A microfluidic chip system for single-stranded DNA library construction

Abstract

The application provides a microfluidic chip system for single-stranded DNA library construction, comprising a chip base body internally provided with a microchannel and a plurality of microvalves, the chip base body is sequentially connected with a sample input port, a thermal denaturation chamber, a first connection reaction chamber, a primer extension chamber, a second connection reaction chamber, an amplification chamber and an outlet, the thermal denaturation chamber is provided with a first temperature control element, the first connection reaction chamber is preloaded with single-stranded DNA ligase and first linker lyophilized reagents and is provided with a second temperature control element, the primer extension chamber is preloaded with DNA polymerase, dNTP and extension primer lyophilized reagents and is provided with a third temperature control element, the second connection reaction chamber is preloaded with second linker and ligase lyophilized reagents, and the amplification chamber is provided with a fourth temperature control element; after the sample is dissociated into single-stranded DNA by the microfluidic chip system, linker connection and complementary strand synthesis are sequentially completed. The application avoids the bias of double-stranded library construction, closes the pipeline and isolates the physical space, cuts off the pollution path, and improves the conversion rate.

Description

Technical Field

[0001] This invention relates to the field of microfluidics, specifically to a microfluidic chip system for single-stranded DNA library construction. Background Technology

[0002] With the widespread adoption of high-throughput sequencing technology, DNA library construction has become an indispensable preprocessing step in the sequencing workflow. The main purpose of library construction is to add specific known sequence adapters to both ends of the target DNA fragment so that the sequencing platform can recognize them and perform subsequent sequence amplification and sequencing reads.

[0003] Existing routine library construction procedures typically use double-stranded DNA as the starting template and are performed in separate reaction tubes. After extracting nucleic acids from the sample and obtaining double-stranded DNA fragments, operators sequentially add a specific enzyme mixture to the reaction tube to repair the ends of the double-stranded DNA fragments. Then, ligase is used to simultaneously ligate the adapters to both ends of the DNA fragment. After adapter ligation, the fluid is transferred to an amplification device, where polymerase chain reaction (PCR) is used to amplify and enrich the target nucleic acid fragments containing the adapters, thus completing the construction of the sequencing library.

[0004] Existing conventional library construction methods using double-stranded DNA as a template suffer from low conversion rates and susceptibility to aerosol contamination when handling degraded samples. Because conventional operations rely on the end repair and simultaneous ligation mechanisms of double-stranded DNA, this biochemical reaction pathway is subject to sequence bias, making it difficult to effectively ligate highly fragmented degraded DNA samples and trace amounts of nucleic acid samples. Furthermore, the entire library construction biochemical process relies on open reaction tube systems, frequent manual pipetting, and repeated opening and closing of the tubes for sample addition, making it susceptible to introducing aerosol impurities from the external environment. Additionally, the manual preparation of various enzyme reagents can easily lead to batch-to-batch reaction errors. Therefore, current technology lacks an automated hardware system that can integrate single-stranded library construction biochemical logic with a closed microfluidic network, making it difficult to simultaneously address the sequence bias limitations of conventional double-stranded ligation and the contamination and error problems caused by manual open operations. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a microfluidic chip system for single-stranded DNA library construction. This system solves the problems of loss of target sequences and low library conversion rates caused by end repair and simultaneous ligation of both ends when conventional double-stranded DNA library construction techniques are used to process highly degraded or trace nucleic acid samples. These problems include aerosol contamination and human batch errors caused by frequent manual pipetting operations in traditional open-type systems.

[0006] To achieve the above objectives, the present invention provides the following technical solution: a microfluidic chip system for single-stranded DNA library construction, comprising:

[0007] The chip substrate contains multiple chambers and microchannels; the sample input port is located at one end of the chip substrate and is used to introduce DNA samples.

[0008] The thermal denaturation chamber is connected to the sample input port via a microchannel, and a first temperature control element is installed in the thermal denaturation chamber;

[0009] The first connection reaction chamber is connected to the heat denaturation chamber through a microchannel. The first connection reaction chamber is pre-loaded with a single-stranded DNA ligase and a lyophilized reagent for the first adapter. A second temperature control element is configured in the first connection reaction chamber.

[0010] The primer extension chamber is connected to the first ligation reaction chamber via a microchannel. The primer extension chamber is pre-loaded with DNA polymerase, dNTPs and lyophilized reagents for extension primers. A third temperature control element is installed in the primer extension chamber.

[0011] The second ligation reaction chamber is connected to the primer extension chamber via a microchannel. The second ligation reaction chamber is pre-loaded with the second adapter and lyophilized reagents of the ligase.

[0012] The amplification chamber is connected to the second connecting reaction chamber via a microchannel, and a fourth temperature control element is configured in the amplification chamber.

[0013] Multiple microvalves are installed in each microchannel.

[0014] The outlet is located at one end of the chip substrate and is connected to the amplification chamber.

[0015] Preferably, the chip substrate has a multilayer bonding structure, comprising a capping layer, a channel layer, and a substrate layer stacked sequentially; the capping layer, channel layer, and substrate layer are fixedly connected by a bonding process to form a seamless integral structure; the channel layer has microstructures inside, including grooves and through holes penetrating the thickness direction of the channel layer; the channel layer is bonded to the capping layer and the substrate layer, and the grooves and through holes on the channel layer are sealed by the capping layer and the substrate layer to form closed cavities and channels, corresponding to multiple chambers and microchannels inside the chip substrate.

[0016] Preferably, the cover plate layer is provided with a through hole that penetrates the thickness direction of the cover plate layer, and the through hole on the cover plate layer corresponds to the sample input port and the output port; the sample input port is connected to the starting end of the internal microchannel, and the output port is a physical through hole that penetrates the surface of the chip substrate and is connected to the end of the internal microchannel, which is used to allow external fluid driving force to push the fluid away from the chip substrate.

[0017] Preferably, the microvalve includes a deformable thin film structure located above the microchannel; when not subjected to external force, the deformable thin film structure is in a flat state and the microchannel is in a conductive state; when an external force is applied above the deformable thin film structure, the deformable thin film structure undergoes bending deformation, and the bent and deformed thin film structure adheres to the bottom wall of the microchannel, sealing the internal cross-section of the microchannel and putting the microchannel in a closed state.

[0018] Preferably, the microchannel forms a unidirectional fluid conduit inside the chip substrate. The unidirectional fluid conduit starts at the sample inlet, sequentially connects each chamber, and ends at the outlet. When a biochemical reaction occurs in a single chamber, the microvalve at the front and rear ends of the single chamber remains closed, so that the chamber forms a closed physical space. After the biochemical reaction is completed, the microvalve at the rear end of the single chamber opens, and the fluid enters the next chamber.

[0019] Preferably, the first temperature control element is located at an external position directly below the thermal denaturation chamber, and the top surface of the first temperature control element is attached to the bottom surface of the chip substrate; the first temperature control element outputs heat, which raises the fluid temperature inside the thermal denaturation chamber and maintains it at 98°C, so that the hydrogen bonds between the base pairs of the DNA sample break and the DNA strands are destranded to form single-stranded DNA free in the liquid buffer.

[0020] Preferably, the second temperature control element is located inside or near the outside of the first connection reaction chamber; the single-stranded DNA ligase and the lyophilized reagent of the first adapter are pre-cured on the inner wall of the first connection reaction chamber; the second temperature control element maintains the temperature of the fluid inside the first connection reaction chamber within the range of 20°C to 30°C, the liquid buffer dissolves the cured lyophilized reagent, the single-stranded DNA ligase catalyzes the covalent binding of the ends of the single-stranded DNA to the first adapter.

[0021] Preferably, the DNA polymerase, dNTPs, and lyophilized reagents for the extension primers are pre-fixed on the inner wall of the primer extension chamber; a third temperature control element maintains the temperature of the fluid inside the primer extension chamber within the range of 37°C to 50°C; the liquid buffer dissolves the fixed lyophilized reagents; the extension primers bind to the first adapter sequence on the single-stranded DNA through complementary base pairing; the DNA polymerase uses the single-stranded DNA as a template and the dNTPs in the fluid to synthesize the complementary strand, converting the single-stranded DNA into a double-stranded DNA structure.

[0022] Preferably, the lyophilized reagents of the second adapter and ligase are pre-fixed on the inner wall of the second ligation reaction chamber; the fixed lyophilized reagents are dissolved in the liquid buffer, and the ligase catalyzes the covalent binding of the second adapter to the other end of the double-stranded DNA that is not ligated, so that the two ends of the double-stranded DNA are respectively connected to the first adapter and the second adapter, forming a library construction intermediate with double-ended adapters.

[0023] Preferably, the fourth temperature control element is located at an external position below the amplification chamber, and the surface of the fourth temperature control element is attached to the bottom surface of the chip substrate. The fourth temperature control element executes the temperature cycling program required for the polymerase chain reaction. The temperature cycling program controls the fluid inside the amplification chamber to undergo periodic temperature alternation of denaturation, annealing and extension. The library preparation intermediate with double-ended connectors is targeted and enriched under periodic temperature alternation to generate the final library preparation product.

[0024] This invention provides a microfluidic chip system for single-stranded DNA library construction. It offers the following advantages:

[0025] 1. This invention, by setting up a thermal denaturation chamber and a reaction chamber inside the microfluidic chip substrate and configuring independent first to fourth temperature control elements, first deconjugates the sample into single-stranded DNA, and then sequentially flows through the first ligation reaction chamber and the primer extension chamber to perform stepwise single-end adapter covalent binding and complementary strand synthesis operations. This avoids the bias limitations of conventional double-stranded library construction end repair and simultaneous double-end ligation processes, and achieves the technical effect of effectively improving the library construction conversion rate for highly fragmented and degraded samples and extremely small amounts of nucleic acid samples.

[0026] 2. This invention constructs a unidirectional fluid pipeline using a multilayer bonding process and connects multiple chambers undergoing different biochemical reactions in series within a closed network of a multilayer bonded chip substrate. Combined with deformable thin-film microvalve structure set on the microchannel, the internal sections are sequentially closed or restored to a conductive state according to a set time sequence. This replaces the pipette sample addition and cap opening steps in traditional library preparation operations and isolates each independent biochemical reaction area, achieving the effect of cutting off the physical pathway of external aerosol contamination of DNA samples and ensuring the purity of the library preparation products.

[0027] 3. This invention pre-solidifies lyophilized reagents containing ligase, polymerase, and adapter components on the inner walls of the first connection reaction chamber, primer extension chamber, and second connection reaction chamber. The sample output from the previous reaction, carrying liquid buffer, is guided into the current closed physical space by a one-way fluid pipeline to directly complete the reconstitution of biochemical reaction substances. This eliminates the need for manual reagent preparation and uses a microchannel network to automatically guide fluid flow, achieving stable operation of the single-chain library construction catalytic system and reducing batch-to-batch errors. Attached Figure Description

[0028] Figure 1 This is a schematic diagram of the overall flow path layout of the microfluidic chip system of the present invention. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Please see the appendix Figure 1 This invention provides a microfluidic chip system for single-stranded DNA library construction, comprising:

[0031] The chip substrate has a multi-layer bonding structure and multiple chambers and microchannels are arranged inside the chip substrate;

[0032] A sample input port is located at one end of the chip substrate and is used to introduce DNA samples;

[0033] A heat-denaturing chamber is connected to the sample input port through the microchannel, and a first temperature control element is provided below the heat-denaturing chamber;

[0034] The first connection reaction chamber is connected to the heat denaturation chamber through the microchannel. The first connection reaction chamber is pre-loaded with a single-stranded DNA ligase and a lyophilization reagent for the first adapter. A second temperature control element is provided inside or near the outside of the first connection reaction chamber.

[0035] The primer extension chamber is connected to the first connection reaction chamber through the microchannel. The primer extension chamber is pre-loaded with DNA polymerase, dNTPs and lyophilization reagents for extension primers. A third temperature control element is provided at the primer extension chamber.

[0036] The second ligation reaction chamber is connected to the primer extension chamber through the microchannel, and the second ligation reaction chamber is pre-loaded with a second adapter and lyophilized reagents of the ligase;

[0037] The amplification chamber is connected to the second connecting reaction chamber through the microchannel, and a fourth temperature control element is provided below the amplification chamber;

[0038] Multiple microvalves are disposed in each of the aforementioned microchannels;

[0039] The outlet is located at one end of the chip substrate and is connected to the amplification chamber.

[0040] During the operation of the microfluidic chip system, each module works collaboratively according to a pre-set biochemical reaction sequence. First, an initial DNA sample containing an aqueous buffer solution is introduced into the chip substrate through the sample inlet under the action of external fluid driving force. A microvalve within the microchannel at the rear end of the sample inlet opens, allowing the fluid to carry the DNA sample into the thermal denaturation chamber. Subsequently, the microchannel at the inlet closes to isolate the thermal denaturation chamber. A first temperature control element provides a thermal denaturation temperature of 98°C to the thermal denaturation chamber, causing the DNA sample to degenerate under heat, forming single-stranded DNA.

[0041] After heat denaturation, the microvalve between the heat denaturation chamber and the first ligation reaction chamber opens. Single-stranded DNA enters the first ligation reaction chamber with the fluid, and the aqueous buffer carried by the single-stranded DNA reconstitutes the pre-loaded single-stranded DNA ligase and the first adapter lyophilization reagent in the first ligation reaction chamber. At this time, the second temperature control element provides a temperature environment of 20°C to 30°C for the first ligation reaction chamber, completing the ligation reaction between the single-stranded DNA and the first adapter.

[0042] After the first ligation reaction is completed, the corresponding microvalve opens, allowing fluid to enter the primer extension chamber. The fluid reconstitutes the pre-loaded DNA polymerase, dNTPs, and lyophilized extension primer reagents within the chamber. A third temperature control element provides an extension reaction temperature ranging from 37°C to 50°C. Based on these temperature conditions, the DNA polymerase uses single-stranded DNA as a template to complete the extension synthesis of the complementary strand, generating a double-stranded DNA structure.

[0043] After the extension reaction is complete, the microchannel is reopened, and the double-stranded DNA generated during the extension reaction enters the second ligation reaction chamber with the fluid. The fluid re-dissolves the second adapter and lyophilized ligase reagent in the second ligation reaction chamber, completing the second adapter ligation reaction and attaching the second adapter to the other end of the nucleic acid fragment.

[0044] After the second connector is connected, fluid enters the amplification chamber. The fourth temperature control element initiates and executes the temperature cycling program required for polymerase chain reaction amplification, amplifying the library preparation product. After the amplification process is completed, the microvalve located at the rear end of the amplification chamber opens, and the final single-stranded DNA library preparation product is collected through microchannels to the outlet and output from the chip substrate. Multiple microvalves operate sequentially according to the time nodes of each reaction step throughout the process, achieving fluid communication and physical isolation between adjacent chambers, ensuring that each biochemical reaction is completed sequentially in an independent space.

[0045] Compared to conventional double-stranded DNA library construction procedures, single-stranded DNA library construction procedures first involve thermal denaturation and melting, followed by ligation of the first adapter in single-stranded form. This single-strand ligation method avoids the bias limitations inherent in the end-repair and ligation processes of double-stranded DNA. This reaction logic improves the library conversion rate for highly fragmented, degraded DNA samples and trace amounts of DNA. The microfluidic chip system integrates the single-stranded library construction biochemical process within a closed circuit, leveraging the advantages of single-stranded library conversion while eliminating the pathway of nucleic acid aerosol contamination from the external environment.

[0046] The chip substrate included in the microfluidic chip system includes:

[0047] Cover layer;

[0048] The channel layer is located below the cover layer;

[0049] The film layer is located below the channel layer.

[0050] The cover layer, channel layer, and film layer are stacked sequentially. The cover layer, channel layer, and film layer are fixedly connected by a bonding process to form a seamless integral structure.

[0051] The channel layer contains microstructures, including grooves and through-holes extending through its thickness. The channel layer is bonded to the cover layer and the substrate layer. The grooves and through-holes in the channel layer are sealed by the cover and substrate layers, forming closed cavities and channels.

[0052] The enclosed cavities and conduits constitute multiple chambers and microchannels within the chip substrate. Specifically, the multiple chambers include a thermal denaturation chamber, a first ligation reaction chamber, a primer extension chamber, a second ligation reaction chamber, and an amplification chamber. The microchannels spatially connect the individual chambers.

[0053] The cover plate has through-holes extending through its thickness. These through-holes form the sample input and output ports of the microfluidic chip system. The sample input port connects to the beginning of an internal microchannel, and the output port connects to the end of an internal microchannel.

[0054] The chip substrate is made of biocompatible materials. The bonding process ensures the airtightness of the microchannels and multiple chambers. The multilayered bonding structure establishes a closed microfluidic network, isolating each independent biochemical reaction region, preventing external environmental contamination of the single-stranded DNA library construction process, and avoiding leakage of internal fluids during operation.

[0055] The system's internal microchannel network and microvalve control mechanism include:

[0056] Microchannels are distributed inside the chip substrate;

[0057] Multiple microvalves are installed in the microchannel.

[0058] The microchannels form a unidirectional fluid conduit within the chip substrate. This unidirectional fluid conduit begins at the sample inlet and sequentially connects to the thermal denaturation chamber, the first connection reaction chamber, the primer extension chamber, the second connection reaction chamber, and the amplification chamber, terminating at the outlet. The unidirectional fluid conduit connects all of the aforementioned chambers.

[0059] Multiple microvalves are positioned within the microchannels between adjacent chambers. Microvalves are also positioned within the microchannel between the sample inlet and the thermal denaturation chamber, and within the microchannel between the amplification chamber and the outlet. These microvalves either block or open the microchannels.

[0060] The microvalve comprises a deformable thin-film structure located above a microchannel. When no external force is applied, the deformable thin-film structure is in a flat state, and the microchannel is conductive. When an external force is applied above the deformable thin-film structure, it undergoes bending deformation. The bent thin-film structure adheres to the bottom wall of the microchannel, sealing the internal cross-section of the microchannel, and the microchannel is in a closed state. After the external force is removed, the deformable thin-film structure returns to its flat state, and the microchannel returns to its conductive state.

[0061] In the single-stranded DNA library preparation process, the microvalves alternately switch between open and closed states. During the biochemical reaction in a single chamber, both the front and rear microvalves of that chamber remain closed. After the biochemical reaction is complete, the rear microvalves of the single chamber open, allowing fluid to enter the next chamber.

[0062] The sample input and thermal denaturation chamber sections include:

[0063] Sample input port, thermal denaturation chamber, first temperature control element.

[0064] The sample inlet is located at one end of the chip substrate. The sample inlet connects the outside of the chip substrate to the microchannels inside the chip substrate. The initial sample, containing double-stranded DNA and buffer solution, is injected into the chip substrate through the sample inlet.

[0065] The thermodegradation chamber is located inside the chip substrate. A microchannel connects the sample input port to the thermodegradation chamber. The microchannel guides the initial sample into the thermodegradation chamber. When the microvalves located at the front and rear ends of the thermodegradation chamber are closed, the thermodegradation chamber forms a sealed fluid space.

[0066] The first temperature control element is located externally, directly below the thermal denaturation chamber. The top surface of the first temperature control element is attached to the bottom surface of the chip substrate. The first temperature control element is an externally powered heating device. The heat generated by the first temperature control element passes through the bottom material of the chip substrate and is physically conducted to the fluid inside the thermal denaturation chamber.

[0067] The first temperature control element outputs heat, raising and maintaining the fluid temperature inside the thermal denaturation chamber at 98°C. At 98°C, the hydrogen bonds between base pairs in the initial double-stranded DNA sample break. The double-stranded structure unwinds, forming two independent single-stranded DNA molecules. These single-stranded DNA molecules are released into the liquid buffer. The thermal denaturation of the DNA sample is completed through heat transfer and molecular unwinding. After the thermal denaturation process is complete, a microvalve at the rear end of the thermal denaturation chamber opens, allowing the single-stranded DNA molecules to be carried by the liquid fluid to the next reaction area.

[0068] The first connecting reaction chamber portion includes:

[0069] The first connection is to the reaction chamber, the lyophilized reagent, and the second temperature control element.

[0070] The first connection reaction chamber is located inside the chip substrate. A microchannel connects the thermal denaturation chamber and the first connection reaction chamber. Fluid output from the thermal denaturation chamber enters the first connection reaction chamber via the microchannel. Microvalves at the front and rear ends of the first connection reaction chamber are closed, forming a closed physical space.

[0071] The lyophilized reagent is pre-fixed on the inner wall of the first ligation reaction chamber. The lyophilized reagent contains a single-stranded DNA ligase and a first adapter. Fluid from the heat-denaturing chamber contains single-stranded DNA and a liquid buffer. Upon entering the first ligation reaction chamber, the fluid contacts the lyophilized reagent. The liquid buffer dissolves the fixed lyophilized reagent. The single-stranded DNA ligase and the first adapter are released and mixed into the fluid.

[0072] The second temperature control element is located inside or near the outside of the first connecting reaction chamber. The second temperature control element exchanges heat with the first connecting reaction chamber through physical contact. The second temperature control element regulates the temperature of the fluid inside the first connecting reaction chamber.

[0073] The second temperature control element maintains the temperature of the fluid inside the first ligation reaction chamber within the range of 20°C to 30°C. At this temperature, single-stranded DNA ligase catalyzes the covalent binding of the ends of the single-stranded DNA to the first adapter. The fluid completes the first step of the adapter ligation reaction inside the first ligation reaction chamber. After the adapter ligation reaction is complete, the microvalve at the rear end of the first ligation reaction chamber opens, and the fluid exits to the next reaction area.

[0074] The primer extension chamber includes: primer extension chamber, lyophilized reagent, and third temperature control element.

[0075] The primer extension chamber is located inside the chip substrate. A microchannel connects the first connection reaction chamber and the primer extension chamber. Fluid output from the first connection reaction chamber enters the primer extension chamber via the microchannel. The microvalves at the front and rear ends of the primer extension chamber are closed, forming a closed physical space within the primer extension chamber.

[0076] The lyophilized reagent is pre-fixed on the inner wall of the primer extension chamber. The lyophilized reagent contains DNA polymerase, dNTPs, and extension primers. The fluid exiting the first ligation reaction chamber contains single-stranded DNA ligated with the first adapter and a liquid buffer. The fluid entering the primer extension chamber contacts the lyophilized reagent fixed on the inner wall of the chamber. The liquid buffer dissolves the lyophilized reagent. The DNA polymerase, dNTPs, and extension primers are released and mixed within the fluid.

[0077] The third temperature control element is located adjacent to the exterior of the primer extension chamber. The surface of the third temperature control element is attached to the chip substrate. The third temperature control element generates heat and conducts it into the primer extension chamber. The third temperature control element regulates the temperature of the fluid inside the primer extension chamber.

[0078] The third temperature control element maintains the temperature of the fluid inside the primer extension chamber within the range of 37°C to 50°C. At this temperature, the extension primer binds to the first adapter sequence on the single-stranded DNA through complementary base pairing. DNA polymerase then catalyzes this process. Using single-stranded DNA as a template, DNA polymerase synthesizes the complementary strand using dNTPs in the fluid. The single-stranded DNA is thus converted into a double-stranded DNA structure.

[0079] The complementary strand extension synthesis reaction is completed within the primer extension chamber. After the extension reaction is complete, a microvalve at the rear end of the primer extension chamber opens, and fluid containing the double-stranded DNA structure is discharged to the next reaction region.

[0080] The second connecting reaction chamber includes: a second connecting reaction chamber and a lyophilized reagent.

[0081] The second connection reaction chamber is located inside the chip substrate. A microchannel connects the primer extension chamber and the second connection reaction chamber. Fluid output from the primer extension chamber enters the second connection reaction chamber via the microchannel. Microvalves at the front and rear ends of the second connection reaction chamber are closed, forming a closed physical space.

[0082] The lyophilized reagent is pre-fixed on the inner wall of the second ligation reaction chamber. The lyophilized reagent contains a ligase and a second adapter. The fluid exiting the primer extension chamber contains double-stranded DNA and a liquid buffer. The fluid entering the second ligation reaction chamber contacts the lyophilized reagent fixed on the inner wall of the chamber. The liquid buffer dissolves the lyophilized reagent. The ligase and second adapter are released and mixed within the fluid.

[0083] One end of the double-stranded DNA has been ligated to the first adapter. Ligase catalyzes this process within the fluid. The ligase then catalyzes the covalent binding of the second adapter to the unligated end of the double-stranded DNA. The fluid then completes the second adapter ligation reaction within the second ligation chamber. The two ends of the double-stranded DNA are now connected to the first and second adapters, respectively, forming a library-building intermediate with paired-end adapters.

[0084] After the connector connection reaction is completed, the micro-valve at the rear end of the second connection reaction chamber opens, and the fluid containing the library construction intermediate product is output to the next reaction area.

[0085] The amplification chamber includes:

[0086] Amplification chamber, fourth temperature control element.

[0087] The amplification chamber is located inside the chip substrate. A microchannel connects the second connecting reaction chamber and the amplification chamber. Fluid output from the second connecting reaction chamber enters the amplification chamber via the microchannel. Microvalves at the front and rear ends of the amplification chamber are closed, forming a closed physical space within the amplification chamber.

[0088] The fluid output from the second connecting reaction chamber not only contains library preparation intermediates with dual-end connectors, but also enters the amplification chamber with the fluid to form a nucleic acid template for the targeted enrichment reaction.

[0089] The fourth temperature control element is located externally below the amplification chamber. The top surface of the fourth temperature control element is in contact with the bottom surface of the chip substrate. The heat generated by the fourth temperature control element is conducted through the chip substrate to the interior of the amplification chamber. The fourth temperature control element regulates the temperature of the fluid inside the amplification chamber.

[0090] The fourth temperature control element executes the temperature cycling program required for the polymerase chain reaction. The temperature cycling program controls the fluid inside the amplification chamber to undergo periodic temperature alternations of denaturation, annealing, and extension.

[0091] Under periodic temperature alternation, the library preparation intermediates undergo exponential targeted enrichment. The quantity of amplified products conforms to the following mathematical relationship:

[0092]

[0093] In the formula, This represents the quantity of library-built products after amplification. This represents the initial quantity of library preparation intermediates entering the amplification chamber. Represents amplification efficiency. This represents the number of cycles in the temperature cycling program.

[0094] The amplification reaction is completed inside the amplification chamber, generating the final library preparation product. After the amplification process is complete, the microvalve at the rear end of the amplification chamber opens, and the fluid containing the library preparation product is output to the next region via a microchannel.

[0095] The product output port section includes: the outlet.

[0096] The outlet is located at one end of the chip substrate. The outlet is a physical via that penetrates the surface of the chip substrate. A microchannel connects the amplification chamber and the outlet. After the amplification reaction is completed inside the amplification chamber, a microvalve located at the rear end of the amplification chamber switches to the open state.

[0097] The fluid contains the final library preparation product after amplification. External fluid driving force propels the fluid out of the amplification chamber. The fluid flows along the microchannels towards the outlet. After reaching the end of the microchannel, the fluid flows out of the chip substrate through the outlet.

[0098] The fluid discharge action marks the end of the single-stranded DNA library preparation process. The chip substrate, relying on pre-loaded reagents and a pre-defined microchannel network, along with external temperature control elements, completes the reaction process from the introduction of the initial DNA sample to the output of the final library preparation product.

[0099] For the specific implementation parameters of the multilayer bonding process, the specific structure of the external driving device of the microvalve, and the internal circuit layout of the temperature control element, those skilled in the art can implement them in combination with conventional microfluidic technology. The aforementioned technical details are well known in the field and will not be repeated here.

[0100] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A microfluidic chip system for single-stranded DNA library construction, characterized in that, include: A chip substrate, wherein multiple chambers and microchannels are provided inside the chip substrate; A sample input port is located at one end of the chip substrate and is used to introduce DNA samples; A heat-denaturing chamber is connected to the sample input port through the microchannel, and a first temperature control element is disposed in the heat-denaturing chamber; The first connection reaction chamber is connected to the heat denaturation chamber through the microchannel. The first connection reaction chamber is pre-loaded with a single-stranded DNA ligase and a lyophilization reagent for the first adapter. A second temperature control element is configured in the first connection reaction chamber. The primer extension chamber is connected to the first connection reaction chamber through the microchannel. The primer extension chamber is pre-loaded with DNA polymerase, dNTPs and lyophilized reagents for extension primers. A third temperature control element is configured in the primer extension chamber. The second ligation reaction chamber is connected to the primer extension chamber through the microchannel, and the second ligation reaction chamber is pre-loaded with a second adapter and lyophilized reagents of the ligase; An amplification chamber is connected to the second connecting reaction chamber via the microchannel, and a fourth temperature control element is disposed in the amplification chamber; Multiple microvalves are disposed in each of the aforementioned microchannels; The outlet is located at one end of the chip substrate and is connected to the amplification chamber.

2. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The chip substrate has a multilayer bonding structure, comprising a cover layer, a channel layer, and a substrate layer stacked sequentially. The cover layer, the channel layer, and the substrate layer are fixedly connected by a bonding process to form a seamless integral structure; The channel layer has a microstructure inside, including grooves and through holes that penetrate the thickness direction of the channel layer. The channel layer is attached to the cover layer and the substrate layer. The grooves and through holes on the channel layer are closed by the cover layer and the substrate layer to form closed cavities and channels, which correspond to multiple chambers and microchannels inside the chip substrate.

3. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The microvalve includes a deformable thin film structure located above the microchannel; When not subjected to external force, the deformable film structure is in a flat state and the microchannel is in a conductive state; when an external force is applied above the deformable film structure, the deformable film structure bends and deforms, and the bent film structure adheres to the bottom wall of the microchannel, sealing the internal cross section of the microchannel and putting the microchannel in a closed state.

4. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The first temperature control element is located at an external position directly below the thermal denaturation chamber, and the top surface of the first temperature control element is attached to the bottom surface of the chip substrate; The first temperature control element outputs heat, which raises the fluid temperature inside the thermal denaturation chamber and maintains it at 98°C, so that the hydrogen bonds between the base pairs of the DNA sample break and the DNA strands are unwound to form single-stranded DNA free in the liquid buffer.

5. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The second temperature control element is located inside the first connecting reaction chamber or adjacent to the outside of the first connecting reaction chamber; The single-stranded DNA ligase and the lyophilized reagent of the first adapter are pre-fixed on the inner wall of the first ligation reaction chamber; The second temperature control element maintains the temperature of the fluid inside the first connected reaction chamber within the range of 20°C to 30°C. The liquid buffer dissolves and solidifies the lyophilized reagent, and the single-stranded DNA ligase catalyzes the covalent binding of the ends of the single-stranded DNA to the first adapter.

6. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The DNA polymerase, dNTPs, and lyophilization reagents for the extension primers are pre-fixed on the inner wall of the primer extension chamber; The third temperature control element maintains the temperature of the fluid inside the primer extension chamber within the range of 37°C to 50°C. The liquid buffer dissolves and solidifies the lyophilized reagent. The extension primer binds to the first adapter sequence on the single-stranded DNA through complementary base pairing. The DNA polymerase uses the single-stranded DNA as a template and utilizes dNTPs in the fluid to synthesize a complementary strand, converting the single-stranded DNA into a double-stranded DNA structure.

7. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The second connector and the lyophilized reagent of the ligase are pre-fixed on the inner wall of the second ligation reaction chamber; The lyophilized reagent is dissolved and solidified in a liquid buffer solution. The ligase catalyzes the covalent binding of the second adapter to the other end of the unattached adapter of the double-stranded DNA, thereby attaching the two ends of the double-stranded DNA to the first adapter and the second adapter, respectively, to form a library construction intermediate with paired adapters.

8. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The fourth temperature control element is located at an external position below the amplification chamber, and the top surface of the fourth temperature control element is attached to the bottom surface of the chip substrate; The fourth temperature control element executes the temperature cycling program required for the polymerase chain reaction. The temperature cycling program controls the fluid inside the amplification chamber to undergo periodic temperature alternation of denaturation, annealing and extension. The library preparation intermediate with double-ended connectors is targeted and enriched under periodic temperature alternation to generate the final library preparation product.

9. The microfluidic chip system for single-stranded DNA library construction according to claim 1, characterized in that, The microchannels form a unidirectional fluid conduit inside the chip substrate. The unidirectional fluid conduit starts at the sample input port, sequentially connects to each of the chambers, and terminates at the outlet. During a biochemical reaction in a single chamber, the microvalves at both the front and rear ends of the chamber remain closed, creating a closed physical space within the chamber. Once the biochemical reaction is complete, the microvalves at the rear end of the chamber open, allowing fluid to enter the next chamber.

10. The microfluidic chip system for single-stranded DNA library construction according to claim 2, characterized in that, The cover plate layer is provided with through holes that penetrate the thickness direction of the cover plate layer, and the through holes on the cover plate layer correspond to the sample input port and the output port; The sample input port is connected to the start end of the internal microchannel, and the outlet is a physical through hole that penetrates the surface of the chip substrate and is connected to the end end of the internal microchannel, used to allow external fluid driving force to flow out of the chip substrate.

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