Method for increasing fluid transmission rate of paper-based micro-channel and paper-based micro-fluidic chip

Abstract

The invention relates to a method for increasing the fluid transmission rate of a paper-based micro-channel and a paper-based micro-fluidic chip. The method for increasing the fluid transmission rate of the paper-based micro-channel in the paper-based micro-fluidic chip comprises the following steps: S1, carrying out oxygen plasma treatment on the paper-based micro-channel for 2-6 minutes; s2, a PVP solution with the concentration being 1%-17% is added into the paper-based micro-channel subjected to oxygen plasma treatment, and the paper-based micro-channel with the fluid transmission rate improved is obtained after drying. The PVP molecules are introduced to the surface of the paper-based micro-channel, so that the hydrogen bond acting force is increased for fluid transmission of the high-viscosity liquid, the transmission rate of the high-viscosity liquid is increased through the synergistic effect of the hydrogen bond acting force and capillary force, and the RPA detection efficiency of the paper-based micro-fluidic chip can be greatly improved.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic detection, and in particular to a method for improving the fluid transmission rate of a paper-based microfluidic channel and a paper-based microfluidic chip. Background Art

[0002] Microfluidic devices are a technology that manipulates fluids at the micrometer scale and are widely used in chemistry, biology, medicine and other fields. Paper has many ideal properties, such as high affinity for liquids, swelling when absorbing liquids, good biocompatibility and biodegradability. Paper-based microfluidic devices can use the inherent wicking ability of paper to drive liquid flow, eliminating the need for external pumping. Compared with traditional silicon-based, glass-based or PDMS-based microfluidic devices, paper-based microfluidic devices are also more convenient to process and prepare. Microfluidic paper-based analytical devices (μPADs) have been developed for a variety of applications.

[0003] Paper-based microfluidic devices can use the capillary action of the internal microchannel to drive the sample to the reaction area without the help of external force. However, when the liquid viscosity is high, due to the weak capillary force of the microchannel, the liquid flow rate is slow, and it takes a long time for the liquid to reach the reaction area. Excessive flow time will cause the liquid to dry and lose due to evaporation during transportation. These factors, coupled with the sample retention in the paper matrix, will reduce the efficacy of μPAD, thereby increasing the detection cycle and reducing sensitivity. Long-term exposure of the liquid to the environment will also increase the risk of environmental contamination. For recombinase polymerase amplification (RPA) detection, it is generally necessary to add polymer aggregators to the activation solution, such as the most common polyethylene glycol (PEG) to increase the activity of the enzyme and improve the reaction efficiency. However, polymer aggregators have greater viscosity, which further slows down the transmission rate of the activation solution in the microchannel, which limits the development and application of paper-based microfluidic devices in RPA detection.

[0004] Therefore, how to enhance the fluid transfer rate in paper-based microfluidics to meet the detection needs of rapid transport of high-viscosity liquids is an urgent problem to be solved. Summary of the invention

[0005] The invention aims to solve the problem that traditional paper-based microfluidic chips are difficult to quickly transport high-viscosity liquids.

[0006] To achieve the above objectives, the present application provides a method for improving the fluid transmission rate of a paper-based microfluidic channel and a paper-based microfluidic chip.

[0007] In a first aspect, the present application provides a method for improving the fluid transmission rate of a paper-based microchannel in a paper-based microfluidic chip, comprising the following steps:

[0008] S1, treating the paper-based microfluidic channel with oxygen plasma for 2 to 6 minutes;

[0009] S2. Adding a PVP solution with a concentration of 1% to 17% into the paper-based microfluidic channel treated with oxygen plasma, and obtaining a paper-based microfluidic channel with improved fluid transfer rate after drying.

[0010] More specifically, during the oxygen plasma treatment, the treatment power is 100 W and the frequency is 13.65 MHz.

[0011] More specifically, the oxygen plasma treatment time is 4 minutes.

[0012] More specifically, the concentration of the PVP solution is 13%.

[0013] More specifically, the material of the paper-based microfluidic channel is selected from any one of printing paper, chromatography paper, filter paper, and nitrocellulose paper.

[0014] In a second aspect, the present application provides a paper-based microfluidic chip, comprising a paper-based microfluidic channel processed by the above method.

[0015] More specifically, it also includes a hydrophobic region, and the hydrophobic region is made of any one of wax, AKD, PDMS, and OTS.

[0016] More specifically, one end of the paper-based microfluidic channel is connected to the sample addition area, and the other end is connected to the reaction area.

[0017] More specifically, the paper-based microfluidic channel is also connected to any one or more of the substrate storage area, reaction area, color development area, buffer solution storage area, and waste liquid storage area.

[0018] In a third aspect, the present application also provides the application of the above-mentioned paper-based microfluidic chip in RPA detection.

[0019] The method for improving the fluid transmission rate of paper-based microfluidic channels provided in the present application firstly uses oxygen plasma to bombard the surface of the paper-based microfluidic channels to roughen the paper fibers (such as Figure 1 As shown in the figure, the paper fiber is significantly roughened after being treated with oxygen plasma for 4 minutes, and the number of hydroxyl groups of the paper fiber is greatly increased. Then, an appropriate amount of PVP solution is added to fix the PVP with the abundant hydroxyl groups on the surface of the paper fiber, thereby obtaining a paper-based microfluidic channel with PVP fixed on the surface. The paper-based microfluidic channel treated by this method has a higher fluid transfer rate, and is particularly suitable for high-viscosity activation fluids in RPA detection.

[0020] The principle of improving the fluid transmission rate of the present application is as follows: the treated paper-based microfluidic channel surface is combined with PVP polymer, and the abundant -C=O groups in the PVP molecules are used to form hydrogen bonds with the polymer aggregating agent (PEG) with higher viscosity in the sample, and the transmission rate of the high-viscosity liquid is increased by the formation force of hydrogen bonds. The amide groups (-C=O and CN) in the PVP molecules have the ability to accept hydrogen bonds, and can interact with hydrogen bond donors (such as -OH groups in PEG molecules) of other molecules (such as PEG) in the high-viscosity liquid. At the same time, PVP and PEG are both polar polymers, PVP has a polar amide structure, and PEG has -OH hydroxyl groups, and the polarities of the two are similar. At the same time, both PVP and PEG have longer chain structures and have certain molecular chain structure complementarity. Their molecular chains will be intertwined or form a network structure, thereby increasing their compatibility. The compatibility between PVP and PEG makes it easier for liquids to move on the paper surface. Through this molecular-level interaction, the viscosity and resistance of the liquid in the paper channel can be reduced, thereby accelerating the transportation speed of the high-viscosity liquid.

[0021] The beneficial effects of this application are:

[0022] This application uses oxygen plasma to treat the surface of paper-based microfluidic channels, and fixes an appropriate amount of PVP material on the treated microfluidic channel surface, and uses the synergistic driving force of PVP material and paper fiber on high-viscosity liquids to significantly improve the transmission rate. Especially for high-viscosity activation liquids in RPA detection, this method can effectively accelerate the transportation of samples and increase the transmission rate by 300%. More efficient transmission can reduce liquid evaporation loss and obtain more stable test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 is an electron microscope schematic diagram of the paper-based flow channel in this application after being treated with oxygen plasma;

[0024] Figure 2 is a schematic diagram of a paper-based microfluidic chip in this application;

[0025] Figure 3 is the performance test result of the embodiment in this application;

[0026] Figure 4 It is a graph of the PRV and ASF test results in this application.

[0027] In the figure: 1. Reagent dropping area; 2. Hydrophobic area; 3. Paper-based microfluidic channel; 4. Reaction area. DETAILED DESCRIPTION

[0028] In order to make the purpose, technical scheme and advantages of the implementation of the present invention clearer, the specific implementation methods of the present invention are clearly and completely described below. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] As Figure 2 The paper-based microfluidic chip shown in the figure was tested in a specific embodiment. Microfluidic chip production process: The paper-based substrate uses Whatman No. 4 filter paper, and a microchannel with a width of 2 mm and a length of 18 cm is drawn on the filter paper. The paper is printed using a wax jet printer (8580DN Xerox). After printing, the paper-based chip is placed on a heating plate (CS15956-31, Cole-Parmer), and heated at 120°C for 120 seconds to melt the wax and penetrate the entire paper to form a reagent drop area 1, a hydrophobic area 2, a paper-based microchannel 3, and a reaction area 4. Finally, the paper-based microfluidic chip is obtained by laser cutting. Microfluidic chip treatment: The paper-based microfluidic chip is placed in an oxygen plasma machine at room temperature, and oxygen plasma treatment is performed for 2 to 6 minutes at a power of 100 W and a frequency of 3.56 MHz. A PVP solution with a concentration of 1% to 17% is added to the treated paper-based microchannel 3, and the paper-based microchannel 3 with improved sample transmission rate is obtained after natural drying.

[0030] like Figure 3 As shown, the present application uses the above-mentioned paper-based microfluidic device to carry out 150 sets of embodiments respectively, and the time for the test sample to reach the reaction zone 4 from the reagent dripping area 1 through the paper-based microfluidic channel 3. PVP solutions with concentrations of 1%, 5%, 9%, 13%, and 17% are added to the paper-based microfluidic channel 3 after oxygen plasma treatment for 2min, 3min, 4min, 5min, and 6min, respectively, to obtain 25 paper-based microfluidic chips with different treatment processes, and each process is provided with 6 chips stored for 1 day, 3 days, 5 days, 7 days, 9 days, and 11 days, to verify that the treatment method of the present application has long-term effectiveness. Storage method: The prepared paper-based microfluidic chip is dried and placed in a sealed bag for storage, wherein the chip with a storage time of 1 day is directly used for testing without being placed in a sealed bag after drying, and the chip with a storage time of 3 days is placed in a sealed bag for 2 days after drying, and tested on the 3rd day. During the test, 25 microliters of African swine fever virus (ASF) fluorescent isothermal amplification detection reagent (the viscous component of which is 10% PEG 20000) is dripped into the reagent dripping area 1, and the time required for the reagent to flow through the paper-based microfluidic channel 3 to reach the reaction area 4 is recorded. The specific test results are as follows: Figure 3 shown.

[0031] In the untreated paper flow channel, it takes at least 80 seconds for the reagent to flow through the paper-based microchannel 3 to reach the reaction zone 4, while the transmission rate of the treated paper flow channel for high-viscosity reagents is significantly improved. With the increase of oxygen plasma treatment time, the degree of coarsening of the paper fiber surface is improved, and the number of hydroxyl groups on the paper fiber surface is also increased, but too long oxygen plasma treatment time will cause the surface energy of the paper-based flow channel to be too high, and at the same time, it will destroy the stable paper fiber structure and increase the water absorption of the paper-based flow channel, which will reduce the sample transmission rate. In this application, the preferred oxygen plasma treatment time is 4 minutes. The added PVP can combine with the hydroxyl groups on the surface of the paper fiber, and the combined PVP helps the transmission of high-viscosity liquids. However, the lower concentration of PVP solution is less combined with the paper fiber surface, the transmission rate is not significantly improved, and the low concentration of PVP is not stable enough to combine with the paper fiber. As the storage time of the paper chip increases, the sample transmission time increases. For example, in the experimental group treated with oxygen plasma for 4 minutes, the paper-based flow channel treated with a 5% PVP solution showed the shortest sample transmission time (38 seconds) on the 3rd day, but as the storage time increased, the sample transmission time was extended to 48 seconds on the 5th day, and the sample transmission time gradually increased during the subsequent storage time (54.3 seconds on the 7th day, 55.1 seconds on the 9th day, and 58.6 seconds on the 11th day). The paper-based flow channel treated with a 9% PVP solution showed a stable and shorter sample transmission time on the 3rd, 5th, and 7th days (25 seconds, 26.3 seconds, and 26.5 seconds, respectively), and the sample transmission time increased to 37.9 seconds on the 9th day, and the transmission time increased to 45.7 seconds on the 11th day. In the present application, the optimal PVP treatment concentration is 13%, and the sample transmission time after treatment is greatly reduced, and it still has a stable transmission rate improvement effect as the storage time increases. Among them, 13% PVP solution was added after oxygen plasma treatment for 4 minutes, and the sample transmission time was 25.5 seconds, 24.6 seconds, 26.5 seconds, 23.8 seconds, and 26.4 seconds when the paper chip was stored for 3, 5, 7, 9, and 11 days, respectively. When the added PVP concentration is too high, PVP will penetrate into the inside of the paper and affect the hydrophobic area. In severe cases, the sample cannot be transmitted along the preset paper-based microfluidic channel 3, but instead penetrates divergently into the reagent drop area 1 and the surrounding hydrophobic area of ​​the paper-based microfluidic channel 3, and the detection cannot be carried out normally.

[0032] This application is suitable for the rapid transportation of high-viscosity activation liquid in RPA detection. The paper-based microfluidic channel treated by the treatment method provided in this application has a nearly 300% increase in the transmission rate of high-viscosity activation liquid. The treatment method of this application can also be applied to paper-based microfluidic chips designed based on other detection principles, such as ELISA detection principle. This application can not only be used to process Figure 2The microfluidic chip shown can also be applied to microfluidic chips of other structures including functional areas such as substrate storage area, reaction area, color development area, buffer solution storage area, waste liquid storage area, etc. The improvement of the transmission rate of the paper-based microfluidic channel in this application mainly lies in the oxygen plasma treatment step implemented and the combination of the PVP solution with an appropriate concentration, while the style of the microfluidic chip and the microfluidic channel configuration do not affect the improvement of the transmission efficiency.

[0033] The present application can be used to improve the RPA detection rate of porcine pseudorabies virus (PRV). The RPA detection of porcine pseudorabies virus is specifically as follows: 8 μL of sample is dropped into the reaction area 4, and then enzyme dry powder is added to the reaction area 4, and the sample and enzyme dry powder are mixed in the reaction area 4 using a mixing device. Add the fluorescent activation liquid to the reagent dropping area 1, heat the reaction area 4 to keep it at 42°C, and after the fluorescent activation liquid flows into the reaction area 4, detect the fluorescent signal to quantify the concentration of porcine pseudorabies virus. The present application performs RPA detection of porcine pseudorabies virus (PRV) and African swine fever virus (ASF) in untreated and treated paper chips, respectively. The test results are as follows. Figure 4 shown.

[0034] The present application can also be used to improve the SERS detection rate of PCR products. The specific detection method is: cut the nanosilver particles into 0.5 cm square fragments, and then stack them in the reaction area 4. Drop 8 μL of the amplified target gene into the reagent drop area 1, add 2 microliters of dye to the reaction area 4, and measure the Raman signal after the target gene flows into the reaction area 4.

[0035] The embodiments of the present invention are described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions and variations of these embodiments are made without departing from the principles and spirit of the present invention, and still fall within the protection scope of the present invention.

Claims

1. A method for improving the fluid transmission rate of a paper-based microchannel in a paper-based microfluidic chip, characterized in that: The following steps are involved: S1, treating the paper-based microfluidic channel with oxygen plasma for 2 to 6 minutes; S2. Adding a PVP solution with a concentration of 1% to 17% into the paper-based microfluidic channel treated with oxygen plasma, and obtaining a paper-based microfluidic channel with improved fluid transfer rate after drying.

2. The method according to claim 1, characterized in that During the oxygen plasma treatment, the treatment power is 100 W and the frequency is 13.65 MHz.

3. The method according to claim 2, characterized in that The oxygen plasma treatment time is 4 minutes.

4. The method according to claim 1, characterized in that: The concentration of the PVP solution is 13%.

5. The method according to claim 1, characterized in that The material of the paper-based microfluidic channel is selected from any one of printing paper, chromatography paper, filter paper and nitrocellulose paper.

6. A paper-based microfluidic chip, characterized in that: A paper-based microfluidic channel treated by the method according to any one of claims 1 to 4.

7. The paper-based microfluidic chip according to claim 6, characterized in that: It also includes a hydrophobic region, which is made of any one of wax, AKD, PDMS, and OTS.

8. The paper-based microfluidic chip according to claim 6, characterized in that: One end of the paper-based microfluidic channel is connected to the sample adding area, and the other end is connected to the reaction area.

9. The paper-based microfluidic chip according to claim 8, characterized in that: The paper-based microfluidic channel is also connected to any one or several of the substrate storage area, the reaction area, the color development area, the buffer solution storage area, and the waste liquid storage area.

10. Use of the paper-based microfluidic chip according to any one of claims 6 to 8 in RPA detection.

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