Photo-thermal cavitation micro-nano needle tip array, manufacturing method thereof and cell transfection method

Abstract

The invention discloses a photo-thermal cavitation micro-nano needle tip array, a manufacturing method thereof and a cell transfection method, and belongs to the field of cell transfection, the photo-thermal cavitation micro-nano needle tip array comprises a plurality of needle tip structures arranged on a substrate, the needle tip structures are located on the same side of the substrate, each needle tip structure comprises a main body, the main body is conical, an optical cladding and a photo-thermal layer are arranged on the outer surface of the main body, the photo-thermal layer is located at the top of the main body, the optical cladding is located between the photo-thermal layer and the main body, the main body is completely wrapped by the optical cladding and the photo-thermal layer, the photo-thermal layer is made of a photo-thermal material, the needle tip of the needle tip structure is in close contact with cells, and when photo-thermal cavitation bubbles are generated in the photo-thermal layer, the trepanning efficiency is higher, so that the cell transfection efficiency is improved; the positioning trepanning of cells is controlled through the arrangement and density of the needle tip array, and the functions of drug delivery, transfection, physical treatment and the like are achieved.

Description

Technical Field

[0001] The invention relates to the technical field of cell transfection, and in particular to a photothermal cavitation micro-nano needle tip array, a method for manufacturing the photothermal cavitation micro-nano needle tip array, and a cell transfection method. Background Art

[0002] Cell transfection technology is an important method in life science and medical research. It is to introduce foreign genes, drugs or other biological molecules into cells. Cell transfection technology is of great significance in gene therapy, cell therapy, disease model establishment and basic biological research. At present, the methods of cell transfection mainly include physical methods (electroporation, microinjection), chemical methods (liposome transfection, polymer transfection) and biological methods (such as viral vector transfection). Traditional cell transfection methods often cause a certain degree of damage to cells, affecting cell survival and function.

[0003] Micro-nano structure substrates, as a perfect combination of biotechnology and nanotechnology, have shown unprecedented potential and broad application prospects in the field of cell transfection in recent years, and have also provided new ideas and methods for cell transfection. Micro-nano structure substrates can achieve fine regulation of the cell transfection process by precisely controlling the shape, size and arrangement of the micro-nano structures on their surface. This controllability not only helps to improve the transfection efficiency, but also achieves the repeatability of the transfection process, making it possible for the standardization and large-scale application of cell transfection. In order to achieve efficient cell transfection, it is necessary to design and prepare substrates with specific micro-nano structures. These structures include nanopores, nanopillars, nanofibers, etc. By changing the size, shape and arrangement of these structures, the cell transfection process can be finely regulated.

[0004] However, the existing published cell transfection technologies based on micro-nano structures all adopt a passive method, that is, relying on the shape, size and arrangement of the micro-nano structure to affect the cells to achieve the purpose of transfection. Although there are active transfection micro-nano structures in the prior art, the existing structure size is comparable to the size of the cell, the number of opening points is limited, the opening efficiency is low, and the function is relatively single. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, one of the objectives of the present invention is to provide a photothermal cavitation micro-nano needle tip array with high opening efficiency and capable of positioning and opening holes in cells.

[0006] In order to overcome the shortcomings of the prior art, the second purpose of the present invention is to provide a photothermal cavitation micro-nano needle tip array manufacturing method with high opening efficiency and the ability to position and open holes in cells.

[0007] In order to overcome the deficiencies of the prior art, the third object of the present invention is to provide a cell transfection method with high pore opening efficiency and the ability to perform targeted pore opening on cells.

[0008] One of the purposes of the present invention is achieved by the following technical solution:

[0009] A photothermal cavitation micro-nano needle tip array includes a substrate, which is made of a light-transmitting material. The photothermal cavitation micro-nano needle tip array also includes a plurality of needle tip structures arranged on the substrate, and the plurality of needle tip structures are located on the same side of the substrate. Each of the needle tip structures includes a main body, which is conical, and an optical cladding and a photothermal layer are provided on the outer surface of the main body. The photothermal layer is located on the top of the main body, and the optical cladding is located between the photothermal layer and the substrate. The optical cladding and the photothermal layer completely wrap the main body, and the photothermal layer is made of a photothermal material.

[0010] Furthermore, a plurality of the needle tip structures are arranged in an array.

[0011] Furthermore, the substrate is made of glass, and the thickness of the substrate is 0.05 mm-1 mm.

[0012] Furthermore, the main body is made of photoresist, and the height of the photoresist is 1 to 50 μm.

[0013] Furthermore, the main body is conical.

[0014] Furthermore, the optical cladding has a thickness of 50 to 500 nm, and the photothermal layer has a height of 0.2 to 5 μm.

[0015] The second object of the present invention is achieved by adopting the following technical solution:

[0016] A method for manufacturing a photothermal cavitation micro-nano needle tip array, used to manufacture any of the above-mentioned photothermal cavitation micro-nano needle tip arrays, comprising the following steps:

[0017] Spin coating photoresist on the surface of the substrate;

[0018] Performing spot exposure on the photoresist;

[0019] Developing the exposed photoresist to obtain a plurality of bodies formed by photocured photoresists, wherein the plurality of bodies form an array structure;

[0020] Performing optical coating on the surfaces of the plurality of bodies to form an optical cladding;

[0021] Spin-coat a protective layer on the substrate that is thicker than the main body;

[0022] The protective layer is etched by a controllable etching process to expose the tip portion of the array structure;

[0023] The optical cladding of the exposed tip portion is removed by wet etching to expose the tip of the main body;

[0024] The exposed tip of the main body is modified with photothermal material by chemical modification to form a photothermal layer;

[0025] The remaining protective layer is removed to form the photothermal cavitation micro-nano needle tip array.

[0026] Furthermore, the point exposure of the photoresist adopts any one of ultraviolet lithography, electron beam lithography or two-photon 3D printing.

[0027] The third object of the present invention is achieved by adopting the following technical solution:

[0028] A cell transfection method, implemented by any one of the above-mentioned photothermal cavitation micro-nano needle tip arrays, comprises the following steps:

[0029] The excitation light is irradiated from the bottom of the substrate;

[0030] The excitation light propagates from the bottom of the main body toward the tip and converges on the photothermal layer at the tip of the needle tip structure;

[0031] The photothermal layer generates photothermal cavitation bubbles under the action of the excitation light;

[0032] The cavitation bubbles cause the cell membrane at the top of the needle tip structure to rupture instantly;

[0033] The nucleic acid plasmid in the solution is introduced into the cells to achieve cell transfection.

[0034] Compared with the prior art, the photothermal cavitation micro-nano needle tip array of the present invention includes a plurality of needle tip structures arranged on a substrate, and the plurality of needle tip structures are located on the same side of the substrate. Each needle tip structure includes a main body, which is conical, and an optical cladding and a photothermal layer are provided on the outer surface of the main body. The photothermal layer is located on the top of the main body, and the optical cladding is located between the photothermal layer and the main body. The optical cladding and the photothermal layer completely wrap the main body, and the photothermal layer is made of photothermal material. The needle tip of the needle tip structure is in close contact with the cells, and the opening efficiency of the photothermal layer is higher when photothermal cavitation bubbles occur, thereby improving the cell transfection efficiency. The positioning and opening of the cells are controlled by the arrangement and density of the needle tip array, and the array has the functions of drug administration, transfection, physical therapy, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 It is a schematic diagram of the structure of the photothermal cavitation micro-nano needle tip array of the present invention;

[0036] Figure 2 for Figure 1 Schematic diagram of the fabrication process of the photothermal cavitation micro-nano tip array;

[0037] Figure 3 for Figure 1 Schematic diagram of the usage status of the photothermal cavitation micro-nano tip array.

[0038] In the figure: 10, substrate; 20, needle tip structure; 21, main body; 22, optical cladding; 23, photothermal layer; 30, excitation light; 40, cavitation bubble. DETAILED DESCRIPTION

[0039] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0040] It should be noted that when a component is referred to as being "fixed to" another component, it may be directly on the other component or there may be another intermediate component through which it is fixed. When a component is considered to be "connected to" another component, it may be directly connected to the other component or there may be another intermediate component at the same time. When a component is considered to be "set on" another component, it may be directly set on the other component or there may be another intermediate component at the same time. The terms "vertical", "horizontal", "left", "right" and similar expressions used herein are for illustrative purposes only.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0042] See also Figure 1 A photothermal cavitation micro-nano needle tip array includes a substrate 10 and a plurality of needle tip structures 20 disposed on the substrate 10. The plurality of needle tip structures 20 are distributed in an array on one side of the substrate 10. Two adjacent needle tip structures 20 are connected so that there is no gap between the two adjacent needle tip structures 20.

[0043] The substrate 10 is made of glass, and the thickness of the substrate 10 is 0.05 mm-1 mm. Preferably, the thickness of the substrate 10 is 0.17 mm. Specifically, the substrate 10 is made of quartz glass or other optical glass with high ultraviolet transmittance.

[0044] Each needle tip structure 20 is conical. In this embodiment, each needle tip structure 20 is conical. The needle tip structure 20 includes a main body 21, an optical cladding 22 and a photothermal layer 23. The optical cladding 22 and the photothermal layer 23 are arranged on the surface of the main body 21 to form a film layer. The optical cladding 22 is located at the bottom and middle of the main body 21, and the photothermal layer 23 is located at the top of the main body 21. The optical cladding 22 and the photothermal layer 23 completely cover the surface of the main body 21, and the optical claddings 22 of two adjacent needle tip structures 20 intersect.

[0045] Specifically, the main body 21 is formed on the substrate 10, and the number of the main body 21 is multiple, and the multiple main bodies 21 are distributed in an array. Each main body 21 is conical, and the main body 21 is formed by spin coating, exposure, and development of photoresist. Specifically, the photoresist is SU-8 photoresist. The height of the main body 21 is 1 to 50 μm.

[0046] The optical cladding 22 is made of silicon dioxide (SiO2) material, and has a thickness of 50-500 nm. The optical cladding 22 is formed by sputtering or electron beam evaporation of the silicon dioxide material.

[0047] The photothermal layer 23 is a noble metal nanoparticle. Specifically, the photothermal layer 23 is the top of the optical cladding 22 is corroded and removed, and then the exposed SU-8 needle tip is immersed in a commercial nanoparticle solution, the material is gold or silver, the particle size is 2 to 50nm, the concentration is 1 to 10nM, and the immersion time is 1 to 12 hours to obtain a noble metal nanoparticle modified exposed tip. The photothermal layer 23 height is 0.2 to 5μm.

[0048] See also Figure 2 The present application also discloses a method for manufacturing a photothermal cavitation micro-nano needle tip array, which is used to manufacture the above-mentioned photothermal cavitation micro-nano needle tip array, comprising the following steps:

[0049] Spin coating photoresist on the surface of substrate 10;

[0050] spot-exposing the photoresist;

[0051] Developing the exposed photoresist to obtain a plurality of main bodies 21 formed of photocured photoresists, wherein the plurality of main bodies 21 form an array structure;

[0052] Performing optical coating on the surfaces of the plurality of main bodies 21 to form an optical cladding 22;

[0053] Spin coating a protective layer on the substrate 10 with a thickness higher than that of the main body;

[0054] The protective layer is etched by a controllable etching process to expose the tip portion of the array structure;

[0055] The optical cladding 22 of the exposed tip portion is removed by wet etching to expose the tip of the main body 21;

[0056] The photothermal material is modified on the tip of the exposed main body 21 by a chemical modification method to form a photothermal layer 23;

[0057] The remaining protective layer is removed to form a photothermal cavitation micro-nano needle tip array.

[0058] The specific step of spin coating the photoresist on the surface of the substrate 10 is: select optical glass with excellent optical properties as the substrate 10. Preferably, the substrate material is quartz glass with a thickness of 0.05 to 1 mm. Preferably, a quartz sheet with a thickness of 0.17 mm or other optical glass with high ultraviolet transmittance is selected. The substrate 10 is a flat plate structure. The substrate 10 is ultrasonically cleaned in acetone and isopropanol for 10 to 20 minutes, and after being dried with nitrogen, the photoresist adhesion layer is treated with hexamethyldisilane (HMDS); spin-coat SU-8 photoresist with a thickness of 1 to 50 μm, and then heat it on a hot plate at 65°C for 0 to 5 minutes, and then heat it on a hot plate at 95°C for 5 to 15 minutes.

[0059] The specific steps of spot exposure of the photoresist are: photolithography on a SUSS MA6 double-sided alignment photolithography machine, or using a 100kV electron beam photolithography machine, or two-photon 3D printing technology to spot expose the SU-8 photoresist, and the exposure dose is 300 to 1000mJ / cm2. The exposed SU-8 photoresist is heated on a hot plate at 65°C for 1 to 5 minutes, and then heated on a hot plate at 95°C for 2 to 8 minutes.

[0060] The exposed photoresist is developed to obtain a main body 21 formed by multiple photocured photoresists, and the steps of forming an array structure of multiple main bodies 21 are specifically as follows: the exposed SU-8 photoresist is developed, the developer is polyethylene glycol dimethacrylate (PEGMA) solution, and the development time is to obtain a needle tip array of photocured SU-8 material, that is, the array structure formed by the main body 21.

[0061] The specific steps of performing optical coating on the surfaces of multiple main bodies 21 to form the optical cladding 22 are: sputtering or electron beam evaporating a layer of silicon dioxide (SiO2) material with a thickness of 50 to 500 nm on the surface of the SU-8 needle tip array (array structure formed by the main body 21).

[0062] The step of spin coating a protective layer thicker than the main body on the substrate 10 is specifically as follows: spin coating a protective layer with a thickness of 2 to 55 μm, the material of which is a conventional polymer such as PMMA and PS.

[0063] The protective layer is etched by a controllable etching process to expose the tip portion of the array structure. Specifically, the steps are as follows: etching the polymer-covered substrate by an oxygen plasma process with a power of 200 to 400 W and a processing time of 30 to 90 minutes to expose the tip portion (optical cladding 22) of the array structure; or etching by a reactive ion etching (RIE) process to expose the tip portion (optical cladding 22) of the array structure; the height of the exposed portion of the tip of the optical cladding 22 is controlled to be between 0.2 and 5 μm.

[0064] The optical cladding 22 of the exposed tip is corroded away by wet etching to expose the tip of the main body 21. The specific steps are: immersing the exposed optical cladding 22 in a 10% HF solution or BOE solution for 1 to 5 minutes, corroding and removing the cladding material SiO2 of the exposed part, and obtaining the exposed SU-8 material needle tip part (main body 21).

[0065] The steps of modifying the exposed tip of the main body 21 with photothermal materials by chemical modification to form the photothermal layer 23 are as follows: subjecting the substrate to an oxygen plasma treatment process with a power of 100 to 200 W and a treatment time of 30 to 60 seconds; subsequently immersing it in a 1 to 10 mg / mL polyethyleneimine solution for 5 to 60 minutes; then washing it thoroughly with deionized water; immersing the substrate in a commercial nanoparticle solution made of gold or silver with a particle size of 2 to 50 nm and a concentration of 1 to 10 nM for 1 to 12 hours to obtain an exposed tip (photothermal layer 23) modified with precious metal nanoparticles.

[0066] The steps of removing the remaining protective layer and forming a photothermal cavitation micro-nano needle tip array are as follows: immersing the above-mentioned substrate in a mixed solution of 0.1-1mM chloroauric acid and 0.2-4mM hydroxylamine hydrochloride in a mixing ratio of 1:1, reacting for 1-10 minutes, and repeating 1-10 times; immersing the above-mentioned substrate in an acetone solution for 1-5 minutes to remove the protective layer material, and obtaining a micro-nano needle tip array structure with only the tip part having photothermal material.

[0067] See also Figure 3 The present application also discloses a cell transfection method, which is implemented by the above-mentioned photothermal cavitation micro-nano needle tip array, comprising the following steps:

[0068] The excitation light is irradiated from the bottom of the substrate 10;

[0069] The excitation light propagates from the bottom of the main body 21 toward the tip, and converges on the photothermal layer 23 at the tip of the needle tip structure 20;

[0070] The photothermal layer 23 generates photothermal cavitation bubbles under the action of the excitation light;

[0071] The cavitation bubbles cause the cell membrane on the top of the needle tip structure 20 to rupture instantly;

[0072] The nucleic acid plasmid in the solution is introduced into the cells to achieve cell transfection.

[0073] Compared with the prior art, the photothermal cavitation micro-nano needle tip array of the present invention includes a plurality of needle tip structures 20 arranged on a substrate 10, and the plurality of needle tip structures 20 are located on the same side of the substrate 10, and each needle tip structure 20 includes a main body 21, and the main body 21 is conical, and an optical cladding 22 and a photothermal layer 23 are provided on the outer surface of the main body 21, and the photothermal layer 23 is located on the top of the main body 21, and the optical cladding 22 is located between the photothermal layer 23 and the substrate 10, and the optical cladding 22 and the photothermal layer 23 completely wrap the main body 21, and the photothermal layer 22 is made of photothermal material, and the needle tip of the needle tip structure 20 is in close contact with the cell, and the opening efficiency of the photothermal layer 22 is higher when photothermal cavitation bubbles occur, thereby improving the cell transfection efficiency; the positioning and opening of the cell are controlled by the arrangement and density of the needle tip array 20, and it has the functions of drug administration, transfection, physical therapy, etc.

[0074] The above embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which are equivalent modifications and improvements made to the above embodiments based on the essential technology of the present invention, and all of them belong to the protection scope of the present invention.

Claims

1. A photothermal cavitation micro-nano tip array, comprising a substrate made of a light-transmitting material, characterized in that: The photothermal cavitation micro-nano needle tip array also includes a plurality of needle tip structures arranged on the substrate, wherein the plurality of needle tip structures are located on the same side of the substrate, and each of the needle tip structures includes a main body, wherein the main body is conical, and an optical cladding and a photothermal layer are provided on the outer surface of the main body, wherein the photothermal layer is located on the top of the main body, and the optical cladding is located between the photothermal layer and the substrate, and the optical cladding and the photothermal layer completely wrap the main body, and the photothermal layer is made of a photothermal material.

2. The photothermal cavitation micro-nano tip array according to claim 1, characterized in that: The plurality of needle tip structures are arranged in an array.

3. The photothermal cavitation micro-nano tip array according to claim 1, characterized in that: The substrate is made of glass, and the thickness of the substrate is 0.05 mm-1 mm.

4. The photothermal cavitation micro-nano tip array according to claim 1, characterized in that: The main body is made of photoresist, and the height of the photoresist is 1-50 μm.

5. The photothermal cavitation micro-nano tip array according to claim 1, characterized in that: The main body is conical.

6. The photothermal cavitation micro-nano tip array according to claim 1, characterized in that: The optical cladding layer has a thickness of 50 to 500 nm, and the photothermal layer has a height of 0.2 to 5 μm.

7. A method for manufacturing a photothermal cavitation micro-nano needle tip array, used to manufacture the photothermal cavitation micro-nano needle tip array according to any one of claims 1 to 6, characterized in that: The following steps are involved: Spin coating photoresist on the surface of the substrate; Performing spot exposure on the photoresist; Developing the exposed photoresist to obtain a plurality of bodies formed by photocured photoresists, wherein the plurality of bodies form an array structure; Performing optical coating on the surfaces of the plurality of bodies to form an optical cladding; Spin-coat a protective layer on the substrate that is thicker than the main body; The protective layer is etched by a controllable etching process to expose the tip portion of the array structure; The optical cladding of the exposed tip portion is removed by wet etching to expose the tip of the main body; The exposed tip of the main body is modified with photothermal material by chemical modification to form a photothermal layer; The remaining protective layer is removed to form the photothermal cavitation micro-nano needle tip array.

8. The method for manufacturing a photothermal cavitation micro-nano needle tip array according to claim 1, characterized in that: The point exposure of the photoresist adopts any one of ultraviolet lithography, electron beam lithography or two-photon 3D printing.

9. A cell transfection method, implemented by the photothermal cavitation micro-nano needle tip array according to any one of claims 1 to 6, characterized in that: The following steps are involved: The excitation light is irradiated from the bottom of the substrate; The excitation light propagates from the bottom of the main body toward the tip and converges on the photothermal layer at the tip of the needle tip structure; The photothermal layer generates photothermal cavitation bubbles under the action of the excitation light; The cavitation bubbles cause the cell membrane at the top of the needle tip structure to rupture instantly; The nucleic acid plasmid in the solution is introduced into the cells to achieve cell transfection.

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