Bioreactor and use thereof

Abstract

The application provides a bioreactor, which is a self-mixing device and comprises an extrusion module and a flexible reaction cavity, wherein the flexible reaction cavity is internally provided with DNA hydrogel microparticles with magnetic cores; the extrusion module can extrude the flexible reaction cavity to produce deformation by mechanical extrusion or air pressure extrusion; the extrusion module repeatedly extrudes the flexible reaction cavity to mix the reaction system in the flexible reaction cavity, and the bioreactor can be used for RNA in vitro transcription. According to the technical scheme of the application, the reaction liquid in the reaction cavity is automatically mixed by the self-mixing mode of mechanical extrusion or air pressure extrusion, the contact of other mixing machines with the reaction system is reduced, and pollution is avoided; meanwhile, the product in the reaction cavity is discharged by extrusion after the reaction, so that the contact of the reaction cavity and the hydrogel with the outside world is effectively reduced.

Description

Technical Field

[0001] This invention relates to the field of RNA in vitro transcription technology, and more specifically, to a bioreactor and its applications. Background Technology

[0002] mRNA is transcribed from DNA and has the function of transmitting genetic information from DNA to proteins. In recent years, mRNA vaccines have been regarded as a promising vaccine technology due to their biocompatibility and the shorter production time and lower cost compared to traditional vaccines. mRNA vaccines have great potential in preventing and treating infectious diseases, tumors, and other health risks. The first step in traditional mRNA vaccine production technology is to amplify plasmid DNA in large quantities in E. coli cells, lyse and extract the corresponding plasmid DNA, and then use restriction endonucleases to cut the required DNA fragments within the plasmid to obtain the desired linear template DNA. Because large-scale in vitro production of mRNA vaccines requires a large amount of template DNA, the time and raw material costs of corresponding bacterial culture and plasmid linearization account for a high proportion. Chinese invention patent CN112334579A discloses a bioreactor for in vitro RNA transcription. This patent uses magnetic particles as the core to fix linear DNA onto the magnetic particles for reuse. In this automated device, the reaction chamber has an ellipsoidal internal geometry and is surrounded by an electromagnetic array. This reactor achieves the mixing of liquids within the reaction chamber and the removal of magnetic particles by altering the magnetism of the electromagnetic array. This process effectively removes magnetic particles by adsorbing them and discharging other liquids from the chamber. The technical solution uses an electromagnetic array to act on magnetic microparticles to mix and stir the reaction solution. In other words, the DNA template with fixed magnetic particles acts as the magnetic core for stirring. However, the shear force generated by the eddy currents or spiral motion of the magnetic particles within the reactor is relatively high. Chinese invention patent CN 110272982 A discloses a DNA composite hydrogel and its preparation method. The prepared gel microparticles with magnetic cores are used in the production of cell-free proteins and can be reused. Clearly, the reusability of the magnetic core DNA template particles significantly reduces costs.

[0003] In view of this, based on the existing technology, this application provides a flexible material as an in vitro transcription reactor for mRNA in order to further optimize the process and reduce the cost of in vitro transcription of mRNA. By utilizing the flexible characteristics of the outer wall of the reactor, the reaction system inside the reactor is self-mixed by external pressure extrusion, which avoids contact between the reaction system and the DNA template and the outside world, and effectively reduces the damage to the reaction system caused by internal shear force. Summary of the Invention

[0004] In view of this, in order to solve the above problems, the present invention provides a bioreactor, which is a self-mixing flexible reactor. It uses a self-mixing mode of mechanical extrusion or pneumatic extrusion to automatically mix the reaction liquid in the reaction chamber, effectively reducing the contact between the reaction chamber and hydrogel and the outside world. While effectively mixing, it can also reduce the damage of internal shear force to the reaction system.

[0005] To achieve the above objectives, the present invention provides a bioreactor, a self-mixing device or a self-mixing flexible reactor, comprising: a squeezing module; a driving module capable of driving the squeezing module to reciprocate; a reaction module including a flexible reaction chamber, wherein the flexible reaction chamber contains DNA hydrogel microparticles with magnetic cores, the squeezing module being able to repeatedly squeeze the flexible reaction chamber to mix the reaction system within the flexible reaction chamber, thereby achieving in vitro RNA transcription; and a magnet trapping module for trapping the DNA hydrogel microparticles with magnetic cores within the flexible reaction chamber, preventing them from detaching from the flexible reaction chamber, thus enabling the DNA hydrogel microparticles to be reused. In a preferred embodiment, the flexible reaction chamber is made of PDMS material.

[0006] Preferably, the extrusion module deforms the flexible reaction chamber by mechanical extrusion or pneumatic extrusion, thereby enabling the reaction system to self-mix.

[0007] Preferably, the drive module includes a mechanical operating component and a drive component; the drive component drives the mechanical operating component to perform circular motion, which is converted into linear reciprocating motion of the extrusion module, which can approach or move away from the flexible reaction chamber, thereby achieving repeated extrusion deformation of the flexible reaction chamber and realizing self-mixing of the reaction liquid in the flexible reaction chamber.

[0008] Preferably, the driving module includes a mechanical rotating component and a driving component; the driving component drives the mechanical rotating component to perform circular motion; the surface of the mechanical rotating component is provided with a guide groove; the limiting module includes at least a limiting component, which is movably disposed on the guide groove; the extrusion module is disposed between the limiting component and the flexible reaction chamber, and is fixed on the limiting component; the limiting component can drive the extrusion module to reciprocate along the limiting groove, and the circular motion of the mechanical rotating component is converted into the linear reciprocating motion of the extrusion module, repeatedly extruding the outer wall of the flexible reaction chamber.

[0009] Preferably, the guide groove is an irregularly shaped closed-loop track; as the mechanical moving part rotates, the limiting part moves linearly along the track;

[0010] Preferably, the guide groove includes a plurality of limiting points. When the mechanical operating component operates, and the limiting points rotate to the position of the limiting component, the extrusion module can extrude the outer wall of the flexible reaction chamber; the limiting component can reciprocate along the track of the guide groove; the extrusion module includes a limiting groove, a limiting member, a fixing member, and an extrusion member, and the limiting groove can limit the movement range of the limiting member.

[0011] Preferably, the limiting module further includes a limiting plate; the limiting plate is provided with limiting grooves corresponding to the number of limiting components; both ends of the limiting component are movably disposed between the limiting groove and the guide groove, respectively; the limiting groove can limit the movement range of the limiting component, the movement range is equal to the difference between the maximum radius and the minimum radius of the irregular track, the limiting component can reciprocate along the track of the guide groove, and the limiting groove can limit the movement range of the limiting component.

[0012] Preferably, the guide groove is provided with a plurality of limiting points, and the distances of two adjacent limiting points from the outer wall of the flexible reaction chamber are not equal; when the limiting rod moves on the track between two limiting points, the squeezing module moves on the track between the two limiting points to realize that the flexible reaction chamber continuously switches between squeezing and relaxing modes, rather than being in a continuous squeezing or relaxing state.

[0013] Preferably, the pneumatic compression method includes the driving module driving the compression module to periodically apply pressure to the flexible reaction chamber, thereby repeatedly compressing the flexible reaction chamber and achieving self-mixing of the reaction liquid within the flexible reaction chamber; the compression module is capable of inflating and deforming a gas chamber, and the driving module periodically inflates / deflates the gas chamber to deform it, thereby achieving periodic and repeated compression of the flexible reaction chamber.

[0014] The in vitro RNA transcription method using the bioreactor provided by this invention uses magnetic core DNA hydrogel microparticles as templates and T7 RNA polymerase as the catalytic enzyme. The reaction is carried out in the above-mentioned bioreactor in a system containing buffer and NTP to produce mRNA products.

[0015] By capturing magnetic core DNA hydrogel microparticles with magnetic components, they can be separated from the reaction solution and retained in the flexible reaction chamber to prevent them from escaping. They can then be reused after cleaning.

[0016] Furthermore, the method for preparing magnetic core DNA hydrogel microparticles includes using a polymer compound with functionalized groups, magnetic particles, and a DNA template. The DNA template may be selectively modified with functionalized groups, which are any one or more combinations selected from amide, thiol, amino, or azide groups.

[0017] The beneficial technical effects obtained by this invention are as follows:

[0018] 1. The technical solution of this invention automatically mixes the reaction liquid in the reaction chamber using a self-mixing mode of mechanical extrusion or pneumatic extrusion, reducing the contact of other mixing machinery with the reaction system and avoiding contamination; at the same time, after the reaction is completed, the product in the reaction chamber is discharged by extrusion, effectively reducing the contact between the reaction chamber and the hydrogel and the outside world, and in particular, reducing the damage of internal shear force to the reaction system while effectively mixing.

[0019] 2. This invention uses PDMS as a flexible reaction chamber, which has a flexible outer wall. Combined with external force extrusion, it achieves self-mixing of the liquid in the reaction chamber. At the same time, through the characteristics of its flexible outer wall, the lifting motor extrudes the liquid. The structure is simple and the operation is convenient.

[0020] 3. The technical solution of the present invention uses magnetic core DNA hydrogel microparticles as templates to produce mRNA products. The template can be reused by using magnetic components to capture the magnetic core DNA hydrogel microparticles. It can also protect the DNA fragments inside the hydrogel. In particular, by adding a magnetic core inside the DNA hydrogel, the recovery rate of the DNA hydrogel after each reaction can be increased.

[0021] 4. By using the technical solution of the present invention, the reaction efficiency is improved by using magnetic core DNA hydrogel microparticles instead of a whole hydrogel, and by miniaturizing the magnetic core DNA hydrogel. Attached Figure Description

[0022] Figure 1 This is a front view of the mechanically flexible bioreactor in Embodiment 1 of the present invention.

[0023] Figure 2 This is a cross-sectional view of the mechanically flexible bioreactor in Embodiment 1 of the present invention.

[0024] Figure 3 This is a top view of the mechanically flexible bioreactor of Embodiment 1 of the present invention.

[0025] Figure 4 This is an exploded view of the limiting component in Embodiment 1 of the present invention.

[0026] Figure 5This is a schematic diagram of the limiting plate in Embodiment 1 of the present invention.

[0027] Figure 6a and Figure 6b These are schematic diagrams showing the structure of the limiting component at different movement positions according to Embodiment 1 of the present invention.

[0028] Figure 7 This is a schematic diagram of the structure of the substrate in Embodiment 1 of the present invention.

[0029] Figure 8 This is a schematic diagram of the functional modules in Embodiment 1 of the present invention.

[0030] Figure 9 This is a layered top view of the pneumatic device in Embodiment 2 of the present invention.

[0031] Figure 10 This is a diagram showing the operational relationship of the pneumatic device in Embodiment 2 of the present invention.

[0032] Figure 11 This is a comparison chart of RNA expression levels in the template DNA composite hydrogel used in Example 1 of the present invention and RNA expression levels in ordinary linear templates.

[0033] Figure 12 This is a histogram showing the RNA expression levels obtained from repeated use of the DNA hydrogel in Example 1 of the present invention.

[0034] Figure 13 The above are comparative histograms showing the mRNA repetitive expression levels obtained using the flexible bioreactor provided in Example 1 and other bioreactors used in Comparative Example 2, respectively.

[0035] The mechanical module 100 includes a first cover plate 1001, a second cover plate 1002, a limiting plate 1003, a fourth cover plate 1004, a pressing module 1005, a driven gear 1006, a limiting rod 1007, a guide groove 1008, a limiting groove 1009, a fastener 1010, and a limiting point 1011; the drive module 110 includes a drive motor 1101, a driving gear 1111, a heater 1103, and a lifting motor 1104. , drive gear 1111; electromagnet 1105, heater switch 1106, lifting motor switch 1107, electromagnet switch 1108; flexible reaction chamber 1201, discharge pipe 1202, product storage chamber 1203; liquid reaction layer 200, liquid reaction chamber 2001, pressurized air inlet 2002, reaction liquid inlet 2003, gas pressurization layer 210, pressurization chamber 2101, gas pressurization channel 2102. Detailed Implementation

[0036] To make the objectives, technical solutions, and advantages of the embodiments of this application clearer, the technical solutions of the embodiments of this application will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are some embodiments of this application, but not all embodiments.

[0037] Example 1

[0038] This embodiment provides a mechanically flexible bioreactor for in vitro RNA transcription, which is a self-mixing device; see the attached diagram for details. Figure 1-3 .

[0039] The mechanical flexible bioreactor mainly includes a mechanical module 100, a drive module 110, a magnet trapping module, a reaction module 120, a substrate, and a functional module.

[0040] The reaction module 120 comprises a flexible reaction chamber 1201, a liquid discharge pipe 1202, and a product storage chamber 1203. The flexible reaction chamber 1201 contains DNA hydrogel microparticles with a magnetic core. Preferably, the flexible reaction chamber 1201 is made of PDMS material and is detachably fixed inside the reactor. PDMS has flexible and breathable properties, providing a good environment for the reaction. The flexible reaction chamber 1201 allows for external compression to mix the liquid within the reaction chamber, and after the reaction, the liquid is discharged through the liquid discharge pipe 1202 to the product storage chamber 1203 by compression. The flexible reaction chamber 1201 is a columnar body without sharp edges on its outer surface; as the most preferred embodiment, the flexible reaction chamber 1201 has a cylindrical structure.

[0041] The functional modules include a heating module, a magnet trapping module, and a lifting module. The heating module can control the temperature of the reaction system inside the flexible reaction chamber 1201. The magnet trapping module is used to trap the DNA hydrogel microparticles with magnetic cores inside the flexible reaction chamber 1201, preventing them from detaching from the flexible reaction chamber 1201 and enabling the DNA hydrogel microparticles to be reused.

[0042] The lifting module can raise or lower the position of the flexible reaction chamber 1201, enabling the installation, cleaning, and replacement of the flexible reaction chamber 1201.

[0043] The drive module 110 includes a motor 1101 and a drive gear 1111, with the motor 1101 driving the drive gear 1111 to rotate. Preferably, the drive module can also implement the technical solution of this embodiment using other components capable of circular motion, such as a turbine.

[0044] The mechanical module 100 includes, from top to bottom, a first cover plate 1001, a second cover plate 1002, and a fourth cover plate 1004 arranged in parallel. A cavity is provided between the first cover plate 1001 and the second cover plate 1002, and between the second cover plate 1002 and the fourth cover plate 1004. The three cover plates are fixed in place by fasteners 1010. Preferably, the fasteners 1010 can be bolts, clips, or other mechanical fastening components that can fix the cover plates. The first cover plate 1001 is the cover plate for the flexible reaction chamber 1201, and its function is to fix the flexible reaction chamber 1201 and prevent it from dislodging during compression. The second cover plate 1002 and the fourth cover plate 1004 are used to fix the entire mechanical module 100.

[0045] The mechanical module 100 includes a mechanical operating component, a limiting component, and a pressing module. The driving component drives the mechanical operating component to perform circular motion. In a preferred embodiment, the mechanical operating component is a driven gear 1006, which rotates accordingly.

[0046] A limiting plate 1003 is fixed between the second cover plate 1002 and the fourth cover plate 1004, and the extrusion module and the limiting component are located between the limiting plate 1003 and the fourth cover plate 1004.

[0047] A guide groove 1008 is provided on the surface of the driven gear 1006. The guide groove is a closed loop track with an irregular shape, which is arranged around the flexible reaction chamber as the center. The guide groove can be formed on the surface of the driven gear 1006 or a track fixed on the surface of the transmission gear 1006. The guide groove 1008 rotates with the circumferential movement of the driven gear 1006, so that the limiting component can move linearly along the irregular shape of the guide groove, thereby driving the extrusion module to make linear reciprocating motion, so that the extrusion module can approach or move away from the flexible reaction chamber 1201, realizing repeated horizontal extrusion of the outer wall of the flexible reaction chamber 1201.

[0048] The limiting component is movably disposed between the limiting plate 1003 and the guide groove 1008. Preferably, the limiting component includes the limiting plate 1003 and a plurality of limiting rods 1007. The limiting plate 1003 is provided with limiting grooves 1009 corresponding to the number of limiting rods 1007. The two ends of the limiting rods are movably disposed between the limiting grooves 1009 and the guide grooves 1008, respectively.

[0049] The extrusion module includes several extrusion blocks 1005, which are fixedly mounted on the limiting rod 1007 and can move closer to or further away from the flexible reaction chamber as the limiting rod moves linearly.

[0050] Specifically, the function of the limiting rod 1007 is to convert the circular motion of the driven gear 1006 through the guide groove 1008 into the linear motion of the limiting rod 1007, thereby realizing the conversion of the circular motion into the reciprocating linear motion of the extrusion block 1005 to vertically and repeatedly extrude the outer wall of the flexible reaction chamber 1201.

[0051] Referring to Figures 4-6, the limiting plate 1003 is provided with limiting grooves 1009 corresponding to the number of limiting rods 1007. The guide groove 1008 is a closed loop track with an irregular shape arranged around the flexible reaction chamber. As the driven gear 1006 rotates, the limiting rods 1007 can slide along the limiting groove 1009.

[0052] Specifically, the limiting groove 1009 can limit the range of motion of the limiting rod, which is equal to the difference between the maximum and minimum radii of the irregular track of the guide groove 1008; the limiting rod 1007 can reciprocate along the track of the guide groove 1008, and the limiting groove 1009 can limit the range of motion of the limiting member 1007.

[0053] The guide groove 1008 is provided with several limiting points 1011, and the distances between two adjacent limiting points 1011 and the outer wall of the flexible reaction chamber 1201 are not equal. When the limiting rod 1007 passes through the track between two limiting points 1011, the extrusion block 1005 switches between extrusion and relaxation modes on the outer wall of the flexible reaction chamber 1201 as it moves on the track between the two limiting points. This achieves reciprocating extrusion of the flexible reaction chamber 1201, rather than being in a continuous extrusion or relaxation state. Preferably, the number of limiting points 1011 is even, and the guide groove 1008 has an axisymmetric structure, with the axis of symmetry being the line connecting two oppositely arranged limiting points. The axisymmetric structure of the guide shaft can simultaneously provide relative mechanical extrusion to the flexible reaction chamber 1201, enabling the reaction system in the reaction chamber to mix more uniformly and efficiently.

[0054] Specifically, when the limiting rod 1007 passes the limiting point 1011, the distance between the extrusion block 1005 and the outer wall of the flexible reaction chamber 1201 is less than the distance between the limiting point and the outer wall of the flexible reaction chamber. The extrusion block extrudes the outer wall of the flexible reactor, causing it to deform. After leaving the limiting point, the distance gradually increases, and the flexible reaction chamber 1201 returns to its shape. As the driven gear 1006 rotates, the extrusion block 1005 continuously switches between approaching and moving away from the flexible reaction chamber 1201, thereby continuously extruding and deforming the outer wall of the flexible reaction chamber 1201. This allows the liquid reaction system inside the flexible reaction chamber 1201 to be in motion, achieving self-mixing.

[0055] See Figures 7-8The base 1102 is a cavity that can accommodate the drive module and the functional modules. The functional modules include a heating module, a magnet capture module, and a lifting module.

[0056] The drive module includes a drive motor 1101 and a drive gear 1111. The drive motor 1101 drives the drive gear 1111 to rotate, and the drive gear 1111 drives the driven gear 1006 to perform circular motion.

[0057] The heating module includes a heater 1103 and a magnet trapping module, which is an electromagnet 1105, respectively positioned on the sides of the flexible reaction chamber 1201; the lifting module is a lifting motor 1104, positioned at the bottom of the flexible reaction chamber 1201. The upward movement of the lifting motor 1104 can compress the flexible reaction chamber 1201.

[0058] The heater 1103 provides a constant temperature for the mRNA reaction; the lifting motor 1104 automatically extracts the reaction product by squeezing the reaction product from the flexible reaction chamber to the product storage chamber 1203 after the reaction is complete. When discharging the reaction liquid from the reaction chamber, the electromagnet 1105 is activated, which adsorbs the DNA hydrogel with a magnetic core, allowing the reaction product to be discharged while the DNA template is automatically retained in the reaction chamber for reuse in the next reaction.

[0059] The outer surface of the substrate 1102 is provided with a heater switch 1106, a lifting motor switch 1107, and an electromagnet switch 1108, which are used to control the heater 1103, the lifting motor 1104, and the electromagnet 1105, respectively.

[0060] Operating principle: After adding the mixed reaction system into the flexible reaction chamber 1201, the first cover plate 1001 is placed on top and secured with fasteners 1010. The drive motor 1101 of the mechanical module is then turned on. At this time, the extrusion module 1005 inside the mechanical module reciprocates against the outer wall of the flexible reactor 1201 to continuously mix the reaction liquid within the flexible reaction chamber 1201.

[0061] Turn on heater switch 1106 to maintain the reaction system temperature at 37°C. After 2 hours of reaction, turn off heater switch 1106 and turn on electromagnet switch 1108, then turn on lifting motor switch 1107. The lifting motor will then compress the flexible reaction chamber 1201, and the liquid inside will flow through drain pipe 1202 to product storage chamber 1203, from which the reaction product is obtained. After lowering lifting motor 1104, clean the flexible reaction chamber 1201 with PBS. After turning off the electromagnet, add all components except the template back into the flexible reaction chamber 1201 to restart the reaction, allowing for the reuse of the DNA template.

[0062] The principle behind in vitro RNA transcription using the aforementioned mechanically flexible reactor is as follows: By adding two DNA hydrogel cementation components in portions to an organic phase (n-hexane) while stirring, DNA hydrogel microparticles with magnetic cores are obtained. These magnetic cores endow the DNA hydrogel microparticles with magnetic properties. Firstly, the magnetic microparticles can be captured by an electromagnet, reducing their loss during product separation. Secondly, using DNA hydrogel microparticles instead of a monolithic hydrogel as a template for protein expression increases the contact area between the microparticle surface and the reaction system, thus promoting the reaction. Thirdly, the hydrogel provides some protection for the DNA within, reducing the risk of DNA degradation during template reuse.

[0063] Meanwhile, a flexible reactor is used as the reaction carrier. The flexible reactor is made of PDMS material, which is a soft and breathable material. With the help of an external mechanical structure, the reaction liquid inside the flexible reactor is mixed by extrusion to accelerate the reaction efficiency and promote the formation of products.

[0064] In this reaction, a DNA hydrogel is used as a template, and T7 RNA polymerase is used as the catalytic enzyme. The template can be reused to produce mRNA products in a system containing buffer and NTPs. Because the DNA template can be reused, costs can be reduced by minimizing the initial DNA template preparation.

[0065] Specifically, the method for achieving in vitro transcription of RNA includes the following steps:

[0066] I. Preparation of thiol-modified DNA (HS-DNA) templates

[0067] The pIJ8660-GFP plasmid was obtained by synthesizing the full DNA sequence. PCR primers were designed based on the GFP gene, and the PCR product HS-DNA of the GFP gene with functional groups modified at the 5' end of the primers was used as a template for cell-free protein synthesis (CFPS) in this embodiment.

[0068] Table 1 PCR primer sequences

[0069] Primers sequence sfGFP-FOR 5-HS-TGGAGCGGATCGGGGATTGT-3 sfGFP-REV 5-HS-CCGGTCGACTCTAGCTAGAG-3

[0070] II. Preparation of Magnetic Core DNA Hydrogel Microparticles

[0071] (1) Preparation of solution 1: Dissolve four-armed polyethylene glycol maleimide (number average molecular weight of 20,000) in PBS (pH=7), mix well, wherein the mass fraction of four-armed polyethylene glycol maleimide is 10%, and the four-armed polyethylene glycol maleimide formed is solution 1;

[0072] (2) Preparation of solution 2: Dissolve the four-arm polyethylene glycol thiol (number average molecular weight of 20,000) in PBS (pH=7) to prepare a 10% mass fraction four-arm polyethylene glycol thiol solution, which is used as solution 2.

[0073] (3) Preparation of solution 3: Add 20 mg of iron oxide to 1 mL of PBS solution to prepare a 10X iron oxide solution; the iron oxide solution is used as solution 3;

[0074] (4) Preparation of solution 4: Add HS-DNA, 6 μL of solution 1 and 2 μL of solution 3 to the EP tube, mix well, and use PBS to make up the volume of the EP tube to 20 μL. The final concentration of HS-DNA is 200 ng / µL HS-DNA. The HS-DNA mixed solution prepared is solution 4.

[0075] (5) Preparation of solution 5: Add HS-DNA, 6 μL of solution 2 and 2 μL of solution 3 to the EP tube, mix well, and use PBS to make up the volume of the EP tube to 20 μL, wherein the final concentration of HS-DNA is 200 ng / µL HS-DNA; the mixed solution obtained is solution 5;

[0076] (6) Add 2 mL of n-hexane to a stirring flask and add 40 μL of Span-80 solution to it;

[0077] (7) Turn on the stirrer to stir the n-hexane and add 20 μL of solution 4 dropwise.

[0078] (8) After stirring for 5 minutes, add 20 μL of solution 5 dropwise to the stirring flask and continue stirring for 24 hours to obtain DNA hydrogel microparticles with magnetic cores.

[0079] III. In vitro transcription of RNA

[0080] The components of in vitro RNA transcription are as follows:

[0081] 40mM Tris-HCl (pH 8.0), 6mM magnesium chloride, 10mM DTT, 50mM sodium chloride, 2mM spermidine, 0.5mM NTP, 1U / µL T7 RNA polymerase; add 100 ng / µL of the above DNA hydrogel template, and add an additional 1mM molecular beacon, mix well, and perform real-time transcription of RNA in vitro.

[0082] Add the liquid to the mechanically flexible reactor, cover it with the cover plate and secure it with nuts.

[0083] Turn on the mechanical module drive motor. The extrusion module within the mechanical module will then reciprocate against the outer wall of the flexible reactor, continuously mixing the reaction liquid within the flexible reaction chamber. Turn on the heater switch to maintain the reaction system temperature at 37°C. After the reaction has completed in 2 hours, turn off the heating switch and turn on the electromagnet switch, then turn on the lifting motor switch. The lifting motor will then move upwards, extruding the flexible reaction chamber 1201 upwards. The liquid within the flexible reaction chamber is forced through the discharge pipe 1202 to the product storage chamber 1203.

[0084] Finally, the reaction product was obtained from the product storage chamber 1203.

[0085] The RNA expression levels were obtained using the standard curve method.

[0086] Using an ELISA reader, the fluorescence value was measured under the conditions of excitation light at 485 nm, emission light at 535 nm, and exposure time of 0.1 s. The amount of mRNA was determined based on the fluorescence value.

[0087] IV. Reuse of DNA Hydrogels

[0088] The purpose of this embodiment is to confirm whether the DNA hydrogel prepared in this application has reusable characteristics. After the reaction, the lifting motor is lowered first, and then the reaction chamber is cleaned with PBS. After cleaning, the electromagnet is turned off, and then all components except the template are added to the flexible reaction chamber, and the reaction is started again. The reaction product is obtained after 2 hours of reaction, and the fluorescence intensity of the system is measured using the same method to estimate the concentration of the reaction product. The same hydrogel will be reused 10 times, and the amount of transcribed mRNA obtained each time will be recorded. See [link to specific results] for details. Figure 12 ,Depend on Figure 12 It is evident that high levels of mRNA expression can still be obtained after 10 reuses. Clearly, using the self-mixing device provided in this embodiment as a bioreactor offers several advantages. First, the physical extrusion method does not damage the hydrogel particles and maintains a reusability of at least 10 times, significantly reducing costs. Second, the mechanical mixing method is easier to operate and controllable than the electromagnetic array method in existing technologies, resulting in better mixing effects. Furthermore, multiple physical mixing methods can be combined. Finally, using flexible materials as the reactor material allows for physical extrusion methods for both mixing and discharging, preventing contact between the reaction chamber and hydrogel particles and the external environment, thus avoiding contamination of the reaction system and magnetic particles by the external environment.

[0089] In particular, the physical extrusion mode can reduce the internal shear force on the reaction system caused by other mechanical stirring systems or electromagnetic array mixing methods, and reduce the impact of the mixing operation on the solution viscosity of the reaction system.

[0090] Example 2

[0091] This embodiment provides a self-mixing reactor. Unlike the mechanical module extrusion method in Embodiment 1, this embodiment uses a pneumatic extrusion method to achieve self-mixing of the reaction system.

[0092] In this embodiment, similar to Example 1, a DNA hydrogel with magnetic particles as its core is used as the reaction template. For the specific structure of the gas pressure extrusion mode, please refer to Example 1. Figure 9-10 This embodiment consists of two parts: a liquid reaction layer 200 and a gas pressurization layer 210. The liquid reaction layer 200 is the upper chamber, and the gas pressurization layer 210 is the lower chamber.

[0093] The liquid reaction layer 200 is mainly made of a flexible material and has three cavity structures: a liquid reaction chamber 2001, a pressurized air inlet 2002, and a reaction liquid inlet 2003. The liquid reaction chamber 2001 is a cylindrical cavity, which is the cavity where the reaction takes place. Preferably, the flexible material is PDMS.

[0094] The gas pressurization layer 210 includes a pressurization chamber 2101 and a gas pressurization channel 2102. The pressurization inlet 2002 is connected to the lower gas pressurization channel 2102, and the pressurization chamber 2101 is periodically pressurized and depressurized through an external air pump.

[0095] During the reaction, the lower chamber rises and falls due to the periodic pressurization and depressurization of the gas in the pressurized chamber 2101, thereby achieving the effect of homogenizing the reaction liquid. The reaction liquid is added to or removed from the liquid reaction chamber 2001 through the reaction liquid inlet 2003.

[0096] The main body of the gas pressurization layer 210 is also made of flexible material, and it has two parts: a gas pressurization chamber 2101 and a gas pressurization channel 2102. The gas pressurization chamber 2101 is a cylindrical cavity, and the gas pressure inside it will periodically increase and decrease as the external air pump operates. The gas pressurization channel is a structure that connects the external air pump and the gas pressurization chamber.

[0097] The functional modules in this embodiment are the same as those in embodiment 1.

[0098] Mixing Principle: After adding the pre-mixed reaction system into the liquid reaction chamber 2001 through the reaction liquid inlet 2003, the external gas pump conduit is inserted into the pressurized gas inlet 2002. This pressurized gas inlet 2102 causes the pressure inside the gas pressurization chamber 2101 to periodically increase and decrease. Due to the periodic changes in gas pressure within the gas pressurization chamber 2101, the upper wall of the gas pressurization chamber 2101 periodically rises or falls. Correspondingly, the lower wall of the liquid reaction chamber 2001 also periodically rises or falls. The periodic movement of the lower wall of the liquid reaction chamber 2001 mixes the liquid within the reaction chamber with low shear force. After the reaction is complete, the liquid in the reaction chamber 2001 is removed through the reaction liquid inlet 2003 to obtain the product.

[0099] Comparative Example 1

[0100] The difference between this comparative example and Example 1 is that a standard linear template (PCR product) was used for mRNA transcription; all other aspects are the same.

[0101] like Figure 11 As shown in the figure, the mRNA expression levels obtained using magnetic hydrogel and ordinary linear template in Example 1 and Comparative Example 1, respectively, are as follows: As can be seen from the figure, the RNA expression level of the DNA hydrogel template provided in this application is much higher than that of the RNA expression level using ordinary linear template.

[0102] Comparative Example 2

[0103] This comparative example used a shaker and a 96-well plate as the bioreactor. The shaker speed was 200 rpm, and the temperature was maintained at 37°C. The single reaction time was 2 hours, and other parameters were the same as in this example. After each reaction, the reaction liquid (excluding the colloid) was aspirated, washed twice with PBS, and then added back into the reaction system for repeated reactions. Six reuse experiments of the colloid were conducted under the conditions of a shaker and a 96-well plate as the bioreactor.

[0104] like Figure 13 As shown, the mRNA repetitive expression levels were obtained using a flexible bioreactor and a different bioreactor, respectively, in Example 1 and Comparative Example 2. The reason for this is that under normal shaking conditions, the rigid magnetic core within the gel easily disrupts the gel's integrity, leading to gel loss during the washing step and a decrease in expression levels when the gel is reused. As can be seen from the figure, the mixing method of the flexible reaction system provided by this invention can improve the repetitive expression levels of the gel prepared under magnetic core conditions.

[0105] The above are merely preferred embodiments of the present invention and do not limit the scope of protection of the present invention. For those skilled in the art, the present invention can have various modifications and variations. Any changes, modifications, substitutions, integrations, and parameter alterations to these embodiments within the spirit and principles of the present invention, achieved through conventional substitutions or by achieving the same function without departing from the principles and spirit of the present invention, fall within the scope of protection of the present invention.

Claims

1. A method for in vitro transcription of RNA, characterized in that, The method is implemented based on a bioreactor for in vitro transcription of RNA, the bioreactor being a self-mixing reactor and comprising a squeezing module, a flexible reaction chamber, and a magnetic trapping module; the flexible reaction chamber contains magnetic core DNA hydrogel microparticles. The extrusion module deforms the flexible reaction chamber through mechanical extrusion. This mechanical extrusion method involves converting circular motion into linear reciprocating motion of the extrusion module via a mechanical rotating component and a limiting component to extrude the flexible reaction chamber. The surface of the mechanical rotating component is provided with a guide groove. The limiting component is movably mounted on the guide groove. The guide groove has an irregularly shaped closed loop centered on the flexible reaction chamber, and it is provided with several limiting points. The distances of adjacent limiting points from the outer wall of the flexible reaction chamber are unequal. When the mechanical rotating component operates, the limiting points rotate to the limiting positions. When the component is positioned, the extrusion module can extrude the outer wall of the flexible reaction chamber; the extrusion module is located between the flexible reaction chamber and the limiting component, and is fixedly installed with the limiting component; the limiting component is used to restrict the range of motion of the extrusion module; and, as the mechanical operating component rotates, the limiting component moves linearly along the track, while simultaneously driving the extrusion module to reciprocate along the guide groove; the magnet capturing module is used to capture the magnetic core DNA hydrogel microparticles in the flexible reaction chamber, preventing the DNA hydrogel microparticles from detaching from the flexible reaction chamber, so that the DNA hydrogel microparticles can be reused; The method includes: using the magnetic core DNA hydrogel microparticles as a template, using T7 RNA polymerase as a catalytic enzyme, and reacting in the bioreactor in a system containing buffer and NTP to produce mRNA products. Furthermore, during the reaction process, the extrusion module repeatedly extrudes the flexible reaction chamber to achieve self-mixing of the reaction system within the flexible reaction chamber.

2. The RNA in vitro transcription method according to claim 1, characterized in that, The mechanical operating component is a gear or a turbine. By driving the circular motion of the mechanical operating component, the extrusion module moves linearly within the limited range of motion of the limiting component, thereby causing the extrusion module to approach or move away from the flexible reaction chamber, and realize repeated extrusion of the outer wall of the flexible reaction chamber.

3. The RNA in vitro transcription method according to claim 1, characterized in that, The limiting component includes a limiting plate and a plurality of limiting rods; the limiting plate is provided with limiting grooves corresponding to the number of limiting rods; the two ends of the limiting rods are respectively movably disposed between the limiting grooves and the guide grooves; The limiting groove can limit the range of motion of the limiting rod, and the range of motion is equal to the difference between the maximum and minimum radii of the irregular track; The limiting component can reciprocate along the track of the guide groove, and the limiting groove can limit the range of motion of the limiting component.

4. The RNA in vitro transcription method according to claim 3, characterized in that, When the limiting rod moves along the track between the two limiting points, the squeezing module moves along the track between the two limiting points to realize that the flexible reaction chamber continuously switches between squeezing and relaxing modes, rather than being in a continuous squeezing or relaxing state.

5. The RNA in vitro transcription method according to claim 1, characterized in that, The method for preparing the magnetic core DNA hydrogel microparticles includes: preparing them using a polymer compound with functionalized groups, magnetic particles, and a DNA template; the DNA template may be selectively modified with functionalized groups, wherein the functionalized groups are any one or more combinations selected from amide, thiol, amino, or azide groups.

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