CN115029384B - Anodic aluminum oxide biochip, preparation method thereof, intracellular delivery system and application method thereof - Google Patents
Anodic aluminum oxide biochip, preparation method thereof, intracellular delivery system and application method thereof Download PDFInfo
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Abstract
The invention provides a technology for preparing an anodic aluminum oxide-based biochip and accurately delivering substances into cells. The preparation method of the anodic aluminum oxide biochip comprises the following steps: preparing a nano stamping die plate, stamping a high-purity aluminum sheet nano needle, performing surface treatment, performing anodic oxidation and performing surface treatment. The anodic aluminum oxide biochip prepared by the invention has high-density nano-needles, and the density and the morphology of the nano-needles are controlled by a stamping die plate. The surface of the anodic aluminum oxide nanoneedle is provided with a high specific surface area nanohole channel. The surface of the nanoneedle is chemically modified, so that the polarity of the surface can be changed, and the nanoneedle is suitable for loading high-quantity biological small molecules, macromolecules, compounds and the like. When the nanoneedle pierces the cell membrane (cell wall) under external force (including but not limited to squeezing force, centrifugal force, etc.), the delivery of biomolecules into the cytoplasm or/and nucleus can be precisely achieved.
Description
Technical Field
The invention relates to the technical field of biochip preparation, in particular to an anodic aluminum oxide-based biochip, a preparation method thereof, an intracellular delivery system and a use method thereof.
Background
Intracellular substance delivery is an important technique for achieving cell manipulation and is widely used in cell therapy, such as immune cell therapy, stem cell therapy, and the like. Cell transduction methods include viral vector transfection (e.g., retrovirus, lentivirus vectors, etc.) and nonviral vector transfection (transposon transfection, electroporation, etc.). For example, CAR-T cell therapy, i.e., chimeric Antigen Receptor (CAR) -modified T cell therapy, is an engineered T cell therapy that is artificially constructed to specifically recognize and bind tumor-associated antigens. The construction of the CAR-T cells is to transfer the structure of the CAR into the T cells through a genetic engineering technology and obtain stable expression, so that the CAR-T cells have the characteristic of specifically binding tumor antigens and have the activity of targeting and killing the tumor cells. Currently marketed CAR-T products are mainly based on viral vector transduction, so that the final therapeutic effect and application of CAR-T are directly affected by the large-scale production of viral vectors, quality control and cost factors. The existing virus vector still has the problems of high production cost, low yield, activity loss and the like; at the same time viral nucleic acid intercalation into host cell chromosomes can lead to long-term lesions and the risk of carcinogenesis. Electroporation is the earliest and most widely used non-viral transfection technique. The technique uses short electric pulses to act on cells, creating temporary permeabilities allowing injection of the gene DNA or RNA, but with low transfection efficiency, and at the same time having a certain impact on cell activity. More recently, nano-drug carriers have been successfully applied to cell therapy. The nano-drug carrier for wrapping mRNA, such as liposome, can enter cells through cell endocytosis and the like, and then release nucleic acid substances into cytoplasm for expression. The nano-drug will enter the cell first to enter the autophagosome and lysosome, so the nano-drug carrier needs to be subjected to complex structural design to escape from the lysosome. The efficiency of nano-drug carrier gene escape was reported to be no more than 5%. Thus, new cellular drug delivery systems are urgently needed to be developed and applied.
Nanomicroneedle delivery systems can utilize physical methods to actively deliver biomolecules to the interior of cells and even to the nuclear area. The cell membrane has strong self-repairing capability, and after being pierced by the nano-scale microneedle, the phospholipid of the cell membrane can be repaired completely again, so that the cell activity is ensured. As a delivery mode of non-viruses, the delivery mode of the nanoneedle actively delivers biomolecules to a specific area in a cell while perforating the cell membrane, thereby solving the problems of escape of a nano drug carrier from autophagy and lysosome and solving the problem of low transfection efficiency of electroporation technology. The nanometer needle material for delivery is preferably nontoxic, has good mechanical property, has high specific surface area, can load biological molecules, can be industrially produced in large scale, and the like. However, the existing nanometer microneedles have limited specific surface area and loading biological molecular weight, and cannot meet the loading capacity required by nanometer drug carriers.
Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide an anodic aluminum oxide-based biochip for cell transduction, a preparation method thereof, an intracellular delivery system and a use method thereof.
In order to solve the technical problems, the invention is realized by the following technical scheme:
an anodic aluminum oxide biochip comprises a high-purity aluminum sheet, wherein the surface of the high-purity aluminum sheet is covered with a high-molecular material and comprises array-type nanometer needle heads protruding outwards from the surface of the high-purity aluminum sheet; the surface of the nanometer needle head is provided with a nanometer hole with a modified surface.
The invention also discloses a preparation method of the anodic aluminum oxide biochip, comprising the following steps:
s1, taking high-purity aluminum sheets with the sizes of chips, wherein the purity of the high-purity aluminum sheets is over 99.99 percent, and the thickness of the high-purity aluminum sheets is 0.1-10mm;
s2, preparing a nano stamping template, wherein a female mold matrix of nano needles is arranged in the nano stamping template and is used for controlling the appearance of the nano needles after stamping of the nano stamping template, and the spacing of the nano needles in the female mold matrix can be designed to be 1-500 micrometers. The diameter of the nanometer needle is 100-1000nm, and the length is 500-5000nm;
s3, placing the high-purity aluminum sheet into a clamp, and heating to 400-600 ℃ in a high-temperature furnace under the protection of protective gas, wherein the heating temperature is 1-20 ℃ per minute; the pressure is applied at 1-1000MPa, and the stepping distance is 0.1-1 mm; then rapidly taking out the clamp, fixing the high-purity aluminum sheet on the nano stamping die plate, and then pressing the nano stamping die plate into the surface of the high-purity aluminum sheet to form a nano needle; when the high-purity aluminum sheet is cooled to below 100 ℃, lifting the nano stamping template; standing until the high-purity aluminum sheet is cooled;
s4, performing ultrasonic degreasing on the high-purity aluminum sheet treated in the step S3, and drying the high-purity aluminum sheet, wherein the ultrasonic duration is 10-30min;
s5, carrying out surface modification on the high-purity aluminum sheet treated in the step S4, so that the high-purity aluminum sheet surface is covered with a high polymer material to insulate the bottom of the high-purity aluminum sheet, and only exposing the nano needle head part;
s6, after the surface modification in the step S5, placing the obtained high-purity aluminum sheet into a plasma device for treatment for 5-20min to remove the residue of the high polymer material at the tip of the nanometer needle;
s7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained after the treatment in the step S6 as an anode, and performing anodic oxidation in oxalic acid solution or sulfuric acid solution to generate nanopores on the surface of the nanometer needle head, wherein the aperture is 10-40nm; after anodic oxidation, cleaning and drying the high-purity aluminum sheet to obtain an anodic aluminum oxide biochip;
in the step S7, the reaction condition of the oxalic acid electrolyte is 40-50V constant voltage, the reaction temperature is 5 ℃, the concentration is 0.1-0.3mol/L, and the reaction time is 5-10min; the reaction condition of sulfuric acid electrolyte, constant voltage of 20-25V, reaction temperature of 5 ℃, concentration of 0.01-0.3mol/L and reaction time of 5-10min.
The length and the width of the high-purity aluminum sheet in the S1 step are formulated according to the size of a designed chip, for example, 10cm is 10cm;
preferably, the nano-imprint template should have dimensions consistent with the chip dimensions, such as 10cm x 10cm.
The high temperature furnace in step S3 is preferably a muffle furnace or a forging furnace, and the shielding gas is preferably nitrogen.
The solution for ultrasonic degreasing in step S4 is preferably acetone.
The surface modification method in step S5 is a chemical vapor deposition method or a spin coating method. The surface of the aluminum sheet is covered with a polymer material of about 100 nm.
In step S6, the plasma device is an air or oxygen plasma device.
The invention also discloses a surface modification method of the anodic aluminum oxide biochip, which comprises the steps of firstly soaking the anodic aluminum oxide biochip in saturated hydrogen peroxide solution for 1h at room temperature, and then crosslinking aminopropyl triethoxysilane (APTES) by a chemical vapor deposition or liquid phase reaction method.
The invention also discloses an intracellular delivery method, which adopts the anodic aluminum oxide biochip to pierce cells and delivers active ingredients into the cells or nuclei through a nanoneedle.
The invention also discloses an intracellular delivery system of the anodic aluminum oxide biochip, which comprises a cell driving device, a cell placing groove and a cell driving device lifter; the cell driving device comprises a lifting plate and a pressurizing cavity arranged at the front end of the lifting plate; the cell placing groove is arranged right below the pressurizing cavity and forms a closed cavity with the pressurizing cavity cover; the lifter of the cell driving device comprises a screw rod connected with the lifting plate and a rotating motor connected to the bottom of the screw rod. After the pressurizing cavity is covered on the upper surface of the cell placing groove for sealing after the pressurizing cavity is descended by the lifting plate, the air valve applies specified air pressure to the pressurizing cavity, so that cells are pressed on the surface of the anodic aluminum oxide biochip, and the nanometer needle is forced to pierce the cells, thereby realizing intracellular delivery of biomolecules.
The invention adopts a motor at the bottom to drive the ball screw to rotate, a screw nut arranged on the ball screw is lifted along the vertical direction of the ball screw, the screw nut is fixedly connected with a lifting plate, an optical axis penetrates through the lifting plate, the lifting plate is limited by the optical axis and the ball screw to slide vertically and upwards, and a cell dish is arranged in a cell placing groove. After the lifting plate descends and covers the cell placing groove to realize the sealing of the whole inner cavity, the cells in the cell placing groove are pressurized to be pierced by the nanometer needle on the anodic aluminum oxide biochip arranged at the top of the pressurizing cavity, so that the directional drug delivery is realized.
The directional drug delivery method of the invention needs to be pressed on the cells by external force, and can apply pressure to the cells and ensure that the nanoneedle pierces the cells.
The technical scheme of the invention has the following beneficial effects:
1. the anodic aluminum oxide nanoneedle is prepared by a micro-nano processing technology, so that the appearance, structure and density of the nanoneedle are controllable.
2. The loading of bioactive substances (such as DNA and mRNA) is realized by surface modification of the anodic aluminum oxide nano needle.
3. The array type anodic aluminum oxide nano needle is prepared by a micro-nano processing technology, so that the medicine is delivered by multiple needles simultaneously.
4. By surface modification of the anodized aluminum nanoneedle, the chance of the nanomedicine carrier delivered into the cell escaping cell autophagy and lysosomes is increased.
5. The nanometer needle has large specific surface area, can load more biological molecular weight, and has strong effectiveness in directional drug delivery.
Drawings
FIG. 1 is a microscopic view of a nanopore created by anodic oxidation of a high purity aluminum-based chip;
FIG. 2 is a Fourier transform infrared absorption spectrum of an anodized aluminum chip modified by APTES and a fluorescent labeling dye-fluorescein isothiocyanate (FTIC) according to the present invention;
FIG. 3 is a perspective view of the intracellular delivery system of the present invention;
fig. 4 is a side view of the intracellular delivery system of the present invention.
Specific reference numerals are as follows:
1 a cell driving device; 2 a cell placement groove; 3 a cell driving device lifter; 11 lifting plates; 12 pressurizing the cavity; 31 screw rods; 32 a rotating electric machine.
Detailed Description
In order to make the technical problems, technical solutions and advantages to be solved by the present invention more apparent, the following detailed description will be made with reference to specific embodiments.
The anodic aluminum oxide biochip comprises a high-purity aluminum sheet, wherein the surface of the high-purity aluminum sheet is covered with a high-molecular material and comprises array-type arranged nanometer needles protruding outwards from the surface of the high-purity aluminum sheet; the surface of the nanometer needle head is provided with a nanometer hole with a modified surface.
The preparation method of the anodic aluminum oxide biochip comprises the following steps:
s1, taking high-purity aluminum sheets with the sizes of chips;
the purity of the high-purity aluminum sheet in the S1 step is more than 99.99%, the thickness is 0.1-10mm, and the length and the width are formulated according to the size of a design chip, such as 10cm x 10cm; high purity aluminum flakes are used because they form dense anodized aluminum porous films on the surface of metallic aluminum under precisely controlled reaction conditions. The anodic aluminum oxide film formed by the method has the advantages of high porosity, controllable nano structure and the like. Anodized aluminum is a nanomaterial with good biocompatibility. The anodic oxidation as the processing technology of the aluminum surface has the advantages of no toxicity, high school, low cost, accurate control of the shape of the nano-pores, and the like.
S2, preparing a nano stamping template, wherein a female mold matrix of a nano needle is arranged in the nano stamping template and is used for controlling the appearance of the nano needle after stamping of the nano stamping template;
the pitch of the nanoneedles in the negative matrix in step S2 may be designed to be 1-500 microns. The diameter of the nanometer needle is 100nm-1000nm, and the length is 500-5000nm. The size of the nano-imprint template should be consistent with the chip size, e.g., 10cm by 10cm. The shape of the nanoneedle may be expanded to a cone or other rectangle or polygon.
S3, placing the high-purity aluminum sheet into a clamp, placing the clamp into a high-temperature furnace, and heating to 400-600 ℃ under the protection of protective gas; then rapidly taking out the clamp, fixing the high-purity aluminum sheet on the nano stamping die plate, and then pressing the nano stamping die plate into the surface of the high-purity aluminum sheet to form a nano needle; when the high-purity aluminum sheet is cooled to below 100 ℃, lifting the nano stamping template; standing until the high-purity aluminum sheet is cooled;
in the step S3, the high-temperature furnace is preferably a muffle furnace or a forging furnace, the shielding gas is preferably nitrogen, and the heating temperature rising frequency is 1-20 ℃ per minute; the pressure is 1-1000MPa, and the stepping distance is 0.1-1 mm.
S4, ultrasonically removing grease from the high-purity aluminum sheet treated in the step S3, and drying;
the solution for ultrasonically removing the self-carried grease on the surface of the aluminum sheet is preferably acetone, and the ultrasonic duration is preferably 10-30min.
S5, carrying out surface modification on the high-purity aluminum sheet treated in the step S4, so that the high-purity aluminum sheet surface is covered with a high polymer material to insulate the bottom of the high-purity aluminum sheet, and only exposing the nano needle head part;
the surface modification method in step S5 is a chemical vapor deposition method or a spin coating method. The surface of the aluminum sheet is covered with a polymer material of about 100 nm. The high polymer material is an insulating material such as polystyrene and is mainly used for insulating the bottom.
SU-8 photoresist having a thickness of about 100nm can also be spin coated on the surface using spin coating, followed by photo-curing.
S6, after the surface modification in the step S5, placing the obtained high-purity aluminum sheet into a plasma device for treatment for 5-20min to remove the residue of the high polymer material at the tip of the nanometer needle;
the plasma may be selected from air and oxygen plasmas commonly used in the industry, avoiding the use of highly corrosive plasma treatments.
S7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained after the treatment in the step S6 as an anode, and performing anodic oxidation in electrolyte to generate nanopores on the surface of the nanometer needle; and after anodic oxidation, cleaning and drying the high-purity aluminum sheet to obtain the anodic aluminum oxide biochip. Anodic oxidation can generate nano holes on the surface of the aluminum nano needle, and the aperture is 10-40nm. After the anodic oxidation is completed, the aluminum sheet is washed with deionized water and dried. The nanopores do not extend completely through. The nanopores preferably grow inward perpendicular to the surface, but without penetration.
The electrolyte in the step S7 is an oxalic acid solution or a sulfuric acid solution. The reaction condition of the oxalic acid electrolyte is 40-50V constant voltage, the reaction temperature is 5 ℃, the concentration is 0.1-0.3mol/L, and the reaction time is 5-10min; the sulfuric acid electrolyte has the reaction conditions of constant voltage of 20-25V, reaction temperature of 5 ℃, concentration of 0.1mol/L and reaction time of 5-10min, the current density and the voltage density need to be accurately calculated according to the total surface area of the actual nanoneedle, so that the current density is not more than 20mAcm < -2 >, and the anodic oxidation process enables the surface of the nanoneedle to radially grow into the nanohole, so that the specific surface area of the nanoneedle can be increased, and the load capacity is increased.
The invention also discloses an anodic aluminum oxide biochip, which is prepared according to the preparation method of the anodic aluminum oxide biochip.
The anodic aluminum oxide biochip can be subjected to surface modification and is specifically connected with bioactive molecules. The surface modification method of the anodic aluminum oxide biochip can be carried out by immersing the anodic aluminum oxide biochip in saturated hydrogen peroxide solution for 1h at room temperature, and then crosslinking aminopropyl triethoxysilane (APTES) by chemical vapor deposition or liquid phase reaction method. The amino group carried by APTES can be combined with various degradable groups such as sulfhydryl and carboxyl, so as to achieve the aim of crosslinking biomolecules. In this example, the FTIR characterization data of a modified APTES and fluorescent labeling dye-fluorescein isothiocyanate (FTIC) -modified anodized aluminum chip is shown in FIG. 2.
The invention also discloses an intracellular delivery system of the anodic aluminum oxide biochip, which comprises a cell driving device, a cell placing groove and a cell driving device lifter; the cell driving device comprises a lifting plate and a pressurizing cavity arranged at the front end of the lifting plate; the cell placing groove is arranged right below the pressurizing cavity and forms a closed cavity with the pressurizing cavity cover; the lifter of the cell driving device comprises a screw rod connected with the lifting plate and a rotating motor connected to the bottom of the screw rod. After the pressurizing cavity is covered on the upper surface of the cell placing groove for sealing after the pressurizing cavity is descended by the lifting plate, the air valve applies specified air pressure to the pressurizing cavity, so that cells are pressed on the surface of the anodic aluminum oxide biochip, and the nanometer needle is forced to pierce the cells, thereby realizing intracellular delivery of biomolecules.
The invention adopts a motor at the bottom to drive the ball screw to rotate, a screw nut arranged on the ball screw is lifted along the vertical direction of the ball screw, the screw nut is fixedly connected with a lifting plate, an optical axis penetrates through the lifting plate, the lifting plate is limited by the optical axis and the ball screw to slide vertically and upwards, and a cell dish is arranged in a cell placing groove. After the lifting plate descends and covers the cell placing groove to realize the sealing of the whole inner cavity, the cells in the cell placing groove are pressurized to be pierced by the nanometer needle on the anodic aluminum oxide biochip arranged at the top of the pressurizing cavity, so that the directional drug delivery is realized.
The anodic aluminum oxide biochip prepared by the invention has high-density nano-needles, and the density and the morphology of the nano-needles are controlled by a stamping die plate. The surface of the anodic aluminum oxide nano needle is provided with a nano pore canal with high specific surface area, and the specific surface area of the nano needle is increased. The surface of the nanoneedle is chemically modified, so that the polarity of the surface can be changed, and the nanoneedle is suitable for loading high-quantity biological small molecules, macromolecules, compounds and the like. When the nanoneedle pierces the cell membrane (cell wall) under external force (including but not limited to squeezing force, centrifugal force, etc.), the delivery of biomolecules into the cytoplasm or/and nucleus can be precisely achieved. The cell delivery system protected by the patent of the invention is characterized in that the anodic aluminum oxide nanoneedle is precisely injected into target cells by a microfluidic technology, so that the intracellular precise delivery and high-flux delivery of biomolecules and chemical molecules are realized.
Example 1
A biochip for preparing anodized aluminum, comprising the steps of:
s1, taking a high-purity aluminum sheet with the purity of more than 99.99%, wherein the thickness is 0.5mm, the length and the width are formulated according to the size of a design chip, and the embodiment is 10cm x 10cm.
S2, preparing a nano stamping template, wherein the template comprises a female template matrix of nano needles and is used for controlling the shape of the nano needles after nano stamping. The nanoneedle pitch in the matrix can be designed to be 500 microns. The diameter of the nanoneedle is 1000nm and the length is 500nm. The size of the nano-imprint template should be consistent with the chip size, in this embodiment 10cm x 10cm.
S3, placing the high-purity aluminum sheet into a custom fixture, placing into a high-temperature furnace (a muffle furnace or a forging furnace), and heating to 600 ℃ at a rate of 10 ℃ per minute under the protection of nitrogen. The mold is then quickly removed and fixed to a custom fixture, and the nano-imprint template is then pressed into the high purity aluminum surface. The applied pressure was 1000MPa and the step distance was 0.1mm. And slowly lifting the nano stamping template at a speed of 0.1mm/min when the aluminum sheet is cooled to below 100 ℃. Until the aluminum sheet cools.
S4, placing the aluminum sheet into acetone, removing grease by ultrasonic for 10min, and then drying.
S5, performing surface modification on the obtained aluminum sheet, so that the surface of the aluminum sheet is covered with a high polymer material with the thickness of about 100nm, the bottom of the aluminum sheet is insulated, and only the nano needle head part is exposed. A layer of polymer material can be deposited by using a chemical vapor deposition method; SU-8 photoresist having a thickness of about 100nm can also be spin coated on the surface using spin coating, followed by photo-curing.
S6, after finishing surface modification, placing the obtained aluminum sheet into a plasma device for treatment for 10min so as to remove polymer residues at the tip of the nanoneedle. The plasma may be selected from air and oxygen plasmas commonly used in the industry, avoiding the use of highly corrosive plasma treatments.
S7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained in the steps as an anode, and performing anodic oxidation in electrolyte; the electrolyte comprises oxalic acid solution. Anodic oxidation can generate nano holes on the surface of the aluminum nano needle, and the aperture is 20nm. After the anodic oxidation is completed, the aluminum sheet is washed with deionized water and dried. The reaction condition of the oxalic acid electrolyte is 50V constant voltage, the reaction temperature is 5 ℃, the concentration is 0.3mol/L, and the reaction time is 10min.
Example 2
A biochip for preparing anodized aluminum, comprising the steps of:
s1, taking a high-purity aluminum sheet with the purity of more than 99.99%, wherein the thickness is 0.1mm, the length and the width are formulated according to the size of a design chip, and the embodiment is 10cm x 10cm
S2, preparing a nano stamping template, wherein the template comprises a female template matrix of nano needles and is used for controlling the shape of the nano needles after nano stamping. The nanoneedle pitch in the matrix can be designed to be 200 microns. The diameter of the nanoneedle is 500nm and the length is 1000nm. The size of the nano-imprint template should be consistent with the chip size, in this embodiment 10cm x 10cm.
S3, placing the high-purity aluminum sheet into a custom fixture, placing into a high-temperature furnace (a muffle furnace or a forging furnace), and heating to 500 ℃ at a rate of 10 ℃ per minute under the protection of nitrogen. The mold is then quickly removed and fixed to a custom fixture, and the nano-imprint template is then pressed into the high purity aluminum surface. The applied pressure was 1000MPa and the step distance was 0.1mm. And slowly lifting the nano stamping template at a speed of 0.1mm/min when the aluminum sheet is cooled to below 100 ℃. Until the aluminum sheet cools.
S4, placing the aluminum sheet into acetone, removing grease by ultrasonic for 10min, and then drying.
S5, performing surface modification on the obtained aluminum sheet, so that the surface of the aluminum sheet is covered with a high polymer material with the thickness of about 100nm, the bottom of the aluminum sheet is insulated, and only the nano needle head part is exposed. A layer of polymer material can be deposited by using a chemical vapor deposition method; SU-8 photoresist having a thickness of about 100nm can also be spin coated on the surface using spin coating, followed by photo-curing.
S6, after finishing surface modification, placing the obtained aluminum sheet into a plasma device for treatment for 5min so as to remove polymer residues at the tip of the nanoneedle. The plasma may be selected from air and oxygen plasmas commonly used in the industry, avoiding the use of highly corrosive plasma treatments.
S7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained in the steps as an anode, and performing anodic oxidation in electrolyte; the electrolyte comprises a sulfuric acid solution. Anodic oxidation can generate nano holes on the surface of the aluminum nano needle, and the aperture is 20nm. After the anodic oxidation is completed, the aluminum sheet is washed with deionized water and dried. The reaction condition of the sulfuric acid electrolyte is that the constant voltage is 20-25V, the reaction temperature is 5 ℃, the concentration is 0.3mol/L, and the reaction time is 10min.
Example 3
A biochip for preparing anodized aluminum, comprising the steps of:
s1, taking a high-purity aluminum sheet with the purity of more than 99.99%, wherein the thickness is 0.1mm, the length and the width are formulated according to the size of a design chip, and the embodiment is 10cm x 10cm.
S2, preparing a nano stamping template, wherein the template comprises a female template matrix of nano needles and is used for controlling the shape of the nano needles after nano stamping. The nanoneedle pitch in the matrix can be designed to be 100 microns. The diameter of the nanoneedle is 100nm and the length is 1000nm. The size of the nano-imprint template should be consistent with the chip size, in this embodiment 10cm x 10cm.
S3, placing the high-purity aluminum sheet into a custom fixture, placing into a high-temperature furnace (a muffle furnace or a forging furnace), and heating to 400 ℃ at a rate of 10 ℃ per minute under the protection of nitrogen. The mold is then quickly removed and fixed to a custom fixture, and the nano-imprint template is then pressed into the high purity aluminum surface. The applied pressure was 500MPa and the step distance was 0.1mm. And slowly lifting the nano stamping template at a speed of 0.1mm/min when the aluminum sheet is cooled to below 100 ℃. Until the aluminum sheet cools.
S4, placing the aluminum sheet into acetone, removing grease by ultrasonic for 10min, and then drying.
S5, performing surface modification on the obtained aluminum sheet, so that the surface of the aluminum sheet is covered with a high polymer material with the thickness of about 100nm, the bottom of the aluminum sheet is insulated, and only the nano needle head part is exposed. A layer of polymer material can be deposited by using a chemical vapor deposition method;
s6, after finishing surface modification, placing the obtained aluminum sheet into a plasma device for treatment for 5min so as to remove polymer residues at the tip of the nanoneedle. The plasma may be selected from air and oxygen plasmas commonly used in the industry, avoiding the use of highly corrosive plasma treatments.
S7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained in the steps as an anode, and performing anodic oxidation in electrolyte; the electrolyte comprises a sulfuric acid solution. Anodic oxidation can generate nano holes on the surface of the aluminum nano needle, and the aperture is 20nm. After the anodic oxidation is completed, the aluminum sheet is washed with deionized water and dried. The reaction condition of the sulfuric acid electrolyte is 25V constant voltage, the reaction temperature is 5 ℃, the concentration is 0.1mol/L, and the reaction time is 10min.
The prepared anodized aluminum biochip is finally formed and scanned under a scanning electron microscope to obtain FIG. 1.
The anodic aluminum oxide biochip has a nano needle structure containing nano holes and high specific surface area. Can adsorb bioactive components such as nucleic acid, polypeptide, protein, chemical molecule, etc., and deliver the active components into cell or nucleus when nanoneedle pierces cell
Surface modified anodic aluminum oxide biochip
And carrying out surface modification on the anodic aluminum oxide biochip, and specifically connecting bioactive molecules.
Firstly, soaking an anodic aluminum oxide biochip in saturated hydrogen peroxide solution for 1h at room temperature, and then crosslinking aminopropyl triethoxysilane (APTES) by a chemical vapor deposition or liquid phase reaction method. The amino group carried by APTES can be combined with various degradable groups, so that the aim of crosslinking biological molecules is fulfilled. For example, the amino group carried by APTES is crosslinked with carboxyl groups on the protein by the action of 1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS), thereby immobilizing the protein on the surface of the nanoneedle.
In the process, the anodic aluminum oxide biochip is soaked in saturated hydrogen peroxide solution, and hydroxyl is introduced to the surface of the anodic aluminum oxide biochip. And crosslinking aminopropyl triethoxysilane (APTES) by chemical vapor deposition or liquid phase reaction, introducing amino, and adding protein/1-ethyl- (3-dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) to react and crosslink the protein and the amino.
Methods of using an intracellular delivery system of an anodized aluminum biochip
As shown in fig. 3 and 4, the intracellular delivery system of the anodized aluminum biochip of the present invention comprises a cell driving device 1, a cell placement tank 2, and a cell driving device lifter 3; the cell driving device 1 comprises a lifting plate 11 and a pressurizing cavity 12 arranged at the front end of the lifting plate 11; the cell placing groove 2 is arranged right below the pressurizing cavity 12 and covers the pressurizing cavity 12 to form a closed cavity; the cell driving device lifter 3 includes a screw rod 31 connected to the lifting plate 11, and a rotary motor 32 connected to the bottom of the screw rod 31. After the pressurizing cavity 12 is covered on the upper surface of the cell placing groove 2 for sealing after the lifting plate 11 descends, the air valve applies specified air pressure to the pressurizing cavity 12, so that cells are pressed on the surface of the anodized aluminum biochip, and the nanometer needle is forced to pierce the cells, thereby realizing intracellular delivery of biomolecules.
Workflow of the intracellular delivery system of anodized aluminum biochip:
an anodic alumina biochip is placed on top of the pressurizing chamber 12, and the nanoneedle is faced to the cell placement tank 2, and then the cell dish is placed in the cell placement tank 2. The rotating motor 32 is started to drive the screw rod 31 to rotate, a screw nut arranged on the screw rod 31 is lifted along the vertical direction of the screw rod 31, the screw nut is fixedly connected with a lifting plate 11, an optical axis penetrates through the lifting plate 11, the lifting plate 11 is limited by the optical axis and the screw rod 31 to slide vertically upwards and downwards, and a cell dish is placed in the cell placement groove 2. After the lifting plate 11 descends and covers the cell placing groove 2 to realize the sealing of the whole inner cavity, the pressurizing cavity 12 is pressurized to ensure that the cells are pierced to a designated depth by the nanometer needle on the anodic aluminum oxide biochip arranged at the top of the pressurizing cavity 12 to realize the directional drug delivery.
While the foregoing is directed to the preferred embodiments of the present invention, it will be appreciated by those skilled in the art that various modifications and adaptations can be made without departing from the principles of the present invention, and such modifications and adaptations are intended to be comprehended within the scope of the present invention.
Claims (6)
1. A preparation method of an anodic aluminum oxide biochip is characterized in that: the method comprises the following steps:
s1, taking high-purity aluminum sheets with the sizes of chips, wherein the purity of the high-purity aluminum sheets is over 99.99 percent, and the thickness of the high-purity aluminum sheets is 0.1-10mm;
s2, preparing a nano stamping template, wherein a female mold matrix of nano needles is arranged in the nano stamping template and is used for controlling the appearance of the nano needles after stamping of the nano stamping template, the distance between the nano needles in the female mold matrix can be designed to be 1-500 micrometers, the diameter of the nano needles is 100-1000nm, and the length of the nano needles is 500-5000nm;
s3, placing the high-purity aluminum sheet into a clamp, and heating to 400-600 ℃ in a high-temperature furnace under the protection of protective gas, wherein the heating temperature is 1-20 ℃ per minute; the pressure is applied at 1-1000MPa, and the stepping distance is 0.1-1 mm; then rapidly taking out the clamp, fixing the high-purity aluminum sheet on the nano stamping die plate, and then pressing the nano stamping die plate into the surface of the high-purity aluminum sheet to form a nano needle; when the high-purity aluminum sheet is cooled to below 100 ℃, lifting the nano stamping template; standing until the high-purity aluminum sheet is cooled;
s4, performing ultrasonic degreasing on the high-purity aluminum sheet treated in the step S3, and drying the high-purity aluminum sheet, wherein the ultrasonic duration is 10-30min;
s5, carrying out surface modification on the high-purity aluminum sheet treated in the step S4, so that the high-purity aluminum sheet surface is covered with a high polymer material to insulate the bottom of the high-purity aluminum sheet, and only exposing the nano needle head part;
s6, after the surface modification in the step S5, placing the obtained high-purity aluminum sheet into a plasma device for treatment for 5-20min to remove the residue of the high polymer material at the tip of the nanometer needle;
s7, taking graphite or platinum as a cathode, taking the high-purity aluminum sheet obtained after the treatment in the step S6 as an anode, and performing anodic oxidation in oxalic acid solution or sulfuric acid solution to generate nanopores on the surface of the nanometer needle head, wherein the aperture is 10-40nm; after anodic oxidation, cleaning and drying the high-purity aluminum sheet to obtain an anodic aluminum oxide biochip;
in the step S7, the reaction condition of the oxalic acid electrolyte is 40-50V constant voltage, the reaction temperature is 5 ℃, the concentration is 0.1-0.3mol/L, and the reaction time is 5-10min; the reaction condition of sulfuric acid electrolyte, constant voltage of 20-25V, reaction temperature of 5 ℃, concentration of 0.01-0.3mol/L and reaction time of 5-10min.
2. The method for preparing an anodized aluminum biochip according to claim 1, wherein: in the step S5, the polymer material is polystyrene.
3. The method for preparing an anodized aluminum biochip according to claim 1, wherein: the surface modification method in step S5 is a chemical vapor deposition method or a spin coating method.
4. A surface modification method of an anodized aluminum biochip obtained by the preparation method according to any one of claims 1 to 3, characterized by: firstly, soaking an anodic aluminum oxide biochip in saturated hydrogen peroxide solution for 1h at room temperature, and then crosslinking aminopropyl triethoxysilane (APTES) by a chemical vapor deposition or liquid phase reaction method.
5. A method of intracellular delivery, comprising: the anodized aluminum biochip obtained by the surface modification method of claim 4, which pierces cells and delivers active ingredients into cells or nuclei through nanoneedles.
6. An intracellular delivery system of an anodized aluminum biochip obtained by the method of any one of claims 1-3, characterized in that: comprises a cell driving device, a cell placing groove and a cell driving device lifter; the cell driving device comprises a lifting plate and a pressurizing cavity arranged at the front end of the lifting plate; the cell placing groove is arranged right below the pressurizing cavity and forms a closed cavity with the pressurizing cavity cover; the cell driving device lifter comprises a screw rod connected with a lifting plate and a rotating motor connected to the bottom of the screw rod, the pressurizing cavity is covered on the upper surface of the cell placing groove after being descended by the lifting plate, and the air valve applies specified air pressure to the pressurizing cavity, so that cells are pressed on the surface of the anodic aluminum oxide biochip, and the nanometer needle head is forced to pierce the cells, so that intracellular delivery of biomolecules is realized.
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