Integrated bio-automated reaction chip and its application method

Abstract

This invention provides an integrated bio-automated reaction chip. Specifically, it includes a reaction device unit, a first control device, a second control device, and a housing (1); the reaction device unit includes: a first storage chamber (2), a second storage chamber (3), a reaction chamber (4), and an outlet (14) located at the bottom of the reaction chamber; and a thin-film structure that keeps the storage chambers, reaction chamber, and outlet in a fluidly non-communicating state. The invention also includes a method for in vitro cell-free protein synthesis using the reaction chip.

Description

[0001] This case is a divisional application of an earlier application, which was filed on April 28, 2018, with application number 201810404885X, and the invention title is: Array-type integrated bio-automatic reaction chip and its usage method. Technical Field

[0002] This invention relates to the field of biotechnology, and more specifically to an automated reaction chip for biological reactions and a method for in vitro cell-free protein synthesis using the reaction chip. Background Technology

[0003] Microfluidics, one of the most important cutting-edge technologies of this century, plays a vital role in fields such as biology, chemistry, and medicine, gradually becoming the fluid "chip" in the hands of scientists. Microfluidic chips possess characteristics such as controllable fluidity, low sample loss, easy integration, rapid reaction, and low cost, and have wide applications in the in vitro synthesis, separation, and detection of biomolecules. Microfluidic chips can be fabricated from various types of materials, such as polydimethylsiloxane (PDMS), 3D printing materials, glass, paper-based materials, and plastics. We can utilize different material preparation methods to create reaction chips with diverse structures and functions, and we can select appropriate preparation materials according to different types of biological reactions. However, reaction chips with multifunctional regions often require strictly standardized and specialized operation by professionals, along with relevant specialized equipment. Furthermore, biological reactions typically involve numerous reaction raw materials and reagents; multi-step experimental operations and repetitive multi-set experiments increase the professional requirements of operators and also increase the probability of operational errors.

[0004] Therefore, the development of integrated bio-automatic reaction and detection chips is the future trend of microfluidic chip development, in order to reduce the professional requirements during chip use, make them easy to operate, improve reaction and detection efficiency, and facilitate the commercialization of such chips.

[0005] In summary, there is an urgent need in this field to develop an integrated reaction chip that can simultaneously perform multiple reproducible experiments (e.g., start the reaction, add reagents, etc.) with a single manual operation. This would effectively reduce human error in reproducible experiments or allow for the simultaneous and simple synthesis of multiple different proteins through an in vitro cell-free system. Summary of the Invention

[0006] The purpose of this invention is to develop an integrated reaction chip that can simultaneously perform multiple reproducible experiments (e.g., start the reaction, add reagents, etc.) with a single manual operation. This can effectively reduce human error in reproducible experiments or simultaneously and easily synthesize multiple different proteins in an in vitro cell-free system.

[0007] A first aspect of the present invention provides an array-type integrated bio-automated reaction chip, comprising a reaction device unit, a first control device capable of simultaneously activating each of the reaction device units, a second control device, and a housing;

[0008] The reaction device unit is located inside the outer shell, and the first control device and the second control device are movably fixed to the outer shell.

[0009] The reaction apparatus unit includes: a first storage chamber, a second storage chamber, a reaction chamber, and an outlet located at the bottom of the reaction chamber; a first membrane structure is provided between the first storage chamber and the second storage chamber, a second membrane structure is provided between the second storage chamber and the reaction chamber, and a third membrane structure is provided between the reaction chamber and the outlet; each membrane structure is used to ensure that the storage chambers, the reaction chamber, and the outlet are in a fluid-disconnected state; and the first storage chamber is located above the second storage chamber, and the second storage chamber is located above the reaction chamber;

[0010] The first control device includes: a first puncture structure and a first linkage structure; wherein, the first puncture structure is fixed on the first linkage structure and the linkage structure is used to achieve linkage control, and the first puncture structure is used to destroy the first film structure and the second film structure, so that the first storage chamber and the second storage chamber, and the second storage chamber and the reaction chamber are changed from a state of non-fluid communication to a state of fluid communication.

[0011] The second control device includes a second puncture structure and a second linkage structure; wherein the second puncture structure is fixed on the first linkage structure and the linkage control is achieved through the second linkage structure, and the second puncture structure is used to destroy the third membrane structure, so that the reaction chamber and the outlet change from a state of non-fluid communication to a state of fluid communication.

[0012] In another preferred embodiment, the number of the first puncture structure and / or the second puncture structure is equal to the number of the reaction device units and they correspond one-to-one.

[0013] In another preferred embodiment, the reaction chip is used for the in vitro synthesis of cell-free proteins.

[0014] In another preferred embodiment, the first storage chamber is provided with a first inlet, the second storage chamber is provided with a second inlet, and the reaction chamber is provided with a third inlet.

[0015] In another preferred embodiment, the reaction chip further includes a first sealing cap that can be sealed to the first injection port and the third injection port, and the first sealing cap has a thin film structure at its center; and / or

[0016] The reaction chip also includes a second sealing cap that can be sealed to the second injection port.

[0017] In another preferred embodiment, the first puncture structure is also used to disrupt the thin film structure on the first sealing cap that is sealed to the first injection port; and / or

[0018] The second puncture structure is also used to disrupt the thin film structure on the first sealing cap that is sealed to the third injection port.

[0019] In another preferred embodiment, the various reaction device units, the first sealing cap, and the second sealing cap are manufactured using 3D printing integral molding technology.

[0020] In another preferred embodiment, after each sample inlet in the reaction device unit of the reaction chip is connected to its corresponding sealing cap, each reaction device unit is in a sealed state before the first control device is turned on.

[0021] In another preferred embodiment, the thin film structure, the first thin film structure, and the second thin film structure on the first sealing cap, which is sealed to the first injection port, are partially or entirely located on the same axis; and / or

[0022] The thin film structure on the first sealing cap, which is sealed to the third injection port, and the third thin film are partially or entirely located on the same axis.

[0023] In another preferred embodiment, the first puncture structure includes: a cylindrical body and a pointed structure; the pointed structure is disposed at one end of the cylindrical body, and the other end of the cylindrical body is connected to the first linkage structure; and / or

[0024] The second puncture structure includes: a column and a pointed structure; the pointed structure is located at one end of the column, and the other end of the column is connected to the second linkage structure.

[0025] In another preferred embodiment, the reaction device unit further includes an internal pressure balancing device.

[0026] In another preferred embodiment, the internal pressure balancing device is a vent pipe, which is located between the first storage chamber and the reaction chamber.

[0027] In another preferred embodiment, the connection method between the sealing cap and the injection port is selected from the group consisting of: threaded engagement structure, plug seal structure, snap-on seal structure, rotary snap-on seal structure, or a combination thereof.

[0028] In another preferred embodiment, the first sealing cap is connected to the first injection port and / or the third injection port by a rotary snap-on sealing structure;

[0029] The structure includes: two or more (2-6) first boss structures disposed on the first injection port and / or the third injection port, and a first empty rail A, a first empty rail B and a first baffle disposed on the first sealing cover and corresponding to the first boss structures;

[0030] The first protrusion can move along the first empty rail A and the first empty rail B, and the maximum rotation angle of the sealing cover is limited by the first baffle.

[0031] In another preferred embodiment, the second sealing cap is connected to the second sample inlet by a rotary snap-on sealing structure;

[0032] The structure includes: two or more (e.g., 2-6) second protrusion structures provided at the second injection port, and a second air rail A, a second air rail B, and a second baffle provided on the second sealing cover and corresponding to the second protrusion structures;

[0033] The second protrusion can move along the second empty rail A and the second empty rail B, and the maximum rotation angle of the sealing cover is limited by the second baffle.

[0034] In another preferred embodiment, the first or second control device is movably fixed to the housing by a sheet structure mounted inside the housing; when no additional force is applied to the control device, the control device is fixed to the housing; when an additional force is applied to the control device, the control device moves downward, and each of the puncture structures therein simultaneously moves downward and destroys the corresponding thin film structure.

[0035] In another preferred embodiment, the sheet structure comprises 3 to 8 sheet structure units uniformly distributed around a cylinder.

[0036] In another preferred embodiment, the bottom surfaces of the first storage chamber, the second storage chamber, and / or the reaction chamber are inclined structures with an inclination angle of 20 to 40°.

[0037] In another preferred embodiment, a shaking device is also provided inside the housing, and / or a shaking device is also provided outside the reaction chip.

[0038] In another preferred embodiment, the thickness of the thin film structure is 20~1000 μm; preferably, it is 60~300 μm.

[0039] In another preferred embodiment, the volume of the first storage chamber is 1 mL to 50 mL, the volume of the second storage chamber is 100 µL to 1000 µL, and / or the volume of the reaction chamber is 2 mL to 80 mL.

[0040] In another preferred embodiment, the first storage chamber in each reaction unit may or may not contain reagents or raw materials for the reaction.

[0041] In another preferred embodiment, the second storage chamber in each reaction unit may or may not contain reagents or raw materials for the reaction.

[0042] In another preferred embodiment, the reaction chambers in each reaction unit may or may not contain reagents or raw materials for the reaction.

[0043] The present invention also provides a method for in vitro cell-free protein synthesis, comprising the steps of:

[0044] (1) Provide a reaction chip as described in the first aspect; wherein,

[0045] Each reaction unit's first storage chamber contains the same or different buffer solutions;

[0046] Each reaction unit's second storage chamber contains a liquid containing DNA and / or RNA, which may contain the same or different DNA and / or RNA; and

[0047] The reaction chambers in each reaction unit have the same or different biological reaction raw materials;

[0048] (2) Press the first control device to simultaneously allow the buffer solution and liquid containing DNA and / or RNA in each reaction device unit to flow into the corresponding reaction chamber and mix with the biological reaction raw materials in the reaction chamber to form a reaction system and carry out the reaction;

[0049] (3) After the reaction is completed, a liquid containing the same or different proteins is formed. Press the second control device to simultaneously cause the liquid containing the same or different proteins in each reaction unit to flow out, and obtain a liquid containing the synthesized target protein.

[0050] In another preferred embodiment, the first storage chambers in each reaction unit contain the same buffer solution;

[0051] The second storage chamber in each reaction unit contains a liquid containing DNA and / or RNA of different types; and / or

[0052] The reaction chambers in each reaction unit have the same biological reaction raw materials.

[0053] In another preferred embodiment, the liquid containing DNA and / or RNA may be added to the second storage chamber of each reaction unit before the reaction, or the liquid containing DNA and / or RNA may be stored in the second storage chamber of each reaction unit during chip manufacturing.

[0054] In another preferred embodiment, the buffer solution may be added to the first storage chamber of each reaction unit before the reaction, or the buffer solution may be stored in the first storage chamber of each reaction unit during chip manufacturing.

[0055] In another preferred embodiment, the bioreactor material may be added to the reaction chamber of each reaction unit before the reaction, or the bioreactor material may be present in the reaction chamber of each reaction unit during chip manufacturing.

[0056] In another preferred embodiment, the liquid containing DNA and / or RNA is added through a second inlet.

[0057] In another preferred embodiment, the buffer solution is added through a first injection port.

[0058] In another preferred embodiment, bioreactor feedstock is added through a third injection port.

[0059] In another preferred embodiment, the DNA and / or RNA is an exogenous DNA and / or RNA molecule used to guide protein synthesis; preferably, it is selected from DNA encoding luciferin, green fluorescent protein, yellow fluorescent protein, or combinations thereof.

[0060] In another preferred embodiment, the buffer solution is a buffer solution in which inorganic salts, nucleoside triphosphates and amino acids are dissolved in a basic buffer solution.

[0061] In another preferred embodiment, the basic buffer solution is selected from: 5-100 mM 4-hydroxyethylpiperazine ethanesulfonic acid solution, 1-100 mM creatine phosphate solution, 0.1-5 mM dithiothreitol solution, 0.1wt%-10wt% polyethylene glycol (PEG) 3350 or (PEG) 8000 solution, 0.1wt%-5wt% aqueous solution of sucrose, or combinations thereof.

[0062] In another preferred embodiment, the inorganic salt is selected from the group consisting of potassium acetate, magnesium acetate, or combinations thereof.

[0063] In another preferred embodiment, the concentration of the inorganic salt in the buffer solution is 1 mM to 200 mM, the concentration of the nucleoside triphosphate is 0.5 mM to 10 mM, and / or the concentration of the amino acid is 0.01 mM to 0.5 mM.

[0064] In another preferred embodiment, the bioreaction raw materials are selected from the group consisting of: DNA polymerase, DNA helicase, RNA polymerase, tRNA, ribonucleic acid, deoxyribonucleic acid, creatine phosphokinase, ribosomes, aminoacyl-tRNA synthetase, initiation factors and elongation factors required for protein synthesis, and termination release factors, or combinations thereof.

[0065] In another preferred embodiment, the first storage chamber contains 1 mL to 50 mL of the buffer solution.

[0066] In another preferred embodiment, the second storage chamber contains 100 µL to 1000 µL of the liquid containing DNA and / or RNA.

[0067] In another preferred embodiment, the concentration of DNA and / or RNA in the liquid containing DNA and / or RNA is 1 to 100 ng / µL.

[0068] In another preferred embodiment, the reaction chamber contains 100 mg to 10 g of the bioreactor material.

[0069] In another preferred embodiment, the method is performed at 15-30°C.

[0070] In another preferred embodiment, step (2) further includes activating an external shaking device and / or a shaking device located in the reaction chip.

[0071] In another preferred embodiment, the shaking device is used to help thoroughly mix the buffer solution, the liquid containing DNA and / or RNA, and the bioreactor ingredients.

[0072] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0073] Figure 1 This is the overall left view.

[0074] Figure 2 This is a left-side cross-sectional view, where the dashed lines represent the thin-film structure.

[0075] Figure 3 This is a perspective view of the first sealing cap.

[0076] Figure 4 This is a front view of the cross-section of the first sealing cap.

[0077] Figure 5 This is a perspective view of the second sealing cap.

[0078] Figure 6This is a front view of the cross-section of the second sealing cap.

[0079] Figure 7 This is a top view of the fixed structure located within the outer shell.

[0080] Figure 8 This is a left view of the fixed structure located within the outer shell.

[0081] In the accompanying figures, the reference numerals are as follows:

[0082] 1 is the outer shell; 2 is the first storage chamber; 3 is the second storage chamber; 4 is the reaction chamber; 5 is the first thin film structure (dashed line); 6 is the second thin film structure (dashed line); 7 is the third thin film structure (dashed line); 8 is a thin film structure; 9 is the column of the first control device; 10 is the pointed structure of the first control device; 11 is the column of the second control device; 12 is the pointed structure of the second control device; 13 is the first sheet structure; 14 is the outlet; 15 is the first inlet of the first storage chamber; 16 is the second inlet of the second storage chamber; 17 is the third inlet of the reaction chamber. 18 is the first sealing cover of the first storage chamber and the reaction chamber; 19 is the second sealing cover of the second storage chamber; 20 is the first empty track A of the first sealing cover; 21 is the first empty track B of the first sealing cover; 22 is the first baffle of the first sealing cover; 23 is the second empty track A of the second sealing cover; 24 is the second empty track B of the second sealing cover; 25 is the second baffle of the second sealing cover; 26 is the first protrusion of the first sample inlet; 27 is the second protrusion of the second sample inlet; 28 is the first protrusion of the third sample inlet; 29 is the vent pipe; 30 is the inclined structure; and 31 is the second thin sheet structure. Detailed Implementation

[0083] In this invention, the yeast cell extract does not contain intact cells. A typical yeast cell extract includes ribosomes for protein translation, transfer RNA, aminoacyl-tRNA synthetase, initiation and elongation factors required for protein synthesis, and termination release factors. Furthermore, the yeast extract also contains other proteins derived from the cytoplasm of yeast cells, especially soluble proteins.

[0084] In this invention, the preparation method of the yeast cell extract is not limited, and a preferred preparation method includes the following steps:

[0085] (i) Provide yeast cells;

[0086] (ii) Wash the yeast cells to obtain washed yeast cells;

[0087] (iii) The washed yeast cells are subjected to cell-breaking treatment to obtain crude yeast extract;

[0088] (iv) The crude yeast extract is subjected to solid-liquid separation to obtain the liquid fraction, which is the yeast cell extract.

[0089] In this invention, the amino acids may include natural or non-natural amino acids, and may include D-type or L-type amino acids. Representative amino acids include (but are not limited to) 20 natural amino acids: glycine, alanine, valine, leucine, isoleucine, phenylalanine, proline, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, and histidine.

[0090] Integrated bioreactor chip

[0091] like Figure 1 , 2 As shown in Figures 3, 4, and 5, the present invention provides an integrated bioreactor chip, the chip comprising:

[0092] An outer casing 1 is provided, in which a first storage chamber 2, a second storage chamber 3, and a reaction chamber 4 are fixedly disposed; wherein, the second storage chamber is located directly below the first storage chamber and is separated by a first membrane structure 5; the reaction chamber is located directly below the second storage chamber and is separated by a second membrane structure 6; the lower outlet of the reaction chamber is provided by a third membrane structure 7;

[0093] Both the first and second storage chambers contain liquid samples; the first and second storage chambers and the reaction chamber are each equipped with injection ports 15, 16, and 17, which can be sealed with a sealing cap (e.g., Figure 3 , 4 As shown in Figures 5 and 6), a thin film structure 8 is provided on the sealing cover; the first storage chamber, the second storage chamber, and the reaction chamber are in a sealed and mutually isolated state when not in use;

[0094] It also includes a first control device, which is used to puncture the thin film structure 8, the first thin film structure 5, and the second thin film structure 6, so that the reaction liquid in the first storage chamber and the second storage chamber flows into the reaction chamber simultaneously and mixes with the sample in the reaction chamber;

[0095] It also includes a second control device, which is used to puncture the thin film structure 8 and the third thin film structure 7, thereby opening the pipeline between the reaction chamber and the outlet.

[0096] In another preferred embodiment, the main body of the first control device is a column 9, with a pointed end 10. The column 9 is connected by a first thin sheet structure 13 located on the outer shell (e.g., Figure 7 and 8 (As shown) is fixed to the outer shell, and the first control device is triggered by pressing with a finger to puncture the thin film structure 8, the first thin film structure 5, and the second thin film structure 6.

[0097] In another preferred embodiment, the main body of the second control device is a column 11 with a pointed end 12. The column 11 is fixed to the outer shell. The second control device is triggered by pressing with a finger to puncture the thin film structure 8 and the third thin film structure 7.

[0098] In another preferred embodiment, the column 9 is connected to the outer shell via four centrally symmetrical, 80-micrometer-thick first sheet structures 13.

[0099] This invention provides a bioreactor chip with a reasonable structural design, high degree of automation, convenient operation and use, and no need for additional equipment, to address the problem of high demand for professional personnel and specialized equipment in the use of bioreactor chips.

[0100] To achieve the above objectives, the present invention provides the following technical solution:

[0101] This in vitro reaction system includes multiple independent reaction chip units consisting of a first storage chamber 2, a second storage chamber 3, and a reaction chamber 4, as well as a control device with two integrated switches. The two storage chambers are located above the reaction chamber, and are separated by 80-micrometer-thick thin-film structures. Each storage chamber and the reaction chamber has its own inlet and sealing caps 18 and 19. The center of the sealing caps of the first storage chamber and the reaction chamber is an 80-micrometer-thick thin-film structure. The thin-film structure directly below the sealing cap of the first storage chamber corresponds to the thin-film structure between the first and second storage chambers and between the second storage chamber and the reaction chamber; the thin-film structure directly below the sealing cap of the reaction chamber corresponds to the thin-film structure between the reaction chamber and the outlet. Above the sealing cap membrane structure of the first storage chamber is a reaction trigger switch, a pointed structure 10, used to simultaneously puncture the three-layer membrane structures 5, 6, and 8 between the sealing cap, the upper and lower storage chambers, and the second storage chamber and the reaction chamber. This allows the first storage chamber to connect to the atmosphere, while simultaneously allowing liquid samples from both storage chambers to flow into the reaction chamber, thus triggering the reaction. The reaction trigger switches of each independent reaction device unit are integrated into the first control device of the overall switch. Above the sealing cap membrane structure of the reaction chamber is a reaction termination / output switch (i.e., the puncture structure of the second control device), a pointed structure 12, used to puncture the two-layer membrane structures 7 and 8 between the sealing cap, the reaction chamber, and the outlet, allowing the reactants in the reaction chamber to flow out through the outlet. The reaction termination / output switches of each independent reaction device unit (i.e., the puncture structure of the second control device) are integrated into the second control device of the overall switch. The first and second control devices of the overall switch are connected to the outer casing through multiple 80-micron thin sheets to fix the switch and maintain its initial state. The first or second control device is triggered by pressing with a finger. An additional thin vent tube 29 connects the first storage chamber to the reaction chamber to balance the air pressure, allowing the liquid sample in the storage chamber to flow smoothly into the reaction chamber after the reaction trigger switch is turned on. The bottom surfaces of the first storage chamber and the reaction chamber are designed with a small-angle inclination (i.e., inclined structure 30) to minimize liquid residue. The three sample inlets and their corresponding sealing caps use a snap-fit ​​structure; the sealing caps can be opened and closed by rotating and moving them. The use of a small amount of vacuum grease can achieve an overall sealing effect for the device.

[0102] The main advantages of this invention include:

[0103] (1) The reaction chip of this application only needs to be controlled by two switches to control the occurrence, termination and output of biological reaction. No additional internal circuits, external controllers or other professional equipment are required. The chip is simple to operate, easy to use, low cost and no professional operator required, which makes it easy to promote and popularize.

[0104] (2) The thin film structure in the reaction chip of this application is formed by 3D printing, which effectively ensures that the reagents stored inside the chip are not contaminated by the outside when the reaction is not turned on.

[0105] (3) The reaction chip of this application can be used to synthesize different proteins simultaneously by placing different RNA or DNA in the second storage chamber through simple operation.

[0106] (4) The reaction chip of this application has an internal pressure balancing device, which allows the liquid to flow smoothly without the need for additional external air (which reduces contamination of reagents).

[0107] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0108] Example 1

[0109] The entire array-type integrated bio-automated reaction chip is directly printed by a 3D printer.

[0110] The first storage chamber 2 (also called the upper storage chamber) is the main liquid sample storage chamber, and the second storage chamber 3 (also called the lower storage chamber) is the auxiliary liquid sample storage chamber. The upper and lower storage chambers are separated by a first thin film structure 5 (a circular thin film) with a thickness of 80 micrometers. This film is fixed inside the device and completely blocks the upper and lower storage chambers. The reaction chamber 4 can be used to store solid samples and is separated from the second storage chamber 3 and the outlet 14 by the second thin film structure 6 and the third thin film structure 7 (which adopt the same design as the first thin film structure 5), respectively. The first storage chamber 2, the second storage chamber 3, and the reaction chamber 4 all have sample inlets. Each sample inlet contains six centrally symmetrical protrusions. The sample inlet 16 of the second storage chamber is located on the back of the system and can be used to input a small amount of liquid sample with a series of adjustable reaction parameters. The sample inlet 15 of the first storage chamber and the sample inlet 17 of the reaction chamber adopt the same design and specifications. The sample inlets are located above the chambers. Each first storage chamber can input the same liquid sample through the sample inlet, and each reaction chamber can input the same solid sample through the sample inlet.

[0111] The inlet 15 of the first storage chamber and the inlet 17 of the reaction chamber are equipped with the same sealing cap 18. The outer ring of the sealing cap contains an empty track 20, an empty track 21 and a baffle 22. The empty track 20 is used to allow the protrusions 26 and 28 of the inlets 15 and 17 to pass vertically. The empty track 21 is used to allow the protrusions 26 and 28 to pass when the sealing cap 18 rotates horizontally and to prevent the protrusions 26 and 28 from moving vertically. The baffle 22 is used to block the protrusions 26 and 28 and limit the maximum angle of rotation of the sealing cap 18. With a small amount of vacuum grease, a good sealing effect can be achieved. The center of the sealing cap 18 contains a circular thin film structure 8 with a thickness of 80 micrometers. After the two sealing caps are screwed on, the two thin film structures are located directly above the thin film structure 7 and directly above the thin film structure 5 and the thin film structure 6, respectively.

[0112] Above the injection port 15 is a reaction trigger switch (i.e., the first puncture structure). The end of the trigger switch has a pointed structure 10, and the main body of the switch is a cylindrical body 9. The cylindrical body 9 is connected to the outer shell through four centrally symmetrical 80-micrometer-thick first thin sheet structures 13 to fix the switch and maintain its initial state. The reaction trigger switches of the five independent reaction systems are integrated into a control device (i.e., the first control device) of a single switch. The switch control device is triggered by pressing with a finger. The five reaction trigger switches simultaneously puncture the thin film structure 8, the first thin film structure 5, and the second thin film structure 6, so that the reaction liquid in the upper and lower storage chambers flows into the reaction chamber at the same time and mixes with the solid sample in the reaction chamber, thereby realizing the simultaneous activation of the five independent reaction systems.

[0113] The reaction termination / output switch (i.e., the second puncture structure) is located directly above the injection port 17. The end of the switch is a pointed structure 12, and the main body of the switch is a cylindrical body 11. The reaction termination / output switches of the five independent reaction systems are integrated into a single switch control device (i.e., the second control device). The control device is connected to the outer shell through four centrally symmetrical 80-micrometer-thick second thin film structures 31 to fix the switch and maintain its initial state. The switch control device is triggered by pressing with a finger, and the five reaction termination / output switches simultaneously puncture the thin film structure 8 and the third thin film structure 7, allowing the reaction liquid in the reaction chamber to be output from the outlet, thus achieving simultaneous termination and output of the five independent reaction systems.

[0114] In addition, a venting tube 29 is connected between the first storage chamber and the reaction chamber to balance the air pressure between the two chambers, allowing the liquid sample to flow smoothly into the reaction chamber. The bottom surfaces of both the first storage chamber and the reaction chamber are designed with a small-angle inclination 30 to minimize liquid residue when the liquid sample flows out of the first storage chamber and the reaction fluid flows out of the reaction chamber.

[0115] Example 2: In vitro cell-free synthesis of various proteins

[0116] A reaction chip with five reaction unit units is used.

[0117] Reagents:

[0118] Buffer solution: The concentrations of each component in the buffer solution are as follows:

[0119] 22 mM 4-hydroxyethylpiperazine ethanesulfonic acid at pH 7.4, 30-150 mM potassium acetate, 1.0-5.0 mM magnesium acetate, 1.5-4 mM nucleoside triphosphate mixture, 0.08-0.24 mM amino acid mixture, 25 mM creatine phosphate, 1.7 mM dithiothreitol, 1%-4% polyethylene glycol, and 0.5%-2% sucrose;

[0120] Bioreactor raw materials: 50% by volume yeast cell extract;

[0121] DNA / RNA: Green fluorescent protein DNA, yellow fluorescent protein DNA, luciferase DNA, catalase DNA, and luciferase mutant DNA.

[0122] Then, 10 mL of buffer solution was added to the first storage chamber of each reaction unit through the first injection port on each reaction unit, and the sealing cap was closed.

[0123] Add 5 g of yeast cell extract to the reaction chamber of each reaction unit through the third injection port on each reaction unit, and then seal the container.

[0124] Each reaction unit's second storage chamber was filled with a liquid containing different DNA / RNA molecules through its second inlet port. These liquids were: 300 µL of green fluorescent protein DNA (DNA concentration 10 ng / µL), 300 µL of yellow fluorescent protein DNA (DNA concentration 10 ng / µL), 300 µL of luciferase DNA (DNA concentration 10 ng / µL), 300 µL of catalase DNA (DNA concentration 10 ng / µL), and 300 µL of luciferase mutant DNA (DNA concentration 10 ng / µL), and the containers were then sealed.

[0125] Pressing the first control device simultaneously breaks the membrane structure on the sealing cover of the first storage chamber in each reaction device unit, the membrane structure between the first and second storage chambers, and the membrane structure between the second storage chamber and the reaction chamber, allowing the buffer solution and DNA / RNA to flow into the reaction chamber, mix with the yeast cell extract in the reaction chamber, and turn on the shaker to make the mixing more uniform, and carry out the reaction at room temperature.

[0126] After 2-6 hours, the second control device is pressed. This device simultaneously breaks the membrane structure on the sealing cover of the reaction chamber in each reaction unit, as well as the membrane structure between the reaction chamber and the outlet, allowing the protein-containing liquid to flow out. Liquids containing green fluorescent protein, yellow fluorescent protein, luciferase, catalase, and a luciferase mutant are obtained, respectively.

[0127] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An integrated bioreactor chip, the chip comprising: An outer shell (1) is provided, in which a first storage chamber (2), a second storage chamber (3) and a reaction chamber (4) are fixed; wherein, the second storage chamber (3) is located directly below the first storage chamber (2) and is separated by a first thin film structure (5); the reaction chamber (4) is located directly below the second storage chamber (3) and is separated by a second thin film structure (6); the outlet below the reaction chamber (4) is a third thin film structure (7); The first storage chamber (2), the second storage chamber (3) and the reaction chamber (4) are all equipped with sample inlets, and the sample inlets are equipped with sealing caps, and the sealing caps are equipped with thin film structures (8); It also includes a first control device, which is used to puncture the thin film structure (8), the first thin film structure (5), and the second thin film structure (6) so that the reaction liquid in the first storage chamber and the second storage chamber flows into the reaction chamber at the same time and mixes with the sample in the reaction chamber; It also includes a second control device, which is used to puncture the thin film structure (8) and the third thin film structure (7) so that the pipeline between the reaction chamber and the outlet is in an open state; It also includes an internal pressure balancing device; the internal pressure balancing device is a vent pipe, which is located between the first storage chamber and the reaction chamber; The bottom surfaces of the first storage chamber (2), the second storage chamber (3) and / or the reaction chamber (4) are inclined structures; the volume of the first storage chamber is 1 mL to 50 mL, the volume of the second storage chamber is 100 μL to 1000 μL, and the volume of the reaction chamber is 2 mL to 80 mL. The width or diameter of the second storage chamber is smaller than that of the first storage chamber; the width or diameter of the reaction chamber is larger than that of the first or second storage chamber.

2. The reaction chip as described in claim 1, characterized in that: The first control device is a column with a pointed end.

3. The reaction chip as described in claim 1, characterized in that: The main body of the second control device is a column with a pointed end.

4. The reaction chip as described in claim 2 or 3, characterized in that: The first or second control device is movably fixed to the outer shell by means of a thin sheet structure installed inside the outer shell. The column is connected to the outer shell by four centrally symmetrical thin sheet structures.

5. A method for in vitro cell-free protein synthesis, characterized in that, Including the following steps: (1) A reaction chip as described in any one of claims 1-4 is provided; wherein... The first storage chamber contains the same or different buffer solutions; The second storage chamber contains a liquid containing DNA and / or RNA, which may be identical or different; and The reaction chambers contain the same or different biological reaction raw materials; (2) Press the first control device to simultaneously allow the buffer solution and liquid containing DNA and / or RNA to flow into the corresponding reaction chamber and mix with the biological reaction raw materials in the reaction chamber to form a reaction system and carry out the reaction; (3) After the reaction is complete, a liquid containing the same or different proteins is formed. Press the second control device to make the liquid containing the same or different proteins flow out, and obtain a liquid containing the synthesized target protein.

6. The method as described in claim 5, characterized in that, The first storage chamber contains the same buffer solution; the second storage chamber contains liquids containing different DNA and / or RNA; and / or the reaction chamber has the same biological reaction raw materials.

Images