A biomolecule delivery device based on microfluidics and nanotechnology and its application

Abstract

The present invention provides a biomolecule delivery device based on microfluidics and nanotechnology, the biomolecule delivery device comprising a microfluidic unit for accurately controlling output fluid characteristics, wherein the fluid characteristics include at least one of fluid composition, concentration and speed; and a delivery unit group, wherein each fluid output port of the microfluidic unit is connected to a delivery unit for delivering the fluid output by the microfluidic unit into cells; the biomolecule delivery device of the present invention has high-throughput screening capabilities, can simultaneously deliver molecules of multiple concentrations and multiple formulations, can be accurately, conveniently, quickly and efficiently used to screen the optimal induction reprogramming molecule formula, thereby facilitating the establishment of a high-efficiency reprogramming hiPSCs preparation method without genetic modification residues.

Description

Technical Field

[0001] The present invention belongs to the technical field of microfluidic technology and nanotechnology application, and specifically relates to a biomolecule delivery device based on microfluidic technology and nanotechnology and its application. Background Art

[0002] Human induced pluripotent stem cells (hiPSCs) are pluripotent stem cells formed by dedifferentiation of mammalian adult cells through the transfer of transcription factors and other means. They were first discovered by the research team of Japanese scholar Shinya Yamanaka in 2006. The current induction method of hiPSCs is to use viral vectors or plasmids to transfect DNA information containing Oct4, Sox2, Klf4 and c-Myc transcription factors into somatic cells, increase the expression of these four transcription factors in cells and induce somatic cell reprogramming, so that they can obtain pluripotency. Since this hiPSCs induction method does not require the use of eggs or embryos, it has great potential medical value in disease mechanism research, new drug screening, and personalized cell replacement therapy.

[0003] At present, biomolecule introduction technology is the main technical means for preparing hiPSCs. According to the introduction mechanism and the type of introduced molecules, it can be divided into integrating virus vector induction, non-integrating virus induction, transposable element induction, mRNA and protein induction. However, these induction methods have high safety risks or low reprogramming efficiency (0.001%-10%) and other problems. Among them, the safety risks mainly come from virus-related induction methods. Regardless of whether it is genome integration induction, there is a risk of gene mutation caused by randomly embedding DNA sequences into the host genome. Non-viral vectors are limited by the current transfection methods (including transposable element vectors, proteins, plasmid vectors and mRNA), which cannot guarantee the delivery of equal amounts of DNA molecules into each cell, and the transfection experiment is highly toxic to cells, which makes the efficiency of transcription factor expression in each cell after transfection vary greatly, but generally low.

[0004] At present, the regenerative medicine community believes that establishing a method for preparing hiPSCs (human iPSCs) without genetic modification residues (such as using mRNA and protein to reprogram human somatic cells into iPS cells) is an effective means to avoid introducing safety risks from viral vectors. In order to completely eliminate the risk of exogenous gene integration, the use of mRNA transcription factors or in vitro purified protein reprogramming transcription factors to establish a scarless hiPSCs preparation strategy is considered to be the preparation method with the lowest safety risk. In 2010, scientists successfully completed the preparation of hiPSCs using mRNA for the first time (Warren, L. et al. Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-630, doi: 10.1016 / j.stem.2010.08.012 (2010); Warren, L. & Lin, C. mRNA-Based Genetic Reprogramming. Mol Ther 27, 729-734, doi: 10.1016 / j.ymthe.2018.12.009 (2019)). Some scholars have also proved that the use of reprogramming proteins can successfully induce the reprogramming of mouse and human fibroblasts. However, mRNA and protein have a limited survival cycle in cells, and it is necessary to repeatedly deliver related biological molecules into cells. However, the current intracellular delivery methods are limited (including electroporation technology and liposome transfection), and cannot completely achieve multiple, real-time, and long-term delivery without damage to cells. Therefore, the reprogramming efficiency using mRNA and protein molecule induction methods is extremely low and cannot be used in practice. The key to realizing the clinical application of hiPSCs is to develop new non-viral intracellular delivery methods to achieve repeated, long-term, efficient, real-time, and non-damaging intracellular delivery, and to establish a new, efficient, and non-genetically modified hiPSCs preparation strategy based on it.

[0005] In summary, there is an urgent need to develop a new type of efficient delivery technology for intracellular induction factors such as mRNA, proteins and other biological molecules, so as to achieve repeated, long-term, efficient and quantitative delivery of biological molecules into cells. Summary of the invention

[0006] In view of the shortcomings of the prior art, the present invention aims to provide a biomolecule delivery device based on microfluidics and nanotechnology, which is based on microfluidics and nanotechnology for manipulating microfluids at microscopic size, and can achieve repeated, efficient, quantitative, and continuous long-term delivery of biomolecules (such as DNA, mRNA or protein) to cells without damage to cells, and has the characteristics of semi-automation and strong operability. The combination ratio, expression amount, and continuity of expression cycle of reprogramming transcription factors will directly affect the efficiency of cell reprogramming, but due to experimental conditions, the current optimization of experimental conditions for reprogramming and the testing process of reagent ratio are cumbersome and time-consuming due to experimental conditions. The delivery device of the present invention has high-throughput screening capabilities, and can simultaneously carry out molecular delivery of multiple concentrations and multiple formulas, which can be accurately, conveniently, quickly, and efficiently used to screen the optimal induction reprogramming molecular formula, thereby facilitating the establishment of a hiPSCs preparation method with high efficiency and no genetic modification residues for reprogramming.

[0007] In order to achieve the above object, the present invention provides a biomolecule delivery device based on microfluidics and nanotechnology, comprising:

[0008] a microfluidic unit for precisely controlling output fluid characteristics, the fluid characteristics comprising at least one of fluid composition, concentration, and velocity; and

[0009] A delivery unit group, each fluid output port of the microfluidic unit is connected to a delivery unit for delivering the fluid output by the microfluidic unit into cells.

[0010] In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the delivery unit includes: a fluid receiver, a micro-nanostructure membrane arranged at the upper opening of the fluid receiver, and a cell culture well plate arranged on the micro-nanostructure membrane; wherein the micro-nanostructure membrane includes a thin film and a plurality of nanotubes arranged on the thin film, the nanotubes pass through the thin film, and the nanotube ports on the nanotubes close to the fluid receiver are configured to be able to contact the fluid in the fluid receiver; the nanotube ports on the side away from the fluid receiver extend out of the thin film and extend into the cell culture wells of the cell culture well plate so as to be able to contact the cells in the cell culture wells.

[0011] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the delivery unit further comprises: a first electrode disposed below the fluid receiver, and a second electrode disposed above the cell culture well plate.

[0012] In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the delivery unit is stacked with a first electrode, a fluid receiver, a micro-nanostructure membrane, a cell culture well plate and a second electrode from bottom to top; preferably, the first electrode is adhered to the lower bottom surface of the fluid receiver to form a fluid delivery channel with the fluid receiver, the cell culture well on the cell culture well plate is a through hole opened both above and below, the micro-nanostructure membrane is arranged on the lower bottom surface of the cell culture well and the upper opening of the fluid receiver, and the second electrode cover is arranged on the cell culture well plate; preferably, the first electrode is one of an ITO electrode, a titanium electrode, an aluminum electrode or a gold electrode, and the second electrode is one of a Pt electrode, a titanium electrode, an aluminum electrode or a gold electrode.

[0013] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the microfluidic unit is a microfluidic system that forms a concentration gradient based on a mono-phase method or a microfluidic system that forms a concentration gradient based on a droplet-based method.

[0014] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the microfluidic unit includes a tree network structure or a Y-shaped junction structure.

[0015] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the microfluidic unit of the tree-shaped network structure comprises:

[0016] Input microfluidic channels, each of which has a fluid input port at its starting end, and there are at least two input microfluidic channels;

[0017] A multi-stage separation and mixing microfluidic channel, wherein the number of stages of the multi-stage separation and mixing microfluidic channel is n, where n≥1, wherein the first-stage separation and mixing microfluidic channel is connected to the input microfluidic channel; when n≥2, the n-th-stage separation and mixing microfluidic channel is connected to the n-1-th-stage separation and mixing microfluidic channel, and the fluid output port of the n-th-stage separation and mixing microfluidic channel (i.e., the fluid output port of the microfluidic unit) is connected to the delivery unit; the multi-stage separation and mixing microfluidic channel plays the role of separating and mixing fluids step by step; preferably, n=2-5 (for example, n=3, n=4, n=5).

[0018] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, each stage of separation and mixing microchannel includes:

[0019] The fluid separation part includes a plurality of groups of fluid separation branches, which are used to separate the fluid from each input microfluidic channel or the fluid from each fluid output port of the previous separation and mixing microfluidic channel in a specific ratio, and each input microfluidic channel or each fluid output port of the previous separation and mixing microfluidic channel is provided with a group of fluid separation branches, and

[0020] The fluid mixing section includes a plurality of fluid mixing branch flow channels, and adjacent and oppositely flowing fluid separation branches merge into one fluid mixing branch flow channel.

[0021] In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the number of fluid mixing branch channels in the first-stage separation and mixing microfluidic channel is greater than the number of input microfluidic channels; preferably, the number of fluid mixing branch channels in the first-stage separation and mixing microfluidic channel is greater than the number of input microfluidic channels by 1.

[0022] In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the number of fluid mixing branch channels in each level of separation and mixing microfluidic channel is greater than the number of fluid mixing branch channels in the previous level of separation and mixing microfluidic channel; preferably, the number of fluid mixing branch channels in each level of separation and mixing microfluidic channel is greater than the number of fluid mixing branch channels in the previous level of separation and mixing microfluidic channel by 1.

[0023] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the fluid mixing branch channel is a serpentine structure.

[0024] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the delivery device further comprises a substrate, and the microfluidics unit and the delivery unit group are arranged in the substrate.

[0025] Preferably, the substrate includes a bottom plate and a cover plate, the microfluidic unit is arranged on the bottom plate, the fluid receiver is also arranged on the bottom plate, each fluid output port of the microfluidic unit is connected to each fluid receiver, and the fluid receiver is a groove structure formed on the bottom plate; a plurality of the cell culture well plates corresponding to the plurality of the delivery units are integrated on the cover plate, and the cell culture wells are formed on the cover plate; the first electrode is arranged on the lower bottom surface of the fluid receiver, the micro-nanostructure membrane is at least attached to the lower bottom surface of the cell culture well and the upper opening of the fluid receiver, and the second electrode is arranged on the upper surface of the cell culture well plate. A plurality of cell culture well plates are integrated on the cover plate.

[0026] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the substrate material is selected from one of polymethyl methacrylate, polyethylene terephthalate, and polyvinyl chloride; more preferably, the substrate material is polymethyl methacrylate.

[0027] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the input microchannel and the multi-stage separation and mixing microchannel of the microfluidic unit are formed by etching the substrate using laser cutting technology.

[0028] In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred embodiment, the material of the film is polycarbonate.

[0029] In the biomolecule delivery device based on microfluidics and nanotechnology, as an embodiment, the distribution density of the nanotubes in the film is 1×10 6 -5×10 8 / cm 2 (For example, 5×10 6 / cm 2 , 1×10 7 / cm 2 , 5×10 7 / cm 2 ).

[0030] In a biomolecule delivery device based on microfluidics and nanotechnology, as an embodiment, the nanotube has a diameter of 50-800nm ​​(e.g., 100nm, 300nm, 500nm, 700nm) and a height of 0.5-20μm (e.g., 1μm, 5μm, 8μm, 12μm, 15μm).

[0031] In the biomolecule delivery device based on microfluidics and nanotechnology, as an embodiment, the surface component of the nanotube is at least one of SiO2, Al2O3, and HfO2.

[0032] The above-mentioned biomolecule delivery device based on microfluidics and nanotechnology is used in establishing a method for reprogramming hiPSCs without genetic modification residues.

[0033] In the above application, preferably, the types, combinations and concentrations of biomolecules delivered to cells are screened and optimized by the delivery device; and / or the delivery frequency of the biomolecules is screened and optimized.

[0034] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0035] (1) Based on microfluidics and nanotechnology, the present invention obtains biomolecules (including DNA, mRNA, protein, mircoRNA and other small molecules) with different concentration gradients by manipulating microfluids at a microscopic scale, thereby achieving semi-automatic high-throughput screening of the concentration ratio of biomolecules that induce reprogramming, and then finding the optimal reprogramming formula and the optimal or minimum concentration of the mixture of the optimal reprogramming formula for reprogramming;

[0036] (2) In the present invention, by setting a delivery unit group, the fluid output by the microfluidic unit (i.e., the optimal or lowest concentration of biomolecules for reprogramming) is delivered into the cell under the action of the electric field, which can achieve repeated, efficient, quantitative, and continuous long-term delivery of biomolecules into the cell without damaging the cell, and has the characteristics of semi-automation and strong operability;

[0037] (3) In the present invention, the microfluidic unit is arranged into a tree-shaped network structure, including an input microchannel and a multi-stage separation and mixing microchannel that can perform step-by-step diversion and mixing of the fluid in the input microchannel, so as to achieve manipulation of the microfluid at a microscopic scale, thereby obtaining biomolecules with a target concentration gradient, thereby achieving high-throughput screening of biomolecules;

[0038] (4) In the present invention, by further limiting the distribution density, diameter, height and surface chemical composition of the nanotubes in the micro-nanostructure membrane, it is possible to efficiently and accurately deliver biological molecules into cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] In order to describe the present invention more clearly, the present invention is further described here with reference to the accompanying drawings.

[0040] Figure 1 A top view of a biomolecule delivery device based on microfluidics and nanotechnology according to an embodiment of the present invention;

[0041] Figure 2 yes Figure 1 A schematic diagram of the structure of the delivery unit in Part A;

[0042] Figure 3 yes Figure 2 The structural diagram of part B;

[0043] Figure 4 It is a microscopic photograph of the micro-nanostructured membrane;

[0044] Figure 5 is a schematic structural diagram of a microfluidic unit according to an embodiment of the present invention;

[0045] Figure 6 It is a schematic diagram of the separation and mixing process of fluids in a certain stage of separation and mixing microfluidic channel of an embodiment of the present invention;

[0046] Figure 7 is a schematic structural diagram of a bottom plate in an embodiment of the present invention;

[0047] Figure 8 Schematic diagram of the structure of the cover plate in an embodiment of the present invention.

[0048] Explanation of the reference numerals: 1. delivery device; 11. microfluidic unit; 12. delivery unit group; 13. substrate; 111. input microfluidic channel; 1111. first input microfluidic channel; 1112. second input microfluidic channel; 1113. third input microfluidic channel; 112. first stage separation and mixing microfluidic channel; 113. second stage separation and mixing microfluidic channel; 1131. fluid separation branch; 1132. fluid mixing branch channel; 121. first electrode; 122. fluid receiver; 123. micro-nanostructure membrane; 1231. thin film; 1232. nanotube; 124. cell culture well plate; 1241. cell culture well; 125. second electrode; 131. bottom plate; 132. cover plate. DETAILED DESCRIPTION

[0049] 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.

[0050] In the description of the present invention, the terms "longitudinal", "lateral", "upper", "lower", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the accompanying drawings. They are only for the convenience of describing the present invention and do not require that the present invention must be constructed and operated in a specific orientation. Therefore, they should not be understood as limitations on the present invention. The terms "connected" and "connected" used in the present invention should be understood in a broad sense. For example, they can be fixed connections or detachable connections; they can be directly connected or indirectly connected through intermediate components. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0051] The specific embodiment of the present invention provides a biomolecule delivery device 1 based on microfluidics and nanotechnology, and its top view is shown in FIG. Figure 1 , including: a microfluidic unit 11 for accurately controlling the output fluid characteristics, the fluid characteristics include at least one of the fluid composition, concentration and speed; the microfluidic unit 11 can provide one or more formulas of fluid at the same time, with the characteristics of high efficiency, convenience and precision; and also includes a delivery unit group 12, each fluid output port of the microfluidic unit 11 is connected to a delivery unit for delivering the fluid output by the microfluidic unit 11 into the cell. Figure 2-8 The delivery device 1 of the present invention is described in more detail.

[0052] The delivery unit group 12 of the present invention includes one or more delivery units, and each fluid output port of the microfluidic unit 11 is connected to a delivery unit, and each delivery unit includes: a fluid receiver 122, a micro-nanostructure membrane 123 arranged at the upper opening of the fluid receiver 122, and a cell culture well plate 124 arranged on the micro-nanostructure membrane 123; wherein the micro-nanostructure membrane 123 includes a thin film 1231 and a plurality of nanotubes 1232 arranged on the thin film 1231, the nanotubes 1232 pass through the thin film 1231, and the nanotube ports on the nanotubes 1232 close to the side of the fluid receiver 122 are configured to be able to contact the fluid in the fluid receiver 122; the nanotube ports on the side away from the fluid receiver 122 extend out of the thin film 1231 and extend into the cell culture well 1241 of the cell culture well plate 124 so as to be able to contact the cells in the cell culture well 1241.

[0053] Specifically, each delivery unit of the present invention can be configured to include: a fluid receiver 122, a cell culture well plate 124 disposed at the upper opening of the fluid receiver 122; a micro-nano structure membrane 123 is disposed on the lower bottom surface of each cell culture well 1241 of the cell culture well plate 124, and no micro-nano structure membrane 123 is disposed at other positions on the bottom of the cell culture well plate 124, the micro-nano structure membrane 123 separates the cell culture well 1241 from the fluid receiving portion of the fluid receiver 122, and the micro-nano structure membrane 123 includes a thin film 1231 and a plurality of nanotubes 123 disposed on the thin film 1231. 232, the nanotube 1232 passes through the film 1231, and the nanotube port on the nanotube 1232 close to the fluid receiver 122 (i.e., the nanotube lower port) is configured to be able to contact the fluid in the fluid receiver 122, and the nanotube lower port may extend downward from the film 1231 or may not extend from the film 1231, but is flush with the lower surface of the film 1231; the nanotube port on the side away from the fluid receiver 122 (i.e., the nanotube upper port) extends out of the film 1231 and extends into the cell culture hole 1241, so as to be able to contact the cells in the cell culture hole 1241. Figure 2 This solution can save the usage of the micro-nanostructure film 123 and reduce unnecessary waste.

[0054] The micro-nanostructure membrane 123 of each delivery unit of the present invention can also be arranged or adhered to the entire bottom of the cell culture well plate 124, so as to avoid leakage of cell culture solution or fluid.

[0055] The present invention can adopt any technology in the prior art that can transport fluid from one side of the micro-nanostructure membrane 123 to the other side, but in order to ensure the transport effect, the present invention preferably adopts electrical transport, and the delivery unit also includes: a first electrode 121 arranged on the lower bottom surface of the fluid receiver 122, and a second electrode 125 arranged on the cell culture well plate 124, and the lower surface of the second electrode 125 can directly contact the cell culture fluid in the cell culture well 1241.

[0056] Exemplarily, the nanotube port of the nanotube 1232 close to the fluid receiver 122 is flush with the upper surface (i.e., the upper opening) of the fluid receiver 122, and the nanotube port of the nanotube 1232 away from the fluid receiver 122 extends out of the film 1231 and penetrates into the cell culture hole 1241.

[0057] The fluid receiver 122 is used to contain the fluid transmitted by the microfluidic unit 11. The fluid receiver 122 can be a fluid delivery channel. In a certain embodiment of the present invention, it can be understood that the upper wall of the fluid receiver 122 is the lower surface of the micro-nanostructure membrane 123, and its lower wall is the upper surface of the first electrode 121, that is, the main body of the fluid receiver 122 is a groove-shaped structure with upper and lower openings, the upper opening is covered by the micro-nanostructure membrane 123, and the lower opening is covered by the first electrode 121, thereby forming a closed fluid delivery channel, and the fluid from the microfluidic unit 11 directly contacts the first electrode 121. In another embodiment of the present invention, the fluid receiver 122 is a groove-shaped structure with a lower bottom surface, and the first electrode 121 is adhered to the outer surface of the lower bottom surface of the groove-shaped structure.

[0058] The cell culture well plate 124 corresponding to each delivery unit in the present invention is provided with one or more rows of cell culture wells 1241. In the embodiment of the present invention, it is set to one row. The number of cell culture wells 1241 in each row can be set as needed. In a certain embodiment of the present invention, each cell culture well 1241 is a through hole with upper and lower openings, and the lower opening is provided with a micro-nano structure membrane 123 for dividing the fluid receiver 122 and the cell culture well 1241. Thus, the cell culture well 1241 can be filled with cell culture fluid, and the cells are immersed in the cell culture fluid. At the beginning of delivery, the second electrode 125 will cover the upper surface of the cell culture well plate 124 and contact with the upper surface of the cell culture fluid in the cell culture well 1241. The whole system will form a loop, so that the fluid output by the microfluidic unit 11 is delivered to the cell under the action of the electric field. The multiple cell culture well plates 124 in the multiple delivery units can be integrally formed to form a complete cell culture well plate group, see the attached Figure 8 .

[0059] Exemplarily, the first electrode 121 in the present invention may be an ITO electrode, a titanium electrode, an aluminum electrode or a gold electrode, etc., and the first electrode 121 in the present invention is preferably an ITO electrode; the second electrode 125 in the present invention may be a Pt electrode, a titanium electrode, an aluminum electrode or a gold electrode, etc., and the second electrode 125 in the present invention is preferably a Pt electrode.

[0060] In an embodiment of the present invention, each delivery unit is stacked from bottom to top with a first electrode 121, a fluid receiver 122, a micro-nanostructure membrane 123, a cell culture well plate 124 (the lower part of each cell culture well 1241 is an opening, and the micro-nanostructure membrane 123 serves as the bottom wall of the cell culture well 1241) and a second electrode 125; the fluid in the fluid receiver 122 is not leaked upward at all except when voltage is applied and is transported upward through the micro-nanostructure membrane 123.

[0061] That is to say, each delivery unit includes two groups of first electrodes 121 and second electrodes 125 arranged in parallel with each other, and a micro-nanostructure membrane 123 and a cell culture well plate 124 arranged between the first electrode 121 and the second electrode 125, and a fluid receiver 122 (i.e., a fluid delivery channel) is arranged between the micro-nanostructure membrane 123 and the first electrode 121; nanotubes 1232 (i.e., nanoneedle-shaped hollow channels) are distributed on the micro-nanostructure membrane 123 and vertically penetrate the membrane layer, and the cell culture well plate 124 is arranged between the second electrode 125 and the micro-nanostructure membrane 123; the nanotube port located above the micro-nanostructure membrane 123 (i.e., the side close to the second electrode 125) can contact the cells in the cell culture well 1241 of the cell culture well plate 124, and the nanotube port located at or below the lower surface of the micro-nanostructure membrane 123 (i.e., the side close to the first electrode 121) can contact the fluid in the fluid receiver 122. One delivery unit corresponds to a fluid output port of the microfluidic unit 11, and the fluid output port of the microfluidic unit 11 is connected to the fluid input port of the fluid receiver 122. Figure 3 When voltage is applied, the fluid in the fluid receiver 122 is transported to the cells through the nanotubes 1232 of the micro-nanostructure membrane 123, thereby completing the delivery of biological molecules of different formulations.

[0062] Exemplarily, the base material, ie, the original material, of the micro-nanostructure film 123 in the present invention is polycarbonate.

[0063] In the present invention, polycarbonate materials with different pore sizes and densities are selected as the base material of the micro-nanostructure membrane 123, and a layer of aluminum oxide is covered on the surface of the base material and the pores using atomic layer deposition technology. Then, the aluminum oxide on the surface of the base material is removed using reactive ion etching technology. Finally, oxygen plasma etching is used to selectively remove part of the polycarbonate in the base material to prepare nanotubes 1232 (i.e., nano-needle-shaped hollow channels) with different lengths and densities. The base membrane with part of the polycarbonate removed becomes a thin film 1231. In addition to being prepared by the above-mentioned method, the micro-nanostructure membrane 123 composed of the thin film 1231 on which the nanotubes 1232 are distributed used in the present invention can also be prepared by other feasible methods.

[0064] When the cells are urine cells, the distribution density of the nanotubes 1232 (i.e., the nano-needle-shaped hollow channels) in the micro-nanostructure membrane 123 is 1×10 6 -5×10 8 / cm 2 .

[0065] The diameter of the nanotube 1232 is 50-800 nm, and the height is 0.5-20 μm.

[0066] The surface material of the nanotube 1232 is at least one of SiO2, Al2O3, and HfO2.

[0067] In the present invention, by further limiting the distribution density, diameter, height and surface chemical composition of the nanotubes 1232, it is more conducive to the entry of biological molecules (such as DNA, mRNA, protein, mircoRNA and other small molecules) from the nanotubes 1232 into cells under the action of the electric field, thereby achieving efficient and accurate delivery of biological molecules into urine cells.

[0068] The microfluidic unit 11 of the present invention can be a microfluidic system that forms a concentration gradient based on a mono-phase method or a microfluidic system that forms a concentration gradient based on a droplet-based method. It should be noted that the microfluidic unit 11 is used to form biomolecules with different concentration gradients from the initial concentration of biomolecules, to achieve high-throughput screening of biomolecules, and thus find the optimal reprogramming formula; the delivery unit group 12 is used to deliver the biomolecules of the target concentration gradient obtained by the microfluidic unit 11 to the cells in a targeted manner.

[0069] The microfluidic unit 11 includes a tree network structure or a Y-shaped confluence structure or other existing structures that can obtain a variety of concentration gradients or formulas. The microfluidic unit 11 with a tree network structure has the characteristics of simple design and easy concentration calculation, and is suitable for wide use. The present invention focuses on the microfluidic unit 11 with a tree network structure, but the present invention is not limited thereto.

[0070] The microfluidic unit 11 of the tree network structure includes: an input microfluidic channel 111, each of which is provided with a fluid input port at the starting end, and there are at least two input microfluidic channels 111; and a multi-stage separation and mixing microfluidic channel, the number of stages of the multi-stage separation and mixing microfluidic channel is n, n≥1, wherein the first stage separation and mixing microfluidic channel 112 is connected to the input microfluidic channel 111; when n≥2, the nth stage separation and mixing microfluidic channel is connected to the n-1th stage separation and mixing microfluidic channel, and the fluid output port of the nth stage separation and mixing microfluidic channel (i.e., the fluid output port of the microfluidic unit) is connected to the delivery unit; the multi-stage separation and mixing microfluidic channel plays the role of separating and mixing fluids step by step. In the present invention, n is preferably 2-5.

[0071] Each level of separation and mixing microfluidic channel includes: a fluid separation part, including a plurality of groups of fluid separation branches 1131, which are used to separate the fluid from each input microfluidic channel 111 or the fluid from each fluid output port of the previous level separation and mixing microfluidic channel in a specific ratio, and each input microfluidic channel 111 or each fluid output port of the previous level separation and mixing microfluidic channel is correspondingly provided with a group of fluid separation branches 1131; and a fluid mixing part, including a plurality of fluid mixing branch channels 1132, and the adjacent and oppositely flowing fluid separation branches 1131 merge into one fluid mixing branch channel 1132, see Figure 6 . In order to obtain fluids with more gradient concentrations, the number of fluid mixing branch channels 1132 in the first-stage separation and mixing microfluidic channel 112 is greater than the number of input microfluidic channels 111; preferably, the number of fluid mixing branch channels 1132 in the first-stage separation and mixing microfluidic channel 112 is 1 greater than the number of input microfluidic channels 111. The number of fluid mixing branch channels 1132 in each stage of separation and mixing microfluidic channels is greater than the number of fluid mixing branch channels 1132 in the previous stage of separation and mixing microfluidic channels; preferably, the number of fluid mixing branch channels 1132 in each stage of separation and mixing microfluidic channels is 1 greater than the number of fluid mixing branch channels 1132 in the previous stage of separation and mixing microfluidic channels.

[0072] The microfluidic unit 11 of the tree-shaped mesh structure includes: an input microfluidic channel 111 and a multi-stage separation and mixing microfluidic channel connected to the input microfluidic channel 111, and the multi-stage separation and mixing microfluidic channel has at least one stage. Specifically, the input microfluidic channel 111 is connected to the inlet of the first-stage separation and mixing microfluidic channel 112, and the inlet of the second-stage separation and mixing microfluidic channel 113 is connected to the outlet of the first-stage separation and mixing microfluidic channel 112. By analogy, the inlet of the next-stage separation and mixing microfluidic channel is connected to the outlet of the previous-stage separation and mixing microfluidic channel. Starting from the first-stage separation and mixing microfluidic channel 112, in each stage of the separation and mixing microfluidic channel, each fluid flow is split or separated into multiple branch flows at a specific ratio, and the separated branch flows will merge with the adjacent branch flows in the relative flow direction at a specific ratio at the intersection position and flow into the fluid mixing branch channel 1132 of this stage. A new fluid concentration is obtained by step-by-step diversion and merging. At the same time, the concentration of the fluid in the two outermost fluid mixing branch channels 1132 is kept the same as the fluid concentration in the outermost input microchannel 111. After repeated separation, combination and mixing at each level or stage, the number of fluid mixing branch channels 1132 at the next level will increase compared with the number of fluid mixing branch channels 1132 at the previous level, such as increasing by 1, 2 or 3, or even more, gradually increasing the branch channels, thereby producing fluids with higher concentrations.

[0073] In a specific embodiment of the present invention, the microfluidic unit 11 refers to Figure 5 and Figure 6 , each fluid mixing branch channel 1132 at each level is separated into a group of fluid separation branches 1131 at the next level, and each group of fluid separation branches 1131 includes two branches with opposite flow directions. Adjacent fluid mixing branch channels 1132 at this level are separated in the next-level microfluidic channel to form their own fluid separation branch groups. In these two groups of fluid separation branch groups, two adjacent branches from different groups merge to form a fluid mixing branch channel 1132 in the next-level separation and mixing microfluidic channel. The fluid mixing branch channel 1132 is a serpentine structure or a zigzag structure. In a specific embodiment of the present invention, it includes 3 input microfluidic channels 111 and 2-level separation and mixing microfluidic channels, wherein the 3 input microfluidic channels 111 are respectively a first input microfluidic channel 1111, a second input microfluidic channel 1112, and a third input microfluidic channel 1113, see Figure 5, the first-stage separation and mixing microfluidic channel 112 includes 4 fluid mixing branch channels 1132 and 3 groups of fluid separation branches 1131, each group of fluid separation branches 1131 includes two branches with completely opposite flow directions, and in two adjacent groups of fluid separation branches 1131, the adjacent branches merge together to form the fluid mixing branch channels 1132 of the first-stage separation and mixing microfluidic channel 112, and the two outermost fluid separation branches 1131 do not merge with other branches, but directly extend downward to form the two outermost fluid mixing branch channels 1132 of the first stage. Since there are 4 fluid mixing branch channels 1132 in the first-stage separation and mixing microfluidic channel 112, according to the setting principle of the first-stage separation and mixing microfluidic channel, the second-stage separation and mixing microfluidic channel 113 includes 5 fluid mixing branch channels 1132 and 4 groups of fluid separation branches 1131. The microfluidic unit 11 of this embodiment can provide 5 fluids with different characteristics.

[0074] In another embodiment of the present invention, see Figure 1 or Figure 7 The microfluidic unit 11 includes two input microfluidic channels and three separation and mixing microfluidic channels, and the specific structure is not repeated here. The specific number of input microfluidic channels and the number of separation and mixing microfluidic channels can be set according to actual needs.

[0075] In this embodiment, the multi-stage separation and mixing microfluidic channel can also be said to be: each stage of the separation and mixing microfluidic channel (also referred to as a microfluidic channel) includes a plurality of serpentine-structured fluid mixing branch channels 1132 and a plurality of inverted T-shaped fluid separation branches 1131, the horizontal ends of two adjacent inverted T-shaped structures merge and extend downward to form a serpentine-structured (or a structure formed by orderly connecting a plurality of S-shaped bends) fluid mixing branch channel 1132, and the horizontal ends of the inverted T-shaped structures on both sides directly extend downward to form serpentine-structured fluid mixing branch channels 1132.

[0076] The microfluidic unit 11 of the tree network structure requires a pressure driving system to complete the delivery of the fluid. Therefore, the biomolecule delivery device 1 based on microfluidics and nanotechnology preferably also includes a fluid driving system, which is connected to the fluid input port to provide input fluid pressure.

[0077] When the pressure drop and channel cross-section of the fluid mixing branch channel 1132 at each stage or each level are equal, the channel fluid dynamic resistance is proportional to the length of the fluid mixing branch channel 1132. In order to ensure sufficient complete mixing time, there is a corresponding relationship between the critical fluid mixing branch channel length and the flow rate. The length of the fluid mixing branch channel 1132 should be greater than the critical value of the designed flow rate, or in other words, for the specified length of the fluid mixing branch channel 1132, the flow rate should be maintained within a specific range.

[0078] For ease of operation, the delivery device 1 further includes a substrate 13, which includes a bottom plate 131 and a cover plate 132. Figure 7 and 8 The microfluidic unit 11 is arranged on the bottom plate 131, and the microchannel is formed on the bottom plate 131. A plurality of fluid receivers 122 are also arranged on the bottom plate 131. Each fluid output port of the microfluidic unit 11 is connected to each fluid receiver 122 for receiving the fluid flowing out of the microfluidic unit 11. The fluid receiver 122 is a groove structure formed on the bottom plate 131; the plurality of cell culture well plates 124 included in the plurality of delivery units can be centrally arranged on the cover plate 132, that is, the plurality of cell culture well plates 124 are integrated on the cover plate 132, the cover plate 132 is the substrate of the cell culture well plate 124, and the cell culture well 1241 is formed on the cover plate 132; the first electrode 121 is arranged (preferably adhered) on the lower bottom surface of the fluid receiver 122 (or adhered to the lower bottom surface of the bottom plate 131), the micro-nano structure membrane 123 is attached to the lower bottom surface of the cover plate 132 and the upper opening of the fluid receiver 122, and the second electrode 125 is covered on the cell culture well plate 124; the microfluidic unit 11 and the delivery unit group 12 can also be assembled by other methods.

[0079] In the present invention, the microfluidic unit 11 is set into a tree-shaped network structure, and the microchannel network configuration, length, separation ratio, etc. are limited, so as to obtain a combination of biological molecules (including DNA, mRNA, protein, mircoRNA and other small molecules) with different concentration gradients and formulas, which is more conducive to efficiently screening out the optimal inducing reprogramming molecule formula, concentration and other experimental conditions.

[0080] The material of the substrate 13 is selected from one of polymethyl methacrylate, polyethylene terephthalate, and polyvinyl chloride; more preferably, the material of the substrate 13 is polymethyl methacrylate.

[0081] The polymethyl methacrylate, polyethylene terephthalate, and polyvinyl chloride materials selected in the present invention are not only cheap and have low production costs, but also have excellent optical properties, high biocompatibility and gas permeability; in addition, the substrate 13 material selected in the present invention can be fixed to the first electrode 121, thereby forming a closed channel with the delivery unit group 12.

[0082] As the above-mentioned biomolecule delivery device 1 based on microfluidics and nanotechnology, preferably, the input microchannel 111 and the multi-stage separation and mixing microchannel of the microfluidic unit 11 are formed by etching the bottom plate 131 by laser cutting technology; preferably, the cell culture hole 1241 is formed by etching the cover plate 132 by laser cutting technology. The laser cutting technology used in the present invention has the advantages of fine cutting cuts and smooth cutting surfaces due to the small laser spot, and has the advantages of good cutting size and high cutting accuracy. In the present invention, a microfluidic channel, a fluid receiver 122 and a residual fluid collection channel (fluid flowing through the fluid receiver 122) with a tree-shaped mesh structure are etched on the bottom plate 131, and then a cell culture hole 1241 and a corresponding fluid inlet (also called a fluid input port) and a residual fluid collection port are cut on the cover plate 132. Finally, the first electrode 121, the bottom plate 131, the micro-nanostructure membrane 123, the cover plate 132 and the second electrode 125 are assembled, and at the same time, it is ensured that the fluid inlet of the input microfluidic channel 111 on the bottom plate 131 corresponds to the fluid input port on the cover plate 132, the fluid receiver 122 on the bottom plate 131 corresponds to the cell culture hole 1241 on the cover plate 132, and the residual fluid collection channel on the bottom plate 131 corresponds to the residual fluid collection port on the cover plate 132, thereby forming a delivery device 1.

[0083] The above-mentioned biomolecule delivery device 1 based on microfluidics and nanotechnology is used in the establishment of a method for reprogramming hiPSCs without genetic modification residues. The delivery device 1 is used to screen and optimize the types, combinations and concentrations of biomolecules delivered to cells; and / or the delivery frequency of the biomolecules.

[0084] In the present invention, a microfluidic unit 11 is provided, which has a microchannel with a size similar to that of microvessels in the body, which is conducive to achieving high-throughput, low-sample-volume, and low-reagent-consumption fluid control, thereby solving the problem that the traditional macro-scale concentration generation method is difficult to achieve precise flow control; a plurality of input microchannels 111 are provided at the starting end of the microfluidic unit 11, which can input initial biomolecules of different concentrations and combinations, and obtain biomolecules of various concentration gradients and formulations by changing the length, separation ratio, cross-sectional area and network configuration of the microchannel, thereby achieving high-throughput screening of biomolecules; in the present invention, a delivery unit group 12 is provided, and the cell first First, it will be closely attached to the micro-nano structure membrane 123 by wall adhesion, and then the biomolecules of the target concentration gradient will contact the port of the nanotube 1232 in the micro-nano structure membrane 123 through the fluid receiver 122, and finally, under the action of the electric field, the biomolecules of the target concentration gradient will pass through the nanotube 1232 and enter the cell in a directional manner; based on microfluidics and nanotechnology, the present invention can manipulate microfluids at a microscopic size to obtain biomolecules of different concentration gradients, and then under the action of the electric field, the biomolecules of the target concentration gradient are delivered into the cell by the delivery unit, so as to achieve efficient, quantitative, and continuous long-term delivery of biomolecules to the cell. Since the delivery device 1 has high-throughput screening capabilities, it can simultaneously deliver molecules of multiple concentrations and multiple formulas, and can be accurately, conveniently, quickly, and efficiently used to screen the optimal induction reprogramming molecular formula, thereby facilitating the establishment of a hiPSCs preparation method with high reprogramming efficiency and no genetic modification residues, and improving the reprogramming efficiency in the hiPSCs preparation process.

[0085] The preparation method of the delivery device 1 of the present invention is as follows:

[0086] S1. Preparation of microfluidic unit 11, fluid receiver 122 and residual fluid collection channel on bottom plate 131: Select polymethyl methacrylate (PMMA) plate as the material of bottom plate 131, and use laser cutting technology to etch the PMMA plate to form microchannels with specific length, partition, direction and configuration to prepare microfluidic unit 11, refer to Figure 7 The microfluidic unit 11 includes two input microfluidic channels 111 and three separation and mixing microfluidic channels. The microfluidic unit 11 eventually forms five fluid output ports. At the position corresponding to each fluid output port of the microfluidic unit 11, the PMMA plate is etched into a rectangular groove (i.e., a fluid receiver 122) with a specific length and width and a residual fluid collection channel connected to the rectangular groove by laser cutting technology; the PMMA plate is etched into a structure in which the microfluidic unit 11, the fluid receiver 122 and the residual fluid collection channel are sequentially connected by laser cutting technology;

[0087] S2. Preparation of cell culture wells 1241, fluid input ports, and residual fluid collection ports on the cover plate 132: A polymethyl methacrylate (PMMA) plate is selected as the cover plate 132 material, and double-sided tape is adhered to one surface of the PMMA plate. The PMMA plate is cut into multiple rows of cell culture wells 1241 (5 rows), fluid input ports (2 ports), and residual fluid collection port (1 port) by laser cutting technology, see Figure 8 , each row of cell culture wells 1241 corresponds to the size and position of the rectangular groove (i.e., the fluid receiver 122) in the bottom plate 131, the fluid input port corresponds to the position of the fluid inlet of the input microchannel 111 in the bottom plate 131, and the residual fluid collection port corresponds to the position of the residual fluid collection channel in the bottom plate 131; the PMMA plate is etched into the structure of the cell culture wells 1241, the fluid input port and the residual fluid collection port by laser cutting technology;

[0088] S3. Preparation of micro-nanostructure membrane 123: Select pore size of 50-800 nm and distribution density of 1x10 6 -5x10 8 / cm 2 The polycarbonate material is used as the base material of the micro-nanostructure membrane 123, and a layer of aluminum oxide is coated on the surface of the base material and the pores by atomic layer deposition technology, and then the aluminum oxide on the surface of the base material is removed by reactive ion etching technology, and finally, oxygen plasma etching is used to selectively remove part of the polycarbonate in the base material to prepare a microstructure membrane with a diameter of 50-800nm, a height of 0.5-20μm, and a distribution density of 1x10 6 -5x10 8 / cm 2 The nanotube 1232 is formed so that the port at one end of the nanotube extends out of the micro-nanostructure film 123 of the base material;

[0089] S4. Integrated delivery device 1: First, adhere the first electrode 121 to the lower bottom surface of each rectangular groove (i.e., fluid receiver 122) of the base plate 131, and then adhere the side of the nanotube port in the micro-nanostructure membrane 123 extending out of the base material to the side of the cover plate 132 with double-sided tape, so that the nanotube 1232 can extend into the cell culture hole 1241, and then bond the surface of the base plate 131 etched with the microfluidic unit 11 to the corresponding position of the surface of the cover plate 132 adhered with the micro-nanostructure membrane 123, ensuring that the fluid input port in the cover plate 132 is aligned with the fluid inlet of the input microchannel 111 in the base plate 131 during the bonding process, and the residual fluid collection port in the cover plate 132 corresponds to the residual fluid collection channel in the base plate 131, and finally place the second electrode 125 on top of the cover plate 132 to form a delivery device 1.

[0090] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention are within the scope of protection of the pending claims of the present invention.

Claims

1. A biomolecule delivery device based on microfluidics and nanotechnology, characterized in that: include: A microfluidic unit for precisely controlling output fluid characteristics, wherein the fluid characteristics include at least one of fluid composition, concentration, and velocity; and a delivery unit group, wherein each fluid output port of the microfluidic unit is connected to a delivery unit for delivering the fluid output by the microfluidic unit into the cell: The microfluidic unit is a microfluidic system that forms a concentration gradient based on a unidirectional method or a microfluidic system that forms a concentration gradient based on a droplet method; The delivery unit comprises: a fluid receiver, a micro-nano structure membrane disposed at an upper opening of the fluid receiver, and a cell culture well plate disposed on the micro-nano structure membrane; The micro-nanostructure membrane includes a thin film and a plurality of nanotubes arranged on the thin film, wherein the nanotubes pass through the thin film, and the nanotube ports on the nanotubes close to the fluid receiver are configured to be able to contact the fluid in the fluid receiver; the nanotube ports on the side away from the fluid receiver extend out of the thin film and into the cell culture wells of the cell culture well plate so as to be able to contact the cells in the cell culture wells.

2. The biomolecule delivery device according to claim 1, characterized in that The delivery unit further includes: a first electrode disposed below the fluid receiver, and a second electrode disposed above the cell culture well plate.

3. The biomolecule delivery device according to claim 2, characterized in that: The delivery unit is stacked with a first electrode, a fluid receiver, a micro-nanostructure membrane, a cell culture well plate and a second electrode in sequence from bottom to top.

4. The biomolecule delivery device according to claim 3, characterized in that The first electrode is adhered to the lower bottom surface of the fluid receiver to form a fluid delivery channel with the fluid receiver. The cell culture well on the cell culture well plate is a through hole with openings at the top and bottom. The micro-nanostructure membrane is arranged on the lower bottom surface of the cell culture well and the upper opening of the fluid receiver. The second electrode cover is arranged on the cell culture well plate.

5. The biomolecule delivery device according to claim 2, characterized in that: The first electrode is one of an ITO electrode, a titanium electrode, an aluminum electrode or a gold electrode, and the second electrode is one of a Pt electrode, a titanium electrode, an aluminum electrode or a gold electrode.

6. The biomolecule delivery device according to any one of claims 1 to 5, characterized in that: The microfluidic unit includes a tree-shaped network structure or a Y-shaped confluence structure.

7. The biomolecule delivery device according to claim 6, characterized in that: The microfluidic unit of the tree-shaped network structure comprises: Input microfluidic channels, each of which has a fluid input port at its starting end, and there are at least two input microfluidic channels; A multi-stage separation and mixing microfluidic channel, wherein the number of stages of the multi-stage separation and mixing microfluidic channel is n, where n≥1, wherein the first-stage separation and mixing microfluidic channel is connected to the input microfluidic channel; when n≥2, the n-th-stage separation and mixing microfluidic channel is connected to the n-1-th-stage separation and mixing microfluidic channel, and the fluid outlet of the n-th-stage separation and mixing microfluidic channel is connected to the delivery unit; the multi-stage separation and mixing microfluidic channel plays the role of separating and mixing fluids step by step.

8. The biomolecule delivery device according to claim 7, characterized in that: n＝2-5。 9. The biomolecule delivery device according to claim 7 or 8, characterized in that: Each stage of separation and mixing microfluidic channel includes: The fluid separation part includes a plurality of groups of fluid separation branches, which are used to separate the fluid from each input microfluidic channel or the fluid from each fluid output port of the previous separation and mixing microfluidic channel in a specific ratio, and each input microfluidic channel or each fluid output port of the previous separation and mixing microfluidic channel is provided with a group of fluid separation branches, and The fluid mixing section includes a plurality of fluid mixing branch flow channels, and adjacent fluid separation branches with opposite flow directions merge into one fluid mixing branch flow channel.

10. The biomolecule delivery device according to claim 9, characterized in that The number of fluid mixing branch flow channels in the first-stage separation and mixing microfluidic channel is greater than the number of input microfluidic channels.

11. The biomolecule delivery device according to claim 10, characterized in that: The number of fluid mixing branch flow channels in the first-stage separation and mixing microfluidic channel is greater than the number of input microfluidic channels by one.

12. The biomolecule delivery device according to claim 9, characterized in that: The number of fluid mixing branch flow channels in each level of separation and mixing microfluidic channel is greater than the number of fluid mixing branch flow channels in the previous level of separation and mixing microfluidic channel.

13. The biomolecule delivery device according to claim 12, characterized in that: The number of fluid mixing branch flow channels in each level of separation and mixing microfluidic channel is one greater than the number of fluid mixing branch flow channels in the previous level of separation and mixing microfluidic channel.

14. The biomolecule delivery device according to claim 9, characterized in that: The fluid mixing branch channel is a serpentine structure.

15. The biomolecule delivery device according to claim 7 or 8, characterized in that: The delivery device further comprises a substrate, wherein the microfluidic unit and the delivery unit group are arranged in the substrate.

16. The biomolecule delivery device according to claim 15, characterized in that The substrate includes a bottom plate and a cover plate, the microfluidic unit is arranged on the bottom plate, the fluid receiver is also arranged on the bottom plate, each fluid output port of the microfluidic unit is connected to each fluid receiver, and the fluid receiver is a groove structure formed on the bottom plate; multiple cell culture well plates corresponding to multiple delivery units are integrated on the cover plate, and the cell culture wells are formed on the cover plate; the first electrode is arranged on the lower bottom surface of the fluid receiver, the micro-nanostructure membrane is at least attached to the lower bottom surface of the cell culture well and the upper opening of the fluid receiver, and the second electrode is arranged on the upper surface of the cell culture well plate.

17. The biomolecule delivery device according to claim 15, characterized in that The substrate material is selected from one of polymethyl methacrylate, polyethylene terephthalate and polyvinyl chloride.

18. The biomolecule delivery device according to claim 17, characterized in that The substrate material is polymethyl methacrylate.

19. The biomolecule delivery device according to claim 15, characterized in that The input microchannel and the multi-stage separation and mixing microchannel of the microfluidic unit are formed by etching a substrate using a laser cutting technique.

20. The biomolecule delivery device according to claim 15, characterized in that The material of the film is polycarbonate.

21. The biomolecule delivery device according to claim 15, characterized in that The distribution density of the nanotubes in the film is 1×10 6 -5×10 8 / cm 2 .

22. The biomolecule delivery device according to claim 15, characterized in that The nanotube has a diameter of 50-800 nm and a height of 0.5-20 μm.

23. The biomolecule delivery device according to claim 15, characterized in that The surface component of the nanotube is at least one of SiO2, Al2O3 and HfO2.

24. Use of a biomolecule delivery device based on microfluidics and nanotechnology as claimed in any one of claims 1 to 23 in establishing a method for preparing hiPSCs by reprogramming without genetic modification residues.

25. The use according to claim 24, characterized in that The delivery device is used to screen and optimize the types, combinations and concentrations of biomolecules delivered to cells; and / or the delivery frequency of the biomolecules is screened and optimized.

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