Digital micro-fluidic chip for preparing NGS methylation library

Abstract

The invention relates to the technical field of microfluidics and molecular diagnosis, and discloses a digital micro-fluidic chip for preparing an NGS methylation library. Comprising a bearing substrate, an electrode array used for electrowetting driving is formed on the upper surface of the bearing substrate to serve as a liquid drop conveying channel, the bearing substrate is divided into a plurality of functional areas which are sequentially arranged from the first side to the second side, the areas are communicated with one another through the liquid drop conveying channel, and micro-liquid drops are transferred and distributed among the areas. The upper cover is arranged opposite to the bearing substrate, a micro-droplet containing cavity is defined between the upper cover and the bearing substrate through a spacing frame arranged around the electrode array, and the upper cover is provided with a plurality of liquid inlet and outlet holes corresponding to the functional areas respectively. The chip is driven by the electrode array, so that micro-droplets entering the reagent sample introduction area and the sample introduction area through the liquid inlet and outlet holes are sequentially conveyed to the reaction area, and the micro-droplets after reaction are transferred to the taking-out area and are output through the liquid inlet and outlet holes, so that at least part of steps in the NGS methylation library preparation process are completed.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of microfluidics and molecular diagnostics, and in particular to a digital microfluidic chip for NGS methylation library preparation. BACKGROUND

[0002] At present, next-generation sequencing (NGS) technology has been widely used in molecular diagnostics, genetic analysis and precision medicine. Library preparation, as one of the most critical steps in the NGS process, usually includes multiple steps such as DNA fragmentation, end repair, adapter ligation, magnetic bead purification and PCR amplification. The steps are complex, the conditions are complicated, and the automation degree and operation stability are required. The existing mainstream library preparation automation equipment mostly uses mechanical arm pipetting or pipeline type microfluidic system, which has the problems of large reaction system volume, high reagent consumption, complex flow path, large dead volume leading to sample loss, high equipment cost and difficult maintenance, and high risk of cross contamination in the switching scene before and after PCR, which is not conducive to the flexible application of high-throughput or on-site scenarios. SUMMARY

[0003] The purpose of the present application is to provide a digital microfluidic chip for NGS methylation library preparation, which can perform multiple operations such as reagent sampling, sample sampling, reaction and product taking out in the process of NGS methylation library preparation on a single microfluidic chip, and can significantly reduce the reaction system volume and reagent consumption, and reduce sample loss.

[0004] The present application discloses a digital microfluidic chip for NGS methylation library preparation, comprising:

[0005] A carrier substrate 1, the upper surface of which is formed with an electrode array 11 for electrowetting driving as a droplet transport channel, the carrier substrate 1 is divided into functional zones arranged in order along the first side to the second side in the plane, the functional zones include reagent sampling zone 13, taking-out zone 14, reaction zone 16, and sample sampling zone 17, wherein each of the functional zones is connected to each other through the droplet transport channel to transfer and distribute microdroplets between each of the functional zones;

[0006] An upper cover is arranged opposite to the carrier substrate 1, and a containing cavity of the microdroplet is defined between the carrier substrate 1 and the upper cover through a spacing frame arranged around the electrode array 11, the upper cover is provided with a plurality of liquid inlet and outlet holes corresponding to the functional zones respectively:

[0007] The chip is configured to, under the driving of the electrode array 11, sequentially transport at least one microdroplet entering the reagent sample area 13 and the sample sample area 17 through the liquid inlet and outlet hole to the reaction area 16, and transfer the microdroplet after the reaction to the take-out area 14 and output through the liquid inlet and outlet hole, so as to complete at least part of the steps in the NGS methylation library preparation process.

[0008] In a preferred example, the at least part of the steps of the NGS methylation library preparation process includes one or more of sample pretreatment and methylation conversion, magnetic bead-based nucleic acid enrichment and / or purification, washing and / or liquid exchange treatment, linker ligation and / or other enzymatic reactions under temperature control, and nucleic acid amplification reaction.

[0009] In a preferred example, the number of the liquid inlet and outlet holes and the corresponding liquid droplet transport channels corresponding to each of the functional areas is 2-20, preferably 8, to realize multi-connection parallel processing.

[0010] In a preferred example, the back surface of the bearing substrate 1 is provided with an array of electrical connection pads 12, which are used for detachable electrical connection with the pin probe of the external preparation instrument.

[0011] In a preferred example, each of the electrical connection pads is electrically connected to at least one driving electrode or group of driving electrodes in the electrode array 11 through conductive wires, and the driving electrodes are arranged continuously along the extension direction of each of the liquid droplet transport channels.

[0012] In a preferred example, when the external driving device sequentially applies driving voltage to adjacent driving electrodes on the liquid droplet transport channel and cuts off the driving voltage of the driving electrodes through which the liquid droplet has passed, an electro-wetting potential well moving in the target direction can be formed in the accommodation cavity, thereby continuously driving the liquid droplet from the sample sample area 17 and / or the reagent sample area 13 to the predetermined position of the reaction area 16 and / or the take-out area 14.

[0013] In a preferred example, a magnetic bead operation area is further included, which is arranged on the side of the sample sample area 17, so that when the external magnet adsorbs the magnetic beads, the electrode array 11 is used to control the reciprocating movement of the liquid droplet between the sample sample area 17 and the reaction area 16, to complete the magnetic bead purification and liquid exchange process.

[0014] In a preferred example, the upper surface of the chip is provided with a dielectric insulation layer.

[0015] In a preferred example, the upper cover is composed of a transparent glass substrate, and the lower surface of the upper cover is coated with a hydrophobic layer, which is a fluororesin coating.

[0016] In a preferred embodiment, the accommodation cavity is between the dielectric insulation layer and the hydrophobic layer, and the accommodation cavity is filled with dielectric oil.

[0017] In a preferred embodiment, the dielectric insulation layer is arranged on the liquid transport channel, and the dielectric insulation layer comprises a transparent conductive layer and a dielectric layer covering the transparent conductive layer.

[0018] In a preferred embodiment, the transparent conductive layer is an ITO conductive layer, and the dielectric layer is a parylene film with a thickness of 1-2 μm.

[0019] In a preferred embodiment, the liquid inlet and outlet hole comprises: a first sample inlet hole array 21, a second sample inlet hole array 24, and a sample outlet hole array 22.

[0020] The first sample inlet hole array 21 is arranged on a side corresponding to the reagent sample area 13, and is used to inject different reagents into the reagent sample area 13.

[0021] The second sample inlet hole array 24 is arranged on a side corresponding to the sample area 17, and is used to inject a sample to be tested into the sample area 17.

[0022] The sample outlet hole array 22 is arranged above the sample outlet area 14, and is used to collect a droplet of reaction product from the sample outlet area 14.

[0023] In a preferred embodiment, a third sample inlet hole array 23 is further included, and the third sample inlet hole array 23 is arranged in the middle of the upper cover.

[0024] In a preferred embodiment, each electrode array 11 comprises: a receiving electrode area 111, a buffer electrode area 112, a transition electrode area 113, and a metering electrode area 114.

[0025] The receiving electrode area 111 has a planar profile that is at least partially a substantially circular area, and is located in a projection area of the liquid inlet and outlet hole, and is used to receive a droplet falling from the liquid inlet and outlet hole.

[0026] The buffer electrode area 112 is arranged on a lateral side of the receiving electrode area 111, and is in conductive connection with the receiving electrode area 111, and the planar width and length of the buffer electrode area 112 are both greater than the diameter of the substantially circular area, and is used to spread and buffer the shape of the droplet when the droplet continuously falls, so as to prevent the droplet from overflowing the electrode boundary.

[0027] The transition electrode area 113 is arranged between the buffer electrode area 112 and the metering electrode area 114, and the planar profile of the transition electrode area 113 is gradually narrowed in the direction of the metering electrode area 114 close to the buffer electrode area 112, for guiding the liquid droplet in the buffer electrode area 112 to the metering electrode area 114;

[0028] The metering electrode area 114 is arranged at the end of the transport direction of the transition electrode area 113, and is a square electrode with a predetermined planar size, for precisely segmenting the liquid droplet under the electro-wetting driving and limiting the volume of the single-transport micro-droplet.

[0029] In a preferred embodiment, the size of each square electrode is 1.2-1.8 mm.

[0030] In a preferred embodiment, the upper cover comprises a cover plate body, the lower surface of the cover plate body is upwardly recessed to form a receiving cavity for covering the electrode array 11 area, the peripheral edge of the cover plate body is integrally formed with a first flange 25 sealingly fitted with the substrate 1, at least one circle of stepped second flanges 26 is sequentially arranged in the circumferential direction at the area inward of the first flange 25, the glue groove 27 is formed between the first flange 25 and the second flange 26, and each flange is arranged in a closed frame shape around the electrode array 11 area.

[0031] In a preferred embodiment, the sample injection area 17 is configured to transport at least one sample droplet containing nucleic acid to be tested and / or nucleic acid sample droplet after methylation conversion; and the reagent injection area 13 is configured to transport a plurality of functional reagents for methylation library preparation.

[0032] In a preferred embodiment, the sample droplet includes genomic DNA extracted from tissue, blood, plasma / serum, cells or extracellular free DNA and / or methylation conversion DNA after bisulfite conversion treatment.

[0033] In a preferred embodiment, the functional reagent for methylation library preparation is selected from the group consisting of bisulfite, magnetic beads, washing liquid, desulfurization liquid, eluent, SSB, sequencing adapter reagent, polymerase Master Mix or Index Primer.

[0034] In a preferred embodiment, the sequencing adapter reagent is a double-end library adapter reagent with P5 adapter sequence and P7 adapter sequence.

[0035] In the embodiments of the present application, the reagent sample injection area, the taking-out area, the reaction area and the sample injection area are sequentially divided along the first side to the second side on a single bearing substrate, and an electrode array for electrowetting driving is formed thereon as a droplet transport channel. In combination with the upper cover and the liquid inlet / outlet holes respectively arranged corresponding to each functional area, the programmable path control of the microdroplets formed by the sample and multiple reagents is realized in the closed containment cavity defined between the two, and the chip realizes the series integration of multiple steps of library preparation on the same plane, replaces the traditional pipeline and large-volume hole plate operation with microliter-level droplets, significantly reduces the reaction system volume and reagent consumption, reduces the flow path dead volume and sample loss, and at the same time, since the droplets always move in the closed cavity and the predetermined electrode path, the residue and pollution caused by complex mechanical / pipeline switching are avoided, thereby improving the automation stability and reliability of the NGS methylation library preparation process.

[0036] A large number of technical features are described in the specification of the present application, which are distributed in various technical solutions. If all possible combinations of technical features (i.e. technical solutions) of the present application are listed, the specification will be too long. In order to avoid this problem, each technical feature disclosed in the above summary of the present application, each technical feature disclosed in the following embodiments and examples, and each technical feature disclosed in the drawings can be freely combined with each other to form various new technical solutions (these technical solutions are all considered to have been described in the specification), unless such combination of technical features is technically infeasible. For example, features A+B+C are disclosed in one example, features A+B+D+E are disclosed in another example, features C and D are equivalent technical means that play the same role, and only one of them can be used technically, and feature E can be combined with feature C technically. Therefore, the scheme of A+B+C+D should not be considered to have been described because it is technically infeasible, and the scheme of A+B+C+E should be considered to have been described. BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is a structural schematic diagram of a digital microfluidic chip for NGS methylation library preparation according to an embodiment of the present application.

[0038] Figure 2 is a structural schematic diagram of an array of electrical connection pads according to an embodiment of the present application;

[0039] Figure 3 is a structural schematic diagram of a bearing substrate according to an embodiment of the present application;

[0040] Figure 4 is a structural schematic diagram of an upper cover according to an embodiment of the present application;

[0041] Figure 5is a structural schematic diagram of an upper cover according to an embodiment of the present application.

[0042] Figure 6 is a structural schematic diagram of an upper cover lower surface according to an embodiment of the present application.

[0043] Figure 7 is a schematic diagram of a bearing substrate partition according to an embodiment of the present application.

[0044] BRIEF DESCRIPTION OF DRAWINGS

[0045] 1-substrate, 11-electrode array, 111-bear electrode area, 112-buffer electrode area, 113-transition electrode area, 114-measuring electrode area, 12-pad array, 13-reagent injection area, 14-withdrawal area, 15-reagent injection area, 16-reaction area, 17-sample injection area, 2-upper cover, 21-first injection hole array, 22-withdrawal hole array, 23-third injection hole array, 24-second injection hole array, 25-first flange, 26-second flange, 27-gel groove DETAILED DESCRIPTION

[0046] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present application. However, it will be apparent to one skilled in the art that the present application can be practiced without some or all of these specific details. In other instances, well known process steps have not been described in detail in order not to unnecessarily obscure the present application.

[0047] Compared with the prior art, the present application has at least the following advantages:

[0048] 1. High integration: integrating magnetic control, temperature control and optical detection areas on a single chip to realize full-process library construction.

[0049] 2. Low volume consumption: single droplet volume 1-3 μL, reaction volume reduced by more than 90% compared with traditional systems.

[0050] 3. High stability: using an optimized dielectric / hydrophobic double-layer structure, service life more than 10 6 times of droplet operation.

[0051] 4. High-throughput scalability: multiple samples can be processed in parallel through arrayed chips.

[0052] 5. Strong compatibility: supporting DNA, RNA and various enzyme reaction systems, suitable for multi-platform NGS systems (Illumina, MGI, Nanopore, etc.).

[0053] In order to make the purposes, technical solutions and advantages of the present application clearer, the embodiments of the present application will be further described in detail below with reference to the drawings.

[0054] The application relates to a digital microfluidic chip for NGS methylation library preparation, a structural diagram of which is shown in the figure. Figures 1-3 The application relates to a digital microfluidic chip for NGS methylation library preparation, a structural diagram of which is shown in the figure.

[0055] The upper surface of the carrier substrate 1 is formed with an electrode array 11 for electrowetting driving as a droplet transport channel, and the carrier substrate 1 is divided into functional zones arranged in sequence from the first side to the second side in the plane, including a reagent sample introduction zone 13, a take-out zone 14, a reaction zone 16 and a sample introduction zone 17, wherein the functional zones are connected to each other through the droplet transport channel to transfer and distribute microdroplets between the functional zones.

[0056] The upper cover is arranged opposite to the carrier substrate 1 and is limited to form a microdroplet containing cavity between the carrier substrate 1 and the spacing frame arranged around the electrode array 11, and the upper cover is provided with a plurality of liquid inlet and outlet holes corresponding to the functional zones respectively.

[0057] The chip is configured to be driven by the electrode array 11, so that at least one microdroplet entering the reagent sample introduction zone 13 and the sample introduction zone 17 through the liquid inlet and outlet hole is transported to the reaction zone 16 in sequence, and the microdroplet after the reaction is transferred to the take-out zone 14 and output through the liquid inlet and outlet hole, so as to complete at least part of the steps in the NGS methylation library preparation process.

[0058] In an embodiment of the application, the digital microfluidic chip for NGS methylation library preparation adopts an upper and lower two-layer substrate clamped to form a closed microdroplet operation space: the lower layer is the carrier substrate 1 for integrating the electrowetting driving electrode and the matching electrical connection structure, and the upper layer is the upper cover for limiting the upper boundary of the microdroplet and providing the inlet and outlet channels of the reagent / sample.

[0059] The at least part of the steps of the NGS methylation library preparation process includes one or more of sample pretreatment and methylation conversion, magnetic bead-based nucleic acid enrichment and / or purification, washing and / or liquid exchange treatment, linker connection and / or other enzymatic reactions under temperature control, and nucleic acid amplification reaction.

[0060] The upper surface of the carrier substrate 1 is formed with an electrode array 11 for electrowetting driving through processes such as photolithography and electroplating, and each electrode is divided into a reagent sample introduction zone 13, a take-out zone 14, a reaction zone 16 and a sample introduction zone 17 arranged in sequence from the first side to the second side in the plane according to a predetermined layout, and the electrodes in different functional zones are connected to each other through the continuously arranged droplet transport channel electrodes, thereby forming one or more programmable droplet transport paths.

[0061] The upper cover is sealed and attached to the carrier substrate 1 by a spacer frame arranged around the electrode array 11, defining a micro-droplet containing cavity filled with dielectric liquid between the two, and is processed with a plurality of liquid inlet and outlet holes at positions corresponding to each functional area, so that external liquid can be dropped into the corresponding area under the action of gravity or pressure, or extracted from the target product in the take-out area 14 by an external pipetting device.

[0062] In use, the chip is electrically connected to the high-voltage drive circuit of the external preparation instrument through the array of electrical connection pads on the back of the carrier substrate 1, and the preparation instrument loads or unloads the drive voltage on the drive electrodes arranged on each functional area and droplet transport channel in sequence according to the preset NGS methylation library preparation process timing, to form a potential well moving in the target direction in the containing cavity under the action of electrowetting effect, thereby realizing the quantitative transport, mixing and reaction of reagent and sample micro-droplets from the reagent sampling area 13, the sample sampling area 17 to the reaction area 16, and the micro-droplets after the reaction are further transported to the take-out area 14 and output through the corresponding liquid inlet and outlet holes, thereby completing the automatic micro-droplet operation of at least part of the key steps in the NGS methylation library preparation process on a single chip.

[0063] In order to realize the point-by-point independent driving of the electrode array 11 on the carrier substrate 1, in some embodiments, as shown in Figure 2 The back of the carrier substrate 1 is also provided with an array of electrical connection pads 12. Each electrode is respectively led out to the array of electrical connection pads 12 on the back through internal conductive traces. The arrangement form and pitch of the array of electrical connection pads 12 correspond to the pin probe array on the external preparation instrument. In use, the chip is detachably electrically connected to the pin probe of the preparation instrument in a face contact manner through the array of electrical connection pads 12 on the back, so as to reliably connect the electrode array 11 to the high-voltage drive circuit without welding or fixing the socket. Through this structural design, on the one hand, it is convenient for quick mounting and dismounting and replacing of the chip, and reduces the maintenance cost, and on the other hand, it is also conducive to realizing high-density and multi-channel electrical connection layout, and provides a hardware basis for subsequent flexible programming and driving of droplet movement in the reagent sampling area 13, the reaction area 16, the sample sampling area 17 and the take-out area 14 according to the predetermined timing.

[0064] Each electrical connection pad is electrically connected to at least one drive electrode or group of drive electrodes in the electrode array 11 through a conductive trace, and the drive electrodes are arranged continuously along the extension direction of each droplet transport channel.

[0065] When the external driving device applies a driving voltage to the adjacent drive electrodes on the droplet transport channel in sequence, and cuts off the driving voltage of the drive electrodes through which the droplet has passed, an electrowetting potential well moving in the target direction can be formed in the containing cavity, so as to continuously drive the droplet from the sample sampling area 17 and / or the reagent sampling area 13 to the predetermined position of the reaction area 16 and / or the take-out area 14

[0066] In order to better understand the technical solutions of the present application, the driving mode will be described below in combination with a specific example, and the details listed in the example are mainly for the purpose of facilitating understanding and should not be regarded as limiting the protection scope of the present application.

[0067] In some preferred embodiments of the present application, there is a one-to-one or one-to-many correspondence between the array of electrical connection pads 12 and the array of electrodes 11, so as to achieve fine programming control of the droplet movement path. Taking the example of transporting a sample droplet in the sample injection area 17 to the reaction area 16, the back surface of the carrier substrate 1 is provided with electrical connection pads Pad1, Pad2, Pad3, …, which are respectively connected to the driving electrodes E1, E2, E3, … on the transport channel between the sample injection area 17 and the reaction area 16 through conductive traces.

[0068] When the upper cover drops a sample droplet onto the first receiving electrode E1 in the sample injection area 17, the high-voltage driving circuit of the external preparation instrument is electrically connected to Pad1 through the pin probe, and the driving voltage is applied to the electrode E1 corresponding to Pad1, while the downstream electrodes E2, E3 are temporarily kept at a lower potential or in a closed state, so that the droplet is first stably stopped at E1; then, the preparation instrument controls the pin probe to apply a driving voltage to Pad2 while cutting off the driving of Pad1, so that the droplet is pulled from E1 to the adjacent E2 under the action of the change of the electrowetting potential well; and then the driving is applied to Pad3, Pad4, … in sequence and the voltage of the pad corresponding to the previous electrode is turned off, so that the droplet is gradually transported along the predetermined electrode sequence from the sample injection area 17 to the target position in the reaction area 16.

[0069] In this way, the present application can realize the linear path and beat control of the droplet between different functional areas under the fixed chip structure by only changing the on-off timing sequence of the external preparation instrument to each pad.

[0070] The digital microfluidic chip of the present application is suitable for use with an NGS library preparation instrument based on digital microfluidic technology. The array of electrical connection pads 12 on the back surface of the carrier substrate 1 is one-to-one corresponding to the array of pin probes on the driving board of the preparation instrument in terms of planar position and arrangement pitch, and when the chip is loaded on the chip carrying platform of the preparation instrument, the first and second elastic clamping forces are applied to the chip from bottom to top and from top to bottom by means of the first and second pressure releasing parts provided in the preparation instrument, so that the array of electrical connection pads 12 is pressed against the corresponding pin probes in the vertical direction, thereby forming a stable and repeatable detachable electrical connection.

[0071] The compression release mechanism can include a first compression release portion arranged below the top plate and a second compression release portion arranged above the top plate: the first compression release portion exerts an upward elastic thrust on the substrate and the driving plate below it through a plurality of elastic components, so that the pin probe array abuts against the chip back surface from bottom to top; the second compression release portion exerts a downward elastic compression force on the upper surface of the chip through a compression assembly and an elastic element arranged above the top plate, so that the chip is clamped and fixed in the vertical direction. Through the above-mentioned bidirectional elastic compression structure, reliable electrical contact is maintained between each pad and the corresponding pin probe, and since the motor is not continuously locked, the risk of motor fatigue during long-term operation can be reduced.

[0072] In some embodiments of the present application, the reaction area 16 is arranged to adapt to the operation space of the NGS methylation library preparation multi-step reaction, for sequentially introducing, merging, incubating and transporting the sample and various reagents on the same digital microfluidic chip. The working principle is illustrated below with a specific driving process example, but this does not limit the present application.

[0073] For example, when performing end repair and adapter operation, the upstream processed DNA sample can be added in the form of droplets to the corresponding sample loading electrode through the rightmost column of loading holes of the sample loading area 17; at the same time, the end repair enzyme system or adapter reagent is added to the corresponding reagent loading electrode through the leftmost column of loading holes of the reagent loading area 13. The external preparation instrument is electrically connected to the back surface of the bearing substrate 1 through the pin probe, and sequentially applies driving voltage to the adjacent driving electrodes on the transport channel between the sample loading area 17 and the reagent loading area 13, and cuts off the driving voltage of the droplets that have passed through the electrodes, so that the sample droplets are transported from right to left and the reagent droplets are transported from left to right along the respective channels to the electrode in the middle of the reaction area 16, and the merging occurs at the predetermined convergence position of the reaction area 16, forming a single reaction droplet.

[0074] After the reaction droplet is formed, the instrument can continue to control the on-off timing of the electrode array 11 in the reaction zone 16 according to the preset NGS methylation library preparation program, so that the reaction droplet moves back and forth between the metering electrodes in the reaction zone 16 or stays at a specific position, and cooperates with the temperature control module of the external library preparation instrument to realize the temperature gradient or constant temperature incubation required by the end repair, adapter ligation, PCR amplification and other steps at different time periods. After the reaction is completed, the target droplet in the reaction zone 16 can be further transported to the adjacent magnetic bead operation zone under the driving of the electrode array 11, combined with the magnetic bead suspension droplet transported from the reagent sampling zone 13 to complete the magnetic bead enrichment and purification operation, and then after elution, the droplet containing the target library product is retransported back to the reaction zone 16 for subsequent reaction, or directly transported to the electrode corresponding to the take-out zone 14 and stays at the position corresponding to the take-out hole of the upper cover. The final library product is collected through the take-out hole by the external pipette or vacuum suction device.

[0075] As can be seen from the above examples, the reaction zone 16 of the chip of the present application is not a single fixed reaction cavity, but is connected with the reagent sampling zone 13, the sample sampling zone 17, the magnetic bead operation zone and the take-out zone 14 through continuous droplet transport channels, and by the precise timing driving of the electrode array 11, the key steps of NGS methylation library preparation such as end repair, adapter ligation, magnetic bead purification and PCR amplification are sequentially completed in the same reaction plane, thereby improving the utilization rate of the reaction space and reducing the loss of samples and reagents.

[0076] It should be noted that the take-out zone 14 of the present application is preferably arranged at the middle position of the arrangement direction of each functional zone from the first side to the second side, that is, between the reagent sampling zone 13 and the reaction zone 16 to form an independent concentrated collection area. The result of such arrangement is that on the one hand, the target product droplet from the reaction zone 16 can be driven to stay under the electrode array 11 corresponding to the take-out zone 14 after completing the intended reaction along the shortest transport path, and directly extracted through the take-out hole of the upper cover, without the need to pass through other functional zones, thereby reducing the evaporation, contamination or secondary mixing that may occur during the additional transport process. On the other hand, as an independent middle node, the take-out zone 14 also facilitates the flexible definition of the intermediate sampling point or the end collection point in different library preparation processes, for example, batch collection in the same take-out zone 14 after end repair, adapter ligation or PCR amplification, so that the chip structure can be adapted to various NGS methylation library preparation strategies through programming of the external driving timing without changing the overall wiring of the chip, thereby improving the versatility and expandability of the chip structure for different processes.

[0077] In some embodiments, the chip of the present application further comprises a magnetic bead operation area, wherein the magnetic bead operation area at least partially overlaps with the sample injection area 17 in the planar position, preferably the region of the electrode array 11 in the sample injection area 17 corresponding to the external magnet. When the external magnet is driven to move upwards under the chip and close to the carrier substrate 1, a magnetic field action area is formed in the overlapping region, and at this time the droplet carrying magnetic beads in the sample injection area 17 is stopped or positioned in the region under the driving of the electrode array 11, i.e. as a magnetic bead operation area, for performing magnetic bead enrichment, washing and elution operations; when the external magnet moves downwards away from the carrier substrate 1, the region is no longer under the action of the magnetic field, and returns to the ordinary sample injection area 17 for sample injection and droplet transport.

[0078] In actual use, the mixed droplet containing magnetic beads can be first transported to the magnetic bead operation area through the sample injection hole, and under the condition that the external magnet applies a magnetic field on the back of the chip, the droplet is driven by the electrode array 11 to be positioned at a fixed point in the magnetic bead operation area, and the substances carried by the magnetic beads are enriched to one side or a local area of the droplet with the help of the magnetic field; then the supernatant part is transferred to other paths by driving the electrode array 11, and the washing liquid droplet is then driven to the magnetic bead operation area to combine with the magnetic beads and mix briefly, and then the supernatant is removed again under the continuous or intermittent action of the magnetic field, realizing multiple rounds of washing. After washing is completed, the external magnet can be removed, and the elution buffer droplet is driven to the magnetic bead operation area to combine with the magnetic beads, and the elution operation from the magnetic beads is completed under the condition of a set temperature or room temperature, and then the elution droplet is transported back to the reaction area 16 or to the take-out area 14 by the electrode array 11.

[0079] Therefore, the above preparation instrument can include a magnetic bead control module for use with the magnetic bead operation area of the chip of the present application. The magnetic bead control module can be arranged below the preparation instrument substrate at the lower side of the sample injection area / magnetic bead operation area, and includes a plurality of magnet assemblies arranged along the width direction of the chip. Each magnet assembly can be mounted on a transmission member of a driving mechanism, preferably a vertically liftable structure. When the driving mechanism drives the transmission member to rise, the upper end faces of the plurality of magnet assemblies uniformly approach or adhere to the lower surface of the chip, corresponding to the magnetic bead operation area (the region overlapping with the sample injection area) of the chip of the present application, so as to form a local high magnetic field in the droplet inside the chip, realizing the enrichment, concentration and fixation of the magnetic beads; when the driving mechanism drives the transmission member to descend, the magnet is away from the lower surface of the chip, and the magnetic field decays rapidly, and the magnetic beads in the droplet are released and re-dispersed, so as to complete the steps of magnetic bead purification and elution liquid exchange by cooperating with the reciprocating transport of the droplet on the chip.

[0080] In order to be able to control the temperature, in some embodiments, the reaction area 16 of the present application is arranged in correspondence with the heat-conducting contact area of the temperature control module of the external library preparation instrument. Specifically, the heat-conducting area of the temperature control platform plane or the upper surface of the metal heat-conducting layer is provided with the reaction area 16, so that when the reaction droplet arranged in the reaction area 16 stays in the area driven by the electrode array 11, precise temperature rising, falling and constant temperature control can be realized by the external film heater and / or TEC module to meet the requirements of the temperature curve of the NGS methylation library preparation steps such as end repair, adapter ligation, PCR amplification and the like.

[0081] Correspondingly, the above preparation instrument can also include a temperature control module, and the heat-conducting area of the temperature control platform of the temperature control module corresponds to the reaction area 16 arranged on the chip carrying substrate of the present application. The temperature control module can include a plurality of long strip-shaped heating units arranged below the chip carrying platform, a semiconductor heat exchange module (such as a TEC module) thermally coupled with the heating units, a heat dissipation fin and a fan assembly. The heating units are preferably arranged in rows along the longitudinal or transverse direction of the chip reaction area, for example, divided into a plurality of independent temperature zones such as temperature control A zone to temperature control F zone, and each temperature zone corresponds to the electrode sub-area where different reaction steps are located on the chip. Preferably, the temperature control module also integrates an NTC temperature sensor for real-time detection of the temperature at the corresponding position of the reaction area 16, and forms a closed-loop feedback regulation with the control circuit, so as to stably control the working temperature of the reaction area 16 in the range of about 25℃ to 98℃ to adapt to the requirements of temperature precision and stability for different enzyme reactions and thermal cycling processes.

[0082] It should be noted that the temperature sensor can also be other types of temperature detection elements, such as thermocouple, thermistor (RTD), integrated semiconductor temperature sensor or optical fiber temperature sensor, etc., as long as it can cooperate with the control circuit to realize real-time detection and feedback regulation of the temperature of the reaction area 16, and can be replaced or used equivalently, and is not limited to the NTC temperature sensor.

[0083] In order to ensure the stable driving of the digital microfluidic droplet on the surface of the chip, the surface layer structure of the chip is specially designed in the present application. In some embodiments, a dielectric insulating layer is provided on the upper surface of the substrate 1 of the chip, which is arranged on the liquid transport channel and used to provide electrical insulation between the electrode and the droplet and form the medium interface required for electrowetting.

[0084] In a preferred example, the upper cover of the chip is composed of a transparent glass substrate, and the lower surface of the upper cover is entirely coated with a hydrophobic layer. The hydrophobic layer is preferably a fluororesin coating, such as a hydrophobic coating based on fluoropolymer, to provide a low surface energy interface on the side in contact with the droplet, reduce the adhesion between the droplet and the upper cover, and cooperate to achieve good electrowetting driving characteristics and droplet sphericity.

[0085] In the above structure, a containing cavity is defined between the upper surface of the dielectric insulation layer and the lower surface of the hydrophobic layer, and the containing cavity is used to contain the micro-droplets driven to move by the electrode array 11. Preferably, the containing cavity is filled with a dielectric oil, such as a silicon oil, a fluorinated oil or other insulating liquid suitable for electrowetting applications, for reducing the frictional resistance of the droplets when moving in the cavity, thereby further improving the controllability of the electrowetting driving.

[0086] Preferably, the dielectric insulation layer comprises a transparent conductive layer and a dielectric layer covering the outside of the transparent conductive layer; wherein the transparent conductive layer is preferably an ITO conductive layer, and the dielectric layer is preferably a Parylene C film with a thickness of 1-2 μm.

[0087] It should be understood that the specific material of the dielectric insulation layer, the type of the hydrophobic coating layer and the type of the dielectric oil are not limited to the above examples, and those skilled in the art can make equivalent substitutions according to the actual application requirements.

[0088] Since an important purpose of the present application is to realize the preparation of NGS methylation library with multiple samples and high throughput, the in-out liquid hole structure is preferably arranged in groups on the chip, so as to simultaneously load and recover products in parallel for multiple reagents and multiple samples to be tested. Specifically, as shown in Figure 4 the in-out liquid hole comprises a first sample injection hole array 21, a second sample injection hole array 24 and a take-out hole array 22.

[0089] The first sample injection hole array 21 is arranged on the side corresponding to the reagent injection area 13, and each first sample injection hole is arranged opposite to the corresponding electrode array 11 and droplet transport channel in the reagent injection area 13, for injecting the same or different types, the same or different proportions of reagent droplets into the reagent injection area 13. For example, each first sample injection hole can be used to load fragmentation reaction liquid, end repair / A reagent, ligation reaction reagent, magnetic bead washing liquid and elution buffer, etc., so that these reagents can be selectively split into metering droplets under the digital microfluidic driving, and transported to the reaction area 16 in a predetermined time sequence.

[0090] The second sample injection hole array 24 is arranged on the side corresponding to the sample injection area 17, and each second sample injection hole is matched with the corresponding electrode array 11 in the sample injection area 17, for injecting multiple samples to be tested into the sample injection area 17. By loading different samples into different hole positions in the second sample injection hole array 24, combined with the driving of the electrode array 11, the parallel start and independent scheduling of multiple sample library preparation processes can be realized on the same chip, thereby improving the overall throughput.

[0091] In addition, an array of take-out holes 22 is arranged above the take-out area 14, each take-out hole corresponding to a region of the electrode array 11 in the take-out area 14, for collecting reaction product droplets from the take-out area 14 after the library preparation process is completed. By controlling the final convergence of droplets to positions below the respective corresponding take-out holes, the library products of different samples can be recovered into external collection tubes or sequencing plate wells respectively, avoiding mutual confusion.

[0092] The access hole can further include a third sample injection hole array 23 arranged in the middle of the substrate 1, for injecting reagents that are the same as or different from the first sample injection hole array 21. Specifically, the first sample injection hole array 21 is preferably used to inject basic reagents with large volume requirements, such as methylation conversion reagents, washing buffers, etc.; the third sample injection hole array 23 is preferably used to inject functional reagents with small amounts but many types, such as library linker reagents, index primer reagents containing sample index sequences, and PCR reaction additives, etc. Corresponding to the third sample injection hole array 23 in the planar position, a small volume reagent injection area 15 is divided on the substrate 1, which is also composed of the aforementioned driving electrodes and is in electrical communication with other functional area electrode arrays through droplet transport channels, for receiving small volume reagent droplets dropped from the third sample injection hole array 23, and merging and mixing with droplets from the first sample injection hole array 21 and / or the sample injection area 17 under the electro-wetting driving.

[0093] By arranging the first sample injection hole array 21 and the third sample injection hole array 23 independently on the upper cover, on the one hand, the arrangement density, hole diameter and upstream liquid supply module of the sample injection hole can be designed differently according to the volume requirement of the reagent, improving the quantitative accuracy in the process of injecting large volume reagents and small volume reagents; on the other hand, the third sample injection hole array 23 is arranged close to the middle of the chip, so that the small volume functional reagent can be transported to the reaction area 16 under the electro-wetting driving with a shorter path, reducing evaporation and non-specific adsorption in the transport process.

[0094] It should be noted that the specific arrangement of the first sample injection hole array 21, the second sample injection hole array 24, the third sample injection hole array 23 and the take-out hole array 22 can be linear arrangement, matrix arrangement or other regular array form, and the number of holes can be designed and expanded according to the target throughput requirement, for realizing 2-20, preferably 8, etc. different number of sample and reagent channel parallel operation, without affecting the implementation of the technical scheme of the present application.

[0095] For the electrode array 11 on the substrate 1, the applicant has found through a large number of experiments that if only a single shape of metering electrode is used to directly receive the liquid droplets dropped from the liquid inlet and outlet holes, problems such as large deviation of the liquid droplet incident position, high drop point speed, and uneven liquid droplet spreading are prone to occur, thereby causing part of the liquid droplets to overflow the electrode boundary, affecting the subsequent metering accuracy and transportation stability. Therefore, in some embodiments, as shown in Figure 5 the electrode array 11 on the bearing substrate 1 is designed by partition optimization, and the electrode array 11 corresponding to each liquid inlet and outlet hole is subdivided into a receiving electrode area 111, a buffer electrode area 112, a transition electrode area 113, and a metering electrode area 114 which are sequentially connected.

[0096] Specifically, the planar profile of the receiving electrode area 111 is preferably at least partially a substantially circular patch area, which is arranged in the projection area of the corresponding liquid inlet and outlet hole, and is used to preferentially receive the main body of the liquid droplet when the liquid droplet is dropped from the liquid inlet and outlet hole, thereby reducing the influence of drop point deviation on the subsequent liquid droplet shape. The reason for choosing a circular electrode is that it has better isotropy in all directions than a rectangular or polygonal electrode, which can make the liquid droplet falling on it tend to form an axisymmetric liquid droplet cross-sectional profile under the combined action of gravity and surface tension, thereby avoiding liquid droplet stretching, edge lifting and irregular spreading caused by local field strength or contact line pinning effect at the corners of the electrode, so that a liquid droplet with stable shape and controllable boundary can still be formed in the receiving electrode area 111 even if there is a certain tolerance in the sampling position and drop speed.

[0097] One side of the receiving electrode area 111 is provided with a buffer electrode area 112 connected thereto, and the width and length of the buffer electrode area 112 on the plane are both greater than the diameter of the above-mentioned substantially circular patch area, so that when there is continuous drop or the liquid droplet volume is large, the liquid droplet can be fully spread, flattened and "buffered" in the buffer electrode area 112, thereby avoiding the liquid droplet from exceeding the electrode boundary due to excessive momentum. As shown in Figure 5 the buffer electrode is generally rectangular in shape, and has a substantially U-shaped appearance in the top view.

[0098] The other side of the buffer electrode area 112 is provided with a transition electrode area 113, and the transition electrode area 113 is at least partially arranged in the notch of the U-shaped receiving electrode area 111 to realize the connection between the transition electrode area and the buffer electrode area 112. The planar profile of the transition electrode area 113 gradually narrows from one end close to the buffer electrode area 112 to the metering electrode area 114, forming a funnel-like guiding shape, which is used to stably converge and guide the liquid droplet in the buffer electrode area 112 to the metering electrode area 114 under the action of electrowetting driving.

[0099] The metering electrode region 114 is located at the end of the transport direction and consists of square electrodes with a predetermined planar dimension of 1.2-1.8 mm. The farthest metering square electrode is connected in series with a series of subsequent square electrodes to the electrode array 11 of the reaction zone 16, thus forming a complete droplet transport channel. When the metering electrode region 114 and its adjacent electrodes are switched on and off, the electrowetting effect can be used to precisely divide the droplets within this square region, limiting the volume of microdroplets transported in a single operation. This ensures stable and repeatable droplet metering even with tolerances in the droplet placement and fluctuations in the drop velocity.

[0100] To create a closed, thickness-controllable, and easily assembled droplet channel during chip operation, in some embodiments, such as Figure 6 As shown, the top cover includes a cover plate body. The lower surface of the cover plate body is recessed upward to form a receiving cavity for covering the area of ​​the electrode array 11. The receiving cavity, together with the dielectric insulating layer on the carrier substrate 1, defines the movement space of the droplets. The periphery of the cover plate body is integrally formed with a first flange 25 that seals against the substrate 1. The lower end face of the first flange 25 is used to bond and / or press against the carrier substrate 1 or its peripheral frame, thereby forming a continuous sealing boundary around the chip. At least one ring of stepped second flanges 26 is sequentially arranged circumferentially in the region inward of the first flange 25. A glue groove 27 is formed between the first flange 25 and the second flange 26. There is a predetermined step difference in the height direction between each second flange 26 and the first flange 25, and they are arranged in a closed frame shape around the area of ​​the electrode array 11. During the assembly process, UV-curable adhesive is dotted or injected into the adhesive tank 27. After the top cover and the carrier substrate 1 are aligned and bonded, the whole assembly is placed in a UV lamp box for curing, so that the cured adhesive layer forms a continuous sealing bonding frame between the first and second flanges 26.

[0101] By limiting the height difference between the first and second flanges, and the gap size between the second flange and the dielectric insulating layer on the carrier substrate 1, the cavity height of the receiving cavity can be precisely controlled, so that the thickness of the dielectric oil layer filled in the receiving cavity and the maximum height of the droplet are consistent, thereby ensuring stable electrowetting driving conditions. At the same time, it improves the alignment accuracy and sealing reliability when assembling the top cover and the carrier substrate 1, and avoids liquid leakage from the chip edge or bubble accumulation at uneven cavity height.

[0102] In other embodiments, the number of flanges can be increased as needed to further improve the overall rigidity and assembly positioning accuracy of the cover plate body, and to form a multi-level sealing and flow guiding structure between different flanges, thereby adapting to the packaging requirements of different cavity heights and different types of carrier substrates 1.

[0103] In order to better understand the technical solutions of the present application, a specific example will be described below, which lists details mainly for the purpose of understanding, and does not limit the protection scope of the present application.

[0104] As shown in Figure 7 The reaction zone 16 of the present application is further divided into multiple temperature control sub-zones in the plane, arranged in sequence along the short side direction of the chip, and respectively marked as temperature control A zone, temperature control B zone, temperature control C zone, temperature control D zone, and temperature control F zone. The planar positions of each temperature control sub-zone correspond one-to-one to independent temperature control modules on the external library preparation instrument, such as different Peltier heating / cooling units or thin film heaters, so as to form multiple temperature platforms on the same chip that are spatially separated and do not interfere with each other in temperature, for meeting the requirements of temperature curves for different reaction steps such as methylation conversion, desulfurization, linker ligation, PCR amplification, etc.

[0105] To realize 8-connection high-throughput operation, the chip is provided with S1-S8 eight sample channels in the sample loading area on one side, each sample channel corresponding to a group of independent droplet transport channels and functional zones in sequence. On the other side of the chip, Z1-Z8 reagent tanks are arranged in the large volume reagent tank area, for storing methylation conversion reagents, magnetic bead binding buffer, washing liquid, desulfurization liquid, eluent, SSB reagent, linker reagent, polymerase MasterMix, etc. In the middle, T1-T6, X1-X6, Y1-Y6, etc. are arranged in the small volume reagent tank area, for storing magnetic beads, cleaning liquid, eluent, and IndexPrimer, etc. reagents with small volume and accurate dosage. Corresponding receiving electrode zones 111, buffer electrode zones 112, and metering electrode zones 114 are arranged below each reagent tank, and through the driving of the electrode array 11 described above, accurate metering and transport of different reagents in each sample channel are realized.

[0106] The methylation conversion process can include: first, adding the DNA samples to be tested into the sample injection holes corresponding to S1-S8 respectively; then, metering bisulfite reagent from the Z1 reagent tank through the electrode array 11 in a volume of 30 μL per link, and distributing it to each sample channel in turn, and completing the electro-wetting mixing with about 10 μL of DNA sample droplets near the sample injection area 17. The mixed sample droplets are transported to the corresponding temperature control A area under the driving of the electrodes, and incubated at about 98°C for several minutes (8 min) to complete the DNA denaturation; then the droplets are moved to the temperature control B area, and continue to incubate at about 50-60°C for a period of time (60 min) to complete the conversion of bisulfite to unmethylated cytosine. After the conversion is completed, a part of the magnetic bead-containing binding buffer is divided from the Z2 reagent tank in a preset volume, transported to the sample channel through the electrode array 11 and mixed with the converted sample, and then the droplet is moved to the magnetic bead operation area (i.e. the area corresponding to the position of the external magnet below the sample injection area 17), under the action of the external magnet, the magnetic beads carrying DNA are enriched to one side of the droplet close to the magnet. Then, the supernatant droplet is pulled out from the magnetic beads and transferred to the waste liquid path through electrode control, realizing the removal of impurities and unbound reagents. Similarly, the washing buffer and desulfurization liquid can be sequentially divided from the Z3, Z4, Z3 reagent tanks, and the magnetic beads are subjected to multiple rounds of washing and desulfurization treatment; finally, the elution buffer is metered from the Z5 reagent tank, and the droplet is transported to the temperature control C area, incubated at an appropriate temperature, so that the methylated DNA is eluted from the magnetic beads, forming a methylated DNA elution liquid for subsequent library construction reaction, and again driven by the electrodes to return to the transport main line of the corresponding sample channel.

[0107] After methylation conversion is completed, the methylated library construction process can be continuously completed on the same chip. First, SSB or similar library construction pretreatment reagents are dispensed from reagent tank Z6 at a volume of approximately 2 μL per unit and added to the methylated DNA droplets S1–S8 through the droplet transport channel. The mixed droplets are then transported to temperature-controlled zone A and incubated at approximately 98°C for a short time (5 min) to ensure sufficient denaturation. They are then immediately moved to temperature-controlled zone C and cooled at a low temperature (e.g., 4°C) for 5 min. Subsequently, adapter ligation reagents are dispensed from reagent tank Z7 at a predetermined volume and added to each sample channel. The mixed droplets are incubated in temperature-controlled zone A (e.g., approximately 30°C) for a certain time (60 min) to complete the adapter ligation reaction. Next, polymerase MasterMix was metered from reagent tank Z8 and added to tanks S1-S8. Index Primer reagent was dispensed from reagent tanks X1-Y2. Utilizing the temperature gradients of three control zones (A, B, and C), with zone A at 98°C, zone B at 60°C, and zone C at 72°C, the droplet was repeatedly transported between these zones according to a pre-set PCR temperature cycle to amplify the ligation product. For example, the droplet was first held at 98°C for approximately 45 seconds; then, it underwent 10 cycles of 98°C for 15 seconds, 60°C for 30 seconds, and 72°C for 30 seconds; after each cycle, it was held at 72°C for approximately 1 minute, and finally held at 4°C.

[0108] After PCR amplification, the library can be further purified using magnetic beads in the small-volume reagent tank areas (T1-T6). For example, a certain volume of magnetic bead reagent is dispensed from reagent tank T1 and added to the amplification product droplet. After mixing under electrode drive, the droplet is moved to the corresponding position on the magnet to enrich the magnetic beads. Then, washing buffer is dispensed sequentially from reagent tank T2 to wash the magnetic beads multiple times. Finally, elution buffer is dispensed from reagent tank T3 to elute the library product. The eluted library droplets can be transported to the center of reaction area 16 or to a designated position near the extraction area 14, such as to the area of ​​electrode array 11 below the extraction holes corresponding to X1-Y2. They can then be extracted from the chip through the extraction hole on the top cover using a pipette or an automatic liquid aspiration device for subsequent sequencing.

[0109] Through the above configuration and operation, the digital microfluidic chip of this application integrates multiple functions such as methylation conversion, magnetic bead enrichment and cleaning, reagent heating reaction and PCR amplification in a single chip plane. It can complete the automated preparation process of methylated libraries for 8 or more samples on the same chip, significantly reducing the consumption of reagents per sample and improving parallel processing capabilities.

[0110] In summary, the digital microfluidic chip for NGS methylation library preparation provided in this application achieves stable electrowetting and precise quantitative distribution of droplets within a closed containment cavity by constructing a large-volume reagent injection area 13, an extraction area 14, a small-volume reagent injection area 15, a reaction area 16, a sample injection area 17, and a magnetic bead operation area sequentially arranged on a substrate 1, combined with a zone-optimized electrode array 11, a multi-ring flange on the top cover, and a dielectric and hydrophobic layer combination structure. By correspondingly arranging the reaction area 16 with the heat-conducting area of ​​the external temperature control module, and superimposing the magnetic bead operation function on the sample injection area 17, integrated temperature-controlled reaction and magnetic bead purification are achieved. Simultaneously, the pad array 12, corner guide chamfer structure, and multi-point inlet / outlet array on the back of the substrate 1 enable reliable docking with a library preparation instrument and support for multi-unit high-throughput operation. Therefore, this chip significantly improves the automation level, operational stability, and result repeatability of the NGS methylation library preparation process while maintaining a compact structure and low reagent consumption, demonstrating significant application and promotion value.

[0111] It should be noted that in this patent application, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Without further limitations, an element defined by the phrase "comprising one" does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes said element. In this patent application, if it refers to performing an action according to an element, it means performing the action at least according to that element, including two cases: performing the action only according to that element, and performing the action according to that element and other elements. Expressions such as "multiple," "repeatedly," and "various" include two, two times, two kinds, and more than two, more than two times, and more than two kinds.

[0112] All documents mentioned in this application are considered to be incorporated in their entirety into the disclosure of this application so that they can serve as a basis for modifications if necessary. Furthermore, it should be understood that after reading the foregoing disclosure of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope of protection claimed in this application.

Claims

1. A digital microfluidic chip for the preparation of NGS methylation libraries, characterized in that, include: The substrate (1) has an electrode array (11) for electrowetting drive formed on its upper surface as a droplet transport channel. The substrate (1) is divided into functional areas arranged sequentially from a first side to a second side on a plane. The functional areas include a reagent injection area (13), an extraction area (14), a reaction area (16), and a sample injection area (17). The functional areas are interconnected through the droplet transport channel to transfer and distribute microdroplets between the functional areas. The top cover is disposed opposite to the carrier substrate (1) and defines a cavity for receiving the microdroplets between itself and the carrier substrate (1) by a spacer frame disposed around the electrode array (11). The top cover is provided with a plurality of liquid inlet and outlet holes corresponding to the functional areas respectively. The chip is configured to, under the drive of the electrode array (11), cause at least one microdroplet that enters the reagent injection area (13) and the sample injection area (17) through the inlet and outlet liquid holes to be sequentially transported to the reaction area (16), and the microdroplet that has completed the reaction is transferred to the extraction area (14) and output through the inlet and outlet liquid holes, so as to complete at least part of the steps in the NGS methylation library preparation process. In a preferred embodiment, at least some steps of the NGS methylated library preparation process include one or more of the following: sample pretreatment and methylation conversion, magnetic bead-based nucleic acid enrichment and / or purification, washing and / or solution replacement, adapter ligation and / or other enzymatic reactions under temperature control, and nucleic acid amplification reactions.

2. The digital microfluidic chip prepared from the NGS methylation library as described in claim 1, characterized in that, The back of the carrier substrate (1) is provided with an electrical connection pad array (12), which is used for detachable electrical connection with the pin probes of an external fabrication instrument.

3. The digital microfluidic chip prepared from the NGS methylation library as described in claim 2, characterized in that, Each of the electrical connection pads is electrically connected to at least one driving electrode or driving electrode group in the electrode array (11) via a conductive trace, the driving electrodes being arranged continuously along the extension direction of each droplet transport channel.

4. The digital microfluidic chip prepared from the NGS methylation library as described in claim 2, characterized in that, When the external driving device sequentially applies driving voltage to the adjacent driving electrodes on the droplet transport channel and cuts off the driving voltage of the driving electrodes that the droplet has passed through, an electrowetting potential trap that moves along the target direction can be formed in the receiving cavity, thereby continuously driving the droplet from the sample injection area (17) and / or reagent injection area (13) to the predetermined position of the reaction area (16) and / or extraction area (14).

5. The digital microfluidic chip prepared from the NGS methylation library as described in claim 1, characterized in that, It also includes a magnetic bead operating area, which is arranged on the side of the sample injection area (17) so that when the external magnet adsorbs the magnetic beads, the electrode array (11) controls the droplets to move back and forth between the sample injection area (17) and the reaction area (16) to complete the magnetic bead purification and liquid exchange process.

6. The digital microfluidic chip prepared from the NGS methylation library as described in claim 5, characterized in that, The chip has a dielectric insulating layer on its upper surface. In a preferred embodiment, the top cover is made of a transparent glass substrate, and the lower surface of the top cover is coated with a hydrophobic layer, which is a fluoropolymer coating. In a preferred embodiment, the cavity between the dielectric insulating layer and the hydrophobic layer is filled with dielectric oil.

7. The digital microfluidic chip prepared from the NGS methylation library as described in claim 1, characterized in that, The liquid inlet and outlet ports include: a first sample inlet array (21), a second sample inlet array, and a sample outlet array (22); The first injection port array (21) is arranged on the side corresponding to the reagent injection area (13) for injecting different reagents into the reagent injection area (13); The second injection port array (24) is arranged on one side of the sample injection area (17) for injecting the sample to be tested into the sample injection area (17); The extraction hole array (22) is arranged above the extraction zone (14) for collecting reaction product droplets from the extraction zone (14). In a preferred embodiment, a third sample inlet array (23) is also included, which is disposed in the middle of the upper cover.

8. The digital microfluidic chip prepared from the NGS methylation library as described in claim 1, characterized in that, Each of the electrode arrays (11) includes: a receiving electrode region (111), a buffer electrode region (112), a transition electrode region (113), and a metering electrode region (114); The receiving electrode area (111) has a planar outline that is at least partially a basically circular plate area located within the projection area of ​​the liquid inlet and outlet holes, and is used to receive droplets dripping from the liquid inlet and outlet holes. The buffer electrode area (112) is disposed on one side of the receiving electrode area (111) and is electrically connected to the receiving electrode area (111). The plane width and length of the buffer electrode area (112) are both greater than the diameter of the basic circular plate area. It is used to expand and buffer the droplet shape when the droplet is continuously dripping, so as to prevent the droplet from overflowing the electrode boundary. The transition electrode region (113) is disposed between the buffer electrode region (112) and the metering electrode region (114). The planar profile of the transition electrode region (113) gradually narrows from the direction close to the metering electrode region (114) of the buffer electrode region (112), and is used to guide the droplets in the buffer electrode region (112) to the metering electrode region (114). The metering electrode region (114) is located at the transport direction end of the transition electrode region (113), and is a square electrode with a predetermined planar size, used to accurately divide the droplets under electrowetting drive and limit the volume of the microdroplets transported in a single operation.

9. The digital microfluidic chip prepared from the NGS methylation library as described in claim 1, characterized in that, The upper cover includes a cover plate body. The lower surface of the cover plate body is recessed upward to form a receiving cavity for covering the area of ​​the electrode array (11). The periphery of the cover plate body is integrally formed with a first flange (25) that is sealed and fitted to the substrate (1). At least one ring of stepped second flanges (26) is arranged circumferentially in the area inward of the first flange (25). A glue groove (27) is formed between the first flange (25) and the second flange (26). Each flange is arranged in a closed frame shape around the area of ​​the electrode array (11).

10. The digital microfluidic chip prepared from the NGS methylation library according to any one of claims 1-9, characterized in that, The sample injection area (17) is configured to deliver at least one sample droplet containing the nucleic acid to be tested and / or a nucleic acid sample droplet after methylation conversion; the reagent injection area (13) is configured to deliver a variety of functional reagents for the preparation of methylated libraries. In a preferred embodiment, the sample droplet comprises genomic DNA extracted from tissue, blood, plasma / serum, cells, or extracellular free DNA and / or methylated DNA after bisulfite conversion. In a preferred embodiment, the functional reagents used for methylation library preparation are selected from the group consisting of: bisulfite, magnetic beads, washing solution, desulfurization solution, elution solution, SSB, sequencing adapter reagent, polymerase Master Mix, or index primer. In a preferred embodiment, the sequencing adapter reagent is a paired-end library adapter reagent with P5 adapter sequence and P7 adapter sequence.

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