Four-channel digital micro-fluidic chip for library construction

Abstract

The invention discloses a four-channel digital micro-fluidic chip for library construction, which comprises a public reagent injection area, a temperature control area, a magnetic bead mixing area, a magnetic bead separation area and a waste liquid area, and realizes parallel four channels and low connecting line number through electrode connecting line multiplexing and symmetrical layout; injection is performed according to a volume shunting strategy, magnetic bead separation is completed in a separation area under the action of a magnetic control system along a set flow direction, and an amplification reaction is completed in a temperature control area. The waste liquid area adsorbs waste liquid to reduce the pollution risk. The chip and the method are adaptive to various library building volumes and reaction systems, and have the advantages of high throughput, low volume, low pollution, easiness in integration and the like.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of digital microfluidics, and in particular, to a four-channel digital microfluidic chip for library construction. BACKGROUND

[0002] Digital microfluidics (DMF) is a droplet handling technology that can manipulate discrete liquid droplets on a planar surface, with the droplet volume size generally in the order of nL to μL. Common droplet driving methods include electrowetting on dielectric (EWOD), thermal capillary, light induction, surface acoustic wave, and magnetic driving. DMF technology based on EWOD can precisely manipulate droplets by applying an electric signal to the electrodes on the digital microfluidic chip to form an external electric field, causing the contact angle of the droplet on the hydrophobic surface to change, thereby achieving precise manipulation of the droplet, including driving, merging, dispensing, and splitting. DMF is widely used in the field of rapid separation and analysis of life sciences due to its low reagent consumption, short reaction time, high automation, and high compatibility. DMF can be coupled with different types of detectors for online analysis. Magnetic beads are a kind of superparamagnetic particles with micro-nano scale, which have the characteristics of superparamagnetism, large specific surface area, and rich surface active groups. Combined with DMF system, they are widely used in the fields of immune analysis, nucleic acid separation and extraction, and cell sorting. Digital microfluidic technology can precisely manipulate discrete droplets on a planar surface, from nanoliters to microliters, and the typical implementation is EWOD driving. DMF system has the advantages of low reagent consumption, fast reaction rate, high automation, and coupling with multiple detection methods, and is used in various biomedical application scenarios such as immune analysis, nucleic acid separation and extraction, and cell sorting. The existing library construction process usually relies on large and complex equipment with limited number of channels, making it difficult to achieve multi-channel parallel library construction.

[0003] Existing systems usually use a single or few-channel serial processing architecture of "droplet generation structure-droplet receiving module-interconnection module", which makes it difficult to achieve collaborative scheduling of key steps such as mixing, separation, and amplification in multiple channels on a single chip, resulting in insufficient throughput and high process switching cost. In addition, the number of connections and the complexity of wiring are too high, which also limits the miniaturization and reliability improvement of the system.

[0004] Therefore, one or more methods are needed to solve the above problems.

[0005] It should be noted that the information disclosed in the above BACKGROUND section is only used to strengthen the understanding of the background of the present disclosure, and therefore can include information that does not constitute prior art known to those of ordinary skill in the art. SUMMARY

[0006] The present disclosure aims to provide a four-channel digital microfluidic chip for library construction, thereby at least partially overcoming one or more problems caused by limitations and defects of the related art.

[0007] To achieve the above-mentioned purpose, the four-channel digital microfluidic chip for library construction comprises a common reagent injection area, a temperature control area, a magnetic bead mixing area, and a magnetic bead separation area.

[0008] The chip comprises four channels, each channel comprising at least a temperature control sub-area, a magnetic bead mixing sub-area, and a magnetic bead separation sub-area corresponding to the channel, and being in communication with the common reagent injection area to form a continuous droplet processing path.

[0009] The common reagent injection area is used for injecting reagents and realizing volume split injection.

[0010] The temperature control sub-area is used for keeping droplets at a set temperature and performing amplification reactions.

[0011] The magnetic bead mixing sub-area is used for performing sample fragmentation and linker ligation reactions.

[0012] The magnetic bead separation sub-area is used for performing magnetic bead capture, purification, and supernatant separation operations.

[0013] The pairs of sub-areas in the magnetic bead mixing area, the magnetic bead separation area, and the temperature control area are multiplexed by electrode wiring and geometrically arranged in a symmetrical layout, and the parallel reactions of each step of library construction are completed within the four channels.

[0014] Further, the chip further comprises a control unit that selectively drives the multiplexed electrode groups, thereby realizing four-channel parallel reactions under non-interfering conditions.

[0015] Further, the four channels are arranged in four quadrants around the common reagent injection area, so that the temperature control sub-area, the magnetic bead mixing sub-area, and the magnetic bead separation sub-area are arranged in pairs and symmetrically, and each functional area is centrally symmetrically distributed relative to the common reagent injection area.

[0016] Further, the temperature control area comprises pairs of temperature control sub-areas, and the corresponding electrodes of the pairs of sub-areas are multiplexed and arranged in left-right symmetry; each temperature control sub-area is internally provided with a stable temperature control area for keeping a set temperature and performing amplification reactions.

[0017] Further, the magnetic bead mixing sub-area comprises an electrode array and is provided with a linker injection port and a sample injection port; droplets circulate and mix on the closed driving path formed by the electrode array; the upper and lower corresponding magnetic bead mixing sub-areas are mirror images in geometry and wiring grouping to support electrode wiring multiplexing.

[0018] Further, the magnetic bead separation sub-zone is provided with a sample outlet, and the droplets flow through the magnetic bead separation sub-zone under the action of an external magnetic field to complete magnetic bead capture and supernatant discharge; the magnetic bead separation sub-zones corresponding to the upper and lower positions are mirror images in geometry and connection grouping to support connection multiplexing.

[0019] Further, the waste liquid zone is filled with water-absorbing materials to adsorb and fix the waste liquid, inhibit backflow and cross contamination; the waste liquid zone is arranged at the magnetic bead separation sub-zone of the adjacent channel and is shared by the magnetic bead separation sub-zones of the two adjacent channels.

[0020] Further, the common reagent injection zone includes a large-volume injection zone and a small-volume injection zone.

[0021] The reagent is injected by the large-volume injection zone when the volume of the reagent is greater than or equal to 1.6 microliters, and the reagent is injected by the small-volume injection zone when the volume of the reagent is less than 1.6 microliters.

[0022] The single-electrode carrying volume of the large-volume injection zone is in the range of 1.5-1.7 microliters, and the single-electrode carrying volume of the small-volume injection zone is in the range of 0.9-1.1 microliters.

[0023] Further, the electrode connection multiplexing relationships of the temperature control sub-zone, the magnetic bead mixing sub-zone and the magnetic bead separation sub-zone are respectively as follows:

[0024] The first temperature control zone and the second temperature control zone are left-right symmetrical and multiplexed; the third temperature control zone and the fourth temperature control zone are left-right symmetrical and multiplexed.

[0025] The first mixing zone and the fourth mixing zone are up-down symmetrical and multiplexed; the second mixing zone and the third mixing zone are up-down symmetrical and multiplexed.

[0026] The first separation zone and the second separation zone are up-down symmetrical and multiplexed; the third separation zone and the fourth separation zone are up-down symmetrical and multiplexed.

[0027] Further, the droplet driving paths of the four channels are respectively formed by the respective electrode arrays, and the droplet flow paths between the channels are controlled by the respective electrode arrays to prevent droplet migration or cross-fusion across the channels, so that the four channels can synchronously perform the library construction steps.

[0028] The present disclosure can independently perform different sub-steps (fragmentation, linker connection, purification, amplification and elution) of library construction by arranging the common reagent injection zone, the magnetic bead mixing zone, the magnetic bead separation zone and the temperature control zone on the same chip, so as to realize four-channel parallel reaction, effectively prevent cross contamination, and significantly improve the library construction efficiency and throughput. The corresponding sub-zones of the magnetic bead mixing zone, the magnetic bead separation zone and the temperature control zone adopt electrode connection multiplexing design, which effectively reduces the number of signal lines, the chip area and the wiring complexity, and avoids the problem of electric field interference caused by dense connections.

[0029] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure. BRIEF DESCRIPTION OF DRAWINGS

[0030] The above and other features and advantages of the present disclosure will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which,

[0031] Figure 1 A structural schematic diagram of a four-channel digital microfluidic chip for library construction according to an exemplary embodiment of the present disclosure is shown. DETAILED DESCRIPTION

[0032] Example embodiments now will be described more fully hereinafter with reference to the accompanying drawings. Like reference numerals or characters refer to like elements throughout. The example embodiments can be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and fully convey the concepts of the example embodiments to those skilled in the art. In the drawings, the same reference numbers or characters indicate the same or similar components throughout.

[0033] In addition, the described features, structures, or characteristics can be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are provided to give a thorough understanding of embodiments of the disclosure. One skilled in the relevant art will recognize, however, that the

[0034] The block diagrams in the drawings show only the functionality of the embodiments and do not imply that the functions must be implemented in a specific order or that they must be implemented in every possible implementation. Also, the block diagrams do not show the various ways in which the embodiments can be implemented (for example, as a software program, hardware, firmware, or some combination of these). The use of the term "to" or "together" does not imply that the functions must be performed in a specific order or that they must be performed in every possible implementation.

[0035] In the present example embodiment, a four-channel digital microfluidic chip for library construction; referring to Figure 1 The four-channel digital microfluidic chip for library construction includes a common reagent injection area, a temperature control area, a magnetic bead mixing area, and a magnetic bead separation area.

[0036] The chip comprises four channels, each channel comprising at least a temperature control sub-area 3, 8, 9, 10 corresponding to the channel, a magnetic bead mixing sub-area 4, 11, 12, 13, and a magnetic bead separation sub-area 5, 14, 15, 16, and being in communication with the common reagent injection area 1 to form a continuous droplet processing path;

[0037] The common reagent injection area is used for injecting reagents and realizing volume shunt injection.

[0038] The temperature control sub-area 3, 8, 9, 10 is used for keeping droplets at a set temperature and performing amplification reactions.

[0039] The magnetic bead mixing sub-area 4, 11, 12, 13 is used for performing sample fragmentation and linker ligation reactions.

[0040] The magnetic bead separation sub-area 5, 14, 15, 16 is used for performing magnetic bead capture, purification and supernatant separation operations.

[0041] The magnetic bead mixing area 4, 11, 12, 13, the magnetic bead separation area 5, 14, 15, 16 and the paired sub-areas in the temperature control area are multiplexed by electrode wiring and geometrically arranged in a symmetrical layout, and the parallel reactions of each step of library construction are completed in the four channels.

[0042] The present example realizes four-channel parallel reactions by arranging the common reagent injection area, the magnetic bead mixing area, the magnetic bead separation area and the temperature control area on the same chip, and the four channels can independently perform different sub-steps (fragmentation, linker ligation, purification, amplification and elution) of library construction, effectively preventing cross contamination and significantly improving library construction efficiency and throughput. The corresponding sub-areas of the magnetic bead mixing area, the magnetic bead separation area and the temperature control area are designed by multiplexing the electrode wiring, which effectively reduces the number of signal lines, reduces the chip area and the wiring complexity, and avoids the problem of electric field interference caused by dense wiring.

[0043] In an embodiment of the present application, the four channels are arranged in four quadrants around the common reagent injection area, so that the temperature control sub-area 3, 8, 9, 10, the magnetic bead mixing sub-area 4, 11, 12, 13 and the magnetic bead separation sub-area 5, 14, 15, 16 are arranged in pairs and symmetrically, and each functional area is distributed in a central symmetric manner relative to the common reagent injection area 1.

[0044] In an embodiment of the present invention, the common reagent injection area 1 includes a large-volume injection area 1 and a small-volume injection area 2; the large-volume injection area 1 and the small-volume injection area 2 are controlled by an external liquid injection system to inject reagents according to the reagent usage amounts in different library construction processes. When the reagent volume is greater than or equal to 1.6 microliters, it is injected by the large-volume injection area, and when the reagent volume is less than 1.6 microliters, it is injected by the small-volume injection area; the single-electrode carrying volume of the large-volume injection area is within the range of 1.5 to 1.7 microliters, and the single-electrode carrying volume of the small-volume injection area is within the range of 0.9 to 1.1 microliters.

[0045] In an embodiment of the present invention, the temperature control area includes mutually paired first temperature control sub-areas 3, second temperature control sub-areas 8, third temperature control sub-areas 9, and fourth temperature control sub-areas 10. In this example, the first temperature control area 3 is composed of 10 electrodes, and the stable temperature control area is the inner 6 electrodes, with a maximum support for 10 μL of reagent; the general process is that the reagent to be amplified is driven to this area, and the external temperature control system controls the temperature of the reagent in the chip to complete the amplification reaction. The electrode connection of the second temperature control area 8 is multiplexed with that of the first temperature control area 3 and is designed to be left-right symmetric; the electrode connection of the third temperature control area 9 and the fourth temperature control area 10 is multiplexed and is designed to be left-right symmetric, effectively reducing the number of connections and combining with the control process to be designed to be left-right symmetric. It should be noted that the number of electrodes in the temperature control area is only an example and is not limited thereto.

[0046] In an embodiment of the present invention, the magnetic bead mixing area includes a first magnetic bead mixing sub-area 4, a second magnetic bead mixing sub-area 11, a third magnetic bead mixing sub-area 12, and a fourth magnetic bead mixing sub-area 13. In this example, the first magnetic bead mixing sub-area 4 is composed of 26 electrodes, including one connector injection port and one sample injection port; it has a maximum support for 20 μL of reagent mixing; the general process is that the magnetic bead mixing reagent is driven to the magnetic bead mixing area, and the driving mixed reagent rotates counterclockwise in a "return" shape. The electrode connection of the first magnetic bead mixing sub-area 4 is multiplexed with that of the fourth magnetic bead mixing sub-area 13 and is designed to be up-down symmetric; the electrode connection of the second magnetic bead mixing sub-area 11 and the third magnetic bead mixing sub-area 12 is multiplexed and is designed to be up-down symmetric, effectively reducing the number of connections and combining with the wiring direction to be designed to be up-down symmetric. It should be noted that the number of electrodes in the magnetic bead mixing area is only an example and is not limited thereto.

[0047] In an embodiment of the present application, the magnetic bead separation zone comprises a first magnetic bead separation sub-zone 5, a second magnetic bead separation sub-zone 14, a third magnetic bead separation sub-zone 15 and a fourth magnetic bead separation sub-zone 16. In this example, the first magnetic bead separation sub-zone 5 is composed of 11 electrodes, including 1 sample outlet. The general process is as follows: before magnetic bead separation, the magnetic bead mixed reagent is located in the magnetic bead mixing zone, the magnetic control system controls the magnet to adhere to the chip, and drives the magnetic bead mixed reagent to flow through the magnetic bead separation zone from left to right to complete the magnetic bead separation; after the reaction is completed during the reaction process, the product reagent is driven to the sample outlet, and then the subsequent experiment is completed. The electrode connection lines of the first magnetic bead separation sub-zone 5 and the second magnetic bead separation sub-zone 14 are multiplexed, and are designed to be symmetrically arranged above and below; the electrode connection lines of the third magnetic bead separation sub-zone 15 and the fourth magnetic bead separation sub-zone 16 are multiplexed, and are designed to be symmetrically arranged above and below, which effectively reduces the number of connection lines and combines the wire direction design to be symmetrically arranged above and below. It should be noted that the number of electrodes in the magnetic bead separation zone is only an example and is not limited thereto.

[0048] In an embodiment of the present application, the chip further comprises a waste liquid zone 7, which is filled with water-absorbing material to adsorb and fix the waste liquid, inhibit backflow and cross contamination; the waste liquid zone 7 is arranged at the adjacent channel near the magnetic bead separation zone and is shared by the magnetic bead separation sub-zones 15, 16 and the magnetic bead separation sub-zones 5, 14 of the two adjacent channels.

[0049] In order for those skilled in the art to more clearly understand the scheme of the present application, the actual library construction reaction process will be introduced as follows:

[0050] According to the actual library construction reaction process, the fragmentation reagent with a dosage higher than 1.6 μL is injected from the large-volume injection port, and the reagent with a dosage lower than 1.6 μL is injected from the small-volume injection port. For example, the reaction fragmentation reagent is 5 μL, which is injected from the large-volume injection port, and the next step is to drive the fragmentation reagent to the adapter port and inject the adapter 1 μL from the adapter port, so that the adapter is mixed with the fragmentation reagent, and the adapter connection reaction is waited to be completed. When the purification reaction is performed, the reagent is driven to the magnetic bead separation zone, the magnetic control system controls the magnet to adhere to the chip, the reagent flows through the magnet, the magnetic bead separation is performed, and the waste liquid is directly driven to the waste liquid zone. 5 μL of enzyme-free water is injected from the large-volume injection port, the reagent is driven to carry the separated magnetic beads to the magnetic bead mixing zone, and the magnetic bead mixing reagent is driven to rotate until the magnetic beads are completely mixed with the enzyme-free water. The magnetic bead separation is performed again, and after the separation, the supernatant is driven to the magnetic bead mixing zone. 5 μL of PCR amplification reagent is injected from the large-volume injection port, the reagent is driven to the magnetic bead mixing zone to be completely mixed with the supernatant, and the amplification reaction is performed in the reagent temperature control zone. After the amplification reaction is completed, the magnetic bead separation is performed again, and finally 6 μL of enzyme-free water is injected to mix with the magnetic beads, the supernatant is taken out from the sampling port, and the library construction reaction is completed. Four channels are simultaneously performed.

[0051] It is noted that while the various steps of the methods of the present disclosure are described in a particular order in the drawings, this is not required or implied as to the order of the steps or that all of the steps shown must be performed to achieve the desired result. Additionally or alternatively, certain steps can be omitted, multiple steps can be combined into one step, one step can be broken into multiple steps, etc.

[0052] Furthermore, the above-described diagrams are merely schematic illustrations of the processes included in the method according to the exemplary embodiments of the present application, and are not intended to be limiting. It is readily understood that the processes shown in the above-described diagrams do not indicate or limit the time order of the processes. In addition, it is readily understood that the processes can be executed synchronously or asynchronously, for example, in multiple modules.

[0053] Other embodiments of the present disclosure will be readily apparent to those skilled in the art upon considering the description herein, the drawings, and the annexed claims. The present application is intended to cover any variations, uses, or adaptations of the present disclosure following, in general, the principles of the present disclosure and including such

[0054] It should be understood that the present disclosure is not limited to the precise structures as herein described and illustrated in the drawings, and that various modifications and changes can be made without departing from its scope. The scope of the present disclosure is limited only by the claims appended hereto.

Claims

1. A four-channel digital microfluidic chip for library construction, characterized in that, It includes a common reagent injection area, a temperature control area, a magnetic bead mixing area, and a magnetic bead separation area; The chip includes four channels, each channel including at least a temperature control sub-region, a magnetic bead mixing sub-region, and a magnetic bead separation sub-region corresponding to that channel, and is connected to the common reagent injection area to form a continuous droplet processing path; The common reagent injection area is used for injecting reagents and to achieve volume split injection; The temperature-controlled sub-region is used to maintain the droplet at a set temperature and to carry out the amplification reaction. The magnetic bead mixing sub-region is used to perform sample fragmentation and connector bonding reactions; The magnetic bead separation sub-region is used to perform magnetic bead capture, purification, and supernatant separation operations; The paired regions in the magnetic bead mixing region, magnetic bead separation region, and temperature control region are multiplexed by electrode connections and arranged in a symmetrical geometric layout, and the parallel reactions of each step of library construction are completed in four channels.

2. The chip as described in claim 1, characterized in that, The chip also includes a control unit that selectively drives the multiplexed electrode group, thereby enabling four-channel parallel reaction under non-interference conditions.

3. The chip as described in claim 1, characterized in that, The four channels are arranged in four quadrants around the common reagent injection area, so that the temperature control sub-area, magnetic bead mixing sub-area, and magnetic bead separation sub-area are arranged in pairs symmetrically, and each functional area is centrally symmetrically distributed relative to the common reagent injection area.

4. The chip as described in claim 1, characterized in that, The temperature control zone includes paired temperature control sub-regions, with the corresponding electrode connections of the paired sub-regions being multiplexed and arranged symmetrically from left to right; each temperature control sub-region has a stable temperature control area inside, used to maintain the set temperature and carry out the amplification reaction.

5. The chip as described in claim 1, characterized in that, The magnetic bead mixing sub-region includes an electrode array and is provided with a connector injection port and a sample injection port; the droplets circulate and mix in the closed driving path formed by the electrode array; the upper and lower corresponding magnetic bead mixing sub-regions are mirrored in geometry and connection grouping to support electrode connection reuse.

6. The chip as described in claim 1, characterized in that, The magnetic bead separation sub-region is equipped with a sample outlet. Under the action of an external magnetic field, droplets flow through the magnetic bead separation sub-region to complete magnetic bead capture and supernatant export. The upper and lower corresponding magnetic bead separation sub-regions are mirrored in geometry and connection grouping to support connection reuse.

7. The chip as described in claim 1, characterized in that, It also includes a waste liquid zone, which is filled with absorbent material to adsorb and fix the waste liquid and inhibit backflow and cross-contamination; the waste liquid zone is arranged in adjacent channels near the magnetic bead separation zone and is shared by the magnetic bead separation sub-zones of two adjacent channels.

8. The chip as described in claim 1, characterized in that, The common reagent injection area includes a large-volume injection area and a small-volume injection area; When the reagent volume is greater than or equal to 1.6 μL, it is injected from the large volume injection area; when the reagent volume is less than 1.6 μL, it is injected from the small volume injection area. The single electrode carrying volume of the large-volume injection region is in the range of 1.5 to 1.7 microliters, and the single electrode carrying volume of the small-volume injection region is in the range of 0.9 to 1.1 microliters.

9. The chip as described in claim 1, characterized in that, The electrode connection reuse relationships of the temperature control sub-region, the magnetic bead mixing sub-region, and the magnetic bead separation sub-region are respectively as follows: The first and second temperature control zones are symmetrical and reused; the third and fourth temperature control zones are symmetrical and reused. The first and fourth mixing zones are symmetrical and reused; the second and third mixing zones are symmetrical and reused. The first separation zone and the second separation zone are symmetrical and reused; the third separation zone and the fourth separation zone are symmetrical and reused.

10. The chip as described in claim 1, characterized in that, The droplet driving paths of the four channels are formed by their respective electrode arrays, and the droplet flow routes between the channels are controlled by their respective electrode arrays to prevent droplets from migrating or merging across channels, so that the four channels can execute the library construction steps synchronously.

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