CN217173764U - Biomolecule delivery device based on micro-fluidic and nano technology - Google Patents

Abstract

The utility model provides a biomolecule delivery device based on micro-fluidic and nano-technology, which comprises a micro-fluidic unit for precisely controlling the output fluid characteristics, wherein the fluid characteristics comprise at least one of fluid components, concentration and speed; each fluid output port of the microfluidic unit is communicated with one delivery unit for delivering the fluid output by the microfluidic unit into a cell; the utility model discloses a biomolecule delivers the device and has high flux screening ability, can carry out the molecule of multiple concentration, multiple prescription simultaneously and deliver, can be accurate, convenient, swift, be used for screening optimum induced reprogramming molecule prescription high-efficiently to do benefit to and establish the efficient no heritage of reprogramming and decorate remaining hiPSCs preparation method.

Description

Biomolecule delivery device based on micro-fluidic and nano technology

Technical Field

The utility model belongs to the technical field of micro-fluidic technique and nanotechnology are used, concretely relates to biomolecule based on micro-fluidic and nanotechnology delivers device.

Background

Human induced pluripotent stem cells (hiPSCs) are pluripotent stem cells formed by dedifferentiation of adult mammalian cells by means of transfer of transcription factors and the like, and were first discovered in 2006 by a research team extended in japan scholars. Most of the current methods for inducing hiPSCs are to transfect DNA information including Oct4, Sox2, Klf4 and c-Myc transcription factors into somatic cells by using viral vectors or plasmids, improve the expression level of the four transcription factors in the cells and induce the reprogramming of the somatic cells, thereby enabling the somatic cells to obtain a pluripotent state. The induction method of the hiPSCs does not need to use ova or embryos, so that the hiPSCs have great potential medical value in disease mechanism research, new drug screening and personalized cell replacement therapy.

Currently, biomolecule introduction technology is the main technical means for preparing hiPSCs, and can be divided into integrated viral vector induction, non-integrated viral induction, transposable element induction, mRNA and protein induction according to the introduction mechanism and the kind of introduced molecules. However, these induction methods have problems such as high safety risk or low reprogramming efficiency (0.001% to 10%). Among these, the safety risk is mainly due to the risk of genetic mutations resulting from random insertion of DNA sequences into the host genome in virus-related induction methods, whether or not induced by genome integration. Non-viral vectors are limited by the fact that current transfection methods (including transposable element vectors, proteins, plasmid vectors, and mRNA) do not guarantee delivery of an equal amount of DNA molecules into each cell, and transfection experiments are highly toxic to cells, so that each cell after transfection expresses transcription factors with far-ranging efficiency, but generally low.

At present, the regenerative medicine field considers that establishing a preparation method of hiPSCs (human iPSCs) without genetic modification residues (such as reprogramming human somatic cells into iPS cells by using mRNA and protein) is an effective means for avoiding the safety risk of introducing virus-derived vectors. In order to completely eliminate the integration risk of exogenous genes, establishing a traceless hiPSCs preparation strategy by using mRNA transcription factors or in-vitro purified protein reprogramming transcription factors is considered to be a preparation method with the minimum safety risk at present. In 2010, scientists were the first to successfully complete the preparation of hiPSCs from mRNA (Warren, l.et al. high effective reproduction to complex and directed differentiation of human cells with synthetic modified mRNA. Cell Stem Cell 7, 618-. It has also been demonstrated by scholars that mouse and human fibroblast reprogramming can be successfully induced using reprogramming proteins. However, mRNA and protein have a limited life cycle in cells, requiring repeated delivery of the relevant biomolecules into the cells. However, the current intracellular delivery means (including electroporation technology and liposome transfection) are limited, and the cell can not be completely delivered in a multiple, real-time and long-term mode without damage, so the reprogramming efficiency by using the mRNA and protein molecule induction method is extremely low, and the practical application can not be carried out. The research and development of a novel non-viral intracellular delivery method can realize repeated, long-term, efficient, real-time and cell-free intracellular delivery, and the establishment of a novel, efficient and genetically-modified hiPSCs preparation strategy is the key to the realization of clinical application of hiPSCs.

In summary, there is an urgent need to develop a novel efficient delivery technology for intracellular induction factors such as mRNA, protein and other biomolecules to achieve efficient and quantitative repeated long-term delivery of biomolecules into cells.

SUMMERY OF THE UTILITY MODEL

The utility model aims at providing a biomolecule based on micro-fluidic and nanotechnology delivers device, this device is based on micro-fluidic technology and the nanotechnology of micro-fluidic of micro-control and nanotechnology under the micro-scale, can realize that repetition, high efficiency, ration, continuous long period deliver biomolecule (for example DNA, mRNA or protein) to the cell in and not damaged to have semi-automatization, the strong characteristic of maneuverability. Reprogramming transcription factor combination ratio, expression quantity, and expression cycle continuity can directly influence cell reprogramming efficiency, but because the experimental conditions restriction, the test process of the experimental conditions optimization and reagent ratio of reprogramming is loaded down with trivial details, time consumption is huge at present, and the utility model discloses delivery device has high flux screening ability, can carry out the molecule delivery of multiple concentration, multiple prescription simultaneously, can be used for screening optimum induced reprogramming molecule prescription accurately, conveniently, swiftly, high-efficiently to do benefit to and establish the high-efficient no-genetic-modification residual hiPSCs preparation method of reprogramming.

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

a microfluidic cell for precisely controlling output fluid characteristics including at least one of fluid composition, concentration and velocity; and

and each fluid output port of the microfluidic unit is communicated with one delivery unit for delivering the fluid output by the microfluidic unit into the cell.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the delivery unit includes: the device comprises a fluid receiver, a micro-nano structure membrane arranged on an upper opening of the fluid receiver and a cell culture pore plate arranged on the micro-nano structure membrane; the micro-nano structure film comprises a film and a plurality of nanotubes arranged on the film, the nanotubes penetrate through the film, and nanotube ports on one side of the nanotubes, which are close to the fluid receiver, are arranged to be capable of contacting fluid in the fluid receiver; the nanotube port on the side away from the fluid receptacle extends out of the membrane and into the cell culture wells of the cell culture well plate so as to be able to contact cells within the cell culture wells.

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the delivery unit further includes: a first electrode disposed below the fluid receptacle, and a second electrode disposed above the cell culture well plate.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, a first electrode, a fluid receiver, a micro-nano structure film, a cell culture pore plate and a second electrode are sequentially stacked from bottom to top in the delivery unit; 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 hole on the cell culture hole plate is a through hole with openings at the upper and lower sides, the micro-nano structure film is arranged on the lower bottom surface of the cell culture hole and the upper opening of the fluid receiver, and the second electrode is covered on the cell culture hole 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.

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferable mode, the microfluidic unit is a microfluidic system for forming a concentration gradient based on a mono-phase method or a microfluidic system for forming a concentration gradient based on a droplet-based method.

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the microfluidic unit comprises a tree network structure or a Y-type confluence structure.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the microfluidic unit of the tree network structure comprises:

the starting end of each input micro-channel is provided with a fluid input port, and the number of the input micro-channels is at least 2;

the multistage separation mixing microchannel is provided with the number of stages n, wherein n is more than or equal to 1, and the 1 st stage separation mixing microchannel is communicated with the input microchannel; when n is more than or equal to 2, the nth-level separation mixing microchannel is communicated with the nth-1-level separation mixing microchannel, and the fluid output port of the nth-level separation mixing microchannel (namely the fluid output port of the microfluidic unit) is communicated with the delivery unit; the multistage separation mixing micro-channel plays a role in separating and mixing fluid stage by stage; preferably, n-2-5 (e.g., n-3, n-4, n-5).

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, each stage of separating the mixed microchannels comprises:

A fluid separation section including a plurality of sets of fluid separation branches for separating a fluid from each input microchannel or a fluid from each fluid output port of a previous-stage separation mixing microchannel in a specific ratio, a set of fluid separation branches being provided for each fluid output port of each input microchannel or previous-stage separation mixing microchannel, and

the fluid mixing part comprises a plurality of fluid mixing branch channels, and adjacent fluid separation branches in opposite directions are converged into one fluid mixing branch channel.

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the number of fluid mixing branch channels in the 1 st separation mixing microchannel is larger than that of input microchannels; preferably, the number of fluid mixing branch channels in the 1 st-stage separation mixing microchannel is 1 greater than the number of input microchannels.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the number of fluid mixing branch channels in each stage of separation mixing microchannel is greater than that of fluid mixing branch channels in the previous stage of separation mixing microchannel; preferably, the number of the fluid mixing branch channels in each stage of the separation and mixing microchannel is 1 greater than that in the previous stage of the separation and mixing microchannel.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the fluid mixing branch flow channel is a serpentine structure.

In the biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the delivery device further comprises a substrate, and the microfluidic unit and the delivery unit group are arranged in the substrate.

Preferably, the substrate comprises a bottom plate and a cover plate, the microfluidic unit is arranged on the bottom plate, the fluid receivers are also arranged on the bottom plate, each fluid output port of the microfluidic unit is communicated with each fluid receiver, and the fluid receivers are in groove-shaped structures formed on the bottom plate; a plurality of cell culture well plates corresponding to the plurality of delivery units are integrally arranged 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-nano structure membrane is at least attached to the lower bottom surface of the cell culture hole and the upper opening of the fluid receiver, and the second electrode is arranged on the upper surface of the cell culture hole plate. A plurality of cell culture well plates are integrated on the cover plate.

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

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, an input micro-channel and a multi-stage separation mixed micro-channel of a microfluidics unit are formed by etching a substrate through a laser cutting technology.

In a biomolecule delivery device based on microfluidics and nanotechnology, as a preferred mode, the material of the thin film is polycarbonate.

In the biomolecule delivery device based on microfluidics and nanotechnology, as one embodiment, the distribution density of the nanotubes in the thin film is 1 × 10 ⁶ -5×10 ⁸ /cm ² (e.g., 5X 10) ⁶ /cm ² 、1×10 ⁷ /cm ² 、 5×10 ⁷ /cm ² )。

In a microfluidic and nanotechnology based biomolecule delivery device, as an embodiment, the nanotubes have a diameter of 50-800nm (e.g., 100nm, 300nm, 500nm, 700nm) and a height of 0.5-20 μm (e.g., 1 μm, 5 μm, 8 μm, 12 μm, 15 μm).

In the biomolecule delivery device based on microfluidics and nanotechnology, as one embodiment, the surface component of the nanotube is SiO ₂ 、Al ₂ O ₃ 、HfO ₂ At least one of (1).

The application of the biomolecule delivery device based on microfluidics and nanotechnology in establishing a method for preparing hiPSCs without genetic modification residues through reprogramming.

In the above application, preferably, the species, combination and concentration of biomolecules that are optimally delivered into the cell are screened by the delivery device; and/or screening to optimize the frequency of delivery of the biomolecule.

Compared with the prior art, the utility model discloses an advantage and beneficial effect lie in:

(1) the utility model discloses in based on micro-fluidic and nanotechnology, through controlling the microfluid under the micro-size, obtain the biomolecule of different concentration gradients (including DNA, mRNA, protein, mircoRNA and other micromolecules) to realize that semi-automatic high flux selects the concentration ratio of induced reprogramming biomolecule, and then find out the mixed liquid of optimum reprogramming prescription and be used for the optimum or minimum concentration of reprogramming;

(2) the delivery unit group is arranged in the utility model, so that the fluid (i.e. the optimal or lowest concentration biomolecule for reprogramming) output by the microfluidic unit is delivered into the cell under the action of the electric field, the repeated, high-efficiency, quantitative and continuous long-period delivery of the biomolecule into the cell without damage to the cell can be realized, and the delivery unit group has the characteristics of semi-automation and strong operability;

(3) The utility model discloses in through setting up the micro-fluidic unit to tree network structure, including input micro-channel and can play the multi-stage separation mixing micro-channel of reposition of redundant personnel and mixing action step by step to the fluid in the input micro-channel, realize controlling the microfluid under the micro-size to can obtain the biomolecule of target concentration gradient, and then realize the high flux screening to biomolecule;

(4) the utility model discloses in through the further injecing to distribution density, diameter, height and the surface chemical composition of the nanotube in the micro-nano structure membrane, can realize high-efficient accurately with biomolecule delivery to the cell.

Drawings

To describe the present invention more clearly, the present invention is further explained herein with reference to the accompanying drawings. Wherein:

fig. 1 is a top view of a biomolecule delivery device based on microfluidics and nanotechnology in accordance with an embodiment of the present invention;

FIG. 2 is a schematic view of the structure of the relevant delivery unit of section A of FIG. 1;

FIG. 3 is a schematic structural view of portion B of FIG. 2;

FIG. 4 is a photomicrograph of a micro-nanostructured film;

fig. 5 is a schematic structural diagram of a microfluidic unit according to an embodiment of the present invention;

FIG. 6 is a schematic diagram of the separation and mixing process of the fluid in a certain stage of the separation and mixing microchannel according to the embodiment of the present invention;

Fig. 7 is a schematic structural diagram of a bottom plate in an embodiment of the present invention;

fig. 8 is a schematic structural diagram of a cover plate in an embodiment of the present invention.

Description of the reference numerals: 1. a delivery device; 11. a microfluidic cell; 12. a group of delivery units; 13. a substrate; 111. inputting a micro-channel; 1111. a first input microchannel; 1112. a second input microchannel; 1113. a third input microchannel; 112. separating and mixing the first stage micro flow channel; 113. separating and mixing the second-stage micro flow channel; 1131. A fluid separation branch; 1132. a fluid mixing branch flow channel; 121. a first electrode; 122. a fluid receptacle; 123. a micro-nano structure film; 1231. a film; 1232. a nanotube; 124. a cell culture well plate; 1241. a cell culture well; 125. a second electrode; 131. a base plate; 132. and (7) a cover plate.

Detailed Description

The technical solutions in the embodiments of the present invention will be described clearly and completely with reference to the accompanying drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments in the present invention, all other embodiments obtained by a person skilled in the art without creative work belong to the protection scope of the present invention.

In the description of the present invention, the terms "longitudinal", "lateral", "up", "down", "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 drawings, and are only for convenience of description of the present invention and do not require that the present invention must be constructed and operated in a particular orientation, and therefore, should not be construed as limiting the present invention. The terms "connected" and "connected" used in the present invention should be understood in a broad sense, and may be, for example, either fixed or detachable; they may be directly connected or indirectly connected through intermediate members, and specific meanings of the above terms will be understood by those skilled in the art as appropriate.

The utility model discloses a specific embodiment provides a biomolecule based on micro-fluidic and nanotechnology and delivers device 1, its top view is seen in figure 1, include: a microfluidic cell 11 for precisely controlling output fluid characteristics including at least one of fluid composition, concentration, and velocity; the microfluidic unit 11 can provide one or more formulas of fluid at the same time, and has the characteristics of high efficiency, convenience and accuracy; and the system also comprises a delivery unit group 12, wherein each fluid output port of the microfluidic unit 11 is communicated with one delivery unit for delivering the fluid output by the microfluidic unit 11 into the cell. The delivery device 1 of the present invention is described in more detail below with reference to figures 2-8.

The delivery unit group 12 of the present invention comprises one or more delivery units, each fluid outlet of the microfluidic unit 11 is connected to one delivery unit, and each delivery unit comprises: the device comprises a fluid receiver 122, a micro-nano structure membrane 123 arranged on the upper opening of the fluid receiver 122 and a cell culture pore plate 124 arranged on the micro-nano structure membrane 123; the micro-nano structure film 123 comprises a film 1231 and a plurality of nanotubes 1232 arranged on the film 1231, the nanotubes 1232 penetrate through the film 1231, and nanotube ports on the nanotubes 1232, which are close to one side of the fluid receiver 122, are configured to be capable of contacting fluid in the fluid receiver 122; the nanotube port on the side away from fluid sink 122 extends out of membrane 1231 and into cell culture well 1241 of cell culture well plate 124 so as to be able to contact cells in cell culture well 1241.

Specifically, each delivery unit of the present invention may be configured to include: a fluid receptacle 122, a cell culture well plate 124 disposed at an upper opening of the fluid receptacle 122; the micro-nano structure membrane 123 is arranged on the lower bottom surface of each cell culture hole 1241 of the cell culture hole plate 124, the micro-nano structure membrane 123 is not arranged at other positions of the bottom of the cell culture hole plate 124, the cell culture hole 1241 is separated from the fluid accommodating part of the fluid receiver 122 by the micro-nano structure membrane 123, the micro-nano structure membrane 123 comprises a film 1231 and a plurality of nanotubes 1232 arranged on the film 1231, the nanotubes 1232 penetrate through the film 1231, a nanotube port (namely a nanotube lower port) on one side of the nanotube 1232, which is close to the fluid receiver 122, is arranged to be capable of contacting fluid in the fluid receiver 122, and the lower port of the nanotube 123can extend downwards out of the film 1231 or not extend out of the film 1231 but is flush with the lower surface of the film 1231; the nanotube port on the side away from fluid sink 122 (i.e., the nanotube upper port) extends out of membrane 1231 and into cell culture well 1241 so as to be able to contact cells within cell culture well 1241. Referring to fig. 2, the scheme can save the usage amount of the micro-nano structure film 123 and reduce unnecessary waste.

The micro-nano structure film 123 of each delivery unit of the utility model can also be arranged or attached to the whole bottom of the cell culture well plate 124, thus avoiding the leakage of the cell culture solution or the fluid.

The utility model discloses can adopt among the prior art any can be with the fluid transport to the technique of opposite side of micro-nano structure membrane 123 one side, nevertheless in order to guarantee to carry the effect, the utility model discloses preferred adoption electrotransport, delivery unit still includes: a first electrode 121 disposed on the bottom surface of the fluid receptacle 122, and a second electrode 125 disposed on the upper surface of the cell culture well plate 124, wherein the lower surface of the second electrode 125 can directly contact the cell culture solution in the cell culture well 1241.

Illustratively, the nanotube port on the side of the nanotube 1232 proximal to the fluid receptacle 122 is flush with the upper surface (i.e., the upper opening) of the fluid receptacle 122, and the nanotube port on the side of the nanotube 1232 distal to the fluid receptacle 122 protrudes through the membrane 1231 and into the cell culture well 1241.

The fluid receiver 122 is used for receiving the fluid transmitted by the microfluidic unit 11, and the fluid receiver 122 may 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-nano structure film 123, and the lower wall thereof 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 an upper opening and a lower opening, the upper opening is covered by the micro-nano structure film 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 with the first electrode 121. In another embodiment of the present invention, the fluid receiver 122 is a groove structure having a lower bottom surface, and the first electrode 121 is adhered to an outer surface of the lower bottom surface of the groove structure.

The cell culture well plate 124 corresponding to each delivery unit of the utility model is provided with one or more rows of cell culture holes 1241, in the embodiment of the present invention, the cells are arranged in a row, the number of the cell culture holes 1241 in each row can be set according to the requirement, in one embodiment of the present invention, each cell culture hole 1241 is a through hole with an upper opening and a lower opening, the lower opening is provided with a micro-nano structure film 123 for dividing the fluid receiver 122 and the cell culture hole 1241, thus, the cell culture medium can be filled in the cell culture hole 1241, the cells are immersed in the cell culture medium, at the beginning of delivery, the second electrode 125 will cover the upper surface of the cell culture well plate 124 and contact the upper surface of the cell culture fluid in the cell culture well 1241, and the whole system will form a loop, so that the fluid outputted through the microfluidic cell 11 is delivered into the cell under the action of the electric field. The plurality of cell culture well plates 124 in the plurality of delivery units may be integrally formed to form a complete cell culture well plate set, see fig. 8.

For example, 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 of the present invention can be a Pt electrode, a titanium electrode, an aluminum electrode, or a gold electrode, etc., and the second electrode 125 is preferably a Pt electrode.

In the embodiment of the present invention, each delivery unit is sequentially stacked from bottom to top with a first electrode 121, a fluid receiver 122, a micro-nano structure film 123, a cell culture well plate 124 (the lower part of each cell culture well 1241 is an opening, and the micro-nano structure film 123 serves as the well 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 for the fluid being transported upward through the micro-nano structure film 123 under the applied voltage.

That is, each delivery unit comprises two groups of first electrodes 121 and second electrodes 125 arranged in parallel, and a micro-nano structure film 123 and a cell culture pore plate 124 arranged between the first electrodes 121 and the second electrodes 125, wherein a fluid receiver 122 (i.e. a fluid delivery channel) is arranged between the micro-nano structure film 123 and the first electrodes 121; nanotubes 1232 (namely, a nano needle-shaped hollow channel) which vertically penetrate through the film layer are distributed on the micro-nano structure film 123, and the cell culture pore plate 124 is arranged between the second electrode 125 and the micro-nano structure film 123; a nanotube port located above the micro-nano structured membrane 123 (i.e., on the side near the second electrode 125) may contact cells in the cell culture well 1241 of the cell culture well plate 124, and a nanotube port located at or below the lower surface of the micro-nano structured membrane 123 (i.e., on the side near the first electrode 121) may contact fluid of the fluid receptacle 122. One delivery unit corresponds to one fluid output port of the microfluidic unit 11, and the fluid output port of the microfluidic unit 11 is communicated with the fluid input port of the fluid receiver 122. Referring to fig. 3, when a voltage is applied, the fluid in the fluid receiver 122 is transported to the cell through the nanotubes 1232 of the micro-nano structure film 123, thereby completing the transport of biomolecules with different formulations.

Exemplarily, the base material, i.e., the original material of the micro-nano structure film 123 in the present invention is polycarbonate.

The utility model discloses in as the base material of micro-nano structure membrane 123 through the polycarbonate material of selecting different aperture sizes and density, utilize atomic layer deposition technique to cover one deck aluminium oxide on base material surface and pore, then utilize the reaction ion etching technique to remove the aluminium oxide on base material surface, thereby utilize oxygen plasma etching selectivity to remove partial polycarbonate in the base material at last and prepare the nanotube 1232 (the acicular hollow channel of nanometer promptly) that has different length, density, the basement membrane of having got rid of partial polycarbonate has become film 1231, the utility model discloses a micro-nano structure membrane 123 that it has the film 1231 of nanotube 1232 to constitute promptly on it can also adopt other feasible methods to prepare except adopting above-mentioned method preparation.

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

The nanotubes 1232 have a diameter of 50 to 800nm and a height of 0.5 to 20 μm.

The surface material of the nanotube 1232 is SiO ₂ 、Al ₂ O ₃ 、HfO ₂ At least one of (1).

The utility model discloses in through the further injeciton to distribution density, diameter, height and the surface chemical composition of nanotube 1232, be favorable to the biomolecule (for example DNA, mRNA, protein, mircoRNA and other small molecules) to enter into the cell from nanotube 1232 under the electric field effect more, realize high-efficient accurately deliver the biomolecule to urine intracellularly.

The utility model discloses micro-fluidic unit 11 can be based on the micro-fluidic system of mono-phase method formation concentration gradient or based on the micro-fluidic system of droplet method formation concentration gradient. It should be noted that the microfluidic unit 11 is configured to form biomolecules with different concentration gradients from biomolecules with initial concentrations, so as to implement high-throughput screening of biomolecules, and thus find out an optimal reprogramming formula; the delivery unit set 12 is used for targeted delivery of biomolecules with a target concentration gradient obtained via the microfluidic unit 11 into the cell.

The microfluidic cell 11 includes a tree network structure or a Y-junction structure or other existing structures that can obtain various concentration gradients or formulations. The microfluidic unit 11 with the tree-shaped mesh structure has the characteristics of simple design and easy concentration calculation, and is suitable for wide application. The utility model discloses the tree network structure's micro-fluidic unit 11 is introduced to the well focus, nevertheless the utility model discloses be not limited to this.

The microfluidic cell 11 of the tree network structure includes: the starting end of each input micro-channel 111 is provided with a fluid input port, and the number of the input micro-channels 111 is at least 2; and a multi-stage separation mixing microchannel, the number of stages of the multi-stage separation mixing microchannel is n, n is more than or equal to 1, wherein the 1 st separation mixing microchannel 112 is communicated with the input microchannel 111; when n is more than or equal to 2, the nth-level separation mixing microchannel is communicated with the nth-1-level separation mixing microchannel, and the fluid output port of the nth-level separation mixing microchannel (namely the fluid output port of the microfluidic unit) is communicated with the delivery unit; the multi-stage separation mixed micro-channel plays a role in separating and mixing fluid stage by stage. In the present invention, n is preferably 2 to 5.

Each stage of separating and mixing micro flow channel comprises: the fluid separation part comprises a plurality of sets of fluid separation branches 1131, and is used for separating the fluid from each input micro-channel 111 or the fluid from each fluid output port of the previous-stage separation mixing micro-channel in a specific proportion, and each fluid output port of each input micro-channel 111 or the previous-stage separation mixing micro-channel is correspondingly provided with one set of fluid separation branches 1131; and a fluid mixing portion including a plurality of fluid mixing sub-channels 1132, and the adjacent and opposite flow direction fluid separation branches 1131 merge into one fluid mixing sub-channel 1132, see fig. 6. In order to obtain more fluid with gradient concentration, the number of the fluid mixing sub-channels 1132 in the 1 st-stage separation mixing microchannel 112 is larger than that of the input microchannel 111; preferably, the number of the fluid mixing sub-channels 1132 in the 1 st-stage separating and mixing micro-channel 112 is 1 larger than the number of the input micro-channels 111. The number of the fluid mixing branch channels 1132 in each stage of separation mixing micro channel is greater than that of the fluid mixing branch channels 1132 in the previous stage of separation mixing micro channel; preferably, the number of the fluid mixing branch flow channels 1132 in each stage of the separation and mixing micro flow channel is 1 larger than the number of the fluid mixing branch flow channels 1132 in the last stage of the separation and mixing micro flow channel.

The microfluidic cell 11 of the tree network structure includes: the multi-stage separation mixing microchannel comprises an input microchannel 111 and a multi-stage separation mixing microchannel communicated with the input microchannel 111, wherein the stage number of the multi-stage separation mixing microchannel is at least 1 stage, specifically, the input microchannel 111 is connected with an inlet of a 1 st stage separation mixing microchannel 112, an inlet of a 2 nd stage separation mixing microchannel 113 is connected with an outlet of the 1 st stage separation mixing microchannel 112, and by analogy, an inlet of a next stage separation mixing microchannel is connected with an outlet of a previous stage separation mixing microchannel. Starting from the first-stage separation mixing microchannel 112, in each stage of separation mixing microchannel, each fluid flow is split or separated into a plurality of branch flows at a specific ratio, and the separated branch flows join with the adjacent branch flows of the opposite flow direction at a crossing point position at a specific ratio and flow into the fluid mixing branch channel 1132 of the stage. And the new fluid concentration is obtained by stepwise diversion and confluence. While the concentration of the fluid in the two outermost fluid mixing sub-channels 1132 remains the same as the concentration of the fluid in the outermost input micro-channel 111. Through repeated separation, combination, and mixing at each stage or stage, the number of fluid mixing sub-channels 1132 for the next stage may be increased from the number of fluid mixing sub-channels 1132 for the previous stage, such as by 1, 2, or 3, or even more, by gradually increasing the number of branch channels, thereby producing a greater concentration of fluid.

In a specific embodiment of the present invention, referring to fig. 5 and fig. 6, each fluid mixing branch channel 1132 of each level is separated into a set of fluid separation branches 1131 at the next level, each set of fluid separation branches 1131 includes two branches with opposite flow directions, adjacent fluid mixing branch channels 1132 in this level are separated into respective fluid separation branch groups in the next level micro-channel, and in these two sets of fluid separation branch groups, two adjacent branches from different groups are merged to form one fluid mixing branch channel 1132 in the next level separation mixing micro-channel. The fluid mixing sub-channel 1132 has a serpentine or zigzag structure. In a specific embodiment of the present invention, the separation mixing micro flow channel includes 3 input micro flow channels 111 and 2 levels, wherein the 3 input micro flow channels 111 are respectively a first input micro flow channel 1111, a second input micro flow channel 1112, and a third input micro flow channel 1113, referring to fig. 5, the 1 st level separation mixing micro flow channel 112 includes 4 fluid mixing sub flow channels 1132 and 3 sets of fluid separation sub flow channels 1131, each set of fluid separation sub flow channel 1131 includes two branches with completely opposite flow directions, in the two adjacent sets of fluid separation sub flow channels 1131, the adjacent branches merge together to form the fluid mixing sub flow channel 1132 of the 1 st level separation mixing micro flow channel 112, and the two fluid separation sub flow channels 1131 located at the outermost side do not merge with other branches, but directly extend downward to form the two fluid mixing sub flow channels 1132 at the outermost side of the 1 st level. Since there are 4 fluid mixing sub-channels 1132 in the 1 st separating and mixing microchannel 112, according to the setting principle of the 1 st separating and mixing microchannel, the 2 nd separating and mixing microchannel 113 includes 5 fluid mixing sub-channels 1132 and 4 sets of fluid separating branches 1131. The microfluidic cell 11 of this embodiment can provide 5 different characteristics of the fluid.

In another embodiment of the present invention, referring to fig. 1 or fig. 7, the microfluidic unit 11 includes 2 input microchannels and 3 separation mixing microchannels, and the detailed structure is not described herein. Specifically, several input microchannels and several stages of separating and mixing microchannels can be set according to actual needs.

In this embodiment, the multi-stage separation mixing microchannel can be said to be: each of the separating and mixing microchannels (also referred to as microchannels) includes a plurality of fluid mixing sub-channels 1132 having serpentine structures and a plurality of fluid separating branches 1131 having inverted T-shaped structures, the horizontal ends of two adjacent inverted T-shaped structures are merged and extend downward to form the fluid mixing sub-channel 1132 having a serpentine structure (or a structure formed by orderly connecting a plurality of S-shaped curves), and the horizontal ends of the inverted T-shaped structures located at both sides respectively extend downward to form the fluid mixing sub-channels 1132 having serpentine structures.

The microfluidic cells 11 of the tree network structure require a pressure driven system to accomplish the fluid transfer. Therefore, the microfluidic and nanotechnology based biomolecule delivery device 1 preferably further comprises a fluid driving system connected to the fluid input port to impart pressure to the input fluid.

When the pressure drop and the flow channel cross-section of the fluid mixing sub-flow channel 1132 at each stage or each level are equal, the flow channel hydrodynamic resistance is proportional to the length of the fluid mixing sub-flow channel 1132, and in order to ensure sufficient thorough mixing time, the critical fluid mixing sub-flow channel length is in correspondence with the flow rate, and the length of the fluid mixing sub-flow channel 1132 should be greater than the critical value of the designed flow rate, or the flow rate should be maintained within a specific range for the limited length of the fluid mixing sub-flow channel 1132.

For convenience of operation, the delivery device 1 further includes a substrate 13, the substrate 13 includes a bottom plate 131 and a cover plate 132, referring to fig. 7 and 8, the microfluidic units 11 are arranged on the bottom plate 131, the microchannels are formed on the bottom plate 131, the bottom plate 131 is further provided with a plurality of fluid receivers 122, each fluid output port of the microfluidic unit 11 is communicated with each fluid receiver 122 for receiving the fluid flowing out from the microfluidic unit 11, and the fluid receivers 122 are groove-shaped structures formed on the bottom plate 131; the plurality of cell culture well plates 124 included in the plurality of delivery units may be collectively 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 a 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 on the lower bottom surface of the bottom plate 131), the micro-nano structure film 123 is adhered on 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 assembly of the microfluidic cell 11 with the delivery unit group 12 may also be achieved by other methods.

The utility model discloses in through setting micro-fluidic unit 11 to tree-like network structure, to injecing such as microchannel network configuration, length, separation ratio to obtain the biomolecule combination (including DNA, mRNA, albumen, mircoRNA and other micromolecules) of different concentration gradients and prescription, it more does benefit to the high efficiency and selects optimum induced reprogramming molecule prescription, concentration and other experimental conditions etc..

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

The polymethyl methacrylate, the polyethylene glycol terephthalate and the polyvinyl chloride materials selected in the utility model are not only cheap, but also have excellent optical characteristics, high biocompatibility and gas permeability; furthermore, the material of the substrate 13 selected in the present invention can be fixed to the first electrode 121, so as to form a closed channel with the delivery unit 12.

As in the above-mentioned biomolecule delivery device 1 based on microfluidics and nanotechnology, preferably, the input micro-channel 111 and the multi-stage separation mixing micro-channel of the microfluidic unit 11 are formed by etching the bottom plate 131 by using a laser cutting technology; preferably, the cell culture holes 1241 are formed by etching the cover plate 132 by a laser cutting technique. The laser cutting technology used in the utility model has the advantages of narrow laser cutting cut, smooth cutting surface, good cutting size and high cutting precision due to small laser facula; the utility model discloses in through the micro-flow channel of the tree-like network structure of sculpture on bottom plate 131, fluid receiver 122 and the fluid collection runner of remaining (the fluid that flows through fluid receiver 122), then cut cell culture hole 1241 and corresponding fluid inlet (also called fluid input port) and the fluid collection mouth of remaining on apron 132, assemble first electrode 121, bottom plate 131, micro-nano structure membrane 123 and apron 132 and second electrode 125 at last, guarantee simultaneously that the fluid import of input micro-flow channel 111 on bottom plate 131 corresponds with the fluid input port on apron 132, fluid receiver 122 on bottom plate 131 corresponds with the cell culture hole 1241 on apron 132, the fluid collection runner of remaining on bottom plate 131 corresponds with the fluid collection mouth of remaining on apron 132, thereby form and deliver device 1.

The application of the biomolecule delivery device 1 based on microfluidics and nanotechnology in establishing a method for preparing hiPSCs without genetic modification residues through reprogramming. Screening the species, combination and concentration of biomolecules for optimal delivery into cells by the delivery device 1; and/or screening to optimize the frequency of delivery of the biomolecule.

The micro-fluidic unit 11 is provided with the micro-channel with the size close to that of a micro-blood vessel in a body, so that the fluid control with high flux, low sample volume and low reagent consumption is realized, and the problem that the traditional macroscopic scale concentration generation method is difficult to realize accurate flow control is solved; the starting end of the micro-fluidic unit 11 is provided with a plurality of input micro-channels 111, which can input initial biomolecules with different concentrations and combinations, and obtain biomolecules with different concentration gradients and formulas by changing the length, separation ratio, cross-sectional area of the channels and network configuration, thereby realizing high-throughput screening of the biomolecules; in the utility model, by arranging the delivery unit group 12, the cell can be closely attached to the micro-nano structure membrane 123 through the wall-attaching mode, then the biomolecule with the target concentration gradient can be contacted with 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 biomolecule with the target concentration gradient can pass through the nanotube 1232 and enter the cell in an oriented manner; the utility model discloses in based on micro-fluidic and nanotechnology, can control the micro-fluid under the micro-scale, obtain the biomolecule of different concentration gradients, then under the electric field effect, enter into the cell with the biomolecule of target concentration gradient under the delivery of delivery unit, realize high-efficient, ration, continuous long period with biomolecule deliver to the cell in. The delivery device 1 has high-throughput screening capacity, can simultaneously carry out molecule delivery of various concentrations and formulas, and can be accurately, conveniently, quickly and efficiently used for screening optimal induced reprogramming molecular formulas, so that the method for preparing the hiPSCs without genetic modification residues with high reprogramming efficiency is favorably established, and the reprogramming efficiency in the preparation process of the hiPSCs is improved.

The preparation method of the delivery device 1 of the utility model is as follows:

s1, preparing the microfluidic cell 11, the fluid receiver 122 and the residual fluid collecting channel on the bottom plate 131: selecting a polymethyl methacrylate (PMMA) plate as a material of the bottom plate 131, etching the PMMA plate to form micro channels with specific length, partition, direction and configuration by using a laser cutting technology to prepare the microfluidic unit 11, referring to fig. 7, the microfluidic unit 11 includes 2 input micro channels 111 and 3 separation mixing micro channels, the microfluidic unit 11 finally forms 5 fluid output ports, and the PMMA plate is etched into rectangular grooves (i.e., fluid receivers 122) with specific length and width and residual fluid collecting channels communicated with the rectangular grooves by using the laser cutting technology at positions corresponding to each fluid output port of the microfluidic unit 11; etching the PMMA plate into a structure in which the microfluidic control unit 11, the fluid receiver 122 and the residual fluid collecting flow channel are sequentially communicated by a laser cutting technology;

s2, preparing cell culture holes 1241, fluid input ports and residual fluid collection ports on the cover plate 132: selecting a polymethyl methacrylate (PMMA) plate as a material of the cover plate 132, adhering a double-sided adhesive tape on one surface of the PMMA plate, and cutting a plurality of rows of cell culture holes 1241(5 rows), fluid input ports (2) and residual fluid collection ports (1) in the PMMA plate by using a laser cutting technique, referring to fig. 8, each row of cell culture holes 1241 corresponds to the size and position of a rectangular groove (i.e., the fluid receiver 122) in the bottom plate 131, the fluid input ports correspond to the fluid inlet positions of the input microchannels 111 in the bottom plate 131, and the residual fluid collection ports correspond to the residual fluid collection channels in the bottom plate 131; etching the PMMA plate into a cell culture hole 1241, a fluid input port and a residual fluid collecting port by a laser cutting technology;

S3, preparing the micro-nano structure film 123: selecting pore size of 50-800nm and distribution density of 1x10 ⁶ -5x10 ⁸ /cm ² The polycarbonate material is used as a substrate material of the micro-nano structure film 123, a layer of alumina is covered on the surface and pore channels of the substrate material by utilizing an atomic layer deposition technology, then the alumina on the surface of the substrate material is removed by utilizing a reactive ion etching technology, and finally partial polycarbonate in the substrate material is selectively removed by utilizing oxygen plasma etching so as to prepare the polycarbonate material with the diameter of 50-800nm, the height of 0.5-20 mu m and the distribution density of 1x10 ⁶ -5x10 ⁸ /cm ² The nanotube 1232, namely, the micro-nano structure film 123 with a port at one end of the nanotube extending out of the substrate material;

s4, integrated delivery device 1: first, the first electrode 121 is correspondingly adhered to the lower bottom surface of each rectangular groove (i.e., the fluid receiver 122) of the bottom plate 131, then, one surface of the micro-nano structure film 123, which extends out of the base material, is correspondingly adhered to one surface of the cover plate 132, which is provided with the double-sided adhesive tape, so that the nano tubes 1232 can extend into the cell culture holes 1241, then, the surface of the bottom plate 131, on which the micro-fluidic unit 11 is etched, is adhered to the corresponding position of the surface of the cover plate 132, on which the micro-nano structure film 123 is adhered, so as to ensure that the fluid input port of the cover plate 132 is aligned with the fluid inlet of the input micro-channel 111 of the bottom plate 131 in the adhering process, and the residual fluid collection port of the cover plate 132 corresponds to the residual fluid collection channel of the bottom plate 131, and finally, the second electrode 125 is placed above the cover plate 132, thereby forming the delivery device 1.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, but rather as the following claims are intended to cover all modifications, equivalents, and improvements falling within the spirit and principles of the present invention.

Claims (18)

1. A biomolecule delivery device based on microfluidics and nanotechnology, comprising:

a microfluidic cell for precisely controlling output fluid characteristics including at least one of fluid composition, concentration and velocity;

and each fluid output port of the microfluidic unit is communicated with one delivery unit for delivering the fluid output by the microfluidic unit into the cell.

2. The biomolecule delivery device of claim 1, wherein the delivery unit comprises: the device comprises a fluid receiver, a micro-nano structure membrane arranged on an upper opening of the fluid receiver and a cell culture pore plate arranged on the micro-nano structure membrane; the micro-nano structure film comprises a film and a plurality of nanotubes arranged on the film, the nanotubes penetrate through the film, and nanotube ports on one side of the nanotubes, which are close to the fluid receiver, are arranged to be capable of contacting fluid in the fluid receiver; the nanotube port on the side away from the fluid receptacle extends out of the membrane and into the cell culture wells of the cell culture well plate so as to be able to contact cells within the cell culture wells.

3. The biomolecule delivery device of claim 2, wherein the delivery unit further includes: a first electrode disposed below the fluid receptacle, and a second electrode disposed above the cell culture well plate.

4. The biomolecule delivery device of claim 3, wherein the delivery unit is provided with a first electrode, a fluid receiver, a micro-nano structure film, a cell culture well plate and a second electrode in a stacked manner in sequence from bottom to top; 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 hole in the cell culture hole plate is a through hole with openings at the upper part and the lower part, the micro-nano structure film is arranged on the lower bottom surface of the cell culture hole and the upper opening of the fluid receiver, and the second electrode is covered on the cell culture hole plate.

5. The biomolecule delivery device of claim 4, wherein the microfluidic cell is a unidirectional-method-based concentration gradient microfluidic system or a droplet-method-based concentration gradient microfluidic system.

6. The biomolecule delivery device of claim 5, wherein the microfluidic cell comprises a tree network structure or a Y-junction structure.

7. The biomolecule delivery device of claim 6, wherein the microfluidic cells of the tree network structure comprise:

the starting end of each input micro-channel is provided with a fluid input port, and the number of the input micro-channels is at least 2;

the multistage separation mixing microchannel is provided with the number of stages n, wherein n is more than or equal to 1, and the 1 st stage separation mixing microchannel is communicated with the input microchannel; when n is more than or equal to 2, the nth-level separation mixing microchannel is communicated with the nth-1-level separation mixing microchannel, and the fluid output port of the nth-level separation mixing microchannel is communicated with the delivery unit; the multi-stage separation mixed micro-channel plays a role in separating and mixing fluid stage by stage.

8. The biomolecule delivery device according to claim 7, wherein the number of stages n of the multi-stage separation mixing microchannel is 2 to 5.

9. The biomolecule delivery device of claim 7, wherein each stage of separating the mixing microchannels comprises:

a fluid separation section including a plurality of sets of fluid separation branches for separating a fluid from each input microchannel or a fluid from each fluid output port of a previous-stage separation mixing microchannel in a specific ratio, a set of fluid separation branches being provided for each fluid output port of each input microchannel or previous-stage separation mixing microchannel, and

The fluid mixing part comprises a plurality of fluid mixing branch flow passages, and adjacent fluid separation branches in opposite flow directions are converged into one fluid mixing branch flow passage.

10. The biomolecule delivery device of claim 9, wherein the number of the fluid mixing branch channels in the level 1 separation mixing microchannel is larger than the number of the input microchannels.

11. The biomolecule delivery device of claim 10, wherein the number of the fluid mixing sub-channels in the level 1 separation mixing micro-channel is 1 greater than the number of the input micro-channels.

12. The biomolecule delivery device of claim 9, wherein the number of the fluid mixing sub-channels in each stage of the separation and mixing micro-channel is greater than the number of the fluid mixing sub-channels in the previous stage of the separation and mixing micro-channel.

13. The biomolecule delivery device of claim 12, wherein the number of the fluid mixing sub-channels in each stage of the separation mixing micro-channel is 1 greater than the number of the fluid mixing sub-channels in the previous stage of the separation mixing micro-channel.

14. The biomolecule delivery device of claim 9, wherein the fluid mixing sub-flow channel is a serpentine structure.

15. The biomolecule delivery device of claim 9, further comprising a substrate, the microfluidic cell and the set of delivery cells being disposed in the substrate.

16. The biomolecule delivery device of claim 15, wherein the substrate includes a base plate and a cover plate, the microfluidic units are disposed on the base plate, the fluid receptacles are also disposed on the base plate, each fluid output port of the microfluidic units is communicated with each fluid receptacle, and the fluid receptacles are groove-type structures formed on the base plate; a plurality of cell culture well plates corresponding to the plurality of delivery units are integrally arranged 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-nano structure membrane is at least attached to the lower bottom surface of the cell culture hole and the upper opening of the fluid receiver, and the second electrode is arranged on the upper surface of the cell culture hole plate.

17. The biomolecule delivery device according to claim 16, wherein the input micro flow channel and the multi-stage separation mixing micro flow channel of the micro flow control unit are formed by etching a substrate by a laser cutting technique.

18. The biomolecule delivery device of claim 2, wherein the distribution density of the nanotubes in the thin film is 1 x 10 ⁶ -5×10 ⁸ /cm ² (ii) a The diameter of the nanotube is 50-800nm, and the height is 0.5-20 μm.

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