A microalgae and bacteria sorting device based on combination of deterministic lateral displacement and dielectrophoresis technology

Abstract

This invention discloses a microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology, belonging to the field of cell filtration and sorting technology. The device includes a PDMS cover layer, an ITO electrode layer, and a glass substrate layer. The PDMS cover layer includes a deterministic lateral displacement sorting module, a buffer anti-clogging module, and a dielectrophoresis array sorting module. The deterministic lateral displacement sorting module can achieve preliminary separation of microalgae cells and bacteria based on size. The buffer anti-clogging module prevents cell aggregation and backflow through a designed arrow-shaped micropillar array. The dielectrophoresis array sorting module achieves high-throughput sorting through a designed parallel 8-channel dielectrophoresis sorting array, and solves the problem of flow rate mismatch between deterministic lateral displacement separation and dielectrophoresis separation. The designed comb-shaped and wheat-ear-shaped electrodes provide strong and stable dielectrophoretic forces to the cells, thereby achieving high-precision and high-efficiency separation of microalgae cells and residual bacteria.

Description

Technical Field

[0001] This invention belongs to the field of cell filtration and sorting technology, specifically relating to a multi-stage sorting device for microalgae cells and bacteria based on a combination of deterministic lateral displacement and dielectrophoresis technology. Background Technology

[0002] Microfluidic chip technology integrates basic operations such as sample preparation, reaction, sorting, and detection into microscale channels, automating the analysis and processing. It offers advantages such as requiring small sample volumes, small size, low cost, high efficiency, portability, and ease of integration, and has been widely applied in various research fields including biology, medicine, chemistry, agriculture, food, environment, and navigation.

[0003] Microfluidic particle separation technology is mainly divided into two categories: active separation and passive separation. Common active separation methods include dielectrophoresis, optical separation, acoustic separation, and magnetic separation, all of which require an external field to manipulate the particles. Passive separation methods rely primarily on the characteristics of the flow field and the physical properties of the particles themselves, such as size and hardness. These methods mainly include microstructure filtration, inertial separation, and deterministic lateral displacement (DLD) separation. However, each method has its limitations, such as low purity, low throughput, and complex fabrication. For complex cell separation, such as that involving bacteria, a single technique is insufficient for effective separation. Combining multiple techniques can integrate their advantages and is an effective method for solving cell separation problems under complex conditions.

[0004] In conclusion, designing a high-throughput, high-precision multi-stage sorting device and method for microalgae cells and bacteria by combining multiple technologies is of great significance. Summary of the Invention

[0005] To overcome the shortcomings and deficiencies of existing single technologies, this invention provides a high-throughput, high-precision multi-stage sorting device for microalgae cells and bacteria by combining multiple technologies. This invention overcomes the problems of low throughput, low separation accuracy, easy clogging, and high cost in existing microfluidic cell separation technologies, which will have important scientific significance and practical value for the field of microalgae separation and ship ballast water detection applications.

[0006] To achieve the above objectives, the technical solution of the present invention is as follows:

[0007] A microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology mainly includes a PDMS cover layer 1, an ITO electrode layer 2, and a glass substrate layer 3; the ITO electrode layer 2 is deposited on the glass substrate layer 3, and the PDMS cover layer 1 is tightly bonded to the glass substrate layer 3; the PDMS cover layer 1 includes a DLD (deterministic lateral displacement) sorting module 1-1, a buffer anti-clogging module 1-2, and a DEP (dielectrophoresis) array sorting module 1-3 connected in sequence.

[0008] Furthermore, the DLD sorting module includes a DLD sorting channel, a sample inlet 1-1-1, a sheath fluid inlet 1-1-2, two waste liquid outlets 1-1-8 and 1-1-9, and a microalgae cell outflow channel 1-1-5. The sample inlet 1-1-1 merges with the sheath fluid inlet 1-1-2 at the inlet end of the DLD sorting channel via left and right branch pipes. The outlet end of the DLD sorting channel branches into two flow resistance matching channels 1-1-6 and 1-1-7 on the left and right, and the central microalgae cell outflow channel 1-1-5. The flow channels 1-1-6 and 1-1-7 are connected to the two waste liquid outlets 1-1-8 and 1-1-9, respectively; the DLD sorting channel includes a circular micropillar array 1-1-3 at the front and an equilateral triangular micropillar array 1-1-4 at the rear. The equilateral triangular micropillar array 1-1-4 is symmetrically distributed around the central axis of the DLD sorting channel, and the angle between the line connecting each row of equilateral triangular micropillars and the central axis of the DLD sorting channel is 1-10°; the end of the microalgae cell outflow channel 1-1-5 is connected to the buffer anti-clogging module 1-2;

[0009] Furthermore, the diameter of the circular micropillars is 50 μm and the spacing is 200 μm; the side length of the equilateral triangular micropillars is 55 μm and the spacing between the triangles is 40 μm.

[0010] Furthermore, the end of the microalgal cell outflow channel 1-1-5 is connected to the buffer anti-blocking module 1-2 through a sudden expansion structure 1-1-10.

[0011] Furthermore, the buffer anti-clogging module 1-2 includes a fluid channel 1-2-1 and an alternating array of arrows 1-2-2, with the arrows having an angle of 30-60° and the tips of the arrows pointing in the direction of sample inflow. The end of the fluid channel is connected to the DEP array sorting module 1-3.

[0012] Furthermore, the end of the fluid channel is connected to the DEP array sorting module 1-3 via a series of sequentially connected constriction structure 1-2-3 and expansion structure 1-2-4.

[0013] Furthermore, the DEP array sorting module 1-3 includes n parallel sorting channels 1-3-1, waste liquid outlet 1-3-2 and microalgae cell collection outlet 1-3-3. Each sorting channel 1-3-1 is connected to the waste liquid outlet 1-3-2 and the microalgae cell collection outlet 1-3-3 respectively through a pipe, where n is an even number.

[0014] Furthermore, the ITO electrode layer 2 is located directly below the sorting channel 1-3-1 in the DEP array sorting module 1-3, and includes comb-shaped ITO electrodes 2-1 and wheat-ear-shaped ITO electrodes 2-2. The comb-shaped ITO electrodes 2-1 and wheat-ear-shaped ITO electrodes 2-2 are respectively connected to the positive and negative terminals of the external power supply. The comb-shaped electrode contains (0.5n+1) elongated electrodes 2-1-2, which are equidistantly distributed. The wheat-ear-shaped electrode contains 0.5n wheat-ear electrodes, which are equidistantly distributed. Each wheat-ear electrode consists of one elongated electrode 2-2-3 and 10-100 evenly distributed hexagonal electrodes 2-2-2. The elongated electrodes 2-1-2 and wheat-ear electrodes of the comb-shaped ITO electrode 2-1 are arranged alternately to form electrode pairs, located directly below each sorting channel 1-3-1 in the DEP array sorting module 1-3, where n is an even number.

[0015] Furthermore, the width of the elongated electrode is 200 μm.

[0016] The present invention also provides a sorting method for microalgae and bacteria using the above-described microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology, comprising the following steps:

[0017] A. Prepare the sample solution and sheath fluid of the microalgal cells to be separated with the required conductivity;

[0018] B. Use a syringe pump to inject the sample solution into sample inlet 1-1-1 at a certain speed, and inject the sheath fluid into sheath fluid inlet 1-1-2 at a certain speed;

[0019] C. After passing through the DLD sorting module, the microalgae cells enter the buffer anti-clogging module 1-2 through the microalgae cell outflow channel 1-1-5, and the bacteria will flow out from the waste liquid outlets 1-1-8 and 1-1-9;

[0020] D. Microalgae cells pass through the staggered arrow-shaped array in the buffer and anti-blockage module 1-2, which effectively prevents cell aggregation and backflow, and enter the DEP array sorting module 1-3;

[0021] E. In the DEP array sorting module 1-3, microalgae cells are subjected to negative mesophoretic force and flow out from the microalgae cell collection outlet 1-3-3; bacteria are subjected to positive mesophoretic force and flow out from the waste liquid outlet 1-3-2, realizing high-throughput and high-precision sorting of microalgae cells and bacteria.

[0022] The advantages of this invention over the prior art are as follows:

[0023] (1) The sorting device of the present invention achieves high-throughput and high-precision separation of microalgae cells and bacteria by combining deterministic lateral displacement technology and dielectrophoresis technology.

[0024] (2) The sorting device of the present invention uses a set of comb-shaped electrodes and a set of wheat-ear-shaped electrodes to enable multiple dielectric separation channels to run in parallel, thereby improving the separation throughput. The designed wheat-ear-shaped electrodes are composed of multiple hexagonal ITO electrodes. The pointed structure of the hexagon can generate strong dielectric force. The residual bacteria flow out from the middle waste liquid outlet under the action of positive dielectric force, while the target microalgae cells flow out from the upper and lower outlets under the action of negative dielectric force, thereby achieving high-precision sorting.

[0025] (3) The present invention overcomes the disadvantages of bacterial residue and low sorting accuracy when using DLD sorting technology alone, as well as the low throughput of dielectrophoresis technology. In addition, the device has the advantages of simple operation and high degree of automation. Attached Figure Description

[0026] To more clearly illustrate the embodiments of the present invention, the accompanying drawings involved in the embodiments will be briefly described below.

[0027] This invention has a total of appendices Figure 5 , of which:

[0028] Figure 1 A schematic diagram of the overall structure of a microalgae and bacteria sorting device based on a combination of deterministic lateral displacement and dielectrophoresis techniques;

[0029] Figure 2 This is a schematic diagram of the PDMS cover layer.

[0030] Figure 3 This is an enlarged view of the structure of the DLD sorting module.

[0031] Figure 4 This is an enlarged view of the structure of the buffer anti-clogging module.

[0032] Figure 5 This is a schematic diagram of the DEP electrode layer. Detailed Implementation

[0033] The present invention will be described in detail below with reference to the embodiments. However, the implementation of the present invention is not limited thereto. Obviously, the embodiments described below are only some embodiments of the present invention. For those skilled in the art, other similar embodiments can be obtained without creative effort and all fall within the protection scope of the present invention.

[0034] Example 1

[0035] like Figure 1 As shown, a microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology includes a PDMS cover layer 1, an ITO electrode layer 2, and a glass substrate layer 3; the ITO electrode layer 2 is deposited on the glass substrate layer 3, and the PDMS cover layer 1 is tightly bonded to the glass substrate layer 3.

[0036] The PDMS cover layer 1 is fabricated using soft lithography to form the substrate layer, which involves several steps including cleaning, pre-baking, spin coating, intermediate baking, exposure, post-baking, and development. Then, PDMS is cast onto the substrate layer.

[0037] The PDMS cover layer 1 includes a DLD sorting module 1-1, a buffer anti-blocking module 1-2, and a DEP array sorting module 1-3 connected in sequence;

[0038] like Figure 2 As shown, the DLD sorting module of this invention includes a DLD sorting channel, a sample inlet 1-1-1, a sheath fluid inlet 1-1-2, two waste fluid outlets 1-1-8 and 1-1-9, and a microalgae cell outflow channel 1-1-5. The sample inlet 1-1-1 merges with the sheath fluid inlet 1-1-2 at the inlet end of the DLD sorting channel via left and right branch pipes. The outlet end of the DLD sorting channel branches into two flow resistance matching channels 1-1-6 and 1-1-7 on the left and right, and the microalgae cell outflow channel 1-1-5 in the middle. The two flow resistance matching channels 1-1-6 and 1-1-7 are connected to the two waste fluid outlets 1-1-8 and 1-1-9, respectively. The DLD sorting channel includes a circular micropillar array 1-1-3 at the front and an equilateral triangular micropillar array 1-1-4 at the rear. The function of the circular micropillar array 1-1-3 is to prevent cells from... The flow channel is clogged by agglomerates. The circular micropillars have a diameter of 50 μm and a spacing of 200 μm. The equilateral triangular micropillar array 1-1-4 is used to sort larger algal cells and smaller bacteria. Larger algal cells will flow out through the microalgal cell outflow channel 1-1-5, while smaller bacteria will flow out through the two upper and lower waste liquid outlets 1-1-8 and 1-1-9. The equilateral triangular micropillar array 1-1-4 is symmetrically distributed around the central axis of the DLD sorting flow channel. The angle between the line connecting each row of equilateral triangular micropillars and the central axis of the DLD sorting flow channel is 2.9°. The side length of the equilateral triangular micropillars is 55 μm, and the spacing between the triangles is 40 μm. The end of the microalgal cell outflow channel 1-1-5 is connected to the buffer anti-clogging module 1-2 through a sudden expansion structure 1-1-10. The purpose is to ensure that the sample cells can enter the buffer anti-clogging module 1-2 evenly after passing through the sudden expansion structure.

[0039] like Figure 4As shown, the buffer anti-clogging module 1-2 includes a fluid channel 1-2-1 and an alternating array of arrows 1-2-2. The arrows are at an angle of 60° and their tips point towards the direction of sample inflow. This is to prevent cell aggregation and sample backflow. The end of the fluid channel is connected to the DEP array sorting module 1-3 through a series of constriction structures 1-2-3 and expansion structures 1-2-4. The purpose of this is to allow sample cells to generate pressure after passing through the constriction structure and then enter the expansion structure, filling the wider outlet channel, so that the sample can enter the DEP array sorting module evenly.

[0040] The DEP array sorting module 1-3 includes eight parallel sorting channels 1-3-1, four waste liquid outlets 1-3-2, and one microalgae cell collection outlet 1-3-3. The eight parallel sorting channels 1-3-1 increase throughput and reduce flow rate, thereby solving the problem of flow rate mismatch between DEP sorting and DLD sorting. Each sorting channel 1-3-1 is connected to the four waste liquid outlets 1-3-2 and the microalgae cell collection outlet 1-3-3 through pipes. The residual bacteria sorted by the DEP array will flow out of the four waste liquid outlets 1-3-2, and the microalgae cells sorted by the eight parallel sorting channels 1-3-1 will converge at the microalgae cell collection outlet 1-3-3.

[0041] The ITO electrode layer 2 is located directly below the eight parallel sorting channels 1-3-1 in the DEP array sorting module 1-3, and is fabricated using an ITO thin film through a wet etching method. Figure 5 As shown, the device includes a comb-shaped ITO electrode 2-1 and a wheat-ear-shaped ITO electrode 2-2. These two sets of electrodes are connected to the positive and negative terminals of an external power supply, respectively. The comb-shaped electrode comprises five elongated electrodes 2-1-2, each 200 μm wide, which are equidistantly distributed. The wheat-ear-shaped electrode comprises four wheat-ear electrodes, which are equidistantly distributed. Each wheat-ear electrode consists of one elongated electrode 2-2-3 and 45 evenly distributed hexagonal electrodes 2-2-2. The long strip electrode 2-1-2 and the wheat ear electrode of electrode 2-1 are arranged alternately to form an electrode pair, located directly below each sorting channel 1-3-1 in the DEP array sorting module 1-3; the tip structure of the hexagonal electrode 2-2-2 in the wheat ear electrode can generate a strong dielectric force. The residual bacteria flow out from the middle waste liquid outlet 1-3-2 under the action of positive dielectric force, and the target microalgae cells flow out from the upper and lower outlets under the action of negative dielectric force, thus achieving high-precision sorting.

[0042] The sorting method using the microalgae and bacteria sorting device based on the combination of deterministic lateral displacement and dielectrophoresis includes the following steps:

[0043] 1. Place 15 mL of the Platychophyton flocculus cell sample solution into a centrifuge and centrifuge at 8000 rpm for 10 min at room temperature. Discard the supernatant.

[0044] 2. Take out the Platychophyton flocculenta cells obtained by centrifugation and put them into a 1.5 mL test tube. Add the prepared PBS buffer with a conductivity of 3000 uS / cm and shake well.

[0045] 3. Inject the prepared E. coli suspension into the prepared Platychoma cell suspension at a concentration ratio of 1:1, shake well, and obtain the Platychoma cell and bacterial suspension sample (hereinafter referred to as the sample solution).

[0046] 4. Draw the prepared sample solution and PBS buffer into two separate syringes and load them onto two syringe pumps.

[0047] 5. Place the fabricated microalgae and bacteria sorting device chip (hereinafter referred to as the chip) based on deterministic lateral displacement and dielectrophoresis technology described in this invention into a plasma cleaner for 2 minutes to ensure the hydrophilicity of the channel.

[0048] 6. Place the cleaned chip under the microscope, adjust the focus, and connect the external power supply and signal generator;

[0049] 7. Slowly inject the prepared PBS buffer into the chip to remove air from the chip, preventing air bubbles from forming on the electrodes and at the edges of the microchannels. At the same time, flush the channels to prevent cells from adhering to the channel walls.

[0050] 8. Adjust the flow rate control of the two syringe pumps, connect the sample solution to the sample inlet 1-1-1 of the chip through the catheter, and connect the PBS buffer to the sheath fluid inlet 1-1-2 of the chip;

[0051] 9. Turn on the syringe pump, and the sample solution and sheath fluid are injected into the chip at a stable flow rate;

[0052] 10. Turn on the power and adjust the voltage and frequency of the signal generator so that the algae cells are subjected to negative mesophoretic force and the E. coli are subjected to positive mesophoretic force. Finally, the pure algae cells flow out from the microalgae cell collection outlet 1-3-3, and the E. coli flow out from each waste liquid outlet, thereby achieving high-throughput and high-precision sorting of microalgae cells and bacteria.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions 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 microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology, characterized in that, It mainly includes a PDMS cover layer (1), an ITO electrode layer (2), and a glass substrate layer (3); the ITO electrode layer (2) is deposited on the glass substrate layer (3), and the PDMS cover layer (1) is tightly bonded to the glass substrate layer (3); the PDMS cover layer (1) includes a DLD sorting module (1-1), a buffer anti-blocking module (1-2), and a DEP array sorting module (1-3) connected in sequence. The buffer anti-clogging module (1-2) includes a fluid channel (1-2-1) and an array of staggered arrows (1-2-2). The angle of the arrows is 30-60°, and the tips of the arrows point in the direction of sample inflow. The end of the fluid channel (1-2-1) is connected to the DEP array sorting module (1-3). The DEP array sorting module (1-3) includes n parallel sorting channels (1-3-1), a waste liquid outlet (1-3-2), and a microalgae cell collection outlet (1-3-3). Each sorting channel (1-3-1) is connected to the waste liquid outlet (1-3-2) and the microalgae cell collection outlet (1-3-3) respectively through pipes, where n is an even number. The ITO electrode layer (2) is located directly below the sorting channel (1-3-1) in the DEP array sorting module (1-3), and includes comb-shaped ITO electrodes (2-1) and wheat-ear-shaped ITO electrodes (2-2). The comb-shaped ITO electrodes (2-1) and wheat-ear-shaped ITO electrodes (2-2) are respectively connected to the positive and negative terminals of the external power supply. The comb-shaped electrodes contain (0.5n+1) elongated electrodes (2-1-2), which are equidistantly distributed. The wheat-ear-shaped electrode contains 0.5n wheat-ear electrodes, which are equidistantly distributed. Each wheat-ear electrode consists of a long strip electrode (2-2-3) and 10-100 evenly distributed hexagonal electrodes (2-2-2). The long strip electrode (2-1-2) and the wheat-ear electrodes of the comb-shaped ITO electrode (2-1) are arranged alternately to form an electrode pair, located directly below each sorting channel (1-3-1) in the DEP array sorting module (1-3), where n is an even number.

2. The sorting device according to claim 1, characterized in that, The DLD sorting module (1-1) includes a DLD sorting channel, a sample inlet (1-1-1), a sheath fluid inlet (1-1-2), waste liquid outlet I (1-1-8), and waste liquid outlet II (1-1-9), as well as a microalgae cell outflow channel (1-1-5). The sample inlet (1-1-1) merges with the sheath fluid inlet (1-1-2) via left and right branch pipes at the inlet end of the DLD sorting channel. The outlet end of the DLD sorting channel branches into flow resistance matching channel I (1-1-6) and flow resistance matching channel II (1-1-7), and the central microalgae cell outflow channel (1-1-5). Flow channel I (1-1-6) and flow resistance matching flow channel II (1-1-7) are connected to waste liquid outlet I (1-1-8) and waste liquid outlet II (1-1-9) respectively; the DLD sorting flow channel includes a circular micro-pillar array (1-1-3) at the front and an equilateral triangular micro-pillar array (1-1-4) at the rear. The equilateral triangular micro-pillar array (1-1-4) is symmetrically distributed around the central axis of the DLD sorting flow channel. The angle between the line connecting each row of equilateral triangular micro-pillars and the central axis of the DLD sorting flow channel is 1-10°; the end of the microalgae cell outflow channel (1-1-5) is connected to the buffer anti-clogging module (1-2).

3. The sorting device according to claim 2, characterized in that, The circular micropillars have a diameter of 50 μm and a spacing of 200 μm; the equilateral triangular micropillars have a side length of 55 μm and a triangle spacing of 40 μm.

4. The sorting device according to claim 2, characterized in that, The end of the microalgal cell outflow channel (1-1-5) is connected to the buffer anti-blocking module (1-2) through a sudden expansion structure (1-1-10).

5. The sorting device according to claim 1, characterized in that, The end of the fluid channel (1-2-1) is connected to the DEP array sorting module (1-3) through a series of constriction structures (1-2-3) and expansion structures (1-2-4).

6. The sorting device according to claim 1, characterized in that, The width of the elongated electrode is 200 μm.

7. A sorting method using the microalgae and bacteria sorting device based on deterministic lateral displacement and dielectrophoresis technology as described in any one of claims 1-6, characterized in that, Includes the following steps: A. Prepare the sample solution and sheath fluid for the microalgal cells to be separated; B. Use a syringe pump to inject the sample solution into the sample inlet (1-1-1) at a certain speed, and inject the sheath fluid into the sheath fluid inlet (1-1-2) at a certain speed; C. After passing through the DLD sorting module, the microalgae cells enter the buffer anti-clogging module (1-2) through the microalgae cell outflow channel (1-1-5), and the bacteria will flow out from waste liquid outlet I (1-1-8) and waste liquid outlet II (1-1-9); D. Microalgae cells pass through the staggered arrow-shaped array in the buffer and anti-blocking module (1-2), which effectively prevents cell aggregation and backflow, and enter the DEP array sorting module (1-3). E. In the DEP array sorting module (1-3), microalgal cells are subjected to negative mesophoretic force and flow out from the microalgal cell collection outlet (1-3-3); bacteria are subjected to positive mesophoretic force and flow out from the waste liquid outlet (1-3-2), thus achieving high-throughput and high-precision sorting of microalgal cells and bacteria.

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