Cell sorting biochip based on dynamic dielectrophoresis and control method thereof

Abstract

According to the dynamic dielectrophoresis-based cell sorting biochip and the control method thereof, a multi-layer modular design is adopted, a metal electrode array layer is processed on the surface of a transparent substrate layer, and electrodes in the metal electrode array layer are in positive / negative cross arrangement in an array manner; a photoresist micro-well layer with a micro-well array is arranged on the surface of the metal electrode array layer, a soft material fluid layer is bonded on the surface of the photoresist micro-well layer, a cavity is formed, and liquid drops enter the cavity and migrate to a micro-well array area; after redundant liquid drops outside the micro-trap array area are removed, target liquid drops are anchored in micro-traps of the photoresist micro-trap layer through the positive dielectrophoresis effect; the non-target liquid drops are discharged, and finally the target liquid drops are collected; the micro-trap array on the chip and the electronic control technology of the metal electrode array layer are utilized, high-precision and high-flux separation of liquid drops is achieved, efficiency is improved, and the micro-trap array chip can be widely applied to the fields of high-flux screening, single cell analysis, enzyme evolution, drug discovery and the like.

Description

Technical Field

[0001] The present invention relates to droplet microfluidics technology, and particularly to a cell sorting biochip based on dielectrophoresis and its control method. Background Art

[0002] In recent years, with the rapid development of precision medicine and biotechnology, the demand for single-cell biological analysis has shown an explosive growth. Compared with traditional population cell analysis, single-cell technology can reveal cell heterogeneity and provide key information for disease mechanism research, personalized treatment, and the development of new therapies. In this context, the importance of single-cell sorting technology has become increasingly prominent. By precisely separating target cell populations, it lays the foundation for downstream analysis and clinical applications (such as CAR-T cell therapy and enzyme engineering optimization), and plays an irreplaceable role in disease diagnosis, treatment monitoring, and targeted drug development.

[0003] However, there are still significant bottlenecks in existing single-cell sorting technologies, restricting their wide application:

[0004] 1. Micromanipulation technology relies on capillary pipetting and microscope operation, with extremely low throughput and time-consuming.

[0005] 2. Flow cytometry can achieve relatively high throughput, but the instrument cost is high (about tens of millions of yuan), and it relies on pre-labeled antibodies, limiting the research on the functions of living cells.

[0006] 3. Immunomagnetic bead sorting may cause abnormal cell functions due to the interference of magnetic markers, resulting in insufficient sorting purity.

[0007] 4. Laser-related technologies (such as laser capture microdissection and laser-induced transfer) have complex equipment, a risk of laser damage to cells, and are mostly applicable to fixed tissues. Patent application No. CN202110946566.3 discloses a multifunctional cell sorting device based on a laser system, which uses laser cutting-assisted sorting, but the problem is that it is only applicable to fixed cells and has a risk of damaging cells.

[0008] 5. Although optical tweezer-assisted sorting can achieve non-contact operation, the system integration is low and the throughput is limited. Patent application No. CN202010060731.0 discloses a coupling device and a microscopy-optical tweezer single-cell sorting system and its sorting method, which uses a microscope combined with optical tweezer-assisted sorting, but the problem is that the sorting speed is very slow.

[0009] The above defects make it difficult for existing methods to balance the core requirements of high throughput, high precision, low cost, and maintaining cell viability. Therefore, developing a new type of efficient, precise, and living-cell-compatible sorting technology has become a key breakthrough point for promoting biomedical research and clinical transformation. Summary of the Invention

[0010] To overcome the above-mentioned problems of the prior art, the present invention aims to provide a cell sorting biochip based on dielectrophoresis and its control method. Single cells are encapsulated into water-in-oil monodisperse droplets, and then through external fluorescence detection and the electrocontrol of the microtrap array and metal electrode array layer of the chip of the present invention, high-precision and high-throughput droplet (cell) sorting is achieved, greatly improving the screening efficiency, and being able to accurately identify and select target droplets (cells), which can be widely applied to fields such as high-throughput screening, single-cell analysis, enzyme evolution, and drug discovery.

[0011] To achieve the above object, the technical solution of the present invention is as follows:

[0012] A cell sorting biochip based on dielectrophoresis, the chip adopts a multi-layer modular design, including a transparent base layer 1, a metal electrode array layer 2 is processed on the surface of the transparent base layer 1, and the electrodes 2-1 in the metal electrode array layer 2 are arranged in a positive / negative cross pattern in an array manner. A photoresist microtrap layer 3 with a microtrap array is arranged on the surface of the metal electrode array layer 2, a soft material fluid layer 4 is bonded to the surface of the photoresist microtrap layer 3, and a cavity 4-3 is formed between the photoresist microtrap layer 3 and the soft material fluid layer 4. The outer surfaces of the transparent base layer 1 and the soft material fluid layer 4 are encapsulated by an acrylic fixture layer 5, and through holes are provided at both ends of the soft material fluid layer 4 and the acrylic fixture layer 5.

[0013] One end of the through hole is the soft layer opening 4-1 and the acrylic fixture opening 5-1 as the droplet inlet, and the other end is the soft layer outlet 4-2 and the acrylic fixture outlet 5-2 as the droplet outlet.

[0014] The spacing between each electrode 2-1 matches the microtrap spacing of the photoresist microtrap layer 3.

[0015] The diameter of the microtrap of the photoresist microtrap layer 3 is 8-15% larger than the diameter of the target droplet, and the depth of the microtrap is the same as the diameter of the target droplet.

[0016] The soft material fluid layer 4 is made of an elastic polymer material through a casting process, and the materials include polydimethylsiloxane PDMS, thermoplastic elastomer TPE / TPU, photocurable elastomer / resin or other elastic polymers; the two ends of the surface of the soft material fluid layer 4 are cut with a soft layer opening 4-1 and a soft layer outlet 4-2 as the inlet and outlet of the droplet, and are bonded to both ends of the photoresist microtrap layer 3 through surface activation treatment, and the inner wall of the cavity 4-3 is subjected to hydrophobic modification treatment.

[0017] When the transparent base layer 1 is made of glass or a transparent polymer material, it includes polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), or cycloolefin polymer / copolymer (COP / COC).

[0018] The material of the metal electrode array layer 2 includes gold (Au), platinum (Pt), titanium (Ti) / chromium (Cr)+gold (Au) multi-layer system, aluminum (Al), copper (Cu), indium tin oxide (ITO), or indium gallium zinc oxide (IGZO).

[0019] The material of the photoresist microtrap layer 3 includes SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative photoresist, or dry film photoresist.

[0020] A control method for a cell sorting biochip based on dynamic dielectrophoresis includes the following steps:

[0021] When the sorting chip is working, first generate a water-in-oil monodisperse droplet of the biological sample to be sorted, and add it through the opening 4-1 of the soft layer and the opening 5-1 of the acrylic fixture into the cavity 4-3 formed between the photoresist microtrap layer 3 and the soft material fluid layer 4. Use the two-phase density difference to drive the droplet to migrate directionally to the microtrap array area of the photoresist microtrap layer 3; after removing the excess droplets outside the microtrap array area, screen the target droplets in the microtrap array area based on optical detection, apply an alternating electric field to the electrode 2-1 corresponding to the target droplet, and use the positive dielectrophoresis effect to anchor the target droplet in the microtrap of the photoresist microtrap layer 3; through the synergistic action of chip spatial pose regulation and hydrodynamics, make the non-target droplets break away from the microtrap and discharge from the outlet 4-2 of the soft layer and the outlet 5-2 of the acrylic fixture, and finally achieve the purpose of collecting the target droplets.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. During sorting, first generate a water-in-oil monodisperse droplet, so that the reagent loss is small, it is compatible with all types of cells, and can separate single cells while maintaining cell viability.

[0024] 2. Due to the design of the metal electrode array layer 2 and the photoresist microtrap layer 3, through the dynamic cooperation of dielectrophoretic force and microfluidic structure, tens of thousands of droplets can be sorted at one time, realizing the efficient sorting of droplets. The sorting speed is comparable to that of a flow cytometer, but the cost is much lower than that of a flow cytometer. At the same time, it is compatible with the flexible adaptation of different-sized droplets and biological samples.

[0025] 3. The present invention is combined with optical detection, can be adapted to scenarios such as single-cell secreted protein detection, CRISPR-edited cell screening, exosome capture, etc., and supports multi-level sorting.

[0026] As described above, the present invention utilizes the microtrap array on the chip and the electronic control technology of the metal electrode array layer to successfully achieve high-precision and high-throughput sorting of droplets (or cells), significantly improving the efficiency of the screening process while ensuring the accurate identification and selection of target droplets (or cells). The present invention has broad application potential in multiple fields, including important fields such as high-throughput screening, single-cell analysis, enzyme evolution research, and drug discovery. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Schematic diagram of the droplet encapsulating cell device.

[0028] Figure 2 Side view of the overall structure of the present invention.

[0029] Figure 3 Schematic diagram of the principle of the present invention, where Figure 3 (A) in it is a schematic diagram of droplet injection, Figure 3 (B) in it is a schematic diagram of control after droplet injection, Figure 3 (C) in it is a schematic diagram of removing excess droplets.

[0030] Figure 4 Electronically controlled target droplet diagram of the present invention, where Figure 4 (A) in it is a schematic diagram of optical recognition of the target droplet, Figure 4 (B) in it is a diagram of the corresponding circuit opening situation.

[0031] Figure 5 Schematic diagram of the droplet in the microtrap, where Figure 5 (A) in it is a schematic diagram of the droplet entering the microtrap and removing excess droplets, Figure 5 (B) in it is a cross-sectional view of the droplet control circuit.

[0032] In the figure, transparent base layer 1, metal electrode array layer 2, electrode 2-1, photoresist microtrap layer 3; soft material fluid layer 4; soft layer opening 4-1; soft layer outlet 4-2; cavity 4-3; acrylic fixture layer 5; acrylic fixture opening 5-1; acrylic fixture outlet 5-2 DETAILED DESCRIPTION OF THE INVENTION

[0033] The present invention will be described in detail below with reference to the accompanying drawings.

[0034] Referring to Figure 2, a cell sorting biochip based on dielectrophoresis, the chip adopts a multi-layer modular design, including a transparent substrate layer 1. On the surface of the transparent substrate layer 1, a metal electrode array layer 2 is formed by micro-nano processing. The electrodes 2-1 in the metal electrode array layer 2 are arranged in a positive / negative cross pattern in an array manner. Above the metal electrode array layer 2, photoresist is spin-coated and lithographed. After ultraviolet exposure and development, an open micro-well array matching the size of the target droplet is formed, which is the photoresist micro-well layer 3. A soft material fluid layer 4 is bonded to the upper surface of the photoresist micro-well layer 3. A cavity 4-3 is formed between the photoresist micro-well layer 3 and the soft material fluid layer 4. The outer surfaces of the transparent substrate layer 1 and the soft material fluid layer 4 are encapsulated by an acrylic fixture layer 5. Through holes are provided at both ends of the soft material fluid layer 4 and the acrylic fixture layer 5. One end is the soft layer opening 4-1 and the acrylic fixture opening 5-1 as the droplet inlet, and the other end is the soft layer outlet 4-2 and the acrylic fixture outlet 5-2 as the droplet outlet.

[0035] The spacing of each electrode 2-1 matches the micro-well spacing of the photoresist micro-well layer 3 to adapt to the droplet manipulation requirements.

[0036] The micro-well diameter of the photoresist micro-well layer 3 is 10% larger than the target droplet diameter, and the micro-well depth is the same as the target droplet diameter to optimize the droplet residence stability.

[0037] The soft material fluid layer 4 is made of an elastic polymer material through a casting process. The materials include polydimethylsiloxane PDMS, thermoplastic elastomer TPE / TPU, photocurable elastomer / resin, or other elastic polymers. The two ends of the surface of the soft material fluid layer 4 are cut with a soft layer opening 4-1 and a soft layer outlet 4-2 as the inlet and outlet of the droplet, and are bonded to both ends of the photoresist micro-well layer 3 through surface activation treatment. The inner wall of the cavity 4-3 is hydrophobically modified to inhibit droplet adsorption.

[0038] The transparent substrate layer 1 is made of glass or a transparent polymer material, including polyethylene terephthalate PET, polycarbonate PC, polymethyl methacrylate PMMA, or cycloolefin polymer / copolymer COP / COC.

[0039] The material of the metal electrode array layer 2 includes gold (Au), platinum Pt, titanium Ti / chromium Cr + gold Au multi-layer system, aluminum (Al), copper Cu, indium tin oxide ITO, or indium gallium zinc oxide IGZO.

[0040] The material of the photoresist micro-well layer 3 includes SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative photoresist, or dry film photoresist.

[0041] The preparation method of the sorting chip includes: forming a negative mold structure of microchannels and micro-wells on a silicon-based template by lithography, mixing a prepolymer and a curing agent and pouring the mixture onto the template for curing and forming, and obtaining a photoresist micro-well layer 3 with a preset channel size through precision cutting; a soft material fluid layer 4 is bonded to the surface of the photoresist micro-well layer 3, and then the soft material fluid layer 4 is bonded to the transparent substrate layer 1 containing the metal electrode array layer 2 through a high-precision alignment system, and a leak-free encapsulation is achieved through an acrylic fixture layer 5 and an elastic sealing component.

[0042] Based on the above control method of a cell sorting biochip based on dielectrophoresis, the method includes the following steps:

[0043] Referring to Figure 1 , when the sorting chip works, first generate a water-in-oil monodisperse droplet from the biological sample to be sorted. Specifically: perform trypsin digestion and medium termination treatment on adherent cells, then remove the supernatant by medium-speed centrifugation, resuspend the cells with a buffer solution containing a protein stabilizer and a non-ionic surfactant, adjust the cell density to the medium-high concentration range and filter to remove agglomerates to obtain a monodisperse suspension; the droplet diameter is designed to be 5-10 times the cell size, and the two-phase flow rate ratio is regulated to generate uniform droplets to avoid mechanical damage and meet the requirement of the single-cell encapsulation rate of Poisson distribution;

[0044] Referring to Figure 3 , add the water-in-oil monodisperse droplet through the opening 4-1 of the soft layer and the opening 5-1 of the acrylic fixture into the cavity 4-3 formed between the photoresist micro-well layer 3 and the soft material fluid layer 4, and use the two-phase density difference to drive the droplet to migrate directionally to the micro-well array area of the photoresist micro-well layer 3, referring to Figure 5 ; after removing the excess droplets outside the micro-well array area, referring to Figure 4 , screen the target droplets in the micro-well array area based on optical detection, apply an alternating electric field to the electrode 2-1 corresponding to the target droplet, and use the positive dielectrophoresis effect to anchor the target droplet in the micro-well of the photoresist micro-well layer 3; through the synergistic action of chip spatial pose regulation and hydrodynamics, make the non-target droplets break away from the micro-well and discharge from the outlet 4-2 of the soft layer and the outlet 5-2 of the acrylic fixture, and finally achieve the purpose of collecting the target droplets.

[0045] The screening of the present invention first generates single-cell droplets through a two-phase microfluidic device and injects them into the chip, and uses the two-phase density difference to make the droplets migrate directionally to the micro-well array area; then identifies the target droplets through fluorescence labeling, and generates a positive dielectrophoresis force by applying an alternating electric field to the corresponding electrode unit to achieve selective anchoring; after removing the uncaught droplets by hydrodynamic flushing, switch the buffer solution and turn off the electric field to release the target droplets, and finally achieve a sorting purity of >95% and a cell survival rate of >90%.

Claims

1. A cell sorting biochip based on dielectrophoresis, characterized in that, The chip adopts a multi-layer modular design, including a transparent base layer (1). A metal electrode array layer (2) is processed on the surface of the transparent base layer (1). The electrodes (2-1) in the metal electrode array layer (2) are arranged in a positive / negative cross pattern in an array manner. A photoresist micro-well layer (3) with a micro-well array is arranged on the surface of the metal electrode array layer (2). A soft material fluid layer (4) is bonded to the surface of the photoresist micro-well layer (3), and a cavity (4-3) is formed between the photoresist micro-well layer (3) and the soft material fluid layer (4). The outer surfaces of the transparent base layer (1) and the soft material fluid layer (4) are encapsulated by an acrylic fixture layer (5), and through holes are provided at both ends of the soft material fluid layer (4) and the acrylic fixture layer (5).

2. The cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that, For the said through holes, one end is a soft layer opening (4-1) and an acrylic fixture opening (5-1) as the droplet inlet, and the other end is a soft layer outlet (4-2) and an acrylic fixture outlet (5-2) as the droplet outlet.

3. The cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that, The spacing between each electrode (2-1) matches the micro-well spacing of the photoresist micro-well layer (3).

4. A cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that The micro-well diameter of the photoresist micro-well layer (3) is 8-15% larger than the target droplet diameter, and the micro-well depth is the same as the target droplet diameter.

5. The cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that, The soft material fluid layer (4) is made of an elastic polymer material through a casting process. The materials include polydimethylsiloxane PDMS, thermoplastic elastomer TPE / TPU, photocurable elastomer / resin, or other elastic polymers. Soft layer openings (4-1) and a soft layer outlet (4-2) are cut at both ends of the surface of the soft material fluid layer (4) as the inlet and outlet of the droplets. Bonding is achieved with both ends of the photoresist micro-well layer (3) through surface activation treatment, and the inner wall of the cavity (4-3) is subjected to hydrophobic modification treatment.

6. The cell sorting biochip based on dielectrophoresis according to claim 1, wherein, The transparent base layer (1) is made of glass or a transparent polymer material, including polyethylene terephthalate PET, polycarbonate PC, polymethyl methacrylate PMMA, or cycloolefin polymer / copolymer COP / COC.

7. A cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that, The material of the metal electrode array layer (2) includes gold (Au), platinum Pt, titanium Ti / chromium Cr + gold Au multi-layer system, aluminum (Al), copper Cu, indium tin oxide ITO, or indium gallium zinc oxide IGZO.

8. A cell sorting biochip based on dielectrophoresis according to claim 1, characterized in that, The material of the photoresist micro-well layer (3) includes SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative photoresist, or dry film photoresist.

9. A control method for a cell sorting biochip based on dielectrophoresis according to any one of claims 1 to 8, characterized in that, It includes the following steps: When the sorting chip works, first generate a water-in-oil monodisperse droplet of the biological sample to be sorted, and add it through the soft layer opening (4-1) and the acrylic fixture opening (5-1) into the cavity (4-3) formed between the photoresist micro-well layer (3) and the soft material fluid layer (4). Utilize the two-phase density difference to drive the droplet to migrate directionally to the micro-well array area of the photoresist micro-well layer (3). After removing the excess droplets outside the micro-well array region, the target droplets in the micro-well array region are screened based on optical detection. An alternating electric field is applied to the electrode (2-1) corresponding to the target droplets, and the target droplets are anchored in the micro-wells of the photoresist micro-well layer (3) by using the positive dielectrophoresis effect. Through the synergistic effect of chip spatial pose regulation and hydrodynamics, the non-target droplets are detached from the micro-wells and discharged from the outlet (4-2) of the soft layer and the outlet (5-2) of the acrylic fixture, ultimately achieving the purpose of collecting the target droplets.

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