Plasmonic right pyramid array substrate and in vitro intracellular substance delivery method

Abstract

The present application relates to a kind of plasmonic positive pyramid array substrate and in vitro intracellular material delivery method, by combining various micro-nano processing technology, etching out the positive pyramid array structure of adjustable periodicity and size on monocrystalline silicon surface, further depositing metal layer or other composite material layer to make it have excellent in situ plasmonic performance and plasmonic photothermal conversion performance, finally, cell is seeded on the surface of substrate, and the delivery of material to cell is promoted by the way of light source irradiation.Compared with prior art, the plasmonic array structure prepared in the present application is neat and controllable, the material delivery efficiency is high, and the in vitro cell activity is well maintained, which provides a new idea for broad-spectrum and high-throughput intracellular delivery, and has wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of microstructure fabrication and cell biology technology, and relates to a plasmonic positive pyramid array substrate and a method for delivering substances into cells in vitro using the substrate. Background Technology

[0002] For a long time, researchers have been seeking effective methods for delivering substances such as miRNAs into cells in vitro for in vitro cell experiments and drug efficacy studies. However, due to the relatively large size of substances such as miRNAs, their effective intracellular delivery remains a challenge.

[0003] Currently, virus-based methods are widely used for intracellular miRNA delivery. However, the biosafety and cost of virus-based methods limit their further development. In addition, various non-viral in vitro intracellular miRNA delivery methods have been developed, such as those using liposomes, polymers, peptides, and heterogeneous systems. However, these methods are generally only applicable to specific types of target cells and lack universality, thus limiting their application. Summary of the Invention

[0004] The purpose of this invention is to provide a plasmonic positive pyramid array substrate and a method for in vitro intracellular substance delivery. This invention combines various micro-nano fabrication techniques to etch a periodically and dimensionally controllable positive pyramid array structure on a single-crystal silicon surface, achieving precise control over the pyramid array structure on the single-crystal silicon surface. Subsequently, a metal layer or other composite material layer is deposited to impart excellent in-situ plasmonic properties and plasma-photothermal conversion performance. In vitro cells are seeded on this substrate surface, and irradiation with a light source promotes the delivery of substances into the cells.

[0005] The objective of this invention can be achieved through the following technical solutions:

[0006] A method for fabricating a plasmonic positive pyramid array substrate, comprising the following steps:

[0007] 1) A binder layer and a photoresist layer are sequentially coated on the surface of a silicon wafer. Then, a mask containing a dot matrix pattern is placed on the photoresist layer and exposed in a photolithography device. After exposure, the mask is removed and the uncured parts of the photoresist layer are rinsed with a positive photoresist developer to obtain the photolithographic material.

[0008] 2) A silicon dioxide layer is deposited on the surface of the photolithographic material, and then the remaining photoresist in the photolithographic material is removed to obtain a photoresist-free silicon wafer with a silicon dioxide mask.

[0009] 3) Place the silica mask-coated, adhesive-free silicon wafer in a silicon anisotropic etchant for wet etching to obtain a silicon wafer containing a positive pyramid array structure;

[0010] 4) depositing a metal layer or a composite layer with plasmonic properties on the surface of the silicon wafer containing the positive pyramid array structure, thereby obtaining the plasmonic positive pyramid array substrate.

[0011] Further, in step 1), the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the adhesive is hexamethyldisilazane, and the coating method of the adhesive is spin coating, spraying or vacuum evaporation; the photoresist is a positive photoresist, and the coating method of the photoresist is spin coating or spraying; in the dot array pattern of the mask plate, the diameter of the dot is 1-5 microns, and the distance between adjacent dots is 3-15 microns.

[0012] Further, in step 2), the silicon dioxide layer is deposited by electroplating, evaporation or magnetron sputtering, and the thickness of the silicon dioxide layer is 10-1000 nanometers; the remaining photoresist after the photoetching is removed by soaking in an organic solvent, and the organic solvent includes one or more of acetone, toluene or dichloromethane.

[0013] Further, in step 3), the silicon anisotropic etchant includes one or more of tetramethylammonium hydroxide solution, isopropyl alcohol solution of KOH, EDP etchant or hydrazine hydrate etchant, and the temperature of the wet etching is 50-100℃, and the etching time is 1-120 minutes.

[0014] Further, in step 4), the metal layer or the composite layer with plasmonic properties is deposited by electroplating, evaporation or magnetron sputtering, and the thickness of the deposited layer is 20-500 nanometers.

[0015] The plasmonic positive pyramid array substrate is prepared by the method.

[0016] The plasmonic positive pyramid array substrate is used for delivering a substance into an in vitro cell under laser irradiation.

[0017] The in vitro cell substance delivery method is based on the plasmonic positive pyramid array substrate, and the substance delivery method is as follows: the in vitro cell is adhered to the surface of the substrate, then a dispersion liquid containing the substance to be delivered is added to the substrate, and the substrate is irradiated by laser to deliver the substance into the in vitro cell.

[0018] Preferably, the method of adhering the in vitro cell to the surface of the substrate is as follows: the in vitro cell is adhered to the surface of the substrate at a density of about 1×10 6Density seeding to the base surface of the plasmonic positive pyramid array, then cultured in complete medium (RPMI1640: fetal bovine serum: penicillin-streptomycin = 90:9:1); all adherent cells were fixed with 4% paraformaldehyde at 4 DEG C for 5-30 minutes, then dehydrated with alcohol 3 times at each concentration gradient (50%, 70%, 90%, 100%) for 5 minutes each time. Subsequently, chemical reaction was carried out with hexamethyldisilazane for 10 minutes to remove trace amounts of residual moisture.

[0019] Further, the substance includes genetic material, biological macromolecules, nanoparticles or drug molecules, and the dispersion liquid is nuclease-free water (DEPC) with a concentration of preferably 4-6nM.

[0020] Further, the wavelength of the laser is 400-3000nm, and the irradiation time is 1-10min.

[0021] The method based on membrane damage mainly pierces the cell membrane by physical means such as extrusion, fluid shear, electroporation, microinjection, etc. to promote the delivery of biological molecule carriers and other substances into the in vitro cells. Since the transient pores on the cell membrane can heal themselves, the method has little effect on the vitality of the cells.

[0022] The plasmonic phenomenon is a kind of electromagnetic wave generated by the mutual coupling and collective coherent oscillation of free electrons on the surface of a metal and incident photons. The local evanescent field strength generated by excitation has a great enhancement compared with the incident light. The photothermal effect of the plasmonic structure under proper light irradiation helps to produce transient pores on the cell membrane. The present application realizes instant and effective delivery of substances to in vitro cells through the photothermal effect induced by the plasmonic positive pyramid array. After a short laser irradiation, a transient high temperature can be generated on a single plasmonic pyramid, which is high enough to destroy the cell membrane and promote the delivery of large biological particles such as miRNA into the cells. After the laser irradiation is removed, the temperature of the pyramid will quickly decrease to promote the recovery of the cell membrane. The method in the present application can successfully realize the delivery of substances into in vitro cells within a few minutes, and provides a convenient method for intracellular substance delivery.

[0023] Compared with the prior art, the present application has the following characteristics:

[0024] 1) The present application uses photolithography technology to accurately transfer the pattern of the mask plate to the surface of the silicon wafer, which is suitable for flexible processing of complex patterns, and the mask plate can be reused, thereby reducing the production cost.

[0025] 2) The invention obtains neat and uniform pyramid array structure on the surface of single crystal silicon, and then obtains plasmonic substrate by depositing relevant material, so that the obtained pyramid array structure is arranged neatly and the size is controllable, the method is convenient to process and low in cost, and the substrate can be repeatedly used.

[0026] 3) The invention can realize delivery of substances into cells in vitro within several minutes, has high delivery efficiency, and maintains good cell activity after delivery, thereby providing a new idea for wide-spectrum and high-throughput intracellular delivery in vitro, and having wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 It is a schematic diagram of the process for preparing the plasmonic positive pyramid array substrate in the embodiment;

[0028] Figure 2 It is a scanning electron microscope photo of the plasmonic positive pyramid array substrate prepared in the embodiment;

[0029] Figure 3 It is a schematic diagram of laser scanning when delivering substances into cells in vitro in the embodiment;

[0030] Figure 4 It is a distribution diagram of 5-FAM labeled miR-185 (green fluorescence) in KYSE30 / Taxol cells in the embodiment and the comparative example. DETAILED DESCRIPTION

[0031] The invention will be described in detail below in combination with the drawings and specific embodiments. The embodiment is implemented on the premise of the technical scheme of the invention, and detailed implementation mode and specific operation process are given, but the protection scope of the invention is not limited to the following embodiments.

[0032] The invention provides a preparation method of plasmonic positive pyramid array substrate, which comprises the following steps:

[0033] 1) A binder layer and a photoresist layer are coated on the surface of a silicon wafer in sequence, then a mask plate containing a dot matrix pattern is placed on the photoresist layer, and exposure is carried out in a photoetching device, after the exposure is completed, the mask plate is removed, and the unhardened part of the photoresist layer is washed with positive photoresist developer, to obtain a post-etching material;

[0034] 2) A silicon dioxide layer is deposited on the surface of the post-etching material, then the remaining photoresist in the post-etching material is removed, to obtain a silicon wafer without photoresist with a silicon dioxide mask;

[0035] 3) The silicon wafer without photoresist with a silicon dioxide mask is placed in a silicon anisotropic etching agent for wet etching, to obtain a silicon wafer containing a positive pyramid array structure;

[0036] 4) depositing a metal layer or a composite material layer with plasmonic properties on the surface of the silicon wafer containing the positive pyramid array structure, i.e. obtaining an plasmonic positive pyramid array substrate.

[0037] In step 1), the silicon wafer is a polished silicon wafer, and the thickness of the silicon wafer is 100-1000 microns; the adhesive is hexamethyldisilazane, and the coating method of the adhesive is spin coating, spraying or vacuum volatilization; the photoresist is a positive photoresist, and the coating method of the photoresist is spin coating or spraying; in the dot matrix pattern of the mask plate, the diameter of the dot is 1-5 microns, and the distance between adjacent dots is 3-15 microns.

[0038] In step 2), the silicon dioxide layer is deposited by electroplating, evaporation or magnetron sputtering, and the thickness of the silicon dioxide layer is 10-1000 nanometers; the remaining photoresist after photoetching is removed by organic solvent immersion, and the organic solvent includes one or more of acetone, toluene or dichloromethane.

[0039] In step 3), the silicon anisotropic etchant includes one or more of tetramethylammonium hydroxide solution, isopropyl alcohol solution of KOH, EDP etchant or hydrazine hydrate etchant, the temperature of wet etching is 50-100℃, and the etching time is 1-120 minutes.

[0040] In step 4), the metal layer or the composite material layer with plasmonic properties is deposited by electroplating, evaporation or magnetron sputtering, and the thickness of the deposited layer is 20-500 nanometers.

[0041] The application also provides a plasmonic positive pyramid array substrate prepared by the above method.

[0042] The application also provides an application of the above plasmonic positive pyramid array substrate, and the substrate is used to deliver substances into extracorporeal cells under laser irradiation.

[0043] The application further provides an extracorporeal cell substance delivery method based on the above plasmonic positive pyramid array substrate, and the substance delivery method is as follows: adhering extracorporeal cells to the surface of the substrate, then adding a dispersion liquid containing a substance to be delivered to the substrate, and then irradiating the substrate with a laser to deliver the substance into the extracorporeal cells.

[0044] The substance includes genetic material, biological macromolecules, nanoparticles or drug molecules, and the dispersion liquid is nuclease-free water. The wavelength of the laser is 400-3000 nm, and the irradiation time is 1-10 min.

[0045] Embodiment:

[0046] A method for delivering miRNA into cells in vitro using a plasmonic positive pyramid array, a plasmonic positive pyramid array substrate is prepared by the following steps:

[0047] 1) A clean silicon wafer with a thickness of 600 microns is cut into a square with a side length of 3 cm, immersed in concentrated sulfuric acid, heated at 90°C for 3 h. Then the silicon wafer is sequentially ultrasonically cleaned in ethanol, acetone and ultrapure water for 15 min, and dried with nitrogen. Hexamethyldisilazane is added to the surface of the silicon wafer, and spin-coated at a speed of 1000 rpm for 2 min to form a uniform adhesive layer; the bubble-free SU-8 photoresist is poured onto the surface of the silicon wafer and spin-coated at a speed of 3000 rpm for 1 min to form a uniform photoresist layer. A chromium plate containing a 2-micron dot pattern is used as a mask to mask the silicon wafer, and the wafer is exposed to ultraviolet light for 120 s; the exposed silicon wafer is immersed in a positive photoresist developer, and the surface of the unhardened photoresist is continuously washed with the developer;

[0048] 2) A 100-nanometer-thick silicon dioxide layer is deposited on the surface of the material obtained in step 1) by magnetron sputtering, and then the wafer is immersed in acetone for 15 minutes to remove the remaining photoresist on the surface of the wafer, obtaining a silicon wafer with a silicon dioxide mask and no photoresist;

[0049] 3) The silicon wafer with a silicon dioxide mask and no photoresist is placed in a 60°C 30wt% KOH / isopropyl alcohol etchant for wet etching for 5 minutes, obtaining a silicon wafer containing a pyramid array structure;

[0050] 4) A 70-nanometer-thick gold layer is deposited on the surface of the silicon wafer containing the pyramid array structure by evaporation.

[0051] KYSE30 / Taxol cells were seeded onto the surface of the plasmonic positive pyramid substrate at a density of 1×10 6 The cells were then incubated in complete medium (RPMI1640: fetal bovine serum: penicillin-streptomycin = 90:9:1). All adherent cells were fixed with 4% paraformaldehyde at 4°C for 15 minutes, and then dehydrated with alcohol for 3 times, 5 minutes each time, at each concentration gradient (50%, 70%, 90%, 100%). Subsequently, a chemical reaction was carried out with hexamethyldisilazane for 10 minutes to remove trace amounts of residual moisture.

[0052] After the KYSE30 / Taxol cells adhered, 5-FAM miR-185 was added, and immediately irradiated with a 640-nanometer red laser for 20 cycles, 8 s each cycle, and finally washed twice with cold PBS to remove the medium containing miR-185.

[0053] Comparative Example:

[0054] A clean silicon wafer with thickness of 500 microns was cut into a square with side length of 3 cm, and then immersed in concentrated sulfuric acid and heated at 90 °C for 3 h. Subsequently, the silicon wafer was sequentially ultrasonically cleaned in ethanol, acetone and ultrapure water for 15 min, and then dried with nitrogen. Then, a 70 nm-thick gold layer was deposited on the surface of the clean silicon wafer by evaporation.

[0055] The cell culture and the miRNA delivery process were the same as in the example.

[0056] Figure 1 A schematic diagram of the process for preparing the plasmonic positive pyramid array substrate in this example is shown in FIG. 1. Figure 1 It can be seen that the preparation method of the silicon substrate has the characteristics of simple process and low cost, which is conducive to large-scale commercial application.

[0057] Figure 2 A scanning electron microscope image of the plasmonic positive pyramid array substrate prepared in this example is shown in FIG. 2. Figure 2 It can be seen that the prepared uniform silicon substrate pyramid array has a nanoscale sharp tip.

[0058] Figure 3 A schematic diagram of the laser scanning during in vitro intracellular substance delivery in this example is shown in FIG. 3. Figure 3 It can be seen that the laser irradiation forms a transient channel on the cell surface under the action of the gold in-situ plasmonic effect, which promotes the extracellular mi-RNA delivery into the cell.

[0059] Figure 4 A distribution diagram of the 5-FAM labeled miR-185 (green fluorescence) in the KYSE30 / Taxol cells in this example and the comparative example is shown in FIG. 4. Figure 4 It can be seen that the cells attached on the plasmonic positive pyramid array substrate have fluorescence after transfection, while the cells on the flat substrate have weak fluorescence and almost no mi-RNA delivery.

[0060] The above description of the examples is for the convenience of the ordinary skilled person in the art to understand and use the invention. Those skilled in the art can easily make various modifications to these examples, and apply the general principles described herein to other examples without creative labor. Therefore, the present application is not limited to the above examples, and the improvements and modifications made by those skilled in the art without departing from the scope of the present application should be within the scope of protection of the present application.

Claims

1. A method for in vitro intracellular substance delivery based on plasmonic positive pyramid array substrate, characterized by, The method comprises the following steps: adhering cells in vitro to a substrate surface, then adding a dispersion liquid containing a substance to be delivered to the substrate, and irradiating the substrate by using a laser to deliver the substance into the cells in vitro. The substance comprises biomacromolecules, nanoparticles or drug molecules, and the dispersion liquid is nuclease-free water; the wavelength of the laser is 400-3000 nm, and the irradiation time is 1-10 min. The plasmonic positive pyramid array substrate is prepared by the following steps: 1) coating a binder layer and a photoresist layer on the surface of a silicon wafer in sequence, then placing a mask plate containing a dot matrix pattern on the photoresist layer and exposing in a photoetching device, removing the mask plate after the exposure is completed, and washing the uncured part of the photoresist layer with a positive photoresist developer to obtain a photoetched material; 2) depositing a silicon dioxide layer on the surface of the photoetched material, then removing the remaining photoresist in the photoetched material to obtain a silicon wafer without glue with a silicon dioxide mask; 3) placing the silicon wafer without glue with a silicon dioxide mask in a silicon anisotropic etchant for wet etching to obtain a silicon wafer containing a positive pyramid array structure; 4) depositing a metal layer or a composite material layer with plasmonic properties on the surface of the silicon wafer containing the positive pyramid array structure to obtain the plasmonic positive pyramid array substrate; In step 1), the silicon wafer is a polished silicon wafer with a thickness of 100-1000 microns; the binder is hexamethyldisilazane, and the coating method of the binder is spin coating, spraying or vacuum evaporation; the photoresist is a positive photoresist, and the coating method of the photoresist is spin coating or spraying; in the dot matrix pattern of the mask plate, the diameter of the circle is 1-5 microns, and the distance between adjacent circles is 3-15 microns; In step 2), the silicon dioxide layer is deposited by electroplating, evaporation or magnetron sputtering, and the thickness of the silicon dioxide layer is 10-1000 nanometers; the remaining photoresist in the photoetched material is removed by organic solvent soaking, and the organic solvent includes one or more of acetone, toluene or dichloromethane; In step 3), the silicon anisotropic etchant includes one or more of tetramethylammonium hydroxide solution, isopropyl alcohol solution of KOH, EDP etchant or hydrazine hydrate etchant, the temperature of the wet etching is 50-100℃, and the etching time is 1-120 minutes; In step 4), the metal layer or the composite material layer with plasmonic properties is deposited by electroplating, evaporation or magnetron sputtering, and the deposition thickness is 20-500 nanometers.

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