1. The acoustic flow control device for sorting outer vesicles in a plasma sample is characterized by comprising an upper-layer micro-fluidic chip and a lower-layer acoustic wave excitation device, wherein the micro-fluidic chip comprises a sorting chip and a cover plate, and the acoustic wave excitation device comprises a piezoelectric transducer and a liquid film layer; the piezoelectric transducer is reversibly connected with the cover plate through a liquid film layer; the piezoelectric transducer is lead zirconate titanate piezoelectric ceramic, a sorting channel is arranged on the sorting chip, the sorting channel comprises a plasma sample sorting channel, a double-row micro-ridge structure corresponding to the plasma sample sorting channel is arranged on the cover plate, and the section of each row of micro-ridges is a congruent right triangle with two bevel edges opposite to each other; the micro-ridge structure adjusts the sound wave excited by the piezoelectric transducer, so that the sound wave is modulated in the space formed by the sorting chip and the cover plate to form the required sound and flow field; forming a symmetrical vortex field above each row of micro ridges by using an acoustic micro-flow effect, and forming a standing wave sound field above the interval between the two rows of micro ridges;
the sorting channel is positioned on the inner surface of the sorting chip and comprises a sheath flow inlet, a sheath flow channel, a shunt channel, a first sheath flow channel, a plasma sample inlet, a plasma sample channel, a second sheath flow channel, a plasma sample sorting channel, a first outer vesicle outlet, a cell channel, a cell outlet, a second outer vesicle channel and a second outer vesicle outlet; the sheath flow inlet realizes sheath flow split through the sheath flow channel and the split channel; two ends of the shunt channel are respectively communicated with the first sheath flow channel and the second sheath flow channel; the plasma sample inlet is in communication with the plasma sample channel, and the plasma sample channel, the first sheath flow channel, and the second sheath flow channel are in communication at their outlet ends and in communication with the inlet end of the plasma sample sorting channel; the outlet end of the plasma sample separation channel is communicated with the cell outlet through the cell channel in the middle; the outlet end of the plasma sample sorting channel is respectively communicated with the first and the second vesicle outlets at one sides of the first and the second sheath flow channels through the first and the second outer vesicle channels;
the sorting chip is also provided with a sheath flow sample inlet, a plasma sample inlet, a first outer vesicle collecting port, a second outer vesicle collecting port and a cell collecting port in a penetrating way; the sheath flow sample inlet, the plasma sample inlet, the first outer vesicle collecting port, the second outer vesicle collecting port and the cell collecting port are respectively communicated with a sheath flow inlet, a plasma sample inlet, a first outer vesicle outlet, a second outer vesicle outlet and a cell outlet on the sorting channel in sequence; the sheath flow sample inlet and the plasma sample inlet are respectively connected with an external sample injection device; the first outer vesicle collecting port, the second outer vesicle collecting port and the cell collecting port are respectively connected with an external collecting device.
Wherein, one side of the cover plate containing the double-row micro-ridge structure is bonded with one side of the sorting chip containing the sorting channel; the double-row micro-ridge structure is distributed in the region where the plasma sample separation channel is located; cross-sectional width w of the single row of micro-ridges 1 Cross-sectional depth h=150-250 μm of the micro-ridges =400-500 μm, spacing w of two rows of micro-ridges 2 =100-200 μm. The extending directions of the plasma sample sorting channel and the double-row micro-ridge structure are parallel to the long side of the rectangular cover plate.
2. The acoustic flow control device for sorting external vesicles in a plasma sample according to claim 1 wherein the cross section of the plasma sample sorting channel is rectangular with a width of 1000-1200 μm and a depth of 300-500 μm; the included angle between the primary sheath flow channel and the plasma sample channel is 30-60 degrees; the included angle between the second sheath flow channel and the plasma sample channel is 30-60 degrees; the included angle between the cell channel and the first outer vesicle channel is 30-60 degrees; the angle between the cell channel and the second outer vesicle channel is 30-60 degrees.
3. The acoustic flow control device for sorting external vesicles in a plasma sample according to claim 1 wherein the material of the cover sheet is polymethyl methacrylate or cyclic olefin copolymer and is produced by injection moulding; the sorting chip is made of polydimethylsiloxane through reverse molding and thermosetting molding; the sorting chip and the cover plate are cleaned by silanization treatment and oxygen plasma, and bonding is completed at normal temperature; the cover plate is provided with a first positioning block and a second positioning block; the sorting chip is provided with a first positioning hole and a second positioning hole; the first positioning block and the second positioning block are respectively matched with the first positioning hole and the second positioning hole, so that after the chip is bonded, the double-row micro-ridge structure is positioned in the plasma sample sorting channel.
4. The acoustic flow control device for sorting outer vesicles in a plasma sample according to claim 1 wherein the liquid film layer is an aqueous layer and has a thickness of 100 to 300 μm.
5. A method of using an acoustic streaming device for sorting outer vesicles in a plasma sample according to any one of claims 1 to 4 comprising the steps of:
step 1: preparing sheath flow solution; preparing a plasma sample to be measured, properly diluting the plasma sample, and preparing a plasma sample solution; wherein the sheath fluid solution is a phosphate buffer solution;
step 2: sequentially cleaning a sorting channel of the acoustic flow control device by using absolute ethyl alcohol and phosphate buffer solution for 1-2min;
step 3: injecting the phosphoric acid buffer solution prepared in the step 1 from the sheath flow sample inlet by adopting a micro-injection pump, and injecting a plasma sample solution to be detected at the plasma sample inlet by adopting the micro-injection pump; controlling the flow rates of the sheath flow solution and the plasma sample solution in the plasma sample sorting channel such that the plasma sample solution is in the middle of the plasma sample sorting channel;
step 4: sinusoidal alternating current signals are applied to the piezoelectric transducer, a required standing wave sound field and a required vortex field are generated in the plasma sample sorting channel, and cells and outer vesicles are collected at a cell outlet and a first outer vesicle outlet and a second outer vesicle outlet respectively, so that sorting of the cells and the outer vesicles in the plasma sample is realized.
6. The method of claim 5, wherein in step 3, the flow rate of the plasma sample solution is 5-15 μl/min, and the ratio of the sheath flow solution to the plasma sample solution is 3:1 or more.
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