Digital micro-fluidic chip, digital micro-fluidic system and detection method of digital micro-fluidic chip and digital micro-fluidic system

Abstract

The invention discloses a digital micro-fluidic chip, a digital micro-fluidic system and a detection method of the digital micro-fluidic chip, and the digital micro-fluidic chip comprises a top plate and a bottom plate; the bottom plate comprises a printed circuit board, a driving electrode, a dielectric layer and a hydrophobic layer which are arranged from bottom to top; the digital micro-fluidic system comprises a digital micro-fluidic chip, a driving module, a temperature control module, an optical detection module, a display module, an operation module, system software and a matched reagent. Through the design of the digital micro-fluidic chip and the cooperation of all modules of the digital micro-fluidic system, the effects of multi-gene synchronous detection, high-throughput detection, high detection speed, high automation degree and the like are realized.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a digital microfluidic chip and a digital microfluidic system and a detection method thereof. Background Art

[0002] Molecular diagnostic technology plays an important role in the diagnosis of pathogen-infected diseases. Highly sensitive and highly specific nucleic acid amplification tests (NAATs) have become the reference standard for diagnosing some common pathogens and specific drug-resistant genes. PCR-based real-time fluorescence PCR, multiplex PCR, digital PCR and isothermal amplification technology, as well as high-throughput sequencing, gene chips, nucleic acid mass spectrometry, biosensors and other detection technologies are widely used in large-scale laboratory batch testing or on-site nucleic acid instant testing (point-of-care testing, POCT).

[0003] Digital microfluidics (DMF) is a technology that uses electronic circuits to control the surface tension of liquids and precisely control the movement of discontinuous microdroplets. Using a programmable DMF drive system, droplets can move, split, and merge along a specified path, integrating the entire process of biochemical analysis on a microchip. Digital microfluidics has the characteristics of low reagent sample consumption, short detection and analysis time, good airtight isolation of pollution, and automated programming, making it possible to integrate nucleic acid extraction, amplification, and detection in the same device, while achieving a certain analytical throughput to meet actual clinical needs. Compared with other microfluidic devices commonly used for nucleic acid detection (such as paper-based microfluidic chips, centrifugal microfluidic chips, etc.), DMF chips have a typical channel-free structure, eliminating the requirements for external driving forces, microvalves, and complex channel networks. Its flexibility and compatibility with other technologies enable the DMF system to achieve complex biochemical analysis functions without increasing the complexity of the chip architecture, making it suitable for building multiple detection systems.

[0004] Nucleic acid detection based on DMF chips has been widely studied, but there are few multiplex detection methods demonstrated on DMF devices. Multiplex detection and high-throughput detection in digital microfluidic chips still need to be further coordinated through multiple technologies such as chip preparation process and surface treatment process to be better realized. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide a digital microfluidic chip and a digital microfluidic system and a detection method thereof in view of the deficiency of the prior art in lacking multi-detection and high-throughput detection based on a digital microfluidic chip.

[0006] One of the technical solutions adopted by the present invention to solve the technical problem is: providing a digital microfluidic chip, the chip comprising: a top plate and a bottom plate;

[0007] The top plate and the bottom plate are arranged in parallel to form a space for the droplets to run, the top plate and the bottom plate are sealed by double-sided tape, and the distance between the top plate and the bottom plate is 100-1000 microns;

[0008] The bottom plate includes the following layers arranged from bottom to top: a printed circuit board, a driving electrode, a dielectric layer and a hydrophobic layer;

[0009] The top plate includes the following layers arranged from top to bottom: an ITO glass layer and a hydrophobic layer, and the top plate is provided with a liquid injection port for adding a solution;

[0010] According to the arrangement of the driving electrodes, the space between the top plate and the bottom plate for the liquid droplets to run is divided into a liquid storage area and a reaction area;

[0011] The liquid storage areas are respectively arranged on the left side, right side and bottom of the chip, the left and right liquid storage areas respectively include multiple liquid storage tanks, and the bottom liquid storage area includes multiple liquid storage tanks for storing samples, primers or reaction solutions; the reaction area includes multiple reaction tanks; the samples, primers and reaction solutions are respectively obtained from the liquid storage tanks through a cross matrix arrangement to achieve efficient mixing, and then reacted in the reaction tanks.

[0012] Furthermore, the top plate is also provided with an oil filling port.

[0013] Furthermore, the dielectric layer is one or more of Parylene C, SU-8, and PDMS.

[0014] Furthermore, the hydrophobic layer is one or more of Teflon-AF and CYTOP.

[0015] Furthermore, the driving electrode uses metal Au as the electrode layer material, and the specific manufacturing process is: first, a metal Au / Cr layer is evaporated and deposited, wherein the metal Cr is used as an adhesion layer; the dielectric layer is prepared by chemical vapor deposition; and the hydrophobic layer is prepared on the dielectric layer by spin coating and drying the hydrophobic layer material.

[0016] The second technical solution adopted by the present invention to solve its technical problem is: to provide a digital microfluidic system, which includes the above-mentioned digital microfluidic chip, a drive module, a temperature control module, an optical detection module, a display module, an operation module, system software and supporting reagents.

[0017] The third technical solution adopted by the present invention to solve its technical problem is: providing a group of primers using the above-mentioned digital microfluidic chip or the above-mentioned digital microfluidic system, the sequences of the primers are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQID NO: 15 and SEQ ID NO: 16 respectively.

[0018] The fourth technical solution adopted by the present invention to solve the technical problem is: to provide a detection method based on the above-mentioned digital microfluidic system, the detection method comprising the following steps:

[0019] S1. Extract sample DNA solution;

[0020] S2. The sample DNA solution extracted in step S1 and the RPA premix are added to the liquid storage area of ​​the chip respectively, and are moved to the reaction area under the drive of the driving electrode to mix and then perform an amplification reaction. The temperature of the amplification reaction is 4°C to 95°C, and the time of the amplification reaction is 20min to 40min. The RPA premix includes 2×Reaction buffer; 10×Basic e-mix; 0.5μM forward primer, 0.5μM reverse primer, 1.8mM dNTPs; 1×SYBR Green I and nuclease-free sterilized water;

[0021] S3. Perform data analysis on the quantitative amplification results obtained in step S2.

[0022] Furthermore, the method for data analysis in step S3 is: taking images at an absorption wavelength of 497 nm and an emission wavelength of 520 nm, and calculating the grayscale values ​​before and after the reaction by ImageJ.

[0023] The implementation of the present invention has the following beneficial effects:

[0024] The present invention aims to further utilize the programmable capabilities of DMF technology to develop a high-throughput multiplex detection method for multiple pathogens based on RPA and DMF technology. The platform can be used under field conditions, thus providing a powerful tool for batch detection of pathogens or on-site instant detection. This method meets the current market demand for high diagnostic rates of pathogens, while also providing a new means of testing for precision medicine. Based on isothermal amplification technology, the present invention has established a new chip for virulence genes of multiple human and animal pathogens, providing a powerful tool, thereby providing new information for assessing the health risks of zoonotic pathogens in the environment. The beneficial effects of the present invention compared to the prior art are:

[0025] 1. Multi-gene simultaneous detection: It can realize the simultaneous detection of multiple virulence genes in the environment under the same conditions, greatly improving the detection efficiency and accuracy of virulence genes.

[0026] 2. High-throughput detection capability: Multiple amplification reactions can be performed simultaneously in a single test, and multiple samples can be tested, which effectively reduces the detection cost and significantly improves the detection throughput.

[0027] 3. Fast detection speed: The entire detection process takes only 45 minutes, which greatly shortens the detection time and meets the needs of rapid detection. It is particularly suitable for environments that require rapid feedback.

[0028] 4. Reagent saving: Due to high-precision droplet operation, the amount of reagents used is significantly reduced, further reducing the detection cost and improving resource utilization efficiency.

[0029] 5. High degree of automation: The entire detection process is automatically completed by the integrated control platform, which simplifies the operation process, reduces the complexity of manual operation, and improves the consistency and reliability of detection.

[0030] 6. Fully closed operation: The nucleic acid reaction is fully closed in the digital microfluidic chip. There is no need to open the cover, which completely eliminates the risk of aerosol contamination and improves the safety of the experiment and the credibility of the results.

[0031] 7. Provide a stable medium environment: Slowly add silicone oil to the oil filling port of the chip to fill the interlayer gap inside the chip, provide a stable medium environment for the movement of droplets, and prevent chemical contamination. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below in conjunction with embodiments with reference to the accompanying drawings.

[0033] Figure 1 It is a cross-sectional view of the microfluidic chip involved in the present invention.

[0034] Figure 2The present invention provides a design diagram of the bottom plate electrode array of the microfluidic chip.

[0035] Figure 3 The present invention is a schematic diagram of the top plate structure of the microfluidic chip.

[0036] Figure 4 This is a diagram of the specificity verification result of Example 2 of the present invention. A in the diagram is the fluorescence signal result displayed by the chip; B in the diagram is the gray value analysis heat map.

[0037] 1 droplet, 10 top plate, 11 liquid injection port, 12 oil injection port, 13 ITO glass layer, 14 hydrophobic layer on the top plate, 20 bottom plate, 21 printed circuit board, 22 driving electrode, 23 dielectric layer, 24 hydrophobic layer on the bottom plate, 30 liquid storage area, 31 liquid storage tank, 40 reaction area, 41 reaction tank. DETAILED DESCRIPTION

[0038] In order to better understand the present invention, the present invention is further described in detail below in conjunction with the embodiments and drawings. However, those skilled in the art understand that the following embodiments are not limitations on the scope of protection of the present invention, and any changes and modifications made on the basis of the present invention are within the scope of protection of the present invention.

[0039] The embodiments described herein are specific embodiments of the present invention, which are used to illustrate the concept of the present invention, are explanatory and exemplary, and should not be construed as limiting the embodiments of the present invention and the scope of the present invention. In addition to the embodiments described herein, those skilled in the art can also adopt other obvious technical solutions based on the contents disclosed in the claims and the specification of the present application, which include technical solutions for making any obvious replacements and modifications to the embodiments described herein. The drawings of this specification are schematic diagrams, which assist in explaining the concept of the present invention and schematically represent the shapes of the various parts and their mutual relationships. Please note that in order to facilitate the clear representation of the structures of the various parts of the embodiments of the present invention, the drawings are not necessarily drawn in the same proportions. The same or similar reference numerals are used to represent the same or similar parts.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0041] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0042] Example 1

[0043] Reference Figure 1The digital microfluidic chip of the present invention comprises: a top plate 10 and a bottom plate 20; the top plate 10 and the bottom plate 20 are arranged in parallel to form a space for the liquid drop 1 to run, and the top plate 10 and the bottom plate 20 are sealed by double-sided tape to achieve full closed operation. The nucleic acid reaction is fully closed in the digital microfluidic chip, and there is no need to open the cover for operation, which completely eliminates the risk of aerosol contamination and improves the safety of the experiment and the credibility of the results. The distance between the top plate 10 and the bottom plate 20 is 100-1000 microns for the solution to run.

[0044] The base plate 20 includes the following layers arranged from bottom to top: a printed circuit board (PCB) 21 , a driving electrode 22 , a dielectric layer 23 and a hydrophobic layer 24 .

[0045] The top plate 10 includes the following layers arranged from top to bottom: an ITO glass layer 13 and a hydrophobic layer 14 .

[0046] The dielectric layer 23 is evenly coated on the upper surface of the electrode layer and is an insulating material with high dielectric constant, uniform thickness and strong anti-puncture capability, and can be any one of Parylene C, SU-8 and PDMS.

[0047] The hydrophobic layer 24 may be made of any hydrophobic material such as Teflon-AF, CYTOP, etc.

[0048] The driving electrode 22 uses metal Au as the electrode layer material, and the specific manufacturing process is: first, a metal Au / Cr layer is evaporated and deposited, wherein the metal Cr is used as an adhesion layer; the dielectric layer 23 is made by chemical vapor deposition; the hydrophobic layer 24 is made on the dielectric layer 23 by spin coating the hydrophobic layer material and then drying it.

[0049] The top plate 10 is provided with a liquid injection port 11 for adding the solution, and is also provided with an oil injection port 12 for adding silicone oil. Silicone oil is slowly added into the oil injection port 12 to fill the interlayer gap inside the chip, provide a stable medium environment for the movement of droplets, and prevent chemical contamination.

[0050] The bottom plate 20 is provided with a grounding electrode, and the top plate 10 is grounded by contacting the bottom plate 20 through conductive silver paste. The ITO glass layer of the top plate 10 serves as a grounding electrode, and is then connected to the PCB through conductive silver paste, and then connected to the driving circuit board through an interface.

[0051] According to the arrangement of the driving electrodes 22, the space between the top plate 10 and the bottom plate 20 for the droplets to run is divided into a liquid storage area 30 and a reaction area 40; the liquid storage area 30 includes a plurality of liquid storage tanks 31, for example, the left and right liquid storage areas can each have 8 liquid storage tanks 31, and the lower liquid storage area 30 can have 12 liquid storage tanks 31, so that 96 amplification reactions can be carried out simultaneously in a single detection, and 8 virulence genes can be detected for 12 samples (or 12 virulence genes can be detected for 8 samples); the reaction area 40 includes a plurality of reaction tanks 41.

[0052] The liquid storage area 30 is respectively arranged on the left side, right side and bottom of the digital microfluidic chip, and can be used to store samples, primers and reaction solutions respectively. The storage of samples, primers and reaction solutions is not limited to specific locations. For example, samples can be stored in the left liquid storage area, the right liquid storage area or the bottom liquid storage area. The reaction area is the area where primers, samples and reaction solutions react. The cross array is used to achieve efficient mixing of reaction solutions, primers and samples.

[0053] The digital microfluidic chip of the present invention is equipped with a high-precision electrode control system, combined with advanced microcontroller technology and programmed software, to achieve precise movement and processing of droplets, meeting the needs of complex chemical and biological experiments.

[0054] The present invention also provides an integrated digital microfluidics (DMF) system, which aims to realize a fully automatic molecular diagnosis process from sample pretreatment to result display. The system includes the above-mentioned digital microfluidics chip, a drive module, a temperature control module, a magnetic module, an optical detection module, a display module, an operation module, system software and supporting reagents. The digital microfluidics chip is the main platform for droplet reaction; the drive module is used to drive the droplets to move, split and merge according to the specified path; the temperature control module is responsible for maintaining the optimal temperature of all reaction areas on the chip to ensure the consistency of reaction conditions and the reliability of results; the magnetic module is used to operate the magnetic beads; the optical detection module is used to detect the reaction process; the display module is used to display the detection results; the operation module is responsible for operating the moving path of the droplets, etc.; the system software is used to set and calibrate the position and direction of the chip, and can control the precise movement of the droplets on the platform. The system automatically controls the switch of the electrode according to the programmed path to achieve the positioning, movement, distribution, merging and splitting of the droplets.

[0055] The integrated digital microfluidic system of the present invention covers sample pretreatment, biochemical reaction, real-time detection and feedback, data processing and result display. The system uses small, light and highly integrated functional modules; tries to achieve integration, reduces unnecessary functions and necessary connections between functions; and realizes a reasonable overall layout.

[0056] The working principle of the present invention is as follows:

[0057] The sample to be tested, the amplification reagent and the primer are loaded into the liquid storage tank 31 in each liquid storage area 30 through the injection port 11 respectively; the operation module controls the driving electrode to form droplets from the liquid storage area and make the droplets move along the planned path on the chip; the RPA reagent is mixed with the sample and the primer in the reaction tank 41 in the reaction area 40 and then left to stand to complete the amplification reaction, and the temperature control module is responsible for maintaining the optimal temperature of all reaction areas on the chip to ensure the consistency of the reaction conditions and the reliability of the results; after the reaction is completed, the high-sensitivity optical detection module is used to read the fluorescence signal at a specific wavelength, and the detection result is displayed through the display module.

[0058] Example 2

[0059] This embodiment provides a digital microfluidic system for detecting typical zoonotic pathogens.

[0060] Primers were designed for the virulence genes of eight typical pathogens causing diseases to humans and animals, including Legionella, Salmonella, Shigella, Campylobacter jejuni, Listeria, Giardia, Amoeba and Bacteroides. The RPA primer combination that can detect multiple virulence genes in parallel (sequences are shown in Table 1) is as follows:

[0061] Table 1 RPA primer combinations for parallel detection of multiple virulence genes

[0062]

[0063] A detection method based on the digital microfluidic system of the present invention comprises the following steps:

[0064] S1. Extract DNA from the sample, take images at an absorption wavelength of 497nm and an emission wavelength of 520nm, load the images into ImageJ and convert them into grayscale images, draw the region of interest (ROI), and calculate the grayscale value before the reaction;

[0065] S2. Add the sample DNA extracted in step S1, RPA premix and primers of different concentrations (25 / 10 / 5mM) to the liquid storage area of ​​the chip respectively, and move them to the reaction area for mixed reaction under the drive of the driving electrode. The reaction temperature is 39°C and the reaction time is 20min.

[0066] Positive Control (PC): RPA reaction performed in a test tube

[0067] Negative control (NC): sterile water,

[0068] The RPA premix includes 2×Reaction buffer; 10×Basic e-mix; 0.5 μM forward primer, 0.5 μM reverse primer, 1.8 mM dNTPs; 1×SYBR Green I and nuclease-free sterilized water;

[0069] The microfluidic chip RPA reaction was carried out according to the steps of Example 1 above to verify the specificity of the 8 pairs of detection primers, with dd H2O as a blank control and Escherichia coli O157:H7 and its target gene eaeA as negative controls. Figure 4 As shown, 8 pairs of specific primers were used to amplify 8 target strain DNAs. Each pair of primers can generate fluorescent signals from the corresponding target strain DNA, but no fluorescent signals are generated in non-target strain DNAs, indicating that the designed primers and the established microfluidic chip RPA method have good specificity.

[0070] S3. Take an image of the amplification result obtained in step S2 at an absorption wavelength of 497nm and an emission wavelength of 520nm, load the image into ImageJ and convert it into a grayscale image, draw the region of interest (ROI) after black and white inversion, and calculate the grayscale value. The results show that there is a significant difference in the grayscale value before and after the reaction, which verifies the ability to perform RPA reactions based on this platform and the potential to detect typical zoonotic pathogens.

[0071] Although the specific implementation modes of the present invention are described above, those skilled in the art should understand that the specific implementation modes described are only illustrative and are not intended to limit the scope of the present invention. Equivalent modifications and changes made by those skilled in the art in accordance with the spirit of the present invention should be included in the scope of protection of the claims of the present invention.

Claims

1. A digital microfluidic chip, characterized in that: The chip comprises: a top plate and a bottom plate; The top plate and the bottom plate are arranged in parallel to form a space for the droplets to run, the top plate and the bottom plate are sealed by double-sided tape, and the distance between the top plate and the bottom plate is 100-1000 microns; The bottom plate includes the following layers arranged from bottom to top: a printed circuit board, a driving electrode, a dielectric layer and a hydrophobic layer; The top plate includes the following layers arranged from top to bottom: an ITO glass layer and a hydrophobic layer, and the top plate is provided with a liquid injection port for adding a solution; According to the arrangement of the driving electrodes, the space between the top plate and the bottom plate for the liquid droplets to run is divided into a liquid storage area and a reaction area; The liquid storage areas are respectively arranged on the left side, right side and bottom of the chip, the left and right liquid storage areas respectively include multiple liquid storage tanks, and the bottom liquid storage area includes multiple liquid storage tanks for storing samples, primers or reaction solutions; the reaction area includes multiple reaction tanks; the samples, primers and reaction solutions are respectively obtained from the liquid storage tanks through a cross matrix arrangement to achieve efficient mixing, and then reacted in the reaction tanks.

2. The chip according to claim 1, characterized in that: The top plate is also provided with an oil filling port.

3. The chip according to claim 1, characterized in that: The dielectric layer is one or more of Parylene C, SU-8, and PDMS.

4. The chip according to claim 1, characterized in that: The hydrophobic layer is one or more of Teflon-AF and CYTOP.

5. The chip according to claim 1, characterized in that: The driving electrode uses metal Au as the electrode layer material, and the specific manufacturing process is: first, a metal Au / Cr layer is evaporated and deposited, wherein the metal Cr is used as an adhesion layer; the dielectric layer is prepared by chemical vapor deposition; and the hydrophobic layer is prepared on the dielectric layer by spin coating and drying the hydrophobic layer material.

6. A digital microfluidic system, characterized in that: The digital microfluidic system comprises the digital microfluidic chip according to any one of claims 1 to 5, a driving module, a temperature control module, an optical detection module, a display module, an operation module, system software and supporting reagents.

7. A set of primers applied to the digital microfluidic chip according to claims 1 to 5 or the digital microfluidic system according to claim 6, characterized in that: The sequences of the primers are shown as SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, respectively.

8. A detection method based on the digital microfluidic system according to claim 6, characterized in that: The detection method comprises the following steps: S1. Extract sample DNA solution; S2. The sample DNA solution extracted in step S1 and the RPA premix are added to the liquid storage area of ​​the chip respectively, and are moved to the reaction area under the drive of the driving electrode to mix and then perform an amplification reaction. The temperature of the amplification reaction is 4°C to 95°C, and the time of the amplification reaction is 20min to 40min. The RPA premix includes 2×Reaction buffer; 10×Basic e-mix; 0.5μM forward primer, 0.5μM reverse primer, 1.8mM dNTPs; 1×SYBR Green I and nuclease-free sterilized water; S3. Perform data analysis on the quantitative amplification results obtained in step S2.

9. The detection method according to claim 8, characterized in that: The method for data analysis in step S3 is: taking images at an absorption wavelength of 497 nm and an emission wavelength of 520 nm, and calculating the grayscale values ​​before and after the reaction by ImageJ.

Images