Micro-trap capture-based single-cell transfection microfluidic chip

Abstract

The utility model discloses a single cell transfection micro -fluidic chip based on micro trap capture relates to cell transfection technical field, it includes cover and base, cover top is equipped with fluid inlet channel and fluid outlet channel, and the conductive glass is placed in fluid inlet channel and fluid outlet channel all, and the conductive glass bottom all are contacted with base; The micro trap array is equipped on the base, and the micro trap array is used for capturing cells, and the conductive glass is connected with positive electrode, negative electrode respectively, and the cell is captured to the electric transfection of power on -off through the conductive glass. The utility model sets up U type micro trap array at the base, increases the electric transfection flux of cell, and will cell-foreign gene capture to the same area, greatly increases the contact probability of cell and foreign gene, is favorable to increase the import amount, import speed and controllability of foreign gene, promotes the expression of target foreign gene, to realize the efficient controllable electric transfection of cell, can significantly improve the efficiency and controllability of cell electric transfection.

Description

Technical Field

[0001] This invention relates to a microfluidic chip for single-cell transfection based on microtrap capture, belonging to the field of cell transfection technology. Background Technology

[0002] In recent years, research and applications in the biomedical field have received increasing attention, particularly in translational medicine and gene therapy. Breaking down the barriers between the "laboratory" and "clinical" has become a significant trend, and gene therapy is one of the key methods to achieve this breakthrough. Especially in chimeric antigen receptor T (CAR-T) cell immunotherapy, cell transfection technology plays a crucial role. By introducing exogenous genes into target cells, products generated from these genes can treat and correct diseases caused by gene mutations, achieving the goal of a fundamental cure.

[0003] Currently, there are various cell transfection technologies on the market, such as viral transfection, hydrodynamic transfection, and electrotransfection. In CAR-T cell therapy, viral transfection technology is still the main application method, but it also faces some significant problems: (1) the production process of viral vectors is complex and costly, and is accompanied by significant biosafety risks; (2) CAR genes are usually randomly inserted into the host cell genome, which may interfere with normal gene expression and bring potential carcinogenic risks; (3) viral vectors have limitations in delivering large nucleic acid molecules and it is difficult to deliver multiple drugs or therapeutic molecules simultaneously. In contrast, hydrodynamic transfection relies on the deformation of cells when passing through narrow channels to create perforations, allowing nucleic acid substances to enter the cells through these pores, but it faces problems such as low transfection throughput, poor controllability, and low delivery efficiency, which limits its widespread application.

[0004] Recent studies have shown that electroporation technology has demonstrated significant advantages in transfecting difficult-to-transfect cells (such as primary T cells, stem T cells, and NK cells), emerging as a potential alternative to viral transfection. However, current electroporation techniques still face challenges in handling high throughput, cell viability, and transfection controllability, particularly in electroporation regulation, cell population manipulation, and nucleic acid delivery efficiency. With increasing pressure in immunotherapy drug production and rising demands in cell therapy research, improving the throughput, controllability, and efficiency of electroporation has become particularly urgent. Utility Model Content

[0005] The purpose of this invention is to overcome the shortcomings of the existing technology and provide a microfluidic chip for single-cell transfection based on microtrap capture, which can significantly improve the efficiency and controllability of cell electrotransfection and provide theoretical and technical support for the preparation of CAR-T cells.

[0006] To achieve the above objectives, this utility model employs the following technical solution: This invention provides a single-cell transfection microfluidic chip based on microtrap capture, including a cover plate and a base plate. A fluid inlet channel and a fluid outlet channel are provided on the top of the cover plate. Conductive glass is placed in both the fluid inlet channel and the fluid outlet channel, and the bottom of the conductive glass is in contact with the base plate. The base is provided with a micro-trap array, which is used to capture cells. The conductive glass is connected to the positive electrode and the negative electrode respectively, and the captured cells are electrotransfected by passing electricity through the conductive glass.

[0007] Furthermore, the microtrap array is composed of multiple rows of microtrap units, each row of microtrap units comprising multiple microtrap units, with the microtrap units at corresponding positions in adjacent rows of microtrap units arranged alternately.

[0008] Furthermore, the microtrap unit has a symmetrical structure and includes a U-shaped groove that acts to capture cells. A flow channel is provided below the U-shaped groove for cell positioning and introduction.

[0009] Furthermore, the micro-trap array is located in the middle of the base, with a length of 9.8 mm and a width of 8.5 mm.

[0010] Furthermore, the opening side of the U-shaped groove is close to the fluid inlet channel, and the flow channel side is close to the fluid outlet channel.

[0011] Furthermore, the cover plate has a height of 2mm, a length of 15mm, and a width of 12mm; the base has a height of 0.5mm, a length of 15mm, and a width of 12mm.

[0012] Furthermore, the fluid inlet channel and fluid outlet channel are 7 mm long and 1.5 mm wide; the wire glass is 4 mm high, 6 mm long, and 1 mm wide.

[0013] Furthermore, the outer wall of the conductive glass away from the microtrap array is in contact with the inner wall of the fluid inlet channel and fluid outlet channel away from the microtrap array.

[0014] Compared with the prior art, the beneficial effects achieved by this utility model are as follows: This invention consists of a cover plate and a base. Fluid inlet and outlet channels are provided on both sides of the upper part of the cover plate. Conductive glass in the fluid inlet and outlet channels is connected to electrodes. A micro-trap array is provided on the base. The micro-trap array is used to capture cells and trap cells and foreign genes in the same area, which greatly increases the contact probability between cells and foreign genes. This is beneficial to increase the amount, speed and controllability of foreign gene introduction and promote the expression of target foreign genes. When the conductive glass is energized, it is beneficial to break down the cell membrane of the captured cells, thereby allowing the foreign genes to be introduced and electrotransfected, realizing high-throughput and high-efficiency cell electrotransfection. It is applicable to biomedical fields such as cell immunotherapy, tumor vaccine preparation, and targeted drug delivery. Attached Figure Description

[0015] Figure 1 This is a schematic diagram of the structure of a single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention; Figure 2 This is an exploded view of the single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention. Figure 3 This is a schematic cross-sectional view of a single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention. Figure 4 This is a top view of the cover plate structure of a single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention. Figure 5 This is a top view of the substrate of a single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention. Figure 6 This is a schematic diagram of the microtrap unit of a single-cell transfection microfluidic chip based on microtrap capture in one embodiment of the present invention; In the figure: 1-base, 2-microtrap array, 3-conductive glass, 4-cover plate, 5-fluid inlet channel, 6-fluid outlet channel. Detailed Implementation

[0016] The present invention will be further described below with reference to the accompanying drawings. The following embodiments are only used to more clearly illustrate the technical solution of the present invention, and should not be used to limit the scope of protection of the present invention. Example 1

[0017] like Figure 1 As shown, this embodiment of the invention provides a single-cell transfection microfluidic chip based on microtrap capture, combined with... Figure 2 It includes a cover plate 4 and a base 1. The cover plate 4 has a fluid inlet channel 5 and a fluid outlet channel 6 on both sides above it, and the base 1 has a micro-trap array 2 in the middle.

[0018] Combination Figure 4 and Figure 5 In this embodiment, the cover plate 4 has a height of 2mm, a length of 15mm, and a width of 12mm, and the base 1 has a height of 0.5mm, a length of 15mm, and a width of 12mm. The fluid inlet channel 5 and the fluid outlet channel 6 have a length of 7mm and a width of 1.5mm.

[0019] like Figure 3 As shown, conductive glass 3 is placed inside both the fluid inlet channel 5 and the fluid outlet channel 6, such that the bottom of the conductive glass 3 is 4 mm high, the length is 6 mm, and the width is 1 mm. In some embodiments, the outer wall of the conductive glass 3 away from the micro-trap array 2 is in contact with the inner wall of the fluid inlet channel 5 and the fluid outlet channel 6 away from the micro-trap array 2.

[0020] The microtrap array 2 is 9.8 mm long and 8.5 mm wide, and consists of multiple rows of microtrap units. Each row contains multiple microtrap units. It is important to note that the microtrap units at corresponding positions in adjacent rows are staggered, such as... Figure 6 As shown.

[0021] The microtrap units have a symmetrical structure, including U-shaped grooves with flow channels below them. Each microtrap unit has an external dimension of approximately 33 μm in length and 15 μm in width, and an internal, elongated guiding channel approximately 8 μm wide and 50 μm long for cell localization and introduction. The ends of the channel have a pair of rounded corner structures with a minimum inner diameter of approximately 10.5 μm and corner radii of 1 μm and 7 μm, respectively, forming an opening structure that allows cells to reside stably. The microtrap units are evenly spaced, and the overall array configuration is compact, ensuring precise single-cell capture and uniformity and stability of subsequent electroporation while maintaining a high-density arrangement. Example 2

[0022] Based on Example 1, this example also provides a single-cell transfection method based on microtrap capture, including the following steps: Preparing cell samples: The cells were thoroughly washed with sterile buffer to remove extracellular impurities and residual culture medium. The cells were then resuspended in a buffer containing the target nucleic acid (in this example, the exogenous gene) and any possible auxiliary components. The cell concentration was typically controlled at 10⁻⁶. 6 The cell / mL ratio is around 100 cells / mL to ensure uniform distribution of single cells in the trap and to avoid the superposition of multiple cells.

[0023] Chip preprocessing: The chip was sterilized with 70 wt% ethanol, the channel surface was washed with sterile saline, and then incubated with a saline solution containing 2% BSA for 30 minutes. The remaining BSA solution was removed, and the chip was washed again with saline.

[0024] Connect the positive and negative electrodes to the electrical signal source, set the high-frequency signal frequency to 1MHz, the pulse signal frequency to 1kHz, the high-frequency voltage to 10V, and the pulse voltage to 1.5V.

[0025] The purpose of pretreatment is to form a stable electrochemical interface on the electrode surface and remove surface oxides to ensure the electrotransfer effect.

[0026] Capture and localization of cellular and nucleic acid substances: The cell-nucleic acid mixture is introduced through the fluid inlet channel 5, and after flowing through the microtrap array 2, the cells are captured and located by the microtrap units.

[0027] Electroporation: The signal is converted from a high-frequency signal to a pulse signal, and then the voltage is set to a specific value (250~300V in this embodiment) to establish a specific electric field distribution. Under this electric field distribution, the cell membrane will rupture locally, allowing nucleic acids on the membrane to be introduced into the cell, thus achieving electrotransfection of the cell.

[0028] It should be noted that during electroporation, the electrical signal used gradually changes from a high-frequency signal to a pulse signal, with the frequency, amplitude, and pulse width of the electric field decreasing sequentially to meet the specific needs of the cells.

[0029] Cell isolation and analysis: After electroporation, the chip is washed with physiological saline, and the cells are collected and centrifuged for separation and analysis, such as observing changes in cell morphology and detecting gene expression levels.

[0030] The above description is only a preferred embodiment of the present utility model. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present utility model, and these improvements and modifications should also be considered within the protection scope of the present utility model.

Claims

1. A single-cell transfection microfluidic chip based on microtrap trapping, characterized in that, The device includes a cover plate and a base. The cover plate has a fluid inlet channel and a fluid outlet channel. Conductive glass is placed in both the fluid inlet channel and the fluid outlet channel. The bottom of the conductive glass is in contact with the base. The base is provided with a micro-trap array, which is used to capture cells. The conductive glass is connected to the positive electrode and the negative electrode respectively, and the captured cells are electrotransfected by passing electricity through the conductive glass.

2. The single-cell transfection microfluidic chip based on microtrap capture according to claim 1, characterized in that, The microtrap array is composed of multiple rows of microtrap units, each row of microtrap units comprising multiple microtrap units, with the microtrap units at corresponding positions in adjacent rows of microtrap units arranged alternately.

3. The single-cell transfection microfluidic chip based on microtrap capture according to claim 2, characterized in that, The microtrap unit has a symmetrical structure and includes a U-shaped groove for capturing cells. A flow channel is provided below the U-shaped groove for cell localization and introduction.

4. The single-cell transfection microfluidic chip based on microtrap capture according to claim 2, characterized in that, The micro-trap array is located in the middle of the base, with a length of 9.8 mm and a width of 8.5 mm.

5. The single-cell transfection microfluidic chip based on microtrap capture according to claim 3, characterized in that, The opening side of the U-shaped groove is close to the fluid inlet channel, and the flow channel side is close to the fluid outlet channel.

6. The single-cell transfection microfluidic chip based on microtrap capture according to claim 1, characterized in that, The cover plate has a height of 2mm, a length of 15mm, and a width of 12mm; the base has a height of 0.5mm, a length of 15mm, and a width of 12mm.

7. The single-cell transfection microfluidic chip based on microtrap capture according to claim 1, characterized in that, The fluid inlet channel and fluid outlet channel are 7 mm long and 1.5 mm wide; the conductive glass is 4 mm high, 6 mm long and 1 mm wide.

8. The single-cell transfection microfluidic chip based on microtrap capture according to claim 1, characterized in that, The outer wall of the conductive glass away from the microtrap array is in contact with the inner wall of the fluid inlet channel and fluid outlet channel away from the microtrap array.

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