CN107164213A - One kind is based on the cellifugal chip of principle of inertia point - Google Patents
One kind is based on the cellifugal chip of principle of inertia point Download PDFInfo
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- 239000002245 particle Substances 0.000 claims description 50
- 239000000758 substrate Substances 0.000 claims description 21
- 239000007788 liquid Substances 0.000 claims description 13
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 abstract description 6
- 230000015572 biosynthetic process Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 154
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- 210000000601 blood cell Anatomy 0.000 description 6
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- -1 Polydimethylsiloxane Polymers 0.000 description 1
- 239000004809 Teflon Substances 0.000 description 1
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Abstract
The invention discloses one kind based on the cellifugal chip of principle of inertia point, runner (3) including matrix and in the matrix, runner (3) is spiral shape or arc, and with the first side wall (S1) and second sidewall (S2) being oppositely arranged；The tongue being spaced apart along the runner (3) is provided with the first side wall (S1), so that the runner (3) formation be interspersed widen section and compression section, the region that tongue is distributed with section correspondence the first side wall is widened, compression section, which is then corresponded on the first side wall, does not have the region of tongue；The runner (3) is used to separate the cell being mixed with the cell solution of sheath fluid stream by size.The present invention is improved by the structure of the runner crucial to its its set-up mode etc., and the problem of both single helical structure and single unilateral reducing and expansion array structure cell separation efficiency are low is can effectively solve the problem that compared with prior art.
Description
Technical Field
The invention belongs to the field of microfluidics, and particularly relates to a chip for separating cells based on an inertia principle.
Background
The microfluidic chip technology is a scientific technology for controlling fluid in a microscale space, and when the technology is used for separating cells, the method is simple, the separation success rate is high, the popularization is easy, and the technology is widely concerned.
The flow channel structure proposed in the prior documents and patents for separating cells based on the principle of inertia is generally: helical and tapered array structures. Both of these structures have some drawbacks. For the spiral configuration, the FD (i.e., dean force) and FL (i.e., inertial lift) provided by the configuration are small due to the limited pressure that the chip can withstand, limiting the separation efficiency. For the contracted and expanded array structure, although it can provide larger FD and FL by virtue of the contracted and expanded array, FD cannot be provided all the time in the entire flow channel, and cells can be affected by FD only when flowing from the expanded section into the contracted section.
Taking a spiral structure as an example, the prior art document [ 3 ] discloses a spiral structure based on the principle of inertial separation for separating red blood cells from blood. The structure adopts a sample inlet, a spiral flow channel and a two-fork outlet. The spiral flow channel consists of 5 circles of Archimedes spiral micro-channels, the cross section of each channel is a rectangular surface, the width of each rectangular surface is 100um, the height of each rectangular surface is 50um, the total length of a curve is 13cm, and the radius of curvature of the innermost side of the spiral is 3 mm. The student injected blood at a rate of 0.15m/s from the sample inlet, collecting 7.32um particles at the lower outlet of the fork, and 1.9um particles at the upper outlet of the fork.
As shown in fig. 3, the principle of cell separation achieved by the helical structure is: in the curved flow channel, the cell is acted by two forces of inertial lift force FL and dean force FD. The equilibrium between FL and FD determines the equilibrium position of the cell in the microchannel. When FL is larger than or equal to FD, the cells move to the side wall S1, and when FL is smaller than FD, the cells move to the side wall S2; cells of larger size are subject to greater FL and cells of smaller size are subject to greater FD. The large particles and the small particles are separated through a flow channel with a certain length.
In the above method, since the pressure that the chip can withstand is limited, the structure provides smaller FD and FL, limiting the separation efficiency. Furthermore, due to the limitation of the flow channel structure, the distance between the lateral equilibrium positions of different cells is small. Finally, to achieve the desired separation effect, the total length of flow channels required is often long, and there are other references which have designed a single-sided convergent-divergent array structure for cell separation, which requires a shorter total length of flow channels, but in which the cells are subjected to both FD and FL forces only when they enter the compression section from the expansion section. The remaining phases are only affected by the inertial lift FL. The time that FD is acting on cells is very short and it is not guaranteed that all cells are affected by FD in such a short time.
Detailed analysis is performed on a unilateral contraction and expansion array structure, and a prior art document [ 1 ] discloses a unilateral contraction and expansion array structure which adopts a sample inlet, a sheath fluid inlet, two outlets, six unilateral widening cavities and five contraction sections to separate blood cells and plasma (cells with the sizes of 2um and below 2 um); the student injected a blood sample into the sample inlet at a rate of 1.2mL/h, injected a phosphate buffer into the sheath fluid inlet at a rate of 12mL/h, and collected blood cells (cells having a size of 2um or more) and plasma (cells having a size of 2um or less) at both outlets, but the separation efficiency of plasma (cells having a size of 2um or less) was only 62.2%.
The principle of realizing cell separation by the unilateral contraction and expansion array structure is as follows: as shown in FIG. 4, in the rectangular microchannel of the elongated straight type, the cells are mainly influenced by the inertial lift force FL, thereby focusing the cells at two equilibrium positions, upper and lower, near the long side of the rectangle; in the curved flow channel, the cell will be acted upon by both inertial lift FL and dean FD forces. When liquid enters the compression section from the widening section, the liquid in the widening section accelerates in a curved path into the compression section, creating a dean vortex in which cells are affected by dean force FD, so that cells will be affected by both FL and FD forces when entering the compression section from the widening section. The size of both FL and FD is related to the location of the cell in the cross-section of the channel. The equilibrium between FL and FD determines the equilibrium position of the cell in the microchannel. When FL is larger than or equal to FD, the cells move to the side wall S1, and when FL is smaller than FD, the cells move to the side wall S2; cells of larger size are subject to greater FL and cells of smaller size are subject to greater FD. Through a plurality of contraction and expansion arrays, blood cells (cells with the size of more than 2 um) and blood plasma (cells with the sizes of 2um and less than 2 um) are separated.
In the above method, the cells are subjected to both FD and FL forces only when they enter the compression section from the expansion section. The remaining phases are only affected by the inertial lift FL. The time that FD is acting on cells is very short and it is not guaranteed that all cells are affected by FD in such a short time.
Second, since at other stages the cells are only affected by the FL, the FL will pull cells moving toward the sidewalls S1 and S2 toward the center of the flow channel, causing cells that are already at the desired equilibrium position to be offset from the equilibrium position, thereby somewhat reducing the separation efficiency.
The references are as follows:
【1】"Inertial blood plasma separation in a contraction–expansion arraymicrochannel"；
【2】"Enhanced blood plasma separation by modulation of inertial liftforce"；
【3】"Continuous particle separation in spiral microchannels using deanflows and differential migration"；
【4】"Improved understanding of particle migration modes in spiralinertial microfluidic devices"；
【5】"Continuous inertial microparticle and blood cell separation instraight channels with local microstructures"。
disclosure of Invention
In view of the above defects or improvement requirements of the prior art, an object of the present invention is to provide a chip for separating cells based on the inertial principle, wherein the arrangement of the critical flow channel structure (especially, the shapes and related parameters of the widened section and the compressed section in the flow channel) is improved, so that the problem of low cell separation efficiency of the single spiral structure and the single-side shrinking and expanding array structure can be effectively solved, and the length, width, and total length and height of the flow channel of each widened section and the compressed section in the flow channel are controlled, so that the chip can particularly separate cells with the sizes of 3um and 6um as the boundary.
In order to achieve the above object, according to the present invention, there is provided a chip for separating cells based on the principle of inertia, comprising a substrate, and a flow channel (3) located in the substrate, wherein the chip further comprises a sample inlet (1), a sheath fluid inlet (2), a large-sized cell outlet (4), and a small-sized cell outlet (5) connected to the flow channel (3);
wherein,
the sample inlet (1) is used for inputting a cell solution to be separated and treated to the inlet of the flow channel (3);
the sheath fluid inlet (2) is used for inputting a sheath fluid to the inlet of the flow channel (3), and the sheath fluid is used for mixing with the cell solution;
the flow channel (3) is spiral or arc; the flow channel (3) is provided with a first side wall (S1) and a second side wall (S2) which are oppositely arranged, wherein the first side wall (S1) is positioned at the inner side close to the spiral center or the arc center, and the second side wall (S2) is positioned at the outer side far away from the spiral center or the arc center; the first side wall (S1) is provided with convex grooves distributed at intervals along the flow channel (3), so that the flow channel (3) forms a widening section and a compressing section which are distributed in a staggered way, the widening section corresponds to the area on the first side wall where the convex grooves are distributed, and the compressing section corresponds to the area on the first side wall where the convex grooves are not arranged; the flow channel (3) is used for separating the cells in the cell solution mixed with the sheath liquid flow according to the size;
the large-size cell outlet (4) is positioned at one side close to the center of the spiral or the arc center and is used for outputting cell separation liquid containing large-size cells from the outlet of the flow channel (3);
the small-size cell outlet (5) is positioned on one side far away from the center of the spiral or the arc center and is used for outputting cell separation liquid containing small-size cells from the outlet of the flow channel (3);
furthermore, the inlet and the outlet of the flow channel (3) are respectively positioned at two ends of the flow channel (3).
As a further preferred aspect of the present invention, in the flow channel (3), the arc length of any one of the widening sections is 300 to 700um, and the arc length of any one of the compressing sections is 300 to 1200 um; preferably, the width of the widening section is 350um, and the width of the compressing section is 50 um; the height of the flow channel (3) is 20-25 um; preferably, the radius of curvature of the second side wall (S2) of the flow path (3) closest to the center of the spiral or the arc-shaped center portion is 5mm to 7 mm.
As a further preferred aspect of the present invention, in the flow channel (3), the arc length of any one of the widening sections is 700um, and the arc length of any one of the compressing sections is 1200 um; the height of runner (3) is 25um, and the overall length of this runner (3) is 23mm, and the number of required compression section is 11, the number of widening section is 12, and second lateral wall (S2) is the closest on this runner (3) spiral center or arc center part' S radius of curvature is 7mm, the particle diameter of jumbo size cell is not less than 6um, the particle diameter of jumbo size cell is no longer than 3 um.
As a further preferred aspect of the present invention, the ratio of the flow rate of the cell solution to be separated and treated, which is input from the sample inlet (1), to the flow rate of the sheath fluid, which is input from the sheath fluid inlet (2), is 1: 5.
as a further preference of the present invention, when the flow channel (3) is spiral-shaped, the flow channel (3) is preferably distributed in an archimedean spiral.
As a further preferred aspect of the present invention, the base body includes an upper base body and a lower base body which are stacked one on another, and the flow channel (3) is provided between the upper base body and the lower base body.
As a further preferred aspect of the present invention, the sheath fluid stream is a phosphate buffer.
In a further preferred embodiment of the present invention, the cross section of the flow channel (3) is rectangular.
According to the chip for separating cells based on the inertial principle, the flow channel is set to be in a spiral/arc structure with a single-side contraction and expansion array, so that the cell separation efficiency can be effectively improved. The invention arranges convex grooves which are distributed at intervals along the flow channel on the inner side wall which is close to the spiral center or the arc center of the flow channel to form the flow channel with the staggered widened section and the compressed section structures, further excavates the advantages of the two flow channel structures of the contraction and expansion array structure and the spiral flow channel structure (or the arc flow channel structure), and makes full use of the characteristics of dean force and inertial lift force in different areas of the flow channel to organically combine the contraction and expansion array structure and the spiral flow channel structure (or the arc flow channel structure), thereby effectively improving the cell separation efficiency.
The invention arranges convex grooves which are distributed at intervals along the flow channel on the inner side wall which is close to the spiral center or the arc center of the flow channel, and the aim of separating cells can not be achieved if the convex grooves which are distributed at intervals along the flow channel are arranged on the outer side wall. When the convex grooves are arranged along the outer side wall, the inertial lift force borne by the large particles is directed to the side wall S2 from the side wall S1, and the large particles are dragged to the side wall S2 by the inertial lift force; at the same time, when the small particles in the channel enter the compression section from the widening section, dean force is also applied to the small particles from the side wall S1 to the side wall S2, so that the small particles are also pulled to the side wall S2 from the side wall S1, and the expected separation effect cannot be achieved.
Table 1: the 3um and 6um particles are separated by a distance between the inward-bent single-side expansion and contraction array arc structure and the outward-bent single-side expansion and contraction array arc structure
Note: the distance between the lowest end of the 6um particles and the highest end of the 3um particles at the outlet is 7mm, and the curvature radius of the circular arc outer side wall is 7 mm. The 6 spans in the table indicate that the channel has 6 spans and 5 compressed segments (the number of compressed segments is typically one less than the number of extended segments).
The radius of curvature of the part closest to the center of the spiral or the arc center is 5 mm-7 mm. When the radius of curvature of the present invention is less than 5mm, it results in that the lower inlet 1 and the inlet 2 cannot be accommodated in the innermost channel, and the radius of curvature of the present invention is not less than 5mm in terms of structural design rationality. When the radius of curvature is greater than 7mm, the separation efficiency of the present invention is lowered.
Table 2: separation effect of 3um and 6um particles under different curvature radiuses
The height of the structure of the invention is generally 20-25 um. When separating particles of 2um and above 2um, the height of the structure is 25 um. When separating particles below 2um, the height of the structure is 20 um. To ensure that the particles can be focused under the action of inertial lift to form a single-row particle beam, the particle diameter apThe characteristic size of the flow channel, namely the height h of the flow channel, is required to satisfy apThe/h is more than or equal to 0.07. Since large particles are mainly received in the channelThe influence of the inertial lift force is that a must be satisfied for the large particles to flow out of the outlet in a straight line in the channelpThe/h is more than or equal to 0.07. Therefore, as the size of the isolated particles becomes smaller, the channel height should also become shorter.
Table 3: the distance between the 3um and 6um particles at different heights
Note: the radius of curvature of the circular arc-shaped outer side wall is 7 mm.
Compared with a single spiral structure, the single-side contraction and expansion array spiral structure can separate cells in a shorter flow channel. When the cells move to the widened flow channel section, the widening of the flow channel enables wall-induced inertial lift force on the cells to be suddenly reduced, the small-size cells move to the S2 side wall and are stabilized at a new balance position, the large-size cells move to the S1 side wall and are stabilized at the new balance position, and therefore the distance between the balance position of the large-size cells and the balance position of the small-size cells is increased. When liquid enters the compression section from the expansion section, the liquid in the expansion section accelerates in a tortuous path into the compression section, which acceleration will enhance the FL and FD experienced by the cells to some extent, thereby allowing faster cell separation.
Compared with the existing contraction and expansion array structure for separating the cells based on the inertia principle, the chip for separating the cells based on the inertia principle can effectively overcome the defect that the existing contraction and expansion array structure can be influenced by dean force only when the cells flow into the contraction section from the widening section.
In the existing contraction and expansion array structure for separating cells based on the inertia principle, the cells at the rear part in the compression section are only influenced by the inertia lift force, and the particles can be focused on two balance positions close to the center of the long edge as shown in fig. 4 under the action of the pure inertia lift force, so that the cells at the rear part in the compression section can deviate to the center of a flow channel and deviate from the expected focusing balance position; the flow channel with the unilateral contraction and expansion array spiral structure (or arc structure) adopted by the invention can enable cells to be always subjected to the coupling action of dean force and inertial lift force in the whole flow channel, so that the cells are fixed at the expected balance position.
The invention adopts the single-side contraction and expansion array spiral flow passage, and improves the separation efficiency of the spiral structure to a certain extent. For the spiral structure, when the cells are in the equilibrium position as shown in fig. 5, the distance between the lateral equilibrium positions where different cells are located is small, which easily causes mixing of part of the cells, resulting in a decrease in separation efficiency. The special single-side contraction and expansion array in the spiral flow channel with the single-side contraction and expansion array structure can enable small-size cells to move towards the S2 side wall, and large-size cells can move towards the S1 side wall, so that the distance between the balance position of the large-size cells and the balance position of the small-size cells is increased, and the separation efficiency is improved.
For the single spiral structure, the total length of the flow channel is usually longer to achieve the desired separation effect. Under the condition of consistent separation effect, the total length of the flow channel designed by the invention can be shortened to a certain extent.
For a single spiral structure, the pressure that the chip can withstand is limited due to the long channel size, and the structure provides less FD and FL at a given flow rate, limiting the separation efficiency. The contraction and expansion array designed by the invention can solve the defect that when cells flow into the compression section from the widening section, the flow channel suddenly becomes small, so that the fluid is accelerated to enter the compression section, the speed of the fluid is increased by several times, the increase of the flow speed provides larger FD and FL, and the cells can reach the balance position more quickly.
In addition, the chip for separating cells based on the inertia principle also has the following advantages:
(1) and miniaturization. The total chip area is only a few square centimeters. The required reagent volume is only in the microliter range.
(2) The separation efficiency is improved. The micro-fluidic chip can separate cells with different sizes through a plurality of circular arc compressed and widened arrays.
(3) And carrying out online observation. The microfluidic chip can be directly observed under a CCD inverted microscope, and a high-speed camera is used for recording images, so that the operation is convenient.
(4) And the price is low. The chip material can adopt PDMS and organic glass. The reagent dosage is less, and the reagent cost is obviously reduced.
Drawings
FIG. 1 is a schematic diagram of the whole structure and a partial enlarged view of the isolated cell chip with a single-sided contraction and expansion array spiral structure according to the present invention;
FIG. 2 is a schematic diagram of the whole structure and a partial enlarged view of the isolated cell chip with a single-sided array arc structure;
FIG. 3 is a schematic diagram of a chip with a spiral structure for cell separation in the prior art;
FIG. 4 is a schematic diagram of a single particle under inertial lift in a straight channel according to the prior art;
FIG. 5 is a schematic diagram of a chip with a spiral structure for cell separation in the prior art;
FIG. 6A is a simulated view of the distribution of 6um particles in a circular arc structure with grooves distributed along the outer sidewall, and FIG. 6B is a partial enlarged view of FIG. 6A;
fig. 7A is a graph showing a simulation of the distribution of 3um particles in a circular arc structure in which convex grooves are distributed along the outer sidewall, and fig. 7B is a partially enlarged view of fig. 7A.
The meanings of the reference symbols in the figures are as follows: 1 is a sample inlet, 2 is a sheath fluid inlet, 3 is a cell separation unit (i.e., flow channel), 4 is a large-sized cell outlet, 5 is a small-sized cell outlet, S1 is an inner sidewall (i.e., first sidewall), and S2 is an outer sidewall (i.e., second sidewall).
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The invention discloses a micro-fluidic chip for separating cells based on the inertial principle, which comprises an upper matrix and a lower matrix from top to bottom, wherein the upper matrix and the lower matrix can be fixedly connected into a whole through a bonding method; the flow channel convex groove is positioned in the upper substrate (the projection of the flow channel convex groove on the plane of the chip is shown in figure 1; the lower substrate can be provided with no convex groove on the surface contacting with the upper substrate, and the surface contacting with the upper substrate of the lower substrate is parallel to the plane of the chip), and as shown in figure 1, the flow channel layer is a spiral flow channel with a single-side contraction and expansion array structure and comprises a sample inlet 1, a sheath fluid inlet 2, a large-size cell outlet 4 and a small-size cell outlet 5. The cells are subjected to the coupling action of dean force FD and inertial lift force FL in the flow channel, and after flowing through the flow channel with a certain length, the cells with different sizes are focused at different transverse equilibrium positions, and finally the cells with different sizes are separated. The chip can realize cell separation under the condition of high flow rate, and the required flow channel has simple structure, simple operation and extremely high flux, and improves the cell separation efficiency to a certain extent.
The chip for separating cells based on the inertia principle is a cell separation chip with a novel flow channel structure, can separate cells efficiently, simply and quickly, and can be prepared and used by adopting the following steps:
1. and designing a flow channel structure. The flow channel is a spiral flow channel with a unilateral contraction and expansion array structure, one end of the flow channel is a Y-shaped sample and sheath fluid inflow port, 1 is a sample inlet, 2 is a sheath fluid inflow port, the other end of the flow channel is a Y-shaped large cell outlet, 3 is a large cell outlet, and 4 is a small cell outlet. The cross section of the micro-channel is rectangular.
2. And designing the size of the flow passage. In the runner contraction and expansion array, the arc length of the expansion section is 300-700 um, the width is 350um, the arc length of the compression section is 300-1200 um, the width is 50um, and the height of the runner is 20-25 um.
3. And (5) manufacturing a chip. The chip comprises an upper substrate and a lower substrate, wherein the upper substrate can be made of Polydimethylsiloxane (PDMS), and the lower substrate can be made of glass. The two substrates are bonded together by means of a bond. The runner convex groove is arranged in the upper base body.
4. The cell solution is processed. A cell sample diluted to some extent in this protocol would greatly reduce the risk of channel blockage.
5. In the technical scheme, the micro-fluidic chip needs a power system for sample introduction. The cell sample and sheath fluid streams are injected simultaneously from injection port 1 and injection port 2 at appropriate velocities using a powered system.
6. And observing the experimental result.
The following are specific examples:
example 1
Referring to fig. 2, the cell separation chip includes a substrate, and a micro channel on the substrate, wherein the micro channel includes a cell sample inlet 1, a sheath fluid inlet 2, a cell separation unit 3, a large-sized cell outlet 4, and a small-sized cell outlet 5. In the technical scheme, the sample introduction of the microfluidic chip needs a power system, the power system is used for continuously injecting cell solution and sheath solution into the chip from a sample inlet 1 and a sample inlet 2 respectively, and the power system in the embodiment is an injector pump. The sheath flow was used to focus the cells into a beam in the first expanse after the inlet and into the constricting section next to the sidewall of S1.
The cell separation unit 3 is a circular arc-shaped channel which is bent inwards from the cell sample inlet 1. The radius of curvature of the inward bend of the outer side wall S2 is 7mm, the size of the channel is that any widening section of the flow channel has an arc length of 700um and a width of 350um, any compressing section has an arc length of 1200um and a width of 50um, the height of the whole flow channel is 25um, and the length of the cell separation unit 3 is 23mm (including the lengths of the inlet part and the outlet part). The diameters of the cell inlet port 1, the sheath fluid flow inlet port 2, the large-sized particle outlet port 4 may preferably be 400um, and the diameter of the small-sized cell outlet port 5 may preferably be 400 um.
As shown in fig. 2, the principle of separating cells of different sizes by using the flow channel is as follows: in a continuously curved microchannel, cells are subjected to two forces, primarily lift FL and dean FD. The equilibrium between FL and FD determines the equilibrium position of the cell in the microchannel. When FL is larger than or equal to FD, the cells move to the side wall S1, and when FL is smaller than FD, the cells move to the side wall S2; cells of larger size are subject to greater FL and cells of smaller size are subject to greater FD. When the cells move to the widened flow channel section, the widening of the flow channel enables wall-induced inertial lift force on the cells to be suddenly reduced, the small-size cells move to the S2 side wall and are stabilized at a new balance position, the large-size cells move to the S1 side wall and are stabilized at the new balance position, and therefore the distance between the balance position of the large-size cells and the balance position of the small-size cells is increased. When liquid enters the compression section from the expansion section, the liquid in the expansion section accelerates in a tortuous path into the compression section, which acceleration will enhance the FL and FD experienced by the cells to some extent, thereby allowing faster cell separation.
The flow rates of the two samples in the technical scheme can influence the cell separation effect, and the separation efficiency generated by different flow rate ratios is different. The ratio of the cell sample to sheath flow velocity in this example was 1: 5. Cell samples (diluted blood) and sheath fluid streams (phosphate buffer) were injected simultaneously from injection port 1 and injection port 2 at 0.012m/s, 0.06m/s, respectively, using two syringe pumps. (in this case, the maximum velocity in the corresponding channel is 0.8m/s)
During detection, a CCD inverted microscope is adopted for observation, and a high-speed camera is used for video recording. By observation, it was observed that blood cells (cells having a size of 6um or more) gradually moved to the side wall S1, and plasma (cells having a size of 3um or less and 3um or less) gradually moved to the side wall S2.
The working process of the micro-fluidic chip comprises the following steps:
1. two syringe pumps are prepared, the diluted blood cells and the phosphate buffer solution are respectively extracted by the syringes, and the syringes are placed on the syringe pumps and are connected with the chip through Teflon tubes.
2. The chip is placed at a proper position under a CCD inverted microscope, the focal length is adjusted, and clear observation is carried out.
3. And opening the syringe pump, adjusting the flow rate, and observing the separation of the cells with different sizes.
Table 4: under different speeds, the distance separating the 3um and 6um particles from the single-side expansion and contraction array arc structure in the straight channel
Note: the separation distance at this time is the separation distance between the lowest end of the 6um particle and the uppermost end of the 3um particle at the outlet. The radius of curvature of the circular arc-shaped structure is 7 mm. The 6 spans in the table indicate that the channel has 6 spans and 5 compressed segments. The 12 widened segments in the table indicate that the channel has 12 widened segments and 11 compressed segments.
Table 5: distance for separating 2um and 7um particles in unilateral expansion and contraction arc-shaped structure and spiral structure
Note: the distance between the lowest end of the 7um particles and the highest end of the 2um particles at the outlet is 7mm, and the curvature radius of the circular arc structure is 7 mm. The spiral structure is a structure in the literature [ 3 ], namely, a structure model is 50um in height, 100um in width, 3mm in innermost radius of curvature and 13cm. in total length, and the speed in the table is the speed when the separation efficiency of the corresponding model is optimal.
Table 6: distance separating 3um and 7um particles in three different models
Note: the separation distance at this time is the separation distance between the lowest end of the 6um particle and the uppermost end of the 3um particle at the outlet. The curvature radius of the two circular arc structures is 7 mm. The dimensions of the circular arc structure are as follows: the width is 50um, and the arc length is 21.6 mm. The arc lengths of these two circular arcs are equal to the length of the straight channel.
As can be seen from the above table, when the sizes of the three models are consistent and the speeds are consistent, the distance between the 3um and 7um particles is 141.6mm, which is greater than the sum of the separation distances of the other two structures.
In the present invention, the positions of the inlet (including the sample inlet 1 and the sheath fluid inlet 2) and the outlet (including the large-sized cell outlet 4 and the small-sized cell outlet 5) can be interchanged, and at this time, the tongue corresponding to the widened section of the flow channel is still located on the inner sidewall S1, the large-sized cell outlet 4 is still located on the same side of the inner sidewall S1, and the small-sized cell outlet 5 is still located on the same side of the outer sidewall S2.
The flow channel of the present invention may be spiral or arc, and may be determined according to the size of the cell particles to be separated, for example, as can be seen from table 4, when the one-side expansion and contraction spiral structure is longer, the optimal speed required for separating the particles is smaller, for example, the optimal speed required for separating 3um and 6um particles by using the 6 widening section one-side expansion and contraction array arc structures is 1.3m/s, and the optimal speed required for separating 3um and 6um particles by using the 12 widening section one-side expansion and contraction array arc structures is 0.8 m/s. Because the chip size is smaller, and the upper base body and the lower base body are combined together in a key combination mode, the pressure born by the core is limited, and when the optimal speed is reduced, the probability that liquid seeps out from an inlet due to overlarge water pressure in the channel can be effectively reduced. In practical use, if the requirement for speed is not so high, a longer channel can be adopted to reduce the probability of cracking and water seepage caused by excessive pressure in the chip.
The arc shape of the flow channel can be an elliptical arc or a regular circular arc; when the arc is arc-shaped, the center of the arc is the circle center of the arc. In addition, the height of the flow channel in the invention can be flexibly adjusted according to the diameter of the particle to be separated, for example, the height of 25um can be used for separating particles of 10um to 2um, and the height of 20um can be used for separating particles of 1.7um to 0.5 um.
The curvature radius of the flow channel is based on the curvature radius of the second side wall S2 of the flow channel; when the flow channel is arc-shaped, the curvature radius of each part of the flow channel is kept unchanged; when the flow path is spiral, the radius of curvature of the second side wall S2 closest to the center of the spiral is 5mm to 7mm (i.e., corresponding to the end of the flow path close to the center of the spiral).
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (8)
1. A chip for separating cells based on the inertial principle is characterized by comprising a substrate and a flow channel (3) positioned in the substrate, and further comprising a sample inlet (1), a sheath fluid inlet (2), a large-size cell outlet (4) and a small-size cell outlet (5) which are connected with the flow channel (3); wherein,
the sample inlet (1) is used for inputting a cell solution to be separated and treated to the inlet of the flow channel (3);
the sheath fluid inlet (2) is used for inputting a sheath fluid to the inlet of the flow channel (3), and the sheath fluid is used for mixing with the cell solution;
the flow channel (3) is spiral or arc; the flow channel (3) is provided with a first side wall (S1) and a second side wall (S2) which are oppositely arranged, wherein the first side wall (S1) is positioned at the inner side close to the spiral center or the arc center, and the second side wall (S2) is positioned at the outer side far away from the spiral center or the arc center; the first side wall (S1) is provided with convex grooves distributed at intervals along the flow channel (3), so that the flow channel (3) forms a widening section and a compressing section which are distributed in a staggered way, the widening section corresponds to the area on the first side wall where the convex grooves are distributed, and the compressing section corresponds to the area on the first side wall where the convex grooves are not arranged; the flow channel (3) is used for separating the cells in the cell solution mixed with the sheath liquid flow according to the size;
the large-size cell outlet (4) is positioned at one side close to the center of the spiral or the arc center and is used for outputting cell separation liquid containing large-size cells from the outlet of the flow channel (3);
the small-size cell outlet (5) is positioned on one side far away from the center of the spiral or the arc center and is used for outputting cell separation liquid containing small-size cells from the outlet of the flow channel (3);
furthermore, the inlet and the outlet of the flow channel (3) are respectively positioned at two ends of the flow channel (3).
2. The chip for separating cells based on the inertial principle according to claim 1, wherein in the flow channel (3), the arc length of any one of the widening sections is 300-700 um, and the arc length of any one of the compressing sections is 300-1200 um; preferably, the width of the widening section is 350um, and the width of the compressing section is 50 um; the height of the flow channel (3) is 20-25 um; preferably, the radius of curvature of the second side wall (S2) of the flow path (3) closest to the center of the spiral or the arc-shaped center portion is 5mm to 7 mm.
3. The chip for separating cells based on the inertial principle according to claim 1, wherein in the flow channel (3), the arc length of any one of the expanded sections is 700um, and the arc length of any one of the compressed sections is 1200 um; the height of runner (3) is 25um, and the overall length of this runner (3) is 23mm, and the number of required compression section is 11, the number of widening section is 12, and second lateral wall (S2) is the closest on this runner (3) spiral center or arc center part' S radius of curvature is 7mm, the particle diameter of jumbo size cell is not less than 6um, the particle diameter of jumbo size cell is no longer than 3 um.
4. The chip for cell separation based on the inertial principle according to claim 1, wherein the ratio of the flow rate of the cell solution to be separated and processed inputted from the sample inlet (1) to the flow rate of the sheath fluid inputted from the sheath fluid flow inlet (2) is 1: 5.
5. the chip for separating cells on the basis of inertial principle according to claim 1, characterized in that when the flow channel (3) is spiral, the flow channel (3) is preferably distributed in an Archimedes spiral.
6. The inertial principle-based cell separation chip according to claim 1, wherein the substrate comprises an upper substrate and a lower substrate stacked one on top of the other, and the flow channel (3) is disposed between the upper substrate and the lower substrate.
7. The inertial-principle-based cell separation chip of claim 1, wherein the sheath fluid stream is a phosphate buffer.
8. The chip for separating cells based on the inertial principle according to claim 1, wherein the flow channel (3) has a rectangular cross section.
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| CN107159326A (en) * | 2017-05-09 | 2017-09-15 | 西北工业大学 | Inertia based on enhanced Secondary Flow focuses on chip |
| CN108246374A (en) * | 2018-03-28 | 2018-07-06 | 东南大学 | Three-dimensional spiral Constituent cell sorting micro-fluidic chip and preparation method thereof |
| CN108715794A (en) * | 2018-05-08 | 2018-10-30 | 南京师范大学 | A kind of cell accurately manipulates micro-fluidic device |
| CN109666584A (en) * | 2018-12-29 | 2019-04-23 | 北京工业大学 | A kind of experimental provision can be used for carrying out circulating tumor cell sorting experiment |
| CN109967150A (en) * | 2019-04-24 | 2019-07-05 | 河海大学常州校区 | It is a kind of for manipulating the inertia micro-fluidic chip of micro-nano granules |
| CN110124757A (en) * | 2019-04-29 | 2019-08-16 | 南京师范大学 | A kind of micro particles sorting micro-fluidic device |
| CN111139182A (en) * | 2018-11-02 | 2020-05-12 | 青岛华大智造普惠科技有限公司 | Magnetic screening device, micro-droplet screening system and magnetic screening method of micro-droplets |
| CN111690508A (en) * | 2020-05-19 | 2020-09-22 | 东南大学 | Multi-functional unit integrated rare tumor cell multi-stage sorting device |
| CN111909823A (en) * | 2019-05-08 | 2020-11-10 | 清华大学 | Inertial micro-fluidic chip for enriching circulating tumor cells |
| CN112007704A (en) * | 2020-07-08 | 2020-12-01 | 河海大学常州校区 | Micro-fluidic chip and method for sorting micro-nano particles by inertial turbulence |
| CN112354573A (en) * | 2020-10-26 | 2021-02-12 | 深圳亘流科技有限公司 | Step type inertial focusing micro-fluidic chip |
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| CN108246374A (en) * | 2018-03-28 | 2018-07-06 | 东南大学 | Three-dimensional spiral Constituent cell sorting micro-fluidic chip and preparation method thereof |
| CN108246374B (en) * | 2018-03-28 | 2020-09-11 | 东南大学 | Three-dimensional spiral structure cell sorting micro-fluidic chip and manufacturing method thereof |
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| CN111139182B (en) * | 2018-11-02 | 2023-11-14 | 青岛华大智造科技有限责任公司 | Magnetic screening device, micro-droplet screening system and micro-droplet magnetic screening method |
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| CN111909823B (en) * | 2019-05-08 | 2023-04-18 | 清华大学 | Inertial micro-fluidic chip for enriching circulating tumor cells |
| CN111909823A (en) * | 2019-05-08 | 2020-11-10 | 清华大学 | Inertial micro-fluidic chip for enriching circulating tumor cells |
| CN111690508A (en) * | 2020-05-19 | 2020-09-22 | 东南大学 | Multi-functional unit integrated rare tumor cell multi-stage sorting device |
| CN112007704A (en) * | 2020-07-08 | 2020-12-01 | 河海大学常州校区 | Micro-fluidic chip and method for sorting micro-nano particles by inertial turbulence |
| CN112354573B (en) * | 2020-10-26 | 2022-01-04 | 深圳亘流科技有限公司 | Step type inertial focusing micro-fluidic chip |
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