Microalgae cell multistage sorting device based on dielectrophoresis controllable sheath flow

Abstract

The invention provides a microalgae cell multistage sorting device based on dielectrophoresis controllable sheath flow. The microalgae cell multistage sorting device comprises a flow channel structure and an electrode substrate which are packaged in a bonding mode. The flow channel structure comprises a liquid inlet, a spiral flow channel, a sudden expansion structure, a first branch flow channel, a second branch flow channel, a deterministic lateral displacement flow channel, a first liquid outlet, a second liquid outlet and a third liquid outlet; the liquid inlet is communicated with the spiral runner; the first branch flow channel and the second branch flow channel are connected to the two ends of the split sudden expansion structure in series. The deterministic lateral displacement runner is connected to the tail end of the second branch runner in series; a second liquid outlet and a third liquid outlet are formed in the tail end of the deterministic lateral displacement runner; the electrode substrate comprises a substrate body and an electrode group; and the electrode group is arranged at the front end of the deterministic lateral displacement runner. According to the invention, sheath flow is accurately controlled through an electrode structure, and high-flux and high-purity separation of single microalgae cells from a mixed solution of three microalgae with different sizes is realized by combining high flux of inertial spirals and high precision of deterministic lateral displacement.

Description

Technical Field

[0001] The present invention relates to the technical field of microalgae sorting, and in particular to a multi-stage microalgae cell sorting device based on dielectrophoresis controllable sheath flow. Background Art

[0002] Microalgae are the most numerous species of marine organisms. It is estimated that there are more than one million different species of microalgae in the world. Microalgae are at the bottom of the food chain and can provide raw materials for survival for many organisms. Microalgae resources have broad application and development prospects in many fields such as food industry, animal feed, biotechnology, pharmaceutical industry, bioenergy, pollution control, etc., and have received widespread attention. However, the number of microalgae species currently studied is limited, and most of them have not been studied and developed. To develop new microalgae strains from the natural environment, they must first be sorted out from the natural environment. This is a necessary prerequisite for developing new algae species and obtaining excellent algae strains. The problem of sorting microalgae cells has become a bottleneck restricting the utilization of microalgae resources. It is urgent to conduct theoretical and applied research related to microalgae cell sorting.

[0003] Microfluidic technology has received widespread attention since its emergence in the 1990s. Microfluidic chips can integrate basic operations such as sample preparation, reaction, sorting, and detection into microscale channels, and automatically complete the analysis and processing process. It has the advantages of low sample consumption and high manipulation accuracy, and has great potential in cell sorting applications. Depending on whether an external force field is used, microfluidic cell sorting technology can be divided into two categories: passive sorting technology and active sorting technology. Passive technologies include microstructure filtration, inertial sorting, deterministic lateral displacement sorting, affinity sorting, etc.; while active sorting technologies include magnetic sorting, optical sorting, and dielectrophoresis sorting.

[0004] The traditional deterministic lateral displacement technology has great limitations due to the sheath flow it introduces, which dilutes the sample and increases the flow channel design and external equipment. At the same time, inertial sorting, as a passive sorting technology, has the advantage of high throughput but also faces the challenge of slightly insufficient sorting accuracy, especially when the object is complex microalgae cells. Summary of the invention

[0005] According to the technical problem that it is difficult for a single sorting technology to achieve both high throughput and high precision, a multi-stage sorting device for microalgae cells based on dielectrophoresis controllable sheath flow is provided. The present invention can achieve precise control of sheath flow through a unique electrode structure, combining the high throughput advantage of the inertial spiral and the high precision advantage of deterministic lateral displacement, and achieve high throughput and high purity sorting of a single microalgae cell from a mixed solution of three microalgae of different sizes.

[0006] The technical means adopted by the present invention are as follows:

[0007] A multi-stage sorting device for microalgae cells based on dielectrophoresis controllable sheath flow, comprising: a bonded and packaged flow channel structure and an electrode substrate; wherein:

[0008] The flow channel structure includes a liquid inlet, a spiral flow channel, a sudden expansion structure, a first branch flow channel, a second branch flow channel, a deterministic lateral displacement flow channel, a first liquid outlet, a second liquid outlet and a third liquid outlet; wherein:

[0009] The liquid inlet is connected to the spiral flow channel; the first branch flow channel and the second branch flow channel are connected in series at the two ends of the split sudden expansion structure; the deterministic lateral displacement flow channel is connected in series to the end of the second branch flow channel; the end of the deterministic lateral displacement flow channel is provided with a second liquid outlet and a third liquid outlet;

[0010] The electrode substrate comprises a substrate body and an electrode group, wherein the electrode group is arranged at the front end of the deterministic lateral displacement flow channel.

[0011] Furthermore, the selected single-species microalgae cells are introduced into the first liquid outlet through the first branch flow channel through the spiral flow channel and the sudden expansion structure, and the remaining mixed algae cells are introduced into the deterministic lateral displacement flow channel through the second branch flow channel 8.

[0012] Furthermore, the vertical cross-sectional dimension of the first branch channel is smaller than that of the second branch channel, that is, the width of the second branch channel is greater than that of the first branch channel, thereby reducing the volume of fluid to be processed by the first branch channel for sorting, thereby achieving the purpose of reducing the flow rate.

[0013] Furthermore, the cross-section of the spiral flow channel is a rectangle with a low aspect ratio, and the cell diameter and the hydraulic diameter of the spiral flow channel satisfy ap / Dh>0.07, where ap is the cell particle diameter and Dh is the hydraulic diameter of the spiral flow channel, so that different algae species cells with large size differences are focused to different equilibrium positions on the flow channel cross-section.

[0014] Furthermore, a micro-pillar array with upper and lower mirror image distribution is arranged in the deterministic lateral displacement flow channel, and each micro-pillar in the micro-pillar array is evenly distributed with a certain distance between them.

[0015] Furthermore, the cross section of the microcolumn is L-shaped, so that particles larger than the critical size migrate along the inclination direction of the array when flowing through the microcolumn array, while particles smaller than the critical size move with the main flow when flowing through the microcolumn array, and migrate horizontally as a whole.

[0016] Furthermore, the electrode group is in a fishtail shape, and the electrode group has at least one pair of electrode arrays. The electrodes in each pair of electrode arrays are parallel to each other, and one end of the electrodes on a single electrode array is connected. The electrodes in each pair of electrode arrays are spaced apart, and the electrodes have an angle of 60°. A periodic low-frequency negative dielectrophoresis signal is applied to the electrode group to deflect all algae cells to the sides close to the flow channel wall, thereby replacing the sheath flow in the traditional deterministic lateral displacement technology. The purpose of controlling the sheath flow can also be achieved by adjusting the frequency and voltage of the dielectrophoresis signal.

[0017] Furthermore, the flow channel structure and the electrode substrate are both made of polydimethylsiloxane.

[0018] Furthermore, the electrode substrate is made by etching ITO glass, or by sputtering and patterning a metal electrode on glass or silicon material.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The present invention provides a multi-stage sorting device for microalgae cells based on controllable sheath flow by dielectrophoresis, which overcomes the problem that a single sorting technology is difficult to take into account both high throughput and high precision. It cleverly uses dielectrophoretic force to replace the role of traditional sheath flow, achieves precise control of sheath flow through a unique electrode structure, and combines inertial and deterministic lateral displacement technology to achieve high-throughput and high-purity sorting of microalgae cells.

[0021] 2. The present invention provides a multi-stage sorting device for microalgae cells based on dielectrophoresis controllable sheath flow, which has the advantages of low cost, fast response, simple operation, miniaturization and integration. The device of the present invention can be used in the fields of microalgae strain screening, environmental monitoring and restoration, biological research, etc., and is particularly suitable for sorting new microalgae strains from the natural environment. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0023] Figure 1 It is a top view of the overall structure of the device of the present invention.

[0024] Figure 2 It is a schematic diagram of the inertial separation principle of the spiral flow channel in the present invention.

[0025] Figure 3 It is a schematic diagram of the local amplification and sorting principle of the micro-pillar array in the present invention.

[0026] Figure 4 This is a schematic diagram of the principle of fishtail electrode dielectrophoresis sorting in the present invention.

[0027] Figure 5 Schematic diagram of the structure of the electrode substrate of the present invention.

[0028] In the figure: 1. channel structure; 2. liquid inlet; 3. spiral channel; 4. sudden expansion structure; 5. electrode group; 6. first branch channel; 7. first liquid outlet; 8. second branch channel; 9. deterministic lateral displacement channel; 10. second liquid outlet; 11. third liquid outlet; 12. electrode base; 13. outer wall; 14. inner wall; 15. first electrode; 16. second electrode; 17. third electrode; 18. fourth electrode. DETAILED DESCRIPTION

[0029] In order to enable those skilled in the art to better understand the scheme of the present invention, the technical scheme in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work should fall within the scope of protection of the present invention.

[0030] It should be noted that the terms "first", "second", etc. in the specification and claims of the present invention and the above-mentioned drawings are used to distinguish similar objects, and are not necessarily used to describe a specific order or sequence. It should be understood that the data used in this way can be interchanged where appropriate, so that the embodiments of the present invention described herein can be implemented in an order other than those illustrated or described herein. In addition, the terms "including" and "having" and any variations thereof are intended to cover non-exclusive inclusions, for example, a process, method, system, product or device that includes a series of steps or units is not necessarily limited to those steps or units that are clearly listed, but may include other steps or units that are not clearly listed or inherent to these processes, methods, products or devices.

[0031] like Figure 1 As shown, the present invention provides a multi-stage sorting device for microalgae cells based on dielectrophoresis controllable sheath flow, comprising: a bonded and packaged flow channel structure 1 and an electrode substrate 12; wherein:

[0032] The flow channel structure 1 includes a liquid inlet 2, a spiral flow channel 3, a sudden expansion structure 4, a first branch flow channel 6, a second branch flow channel 8, a deterministic lateral displacement flow channel 9, a first liquid outlet 7, a second liquid outlet 10 and a third liquid outlet 11; wherein:

[0033] The liquid inlet 2 is connected to the spiral flow channel 3; the first branch flow channel 6 and the second branch flow channel 8 are connected in series to the two ends of the split sudden expansion structure 4; the deterministic lateral displacement flow channel 9 is connected in series to the end of the second branch flow channel 8; the end of the deterministic lateral displacement flow channel 9 is provided with a second liquid outlet 10 and a third liquid outlet 11;

[0034] The electrode substrate 12 includes a substrate body and an electrode group 5 ; wherein the electrode group 5 is arranged at the front end of the deterministic lateral displacement flow channel 9 .

[0035] In specific implementation, as a preferred embodiment of the present invention, the selected single-species microalgae cells are introduced into the first liquid outlet 7 through the spiral flow channel 3 and the sudden expansion structure 4 respectively, and the remaining mixed algae cells are introduced into the deterministic lateral displacement flow channel 9 through the second branch flow channel 8. In this embodiment, the sorting principle of the spiral flow channel is shown in FIG. Figure 2 , the inner wall 14 and outer wall 13 of the spiral flow channel 3 are shown in the figure. The mixed algae solution is injected into the spiral flow channel 3 through the sample inlet 2 at a specific flow rate. The fluid at the center of the flow channel has a higher flow rate than the fluid near the wall, and flows outward under the action of centrifugal force; due to the closed flow channel, the fluid at the outer wall is squeezed and forced to flow back along the upper and lower walls, thus forming two symmetrical vortices. This phenomenon is called Dean flow or secondary flow. Under the inertial lift F L and the Dean drag F induced by the Dean flow D Under the joint action of , the cells reach stable equilibrium positions A and B, and particles of different sizes and shapes have different equilibrium positions. Through the Dean flow and inertial migration in the curved channel of the spiral channel 3, the Chlorella, Platymonas and Closterium cells of different sizes and shapes are focused to different equilibrium positions. Among them, Chlorella is a spherical cell with a diameter of 3-5μm, and the focusing position is close to the inner wall of the spiral channel, and enters the first branch channel 6 through the sudden expansion structure 4; Platymonas is an oval cell with a long axis of 10-15μm and a short axis of 5-8μm, and Closterium is a crescent-shaped cell with a long axis of 10-14μm and a short axis of 2-3μm. The focusing position of the two in the spiral channel is close to the center of the channel and is introduced into the second branch channel 8 through the sudden expansion structure 4. That is, through the first-level spiral channel 3 sorting, Chlorella cells can be sorted under high-throughput conditions. The Closterium and Platymonas cells with a focusing position close to the middle of the channel enter the second branch channel 8 after passing through the sudden expansion structure 4.

[0036] In specific implementation, as a preferred embodiment of the present invention, the cross-section of the spiral flow channel 3 is a rectangle with a low aspect ratio (for example, 1 / 3 to 1 / 4), and the cell diameter and the hydraulic diameter of the spiral flow channel 3 satisfy ap / Dh>0.07, ap is the cell particle diameter, and Dh is the hydraulic diameter of the spiral flow channel 3, so that different algae species with large size differences are focused to different equilibrium positions of the flow channel cross-section.

[0037] In specific implementation, as a preferred embodiment of the present invention, the vertical cross-sectional dimension of the first branch channel 6 is smaller than the vertical cross-sectional dimension of the second branch channel 8, that is, the width of the second branch channel 8 is greater than the width of the first branch channel 6, thereby reducing the volume of fluid required for sorting in the first branch channel 6, thereby achieving the purpose of reducing the flow rate.

[0038] In specific implementation, as a preferred embodiment of the present invention, the deterministic lateral displacement flow channel 9 is arranged with a micro-pillar array with mirror images of each other, and each micro-pillar in the micro-pillar array is evenly distributed with a certain spacing. The cross section of the micro-pillar is L-shaped, so that particles larger than the critical size migrate along the inclination direction of the array when flowing through the micro-pillar array, while particles smaller than the critical size move with the main flow when flowing through the micro-pillar array, and migrate in the horizontal direction as a whole.

[0039] In this embodiment, the sorting principle of the DLD flow channel is shown in Figure 3 , a local amplification of the micropillar array in the deterministic lateral displacement channel 9 is shown in FIG. Figure 3 As shown, the microcolumn array is arranged at a certain inclination angle to the horizontal direction. Particles smaller than the critical size will move with the main flow and migrate in the horizontal direction as a whole; while particles larger than the critical size will migrate in the inclined arrangement direction. The critical size depends on the inter-column spacing and the inclination angle of the microcolumn array. In this embodiment, the critical size is adjusted between 8-10 μm, so that the flat algae cells (oval cells with a long axis of 10-15 μm and a short axis of 5-8 μm) migrate in the inclination direction of the microcolumn array, while the crescent algae cells (crescent-shaped cells with a long axis of 10-14 μm and a short axis of 2-3 μm) do not migrate in the longitudinal direction with the inclination of the microcolumn array, but move with the main flow and migrate in the horizontal direction as a whole. Finally, the flat algae cells are exported from the second liquid outlet 10, and the crescent algae cells are exported from the third liquid outlet 11, thereby realizing the sorting of crescent algae and flat algae cells.

[0040] In specific implementation, as a preferred embodiment of the present invention, the electrode group 5 is in a fishtail shape, and the electrode group 5 has at least one pair of electrode arrays. The electrodes in each pair of electrode arrays are parallel to each other, and one end of the electrodes on a single electrode array is connected. The electrodes in each pair of electrode arrays are spaced apart, and the electrodes have an angle of 60°. A periodic low-frequency negative dielectrophoresis signal is applied to the electrode group 5 to deflect all algae cells to the sides close to the flow channel wall, thereby replacing the sheath flow in the traditional deterministic lateral displacement technology. The purpose of controlling the sheath flow can also be achieved by adjusting the frequency and voltage of the dielectrophoresis signal.

[0041] In this embodiment, the working principle of electrode group dielectrophoresis is as follows Figure 4 As shown. Figure 4It can be seen that at the front end of the deterministic lateral displacement flow channel 9, an alternating current signal of a specific frequency is applied through the electrode group 5, and a non-uniform electric field will be formed at the front end of the deterministic lateral displacement flow channel 9, and the cells flowing through this place will be induced to generate an asymmetric dipole moment under the action of the non-uniform electric field, thereby generating a dielectrophoretic force. In this embodiment, a negative dielectrophoretic force is applied to the flat algae and crescent algae cells passing through this place, so that they move in the direction with weak electric field intensity, that is, away from the electrode. When an alternating current signal of a specific frequency is applied to the electrode group 5, the cells here are subjected to the fluid drag force F D1 and negative dielectrophoretic force F DEP The combined force F S Under the action of the fishtail electrode, the cells of Closterium and Platyhelminthes can enter the micro-pillar array from both sides of the deterministic lateral displacement channel 9, replacing the role of the sheath flow in the traditional DLD chip, and by adjusting the frequency and voltage of the signal, the dielectrophoretic force received by the Closterium and Platyhelminthes cells can be controlled to achieve precise control of the sheath flow.

[0042] In specific implementation, as a preferred embodiment of the present invention, the flow channel structure 1 and the electrode substrate 12 are both made of polydimethylsiloxane.

[0043] In specific implementation, as a preferred embodiment of the present invention, the electrode substrate 12 is made by etching ITO glass, or by sputtering and patterning metal electrodes on glass or silicon material.

[0044] In this embodiment, the electrode substrate 12, see Figure 5 , including a substrate body and a group of electrode groups 5, wherein the electrode group 5 is located at the bottom of the front end of the deterministic lateral displacement flow channel 9. The electrode group 5 includes a pair of electrode arrays, each electrode array includes two fishtail electrodes. The four fishtail electrodes are parallel to each other and are evenly arranged with equal spacing, and the electrodes have an angle of 60°. The first electrode 15 and the third electrode 17 are connected by two parallel and equal-width inclined electrodes at the upper end, and the second electrode 16 and the fourth electrode 18 are connected by two parallel inclined electrodes at the lower end. In other embodiments, the number of electrodes of the first electrode 15, the second electrode 16, the third electrode 17 and the fourth electrode 18 and the angle between the fishtail electrode and the main flow direction can be redesigned as required. The overall height of the electrode group 5 is greater than the width of the deterministic lateral displacement flow channel 9, and the overall width is only distributed at the front end of the deterministic lateral displacement flow channel. In addition, the first electrode 15 and the third electrode 17, the second electrode 16 and the fourth electrode 18 are respectively connected to different extraction electrodes, and a signal generator is used to apply a low periodic low-frequency negative dielectrophoresis signal to the electrode group 5 through the extraction electrode.

[0045] In this embodiment, after the surface of the flow channel structure and the electrode substrate microstructure is cleaned, the surface of the flow channel structure and the electrode substrate is modified by a plasma cleaning machine to achieve the bonding of the flow channel structure and the electrode substrate. After the chip is manufactured, the sample is introduced from the liquid inlet 2 at a specific flow rate; the samples are collected at the first liquid outlet 7, the second liquid outlet 10 and the third liquid outlet 11 respectively. The introduction of the sample can be achieved by connecting the microtube to the injection pump; the collection of the sample can be achieved by connecting the microtube to the collection tube.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A device for multi-stage sorting of microalgae cells based on dielectrophoresis controllable sheath flow, characterized in that: include: A flow channel structure (1) and an electrode substrate (12) for bonding and packaging; wherein: The flow channel structure (1) comprises a liquid inlet (2), a spiral flow channel (3), a sudden expansion structure (4), a first branch flow channel (6), a second branch flow channel (8), a deterministic lateral displacement flow channel (9), a first liquid outlet (7), a second liquid outlet (10) and a third liquid outlet (11); wherein: The liquid inlet (2) is connected to the spiral flow channel (3); the first branch flow channel (6) and the second branch flow channel (8) are connected in series to the two ends of the split abrupt expansion structure (4); the deterministic lateral displacement flow channel (9) is connected in series to the end of the second branch flow channel (8); the end of the deterministic lateral displacement flow channel (9) is provided with a second liquid outlet (10) and a third liquid outlet (11); The electrode substrate (12) comprises a substrate body and an electrode group (5); wherein the electrode group (5) is arranged at the front end of the deterministic lateral displacement flow channel (9).

2. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The selected single-species microalgae cells are introduced into the first liquid outlet (7) through the spiral flow channel (3) and the sudden expansion structure (4) respectively via the first branch flow channel (6), while the remaining mixed algae cells are introduced into the deterministic lateral displacement flow channel (9) via the second branch flow channel (8).

3. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The vertical cross-sectional dimension of the first branch flow channel (6) is smaller than the vertical cross-sectional dimension of the second branch flow channel (8), that is, the width of the second branch flow channel (8) is greater than the width of the first branch flow channel (6), thereby reducing the volume of fluid to be processed by the first branch flow channel (6) for sorting, thereby achieving the purpose of reducing the flow rate.

4. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The cross section of the spiral flow channel (3) is a rectangle with a low aspect ratio, and the cell diameter and the hydraulic diameter of the spiral flow channel (3) satisfy ap / Dh>0.07, where ap is the cell particle diameter and Dh is the hydraulic diameter of the spiral flow channel (3), so that different algae species with large size differences are focused to different equilibrium positions on the flow channel cross section.

5. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The deterministic lateral displacement flow channel (9) is provided with an array of micro-pillars distributed in an upper and lower mirror image, and each micro-pillar in the micro-pillar array is evenly distributed with a certain spacing between them.

6. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 5, characterized in that: The cross section of the microcolumn is L-shaped, so that particles larger than a critical size migrate along the inclination direction of the array when flowing through the microcolumn array, while particles smaller than a critical size move with the main flow when flowing through the microcolumn array, and migrate horizontally as a whole.

7. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The electrode group (5) is in a fishtail shape, and the electrode group (5) has at least one pair of electrode arrays, the electrodes in each pair of electrode arrays are parallel to each other, one end of the electrodes on a single electrode array is connected, the electrodes in each pair of electrode arrays are spaced apart, and the electrodes have an angle of 60°. A periodic low-frequency negative dielectrophoresis signal is applied to the electrode group (5) to deflect all algae cells to the sides close to the flow channel wall, thereby replacing the sheath flow in the traditional deterministic lateral displacement technology, and the purpose of controlling the sheath flow can also be achieved by adjusting the frequency and voltage of the dielectrophoresis signal.

8. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The flow channel structure (1) and the electrode substrate (12) are both made of polydimethylsiloxane.

9. The device for multi-stage separation of microalgae cells based on dielectrophoresis controllable sheath flow according to claim 1, characterized in that: The electrode substrate (12) is made by etching ITO glass, or by sputtering and patterning a metal electrode on glass or silicon material.

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