Manufacturing method and application of microfluidic device based on biological microreactor

Abstract

The invention discloses a manufacturing method and application of a micro-fluidic device based on a biological micro-reactor, and relates to the technical field of biological engineering. The manufacturing method comprises the following steps: fixing a filter membrane on the first opening through a clamp, injecting a solution containing the biological microreactor into the microfluidic device through the second opening of which the other end is not provided with the filter membrane, and injecting a buffer solution into the microfluidic device from the position of the second opening, and replacing and washing the solvent system in the micro-fluidic device to manufacture the micro-fluidic device based on the biological micro-reactor. Besides, the invention provides application of the microfluidic device, including but not limited to biological activity evaluation and biological activity determination, an on-site rapid evaluation and rapid detection technology can be provided for biological evaluation and control of the quality of the traditional Chinese medicine, and the microfluidic device has the advantages of portability, low cost, easiness in preparation, operation and storage and the like, and is suitable for various use scenes.

Description

Technical Field

[0001] The invention relates to the technical field of bioengineering, and in particular to a manufacturing method and application of a microfluidic device based on a biological microreactor. Background Art

[0002] Quality control of traditional Chinese medicine is an important part of the modernization of traditional Chinese medicine and its internationalization. How to ensure that traditional Chinese medicine and Chinese patent medicine produced by different origins, different varieties and different processing methods maintain consistent quality is a major problem. Most of the existing quality control methods start from the analysis of chemical components, focus on the quality markers in the components of traditional Chinese medicine, and use this as a benchmark to judge the quality of traditional Chinese medicine. However, the composition of traditional Chinese medicine is complex and there are many types of traditional Chinese medicine, which leads to many pharmacological action pathways of traditional Chinese medicine. The relationship between the content and type of the ingredients is difficult to quantify, and it is difficult to comprehensively complete the quality evaluation through the analysis of several components. Therefore, it is very necessary to establish a simple, fast and comprehensive method and system for evaluating the biological activity of traditional Chinese medicine.

[0003] A bioreactor is a device system that uses the biological functions of enzymes or organisms (such as microorganisms) to perform biochemical reactions in vitro. A biomicroreactor refers to a smaller bioreactor with the characteristics of small size, high throughput, and low cost. Among them, immobilized enzymes are used as bioreactors in drug synthesis, food industry, chemical industry and other industries due to their low cost and high activity. Enzyme cross-linked aggregates are a carrier-free immobilization technology that forms enzyme aggregates and uses cross-linking agents to achieve immobilization between enzyme aggregates. It is a type of immobilized protein technology. Compared with other immobilization technologies, this technology has the characteristics of low cost, high enzyme activity, small size, and low operating threshold. However, although the immobilized protein technology, such as enzyme cross-linked aggregates, has advantages such as simple synthesis, preparation, washing, and reuse steps will still cause losses and structural damage to this type of "bioreactor".

[0004] Microfluidic chip is a fluid-driven microfluidic device, which is composed of a substrate with grooves etched and a cover. Its biggest features are small size, fast, portable and low cost. It is now widely used in biomedicine, chemical analysis, environmental testing, drug screening and other fields. By combining enzyme cross-linked aggregates with microfluidic chips, it can be reused repeatedly, easy to carry, easy to prepare, and highly integrated. It can be applied to the fields of traditional Chinese medicine activity evaluation and traditional Chinese medicine ingredient screening.

[0005] Enzyme is a natural biocatalyst that is closely related to the metabolic process in the body. With the in-depth study of the disease mechanism, enzyme inhibitors are gradually used in disease treatment. For example, carbonic anhydrase inhibitors can be used to treat glaucoma, acute mountain sickness and other diseases, lipase inhibitors can be used for weight loss, angiotensin converting enzyme inhibitors can be used to lower blood pressure, and so on. Therefore, research around enzyme inhibitors and enzyme inhibitory activity can promote new drug development, drug activity evaluation, drug quality control and other fields. Among them, around enzyme inhibitory activity, establishing a systematic, stable and convenient bioactivity evaluation method can make up for the missing link in the quality control of traditional Chinese medicine. Summary of the invention

[0006] Aiming at the problems of complicated and time-consuming washing steps and low reuse efficiency of bio-microreactors (enzyme cross-linked aggregates) in the prior art, the present invention provides a manufacturing method and application of a microfluidic device based on a bio-microreactor.

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

[0008] A method for manufacturing a microfluidic device based on a biological microreactor, wherein the microfluidic device is composed of a biological microreactor, a microfluidic chip, a fixture, and a filter membrane; the microfluidic chip comprises a substrate and a cover sheet, and the cover sheet is attached to the upper end surface of the substrate; wherein a groove channel is provided on the microfluidic chip, the groove channel passes through the substrate and the cover sheet, and a first opening and a second opening are formed after the groove channel passes through the cover sheet; a filter membrane is fixed on the first opening / or the second opening by a fixture.

[0009] The manufacturing process includes the following steps: S1, fixing a filter membrane on a first opening through a clamp, and injecting a solution containing a biological microreactor into the interior of a 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 position, replacing and washing the solvent system in the microfluidic device, and completing the manufacture of a microfluidic device based on a biological microreactor.

[0010] Based on the above technical solution, further, the clamp is installed to cover the first opening or the second opening of the cover plate, and each clamp is provided with 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 a sealing rubber ring is provided at the bottom of the clamp, and the sealing rubber ring is in contact with the upper end surface of the filter membrane.

[0011] Based on the above technical solution, further, the pore size range of the filter membrane in S1 is 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 biological microreactor solution in step S1 is 0.5-10mL; the volume of the buffer solution in step S2 is 0.5-10mL.

[0012] Based on the above technical solution, further, the method of injecting the solution containing the biological microreactor into the microfluidic device in step S1 and the structure of injecting the buffer solution into the microfluidic device in step S2 are any one of the structures of a syringe, an injection pump, and a peristaltic pump to pressurize and drive the fluid.

[0013] A microfluidic device based on a biological microreactor is used in the evaluation of the biological activity of traditional Chinese medicine or the screening of traditional Chinese medicine components. The application is to perform biological evaluation on the active components of traditional Chinese medicine, and the active components of traditional Chinese medicine are salvianolic acid components.

[0014] A microfluidic device based on a biomicroreactor is used in the activity evaluation of salvianolic acid components, and comprises the following steps:

[0015] A1. Inject PNPA solution into a microfluidic device equipped with a bio-microreactor, place the microfluidic device at room temperature for a period of reaction time, and then take a photo to observe and record the color A in the groove channel;

[0016] A2, injecting buffer into the microfluidic device to flush the bio-microreactor;

[0017] A3, injecting the mixture of acetazolamide solution and PNPA solution into the microfluidic device to complete the injection; placing the microfluidic device at room temperature for a period of time, taking a photo to observe and record the color B in the groove channel;

[0018] A4, injecting desorption liquid into the microfluidic device to desorb and rinse the bio-microreactor, and then injecting buffer to rinse the bio-microreactor;

[0019] A5. Extract RGB values ​​of color A and color B in the groove channel of the photographed photo, respectively, and make and print an eight-level color chart between color A and color B according to the RGB values, wherein 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 color 1 to color 8, wherein color 1 to color 4 are lighter in green, and color 5 to color 8 are gradually darker in green;

[0020] A6. Inject the mixed solution of the sample solution and the PNPA solution into the microfluidic device to complete the injection; then place the microfluidic device at room temperature for a period of time, and then compare the color C in the groove channel with the color of the eight-level color card. If the color C falls within the color range of color 1-color 3 in the eight-level color card, it can be judged that the sample to be tested meets the qualified quality requirements for biological activity; if the color C falls within the color range of color 6-color 8 in the eight-level color card, it can be judged that the sample solution to be tested meets the unqualified quality requirements for biological activity. If the color C is within the color range of color 4-color 5 in the eight-level color card, repeat steps A1-A6 to re-evaluate the activity; the color comparison method described above can refer to the use of existing colorimetric test paper, such as pH test paper;

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

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

[0023] A microfluidic device based on a biomicroreactor is used in the activity determination of salvianolic acid components, and comprises the following steps:

[0024] C1. Inject buffer into the microfluidic device equipped with the bio-microreactor, place the microfluidic device at room temperature for a period of time, and observe and record the color a in the groove channel;

[0025] C2, injecting buffer into the microfluidic device to flush the bio-microreactor;

[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, injecting buffer into the microfluidic device to flush the bio-microreactor;

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

[0029] C6, injecting desorption liquid into the microfluidic device to desorb and rinse the bio-microreactor, and then injecting buffer to rinse the bio-microreactor for recycling;

[0030] C7. Extract RGB values ​​of colors a, b, and c, record the R values ​​of colors a, b, and c as R1, R2, and R3, record the G values ​​as G1, G2, and G3, and record the B values ​​as B1, B2, and B3, respectively, calculate the inhibition rates using the following formulas, and find the average value to obtain the inhibition 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)] x 100%.

[0034] Based on the above technical scheme, further, the volume of the buffer solution in steps C1, C2, C4, and C6 is 0.1-5mL; the volume of the PNPA solution described in step C3 is 0.1-5mL, and the concentration is 0.1-20mM; the reaction time in steps C3 and C5 is 5-60min; the mixed solution in step C5 is a mixture of the sample solution to be tested and the PNPA solution in a ratio of 1:1, and the injection volume is 0.1-5mL; the desorption solution in step C6 is 50%-100% methanol by volume, and the volume is 0.1-5mL; the RGB extraction method in step C7 is: intercept the unobstructed part in the middle of the groove channel in the photo, and use the tool to perform average RGB extraction to obtain it.

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

[0036] 1. The device provided by the present invention combines a biological microreactor with a microfluidic chip, thereby solving the problem of structural damage and loss that is easily caused by the washing process during the preparation of a biological microreactor, such as enzyme cross-linked aggregates.

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

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

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

[0040] 4. The device provided by the present invention combines the advantages of portability, low cost, easy preparation, easy operation, and easy storage, is suitable for a variety of usage scenarios, and has a very low threshold for use. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 is a cross-sectional schematic diagram of a microfluidic device based on a biological microreactor of the present invention;

[0042] Figure 2 Schematic diagram of a top view of a microfluidic device based on a biological microreactor of the present invention;

[0043] Reference numerals:

[0044] 1. Fixture; 2. Filter membrane; 3. Groove channel; 4. Opening; 5. Cover sheet; 6. Substrate; 7. Microfluidic chip. DETAILED DESCRIPTION

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

[0046] The following sources of raw materials are provided for illustrative purposes:

[0047] Example 1

[0048] This embodiment provides a method for manufacturing a microfluidic device based on a biological microreactor, Figure 1 and Figure 2As shown, the microfluidic device is composed of a bio-microreactor, a microfluidic chip 7, a fixture 1, and a filter membrane 2. In this embodiment, the microfluidic chip 7 includes a substrate 6 and a cover sheet 5, and the cover sheet 5 is attached to the upper end surface of the substrate 6; wherein, a groove channel 3 is provided on the microfluidic chip 7, and the groove channel 3 penetrates the substrate 6 and the cover sheet 5, and an opening 4 structure is formed after the groove channel 3 penetrates the cover sheet 5, and the opening 4 structure includes a first opening and a second opening; a filter membrane 2 is fixed on the first opening and / or the second opening by a fixture 1; wherein, the filter membrane 2 is fixed at the first opening and the second opening at both ends of the microfluidic chip 7 by the fixture 1, and each opening corresponds to a filter membrane 2, which is located between the corresponding opening and the groove channel 3, and plays the role of filtering enzyme cross-linked aggregates. The fixture 1 is installed and covered on the first opening or the second opening of the cover plate, and each fixture 1 is provided with a through hole, and a through hole corresponds to the first opening or the second opening. The filter membrane 2 is installed between the cover plate and the fixture 1, and a sealing rubber ring is provided at the bottom of the fixture 1, and the sealing rubber ring contacts the upper end surface of the filter membrane 2. Further, the microfluidic device is transparent, and the groove channel 3 is provided to limit the solution to the groove space area on the one hand, and to visually observe the change in the color of the solution on the other hand. It should be noted that the biological microreactor 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 sheet 5 contains a groove channel 3 structure formed by an etching process or a molding process, and the cover sheet 5 has holes formed by a molding process, an etching process or punching at both ends of the groove channel 3 corresponding to the substrate 6. After the processed substrate 6 and the cover sheet 5 are assembled together, a single-channel microfluidic chip 7 with an opening structure at both ends can be formed. The number of the clamps 1 in this embodiment is two, which are respectively installed to cover the two hole positions on the cover sheet 5, and the positions aligned with the two openings have holes formed by a molding process, an etching process or punching, and include a sealing rubber ring. The clamp 1 is connected to the microfluidic chip 7 by screws and can be disassembled and replaced.

[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. Fix a filter membrane on the first opening through a fixture 1, and inject the enzyme cross-linked aggregate solution into the microfluidic device through the second opening at the other end where the filter membrane is not installed; in this embodiment, the filter membrane pore size range is 0.1-10μm, preferably 50-500μm, and 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 range of the enzyme cross-linked aggregate solution is 0.5-10mL.

[0052] S2, the buffer is injected into the microfluidic device from the second opening position, the solvent system in the microfluidic device is replaced, washed, and the microfluidic device based on the enzyme cross-linked aggregate is manufactured. In the present embodiment, the buffer volume range of washing is 0.1-5mL, preferably 0.5mL. Preferably, the buffer is 3-morpholinepropanesulfonic acid (MOPS). The microfluidic device sample addition mode is a device that can pressurize the driven fluid such as a syringe, a syringe pump, a peristaltic pump. Preferably, the microfluidic device sample addition mode is a syringe.

[0053] Wherein, the enzyme cross-linked aggregate in this embodiment is a cross-linked carbonic anhydrase, and the preparation process of the cross-linked carbonic anhydrase is: mixing a carbonic anhydrase solution and a precipitant, oscillating and precipitating for a period of time, and the precipitant is ethanol or acetonitrile; adding a cross-linking agent to the mixed solution, oscillating and cross-linking for a period of time, and obtaining a cross-linked carbonic anhydrase. Wherein, 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 and the precipitant is 1-1000 μL: 90-9000 μL. Preferably, the volume ratio of the carbonic anhydrase solution and the precipitant is 100 μL: 900 μL. Preferably, the precipitant is ethanol. Preferably, the conditions of the precipitation reaction are: temperature of 2-10 ° C, time of 0.5-10h. Preferably, the conditions of the precipitation reaction are: temperature of 6 ° C, time of 2h. Preferably, the cross-linking agent is glutaraldehyde, and the final concentration of the glutaraldehyde is 2nM. Preferably, the conditions of the cross-linking reaction are: temperature of 5-45°C, time of 1-3h. Preferably, the conditions of the cross-linking reaction are: temperature of 20-30°C, time of 2-2.5h. Preferably, the conditions of the cross-linking reaction are: temperature of 25°C, time of 2.5h. The centrifugal conditions are relative centrifugal force of 6000-12000×g, time of 5-20min; preferably, the centrifugal conditions are relative centrifugal force of 12000×g, time of 10min.

[0054] The fabricated microfluidic device based on enzyme cross-linked aggregates was verified using PNPA solution, which included the following steps:

[0055] PNPA solution (6mM) was injected into the microfluidic device containing carbonic anhydrase cross-linked aggregates. The microfluidic device was placed at room temperature for 20 minutes and then the color in the groove channel was observed and recorded. The color was compared with the color of the PNPA solution that was not injected and was left for 20 minutes. The comparison results are as follows: the uninjected PNPA solution is clear and transparent with little discoloration; the PNPA solution in the groove channel of the microfluidic chip turns olive green, indicating that it is catalyzed by CA-CLEAs, proving that CA-CLEAs have been successfully stored in the microfluidic chip and the device has been successfully prepared. It should be noted that the PNPA solution refers to p-Nitrophenylacetate solution.

[0056] Example 2

[0057] Based on the microfluidic device based on enzyme cross-linked aggregates manufactured in Example 1, the microfluidic device can be applied to the application of traditional Chinese medicine biological activity evaluation or traditional Chinese medicine component screening. Wherein, the application is the biological evaluation of traditional Chinese medicine active ingredients, and the traditional Chinese medicine active ingredients are salvianolic acid components.

[0058] Example 3

[0059] Based on the microfluidic device based on enzyme cross-linked aggregates manufactured in Example 1, the microfluidic device can be used in the activity evaluation and quality control of traditional Chinese medicine. In this embodiment, the application is implemented in the method for evaluating the inhibition of carbonic anhydrase activity by the salvianolic acid component. Specifically, the following steps are included:

[0060] A1. Inject PNPA solution (6 mM) into a microfluidic device equipped with enzyme cross-linked aggregates, place the microfluidic device at room temperature for a period of reaction time (20 min), take a photo to observe and record the color A in the groove channel; wherein the enzyme cross-linked aggregates are selected from carbonic anhydrase cross-linked aggregates;

[0061] A2, injecting buffer into the microfluidic device to wash the enzyme cross-linked aggregates;

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

[0063] A4, injecting a desorption solution into the microfluidic device to desorb and rinse the enzyme cross-linked aggregates, and then injecting a buffer solution to rinse the enzyme cross-linked aggregates;

[0064] A5. Extract RGB values ​​of color A and color B in the groove channel of the photographed photo, respectively, and make and print an eight-level color chart between color A and color B based on the RGB values, wherein 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 color 1 to color 8, among which color 1 to color 4 are lighter in green, and color 5 to color 6 are gradually darker in green;

[0065] A6. Inject the mixed solution of the sample solution and the PNPA solution into the microfluidic device to complete the injection; then place the microfluidic device at room temperature for a period of time, and then compare the color C in the groove channel with the color of the eight-level color card. If the color C falls within the color range of color 1-color 3 in the eight-level color card, it can be judged that the sample to be tested meets the qualified quality requirements for biological activity; if the color C falls within the color range of color 6-color 8 in the eight-level color card, it can be judged that the sample solution to be tested does not meet the qualified quality requirements for biological activity. If the color C is within the color range of color 4-color 5 in the eight-level color card, repeat steps A1-A6 to re-evaluate the activity; the color comparison method described above can refer to the use of existing colorimetric test paper, such as pH test paper;

[0066] A7. Repeat A4 to complete washing, or re-inject the enzyme cross-linked aggregate for recycling.

[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 fixed photography, studio / box, simple fixed stand, and the specific method is: lay the white printed paper flat on the table, one hand-held chip is 10-20 cm high from the table, and the angle with the horizontal plane is 30°-60°, and the other hand-held chip is 10-20 cm high from the table, and the angle with the horizontal plane is 30°-60°. A handheld mobile phone is used to take pictures at a distance of 10-20 cm from the chip; 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, preferably 0.5 mL; the acetazolamide solution in step A3 is prepared by dissolving acetazolamide in dimethyl sulfoxide to prepare a mother liquor, and then further diluting it with a buffer solution; the concentration of the acetazolamide solution in step A3 is 0.1 μM-100 μM; preferably, the concentration of the acetazolamide solution described in step A3 is 2 μM. Preferably, the mixed solution described in step A3 is obtained by mixing acetazolamide solution and PNPA solution in a ratio of 1:1. Preferably, the concentration of the PNPA solution described in step A3 is 0.2-40 mM. Preferably, the concentration of the PNPA solution described in step A3 is 12 mM.

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

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

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

[0072] Correspondingly, the 1 mg / mL salvianolic acid extract solution in step A6 of this embodiment was replaced with a 0.1 mg / mL salvianolic acid extract solution, and the other steps were the same.

[0073] The experimental process is as follows: 1. Only the PNPA solution is injected into the groove channel of the microfluidic chip; 2. Only the buffer solution is injected into the groove channel of the microfluidic chip. 3. A photo of the groove channel of the microfluidic chip obtained after the mixed solution is injected is obtained.

[0074] Experimental results: According to the information obtained, in this embodiment, the green color in the channel of the mixed solution containing 0.5 mg / mL of salvianolic acid extract is lighter, which is closer to color 3 in the eight-step color card, indicating that it has good carbonic anhydrase inhibition and is of qualified quality; while the green color in the channel finally obtained by the mixed solution containing 0.05 mg / mL of salvianolic acid extract in the embodiment is darker, which is closer to color 6 in the eight-step color card, indicating that its inhibitory activity is weaker and the quality is unqualified.

[0075] The above examples and results show that the microfluidic device can be applied to the evaluation of the biological activity of traditional Chinese medicine, solving the problem that quality control is currently only determined by chemical evaluation.

[0076] Example 4

[0077] Based on the microfluidic device based on enzyme cross-linked aggregates manufactured in Example 1, the microfluidic device can be used in the inhibition activity assay. In this embodiment, the application is implemented in the method for determining the inhibition activity of carbonic anhydrase by the salvianolic acid component. Specifically, the following steps are included:

[0078] C1. Inject 0.5 mL of buffer into the microfluidic device containing the enzyme cross-linked aggregates, place the microfluidic device at room temperature for a period of time (20 min), and observe and record the color a in the groove channel;

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

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

[0083] C6, injecting a desorption liquid into the microfluidic device to desorb and rinse the enzyme cross-linked aggregates, and then injecting a buffer solution to rinse the enzyme cross-linked aggregates, and then recycling them;

[0084] C7. Extract RGB values ​​of colors a, b, and c, record the R values ​​of colors a, b, and c as R1, R2, and R3, record the G values ​​as G1, G2, and G3, and record the B values ​​as B1, B2, and B3, respectively, calculate the inhibition rates using the following formulas, and find the average value to obtain the inhibition 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)] x 100%.

[0088] Preferably, the volume of the buffer solution in steps C1, C2, C4, and C6 is 0.1-5 mL. Preferably, the volume of the buffer solution in steps C1, C2, C4, and C6 is 0.5 mL. Preferably, the color recording method in steps C1, C3, and C5 can be handheld mobile phone photography, mobile phone stand fixed photography, studio, and simple fixed stand. Preferably, the color recording method in steps C1, C3, and C5 is handheld photography, and the specific method is: lay white printed paper flat on the desktop, hold a handheld chip 10-20 cm above the desktop, and an angle of 30°-60° with the horizontal plane, and hold another handheld mobile phone 10-20 cm away from the chip to take pictures.

[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 of steps C3 and C5 is 5-60 min. Preferably, the reaction time of steps C3 and C5 is 20 min.

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

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

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

[0094] In this embodiment, the sample solution to be tested in step C5 is selected as a salvianolic acid extract solution (1 mg / mL). Specifically, C5, 0.5 mL of a mixture of salvianolic acid extract solution (1 mg / mL) and PNPA (12 mM) solution in a ratio of 1:1 is injected into the microfluidic device. The microfluidic device is placed in a constant temperature box and allowed to react for 20 minutes, and then the color c in the groove channel is observed and recorded. The other steps are the same.

[0095] It was calculated that the inhibition rate of 0.5 mg / mL salvianolic acid extract solution was 62%, while the inhibition rate measured by the colorimetric method using a 96-well plate and an ELISA reader was 71%, which is not much different from the previous result. This indicates that the device can be used to more 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, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate 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 biological microreactor, characterized in that: The microfluidic device consists of a biomicroreactor, a microfluidic chip, a fixture, and a filter membrane; The microfluidic chip comprises a substrate and a cover sheet, and the cover sheet is attached to the upper end surface of the substrate; wherein a groove channel is provided on the microfluidic chip, the groove channel passes through the substrate and the cover sheet, and the groove channel passes through the cover sheet to form a first opening and a second opening; A filter membrane is fixed on the first opening and / or the second opening respectively by a clamp; The manufacturing process includes the following steps: S1, fixing a filter membrane on the first opening by a fixture, and injecting the solution containing the bio-microreactor into the microfluidic device through the second opening at the other end where the filter membrane is not installed; S2. Injecting a buffer solution into the microfluidic device from the second opening position, replacing and washing the solvent system in the microfluidic device, and manufacturing a microfluidic device based on a biological microreactor.

2. The method for manufacturing a microfluidic device based on a biological microreactor according to claim 1, characterized in that: The clamp is installed to cover the first opening or the second opening of the cover plate, and each clamp is provided with 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 a sealing rubber ring is provided at the bottom of the clamp, which contacts the upper end surface of the filter membrane.

3. The method for manufacturing a microfluidic device based on a biological microreactor according to claim 1, characterized in that: The pore size of the filter membrane in S1 is in the 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 biomicroreactor solution contained 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 biological microreactor according to claim 1, characterized in that: The method of injecting the solution containing the bio-microreactor into the microfluidic device in S1 and the method of injecting the buffer solution into the microfluidic device in S2 are injection methods with any structure of a syringe, a syringe pump, and a peristaltic pump.

5. Application of a microfluidic device based on a bio-microreactor in the evaluation of the biological activity of traditional Chinese medicine or the screening of traditional Chinese medicine components, characterized in that: The microfluidic device based on the biological microreactor is obtained by the manufacturing method according to any one of claims 1 to 4.

6. The use according to claim 5, characterized in that: The application is to carry out biological evaluation on active ingredients of traditional Chinese medicine, and the active ingredients of traditional Chinese medicine are salvianolic acid components.

7. Application of a microfluidic device based on a biomicroreactor in the activity evaluation of salvianolic acid components, characterized in that: The microfluidic device based on the biomicroreactor is obtained by the manufacturing method according to any one of claims 1 to 4; and comprises the following steps: A1. Inject PNPA solution into the microfluidic device equipped with the bio-microreactor, place the microfluidic device at room temperature for a period of reaction time, and then take a photo to observe and record the color A in the groove channel; A2, injecting buffer into the microfluidic device to flush the bio-microreactor; A3, injecting the mixture of acetazolamide solution and PNPA solution into the microfluidic device to complete the injection; placing the microfluidic device at room temperature for a period of time, taking a photo to observe and record the color B in the groove channel; A4, injecting desorption liquid into the microfluidic device to desorb and rinse the bio-microreactor, and then injecting buffer to rinse the bio-microreactor; A5. Extract RGB values ​​of color A and color B in the groove channel of the photographed image, respectively, and make and print an eight-level color chart between color A and color B according to the RGB values. Starting with color B, the colors of the eight-level color chart are named from light to dark, color 1 to color 8; A6. Inject the mixed solution of the sample solution and the PNPA solution into the microfluidic device to complete the injection; then place the microfluidic device at room temperature for a period of time, and compare the color C in the groove channel with the color of the eight-level color card. If the color C falls within the color range of color 1-color 3 in the eight-level color card, it is judged that the sample to be tested meets the qualified quality requirements for biological activity; if the color C falls within the color range of color 6-color 8 in the eight-level color card, it is judged that the sample solution to be tested does not meet the qualified quality requirements for biological activity. If the color C is within the color range of color 4-color 5 in the eight-level color card, repeat steps A1-A6 to re-evaluate the activity; A7. Repeat A4 to complete washing, or re-inject the solution containing the bio-microreactor for recycling.

8. The use according to claim 7, characterized in that: 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% methanol by volume, 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 ratio of 1:

1.

9. Application of a microfluidic device based on a biomicroreactor in the activity determination of salvianolic acid components, characterized in that: The microfluidic device based on the biomicroreactor is obtained by the manufacturing method according to any one of claims 1 to 4; and comprises the following steps: C1. Inject buffer into the microfluidic device equipped with the bio-microreactor, place the microfluidic device at room temperature for a period of time, and observe and record the color a in the groove channel; C2, injecting buffer into the microfluidic device to flush the bio-microreactor; 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, injecting buffer into the microfluidic device to flush the bio-microreactor; C5. Inject the mixture of the sample solution and the PNPA solution in a ratio of 1:1 into the microfluidic device; place the microfluidic device at room temperature for a period of time, and observe and record the color c in the groove channel; C6, injecting desorption liquid into the microfluidic device to desorb and rinse the bio-microreactor, and then injecting buffer to rinse the bio-microreactor for recycling; C7. Extract RGB values ​​of colors a, b, and c, record the R values ​​of colors a, b, and c as R1, R2, and R3, record the G values ​​as G1, G2, and G3, and record the B values ​​as B1, B2, and B3, respectively, calculate the inhibition rates using the following formulas, and find the average value to obtain the inhibition 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)] x 100%.

10. The use according to claim 9, characterized in that: The volume of the buffer in steps C1, C2, C4, and C6 is 0.1-5 mL; the volume of the PNPA solution 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 mixed solution in step C5 is a mixture of the sample solution and the PNPA solution in a ratio of 1:1, and the injection volume is 0.1-5 mL; the desorption solution in step C6 is 50%-100% methanol by volume, and the volume is 0.1-5 mL; the RGB extraction method in step C7 is: intercept the unobstructed part in the middle of the channel in the photo, and use the tool to perform average RGB extraction to obtain it.

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