A method for manufacturing and application of a microfluidic device based on a biomicroreactor

Abstract

This invention discloses a manufacturing method and application of a microfluidic device based on a biomicroreactor, relating to the field of bioengineering technology. The manufacturing method includes fixing a filter membrane to a first opening using a clamp; injecting a solution containing the biomicroreactor into the microfluidic device through a second opening (without the filter membrane installed) at the other end; injecting a buffer solution into the microfluidic device through the second opening; and replacing and washing the solvent system within the microfluidic device to complete the manufacturing of the biomicroreactor-based microfluidic device. Furthermore, this invention provides applications for the microfluidic device, including but not limited to bioactivity evaluation and bioactivity determination. It can provide on-site rapid evaluation and testing technology for the bio-evaluation and control of traditional Chinese medicine quality, and possesses advantages such as portability, low cost, ease of preparation, ease of operation, and ease of storage, making it suitable for various application scenarios.

Description

Technical Field

[0001] This invention relates to the field of bioengineering technology, specifically to a method for manufacturing and applying a microfluidic device based on a bioreactor. Background Technology

[0002] Quality control of traditional Chinese medicine (TCM) is a crucial aspect of its modernization and internationalization. Ensuring consistent quality across TCM products from different origins, varieties, and processing methods is a significant challenge. Existing quality control methods largely rely on chemical composition analysis, focusing on quality markers within TCM components and using these as a benchmark for quality assessment. However, the complex and diverse composition of TCM components leads to multiple pharmacological pathways, making it difficult to quantify the relationships between component content and type. A comprehensive quality evaluation cannot be achieved solely through the analysis of a few components. Therefore, establishing a simple, rapid, and comprehensive method and system for evaluating the bioactivity of TCM is essential.

[0003] A bioreactor is a device system that utilizes the biological functions of enzymes or organisms (such as microorganisms) to carry out biochemical reactions in vitro. A biomicroreactor refers to an even smaller bioreactor, characterized by its small size, high throughput, and low cost. Immobilized enzymes, due to their low cost and high activity, are used as bioreactors in industries such as drug synthesis, food processing, and chemical engineering. Enzyme cross-linking aggregates are a carrier-free immobilization technology that involves forming enzyme aggregates and using cross-linking agents to immobilize the enzymes between the aggregates; it belongs to the category of immobilized protein technology. Compared to other immobilization technologies, this technology features low cost, high enzyme activity, small size, and low operational barriers. However, despite the advantages of simple synthesis, immobilized protein technology, exemplified by enzyme cross-linking aggregates, still causes losses and structural damage to these "bioreactors" during preparation, washing, and reuse.

[0004] Microfluidic chips are fluid-driven microfluidic devices composed of a substrate with etched grooves and a cover plate. Their main advantages are small size, rapid processing, portability, and low cost, and they are now widely used in biomedicine, chemical analysis, environmental monitoring, and drug screening. By combining enzyme cross-linked aggregates with microfluidic chips, reusability, portability, ease of preparation, and high integration can be achieved, making them applicable to areas such as the evaluation of the activity of traditional Chinese medicine and the screening of its components.

[0005] Enzymes are natural biological catalysts closely related to metabolic processes in living organisms. With in-depth research into disease mechanisms, enzyme inhibitors are increasingly being used in disease treatment. For example, carbonic anhydrase inhibitors can be used to treat glaucoma and acute mountain sickness, lipase inhibitors can be used for weight loss, and angiotensin-converting enzyme inhibitors can be used to lower blood pressure. Therefore, research on enzyme inhibitors and their inhibitory activity can advance new drug development, drug activity evaluation, and drug quality control. In particular, establishing systematic, stable, and convenient bioactivity evaluation methods for enzyme inhibitory activity can fill gaps in the quality control of traditional Chinese medicine. Summary of the Invention

[0006] This invention addresses the problems of cumbersome and time-consuming washing steps and low reusability of existing bioreactors (enzyme cross-linked aggregates), and provides a method for manufacturing and applying a microfluidic device based on a bioreactor.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0008] A method for manufacturing a microfluidic device based on a biomicroreactor, the microfluidic device comprising a biomicroreactor, a microfluidic chip, a clamp, and a filter membrane; the microfluidic chip includes a substrate and a cover plate, with the cover plate attached to the upper surface of the substrate; wherein, the microfluidic chip has a groove channel that penetrates the substrate and the cover plate, and the groove channel forms a first opening and a second opening after penetrating the cover plate; a filter membrane is fixed to the first opening / / or the second opening by a clamp.

[0009] The manufacturing process includes the following steps: S1, fixing a filter membrane to the first opening with a clamp, and injecting a solution containing the bioreactor into the microfluidic device through a second opening at the other end where no filter membrane is installed; S2, injecting a buffer solution into the microfluidic device from the second opening, replacing and washing the solvent system in the microfluidic device, and completing the manufacturing of the microfluidic device based on the bioreactor.

[0010] Based on the above technical solution, further, the clamp is installed on the first opening or the second opening of the cover plate, and each clamp has a through hole, one through hole corresponding to the first opening or the second opening; the filter membrane is installed between the cover plate and the clamp, and the bottom of the clamp is provided with a sealing rubber ring, which contacts the upper end face of the filter membrane.

[0011] Based on the above technical solution, further, the pore size of the filter membrane in S1 is in the range of 0.1-10μm, preferably 50-500μm, and the filter membrane material includes, but is not limited to, polytetrafluoroethylene, polyethersulfone, polypropylene, polyvinylidene fluoride, nylon and regenerated cellulose; the volume of the solution containing the bioreactor in step S1 is 0.5-10mL; and the volume of the buffer solution in step S2 is 0.5-10mL.

[0012] Based on the above technical solution, furthermore, the method of injecting the solution containing the bioreactor into the microfluidic device in step S1 and the structure of injecting the buffer solution into the microfluidic device in step S2 are both any structure of a syringe, injection pump, or peristaltic pump to pressurize and drive the fluid.

[0013] An application of a microfluidic device based on a bioreactor in the evaluation of the bioactivity of traditional Chinese medicine or the screening of its components, wherein the application is to conduct bioevaluation of the active components of traditional Chinese medicine, and the active components of traditional Chinese medicine are tanshinone components.

[0014] An application of a microfluidic device based on a biomicroreactor in the evaluation of the activity of tanshinone components, comprising the following steps:

[0015] A1. Inject PNPA solution into a microfluidic device that has been equipped with a bioreactor. After placing the microfluidic device at room temperature and allowing it to stand for a period of time, take a picture to observe and record the color A in the groove channel.

[0016] A2. Inject buffer solution into the microfluidic device to rinse the bioreactor;

[0017] A3. Inject the mixture of acetazolamide solution and PNPA solution into the microfluidic device to complete the sample injection; after the microfluidic device is placed at room temperature and allowed to react for a period of time, take a picture to observe and record the color B in the groove channel;

[0018] A4. Inject desorption solution into the microfluidic device to desorb and rinse the biomicroreactor, and then inject buffer solution to rinse the biomicroreactor.

[0019] A5. Extract the RGB values ​​of color A and color B from the groove channel of the photograph. Create and print an eight-level color chart between color A and color B using the RGB values. Color A is olive green and color B is grayish white. Starting with color B, name the colors of the eight-level color chart from light to dark as color 1 to color 8. Among them, colors 1 to 4 are lighter green and colors 5 to 8 are gradually darker green.

[0020] A6. Inject the mixture of the sample solution and PNPA solution into the microfluidic device to complete the sample injection. Then, place the microfluidic device at room temperature and allow it to react for a period of time. Compare the color C in the groove channel with the color of the eight-level color chart. If color C falls within the color range of colors 1-3 in the eight-level color chart, the sample solution meets the bioactivity quality requirements. If color C falls within the color range of colors 6-8 in the eight-level color chart, the sample solution does not meet the bioactivity quality requirements. If color C falls within the color range of colors 4-5 in the eight-level color chart, repeat steps A1-A6 to re-evaluate the activity. The color comparison method described above can refer to the usage method of existing colorimetric test strips, such as pH test strips.

[0021] A7. Repeat step A4 to complete the washing process, or re-inject the solution containing the bioreactor for recycling.

[0022] Based on the above technical solution, further, the volume of the PNPA solution in step A1 is 0.1-5 mL, and the concentration is 0.1-20 mM; the reaction time in steps A1, A3, and A6 is 5-60 min, and the volume of the buffer solution used for washing is 0.1-5 mL; the volume of the buffer solution used for washing in steps A2 and A4 is 0.1-5 mL; the concentration of the acetazolamide solution in step A3 is 0.1 μM-100 μM; the volume of the mixed solution in steps A3 and A6 is 0.1-5 mL; the desorption solution in step A4 is 50%-100% volume fraction methanol, and the volume is 0.1-5 mL; wherein, the mixed solution in step A6 is obtained by mixing the sample solution to be tested and the PNPA solution in a 1:1 ratio.

[0023] An application of a microfluidic device based on a biomicroreactor in the determination of the activity of tanshinone components, comprising the following steps:

[0024] C1. Inject buffer solution into a microfluidic device that has been equipped with a biomicroreactor. After placing the microfluidic device at room temperature for a period of time, observe and record the color a in the groove channel.

[0025] C2. Inject buffer solution into the microfluidic device to flush the bioreactor;

[0026] C3. Inject PNPA solution into the microfluidic device, place the microfluidic device at room temperature for a period of time, and observe and record the color b in the groove channel.

[0027] C4. Inject buffer solution into the microfluidic device to flush the bioreactor;

[0028] C5. Inject the mixture of the sample solution and PNPA solution at a 1:1 ratio into the microfluidic device; after the microfluidic device is placed at room temperature and allowed to react for a period of time, observe and record the color c in the groove channel;

[0029] C6. Inject desorption solution into the microfluidic device to desorb and rinse the bioreactor, then inject buffer solution to rinse the bioreactor again for recycling;

[0030] C7. Extract the RGB values ​​from colors a, b, and c. Label the R values ​​of colors a, b, and c as R1, R2, and R3, respectively; the G values ​​as G1, G2, and G3, respectively; and the B values ​​as B1, B2, and B3, respectively. Calculate the suppression rate using the following formula and average them to obtain the suppression rate:

[0031] Inhibition rate 1 = [1 - (R3 - R1) / (R2 - R1)] × 100%;

[0032] Inhibition rate 2 = [1 - (G3 - G1) / (G2 - G1)] × 100%;

[0033] Inhibition rate 3 = [1 - (B3 - B1) / (B2 - B1)] × 100%.

[0034] Based on the above technical solution, furthermore, the buffer volume in steps C1, C2, C4, and C6 is 0.1-5 mL; the PNPA solution in step C3 has a volume of 0.1-5 mL and a concentration of 0.1-20 mM; the reaction time in steps C3 and C5 is 5-60 min; the mixture in step C5 is a 1:1 mixture of the sample solution and the PNPA solution, injected in a volume of 0.1-5 mL; the desorption solution in step C6 is 50%-100% methanol, with a volume of 0.1-5 mL; the RGB extraction method in step C7 is as follows: the unobstructed portion in the middle of the groove channel in the photograph is cropped, and average RGB extraction is performed using a tool.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] 1. The device provided by this invention combines a biomicroreactor with a microfluidic chip, which solves the structural damage and loss that is easily caused by the washing process in the preparation of biomicroreactors, taking enzyme cross-linked aggregates as an example.

[0037] 2. The device provided by this invention combines a biomicroreactor with a microfluidic chip, which solves the problems of easy loss and complicated steps in the recycling of biomicroreactors, such as enzyme cross-linked aggregates, and lowers the threshold for use.

[0038] 3. The device provided by this invention can be used for bioactivity evaluation and can provide on-site rapid evaluation and testing technology for the bio-evaluation and control of traditional Chinese medicine quality.

[0039] 4. The device provided by the present invention can be used for the determination of inhibitory activity, and it is simple to operate and can be reused.

[0040] 4. The device provided by this invention combines the advantages of portability, low cost, easy preparation, easy operation, and easy storage, and is suitable for a variety of use scenarios with a very low barrier to entry. Attached Figure Description

[0041] Figure 1 This is a schematic cross-sectional view of the microfluidic device based on a biomicroreactor according to the present invention;

[0042] Figure 2 This is a top-view schematic diagram of the microfluidic device based on a biomicroreactor according to the present invention;

[0043] Figure label:

[0044] 1. Fixture; 2. Filter membrane; 3. Groove channel; 4. Opening; 5. Cover plate; 6. Substrate; 7. Microfluidic chip. Detailed Implementation

[0045] It is worth noting that the raw materials used in this invention are all commercially available products, and their sources are not specifically limited.

[0046] The following sources of raw materials are provided as examples:

[0047] Example 1

[0048] This embodiment provides a method for manufacturing a microfluidic device based on a bioreactor, combined with... Figure 1 and Figure 2As shown, the microfluidic device consists of a bioreactor, a microfluidic chip 7, a clamp 1, and a filter membrane 2. In this embodiment, the microfluidic chip 7 includes a substrate 6 and a cover plate 5, with the cover plate 5 attached to the upper surface of the substrate 6. The microfluidic chip 7 has a groove channel 3 that penetrates the substrate 6 and the cover plate 5, forming an opening 4 structure after penetrating the cover plate 5. The opening 4 structure includes a first opening and a second opening. A filter membrane 2 is fixed to the first opening and / or the second opening by a clamp 1. The filter membrane 2 is fixed to the first and second openings at both ends of the microfluidic chip 7 by the clamp 1, with each opening corresponding to one filter membrane 2, located between the corresponding opening and the groove channel 3, serving to filter enzyme cross-linked aggregates. The clamp 1 is installed covering the first or second opening of the cover plate, and each clamp 1 has a through hole corresponding to the first or second opening. The filter membrane 2 is installed between the cover plate and the clamp 1, and the bottom of the clamp 1 is provided with a sealing rubber ring, which contacts the upper surface of the filter membrane 2. Furthermore, this microfluidic device is transparent, and the grooved channel 3 serves two purposes: firstly, it confines the solution within the grooved space, and secondly, it allows for direct observation of changes in the solution's color. It should be noted that the bioreactor in this embodiment refers to, but is not limited to, enzyme cross-linked aggregates.

[0049] Specifically, the side of the substrate 6 facing the cover plate 5 contains a groove channel 3 structure formed by etching or molding processes. The cover plate 5 has holes formed by molding, etching, or drilling at both ends of the groove channel 3 corresponding to the substrate 6. After the processed substrate 6 and cover plate 5 are bonded and assembled, a single-channel microfluidic chip 7 with openings at both ends is formed. In this embodiment, two clamps 1 are used, each covering one of the two holes on the cover plate 5. The two openings are aligned with holes formed by molding, etching, or drilling, and each clamp includes a sealing rubber ring. The clamps 1 are connected to the microfluidic chip 7 by screws and are removable and replaceable.

[0050] In this embodiment, taking enzyme cross-linked aggregates as an example, the specific process of the manufacturing method includes the following steps:

[0051] S1. A filter membrane is fixed to the first opening using a clamp 1. The enzyme cross-linked aggregate solution is injected into the microfluidic device through a second opening at the other end where no filter membrane is installed. In this embodiment, the pore size of the filter membrane ranges from 0.1 to 10 μm, preferably 50 to 500 μm. The filter membrane material can be polytetrafluoroethylene (PTFE), polyethersulfone (PES), polypropylene (PP), polyvinylidene fluoride (PVDF), nylon, regenerated cellulose, etc. Preferably, the filter membrane material is polypropylene. The volume of the enzyme cross-linked aggregate solution ranges from 0.5 to 10 mL.

[0052] S2. Inject the buffer solution into the microfluidic device through the second opening to replace and wash the solvent system within the microfluidic device, thus completing the fabrication of the enzyme cross-linked aggregate-based microfluidic device. In this embodiment, the volume of the washing buffer solution ranges from 0.1 to 5 mL, preferably 0.5 mL. Preferably, the buffer solution is 3-morpholinopropanesulfonic acid (MOPS). The sample dispensing method for the microfluidic device is a syringe, injection pump, peristaltic pump, or other device capable of pressurizing and driving the fluid. Preferably, the sample dispensing method for the microfluidic device is a syringe.

[0053] In this embodiment, the enzyme cross-linked aggregate is a cross-linked carbonic anhydrase. The preparation process of this cross-linked carbonic anhydrase is as follows: a carbonic anhydrase solution and a precipitant are mixed, and the mixture is shaken to precipitate for a period of time. The precipitant is ethanol or acetonitrile. A cross-linking agent is added to the mixture, and the mixture is shaken to cross-link for a period of time to obtain the cross-linked carbonic anhydrase. During the preparation process, the carbonic anhydrase is dissolved in a buffer solution, and the concentration of the carbonic anhydrase solution is 0.1-10 mg / mL, preferably 1 mg / mL. Preferably, the volume ratio of the carbonic anhydrase solution to the precipitant is 1-1000 μL:90-9000 μL. Preferably, the volume ratio of the carbonic anhydrase solution to the precipitant is 100 μL:900 μL. Preferably, the precipitant is ethanol. Preferably, the precipitation reaction conditions are: temperature 2-10℃, time 0.5-10 h. Preferably, the precipitation reaction conditions are: temperature 6℃, time 2 h. Preferably, the cross-linking agent is glutaraldehyde, and the final concentration of glutaraldehyde is 2 nM. Preferably, the crosslinking reaction conditions are: temperature 5-45℃, time 1-3h. Preferably, the crosslinking reaction conditions are: temperature 20-30℃, time 2-2.5h. Preferably, the crosslinking reaction conditions are: temperature 25℃, time 2.5h. The centrifugation conditions are: relative centrifugal force 6000-12000×g, time 5-20min; preferably, the centrifugation conditions are: relative centrifugal force 12000×g, time 10min.

[0054] The fabricated microfluidic device based on enzyme cross-linked aggregates was validated for successful fabrication using PNPA solution, including the following steps:

[0055] PNPA solution (6 mM) was injected into a microfluidic device containing carbonic anhydrase cross-linked aggregates. After the microfluidic device was left to stand at room temperature for 20 min, the color in the grooved channels was observed and recorded. This color was compared with the color of PNPA solution without injection and left to stand for 20 min. The comparison results were as follows: the uninjected PNPA solution was clear and transparent with almost no color change; the PNPA solution in the grooved channels of the microfluidic chip turned olive green, indicating that it was catalyzed by CA-CLEAs, proving that CA-CLEAs were successfully preserved in the microfluidic chip, and the device was successfully prepared. It should be noted that the PNPA solution refers to p-nitrophenylacetate solution.

[0056] Example 2

[0057] Based on the enzyme-crosslinked aggregate-based microfluidic device manufactured in Example 1, this microfluidic device can be applied to the evaluation of the bioactivity of traditional Chinese medicine or the screening of its components. Specifically, this application involves the bioevaluation of active components of traditional Chinese medicine, where the active component is a tanshinone component.

[0058] Example 3

[0059] Based on the microfluidic device based on enzyme cross-linked aggregates manufactured in Example 1, this microfluidic device can be applied in the evaluation and quality control of traditional Chinese medicine activity. In this example, its application is demonstrated in a method for evaluating the inhibitory activity of carbonic anhydrase on the tanshinone component. Specifically, it includes the following steps:

[0060] A1. Inject PNPA solution (6mM) into a microfluidic device containing enzyme crosslinking aggregates. After the microfluidic device is placed at room temperature and allowed to stand for a reaction time (20min), take a picture to observe and record the color A in the groove channel; the enzyme crosslinking aggregate is carbonic anhydrase crosslinking aggregate.

[0061] A2. Inject buffer solution into the microfluidic device to rinse the enzyme cross-linked aggregates;

[0062] A3. Inject the mixture of acetazolamide solution and PNPA solution (12mM) in a 1:1 ratio into the microfluidic device to complete the sample injection; after the microfluidic device is placed at room temperature and allowed to react for a period of time (20min), take a picture to observe and record the color B in the groove channel;

[0063] A4. Inject desorption solution into the microfluidic device to desorb and rinse the enzyme cross-linked aggregates, and then inject buffer solution to rinse the enzyme cross-linked aggregates.

[0064] A5. Extract the RGB values ​​of color A and color B from the groove channel of the photograph. Create and print an eight-level color chart between color A and color B using the RGB values. Color A is olive green and color B is grayish white. Starting with color B, name the colors of the eight-level color chart from light to dark as color 1 to color 8. Among them, color 1 to color 4 are lighter green and color 5 to color 6 are gradually darker green.

[0065] A6. Inject the mixture of the sample solution and PNPA solution into the microfluidic device to complete the sample introduction. Then, place the microfluidic device at room temperature and allow it to react for a period of time. Compare the color C in the groove channel with the color of the eight-level color chart. If color C falls within the color range of colors 1-3 in the eight-level color chart, the sample can be judged to meet the bioactivity quality requirements. If color C falls within the color range of colors 6-8 in the eight-level color chart, the sample solution can be judged to not meet the bioactivity quality requirements. If color C falls within the color range of colors 4-5 in the eight-level color chart, repeat steps A1-A6 to re-evaluate the activity. The color comparison method described above can refer to the usage method of existing colorimetric test strips, such as pH test strips.

[0066] A7. Repeat step A4 to complete the washing process, or re-inject the enzyme cross-linked aggregates for reuse.

[0067] The volume of the PNPA solution in step A1 is 0.1-5 mL, preferably 0.5 mL, and the concentration is 0.1-20 mM, preferably 6 mM; the reaction time in steps A1, A3, and A6 is 5-60 min, preferably 20 min. The color recording method can be handheld mobile phone photography, mobile phone stand-mounted photography, photography studio / box, or simple mounting bracket. Specifically, place white printing paper flat on a table, hold the chip 10-20 cm above the table, at an angle of 30°-60° to the horizontal plane, and... The phone is held 10-20cm away from the chip for imaging; the volume of the buffer solution used for washing is 0.1-5mL; the volume of the buffer solution used for washing in steps A2 and A4 is 0.1-5mL, preferably 0.5mL; the acetazolamide solution in step A3 is obtained by dissolving acetazolamide in dimethyl sulfoxide to prepare a stock solution, and then further diluting it with buffer solution; the concentration of the acetazolamide solution in step A3 is 0.1μM-100μM; preferably, the concentration of the acetazolamide solution in step A3 is 2μM. Preferably, the mixture in step A3 is obtained by mixing the acetazolamide solution and the PNPA solution in a 1:1 ratio. Preferably, the concentration of the PNPA solution in step A3 is 0.2-40mM. Preferably, the concentration of the PNPA solution in step A3 is 12mM.

[0068] Preferably, the volume of the mixed solution in steps A3 and A6 is 0.1-5 mL. Preferably, the volume of the mixed solution in steps A3 and A6 is 0.5 mL.

[0069] Preferably, the desorption solution in step A4 is 50%-100% methanol by volume. Preferably, the desorption solution in step A4 is 100% methanol. Preferably, the volume of the desorption solution in step A4 is 0.1-5 mL. Preferably, the volume of the desorption solution in step A4 is 0.5 mL.

[0070] Preferably, the sample solution in step A6 is obtained by dissolving the sample to be tested in dimethyl sulfoxide to prepare a stock solution, and then further diluting it with a buffer solution. Preferably, the mixture in step A6 is obtained by mixing the sample to be tested and PNPA solution in a 1:1 ratio. Preferably, the concentration of the PNPA solution in step A6 is 0.2-40 mM. Preferably, the concentration of the PNPA solution in step A6 is 12 mM. The mixture in step A6 is obtained by mixing the sample solution to be tested and the PNPA solution in a 1:1 ratio.

[0071] In this embodiment, in step A6, the mixture of tanshinone extract solution (1 mg / mL) and PNPA solution (12 mM) at a 1:1 ratio is injected into the microfluidic device to complete the sample injection. After the microfluidic device is placed at room temperature and allowed to react for a period of time, the color C in the groove channel is compared with the color of the color scale card.

[0072] Correspondingly, the 1 mg / mL tanshinone extract solution in step A6 of this embodiment is replaced with a 0.1 mg / mL tanshinone extract solution, and the other steps are the same.

[0073] The experimental procedure was as follows: 1. Microfluidic chip groove channels injected with only PNPA solution; 2. Microfluidic chip groove channels injected with only buffer solution; 3. Photographs of the microfluidic chip groove channels obtained after injecting the mixed solution.

[0074] Experimental Results: According to the information obtained, in this embodiment, the channel of the mixture containing 0.5 mg / mL tanshinone extract showed a lighter green color, which was closer to color 3 in the eight-level color chart. This indicates that it has a good carbonic anhydrase inhibitory effect and is of acceptable quality. However, in the embodiment, the channel of the mixture containing 0.05 mg / mL tanshinone extract showed a darker green color, which was closer to color 6 in the eight-level color chart. This indicates that its inhibitory activity was weak and its quality was unacceptable.

[0075] The above examples and results demonstrate that this microfluidic device can be applied to the evaluation of the bioactivity of traditional Chinese medicine, solving the problem that current quality control relies solely on chemical evaluation.

[0076] Example 4

[0077] Based on the microfluidic device based on enzyme cross-linked aggregates manufactured in Example 1, this microfluidic device can be applied in the assay of inhibitory activity. In this example, its application is implemented in a method for measuring the inhibitory activity of carbonic anhydrase on the tanshinone fraction. Specifically, it includes the following steps:

[0078] C1. Inject 0.5 mL of buffer solution into the microfluidic device containing enzyme crosslinked aggregates. After the microfluidic device has been placed at room temperature for a period of time (20 min), observe and record the color a in the groove channel.

[0079] C2. Inject 0.5 mL of buffer solution into the microfluidic device to rinse the enzyme cross-linked aggregates;

[0080] C3. Inject 0.5 mL of PNPA (6 mM) solution into the microfluidic device, place the microfluidic device at room temperature for a period of time (20 min), and observe and record the color b in the groove channel.

[0081] C4. Inject 0.5 mL of buffer solution into the microfluidic device to rinse the enzyme cross-linked aggregates;

[0082] C5. Inject the mixture of the sample solution and PNPA solution at a 1:1 ratio into the microfluidic device; after the microfluidic device is placed at room temperature and allowed to react for a period of time, observe and record the color c in the groove channel;

[0083] C6. Inject desorption solution into the microfluidic device to desorb and rinse the enzyme cross-linked aggregates, and then inject buffer solution to rinse the enzyme cross-linked aggregates for recycling.

[0084] C7. Extract the RGB values ​​from colors a, b, and c. Label the R values ​​of colors a, b, and c as R1, R2, and R3, respectively; the G values ​​as G1, G2, and G3, respectively; and the B values ​​as B1, B2, and B3, respectively. Calculate the suppression rate using the following formula and average them to obtain the suppression rate:

[0085] Inhibition rate 1 (%) = [1 - (R3 - R1) / (R2 - R1)] × 100%;

[0086] Inhibition rate 2 (%) = [1 - (G3 - G1) / (G2 - G1)] × 100%;

[0087] Inhibition rate 3 (%) = [1 - (B3 - B1) / (B2 - B1)] × 100%.

[0088] Preferably, the volume of the buffer solution described in steps C1, C2, C4, and C6 is 0.1-5 mL. Preferably, the volume of the buffer solution described in steps C1, C2, C4, and C6 is 0.5 mL. Preferably, the color recording method described in steps C1, C3, and C5 can be handheld mobile phone photography, mobile phone stand-fixed photography, photography studio, or simple fixed stand. Preferably, the color recording method described in steps C1, C3, and C5 is handheld photography, specifically as follows: place the white printing paper flat on the table, hold the chip in one hand 10-20 cm above the table, at an angle of 30°-60° to the horizontal plane, and hold the mobile phone in the other hand 10-20 cm away from the chip to take a picture.

[0089] Preferably, the volume of the PNPA solution in step C3 is 0.1-5 mL. Preferably, the volume of the PNPA solution in step C3 is 0.5 mL. Preferably, the concentration of the PNPA solution in step C3 is 0.2-40 mM. Preferably, the concentration of the PNPA solution in step C3 is 12 mM.

[0090] Preferably, the reaction time in steps C3 and C5 is 5-60 minutes. Preferably, the reaction time in steps C3 and C5 is 20 minutes.

[0091] Preferably, the sample solution in step C5 is obtained by dissolving the sample to be tested in dimethyl sulfoxide to prepare a stock solution, and then further diluting it with a buffer solution. Preferably, the mixture in step C5 is obtained by mixing the sample solution to be tested and a PNPA solution (0.2-40 mM, final concentration the same as in step C3) at a 1:1 ratio. Preferably, the concentration of the PNPA solution in step C5 is 0.2-40 mM. Preferably, the concentration of the PNPA solution in step C5 is 12 mM. Preferably, the volume of the mixture in step C5 is 0.1-5 mL. Preferably, the volume of the mixture in step C5 is 0.5 mL.

[0092] Preferably, the desorption solution in step C6 is 50%-100% methanol by volume. Preferably, the desorption solution in step C6 is 100% methanol. Preferably, the volume of the desorption solution in step C6 is 0.1-5 mL. Preferably, the volume of the desorption solution in step C6 is 0.5 mL.

[0093] Preferably, the RGB extraction method described in step C7 is as follows: crop the unobstructed part in the middle of the groove channel in the photo, and use tools such as Photoshop and MATLAB to perform average RGB extraction.

[0094] In this embodiment, the sample solution to be tested in step C5 is selected as tanshinone extract solution (1 mg / mL). Specifically, in C5, 0.5 mL of a mixture of tanshinone extract solution (1 mg / mL) and PNPA (12 mM) solution at a 1:1 ratio is injected into the microfluidic device. After the microfluidic device is placed in an incubator and allowed to react for 20 min, the color c in the groove channel is observed and recorded. Other steps are the same.

[0095] The calculation showed that the inhibition rate of 0.5 mg / mL tanshinone extract solution was 62%, while the inhibition rate measured by colorimetric method using a 96-well plate and microplate reader was 71%. The two results were not significantly different, indicating that the device can accurately determine the carbonic anhydrase inhibition rate.

[0096] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, and is not intended to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions made by those skilled in the art to the technical solution of the present invention do not depart from the essence and scope of the technical solution of the present invention.

Claims

1. A method for manufacturing a microfluidic device based on a bioreactor, characterized in that: The microfluidic device consists of a biomicroreactor, a microfluidic chip, a fixture, and a filter membrane; the biomicroreactor refers to, but is not limited to, enzyme cross-linked aggregates. The microfluidic chip includes a substrate and a cover plate, with the cover plate attached to the upper surface of the substrate; the microfluidic chip has a groove channel that penetrates the substrate and the cover plate, and the groove channel forms a first opening and a second opening after penetrating the cover plate; A filter membrane is fixed to the first opening and / or the second opening by a clamp respectively; The manufacturing process includes the following steps: S1. A filter membrane is fixed to the first opening by a clamp, and the solution containing the bioreactor is injected into the microfluidic device through the second opening at the other end where the filter membrane has not yet been installed. S2. Inject the buffer solution into the microfluidic device through the second opening to replace and wash the solvent system inside the microfluidic device, thus completing the fabrication of the microfluidic device based on the bioreactor.

2. The method for manufacturing a microfluidic device based on a biomicroreactor according to claim 1, characterized in that: The clamp is installed on the first or second opening of the cover plate, and each clamp has a through hole corresponding to the first or second opening; the filter membrane is installed between the cover plate and the clamp, and the bottom of the clamp is provided with a sealing rubber ring, which contacts the upper end face of the filter membrane.

3. The method for manufacturing a microfluidic device based on a biomicroreactor according to claim 1, characterized in that: The filter membrane in S1 has a pore size range of 0.1-10 μm, and the filter membrane material includes, but is not limited to, polytetrafluoroethylene, polyethersulfone, polypropylene, polyvinylidene fluoride, nylon, and regenerated cellulose; the volume of the solution containing the bioreactor in S1 is 0.5-10 mL; the volume of the buffer solution in S2 is 0.5-10 mL.

4. The method for manufacturing a microfluidic device based on a biomicroreactor according to claim 1, characterized in that: The method of injecting the solution containing the bioreactor into the microfluidic device in S1 and the method of injecting the buffer solution into the microfluidic device in S2 can be any of the following injection methods: syringe, injection pump, or peristaltic pump.

5. An application of a microfluidic device based on a bioreactor in the evaluation of the bioactivity of traditional Chinese medicine or the screening of its components, characterized in that: The microfluidic device based on the biomicroreactor is obtained by the manufacturing method described in any one of claims 1-4.

6. The application according to claim 5, characterized in that: The application is for the biological evaluation of active ingredients in traditional Chinese medicine, wherein the active ingredient in traditional Chinese medicine is tanshinone components.

7. The application of a microfluidic device based on a bioreactor in the activity evaluation of tanshinone components, characterized in that: The microfluidic device based on a biomicroreactor is obtained by the manufacturing method according to any one of claims 1-4; and includes the following steps: A1. Inject PNPA solution into a microfluidic device that has been equipped with a bioreactor. After placing the microfluidic device at room temperature and allowing it to stand for a period of time, take a picture to observe and record the color A in the groove channel. A2. Inject buffer solution into the microfluidic device to rinse the bioreactor; A3. Inject the mixture of acetazolamide solution and PNPA solution into the microfluidic device to complete the sample injection; after the microfluidic device is placed at room temperature and allowed to react for a period of time, take a picture to observe and record the color B in the groove channel; A4. Inject desorption solution into the microfluidic device to desorb and rinse the biomicroreactor, and then inject buffer solution to rinse the biomicroreactor. A5. Extract the RGB values ​​of color A and color B from the groove channel of the photograph. Create and print an eight-level color chart between color A and color B using the RGB values. Starting with color B, name the colors of the eight-level color chart from light to dark as color 1 to color 8. A6. Inject the mixture of the sample solution and PNPA solution into the microfluidic device to complete the sample injection. Then, place the microfluidic device at room temperature and allow it to react for a period of time. Compare the color C in the groove channel with the color of the eight-level color chart. If color C falls within the color range of colors 1-3 in the eight-level color chart, the sample is judged to meet the bioactivity quality requirements. If color C falls within the color range of colors 6-8 in the eight-level color chart, the sample solution is judged to not meet the bioactivity quality requirements. If color C falls within the color range of colors 4-5 in the eight-level color chart, repeat steps A1-A6 to re-evaluate the activity. A7. Repeat step A4 to complete the washing process, or re-inject the solution containing the bioreactor for recycling.

8. The application according to claim 7, characterized in that, The volume of PNPA solution used in step A1 is 0.1-5 mL, and the concentration is 0.1-20 mM; the reaction time in steps A1, A3, and A6 is 5-60 min, and the volume of the buffer solution used for washing is 0.1-5 mL; the volume of the buffer solution used for washing in steps A2 and A4 is 0.1-5 mL. In step A3, the concentration of the acetazolamide solution is 0.1 μM-100 μM; The volume of the mixed solution in steps A3 and A6 is 0.1-5 mL; the desorption solution in step A4 is 50%-100% methanol with a volume of 0.1-5 mL; wherein, the mixed solution in step A6 is obtained by mixing the sample solution to be tested and the PNPA solution in a 1:1 ratio.

9. The application of a microfluidic device based on a bioreactor in the determination of the activity of tanshinone components, characterized in that: The microfluidic device based on a biomicroreactor is obtained by the manufacturing method according to any one of claims 1-4; and includes the following steps: C1. Inject buffer solution into a microfluidic device that has been equipped with a biomicroreactor. After placing the microfluidic device at room temperature for a period of time, observe and record the color a in the groove channel. C2. Inject buffer solution into the microfluidic device to flush the bioreactor; C3. Inject PNPA solution into the microfluidic device, place the microfluidic device at room temperature for a period of time, and observe and record the color b in the groove channel. C4. Inject buffer solution into the microfluidic device to flush the bioreactor; C5. Inject the mixture of the sample solution and PNPA solution at a 1:1 ratio into the microfluidic device; after the microfluidic device is placed at room temperature and allowed to react for a period of time, observe and record the color c in the groove channel; C6. Inject desorption solution into the microfluidic device to desorb and rinse the bioreactor, then inject buffer solution to rinse the bioreactor again for recycling; C7. Extract the RGB values ​​from colors a, b, and c. Label the R values ​​of colors a, b, and c as R1, R2, and R3, respectively; the G values ​​as G1, G2, and G3, respectively; and the B values ​​as B1, B2, and B3, respectively. Calculate the suppression rate using the following formula and average them to obtain the suppression rate: Inhibition rate 1 = [1 - (R3 - R1) / (R2 - R1)] × 100%; Inhibition rate 2 = [1 - (G3 - G1) / (G2 - G1)] × 100%; Inhibition rate 3 = [1 - (B3 - B1) / (B2 - B1)] × 100%.

10. The application according to claim 9, characterized in that, The buffer solution volume in steps C1, C2, C4, and C6 is 0.1-5 mL; the PNPA solution volume in step C3 is 0.1-5 mL, and the concentration is 0.1-20 mM. The reaction time in steps C3 and C5 is 5-60 min; the mixture in step C5 is a 1:1 mixture of the sample solution and PNPA solution, injected in a volume of 0.1-5 mL; the desorption solution in step C6 is 50%-100% methanol, in a volume of 0.1-5 mL; the RGB extraction method in step C7 is to crop the unobstructed portion in the middle of the channel of the image and use a tool to perform average RGB extraction.

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