Cell sorting biochip based on dynamic dielectrophoresis and control method thereof

Abstract

The application discloses a cell sorting biochip based on dynamic dielectrophoresis and a control method thereof, adopts a multi-layer modular design, and is characterized in that a transparent substrate layer is provided with a metal electrode array layer on a surface thereof, electrodes in the metal electrode array layer are arranged in an array mode and are positive / negative electrode crossed arrangement, a photoresist microwell layer with a microwell array is arranged on the surface of the metal electrode array layer, a soft material fluid layer is bonded to the surface of the photoresist microwell layer and cavities are formed, droplets enter the cavities and migrate to the microwell array area, after removing the excess droplets except the microwell array area, the target droplets are anchored in the microwells of the photoresist microwell layer by using the positive dielectrophoresis effect, the non-target droplets are discharged, and finally the purpose of collecting the target droplets is achieved; the application utilizes the microwell array on the chip and the electronic control technology of the metal electrode array layer, realizes high-precision and high-throughput sorting of the droplets, improves the efficiency, and can be widely applied to the fields of high-throughput screening, single cell analysis, enzyme evolution, drug discovery and the like.

Description

Technical Field

[0001] This invention relates to droplet microfluidics, and particularly to a cell sorting biochip based on dynamic dielectrophoresis and its control method. Background Technology

[0002] In recent years, with the rapid development of precision medicine and biotechnology, the demand for single-cell bioanalysis has exploded. Compared with traditional population cell analysis, single-cell technology can reveal cellular heterogeneity, providing crucial information for disease mechanism research, personalized treatment, and the development of novel therapies. Against this backdrop, 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), playing an irreplaceable role, especially in disease diagnosis, treatment monitoring, and targeted drug development.

[0003] However, existing single-cell sorting technologies still have significant bottlenecks that limit their widespread application:

[0004] 1. Micromanipulation techniques rely on capillary pipetting and microscope operation, which have extremely low throughput and are time-consuming;

[0005] 2. Although flow cytometry can achieve high throughput, the instrument is expensive (around ten million yuan) and relies on pre-labeled antibodies, which limits the study of live cell function.

[0006] 3. Immunomagnetic bead sorting may result in abnormal cell function and insufficient sorting purity due to interference from magnetic markers.

[0007] 4. Laser-related technologies (such as laser capture microdissection and laser-induced transfer) are complex to equipment, pose a risk of laser-induced cell damage, and are mostly applicable to fixed tissues. Patent application CN202110946566.3 discloses a multifunctional cell sorting device based on a laser system, which uses laser cutting to assist sorting. However, the problem is that it is only applicable to fixed cells and has the risk of cell damage.

[0008] 5. Although optical tweezers-assisted sorting can achieve non-contact operation, the system integration is low and the throughput is limited. Patent application CN202010060731.0 discloses a coupling device and a micro-optical tweezers single-cell sorting system and its sorting method, which uses a microscope combined with optical tweezers-assisted sorting. However, the problem is that the sorting speed is very slow.

[0009] The aforementioned shortcomings make it difficult for existing methods to simultaneously meet the core requirements of high throughput, high precision, low cost, and preservation of cell viability. Therefore, developing a novel, efficient, precise, and live-cell compatible sorting technology has become a key breakthrough for promoting biomedical research and clinical translation. Summary of the Invention

[0010] To overcome the aforementioned problems in the prior art, the present invention aims to propose a cell sorting biochip based on dynamic dielectrophoresis and its control method. This method encapsulates a single cell within a water-in-oil monodisperse droplet, and then, through external fluorescence detection combined with the electronic control of the chip's micro-trap array and metal electrode array layer, achieves high-precision, high-throughput droplet (cell) sorting. This significantly improves screening efficiency and enables precise identification and selection of target droplets (cells), making it widely applicable in high-throughput screening, single-cell analysis, enzyme evolution, drug discovery, and other fields.

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

[0012] A cell sorting biochip based on dynamic dielectrophoresis is disclosed. The chip adopts a multi-layer modular design, including a transparent substrate layer 1, a metal electrode array layer 2 formed on the surface of the transparent substrate layer 1, electrodes 2-1 in the metal electrode array layer 2 arranged in an array of positive and negative electrodes, a photoresist micro-well layer 3 with a micro-well array is formed 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 substrate layer 1 and the soft material fluid layer 4 are encapsulated by an acrylic clamping layer 5, and through-holes are provided at both ends of the soft material fluid layer 4 and the acrylic clamping layer 5.

[0013] The through-type inlet and outlet has a soft layer opening 4-1 and an acrylic clamp opening 5-1 at one end as a droplet inlet, and a soft layer outlet 4-2 and an acrylic clamp outlet 5-2 at the other end as a droplet outlet.

[0014] The spacing between each electrode 2-1 is matched with the spacing between the microwells in the photoresist microwell layer 3.

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

[0016] The soft material fluid layer 4 is made of elastic polymer material through a molding process. The material includes polydimethylsiloxane (PDMS), thermoplastic elastomer (TPE / TPU), photocurable elastomer / resin, or other elastic polymers. The soft material fluid layer 4 has soft layer openings 4-1 and soft layer outlets 4-2 cut at both ends of its surface as droplet inlets and outlets. It is bonded to both ends of the photoresist micro-trap layer 3 through surface activation treatment. The inner wall of the cavity 4-3 is hydrophobically modified.

[0017] The transparent substrate 1 is made of glass or a transparent polymer material, including polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), or cyclic olefin polymers / copolymers (COP / COC).

[0018] The metal electrode array layer 2 is made of materials including gold (Au), platinum (Pt), titanium (Ti / chromium Cr + gold Au) multilayer system, aluminum (Al), copper (Cu), indium tin oxide (ITO) or indium gallium zinc oxide (IGZO).

[0019] The photoresist microwell layer 3 is made of materials including SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative resist, or dry film photoresist.

[0020] The control method for a cell sorting biochip based on dynamic dielectrophoresis, as described above, includes the following steps:

[0021] During the sorting chip's operation, the biological sample to be sorted is first generated into water-in-oil monodisperse droplets, which are then added to the cavity 4-3 formed between the photoresist micro-well layer 3 and the soft material fluid layer 4 through the soft layer opening 4-1 and the acrylic clamp opening 5-1. The droplets are driven to migrate directionally to the micro-well array region of the photoresist micro-well layer 3 by utilizing the two-phase density difference. After removing excess droplets outside the micro-well array region, the target droplets within 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 droplet, and the target droplet is anchored in the micro-well of the photoresist micro-well layer 3 by utilizing the positive mesophoresis effect. Through the synergistic effect of chip spatial pose control and hydrodynamics, non-target droplets are detached from the micro-well and discharged from the soft layer outlet 4-2 and the acrylic clamp outlet 5-2, ultimately achieving 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 water-in-oil monodisperse droplets. This reduces reagent consumption, is compatible with all cell types, and allows for the separation of individual cells while maintaining cell viability.

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

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

[0026] As described above, this invention utilizes electronic control technology based on on-chip micro-well arrays and metal electrode array layers to successfully achieve high-precision and high-throughput sorting of droplets (or cells), significantly improving the efficiency of the screening process while ensuring accurate identification and selection of target droplets (or cells). This invention has broad application potential in multiple fields, including high-throughput screening, single-cell analysis, enzyme evolution research, and drug discovery. Attached Figure Description

[0027] Figure 1 Schematic diagram of a device that encapsulates cells with droplets.

[0028] Figure 2 This is a side view of the overall structure of the present invention.

[0029] Figure 3 This is a schematic diagram illustrating the principle of the present invention, wherein... Figure 3 (A) in the diagram is a schematic of droplet injection. Figure 3 (B) in the diagram is a control diagram after droplet injection. Figure 3 (C) in the diagram is a schematic diagram of the removal of excess droplets.

[0030] Figure 4 This is a diagram of the electrically controlled target droplet of the present invention, wherein... Figure 4 (A) in the diagram is a schematic diagram of optical recognition of the target droplet. Figure 4 (B) in the diagram shows the corresponding circuit's on / off state.

[0031] Figure 5 This is a schematic diagram of a droplet in a micro-trap, where Figure 5 (A) in the diagram shows the droplet entering the micro-trap and the removal of excess droplets. Figure 5 (B) in the diagram is a cross-sectional view of the droplet control circuit.

[0032] In the figure, there is a transparent substrate layer 1, a metal electrode array layer 2, an electrode 2-1, a photoresist micro-trap layer 3, a soft material fluid layer 4, a soft layer opening 4-1, a soft layer outlet 4-2, a cavity 4-3, an acrylic clamp layer 5, an acrylic clamp opening 5-1, and an acrylic clamp outlet 5-2. Detailed Implementation

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

[0034] Reference Figure 2A cell sorting biochip based on dynamic dielectrophoresis is disclosed. The chip employs a multi-layer modular design, including a transparent substrate layer 1. A metal electrode array layer 2 is formed on the surface of the transparent substrate layer 1 using micro / nano fabrication. Electrodes 2-1 in the metal electrode array layer 2 are arranged in an array with alternating positive and negative electrodes. Photoresist is spin-coated onto the metal electrode array layer 2. After ultraviolet exposure and development, the photoresist forms an open micro-well array matching the size of the target droplet, which is the photoresist micro-well layer 3. A soft material fluid layer 4 is bonded to the upper surface of the 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 clamp layer 5. The soft material fluid layer 4 and the acrylic clamp layer 5 are provided with through-holes at both ends. One end is the soft layer opening 4-1 and the acrylic clamp opening 5-1 as a droplet inlet, and the other end is the soft layer outlet 4-2 and the acrylic clamp outlet 5-2 as a droplet outlet.

[0035] The spacing between each electrode 2-1 is matched with the spacing between the microwells in the photoresist microwell layer 3 to meet the droplet manipulation requirements.

[0036] The microwell diameter of the photoresist microwell layer 3 is 10% larger than the diameter of the target droplet, and the depth of the microwell is the same as the diameter of the target droplet, in order to optimize the droplet's residence stability.

[0037] The soft material fluid layer 4 is made of elastic polymer material through a molding process. The material includes polydimethylsiloxane (PDMS), thermoplastic elastomer (TPE / TPU), photocurable elastomer / resin, or other elastic polymers. The soft material fluid layer 4 has soft layer openings 4-1 and soft layer outlets 4-2 cut at both ends of its surface as droplet inlets and outlets. It is bonded to both ends of the photoresist microtrap 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 1 is made of glass or a transparent polymer material, including polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), or cyclic olefin polymers / copolymers (COP / COC).

[0039] The metal electrode array layer 2 is made of materials including gold (Au), platinum (Pt), titanium (Ti / chromium Cr + gold Au) multilayer system, aluminum (Al), copper (Cu), indium tin oxide (ITO) or indium gallium zinc oxide (IGZO).

[0040] The photoresist microwell layer 3 is made of materials including SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative resist, or dry film photoresist.

[0041] The method for fabricating the sorting chip includes: forming a negative mold structure of microchannels and microwells on a silicon-based template by photolithography; mixing a polymer prepolymer with a curing agent and pouring it into the template and curing it; obtaining a photoresist microwell layer 3 with a preset channel size by precision cutting; bonding a soft material fluid layer 4 to the surface of the photoresist microwell layer 3; then bonding the soft material fluid layer 4 to a transparent substrate layer 1 containing a metal electrode array layer 2 by a high-precision alignment system; and achieving leak-free encapsulation by using an acrylic clamp layer 5 and an elastic sealing component.

[0042] The control method for a cell sorting biochip based on dynamic dielectrophoresis, as described above, includes the following steps:

[0043] Reference Figure 1 When the sorting chip is working, the biological samples to be sorted are first generated into water-in-oil monodisperse droplets. Specifically, adherent cells are digested with trypsin and the culture medium is terminated. Then, the supernatant is removed by medium-speed centrifugation. The cells are resuspended in a buffer containing protein stabilizers and nonionic surfactants. The cell density is adjusted to a medium-high concentration range and clumps are removed by filtration to obtain a monodisperse suspension. The droplet diameter is designed to be 5-10 times the cell size. The flow rate ratio of the two phases is controlled to generate uniform droplets to avoid mechanical damage and meet the single-cell encapsulation rate requirements of Poisson distribution.

[0044] Reference Figure 3 A water-in-oil monodisperse droplet is added through the soft layer opening 4-1 and the acrylic fixture opening 5-1 into the cavity 4-3 formed between the photoresist microwell layer 3 and the soft material fluid layer 4. The droplet is then driven to migrate directionally to the microwell array region of the photoresist microwell layer 3 by utilizing the two-phase density difference. Figure 5 After removing excess droplets outside the micro-trap array region, refer to Figure 4 The target droplets in the micro-trap array region are screened based on optical detection. An alternating electric field is applied to the electrode 2-1 corresponding to the target droplet. The target droplet is anchored in the micro-trap of the photoresist micro-trap layer 3 by using the positive mesophoresis effect. Through the synergistic effect of chip spatial pose control and hydrodynamics, non-target droplets are detached from the micro-trap and discharged from the soft layer outlet 4-2 and the acrylic clamp outlet 5-2, ultimately achieving the purpose of collecting the target droplets.

[0045] The screening method of this invention first generates single-cell droplets through a two-phase microfluidic device and injects them into a chip. The density difference between the two phases is used to orient the droplets to the micro-trap array region. Then, the target droplets are identified by fluorescent labeling, and an alternating electric field is applied to the corresponding electrode unit to generate positive mesoelectrophoresis force to achieve selective anchoring. After removing uncaptured droplets by hydrodynamic rinsing, the buffer is switched and the electric field is turned off to release the target droplets, ultimately achieving a sorting purity of >95% and a cell viability of >90%.

Claims

1. A cell sorting biochip based on dynamic dielectrophoresis, characterized in that, The chip adopts a multi-layer modular design, including a transparent substrate layer (1), a metal electrode array layer (2) is processed on the surface of the transparent substrate layer (1), the electrodes (2-1) in the metal electrode array layer (2) are arranged in an array of positive and negative electrodes, a photoresist micro-well layer (3) with micro-well array is provided 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 substrate layer (1) and the soft material fluid layer (4) are encapsulated by an acrylic clamp layer (5), and the soft material fluid layer (4) and the acrylic clamp layer (5) are provided with through entrances and exits at both ends.

2. The cell sorting biochip based on dynamic dielectrophoresis according to claim 1, characterized in that, The through-type inlet and outlet has a soft layer opening (4-1) and an acrylic clamp opening (5-1) at one end as a droplet inlet, and a soft layer outlet (4-2) and an acrylic clamp outlet (5-2) at the other end as a droplet outlet.

3. The cell sorting biochip based on dynamic dielectrophoresis according to claim 1, characterized in that, The spacing between each electrode (2-1) is matched with the spacing between the microwells in the photoresist microwell layer (3).

4. A cell sorting biochip based on dynamic 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 diameter of the target droplet, and the depth of the micro-well is the same as the diameter of the target droplet.

5. A cell sorting biochip based on dynamic dielectrophoresis according to claim 1, characterized in that, The soft material fluid layer (4) is made of elastic polymer material by molding process. The material includes polydimethylsiloxane PDMS, thermoplastic elastomer TPE / TPU, photocurable elastomer / resin or other elastic polymer. The soft material fluid layer (4) has soft layer openings (4-1) and soft layer outlets (4-2) cut at both ends of its surface as droplet inlets and outlets. It is bonded to both ends of the photoresist micro-trap layer (3) through surface activation treatment. The inner wall of the cavity (4-3) is hydrophobically modified.

6. A cell sorting biochip based on dynamic dielectrophoresis according to claim 1, characterized in that, The transparent substrate layer (1) is made of glass or a transparent polymer material, including polyethylene terephthalate (PET), polycarbonate (PC), polymethyl methacrylate (PMMA), or cyclic olefin polymers / copolymers (COP / COC).

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

8. A cell sorting biochip based on dynamic dielectrophoresis according to claim 1, characterized in that, The photoresist microwell layer (3) is made of materials including SU-8 photoresist, AZ series photoresist, S1813 photoresist, epoxy resin-based, acrylic-based negative adhesive, or dry film photoresist.

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

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