A method for constructing a hydrogel-based chip by 3D printing assisted water-phase frontal polymerization

Abstract

The application discloses a method for constructing a hydrogel-based chip by 3D printing assisted water-phase front polymerization, and relates to the technical field of functional polymer material preparation; gelatin is dissolved in water to form uniform ink, a 3D printer is used to print channels and transfer the channels to a culture dish, and gelatin supports are arranged at two ends of the channels; methyl methacrylate gelatin monomers, acrylic acid monomers, a polymerization inhibitor, an initiator and a crosslinking agent are dissolved in water and injected into the culture dish; an electric iron is used to heat to 100 DEG C, and after the polymerization reaction is completed, the gelatin supports are removed, the channels are cleaned in water at 60 DEG C, and the hydrogel-based chip is obtained. The method has the advantages of high speed, high efficiency, low cost, fast molding and the like, provides a path for realizing the preparation of a complex hydrogel chip, and has high application value in the fields of biology and chemical engineering.

Description

Technical Field

[0001] This invention relates to the field of functional polymer material preparation technology, specifically a method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization. Background Technology

[0002] Typically, high-boiling-point organic solvents such as dimethyl sulfoxide and glycerol are used in thermally initiated front-end polymerization to obtain stable and homogeneous polymers. However, the use of organic solvents limits the application of materials, especially in the biological field. Water is an excellent solvent, but its relatively low boiling point leads to a sharp increase in initiator decomposition temperature in aqueous front-end systems, causing boiling or gas release and resulting in the generation of a large number of bubbles in the polymer, which seriously affects the polymer's properties. In addition, the tendency of aqueous systems to self-polymerize also greatly affects the implementation of front-end polymerization reactions. Therefore, it is necessary to find a mild reaction method to achieve front-end polymerization in aqueous systems. However, to date, there are very few research results in this area, especially the use of thermal initiation to achieve aqueous front-end polymerization, which remains a challenge. Potzmann et al. (Macromolects 2015, 48, 24, 8738–8745) synthesized a novel initiator, potassium benzenesulfonyl peroxide, to induce a bubble-free, steady-state end-face reaction of monomers in water, preparing a bubble-free hydrogel material. This provides a foundation for the study of UV-initiated aqueous front-end polymerization reactions. Furthermore, Sun et al. (ACS Appl. Mater. Interfaces 2023, 15, 23, 28618–28625) used the cleavage of a water-soluble photoinitiator as the starting point for front-end polymerization, with potassium benzenesulfonyl peroxide as the thermal initiator and nano-SiO2 promoting the formation of hydrogen bonds between the main chains. This provides a new approach for the rapid synthesis of bubble-free, self-propagating hydrogel anti-corrosion coatings via UV-initiated front-end polymerization.

[0003] Traditional microfluidic chips are mainly based on rigid materials such as polymers, including the widely used polydimethylsiloxane. However, these materials have poor biocompatibility and cannot simulate the microenvironment of the extracellular matrix in vivo, which limits the in-depth application of microfluidic chips in biomedical research. Gel-based chips, on the other hand, can modify their composition according to the needs of the cell culture environment, providing maximum adaptability to the cell culture environment and even nutrients. Ni et al. (Small, 2018, 14, 45, 1802368) developed a novel method for manufacturing gel-based microfluidic chips with secondary cross-linking, capable of constructing gel chips with different complex internal flow channels. Using methacrylamide gelatin as the gel matrix, vascular endothelial cells were seeded to form a vascular model with vascular morphology and function. This method enables the rapid preparation of complex hydrogel chips, possessing both innovative and creative significance, as well as significant potential for widespread application. To address the aforementioned problems, the inventors proposed a 3D printing-assisted aqueous front-end polymerization method for constructing hydrogel-based chips. Summary of the Invention

[0004] To address the issue that traditional microfluidic chips are mainly based on rigid materials such as polymers, like the widely used polydimethylsiloxane, but these materials have poor biocompatibility and cannot simulate the microenvironment of the extracellular matrix in vivo, thus limiting the in-depth application of microfluidic chips in biomedical research, this invention aims to provide a method for constructing hydrogel-based chips using 3D printing-assisted aqueous front-end polymerization. This method has advantages such as high speed, high efficiency, low cost, and rapid molding, providing a pathway for the fabrication of complex hydrogel chips and possessing high application value in both biological and chemical engineering fields.

[0005] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for constructing hydrogel-based chips by 3D printing-assisted aqueous phase front-end polymerization, the specific steps of which are as follows:

[0006] S1. Weigh a certain amount of gelatin and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent, and then add ink cartridges.

[0007] S2. Use a 3D printer to print the ink through the preset path, adjust the parameters to heat to 37°C, and set the cooling plate temperature to 0°C.

[0008] S3. Weigh a certain amount of methacrylamide gelatin monomer and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent.

[0009] S4, acrylic acid monomer, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide monomer are added to the methacrylamide gelatin solution and stirred at room temperature (40-50°C) until the solution becomes clear;

[0010] S5. Add the initiator, polymerization inhibitor and crosslinking agent to the above mixed solution, and after stirring and dissolving, prepare a precursor solution of gel. Use ultrasound to remove air bubbles from the precursor solution.

[0011] S6. Pour the prepared gel precursor solution into a culture dish with a gelatin-supported template.

[0012] S7. Adjust the temperature of the soldering iron to 100-120℃, use the heating gun to heat the center of the solution, and stop heating when a stable end face appears in the thermal imager.

[0013] S8. After the polymerization reaction at the current end is completed, a hydrogel is formed. The gel is removed, the part containing the gelatin gel support is cut off, and it is heated in warm water at 40-60℃. Hot water is injected with a syringe to clean the channels, thus obtaining the hydrogel-based chip.

[0014] Preferably, the ink is a gelatin solution, and the specific gravity of the gelatin in the ink is 15-25 wt%.

[0015] Preferably, the pressure of the 3D printing air pump is 0.5-0.7 MPa.

[0016] Preferably, the diameter of the hydrogel chip is 0.5-1.5 mm.

[0017] Preferably, the diameter of the 3D printing needle is 0.4-1mm.

[0018] Preferably, the specific gravity of the methacrylamide gelatin is 4-6 wt%.

[0019] Preferably, the specific gravity of acrylic acid is 15-20 wt%, the specific gravity of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide is 4-6 wt%, and the solvent used is water with a specific gravity of 60-85 wt%.

[0020] Preferably, the polymerization inhibitor 2,2,6,6-tetramethylpiperidine-1-oxy radical has a specific gravity of 0.1 wt%, and the crosslinking agent is one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate 200 with a specific gravity of 1.5-2.5 wt%.

[0021] Preferably, the initiator is potassium benzenesulfonyl peroxysulfate with a specific gravity of 0.5-0.7 wt%, and the polymerization reaction is a front-end polymerization reaction method in which the polymerization temperature initiated by the soldering iron is 80-120℃, the distance between the soldering iron and the liquid surface of the culture dish is 0.8-1.2 mm, and the polymerization time initiated by the soldering iron is 40-60 s.

[0022] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0023] 1. In this invention, with the support of front-end polymerization technology, the polymerization reaction is ensured to proceed efficiently, the preparation time is shortened, and the mechanical properties and stability of the hydrogel are improved. Furthermore, combined with 3D printing technology, a sacrificial template method is used to prepare the hydrogel-based chip. Fluorescence microscopy reveals that the minimum channel pore size can reach 578 μm. In addition, stability tests on the gel-based chip, including mechanical properties and no leakage or diffusion within the gel after 12 days of injection with Rhodamine B solution, provide evidence supporting its suitability as a cell culture chip.

[0024] 2. In this invention, by combining front-end polymerization technology and 3D printing technology, complex microchannels and structures can be designed and manufactured, improving the performance and application flexibility of the chip. By injecting cells and culture medium into the hydrogel-based chip channel for cultivation, its applicability in the field of cell culture is confirmed, indicating that this method can provide technical reference for the preparation of gel microtube materials and has high promotion and application value. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 This is a schematic diagram illustrating the principle of constructing a hydrogel-based chip using 3D printing-assisted aqueous phase front-end polymerization in Example 1.

[0027] Figure 2 This is a schematic diagram of a gel-based chip used in cell culture. Detailed Implementation

[0028] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0029] Example: Figure 1-2 As shown, this invention provides a method for constructing hydrogel-based chips using 3D printing-assisted aqueous front-end polymerization, the specific steps of which are as follows:

[0030] S1. Weigh a certain amount of gelatin and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent, and then add ink cartridges.

[0031] S2. Use a 3D printer to print the ink through the preset path, adjust the parameters to heat to 37°C, and set the cooling plate temperature to 0°C.

[0032] S3. Weigh a certain amount of methacrylamide gelatin monomer and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent.

[0033] S4, acrylic acid monomer, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide monomer are added to the methacrylamide gelatin solution and stirred at room temperature (40-50°C) until the solution becomes clear;

[0034] S5. Add the initiator, polymerization inhibitor and crosslinking agent to the above mixed solution, and after stirring and dissolving, prepare a precursor solution of gel. Use ultrasound to remove air bubbles from the precursor solution.

[0035] S6. Pour the prepared gel precursor solution into a culture dish with a gelatin-supported template.

[0036] S7. Adjust the temperature of the soldering iron to 100-120℃, use the heating gun to heat the center of the solution, and stop heating when a stable end face appears in the thermal imager.

[0037] S8. After the polymerization reaction at the current end is completed, a hydrogel is formed. The gel is removed, the part containing the gelatin gel support is cut off, and it is heated in warm water at 40-60℃. Hot water is injected with a syringe to clean the channels, thus obtaining the hydrogel-based chip.

[0038] The ink is a gelatin solution with a gelatin specific gravity of 15-25 wt%.

[0039] By adopting the above technical solution, the viscosity of gelatin can be affected by its concentration (15-25wt%). A higher concentration helps to form a stable three-dimensional network structure, which supports the subsequent polymerization reaction.

[0040] The pressure of the 3D printing air pump is 0.5-0.7 MPa.

[0041] By adopting the above technical solution, appropriate air pump pressure can ensure that ink or material is smoothly delivered to the print head, avoiding clogging and uneven flow. Stable air pressure can also reduce material fluctuations and improve printing accuracy and surface smoothness.

[0042] The diameter of the hydrogel chip is 0.5-1.5mm.

[0043] By adopting the above technical solution, a suitable microenvironment can be created with a small diameter, which promotes cell growth and interaction, making it suitable for cell culture and drug screening. Furthermore, the smaller size increases the surface area to volume ratio, which helps to improve the reaction rate and material exchange efficiency.

[0044] The 3D printing speed is 150-250 mm / min.

[0045] By adopting the above technical solutions, printing efficiency can be improved and production time can be shortened by using a moderate speed, which can meet the needs of rapid prototyping. Furthermore, controlling the speed within this range helps to ensure good interlayer adhesion, reduce defects, and improve the overall accuracy and surface quality of printing.

[0046] The diameter of the 3D printing needle is 0.4-1mm.

[0047] By adopting the above technical solution, the smaller diameter is suitable for fine printing, the material flow can be precisely controlled, it is suitable for printing complex details, and the appropriate needle diameter can improve the smoothness of the printed surface and reduce the need for post-processing.

[0048] The specific gravity of methacrylamide gelatin is 4-6 wt%, the specific gravity of acrylic acid is 15-20 wt%, and the specific gravity of [2-(methacryloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide is 4-6 wt%.

[0049] By adopting the above technical solution, the combination of methacrylamide gelatin and acrylic acid can improve the strength and toughness of the material, making the printed parts more durable.

[0050] The solvent used is water with a specific gravity of 60-85 wt%. The polymerization inhibitor used is 2,2,6,6-tetramethylpiperidine-1-oxy free radical with a specific gravity of 0.1 wt%. The crosslinking agent is one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate 200 with a specific gravity of 1.5-2.5 wt%. The initiator is potassium benzenesulfonyl peroxysulfate with a specific gravity of 0.5-0.7 wt%.

[0051] By adopting the above technical solution, the main function of the polymerization inhibitor (2,2,6,6-tetramethylpiperidine-1-oxy radical) is to prevent unnecessary polymerization in the polymerization reaction, ensure the controllability of the reaction process, and thus improve the uniformity and performance of the final product. The crosslinking agent (N,N'-methylenebisacrylamide or polyethylene glycol diacrylate 200) can form a network structure during the polymerization process, improve the mechanical strength and heat resistance of the material, and at the same time improve the stability and durability of the material.

[0052] In the front-end polymerization reaction method, the polymerization temperature initiated by the soldering iron is 80-120℃, the soldering iron is 0.8-1.2mm away from the liquid surface of the culture dish, and the polymerization initiation time is 40-60s.

[0053] By adopting the above technical solution, the rate of polymerization reaction can be effectively adjusted by using appropriate temperature and time, ensuring uniform polymerization of materials. Higher temperatures help to improve the activity of monomers, promote cross-linking reactions, and enhance the mechanical strength and stability of hydrogels.

[0054] Working principle: When it is necessary to test a method for constructing hydrogel-based chips by 3D printing-assisted aqueous phase front-end polymerization;

[0055] Example 1

[0056] Weigh 3g of gelatin and dissolve it in 10mL of deionized water. Heat and stir at 50℃ until the particles dissolve, obtaining a pale yellow transparent liquid. Transfer the liquid to an ink cartridge and use a 0.423cm diameter needle. Print the channel using a 3D printer according to the preset path, adjusting the 3D air pump to 0.5MPa and the printing speed to 200mm / min. Heat to 37℃, and keep the cooling plate at 0℃. Transfer the printed channel to a petri dish, supporting both ends with gelatin gel. Simultaneously, weigh 0.57g of methacrylamide gelatin and dissolve it in 7.18g of deionized water. Heat and stir at 50°C until completely dissolved. Add 1.75g ​​of acrylic acid, 0.5g of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propionic acid)ammonium hydroxide, 0.01g of polymerization inhibitor 2,2,6,6-tetramethylpiperidine-1-oxy radical, and 200μL of crosslinking agent polyethylene glycol diacrylate 200 to a beaker. Stir thoroughly and then add 0.06g of initiator benzenesulfonyl peroxysulfate. After it is completely dissolved, add 200μL of tetramethylethylenediamine. Transfer the solution to a petri dish containing the gelatin support template. Set the soldering iron to 100°C and heat the center of the solution with a heating gun. Stop heating when a stable end face appears in the thermal imager. After the front-end polymerization reaction is complete and a hydrogel forms, the gel is removed, the portion containing the gelatin gel support is cut off, and the gel is heated in 60°C warm water. Hot water is then injected using a syringe to clean the channels, resulting in a hydrogel-based chip. The diameter of the obtained hydrogel chip is 642 μm. The front-end temperature is 62°C, the front-end speed is 0.0095 cm / min, the equilibrium swelling ratio is -22.56%, the tensile strength is 123.4 kPa, the fracture growth rate is 88.56%, the antibacterial effect is 21.23% lower than the control group, and the cell viability is 98.35%.

[0057] Example 2

[0058] Weigh 2.5g of gelatin and dissolve it in 10mL of deionized water. Heat and stir at 50℃ until the particles dissolve, obtaining a pale yellow transparent liquid. Transfer the liquid to an ink cartridge and use a 0.652cm diameter needle. Print the channel using a 3D printer according to the preset path, adjusting the 3D air pump to 0.6MPa and the printing speed to 220mm / min. Heat to 37℃, and keep the cooling plate at 0℃. Transfer the printed channel to a petri dish, supporting both ends with gelatin gel. Simultaneously, weigh 0.55g of methacrylamide gelatin and dissolve it in 7.18g of deionized water. Heat and stir at 50°C until completely dissolved. Add 2g of acrylic acid, 0.5g of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propionic acid)ammonium hydroxide, 0.01g of polymerization inhibitor 2,2,6,6-tetramethylpiperidine-1-oxy radical, and 200μL of crosslinking agent polyethylene glycol diacrylate 200 to a beaker. Stir and mix thoroughly. Add 0.06g of initiator benzenesulfonyl peroxysulfate and wait for it to completely dissolve. Add 200μL of tetramethylethylenediamine and transfer to a petri dish containing a gelatin-supported template. Set the soldering iron to 100°C and heat the center of the solution with a heating gun. Stop heating when a stable end face appears in the thermal imager. After the front-end polymerization reaction is complete and a hydrogel forms, the gel is removed, the portion containing the gelatin gel support is cut off, and the gel is heated in 60°C warm water. Hot water is then injected using a syringe to clean the channels, resulting in a hydrogel-based chip. The diameter of the obtained hydrogel chip is 725 μm. The front-end temperature is 65°C, the front-end speed is 0.011 cm / min, the equilibrium swelling ratio is -20.12%, the tensile strength is 125.2 kPa, the fracture growth rate is 74.81%, the antibacterial effect is 23.85% lower than the control group, and the cell viability is 98.85%.

[0059] Example 3

[0060] Weigh 2.6g of gelatin and dissolve it in 10mL of deionized water. Heat and stir at 50℃ until the particles dissolve, obtaining a pale yellow transparent liquid. Transfer the liquid to an ink cartridge and use a 0.754cm diameter needle. Print the channel using a 3D printer according to the preset path, adjusting the 3D air pump to 0.5MPa and the printing speed to 230mm / min. Heat to 37℃, and keep the cooling plate at 0℃. Transfer the printed channel to a petri dish, supporting both ends with gelatin gel. Simultaneously, weigh 0.54g of methacrylamide gelatin and dissolve it in 7.18g of deionized water. Heat and stir at 50°C until completely dissolved. Add 2.25g of acrylic acid, 0.5g of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonic acid propionic acid)ammonium hydroxide, 0.01g of polymerization inhibitor 2,2,6,6-tetramethylpiperidine-1-oxy radical, and 0.2mg of crosslinking agent N,N'-methylenebisacrylamide to a beaker. Stir and mix thoroughly. Add 0.07g of initiator benzenesulfonyl peroxysulfate and wait for it to completely dissolve. Add 200μL of tetramethylethylenediamine and transfer to a petri dish containing the gelatin support template. Set the temperature of the soldering iron to 110°C and heat the center of the solution with a heating gun. Stop heating when a stable end face appears in the thermal imager. After the front-end polymerization reaction is complete and a hydrogel forms, the gel is removed, the portion containing the gelatin gel support is cut off, and the gel is heated in 60°C warm water. Hot water is then injected using a syringe to clean the channels, resulting in a hydrogel-based chip. The diameter of the obtained hydrogel chip is 892 μm. The front-end temperature is 67.1°C, the front-end speed is 0.012 cm / min, the equilibrium swelling ratio is -17.35%, the tensile strength is 202.17 kPa, the fracture growth rate is 72.92%, the antibacterial effect is 28.85% lower than the control group, and the cell viability is 97.65%.

[0061] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. A method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization, characterized in that, Includes the following steps: S1. Weigh a certain amount of gelatin and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent, and then add ink cartridges. S2. Use a 3D printer to print the ink through the preset path, adjust the parameters to heat to 37°C, and set the cooling plate temperature to 0°C. S3. Weigh a certain amount of methacrylamide gelatin monomer and solvent water and put them into a container. Stir at 40-50℃ until the solution is clear and transparent. S4, acrylic acid monomer, and [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfonylpropyl)ammonium hydroxide monomer are added to the methacrylamide gelatin solution and stirred at room temperature (40-50°C) until the solution becomes clear; S5. Add the initiator, polymerization inhibitor and crosslinking agent to the above mixed solution, and after stirring and dissolving, prepare a precursor solution of gel. Use ultrasound to remove air bubbles from the precursor solution. S6. Pour the prepared gel precursor solution into a culture dish with a gelatin-supported template. S7. Adjust the temperature of the soldering iron to 100-120℃, use the heating gun to heat the center of the solution, and stop heating when a stable end face appears in the thermal imager. S8. After the polymerization reaction at the current end is completed, a hydrogel is formed. The gel is removed, the part containing the gelatin gel support is cut off, and it is heated in warm water at 40-60℃. Hot water is injected with a syringe to clean the channels, thus obtaining the hydrogel-based chip.

2. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The ink is a gelatin solution, and the specific gravity of the gelatin in the ink is 15-25 wt%.

3. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The pressure of the 3D printing air pump is 0.5-0.7 MPa.

4. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The diameter of the hydrogel chip is 0.5-1.5 mm.

5. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The 3D printing speed is 150-250 mm / min.

6. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The diameter of the 3D printing needle is 0.4-1mm.

7. The method for constructing a hydrogel-based chip using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The specific gravity of methacrylamide gelatin is 4-6 wt%.

8. The method for constructing hydrogel-based chips using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The specific gravity of acrylic acid is 15-20 wt%, the specific gravity of [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide is 4-6 wt%, and the solvent used is water with a specific gravity of 60-85 wt%.

9. The method for constructing a hydrogel-based chip using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The polymerization inhibitor 2,2,6,6-tetramethylpiperidine-1-oxy free radical used has a specific gravity of 0.1 wt%, and the crosslinking agent is one of N,N'-methylenebisacrylamide and polyethylene glycol diacrylate 200 with a specific gravity of 1.5-2.5 wt%.

10. The method for constructing a hydrogel-based chip using 3D printing-assisted aqueous phase front-end polymerization as described in claim 1, characterized in that, The initiator is benzenesulfonyl peroxysulfate potassium with a specific gravity of 0.5-0.7wt%. The polymerization reaction is a front-end polymerization reaction method in which the polymerization temperature initiated by the soldering iron is 80-120℃, the distance between the soldering iron and the liquid surface of the culture dish is 0.8-1.2mm, and the polymerization time initiated by the soldering iron is 40-60s.

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