Laser-mediated biological macromolecular cell introduction instrument

Abstract

The invention relates to a laser-mediated biomacromolecular cell introduction instrument, and belongs to the crossing field of biomedical engineering and laser technology. Aiming at the problems that high-precision light perforation equipment special for cells is lacked in the existing market and most of commercially available laser devices are high-energy industrial equipment (which is easy to damage the cells), the equipment realizes rapid and safe introduction of biological macromolecules into animal and plant cells through modular design (a laser light source, a high-precision translation platform and a loading type optical filter). The laser module adopts light sources with wavelengths of 532nm, 561nm, 647nm, 808nm, 1064nm and the like, the energy distribution density (0.00-10J / cm < 2 >) is optimized by combining a loading type optical filter, and the light spot distribution uniformity is + / -3%; the stepping precision of the three-dimensional moving platform is + / -10 [mu] m, the effective stroke is 200 mm, the running speed is 1-250 mm / s, programmable adjustment is achieved, batch sample treatment is supported, and the single-time operation time is smaller than or equal to 2 minutes. The device avoids the dependence problem of a traditional microscope system, can be directly used for suspension cells and living tissues, remarkably reduces the cost by 50%, and fills the market blank.

Description

Technical Field

[0001] The present invention belongs to the cross - field of biomedical engineering and laser technology, and relates to a laser - mediated biomacromolecule cell introducer, which is particularly suitable for the efficient and high - precision delivery of macromolecules into animal and plant cells. It forms transient pores on the cell membrane through the uniform action of laser to achieve a controllable delivery effect. Background Art

[0002] With the rapid development of gene editing technology and cell therapy technology, the development of efficient and safe macromolecule cell introduction methods has become the core challenge in biomedical engineering. Traditional electroporation and chemical transfection technologies are difficult to meet the requirements of high - precision cell engineering due to problems such as strong operation toxicity and significant cell damage. As an emerging biophysical delivery method, photo - poration technology, through the mechanism of laser - mediated transient micropore formation on the cell membrane, exhibits unique non - contact operation and spatio - temporal selection advantages. However, its translational application is restricted by the lag in the development of engineering equipment.

[0003] Most existing photo - poration devices are realized by modifying industrial laser equipment. Such systems have significant limitations at the design principle level. Industrial - grade continuous lasers (typical applications include precision drilling of titanium alloys or micro - hole processing of sapphire substrates) usually use kW - level high - energy laser beams and lack the ability to dynamically adjust the pulse energy according to the characteristics of the liquid bilayer structure of the cell membrane. The existing parameter adjustment range cannot meet the biological requirements of the cell safety threshold.

[0004] At the system integration level, the mechanical structure and control system design of commercially available laser processing equipment (such as large - scale engraving machines equipped with five - axis linkage positioning mechanisms) are mainly oriented towards macroscopic material processing. They not only occupy a large operation space but also are difficult to achieve micron - level aperture control and sub - micron - level beam focusing accuracy for cell - level operations. Summary of the Invention

[0005] In view of the above - mentioned technical gaps, the purpose of the present invention is to provide a laser - mediated biomacromolecule cell introducer, which has functions of micro - scale energy regulation, biocompatibility design, and automated operation.

[0006] The present invention adopts the following technical solutions.

[0007] A laser - mediated biomacromolecule cell introducer, comprising: an introducer body; a mechanical motion assembly, the mechanical motion assembly includes an X - axis motion mechanism, a Y - axis motion mechanism, and a Z - axis motion mechanism; a dynamic focusing optical path system; a mechanical motion control system.

[0008] Furthermore, the introducer body includes a base and Y - axis mounting brackets arranged on both sides. The introducer body is made of aluminum 6061 and its surface is treated with fog - silver technology. The Y - axis motion mechanism is arranged on the Y - axis mounting brackets.

[0009] Furthermore, a Y-axis linear module and a Y-axis servo motor are provided on the top surface of the Y-axis mounting bracket. A Y-axis metal bracket is provided on the side top surface of the Y-axis mounting bracket. The Y-axis moving mechanism includes a Y-axis linear module, a Y-axis servo motor, and a cross beam, and the cross beam is fixed to the slide table of the Y-axis linear module.

[0010] Furthermore, an X-axis linear module and an X-axis servo motor are provided on one side surface of the cross beam, and an X-axis metal bracket is provided on the other side surface of the cross beam. The X-axis moving mechanism includes an X-axis linear module, an X-axis servo motor, and a Z-axis fixing table, and the fixing table is fixed to the slide table of the X-axis linear module.

[0011] Furthermore, a Y-axis cable carrier is provided on the surface of the Y-axis metal bracket. The Y-axis cable carrier carries the lines required for the X-axis and Z-axis servo motors. The Y-axis cable carrier cooperates with the Y-axis linear module, and the lines required for the X-axis and Z-axis servo motors cooperate with the Y-axis cable carrier.

[0012] Furthermore, a Z-axis linear module and a Z-axis servo motor are provided on the Z-axis fixing table. The Z-axis moving mechanism includes a Z-axis linear module, a Z-axis servo motor, and a laser mounting table, and the laser mounting table is fixed to the slide table of the Z-axis linear module.

[0013] Furthermore, an X-axis cable carrier is provided on the surface of the X-axis metal bracket. The X-axis cable carrier carries the lines required for the Z-axis servo motor. The X-axis cable carrier cooperates with the X-axis linear module, and the lines required for the Z-axis servo motor cooperate with the X-axis cable carrier.

[0014] Furthermore, a laser is fixed on the laser mounting table. The dynamic focusing optical path system includes a laser, optical path elements, and a focusing component. The optical path elements are respectively connected to the laser and the focusing component, and the focusing component is a 10x long focal length lens.

[0015] Furthermore, a loading type optical filter is carried on the optical path element, and the loading type optical filter is an optical filter with a series of filtering effects.

[0016] Furthermore, the mechanical motion control system is connected to the X-axis moving mechanism, the Y-axis moving mechanism, and the Z-axis moving mechanism through cables. The mechanical motion control system includes a touch screen, an electric control box, start / stop, emergency stop buttons, cables, relays, air switches, terminal blocks, and control software. The control software cooperates with the X-axis moving mechanism, the Y-axis moving mechanism, and the Z-axis moving mechanism.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The laser-mediated biomacromolecule cell introduction instrument provided by the present invention adopts linear modules for the X-axis motion mechanism and the Y-axis motion mechanism, thereby improving the accuracy and efficiency of the X-axis and Y-axis motions. In order to achieve the processing of different types of samples, a Z-axis motion mechanism is set on the Z-axis, and the laser focal length is dynamically adjustable to meet the diversified size requirements of animal and plant cells.

[0019] The laser-mediated biomacromolecule cell introduction instrument provided by the present invention utilizes high pulse energy ultrafast laser for rapid perforation, thereby improving processing efficiency. By loading a telephoto lens for laser-mediated surface perforation, experimental errors caused by focal length changes caused by shaking during mechanical movement are avoided.

[0020] The laser-mediated biomacromolecule cell introduction instrument provided by the present invention has better control over the laser energy during laser-mediated surface perforation, and can achieve dynamic adjustment of 0.00-10.00 J / cm2, thereby avoiding damage to cells caused by high-energy lasers.

[0021] The laser-mediated biomacromolecule cell introduction instrument provided by the present invention has simple process and high processing efficiency, and is conducive to large-scale cell introduction and practical industrial application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the overall structure of the device provided in an embodiment of the present invention, with the laser module, mobile platform, and control system marked.

[0023] Figure 2 This is an enlarged view of the dynamic focusing optical path system provided by an embodiment of the present invention, showing the arrangement of the dynamic focusing lens group.

[0024] Figure 3 A delivery flow chart (positioning→parameter loading→execution) provided in an embodiment of the present invention.

[0025] Figure 4 This is a diagram showing the delivery effect of fluorescently labeled biomacromolecules in lily pollen cells obtained in Example 1.

[0026] Figure 5 This is a diagram showing the delivery effect of fluorescently labeled biomacromolecules in animal Jurket cells obtained in Example 2.

[0027] In the figure: 1 - Importer body; 10 - Base; 11 - Y-axis mounting bracket; 2 - Y-axis movement mechanism; 21 - Y-axis linear module; 22 - Y-axis servo motor; 23 - Y-axis slide; 24 - Y-axis drag chain; 3 - Cross beam; 4 - X-axis movement mechanism; 41 - X-axis linear module; 42 - 1-axis servo motor; 43 - X-axis slide; 44 - X-axis drag chain; 5 - Z-axis movement mechanism; 51 - Z-axis linear module; 52 - Z-axis servo motor; 53 - Z-axis slide; 6 - Dynamic focusing optical path system; 61 - Laser mounting table; 62 - Laser; 63 - Filter loading table; 64 - Loading type filter; 65 - Focusing component; 7 - Mechanical movement control system; 71 - Power on / off key; 72 - Emergency stop button; 73 - Case opener; 74 - Control panel. Detailed implementation mode

[0028] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative work shall fall within the protection scope of the present invention.

[0029] Embodiment 1:

[0030] In this embodiment, the experimental material is lily pollen cells, and the sample thickness is 50 μm. The laser wavelength generated by the laser is 532 nm, the spot diameter after focusing through the lens is 50 μm, and the energy density after weakening the energy through the loading type filter is 0.97 J / cm 2 . This embodiment uses the proposed laser-mediated biological macromolecule cell importer to achieve the import of biological macromolecules into lily pollen cells. Specifically, it includes the following steps:

[0031] Step 1: Take a sample of lily pollen cell solution containing biological macromolecules and place it in a glass slide with a depth of about 0.1 mm. After covering the cover glass, wipe it with lens paper to ensure that the surface of the sample is clean and free of stains. Place the glass slide on the base to ensure that the processing area of the sample is not blocked.

[0032] Step 2: Turn on the laser and the mechanical movement control system. Adjust the working distance of the laser by operating the Z-axis movement mechanism to match the thickness of the glass slide to reach the focal position of the laser. Set the X-axis movement speed of the laser scan to 250 mm / s, the X-axis movement distance to 10 mm, the Y-axis step to 0.03 mm, and the number of Y-axis steps to 333 times. Adjust the laser energy density to 0.97 J / cm through the loading type filter 2 .

[0033] Step 3: Adjust the initial position of the laser and perform scanning through the built-in program of the mechanical movement control system.

[0034] Step 4: After the scanning is completed, take out the sample for subsequent observation.

[0035] See Figure 4 As shown, in this embodiment, the laser-mediated biomacromolecule cell introducer proposed by the present invention is used to achieve rapid and efficient introduction of biomacromolecules into lily pollen cells of plants, and the delivery efficiency is as high as 15%.

[0036] Example 2:

[0037] In this embodiment, the experimental material is animal Jurket cells, and the sample thickness is 10 μm. The laser wavelength generated by the laser is 532 nm, the spot diameter after focusing through the lens is 50 μm, and the energy density after weakening the energy through the loaded filter is 0.08 J / cm 2 . In this embodiment, the proposed laser-mediated biomacromolecule cell introducer is used to achieve large-scale introduction of biomacromolecules into animal Jurket cells. Specifically, it includes the following steps:

[0038] Step 1: Take a sample of animal Jurket cell solution containing biomacromolecules and place it in a sterile 96-well plate. Wipe it with lens paper to ensure that the surface of the sample is clean and free of stains. Place the sterile 96-well plate on the base to ensure that the processing area of the sample is not blocked.

[0039] Step 2: Turn on the laser and the mechanical motion control system. Adjust the working distance of the laser by operating the Z-axis motion mechanism to match the thickness of the sterile 96-well plate to reach the focal position of the laser. Set the laser scanning X-axis motion speed to 250 mm / s, the X-axis motion distance to 150 mm, the Y-axis step to 0.03 mm, and the Y-axis step number to 3600 times. Adjust the laser energy density to 0.08 J / cm 2 .

[0040] Step 3: Adjust the initial position of the laser and perform scanning through the built-in program of the mechanical motion control system.

[0041] Step 4: After the scanning is completed, take out the sample for subsequent observation.

[0042] See Figure 5 As shown, in this embodiment, the laser-mediated biomacromolecule cell introducer proposed by the present invention is used to achieve rapid and efficient introduction of biomacromolecules into animal Jurket cells, and the delivery efficiency is as high as 90%.

Claims

1. A laser-mediated biomacromolecule cell introduction instrument, characterized in that, Including: A laser module (1) that provides laser sources with wavelengths such as 532nm, 561nm, 647nm, 808nm, 1064nm, etc.; A three-dimensional moving platform (2) with a stepping accuracy of ±10μm, an effective stroke of 200mm, and a running speed that can be programmatically adjusted from 1 - 250mm / s, used for sample positioning and batch processing; Dynamic focusing optical path system (3), comprising a lens group (31), a filter mold (32) and a loaded filter (33), which is used to focus the laser on the surface of the target cell and evenly distribute the light spot (light spot uniformity ±3%), and adjust the energy density to 0.00-10.00 J / cm 2 ; A mechanical motion control system (4) integrated with a programmed operation interface (41), supporting the batch processing function for batch samples, with a single operation time ≤ 2 minutes.

2. The laser-mediated biomacromolecule cell introduction instrument according to claim 1, wherein: The laser module (1) provides 532nm, 561nm, 647nm, 808nm, 1064nm laser light sources with an energy of not less than 80μJ.

3. A laser-mediated biomacromolecule cell introducer according to claim 1, characterized in that: The three-dimensional moving platform (2) includes: X / Y-axis linear motors (21) with a resolution of 10μm; A Z-axis ball screw (22) with an effective stroke of 200mm and a repeat positioning error of ±10μm.

4. A laser-mediated biomacromolecule cell introducer according to claim 1, characterized in that: The lens group (31) of the dynamic focusing optical path system (3) is a 10x telephoto lens.

5. A laser-mediated biomacromolecule cell introducer according to claim 1, characterized in that: The loaded filter (33) of the dynamic focusing optical path system (3) is a set of filters for 532nm, 561nm, 647nm, 808nm, 1064nm, and the filtering effects are 5%, 10%, 20%, 32%, 40%, 50%, 63%, 78% respectively. They can be pairwise matched through the filter mold (32) to achieve filtering effects that can be selected within the range of 100, 78, 63, 50, 49.14, 40, 39, 32, 31.5, 31.2, 25.2, 24.96, 20.16, 20, 16, 15.6, 12.8, 12.6, 10, 8, 7.8, 6.4, 6.3, 5, 4, 3.9, 3.2, 3.15, 2.5, 2, 1.6, 1, 0.5%.

6. The laser-mediated biological macromolecule cell introduction instrument according to claim 1, characterized in that: The three-dimensional moving platform (2) is configured with a living tissue fixation fixture (24), and the surface of the fixture is provided with a micropore array (241), which is adapted to suspension cell culture dishes and animal and plant tissue samples.

7. A laser-mediated biomacromolecule cell introduction instrument according to claim 1, characterized in that: The control unit (4) includes a multi-task parallel processing module (42) that synchronously controls the moving platform path and focal length parameters.

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