Digital micro-fluidic chip for methylation sample pretreatment

Abstract

The digital micro-fluidic chip comprises an upper cover plate and a bottom plate, a cavity is formed between the upper cover plate and the bottom plate in a closed mode, the upper cover plate comprises an upper substrate, a conductive layer and a first hydrophobic layer, and the conductive layer and the first hydrophobic layer are arranged on one side of the upper substrate. A cavity is formed in the bottom plate, a lower substrate, a control electrode layer and a second hydrophobic layer are sequentially arranged on one side, facing the cavity, of the bottom plate, the control electrode layer and the second hydrophobic layer are arranged on one side of the lower substrate, the control electrode layer comprises a plurality of control electrodes, the digital micro-fluidic chip comprises a sample processing module, the sample processing module is provided with a plurality of functional areas, the functional areas are located in the cavity, and the sample processing module is provided with a plurality of functional areas. According to the digital micro-fluidic chip disclosed by the utility model, the automation degree of a methylation sample pretreatment process is high, the working efficiency is improved, the digital micro-fluidic chip is suitable for portable and miniaturized equipment, field detection can be realized, and the rapid detection efficiency can be improved.

Description

Technical Field

[0001] The utility model relates to a microfluidic chip technology, in particular to a microfluidic chip for processing methylation detection samples and a processing method for methylation samples. Background Art

[0002] Abnormal DNA methylation is closely related to the occurrence, development and cell carcinogenesis of tumors. In recent years, detecting the occurrence of cancer by detecting DNA methylation has become an important means, and even a routine technical indicator for medical detection and treatment of certain diseases.

[0003] The methylation detection process of biological samples includes preliminary nucleic acid extraction, methylation treatment of nucleic acid samples (also known as methylation sample pre-treatment) and subsequent PCR amplification.

[0004] Currently, the pretreatment of methylated samples mainly adopts bisulfite treatment. The basic steps of conventional bisulfite conversion are divided into: bisulfite conversion and desulfonation group, purification and recovery.

[0005] The main steps of bisulfite conversion include: (1) adding 100ul bisulfite conversion solution to 20ul DNA sample and mixing, and then performing the reaction in a constant temperature metal bath at 98℃ / 10min and 64℃ / 1h to complete denaturation; (2) transferring 220μL conversion product to a centrifuge tube, adding 600μL binding solution and 20μL magnetic bead suspension, and shaking and mixing at room temperature for 10min to complete binding; adding 500μL washing solution to the centrifuge tube, shaking and mixing, centrifuging instantly, placing on a magnetic stand and letting it stand, and aspirating and discarding the liquid in the tube to complete washing; adding 200μL desulfonation solution to the centrifuge tube, shaking and mixing, and then washing. The tube was mixed and incubated at room temperature for 15 minutes, centrifuged and placed on a magnetic rack for a short time, and the liquid in the tube was discarded to complete the desulfurization; 500 μL of washing solution was added to the centrifuge tube, centrifuged and placed on a magnetic rack for a short time, and the liquid in the tube was discarded to complete the first wash; 200 μL of washing solution was added to the centrifuge tube, centrifuged and placed on a magnetic rack for a short time, and the liquid in the tube was discarded to complete the second wash, and then dried at room temperature for 15 minutes; 30 μL of elution solution was added to the centrifuge tube, mixed and oscillated, warmed at 65°C for 10 minutes, centrifuged and placed on a magnetic rack for a short time, and the eluted product was finally completed to complete the methylation sample pretreatment. The traditional bisulfite conversion process often encounters many problems, such as: (1) low conversion efficiency; (2) many steps that require manual participation, which are prone to cross-contamination and lead to unstable results; (3) long bisulfite conversion and temperature change processes; (4) large amount of sample and reagent input and high cost.

[0006] The pre-treatment of methylation test samples is time-consuming due to the long process, multiple operations and complex procedures. In addition to the initial nucleic acid extraction and subsequent PCR amplification, the entire methylation test generally requires a test cycle of 1 and a half days. Therefore, clinicians and laboratories expect that the processing of methylation test samples can be completed in a shorter time, thereby shortening the time of PCR-based methylation testing. Utility Model Content

[0007] In view of the problems existing in the prior art, the utility model provides a methylation sample pretreatment method based on a digital microfluidic chip, including providing a digital microfluidic chip and a conversion reagent, wherein the digital microfluidic chip includes an upper cover plate, a bottom plate, and a cavity formed by sealing between the upper cover plate and the bottom plate, the upper cover plate includes an upper substrate and a conductive layer and a first hydrophobic layer on one side of the upper substrate, the bottom plate is sequentially provided with a lower substrate, a control electrode layer on one side of the lower substrate, a dielectric layer and a second hydrophobic layer on the side facing the cavity, the control electrode layer includes a plurality of control electrodes, the digital microfluidic chip includes a plurality of sample processing modules, the sample processing module is provided with a plurality of functional areas, the functional areas are located in the cavity, the control electrodes control the liquid distribution in the functional areas, the functional areas include a liquid storage area, a product extraction area, a sample area, a reaction area, a purification area and a waste liquid area; through holes are provided on the upper cover plate corresponding to the functional areas; the steps include: adding the conversion reagent to the liquid storage area through the through holes, adding the sample to the sample area, and the control electrodes control the conversion reagent and the sample to move between the functions to complete the methylation sample pretreatment.

[0008] Furthermore, the sample processing module may be one or more.

[0009] The conversion reagent comprises a conversion solution, magnetic beads, a binding solution, a desulfonation solution, a cleaning solution and an elution solution. The conversion solution, magnetic beads, a binding solution, a desulfonation solution, a cleaning solution and an elution solution are respectively stored in different liquid storage areas through through holes. Control electrodes control the movement of samples, the conversion solution, magnetic beads, a binding solution, a desulfonation solution, a cleaning solution and an elution solution in different functional areas to complete the steps of bisulfite conversion of methylated samples before treatment, binding of bisulfite-converted DNA with magnetic beads, purification, desulfonation, cleaning and elution.

[0010] Furthermore, the bisulfite conversion step includes: separating the DNA sample and the conversion solution required for the reaction by controlling the electrode and moving them to the reaction area, mixing the sample and the conversion solution to form a reaction solution, and incubating at 65 to 90° C. for 30 to 60 minutes.

[0011] Furthermore, the binding step includes separating the required magnetic beads and binding solution by controlling the electrodes and moving them to the reaction zone, and fully mixing them with the reaction solution in the bisulfite conversion step, and incubating at 15 to 30° C. for 5 to 15 minutes.

[0012] Furthermore, the step also includes adding sealing oil into the cavity.

[0013] Furthermore, the methylation sample pre-treatment method includes the following steps:

[0014] S1. The digital microfluidic chip is placed in a control instrument, which is connected and powered by a spring connector and a gold finger on the digital microfluidic chip, and the motion of the droplets is controlled by the control electrode;

[0015] S2. Add sealing oil. Use a pipette to add sealing oil from the waste liquid hole in the waste liquid area.

[0016] S3. Add samples and reagents, and use a pipette to inject DNA samples, magnetic beads, conversion solution, binding solution, cleaning solution, desulfonation solution, and elution solution into the corresponding reagent storage area through the corresponding through holes in the liquid storage area on the digital microfluidic chip;

[0017] S4. Bisulfite conversion: the temperature of the chip reaction zone is controlled to 80°C by controlling the temperature control module of the instrument, the DNA sample and the conversion solution in the liquid storage area are separated by controlling the electrode and moved to the reaction zone, the sample and the conversion solution are mixed to form a reaction solution, incubated at 80°C, and the reaction solution is controlled to move back and forth in the reaction zone every 30 seconds to improve the reaction efficiency, and the bisulfite conversion is completed in 40 minutes;

[0018] S5. Binding, the temperature of the reaction zone is controlled to be 22°C by the temperature control module, and the magnetic beads and the binding solution in the liquid storage area are separated by the control electrode and moved to the reaction zone, and then fully mixed with the reaction solution in the previous step, and incubated at 22°C for 5 minutes to complete the binding of DNA and magnetic beads;

[0019] S6. Purification 1, the reaction solution of the previous step is moved through the purification area by controlling the control electrode, and the magnetic control module is used to control the magnet to adsorb the magnetic beads to complete the separation of the magnetic beads and the liquid, and the separated liquid is moved to the waste liquid area by controlling the electrode, and the magnetic beads are retained in the purification area;

[0020] S7. Purification 2, by controlling the cleaning liquid in the liquid storage area through the control electrode to separate the cleaning liquid and move it to the purification area to complete the redissolution of the magnetic beads, and controlling the redissolved magnetic beads and the cleaning liquid to move to the reaction area to complete the mixing of the cleaning liquid and the magnetic beads; then controlling the magnetic bead mixed liquid to move through the purification area through the control electrode, and simultaneously controlling the magnet to adsorb the magnetic beads through the magnetic control module to complete the separation of the magnetic beads and the liquid, and moving the separated liquid to the waste liquid area through the control electrode, and the magnetic beads are retained in the purification area;

[0021] S8. Desulfonation, the desulfonation solution in the storage area is separated by controlling the electrode to move to the purification area to complete the re-dissolution of the magnetic beads, and then to the reaction area to complete the mixing of the desulfonation solution and the magnetic beads; incubate at 22°C for 5 minutes to complete the desulfonation reaction;

[0022] S9. Cleaning 1, controlling the reaction solution in the reaction zone to move through the purification zone by controlling the electrodes, synchronously adsorbing the magnetic beads to separate the magnetic beads and the liquid, and moving the separated liquid to the waste liquid zone by controlling the electrodes, and retaining the magnetic beads in the purification zone;

[0023] S10. Cleaning 2, controlling the cleaning liquid in the liquid storage area by controlling the electrodes to separate the cleaning liquid and move it to the purification area to complete the re-dissolution of the magnetic beads, and then moving it to the reaction area to complete the mixing of the cleaning liquid and the magnetic beads; then controlling the magnetic bead mixed liquid to move through the purification area by controlling the electrodes, and simultaneously controlling the magnet to adsorb the magnetic beads by the magnetic control module to complete the separation of the magnetic beads and the liquid, and moving the separated liquid to the waste liquid area by controlling the electrodes, and retaining the magnetic beads in the purification area;

[0024] S11. Clean 3, repeat step S10 once;

[0025] S12. Elution, control the eluent in the storage area by controlling the electrodes to separate the eluent and move it to the purification area to complete the re-dissolution of the magnetic beads, and move it to the reaction area to complete the mixing of the eluent and the magnetic beads; incubate at 65°C for 3

[0026] min to complete the reaction; then the reaction liquid is controlled by the control electrode to move in the reverse direction through the purification area, and the synchronous magnet adsorbs the magnetic beads to complete the separation of the magnetic beads and the liquid. The 5μL liquid that has been separated is the reaction product of the sample pretreatment. The reaction product is controlled by the control electrode to move to the product extraction area, and a pipette is used to collect the reaction product from the extraction hole.

[0027] The sulfite conversion solution includes sodium bisulfite and TWEEN-20; the binding solution includes guanidine hydrochloride, TWEEN-20, sodium chloride and anhydrous ethanol; the magnetic beads are hydroxyl-modified magnetic beads; the desulfonation solution includes sodium hydroxide, anhydrous ethanol and TWEEN-20; the elution solution includes Tris-HCl, EDTA and water, with a pH value of 8; and the cleaning solution includes anhydrous ethanol and Tris-HCl.

[0028] The utility model also provides a system for pre-treatment of methylation samples, including a digital microfluidic chip, a bisulfite conversion reagent and a control instrument, wherein the digital microfluidic chip includes an upper cover plate, a bottom plate, and a cavity formed by sealing between the upper cover plate and the bottom plate, wherein the upper cover plate includes an upper substrate and a conductive layer and a first hydrophobic layer on one side of the upper substrate, and the bottom plate is sequentially provided with a lower substrate, a control electrode layer on one side of the lower substrate, a dielectric layer and a second hydrophobic layer on the side of the bottom plate facing the cavity, wherein the control electrode layer includes a plurality of control electrodes, and the digital microfluidic chip includes a sample processing module, wherein the sample processing module is provided with a plurality of functional areas, wherein the functional areas are located in the cavity, and the control electrodes control the liquid distribution in the functional areas, and the functional areas include a liquid storage area, a product extraction area, a sample area, a reaction area, a purification area and a waste liquid area.

[0029] Furthermore, the sample processing module may be one or more.

[0030] Furthermore, the control instrument includes a temperature control module, a magnetic control module and a droplet motion control device for controlling the movement of droplets in the chip.

[0031] In view of the problems existing in the prior art, the utility model provides a digital microfluidic chip for methylation sample pretreatment, comprising an upper cover plate, a bottom plate, and a cavity formed by sealing between the upper cover plate and the bottom plate, wherein the upper cover plate comprises an upper substrate and a conductive layer and a first hydrophobic layer on one side of the upper substrate, and a lower substrate, a control electrode layer on one side of the lower substrate and a second hydrophobic layer are sequentially arranged on the side of the bottom plate facing the cavity, wherein the control electrode layer comprises a plurality of control electrodes, and the digital microfluidic chip comprises a sample processing module, wherein the sample processing module is provided with a plurality of functional areas, wherein the functional areas are located in the cavity, and the control electrodes control the liquid distribution in the functional areas, and the functional areas comprise a liquid storage area, a product extraction area, a sample area, a reaction area, a purification area and a waste liquid area.

[0032] Furthermore, the sample processing module may be one or more.

[0033] Furthermore, the number of the sample processing modules is any one of 1 to 16.

[0034] Furthermore, one side of the lower substrate also includes a dielectric layer.

[0035] Furthermore, the liquid storage area is connected to the product extraction area, the product extraction area is connected to the sample area, the sample area is connected to the reaction area, the reaction area is connected to the purification area, and the purification area is connected to the waste liquid area.

[0036] Furthermore, the liquid storage area includes a conversion liquid storage area, a binding liquid storage area, a magnetic bead storage area, a desulfurization liquid storage area, a cleaning liquid storage area and an elution liquid storage area.

[0037] Furthermore, the reaction zone includes a mixing zone, a sulfite conversion zone, and a desulfonation zone.

[0038] Furthermore, through holes are provided on the upper cover plate corresponding to different functional areas.

[0039] Furthermore, the functional areas are in liquid communication with each other through the transfer channels formed by the control electrodes.

[0040] Furthermore, the digital microfluidic chip also includes a gold finger, and each control electrode is connected to the gold finger.

[0041] Compared with the traditional manual methylation sample pretreatment method, the steps of bisulfite conversion, binding, purification, desulfonation, cleaning and elution of the methylation sample pretreatment are completed in the digital microfluidic chip by controlling the liquid on the chip by controlling the electrode, which can reduce the input of samples and reagents, shorten the direct conversion time, and ensure the stability of the conversion efficiency, and is not easily affected by human and environmental factors. The operation of the methylation sample pretreatment is simple, time-consuming, the amount of reagents used is small, and the cost is low. The DNA sulfite conversion efficiency and purification rate after treatment are high. The digital microfluidic chip can be provided with a processing module for multiple samples to realize the synchronous processing of multiple samples. And the detection sensitivity is high. The whole methylation sample pretreatment process based on the digital microfluidic chip has a high degree of automation, can be fully automated, improve work efficiency, get rid of the restrictions on professionals and laboratories, is suitable for portable and miniaturized equipment, and realizes on-site detection and improves the efficiency of rapid detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 It is a cross-sectional schematic diagram of a digital microfluidic chip used for pre-treatment of methylation samples according to the utility model.

[0043] Figure 2 It is a schematic diagram of the distribution of functional areas of the digital microfluidic chip used for methylation sample pretreatment of the utility model.

[0044] Figure 3 It is a schematic diagram of the connection structure between the digital microfluidic chip and the control instrument. DETAILED DESCRIPTION

[0045] The present invention is described in detail below in conjunction with specific embodiments.

[0046] like Figure 1 and 2 As shown, the digital microfluidic chip 10 for methylation sample pretreatment includes an upper cover plate 1, a bottom plate 2, and a cavity 3 formed by sealing between the upper cover plate and the bottom plate. The upper cover plate and the bottom plate are connected to each other by means of, but not limited to, glue sealing and bonding.

[0047] The upper cover plate 1 includes an upper substrate 11, and a conductive layer 12 and a first hydrophobic layer 13 are sequentially provided on the side of the upper substrate facing the cavity. The bottom plate 2 is sequentially provided with a lower substrate 21, a control electrode layer 22 on one side of the lower substrate, a dielectric layer 24, and a second hydrophobic layer 23 on the side facing the cavity. When the upper cover plate is placed upward, the digital microfluidic chip is sequentially provided with an upper substrate, a conductive layer, a first hydrophobic layer, a cavity, a second hydrophobic layer, a dielectric layer, a control electrode layer on one side of the lower substrate, and a lower substrate from top to bottom.

[0048] The material of the upper substrate 11 is selected from, but not limited to, transparent glass, PC plastic, COC plastic, PDMS plastic, etc., and is manufactured by injection molding, etc. The material of the conductive layer 12 is selected from, but not limited to, transparent conductive film ITO, polymer conductive material, etc., and the conductive layer is formed on the upper substrate 11 by vacuum sputtering, etc.

[0049] The material of the lower substrate 21 is selected from, but not limited to, PCB board, ITO glass, etc. The material of the control electrode layer 22 is selected from, but not limited to, copper foil, and is made using the PCB process of the integrated circuit industry. The material of the dielectric layer 24 is selected from, but not limited to, polyimide, and the dielectric layer can be made on the control electrode by processes such as coating or laminating a polyimide film.

[0050] The hydrophobic material of the hydrophobic layer is selected from but not limited to Teflon, Cytop, fluorocarbon compounds and their derivatives, and is formed on the conductive layer or the dielectric layer by spin coating, baking and curing.

[0051] The cavity is filled with sealing oil, which is selected from but not limited to silicone oil with a viscosity of 1 cst, to assist droplet driving and prevent droplet volatilization and reagent contamination. The sealing oil can be filled into the cavity of the microfluidic chip through the through hole on the waste liquid area.

[0052] The digital microfluidic chip for pre-processing methylation samples may include one or more sample processing modules, and the number of samples processed on the digital microfluidic chip may be set as needed. Figure 2 The digital microfluidic chip shown is provided with 8 sample processing modules in total, which can realize the simultaneous processing of 8 samples. Each sample processing module is provided with multiple functional areas, and the functional areas are located in the cavity. The functional areas include a liquid storage area 31, a product extraction area 32, a sample area 33, a reaction area 34, a purification area 35 and a waste liquid area 36. The flow and distribution of liquids in each functional area are controlled by one or more control electrodes. The liquid storage area 31 is used to store different reaction reagents, the product extraction area 32 is used to collect the reaction products after the conversion is completed, the sample area 33 is used for the storage of samples, the reaction area 34 is used for the mixing and reaction of samples and reagents, the purification area 35 is used for the purification of magnetic beads to separate the magnetic beads and the reaction liquid, and the waste liquid area 36 is used to collect waste liquid.

[0053] The liquid storage area 31 can also be subdivided into different reagent storage areas for storing different reaction reagents. The reagents for pre-treatment of methylated samples include conversion solution, binding solution, magnetic beads, desulfonation solution, cleaning solution and eluent, which are respectively stored in the conversion solution storage area 311, the binding solution storage area 312, the magnetic beads storage area 313, the desulfonation solution storage area 314, the cleaning solution storage area 315 and the eluent storage area 316 in the liquid storage area. When a plurality of sample processing modules are provided on the digital microfluidic chip, in a preferred example, two or more sample processing modules can share the reagents in one reagent storage area.

[0054] According to the different reaction steps, the reaction zone can also be subdivided into different areas, such as a mixing zone, a bisulfite conversion zone, a desulfonation zone and other multiple reaction zones (not shown in the figure). In a preferred embodiment, one or more of the mixing zone, the bisulfite conversion zone, the desulfonation zone, etc. can be shared, for example, the bisulfite conversion zone and the desulfonation zone are combined into one reaction zone to reduce the number of reaction zones set on the microfluidic chip.

[0055] The through hole on the waste liquid area 36 can also be used as a hole for adding sealing oil, and the sealing oil is filled into the cavity 3 after being added.

[0056] The upper cover plate is provided with a plurality of through holes 5 for different types of liquids to enter or be sucked out, and samples or reagents are injected into the corresponding functional areas through the through holes. For example, the upper cover corresponding to the reagent storage area is provided with a conversion liquid hole 51 for adding a conversion liquid, a binding liquid hole 52 for adding a binding liquid, a magnetic bead hole 53 for adding magnetic beads, a desulfurization liquid hole 54 for adding a desulfurization liquid, a cleaning liquid hole 55 for adding a cleaning liquid, and an eluent hole 56 for adding an eluent, the upper cover plate corresponding to the reaction product extraction area is provided with a product extraction hole 57, the upper cover plate corresponding to the sample area is provided with a sample addition hole 58, and the upper cover plate corresponding to the waste liquid area is provided with a closed oil addition hole 59. Different liquids can be injected and stored in different functions of the cavity through the corresponding through holes.

[0057] The distribution of each functional area of ​​the sample processing module can be arranged in sequence according to the reaction steps of the methylation sample pretreatment, for example, the liquid storage area is connected to the sample area, the sample area is connected to the reaction area, the reaction area is connected to the purification area, the purification area is connected to the product extraction area, and the product extraction area is connected to the waste liquid area. Figure 2 In the functional area distribution mode of the digital microfluidic chip shown, the product extraction area is arranged between the liquid storage area and the sample area, that is, the liquid storage area is connected to the product extraction area, the product extraction area is connected to the sample area, the sample area is connected to the reaction area, the reaction area is connected to the purification area, and the purification area is connected to the waste liquid area.

[0058] Depending on the type and amount of reagents, the storage area can be provided with multiple reagent storage areas, e.g. Figure 2The liquid storage area shown is provided with double rows of reagent storage areas, and in order to make full use of the space of the digital microfluidic chip, the two rows of reagent storage areas are arranged at intervals. For each sample processing, a set of reagents can be configured separately, or a set of reagents can be shared by multiple sample processing, that is, the reagents in the liquid storage area can provide reaction reagents for multiple samples at the same time.

[0059] The control electrode 6 refers to the small square copper foil of the control electrode layer, and the movement of the droplet 100 is from one control electrode to an adjacent control electrode. The digital microfluidic chip can include different functional areas formed by multiple control electrodes according to different functional requirements and different reagents, and the functional areas include but are not limited to a liquid storage area 31, a product extraction area 32, a sample area 33, a reaction area 34, a purification area 35 and a waste liquid area 36. The connection between the functional areas is to control the droplets to move along the control electrodes one by one, so that the liquid reaches another functional area from one functional area. Multiple adjacent control electrodes located between the functional areas are interconnected to form a longitudinal pipetting channel 221, a transverse pipetting channel 222 or a pipetting channel in other path directions, and the liquid between the areas is transferred to each other through these pipetting channels formed by the control electrodes.

[0060] like Figure 2 As shown, a gold finger 7 is also provided at the edge of the digital microfluidic chip 10, and each control electrode 6 is connected to the PCB gold finger 7. When the movement of the droplet needs to be controlled, the external electrical control system is electrically connected to the control electrode via the connector and the PCB gold finger.

[0061] like Figure 3 The control instrument 200 of the digital microfluidic chip shown includes but is not limited to a temperature control module 201 for heating the digital microfluidic chip, a magnetic control module 202 and a droplet motion control device for controlling the movement of droplets in the chip. The droplet motion control device includes a connector 203, which can be selected from but is not limited to a spring connector. The control instrument 200 supplies power to the gold finger 7 on the digital microfluidic chip through the spring connector to achieve control of the movement of the droplets by the control electrode.

[0062] Example 1 Methylation sample pretreatment method based on digital microfluidic chip

[0063] This embodiment uses Figures 1 to 3 Taking the digital microfluidic chip shown in the figure as an example, the method of implementing methylation sample pretreatment on the digital microfluidic chip is explained.

[0064] like Figure 1 and Figure 2The digital microfluidic chip 10 for methylation sample pretreatment shown comprises an upper cover plate 1, a bottom plate 2, and a cavity 3 formed between the upper cover plate and the bottom plate. A conductive layer 12 and a first hydrophobic layer 13 are provided on the upper cover plate, and a lower substrate 21, a control electrode layer 22 on one side of the lower substrate, a dielectric layer 24, and a second hydrophobic layer 23 are provided on the bottom plate.

[0065] like Figure 2 As shown, the digital microfluidic chip 10 shown in this embodiment sets up 8 DNA sample areas 33 in parallel, and each sample area is connected to the liquid storage area 31, the product extraction area 32, the reaction area 104, the purification area 35 and the waste liquid area 36 through the liquid transfer channel formed by the control electrode, thereby forming 8 sample processing modules to achieve multi-channel detection. Under the flexible control of the control electrode, the samples or reagents in the 8 channels can be moved synchronously and in parallel, and the pre-treatment experiments of the 8 methylation samples can be completed synchronously.

[0066] In this example, the connection order of the functional areas is: the liquid storage area 31 is connected to the product extraction area 32, the product extraction area 32 is connected to the sample area 33, the sample area 33 is connected to the reaction area 34, the reaction area 34 is connected to the purification area 35, and the purification area 35 is connected to the waste liquid area 36.

[0067] When there are multiple purification steps in the sample processing process, the purification zone is set between the reaction zone and the waste liquid zone, so that the purified waste liquid moves to the adjacent waste liquid zone, and the purified product moves to the adjacent reaction zone or extraction zone, which can effectively shorten the travel distance of the droplets and reduce the sample processing time.

[0068] The extraction area is arranged between the reaction area and the storage area, and at least a purification area and / or a reaction area is arranged between the extraction area and the waste liquid area. Such an arrangement can, on the one hand, shorten the travel distance of the droplets and reduce the sample processing time. On the other hand, due to the spacing between the functional areas such as the purification area, the liquid in the waste liquid area is not easily sucked into the extraction area by mistake during sampling, thereby avoiding the mixing of waste liquid and the influence on the PCR test results.

[0069] Each sample processing module includes a product extraction area 32, a reaction area 34, a purification area 35 and a waste liquid area 36, ​​and different sample processing modules can share the reagent storage area of ​​the liquid storage area 31. The liquid storage area is provided with a conversion liquid storage area 311, a desulfonation liquid storage area 314, a cleaning liquid storage area 315, an eluent storage area 316, a binding liquid storage area 312 and a magnetic bead storage area 313, which respectively store conversion liquid, desulfonation liquid, cleaning liquid, eluent, binding liquid and magnetic beads. In this example, two conversion liquid storage areas, two desulfonation liquid storage areas, two cleaning liquid storage areas, two eluent storage areas, two binding liquid storage areas and two magnetic bead storage areas are provided, forming two groups of reagent supply systems, and each group of reagent supply systems provides the four methylation samples with the reaction reagents required for the pretreatment of the methylation samples. In this example, the conversion liquid storage area, the desulfurization liquid storage area, the cleaning liquid storage area and the elution liquid storage area are arranged in the first row of the liquid storage area, and are in liquid communication with the reaction area and other functional areas through the longitudinal pipetting channel. The binding liquid storage area and the magnetic bead storage area are arranged in the second row of the liquid storage area, and are in liquid communication with the reaction area and other functional areas through the transverse pipetting channel and the longitudinal pipetting channel. The reagent storage areas in the first row and the reagent storage areas in the second row are arranged alternately, so that the arrangement of each functional area on the chip is more compact.

[0070] The multi-channel setting of the microfluidic chip shown in this example can realize parallel control of 8 channels and synchronously complete the reaction process of 8-channel reagents. The layout of different functional areas on the microfluidic chip improves the space utilization of the chip, reduces the area of ​​the chip, and further realizes the miniaturization and high efficiency of the detection equipment.

[0071] The reagents for pre-treatment of methylated samples include conversion solution, binding solution, magnetic beads, desulfonation solution, elution solution and cleaning solution.

[0072] In this embodiment, the conversion solution includes 5M sodium bisulfite and 0.5% TWEEN-20. The binding solution includes 5M guanidine hydrochloride, 0.5% TWEEN-20, 200mM sodium chloride, and 20% anhydrous ethanol. The desulfonation solution includes 0.2M sodium hydroxide, 90% anhydrous ethanol, and 0.5% TWEEN-20. The elution solution includes 10mM Tris-HCl, 0.1mM EDTA, pH 8. The cleaning solution includes 20% anhydrous ethanol, 10mM Tris-HCl, and 100mM Nacl.

[0073] The specific implementation steps of the methylation sample pretreatment based on the digital microfluidic chip of the utility model are as follows:

[0074] S1. The digital microfluidic chip is placed in the control instrument 200, the instrument is connected and powered by the spring connector and the gold finger on the digital microfluidic chip, and the motion control of the droplet is achieved by controlling the electrode;

[0075] S2. Add sealing oil. Use a pipette to add sealing oil from the waste liquid hole in the waste liquid area.

[0076] S3. Add samples and reagents, and use a pipette to inject the DNA sample, magnetic beads, conversion solution, binding solution, cleaning solution, desulfonation solution, and elution solution into the corresponding reagent storage area through the corresponding through holes in the liquid storage area of ​​the digital microfluidic chip;

[0077] S4. Bisulfite conversion: the temperature of the chip reaction zone is controlled to 80°C by controlling the temperature control module of the instrument, and 2 μL DNA sample and 4 μL conversion solution are separated from the storage area DNA sample and conversion solution by controlling the electrode and moving them to the reaction zone, and the sample and conversion solution are mixed to form a reaction solution, which is incubated at 80°C. The reaction solution is controlled to move back and forth in the reaction zone every 30 seconds to improve the reaction efficiency, and the bisulfite conversion is completed in 40 minutes;

[0078] S5. Binding, control the temperature of the reaction zone to 22°C through the temperature control module, separate 2 μL of magnetic beads and 4 μL of binding solution from the storage area through the control electrode and move them to the reaction zone, and mix them with the 6 μL reaction solution in the previous step, and incubate at 22°C for 5 minutes to complete the binding of DNA and magnetic beads;

[0079] S6. Purification 1, control the 12 μL reaction solution from the previous step to move through the purification area through the control electrode, and simultaneously control the magnet to adsorb the magnetic beads through the magnetic control module to complete the separation of the magnetic beads and the liquid, and move the separated liquid to the waste liquid area through the control electrode, and keep the magnetic beads in the purification area;

[0080] S7. Purification 2, the cleaning liquid in the liquid storage area is controlled by the control electrode to separate 4 μL of cleaning liquid and move it to the purification area to complete the redissolution of the magnetic beads, and the control electrode controls the redissolved magnetic beads and the cleaning liquid to move to the reaction area to complete the mixing of the cleaning liquid and the magnetic beads; then the control electrode controls the magnetic bead mixed liquid to move through the purification area, and the magnetic control module is used to control the magnet to adsorb the magnetic beads to complete the separation of the magnetic beads and the liquid, and the separated liquid is moved to the waste liquid area by the control electrode, and the magnetic beads are retained in the purification area;

[0081] S8. Desulfonation, by controlling the desulfonation solution in the storage area through the control electrode, 6 μL of the desulfonation solution is separated and moved to the purification area to complete the re-dissolution of the magnetic beads, and then moved to the reaction area to complete the mixing of the desulfonation solution and the magnetic beads; incubate at 22°C for 5 minutes to complete the desulfonation reaction;

[0082] S9. Cleaning 1, controlling the reaction solution in the reaction zone to move through the purification zone by controlling the electrodes, synchronously adsorbing the magnetic beads to separate the magnetic beads and the liquid, and moving the separated liquid to the waste liquid zone by controlling the electrodes, and retaining the magnetic beads in the purification zone;

[0083] S10. Washing 2, control the washing liquid in the liquid storage area by controlling the electrodes to separate 4 μL of washing liquid and move it to the purification area to complete the re-dissolution of the magnetic beads, and move it to the reaction area to complete the mixing of the washing liquid and the magnetic beads; then control the mixed magnetic bead liquid to move through the purification area by controlling the electrodes, and simultaneously control the magnet to adsorb the magnetic beads by the magnetic control module to complete the separation of the magnetic beads and the liquid, and move the separated liquid to the waste liquid area by controlling the electrodes, and the magnetic beads are retained in the purification area;

[0084] S11. Clean 3, repeat step S10 once;

[0085] S12. Elution, control the eluent in the storage area through the control electrode to separate 5ul of eluent and move it to the purification area to complete the re-dissolution of the magnetic beads, and move it to the reaction area to complete the mixing of the eluent and magnetic beads; incubate at 65°C for 3 minutes to complete the reaction; then control the reaction liquid through the purification area through the control electrode, and synchronously adsorb the magnetic beads with the magnet to complete the separation of the magnetic beads and the liquid. The 5μL liquid that has been separated is the reaction product of the sample pretreatment. The 5μL reaction product is controlled by the control electrode to move to the product extraction area, and the product is extracted from the extraction hole with a pipette to complete the collection of the reaction product.

[0086] In this case, the amount of sample and reagent is very small, which effectively reduces the amount of reaction system. By controlling the power supply of the electrode, the liquid can be moved back and forth, the reaction liquid can be fully mixed, and the chemical reaction efficiency can be improved. The entire pretreatment can be completed in about 1.5 hours, effectively reducing the reaction time. The entire experimental process is carried out in the oil phase, which can also shorten the direct conversion time and ensure the stability of the conversion efficiency.

[0087] Example 2 Comparative experiment of the present invention and traditional manual methylation sample pretreatment

[0088] This experiment used a methylation sample pretreatment method based on a digital microfluidic chip and a traditional manual methylation sample pretreatment method, and compared the effects of the two through a comparative experiment.

[0089] The traditional manual methylation sample pretreatment used a traditional manual methylation kit. The kit used was the human Septin 9 gene methylation detection kit (PCR fluorescent probe method) produced by Boercheng (Beijing) Technology Co., Ltd. The experimental process was carried out according to the kit instructions.

[0090] For plasma samples, the human Septin9 gene methylation detection kit (PCR fluorescent probe method) of Boercheng (Beijing) Technology Co., Ltd. was used to extract DNA samples from plasma to obtain 100 μL of DNA samples. 3.3 μL and 10 μL of DNA samples were taken respectively to complete the sulfite conversion on the digital microfluidic chip for automatic operation and traditional manual operation. After the conversion, 5 μL and 15 μL of completed DNA samples were obtained respectively.

[0091] Two groups of DNA samples were obtained by sulfite conversion of DNA samples in two ways, and 5 μL of the product was taken from each group for real-time fluorescence PCR detection. The fluorescence PCR detection instrument used was ABI 7500 instrument. The default baseline of the instrument was used for the analysis of the results of ABI 7500 instrument. When the SDS V2.3.1 software was used for analysis, the FAM channel (detection of SDC2 gene) value was set to 65000, and the Texas Red channel (detection of ACTB gene) threshold was set to 95000. The Ct value can be calculated only when the amplification curves of the FAM channel and the Texas Red channel show "S" type amplification.

[0092] The test results are shown in Table 1. The results show that under the same sample conditions, the final detection Ct value of the traditional manual method and the sulfiting conversion completed on the digital microfluidic chip differed by 2 Ct values; the shortening of the Ct value is a comprehensive result of high conversion degree and high extraction yield.

[0093] In this experiment, two methods were used to complete the sulfite conversion of DNA samples. On the digital microfluidic chip, the experimental steps S4 sulfitation, S5 binding time, and S12 elution time were all reduced compared to traditional manual methods. The entire DNA sample sulfite conversion experiment took only 80 minutes, saving time compared to the manual method of 2 hours.

[0094] This example shows that using the same extracted DNA sample and the same reagent kit, the methylation sample pre-treatment on the digital microfluidic chip is simple, time-saving, requires small amounts of reagents and is low in cost. The DNA conversion efficiency after treatment is higher than that of the traditional manual method, and the detection sensitivity is high, which is conducive to the full automation and standardized operation of methylation research.

[0095] Table 1

[0096]

Claims

1. A digital microfluidic chip for methylation sample pretreatment, comprising an upper cover plate, a bottom plate, and a cavity formed by sealing between the upper cover plate and the bottom plate, characterized in that: The upper cover plate includes an upper substrate and a conductive layer and a first hydrophobic layer on one side of the upper substrate. The bottom plate facing the cavity is provided with a lower substrate, a control electrode layer on one side of the lower substrate and a second hydrophobic layer in sequence. The control electrode layer includes a plurality of control electrodes. The digital microfluidic chip includes a sample processing module. The sample processing module is provided with a plurality of functional areas. The functional areas are located in the cavity. The control electrodes control the liquid distribution in the functional areas. The functional areas include a liquid storage area, a product extraction area, a sample area, a reaction area, a purification area and a waste liquid area.

2. The digital microfluidic chip according to claim 1, characterized in that: The sample processing module includes one or more.

3. The digital microfluidic chip according to claim 2, characterized in that: The number of sample processing modules is any one from 1 to 16.

4. The digital microfluidic chip according to claim 1, characterized in that: The lower substrate also includes a dielectric layer on one side.

5. The digital microfluidic chip according to claim 1, characterized in that: The liquid storage area is connected to the product extraction area, the product extraction area is connected to the sample area, the sample area is connected to the reaction area, the reaction area is connected to the purification area, and the purification area is connected to the waste liquid area.

6. The digital microfluidic chip according to claim 1, characterized in that: The liquid storage area includes a conversion liquid storage area, a binding liquid storage area, a magnetic bead storage area, a desulfurization liquid storage area, a cleaning liquid storage area and an elution liquid storage area.

7. The digital microfluidic chip according to claim 1, characterized in that: The reaction zone includes a mixing zone, a sulfite conversion zone, and a desulfonation zone.

8. The digital microfluidic chip according to claim 1, characterized in that: Through holes are arranged on the upper cover plate corresponding to different functional areas.

9. The digital microfluidic chip according to claim 1, characterized in that: The functional areas are in liquid communication with each other through the pipetting channels formed by the control electrodes.

10. The digital microfluidic chip according to claim 1, characterized in that: The digital microfluidic chip also includes a gold finger, and each control electrode is connected to the gold finger.

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