Digital micro-fluidic chip and integrated sequencing system and method

Abstract

The invention provides a digital micro-fluidic chip and an integrated sequencing system and method.The digital micro-fluidic chip comprises a chip body and at least one nucleic acid extraction and library construction unit, and the nucleic acid extraction and library construction unit is arranged in the chip body and comprises a nucleic acid extraction module and a library construction module which are communicated; the nucleic acid extraction process and the library construction process can be continuously completed on the same digital micro-fluidic chip, manual or external intervention is not needed, and the constructed library can be transferred to a sequencer for direct sequencing. The whole process of nucleic acid extraction, library construction and sequencing can be automated, the reagent dosage can be reduced, the library construction and sequencing cost can be reduced, and the library construction and sequencing time can be shortened. The integrated sequencing system disclosed by the invention can realize smaller volume and electricity consumption, needs less manual operation, has low requirements on use environments (such as temperature, humidity, ventilation and the like), can be used in environments outside a laboratory, and is suitable for various application scenes.

Description

Technical Field

[0001] This invention belongs to the field of microfluidics and gene sequencing, and relates to a digital microfluidic chip, an integrated sequencing system and method. Background Technology

[0002] There are numerous instruments and devices used for biomedical detection and analysis. When using many of these instruments and devices, a significant proportion of the samples or reagents added do not participate in the reaction or measurement and are thus wasted. In digital microfluidics (DMF) systems, the dead volume (the volume of liquid added to the system but not involved in the reaction or detection) can be significantly reduced, sometimes even to zero. This means that the amount of sample or reagent required for the experiment is the amount used for measurement. This not only greatly reduces the cost of sample and reagent usage, as the smaller reaction volume shortens the mixing and reaction time of reagents and samples, but also significantly reduces the time required for detection and analysis. Moreover, many current biomedical analysis systems require numerous manual steps, while DMF-based systems (including DMF chips and instruments) offer a high degree of integration and automation, greatly reducing potential human error and thus improving the repeatability and reliability of experimental results.

[0003] In droplet-based digital microfluidic systems, liquids are manipulated in a discrete format (droplets) within a two-dimensional space. Each droplet can be controlled independently, hence the name "digital microfluidics." In digital microfluidic devices, the droplet's path can be determined experimentally and modified in real time. Compared to conventional pressure-driven (via vacuum pumps) or centrifugal-driven (via rotating centrifuges) pipe microfluidics, digital microfluidic devices require only low-amplitude voltages to control the droplets. The driving force in digital microfluidics is based on electric field effects, such as electrowetting or dielectrophoresis. This electric field effect is typically not highly correlated with the specific composition of the sample being tested, making the design and control of digital microfluidic systems independent of specific detection methods, thus offering greater versatility.

[0004] In recent years, digital microfluidics technology has attracted widespread attention due to its ability to control individual droplets and its advantages in miniaturization, integration, and automation. Digital microfluidics technology reduces reagent usage, simplifies experimental procedures, and shortens reaction time, offering significant advantages and holding great application value in fields such as medical diagnostics, gene sequencing, gene synthesis, and drug development.

[0005] Gene sequencing refers to the measurement and analysis of the base sequence of a target DNA / RNA fragment, specifically the arrangement of adenine (A), guanine (G), cytosine (C), and thymine (T) / uricil (U). Accurate and rapid interpretation of gene sequences is of great significance in basic biological research and many applied fields, such as medical diagnostics, forensic testing, bio-breeding, and microbial research.

[0006] Gene sequencing technology is an important tool in modern medicine. By analyzing nucleic acid sequences, the genetic factors of diseases can be revealed, providing a valid basis for clinical diagnosis and treatment. It has significant implications for disease diagnosis, prevention, and treatment, for example:

[0007] 1) Revealing the genetic factors of diseases: By determining nucleic acid sequences, information on genetic variations and mutations can be revealed, thereby revealing the genetic factors of diseases;

[0008] 2) Guiding Treatment Plans: Based on gene sequencing results, more targeted treatment plans can be selected, improving treatment outcomes. Many treatments, such as chemotherapy, radiotherapy, targeted therapy, and immunotherapy, are based on specific gene sequences. Different gene sequences require different treatment plans. Molecular markers provided by gene sequencing technology can serve as a basis for clinicians to select correct and personalized treatment plans.

[0009] 3) Prevention of chronic diseases: Personalized preventative measures can be developed based on genetic information. For example, early screening is crucial for cancer prevention, but its effectiveness can only be guaranteed by obtaining appropriate screening guidance through DNA sequencing. Currently, several genetic variations have been found to be significantly associated with cancer risk. Based on this information, more targeted cancer screening programs can be designed. Furthermore, for certain routine diseases caused by gene mutations, such as breast cancer and ovarian cancer, relevant preventative measures can be taken to reduce the risk of developing these diseases. Personalized disease prevention measures can be identified through the sequencing of an individual's nucleic acid sequence.

[0010] There are roughly three categories of sequencing technologies: first-generation sequencing, second-generation sequencing, and third-generation sequencing.

[0011] Gene sequencing technology was reported shortly after the discovery of the DNA double helix structure (Watson and Crick, 1953), but the complex procedures at the time prevented its widespread adoption. In 1977, Sanger et al. invented the landmark end-termination sequencing method. Because of its simplicity and speed, and through continuous improvements, the Sanger sequencing method became one of the mainstream gene sequencing methods at the time.

[0012] Sanger sequencing (also known as first-generation sequencing) has high accuracy (99.999%), but its slow speed, high cost, and low throughput have prevented its widespread application. Sanger sequencing is currently used for sequence analysis of plasmids, PCR products, and single-gene mutations.

[0013] Next-generation sequencing (NGS), also known as high-throughput sequencing (HTS), is a low-cost, high-accuracy (greater than 99%) technology that can simultaneously perform rapid sequencing analysis on hundreds of thousands to millions of DNA molecules from multiple samples. It has quickly gained widespread acceptance and is currently the mainstream sequencing technology. The rapid development of NGS has accelerated research in fields such as genetic diseases and cancer, and it is gradually entering clinical practice as a more advanced gene detection method.

[0014] The main representatives of third-generation sequencing are single-molecule fluorescence sequencing and nanopore sequencing. A representative technology for single-molecule fluorescence sequencing is SMRT (Single-Molecule Real-Time Sequencing) from Pacific Biosciences, which uses an SMRT chip as a carrier for sequencing while synthesis occurs. The SMRT chip has neatly arranged nanoscale micropores, with polymerase immobilized on the substrate of the sequencing wells. During sequencing, the polymerase combines with the DNA template and sequencing primers, and a four-color fluorescent dNTP substrate is added. During synthesis, free dNTPs are captured by the polymerase immobilized on the substrate, and the excited dNTPs emit fluorescence from the bottom of the substrate. The wavelength of this fluorescence peak indicates the type of base that has entered the DNA. When the fluorescently labeled dNTP forms a chemical bond with the DNA strand, its fluorescent group is cleaved by the DNA polymerase, and the fluorescence disappears.

[0015] Nanopore sequencing, with Oxford Nanopore in the UK being a representative company, integrates nanopores into its sequencing chips. Under the influence of an electric field (electrophoresis), negatively charged DNA molecules pass through the nanopores one by one. Because the nanopores are extremely small, only single bases can pass through. Since the individual bases ATCG have different electrical charges, the difference in electrical signals can be measured to detect the type of base that passed through, thus achieving sequencing.

[0016] NGS has been commercially developed for nearly 20 years. For example, in 2005, 454 Biosciences launched the first high-throughput genome sequencing system based on pyrosequencing, the Genome Sequencer 20 System, marking a milestone in the history of gene sequencing technology. In 2006, Solexa also launched its own NGS system, the Genome Analyzer, which is based on core technologies such as DNA clusters, bridge PCR, and reversible terminators, offering advantages such as high throughput and low error rates. Currently, NGS is the mainstream sequencing method, but due to the high cost of the instruments and the complexity of its use, it is mainly used in central laboratories.

[0017] Although the concept of nanopore sequencing was proposed in the 1980s, its product and commercial development history is much shorter than that of NGS. However, it has many advantages, such as high speed and long read length, which can overcome the shortcomings of NGS caused by short read length in areas such as pathogen detection, oncogenic fusion gene analysis, copy number variation (CNV) analysis, and identification of genomes containing a large number of repetitive sequences.

[0018] Nanopore sequencers can be made into small, portable products and applied to cutting-edge biological research and clinical medical fields such as clinical emergency and intensive care units, real-time pathogen detection, gut microbiota metagenomics, immunophenotyping, rapid tumor detection based on structural variations, early cancer screening based on DNA methylation detection, epigenetics, RNA and transcriptomics, etc. In the field of sequencing, they are a good extension and complement to NGS and are a technology of great significance.

[0019] Creating portable point-of-care testing (POCT) products based on sequencing is one of the best ways to achieve decentralized (laboratory-based) sequencing, which can be applied to many scenarios where it is difficult to use large sequencing equipment, such as customs and disease control.

[0020] Second- and third-generation sequencing typically involves the following steps: nucleic acid extraction, library construction, sequencing, and data analysis.

[0021] Nucleic acid extraction is the process of isolating and purifying nucleic acid molecules from biological samples. Nucleic acid extraction typically includes the following steps:

[0022] 1) Disruption and lysis of cells or viruses: Disruption and lysis of cells, viruses, or tissue samples to release nucleic acids. Common methods include mechanical disruption, chemical lysis, and enzymatic lysis.

[0023] 2) Protein removal: The lysed sample is treated with proteases to remove proteins and other impurities. Common methods include proteinase K treatment, phenol, chloroform method, or silica membrane method.

[0024] 3) Alcohol precipitation: Nucleic acids are precipitated by adding an appropriate amount of alcohol, such as ethanol or isopropanol. Salts, such as sodium chloride or sodium acetate, are usually added to adjust the ion concentration of the solution.

[0025] 4) Washing: The nucleic acid precipitate is washed to remove impurities. Common washing methods include ethanol washing, phenol / chloroform washing, or silica membrane washing.

[0026] 5) Dissolution: Dissolve the washed nucleic acid precipitate in an appropriate buffer to obtain high-purity nucleic acid. Commonly used dissolution buffers include Tris-HCl (tris(Hydroxymethyl)aminomethane hydrochloride) buffer or enzyme-free water.

[0027] Magnetic bead-based nucleic acid extraction is a method that uses magnetic beads as carriers to separate nucleic acids from complex samples through specific chemical treatments. The magnetic beads used are made of superparamagnetic nanoparticles with modified and enhanced surfaces that enable them to specifically recognize and efficiently bind to nucleic acid molecules. The steps involved in magnetic bead-based nucleic acid extraction typically include:

[0028] 1) Lysing samples such as cells or viruses: The sample is lysed using a lysis buffer to release nucleic acid molecules from the cells or viruses;

[0029] 2) Nucleic acid adsorption onto magnetic beads: Nucleic acid molecules released from lysed cell or tissue samples are specifically adsorbed onto the surface of magnetic particles;

[0030] 3) Washing and elution: Non-specifically adsorbed impurities, such as proteins and polysaccharides, are washed away by stirring and mixing the liquid and using eluent.

[0031] 4) Purification: The obtained nucleic acid undergoes further purification to ensure its quality and purity.

[0032] The advantages of the magnetic bead method lie in its simplification of the traditional nucleic acid extraction process, reducing manual steps and improving the efficiency and quality of nucleic acid extraction. Furthermore, the magnetic bead method is suitable for nucleic acid extraction needs in various fields, including clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbiology testing, food safety testing, and molecular biology research. Library preparation is a crucial step in the sequencing process. A DNA library (also called a gene library, here simply referred to as a library), strictly speaking, should be called a cDNA library, which refers to the collection of all expressible gene fragments in an organism's genome, obtained by reverse transcription of mRNA to obtain the corresponding cDNA.

[0033] In sequencing, a library typically refers to a collection of DNA fragments of similar length corresponding to the same sample, with adapters at both the 3' and 5' ends. Multiple different libraries (each with unique adapters) are usually combined and sequenced together. Library preparation involves processing the DNA to be sequenced into the format required by the sequencer. This formatting process may include DNA fragmentation, end repair (ER), A-tailing (AT), adapter addition, concentration amplification (usually using PCR), and purification (e.g., magnetic bead washing). Library preparation is a crucial step in gene sequencing, directly impacting sequencing quality.

[0034] Next-generation sequencing DNA library preparation typically involves the following steps:

[0035] 1) DNA fragmentation: DNA is fragmented by sonication, chemical treatment, or fragmentation enzyme treatment (ctDNA and other DNA fragments are relatively short, so they usually do not need to be fragmented). The purpose is to make the DNA fragment length suitable for the sequencer's reading requirements.

[0036] 2) End Repair (ER): DNA polymerase, Klenow enzyme, etc. are used to extend and fill in the 5' protrusions and cut the 3' protrusions. T4PNK enzyme, etc. are used to phosphorylate the 5' protrusions.

[0037] 3) A-tail addition (AT, A-Tailing): Using enzymes such as Klenow, an A-tail is added to the 3' end of the DNA fragment to prepare for subsequent adapter ligation;

[0038] 4) Adapter ligation: DNA ligase is used for TA cloning and ligation. The DNA fragment with the ligated adapter can be bound to a flow cell sequencing chip for NGS sequencing.

[0039] 5) Library amplification: PCR amplification, the purpose of which is to obtain enough DNA for sequencing.

[0040] Traditional next-generation sequencing, taking Illumina sequencing as an example, generally requires random fragmentation, addition of A bases, addition of sequencing primers, addition of indexes or barcodes, addition of adapters, and amplification. Because nanopore sequencing allows DNA molecules to pass through nanopores and identifies bases by reading electrical signals, library preparation does not require this cumbersome process. This not only increases the speed of library preparation but also reduces introduced errors. Nanopore sequencing library preparation mainly involves adding A bases to both sides of the DNA sample to turn blunt ends into sticky ends, followed by the addition of Y-linkers and motor proteins.

[0041] Nanopore sequencing library preparation typically involves the following steps:

[0042] 1) DNA fragmentation: DNA is divided into fragments suitable for specific nanopore sequencers using methods such as sonication, heat treatment, or enzyme digestion. Similar to NGS library construction, fragmentation usually requires end repair and purification.

[0043] 2) DNA modification and ligation: In order to ensure the accuracy of nanopore sequencing, DNA needs to be modified, such as by adding aptamers to the DNA ends, so that the DNA can be ligated to the specific nanopore sequencing device.

[0044] 3) Purification and oligomerization: After DNA ligation, purification and oligomerization are required to remove impurities and reduce low-quality fragments;

[0045] 4) Free aptamer removal: In nanopore sequencing, free aptamers can affect the accuracy of sequencing, so they need to be removed.

[0046] In NGS sequencing-by-synthesis protocols, extensive PCR operations are required, including adapter ligation and clustering. Due to the length limitations of PCR, Sanger and Illumina sequencing cannot perform long reads (over 1000 bp). Nanopore sequencing, on the other hand, can completely eliminate the need for PCR, enabling ultra-long read sequencing. Furthermore, PCR often introduces amplification bias, introducing errors into the sequencing data.

[0047] Of course, depending on the research objectives, some nanopore sequencing library preparation processes may require PCR. For example, if the amount of raw DNA is too small, amplification is necessary; or if the transcript itself is not particularly long, reverse transcription into cDNA is required, which can be done using PCR. Alternatively, if 16S sequencing is being performed and a large amount of data is not needed, pooled library preparation is required, which also necessitates the use of PCR. Currently, nanopore sequencing also supports pooled library preparation, allowing for sequencing of up to 96 samples. Therefore, the appropriate library preparation method should be selected based on the specific research objectives.

[0048] DNA library construction usually includes other steps, such as 1) library sorting and purification: effectively removing dNTPs, primer dimers, salt ions and other impurities; 2) library quantification: measuring the concentration of target DNA in the constructed library.

[0049] RNA library construction typically involves the following steps:

[0050] 1) mRNA extraction;

[0051] 2) RNA fragmentation: cutting RNA into lengths suitable for specific sequencers;

[0052] 3) First-strand cDNA synthesis: Reverse transcription of fragmented RNA is performed using reverse transcriptase and primers;

[0053] 4) Second-strand cDNA synthesis: RNase H endonuclease is used to create a nick, and RNA is used as a primer to extend the cDNA second strand from the nick.

[0054] 5) cDNA library construction – The library construction steps after double-stranded cDNA synthesis are similar to the DNA library construction steps.

[0055] As can be seen from the simple description above, the library construction steps for both NGS and nanopore sequencing are quite complicated.

[0056] Therefore, how to automate the entire process of nucleic acid extraction, library construction and sequencing, reduce reagent consumption, reduce library construction costs and shorten library construction time has become an important technical problem that urgently needs to be solved by those skilled in the art.

[0057] It should be noted that the above description of the technical background is only for the purpose of providing a clear and complete explanation of the technical solutions of the present invention and facilitating understanding by those skilled in the art. It should not be assumed that the above technical solutions are known to those skilled in the art simply because they have been described in the background section of this invention. Summary of the Invention

[0058] In view of the shortcomings of the prior art described above, the purpose of this invention is to provide a digital microfluidic chip, an integrated sequencing system and method to solve the problems of high cost and long time required by existing sequencing methods.

[0059] To achieve the above and other related objectives, the present invention provides a digital microfluidic chip, comprising:

[0060] The chip body includes a base plate and an upper cover plate. The upper cover plate is located above the base plate and forms a gap between the upper cover plate and the base plate to provide space for the movement of liquid or droplets in the digital microfluidic chip.

[0061] At least one nucleic acid extraction and library construction unit is disposed in the chip body. The nucleic acid extraction and library construction unit includes a nucleic acid extraction module and a library construction module connected via the gap. The nucleic acid extraction module is used to extract nucleic acids from the sample, and the library construction module is used to construct a library from the nucleic acids extracted by the nucleic acid extraction module.

[0062] Optionally, the base plate includes a base plate main body layer and a first dielectric layer located on the base plate main body layer, wherein at least a portion of the surface of the first dielectric layer is hydrophobic; the upper cover plate includes a cover plate main body layer and a second dielectric layer located below the cover plate main body layer, wherein at least a portion of the surface of the second dielectric layer is hydrophobic.

[0063] Optionally, the nucleic acid extraction module includes a lysis tank for sample lysis and nucleic acid capture, at least one nucleic acid washing tank for nucleic acid washing, and a nucleic acid elution tank for nucleic acid elution. The lysis tank, the nucleic acid washing tank, and the nucleic acid elution tank are disposed in different areas of the upper cover plate and are all in communication with the gap. The top of the lysis tank, the nucleic acid washing tank, and the nucleic acid elution tank are respectively provided with corresponding liquid filling holes.

[0064] Optionally, the library construction module includes a droplet control electrode assembly, a magnetic bead cleaning tank, and a library collection and storage tank. The droplet control electrode assembly is used to control droplets or liquids in the digital microfluidic chip. The droplet control electrode assembly includes multiple droplet control electrodes disposed between the base plate main body layer and the first dielectric layer and arranged at preset path intervals, and a ground electrode layer disposed between the cover plate main body layer and the second dielectric layer. The preset path starts from the nucleic acid elution tank and includes multiple branch paths. The end region of the branch path is provided with a liquid filling hole that penetrates the upper cover plate and communicates with the gap. The magnetic bead cleaning tank and the library collection and storage tank are disposed in different regions of the upper cover plate and are both in communication with the gap. The top of the magnetic bead cleaning tank and the library collection and storage tank are respectively provided with corresponding liquid filling holes.

[0065] Optionally, the library construction module further includes a quantitative detection reagent loading and storage tank, which is located at the end of one of the branch paths and passes through the upper cover plate to communicate with the gap. The top of the quantitative detection reagent loading and storage tank is provided with a corresponding liquid filling hole.

[0066] Optionally, the number of nucleic acid extraction and library construction units can be single or multiple. When the number of nucleic acid extraction and library construction units is multiple, the multiple nucleic acid extraction and library construction units are arranged side by side and spaced apart in the chip body.

[0067] Optionally, the gaps between two adjacent nucleic acid extraction and library construction units are connected, or a barrier is provided in the gaps between two adjacent nucleic acid extraction and library construction units.

[0068] Optionally, the library constructed by the library construction module is used for nanopore sequencing or next-generation sequencing.

[0069] Optionally, at least a portion of the base plate is optically transparent.

[0070] Optionally, the light transmittance of the optically transparent portion of the base plate is greater than 50%, or greater than 70%, or greater than 80%, or greater than 90% in the visible light range.

[0071] Optionally, the digital microfluidic chip is pre-filled with a filler oil with a specified melting point.

[0072] Optionally, the melting point of the filler oil is greater than 0°C, or greater than 10°C, or greater than 25°C, or greater than 35°C.

[0073] Optionally, the digital microfluidic chip is pre-packaged with reagents, including at least one of the reagents required for nucleic acid extraction and the reagents required for library construction.

[0074] Optionally, the pre-sealed reagent has an independent packaging film layer that can be broken during use to release the pre-sealed reagent into the digital microfluidic chip.

[0075] Optionally, the packaging film may be damaged by heating or mechanical puncture.

[0076] The present invention also provides an integrated sequencing system, comprising:

[0077] A nucleic acid extraction and library construction module, wherein the nucleic acid extraction and library construction module includes a digital microfluidic chip as described in any of the above, for performing nucleic acid extraction and library construction;

[0078] The sequencing module is used to sequence the library constructed by the nucleic acid extraction and library construction module;

[0079] The library transfer module is used to transfer the library constructed by the nucleic acid extraction and library construction module to the sequencing module for sequencing.

[0080] Optionally, the nucleic acid extraction and library construction module includes a droplet control module, which provides electrical signals to the digital microfluidic chip to operate the liquid or droplets in the digital microfluidic chip.

[0081] Optionally, the nucleic acid extraction and library construction module further includes at least one magnet control module, which controls the movement of one or more magnets to drive the magnetic beads in the digital microfluidic chip to move, and each magnet has at least two degrees of freedom of motion.

[0082] Optionally, the magnet includes a focusing magnet.

[0083] Optionally, the nucleic acid extraction and library construction module further includes at least one temperature control module, which is used to heat or cool a designated area of ​​the digital microfluidic chip to a designated temperature.

[0084] Optionally, the nucleic acid extraction and library construction module further includes a library quantification module, which includes an excitation light source and an optical detection module. The excitation light source is used to project excitation light onto the nucleic acid concentration measurement area of ​​the digital microfluidic chip, and the optical detection module is used to perform optical measurement on at least one site of the digital microfluidic chip.

[0085] Optionally, the nucleic acid extraction and library construction module further includes a library transfer module and a sequencing module, wherein the library transfer module is used to transfer the library constructed by the library construction module to the sequencing module for sequencing.

[0086] Optionally, the library transfer module includes a peristaltic pump for powering the transfer of liquids.

[0087] Optionally, the library transfer module includes a flow meter for quantitative monitoring of the transferred liquid.

[0088] Optionally, the flow meter includes an ultrasonic flow meter that is non-contact with the liquid.

[0089] Optionally, the library transfer module includes a defoamer disposed in the liquid transfer path.

[0090] Optionally, the sequencing module includes a nanopore sequencing module, a single-molecule fluorescence sequencing module, or an NGS sequencing module, wherein the nanopore sequencing module employs a biological nanopore or a solid nanopore.

[0091] This invention also provides an integrated sequencing method, comprising the following steps:

[0092] Nucleic acid extraction and library construction steps: Nucleic acid extraction and library construction are performed on any of the digital microfluidic chips described above;

[0093] Library transfer step: Transfer the library constructed in the digital microfluidic chip to the sequencing module;

[0094] Sequencing steps: Sequencing is performed on the sequencing module.

[0095] Optionally, in the nucleic acid extraction and library construction steps, the temperature control module is first set to a specified temperature to melt the filling oil in the digital microfluidic chip, and the sample to be tested is added to the specified liquid tank of the digital microfluidic chip. If the required reagents are not pre-sealed in the digital microfluidic chip, the required reagents are also added to the specified liquid tank of the digital microfluidic chip.

[0096] Optionally, the following steps are also included:

[0097] Analyze the sequencing data;

[0098] The analysis results are delivered to the user.

[0099] Optionally, in the nucleic acid extraction and library construction step, the nucleic acid extraction is performed using a magnetic bead method, including the following steps:

[0100] The sample to be tested, lysis buffer and magnetic beads are added to the lysis tank of the nucleic acid extraction module. The nucleic acid molecules in the sample to be tested are released by the lysis buffer and the nucleic acid molecules are adsorbed by the magnetic beads.

[0101] A magnet is used to gather magnetic beads containing nucleic acid molecules adsorbed in the lysis cell from below the digital microfluidic chip, and the magnetic beads are moved along the bottom plate of the digital microfluidic chip to the nucleic acid cleaning cell of the nucleic acid extraction module for cleaning to remove impurities adsorbed on the magnetic beads.

[0102] The cleaned magnetic beads are moved into the nucleic acid elution tank of the nucleic acid extraction module using a magnet. The nucleic acid elution tank contains an eluent to elute the nucleic acid molecules adsorbed on the magnetic beads into the solution.

[0103] Optionally, the library construction includes one or more of the following steps:

[0104] Fragmenting nucleic acid molecules using enzyme reactions;

[0105] Using enzyme reactions to repair the ends of nucleic acid molecular fragments;

[0106] Adding an A-tail to the ends of nucleic acid fragments using an enzyme reaction;

[0107] Adding linkers to nucleic acid molecular fragments using enzymatic reactions;

[0108] PCR amplification;

[0109] Nucleic acid molecule purification using magnetic beads;

[0110] Quantitative analysis of the document library.

[0111] Optionally, at least a portion of the reagents are added to the digital microfluidic chip via a pre-sealing method. The pre-sealing method includes placing the reagents to be pre-sealed at a designated location on the digital microfluidic chip, and solidifying the filling oil in the digital microfluidic chip by lowering the temperature to fix the position of the reagents to be pre-sealed in the digital microfluidic chip. When nucleic acid extraction and library construction are performed on the digital microfluidic chip, the digital microfluidic chip is first heated to a designated temperature to change the filling oil in the digital microfluidic chip from solid to liquid, so that the pre-sealed reagents can be moved in the digital microfluidic chip.

[0112] Optionally, multiple nucleic acid extraction and library construction units in the digital microfluidic chip can be operated synchronously.

[0113] As described above, in the digital microfluidic chip, integrated sequencing system, and method of the present invention, the digital microfluidic chip includes a chip body and at least one nucleic acid extraction and library construction unit. The nucleic acid extraction and library construction unit is disposed in the chip body and includes a connected nucleic acid extraction module and a library construction module, enabling the nucleic acid extraction and library construction processes to be completed continuously on the same digital microfluidic chip without manual or external intervention. The constructed library can be transferred to a sequencer for direct sequencing. The present invention can automate the entire process of nucleic acid extraction, library construction, and sequencing, reducing reagent consumption, which is beneficial to reducing library construction and sequencing costs and shortening library construction and sequencing time. The integrated sequencing system of the present invention can achieve a smaller size and lower power consumption, requires less manual operation, and has low requirements for the operating environment (such as temperature, humidity, ventilation, etc.), and can be used in environments other than laboratories. It is suitable for applications such as large, medium, and small hospitals, community health clinics, disease control centers, environmental monitoring, customs, and forensic identification. Attached Figure Description

[0114] Figure 1 The diagram shown is a cross-sectional view of a partial structure of the digital microfluidic chip of the present invention in one embodiment.

[0115] Figure 2 The diagram shown is a planar layout of the digital microfluidic chip of the present invention in one embodiment.

[0116] Figure 3 The quantities in the Young-Laplace equation are shown.

[0117] Figure 4 The diagram shown is a planar layout of the digital microfluidic chip of the present invention in another embodiment.

[0118] Figure 5 The diagram shows the modular structure of the integrated sequencing system of this invention.

[0119] Figure 6 The diagram shown is a structural schematic of a magnet control module in an embodiment of the integrated sequencing system of the present invention.

[0120] Figures 7-11 This illustrates the process of manipulating a magnetic bead in a DMF device using a magnet.

[0121] Figure 12 The diagram shown is a structural schematic of the temperature control module in one embodiment of the integrated sequencing system of the present invention.

[0122] Figure 13 The display shows the temperature control module being placed... Figure 2 The diagram below shows the digital microfluidic chip.

[0123] Figure 14 The display shows the temperature control module being placed... Figure 4 The diagram below shows the digital microfluidic chip.

[0124] Figure 15 The diagram shows a top-front-side view of the device structure of the integrated sequencing system of the present invention in one embodiment.

[0125] Figure 16 Shown is a rear view of the device structure of an embodiment of the integrated sequencing system of the present invention.

[0126] Figure 17 The flowchart shown is an embodiment of the integrated sequencing method of the present invention.

[0127] Component designation explanation

[0128] 100 Chip Body

[0129] 101 base plate

[0130] 1011 Base Plate Main Layer

[0131] 1012 First dielectric layer

[0132] 102 Top Cover Plate

[0133] 1021 Cover Plate Main Layer

[0134] 1022 Second dielectric layer

[0135] 103 gap

[0136] 200 Nucleic Acid Extraction and Library Construction Units

[0137] 201 Nucleic Acid Extraction Module

[0138] 2011 pyrolysis tank

[0139] 2012a First Nucleic Acid Washing Tank

[0140] 2012b Second Nucleic Acid Washing Tank

[0141] 2013 Nucleic Acid Elution Tank

[0142] 202 Library Construction Module

[0143] 2021 Droplet Control Electrode Assembly

[0144] 2021a Droplet Control Electrode

[0145] 2021b Grounding Electrode Layer

[0146] 2022a First Magnetic Bead Cleaning Tank

[0147] 2022b Second Magnetic Bead Cleaning Tank

[0148] 2023 Document Collection Storage Slot

[0149] 20201 First filling hole

[0150] 20202 Second filling hole

[0151] 20203 Third liquid filling hole

[0152] 20204 Fourth liquid filling hole

[0153] 20205 Fifth liquid filling hole

[0154] 20206 Sixth filling hole

[0155] 20207 Seventh filling hole

[0156] Liquid filling holes for 20208, 20209, and 20210

[0157] 300 Nucleic Acid Extraction and Library Construction Units

[0158] 301 Nucleic Acid Extraction Module

[0159] 3011 pyrolysis tank

[0160] 3012a First Nucleic Acid Washing Tank

[0161] 3012b Second Nucleic Acid Washing Tank

[0162] 3013 Nucleic Acid Elution Tank

[0163] 30101, 30102, 30103 Liquid filling holes

[0164] 302 Library Building Module

[0165] 3021a Droplet Control Electrode

[0166] 3022a First Magnetic Bead Cleaning Tank

[0167] 3022b Second Magnetic Bead Cleaning Tank

[0168] 3022c Third Magnetic Bead Cleaning Tank

[0169] 3022d Fourth Magnetic Bead Cleaning Tank

[0170] 30201 First filling hole

[0171] 30202 Second liquid filling hole

[0172] 30203 Third liquid filling hole

[0173] 30204 Fourth liquid filling hole

[0174] 30205 Fifth liquid filling hole

[0175] 30206 Sixth filling hole

[0176] 30207 Seventh liquid filling hole

[0177] 30208 Eighth liquid filling hole

[0178] 30209 Ninth liquid filling hole

[0179] 30210 Liquid Filling Hole

[0180] 400 Integrated Sequencing System

[0181] 401 Nucleic Acid Extraction and Library Construction Module

[0182] 402 Document Transfer Module

[0183] 403 Sequencing Module

[0184] 500 Magnet Control Module

[0185] 501 magnet

[0186] 601 First Storage Tank

[0187] 602 Second Storage Tank

[0188] 701 First Liquid

[0189] 702 Second Liquid

[0190] 800 magnetic beads

[0191] 900 Temperature Control Module

[0192] 901 First Temperature Control Unit

[0193] 902 Second Temperature Control Unit

[0194] 903 Third Temperature Control Unit

[0195] 904 Fourth Temperature Control Unit

[0196] 905 Fifth Temperature Control Unit

[0197] 906 Sixth Temperature Control Unit

[0198] 907 Seventh Temperature Control Unit

[0199] 1001 Spring Probe

[0200] 1002 Touchscreen

[0201] 1003 Sliding Tray

[0202] 1004 DMF device

[0203] 1005 Ventilation Opening

[0204] 1006 USB ports

[0205] 1007 Ethernet port

[0206] 1008 AC power port Detailed Implementation

[0207] The following specific examples illustrate the implementation of the present invention. It should be noted that these examples are intended to aid in the explanation and not to limit the intent and spirit of the invention.

[0208] The accompanying drawings and detailed description, used as supplementary materials, further illustrate and explain the principles of the invention, and enable those skilled in the art to manufacture and use the corresponding chips, systems, and methods.

[0209] Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0210] It should be emphasized that the term "including / comprises" as used herein refers to the presence of a feature, whole, step, or component, but does not exclude the presence or addition of one or more other features, wholes, steps, or components.

[0211] Features described and / or illustrated for one embodiment may be used in the same or similar manner in one or more other embodiments, combined with features in other embodiments, or substituted for features in other embodiments.

[0212] In the detailed description of embodiments of the present invention, for ease of explanation, the schematic diagrams illustrating the device structure may be partially enlarged without adhering to the general scale, and the schematic diagrams are merely examples and should not limit the scope of protection of the present invention. Furthermore, in actual manufacturing, the three-dimensional spatial dimensions of length, width, and depth should be included.

[0213] For ease of description, spatial relation terms such as "below," "below," "lower than," "below," "above," and "upper" may be used herein to describe the relationship between one element or feature shown in the accompanying drawings and other elements or features. It will be understood that these spatial relation terms are intended to include directions other than those depicted in the drawings for devices in use or operation. Furthermore, when a layer is referred to as being "between" two layers, it may be the only layer between the two layers, or there may be one or more layers in between.

[0214] In the context of this invention, the described structure of the first feature "above" the second feature may include embodiments in which the first and second features are in direct contact, or embodiments in which additional features are formed between the first and second features, such that the first and second features may not be in direct contact.

[0215] In this specification, terms such as "an embodiment," "an example," or "an aspect" refer to specific features, structures, and characteristics that may be included in one embodiment but not necessarily in all embodiments. Furthermore, the features, structures, and characteristics disclosed in this invention can be applied in one or more embodiments in any suitable combination.

[0216] Unless otherwise stated, in the context of this invention, the term "less than" generally means "equal to or less than", and "greater than" generally means "equal to or greater than".

[0217] It should be noted that the illustrations provided in this embodiment are only schematic representations of the basic concept of the present invention. Therefore, the illustrations only show the components related to the present invention and are not drawn according to the actual number, shape and size of the components in the actual implementation. In the actual implementation, the form, quantity and proportion of each component can be arbitrarily changed, and the layout of the components may also be more complex.

[0218] Example 1

[0219] Please see Figure 1 The image shown is a cross-sectional view of a partial structure of the digital microfluidic chip of the present invention in one embodiment. The digital microfluidic chip includes a chip body 100 and at least one nucleic acid extraction and library construction unit disposed in the chip body 100.

[0220] Specifically, in the specification of this invention, the terms "digital microfluidic chip," "digital microfluidic device," and "DMF chip" are used interchangeably. Additionally, the terms "library construction" and "library building" are also used interchangeably. Library construction refers to the process of processing DNA or RNA samples into the format required for sequencing by a sequencer. This process typically includes (but is not limited to) some or all of the following steps: fragmentation (e.g., enzyme digestion), end repair, addition of A bases, addition of linkers, concentration amplification (usually using PCR), purification (usually using magnetic beads for washing), etc.

[0221] Specifically, the "amplification" mentioned in the above explanation of the term "library construction" refers to the process of increasing the quantity or concentration of the analyte. Non-restrictive examples include PCR and its variants (such as quantitative competitive PCR, immunoPCR, reverse transcription PCR, etc.), strand displacement amplification (SDA), nucleic acid sequence-based amplification (NASBA), loop-mediated isothermal amplification (LAMP), and helicase-dependent amplification (HAD). Among these, PCR, or polymerase chain reaction, is a molecular biology technique used to amplify specific DNA fragments. It is a special form of DNA replication outside of living organisms, and its most significant characteristic is its ability to dramatically increase minute amounts of DNA. PCR requires repeated temperature cycling of the reaction system, which contains specific DNA primers, dNTPs (deoxyribonucleoside triphosphates), and thermostable DNA polymerases (such as Taq polymerase). Each temperature cycle theoretically doubles the number of target DNA molecules, resulting in an exponential increase in the number of target sequences. This technique amplifies trace amounts of DNA or RNA in a sample to measurable and analyzable levels. PCR technology has been applied in many different fields, including viral load testing, quantification of foodborne pathogens, clinical diagnostics, drug resistance analysis, and forensic science. Using PCR, doctors and researchers can determine the source of viral infection by analyzing a single cell. PCR can now detect many infectious organisms, such as COVID-19, HIV, hepatitis B, hepatitis C, SARS virus, West Nile virus, and Mycobacterium tuberculosis.

[0222] Specifically, the chip body 100 includes a base plate 101 and an upper cover plate 102. The upper cover plate 102 is located above the base plate 101 and forms a gap 103 between it and the base plate 101 to provide space for the movement of liquid or droplets in the digital microfluidic chip.

[0223] Specifically, the upper cover plate 102 and the bottom plate 101 are spaced apart by a certain distance, and the gap 103 is defined by the space between the upper cover plate 102 and the bottom plate 101. The distance is sufficient to accommodate droplets placed in the gap 103.

[0224] Specifically, in the specification of this invention, the term "droplet" refers to a certain amount of liquid (a mixture of one or more) separated from other parts by air or other gases, other (usually referring to immiscible) liquids, and solid surfaces (e.g., the inner surface of a DMF chip). The volume of a "droplet" ranges widely, typically from a few picoliters to hundreds of microliters.

[0225] As an example, the gap 103 is sealed at the edge of the base plate 101 and the upper cover plate 102, and the sealing method used may be, for example, UV-curable adhesive, gasket, etc.

[0226] As an example, the base plate 101 may be made of an opaque material, or at least a portion of it may be optically transparent. For example, in some embodiments, the light transmittance of the optically transparent portion of the base plate 101 is greater than 50%, or greater than 70%, or greater than 80%, or greater than 90% in the visible light range.

[0227] As an example, the base plate 101 includes a base plate main layer 1011 and a first dielectric layer 1012 located on the base plate main layer 1011, at least a portion of the surface of the first dielectric layer 1012 being hydrophobic; the upper cover plate 102 includes a cover plate main layer 1021 and a second dielectric layer 1022 located below the cover plate main layer 1021, at least a portion of the surface of the second dielectric layer 1022 being hydrophobic.

[0228] Specifically, in this specification, the terms "layer" and "film" are used interchangeably to refer to the structure of a substrate, which is typically, but not necessarily, planar or substantially planar, and is usually placed on another structure by deposition, formation, coating, or other means. The terms "dielectric layer," "dielectric layer," and "dielectric film" are used interchangeably to refer to a thin film made of a dielectric material on a chip, wherein the dielectric material is an insulating material that can be polarized. Materials used to make dielectric layers include, but are not limited to, Teflon, Cytop, Parylene C, silicon nitride, and silicon oxide. Fabrication methods include spin coating, dip coating, spray coating, sputtering, and PECVD (Plasma Enhanced Chemical Vapor Deposition).

[0229] As an example, the first dielectric layer 1012 and the second dielectric layer 1022 can be a single layer or have two or more layers, one of which can be a hydrophobic material, such as Teflon, Cytop, and FluoroPel.

[0230] It is understood that when a given component, such as a layer, region, or substrate, is referred to as being "placed," "in," or "located" on or formed on another component, the given component may be directly located on the other component, or alternatively, intermediate components (such as one or more buffer layers, interlayers, and electrodes) may be present. It is also understood that the terms "placed on" and "formed on" are used interchangeably to describe how a given component is positioned or disposed relative to another component. Therefore, the terms "placed on" and "formed on" are not intended to impose any limitations on specific methods of material transport, deposition, and fabrication.

[0231] As an example, the base plate main body layer 1011 and the cover plate main body layer 1021 can be any non-conductive material or a conductive material coated with a non-conductive layer, as long as it has sufficient mechanical strength to maintain its shape within the required system operating and storage conditions. In terms of light transmittance, it can be transparent, translucent, or opaque.

[0232] In some embodiments, the base plate main body layer 1011 and the cover plate main body layer 1021 are made of transparent substrates, which can be made of various transparent materials, such as glass, quartz, plastic, transparent ceramics, transparent printed circuit boards, etc.

[0233] In the examples of transparent substrates above, "Printed Circuit Board (PCB)" also refers to a circuit board without soldered components, and mainly consists of the following parts:

[0234] (1) Circuit and Pattern: The material used for circuits is usually copper. Circuits provide a conductive path between electronic components. In addition, large copper surfaces are usually designed as ground and power layers. Circuits and patterns are made at the same time.

[0235] (2) Dielectric layer: used to maintain the insulation between the circuit and the layers, also called substrate.

[0236] (3) Through hole (or Via): Through holes allow two or more layers of circuits to communicate with each other. Larger through holes are used for component insertion. In addition, non-through holes are usually used for surface mount positioning.

[0237] (4) Solder Mask: Not all copper surfaces need to be soldered onto components. Therefore, non-solderable areas are coated with a layer of material (usually epoxy resin) to prevent short circuits between non-solderable traces. Depending on the process, it is divided into green solder mask, red solder mask, and blue solder mask.

[0238] (5) Silk screen: This is a non-essential component. Its main function is to mark the names and locations of each component on the circuit board for easy maintenance and identification after assembly.

[0239] For example, please refer to Figure 2 The image shown is a planar layout diagram of a digital microfluidic chip in one embodiment, the digital microfluidic chip including at least one nucleic acid extraction and library construction unit 200, for example... Figure 2 The example presented is a digital microfluidic chip comprising two of the aforementioned nucleic acid extraction and library construction units 200. In other embodiments, the number of nucleic acid extraction and library construction units in a digital microfluidic chip may also be one, three, or other numbers as needed.

[0240] As an example, when the digital microfluidic chip includes multiple nucleic acid extraction and library construction units 200, the multiple nucleic acid extraction and library construction units 200 are arranged side by side and spaced apart in the chip body 100.

[0241] In some embodiments, the gap 103 between two adjacent nucleic acid extraction and library construction units 200 is connected. In other embodiments, when the spacing between units is small, a barrier (not shown) is provided in the gap 103 between two adjacent nucleic acid extraction and library construction units 200 to prevent liquid in one unit from spreading into another unit.

[0242] Specifically, the nucleic acid extraction and library construction unit 200 is disposed in the chip body 100 and includes a nucleic acid extraction module 201 and a library construction module 202 connected via the gap 103. The nucleic acid extraction module 201 is used to extract nucleic acids from the sample, and the library construction module 202 is used to construct a library from the nucleic acids extracted by the nucleic acid extraction module 201.

[0243] As an example, the nucleic acid extraction module 201 described in this invention utilizes the magnetic bead method for nucleic acid extraction. The advantage of the magnetic bead method is that it simplifies the traditional nucleic acid extraction process, reduces manual steps, and improves the efficiency and quality of nucleic acid extraction. Furthermore, the magnetic bead method is suitable for nucleic acid extraction needs in various fields, including clinical disease diagnosis, blood transfusion safety, forensic identification, environmental microbiology detection, food safety testing, and molecular biology research.

[0244] Specifically, in this invention's specification, the term "bead" can refer to any bead or particle that reacts with a solution. Beads can be of any shape, such as spherical, egg-shaped, cubic, disc-shaped, or irregular. Beads can be inside droplets, on the inner surface of a DMF chip, in the filling solution of a DMF chip, in a liquid tank, etc. Beads can be made of a wide variety of materials, such as resins, polymers, glass, nanomaterials, etc., and can have any size, such as microspheres and nanospheres. Beads can be magnetically responsive; in this case, at least one or part of their components are composed of a magnetically responsive material, while the remaining material may contain polymeric materials, coatings, or groups linked to detection reagents, etc. Examples of beads include quantum dots, polyethylene microspheres, silica microspheres, fluorescent microspheres or nanospheres, magnetic microspheres, magnetic nanospheres, flow cytometry microspheres, etc. The term "magnetic bead" refers to a bead containing a magnetically responsive material. Examples of magnetically responsive materials include ferromagnetic materials, paramagnetic materials, supermagnetic materials, and ferrimagnetic materials, etc. Examples of paramagnetic materials include metals such as nickel, iron, and cobalt, as well as metal oxides such as Fe3O4, Cr2O3, NiO, and Mn2O3. Magnetic-responsive materials can constitute essentially all, part, or a component of the magnetic bead. The remaining portion of the magnetic bead may include polymeric materials that allow target particles to adhere and coating portions. Magnetic beads can be used in a variety of assays where they are typically used to bind one or more target substances, such as analytes or contaminants, to a mixture. Assays typically require an effective magnetic bead cleaning process to reduce the amount of one or more target substances in the mixture that are not bound to the bead surface, thus avoiding interference with the assay results. Validation experiments based on digital microfluidics have shown that magnetic beads coated with anti-human serum albumin antibodies can be used to separate human serum albumin. Validation experiments using magnetic beads to extract DNA from whole blood samples have also been performed on a digital microfluidic platform. DNA extraction on a digital microfluidic platform, similar to conventional methods, typically requires a pretreatment step to achieve cell lysis.

[0245] As an example, the nucleic acid extraction module 201 includes a lysis tank 2011 for sample lysis and nucleic acid capture, at least one nucleic acid washing tank for nucleic acid washing, and a nucleic acid elution tank 2013 for nucleic acid elution. The lysis tank 2011, the nucleic acid washing tank, and the nucleic acid elution tank 2013 are disposed in different areas of the upper cover plate 102 and are all connected to the gap 103.

[0246] Specifically, in the specification of this invention, the term "reservoir" is used to refer to a portion of the DMF chip that can be used to store, hold, and supply liquid, and can be fully enclosed or partially enclosed. The reservoir can be associated with a fluid path that allows liquid to be introduced into the gaps between the DMF chips for droplet manipulation, or to enter the reservoir from between the DMF chips for liquid storage or temporary preservation.

[0247] It should be pointed out that, Figure 2 The illustration shows a nucleic acid extraction module 201 comprising two nucleic acid washing tanks (a first nucleic acid washing tank 2012a and a second nucleic acid washing tank 2012b), which removes as much debris as possible from the magnetic beads through two washing cycles. However, in other embodiments, the number of nucleic acid washing tanks in a nucleic acid extraction module 201 can be one, three, or other numbers as needed.

[0248] As an example, the top of the lysis tank 2011, the nucleic acid washing tank, and the nucleic acid elution tank 2013 are respectively provided with corresponding liquid filling holes. The liquid filling hole 20101 at the top of the lysis tank 2011 is used to load samples, lysis buffer, and magnetic bead solution, etc. The liquid filling hole 20102 at the top of the nucleic acid washing tank is used to load magnetic bead washing solution, and the liquid filling hole 20103 at the top of the nucleic acid elution tank 2013 is used to load elution buffer.

[0249] As an example, the library construction module 202 includes a droplet control electrode assembly 2021, a magnetic bead cleaning tank, and a library collection and storage tank 2023.

[0250] Specifically, the droplet control electrode assembly 2021 is used to control droplets or liquids in the digital microfluidic chip.

[0251] In this invention specification, the terms "droplet control," "droplet drive," and "droplet operation" are used interchangeably and can include droplet generation (from a liquid tank or continuous liquid), movement, merging and mixing, segmentation (symmetric or asymmetric), shaping (forming a specified shape), suspension and dispersion of particles (within a liquid or droplet), etc. The term "particle" is used to refer to entities on the micrometer or nanometer scale, which can be natural or artificial, such as cells, subcellular components, liposomes, viruses, nanospheres and microspheres, or even smaller entities such as biomolecules, proteins, DNA, and RNA. It can also refer to droplets that are immiscible with the suspension medium, or small bubbles in a liquid. The (linear) size of a "particle" can range from a few nanometers to hundreds of micrometers.

[0252] For details, please refer to the following: Figure 1 and Figure 2The droplet control electrode assembly 2021 includes a plurality of droplet control electrodes 2021a disposed between the base plate main body layer 1011 and the first dielectric layer 1012 and arranged at preset path intervals, and a ground electrode layer 2021b disposed between the cover plate main body layer 1021 and the second dielectric layer 1022. The preset path starts from the nucleic acid elution tank 2013 and includes multiple branch paths. The end region of the branch path is provided with a liquid filling hole that penetrates the upper cover plate 102 and communicates with the gap 103.

[0253] As an example, the plurality of said droplet control electrodes 2021a may be in different layers (typically electrically insulated from each other), as described in patent WO2008 / 147568 entitled “Electrowetting Based Digital Microfluidics” (inventor: Chuanyong Wu).

[0254] It should be noted that when arranging a covering dielectric layer, the spaces between adjacent electrodes in the same layer are typically filled with dielectric material. These spaces may also be left empty or filled with gases such as air, nitrogen, helium, and argon. All electrodes in the same layer and electrodes in different layers are preferably electrically insulated.

[0255] Specifically, the digital microfluidic chip is hydrophobic to droplets, and droplets on the electrodes can be manipulated (moved, split, merged, mixed, etc.) through electric field effects such as electrowetting.

[0256] Specifically, "microfluidics" refers to devices, systems, or methods capable of manipulating at least one liquid with a cross-sectional size of a few micrometers to a few millimeters. As a subfield of microfluidics, "digital microfluidics" refers to devices, systems, and methods that utilize some control mechanism, such as electrowetting, dielectrophoresis, or light, to manipulate one or more individual droplets. In this invention, electrowetting and dielectrophoresis, two common droplet control mechanisms, can be employed, but the possibility of using other control mechanisms is not limited, such as electrophoresis, electroosmosis, opto-electrowetting, or combinations thereof.

[0257] Specifically, in the explanation of the droplet control mechanism above, the term "dielectrophoresis" refers to the effect of a neutral particle under the influence of a non-uniform electric field. When a particle suspended in a liquid medium is subjected to a non-uniform electric field, it may move towards a region with a stronger electric field (positive dielectrophoresis) or towards a region with a weaker electric field (negative dielectrophoresis). Unlike electrophoresis, particles subjected to dielectrophoretic forces can be uncharged neutral particles, and the dielectrophoretic force is insensitive to the polarity of the electric field. Both direct current and alternating current can be used to generate the dielectrophoretic effect. All particles will exhibit some degree of dielectrophoretic effect under the influence of a non-uniform electric field. The strength of the dielectrophoretic force is related to the size and shape of the particle, the electrical properties of the particle and the medium, the gradient distribution of the electric field, and the frequency of the electric field. Due to its ease of operation, the frequency of the electric field is often used as an adjustment parameter to control different particles in liquids. The relevant scheme is specifically described in the document "Dielectrophoresis based apparatuses and methods for the manipulation of particles in liquids" with patent number WO 2014 / 036915A1.

[0258] Specifically, in the above explanation of the droplet control mechanism, the term "electrowetting" refers to the effect of the contact angle between a liquid and a solid surface changing with the applied electric field. It should be noted that when the applied voltage or electric field is alternating current (AC), both the "electrowetting" and "dielectrophoresis" effects exist simultaneously. As the frequency of the voltage or electric field increases, the relative weight of the "dielectrophoresis" effect usually increases accordingly. In this invention, the "electrowetting" and "dielectrophoresis" effects are not strictly distinguished; both refer to the mechanism of manipulating droplets using an electric field.

[0259] Specifically, in the above explanation of the electrowetting effect, the term "contact angle" refers to the angle formed when the liquid-vapor interface comes into contact with a solid surface. When the three phases—liquid, solid, and gas (which can be a mixture of ambient atmospheric and liquid equilibrium concentrations of vapor)—reach thermodynamic equilibrium, the shape of the liquid-vapor interface is determined by the Young-Laplace equation.

[0260] γ SG -γ SL -γ LG cosθ c =0 where γ SG Represents the solid-gas interface energy, γ SL γ represents the solid-liquid interfacial energy. LGθ represents the liquid-gas interfacial energy (i.e., surface tension), and θ represents the contact angle at equilibrium. Please refer to [link to relevant documentation]. Figure 3 This shows the quantities in the Young-Laplace equation. It should be noted that the above equation also applies if the gas phase is replaced by another immiscible liquid phase.

[0261] Specifically, within a pure liquid, each molecule is equally pulled by its neighboring liquid molecules in every direction, resulting in a net force of zero. However, molecules on the liquid surface do not have neighboring liquid molecules in all directions and do not possess a net force providing equilibrium. They are pulled inward by their neighbors, generating internal pressure. Consequently, the liquid surface area contracts to maintain its minimum surface free energy. This intermolecular force causing surface contraction is known as the liquid-gas interfacial energy γ. LG The surface tension, or t-coefficient of mass, determines the shape of a droplet. Other external forces, such as gravity, can also deform the droplet. Therefore, the contact angle is determined by both surface tension and external forces (usually gravity). The contact angle is also a characteristic parameter of a solid-liquid system under specific conditions.

[0262] Specifically, hydrophobic surfaces repel liquids, while hydrophilic surfaces attract liquids. In this invention, the "hydrophobic surface" has a contact angle greater than 90°, while the "hydrophilic surface" has a contact angle less than 90°.

[0263] In this invention, it is understood that when a liquid of any form (such as a droplet or a continuum, which may be in motion or stationary) is described as being “on,” “at,” or “above” an electrode, array, matrix, or surface, the liquid may be in direct contact with the electrode, array, matrix, or surface, or may be in contact with one or more layers or films inserted between the liquid and the electrode, array, matrix, or surface.

[0264] Specifically, in the specification of this invention, the term "Ground" (e.g., for "ground electrode" or "ground voltage") refers to a voltage on the corresponding electrode that is zero or sufficiently close to zero, such that the area on the DMF chip corresponding to the electrode to which the ground voltage is applied does not exhibit a noticeable electrowetting effect. All other voltage values, although typically less than 300 volts, should be high enough to allow a substantial observation of the electrowetting effect.

[0265] As an example, the droplet control electrode 2021a can have different shapes, such as rectangles, squares, trapezoids, pentagons, hexagons, and irregular shapes, and can be arranged in straight lines or other shapes. For example, in Figure 2 In the present invention, the droplet control electrode 2021a is square or rectangular. In actual implementation, the electrode can be other sizes and shapes.

[0266] As an example, the droplet control electrode 2021a can be made of any conductive material, such as metals, alloys and conductive polymers.

[0267] As an example, the droplet control electrode 2021a may be made of one material or a mixture of different materials.

[0268] As an example, the droplet control electrode 2021a and the ground electrode layer 2021b of the droplet control electrode assembly 2021 can be made of transparent conductive materials, such as indium tin oxide (ITO), aluminum-doped zinc oxide (AZO), transparent conductive polymers (polyacetylene, polyaniline, etc.) or transparent nanomaterials.

[0269] Specifically, in Figure 2 In the digital microfluidic chip shown, the library constructed by the library construction module 202 can be used for nanopore sequencing. In one nanopore sequencing library construction method, the library construction module 202 is provided with a first liquid inlet 20201, a second liquid inlet 20202, a third liquid inlet 20203, a fourth liquid inlet 20204, a fifth liquid inlet 20205, a sixth liquid inlet 20206, and a seventh liquid inlet 20207 at the ends of multiple branch paths of the preset path. The first liquid inlet 20202... Well 01 is used to load enzymes for nucleic acid molecule fragmentation; well 20202 is used to load buffer for nucleic acid molecule fragmentation; well 20203 is used to load fragmentation termination buffer; well 20204 is used to load magnetic bead solution; well 20205 is used to load nucleic acid molecule elution buffer (e.g., deionized water); well 20206 is used to load sequencing connectors; and well 20207 is used to load sequencing buffer.

[0270] Specifically, the magnetic bead cleaning tank and the library collection and storage tank 2023 are disposed in different areas of the upper cover plate 102 and are both connected to the gap 103. The magnetic bead cleaning tank is used for cleaning magnetic beads during the library construction process, and the library collection and storage tank 2023 is used for collecting and storing mixed nanopore sequencing libraries.

[0271] It should be pointed out that, Figure 2 The example presented shows a library construction module 202 comprising two magnetic bead cleaning tanks (a first magnetic bead cleaning tank 2022a and a second magnetic bead cleaning tank 2022b), achieving a better cleaning effect through two cleaning cycles. However, in other embodiments, the number of magnetic bead cleaning tanks included in a library construction module 202 can also be set to 1, 3, 4, or other numbers as needed.

[0272] As an example, the top of the magnetic bead cleaning tank and the library collection and storage tank 2023 are respectively provided with corresponding liquid filling holes. The liquid filling hole 20208 at the top of the magnetic bead cleaning tank is used to load magnetic bead cleaning solution (e.g., 80% ethanol), and the liquid filling hole 20209 at the top of the library collection and storage tank 2023 is used to load auxiliary reagents, such as conduction buffer, deionized water, etc.

[0273] In some embodiments, the library construction module 202 further includes a quantitative detection reagent loading and storage tank 2024, which is located at the end of one of the branch paths and passes through the upper cover plate 102 to communicate with the gap 103. The top of the quantitative detection reagent loading and storage tank 2024 is provided with a liquid addition hole 20210 for loading and storing quantitative detection reagents.

[0274] Specifically, in the specification of this invention, the term "reagent" refers to any substance added to a sample for reacting with the sample, diluting the sample, suspending the sample, emulsifying the sample, encapsulating the sample, interacting with the sample, etc.

[0275] As mentioned above, Figure 2 The library construction module 202 of the digital microfluidic chip shown constructs libraries for nanopore sequencing. In other embodiments, the layout of the digital microfluidic chip library construction module 202 can also be changed to make it suitable for constructing libraries required for next-generation sequencing.

[0276] Please see Figure 4 The diagram shown is a planar layout of the digital microfluidic chip in another embodiment, from which libraries can be constructed for next-generation sequencing.

[0277] Specifically, such as Figure 4 As shown, the digital microfluidic chip includes two nucleic acid extraction and library construction units 300. Each nucleic acid extraction and library construction unit 300 includes a nucleic acid extraction module 301 and a library construction module 302 connected via the gap 103.

[0278] Specifically, the nucleic acid extraction module 301 includes a lysis tank 3011 for sample lysis and nucleic acid capture, at least one nucleic acid washing tank for nucleic acid washing, and a nucleic acid elution tank 3013 for nucleic acid elution. The lysis tank 3011, the nucleic acid washing tank, and the nucleic acid elution tank 3013 are disposed in different areas of the upper cover plate 102 and are all connected to the gap 103.

[0279] It should be pointed out that, Figure 4The illustration shows a nucleic acid extraction module 301 comprising two nucleic acid washing tanks (a first nucleic acid washing tank 3012a and a second nucleic acid washing tank 3012b), which removes as much debris as possible from the magnetic beads through two washing cycles. However, in other embodiments, the number of nucleic acid washing tanks in a nucleic acid extraction module 301 can be set to one, three, or other numbers as needed.

[0280] As an example, the top of the lysis tank 3011, the nucleic acid washing tank, and the nucleic acid elution tank 3013 are respectively provided with corresponding liquid filling holes. The liquid filling hole 30101 at the top of the lysis tank 3011 is used to load samples, lysis buffer, and magnetic bead solution, etc. The liquid filling hole 30102 at the top of the nucleic acid washing tank is used to load magnetic bead washing solution, and the liquid filling hole 30103 at the top of the nucleic acid elution tank 3013 is used to load elution solution.

[0281] As an example, the library construction module 302 includes a droplet control electrode assembly 3021, a magnetic bead cleaning tank, and a library collection and storage tank.

[0282] Specifically, the droplet control electrode assembly 3021 is used to control the droplets or liquid in the digital microfluidic chip. Figure 4 The droplet control electrode assembly 3021 includes a plurality of droplet control electrodes 3021a disposed between the base plate main body layer 1011 and the first dielectric layer 1012 and arranged at intervals according to a preset path, and a ground electrode layer disposed between the cover plate main body layer 1021 and the second dielectric layer 1022. The droplet control electrodes 3021a can be square, rectangular or other suitable shapes, and the area of ​​the droplet control electrodes 3021a can be designed according to actual needs. The preset path starts from the nucleic acid elution tank 3013 and includes multiple branch paths. The end area of ​​the branch path is provided with a liquid filling hole that penetrates the upper cover plate 102 and communicates with the gap 103.

[0283] As an example, in a second-generation sequencing library construction method, the library construction module 302 is provided with a first liquid addition well 30201, a second liquid addition well 30202, a third liquid addition well 30203, a fourth liquid addition well 30204, a fifth liquid addition well 30205, a sixth liquid addition well 30206, a seventh liquid addition well 30207, an eighth liquid addition well 30208, and a ninth liquid addition well 30209 at the end of multiple branch paths of the preset path. The first liquid addition well 30201 is used to load fragment 30 reagents, and the second liquid addition well 30202 is used to load reagents for linking fragments. The adapter reagent is used as follows: the third well 30203 is used to load ER / AT (terminal repair / A-tail addition) reagent; the fourth well 30204 is used to load ligase reagent; the fifth well 30205 is used to load magnetic bead solution; the sixth well 30206 is used to load elution buffer (e.g., deionized water); the seventh well 30207 is used to load elution buffer (e.g., deionized water); the eighth well 30208 is used to load PCR reaction solution; and the ninth well 30209 is used to load magnetic bead solution.

[0284] Specifically, the magnetic bead cleaning tank and the library collection and storage tank are located in different areas of the upper cover plate 102 and are both connected to the gap 103. The magnetic bead cleaning tank is used for cleaning magnetic beads during the library construction process, and the library collection and storage tank is used for collecting and storing mixed next-generation sequencing libraries.

[0285] It should be pointed out that, Figure 4 The illustration shows a library construction module 302 comprising four magnetic bead cleaning tanks (first magnetic bead cleaning tank 3022a, second magnetic bead cleaning tank 3022b, third magnetic bead cleaning tank 3022c, and fourth magnetic bead cleaning tank 3022d), achieving a better cleaning effect through four cleaning cycles. However, in other embodiments, the number of magnetic bead cleaning tanks in a library construction module 302 can be set to one, two, three, or other numbers as needed.

[0286] As an example, the top of the magnetic bead cleaning tank is provided with a corresponding liquid filling hole, wherein the liquid filling hole 30210 at the top of the magnetic bead cleaning tank is used to load magnetic bead cleaning fluid (e.g., 80% ethanol).

[0287] As an example, the library collection and storage tank is located below the sixth liquid addition well 30206 and has the same area as the sixth liquid addition well 30206. After the droplets after the PCR reaction are mixed with the magnetic beads and moved to the library collection and storage tank below the sixth liquid addition well 30206, the eluted magnetic beads are removed after elution. The elution solution at this time is a library that can be used for sequencing.

[0288] In some embodiments, the library construction module 302 further includes a quantitative detection reagent loading and storage tank 3024, which is located at the end of one of the branch paths and passes through the upper cover plate 102 to communicate with the gap 103. The top of the quantitative detection reagent loading and storage tank 3024 is provided with a liquid addition hole 30211 for loading and storing quantitative detection reagents.

[0289] Specifically, the reactants used in the microfluidic chip (all substances on the chip involved in the experiment, including samples and reagents) are usually added to the chip in liquid form, but can also be added in solid form, such as by adding lyophilized reagents. Here, "lyophilized reagents" refers to reagents prepared using a lyophilization method, typically used for reagents containing heat-sensitive active substances.

[0290] Specifically, the preservation of biological reagents is a crucial step in medical diagnostics. From a reagent preservation perspective, many point-of-care testing (POCT) kits for nucleic acid tests need to be stored at room temperature. This necessitates that the reagents undergo dehydration to exist in solid form, requiring reconstitution with buffer solution before use. For example, liquid reagents dropped into liquid nitrogen solidify into microspheres within a very short time. These solid microspheres are then placed in a pre-cooled freeze dryer, and a freeze-drying curve is designed to complete the freeze-drying process. The freeze-dried microspheres maximize enzyme activity, and their loose network structure allows for rapid reconstitution. After encapsulation, the freeze-dried solid microspheres can be stored and transported at room temperature, significantly reducing transportation costs and shelf life. These outstanding advantages of freeze-dried bead technology are suitable not only for nucleic acid reagent POCT production processes but also for the production processes of other biological products requiring preservation, protection of active substances, and room-temperature storage and transportation. This new technology has been rapidly adopted in the past few years.

[0291] Specifically, reactants can be used immediately after being added to the microfluidic chip, or they can be pre-encapsulated in the chip for use at a later time. For example, during chip manufacturing, specified reagents (which can be liquid or lyophilized reagents) can be pre-encapsulated. The chip is then transported to the location required for the user's experiment, where the user adds the sample to the chip with the pre-encapsulated reagent and begins the corresponding experiment.

[0292] Specifically, reagent pre-encapsulation is crucial in decentralized laboratory applications, namely point-of-care testing (POCT). In recent years, reagent pre-encapsulation storage technology has seen significant development, with various reagent types applied to nucleic acid detection and analysis, including sequencing, such as enzymes, primers, substrates, and buffer solutions. Different types of reagents require different encapsulation methods. For example, protein reagents are sensitive to temperature and solution pH; fluorescent probes or photoexcitation materials require light-protected storage; nanoparticles need to be stored in solutions with specific salt concentrations and surfactants to maintain good suspension and dispersion; gel-like storage, such as agarose, requires humidity to maintain its pore morphology, and so on. Furthermore, the volume of reagents integrated into microfluidic chips varies greatly, with complex requirements. For instance, liquid reagent volumes can range from nanoliters to milliliters, and solid reagent masses from picograms to micrograms. Overall, portable analytical systems need to stably store all (at least most) the liquid and solid reactants required for reactions and enable controlled release and reaction of reagents, achieving the goal of "sample in - result out."

[0293] Specifically, reagent pre-encapsulation includes two methods: liquid reagent pre-encapsulation and solid reagent pre-encapsulation. Liquid reagent storage can be further categorized into two main types based on its storage location: internal and external. The first type involves liquid reagents stored outside the microfluidic chip. The chip is connected to an external reservoir via a conduit or liquid storage tube interface. When needed, the reagent is introduced into the chip through external control equipment (e.g., squeezing or puncturing the reservoir). Alternatively, blister packs made of aluminum foil or polymer films can be used to seal the reagent. The seal is broken by squeezing or puncturing to release the reagent. The disadvantages of this method are the need for external conduits, pumps, valves, squeezing / puncturing devices, liquid flow paths, and control units, and the potential for leaks, contamination, air bubbles, and liquid stagnation (dead volume) at the interface. The second type involves storing reagents within a microfluidic chip. Reagents are released into the reaction region of the chip through actively controlled release methods. Examples include using paraffin wax, hydrogels, thermally soluble films, or sealing the liquid reservoir outlet on the chip with single-sided adhesive. During use, local heating melts the paraffin wax, dehydrates the hydrogel, melts the film with electricity, or opens the single-sided adhesive sealing valve, ultimately releasing the stored liquid within the chip. Solid reagent storage methods are also divided into two main categories based on their placement. The first method involves fixing the reagent onto a carrier and then integrating the carrier into the microfluidic chip. The most commonly used carrier is microbeads, which can be made of polymers, glass, metals, gels, etc., and range in size from nanometers to millimeters. Microbeads can fix reagents through passive adsorption or through chemical cross-linking. Compared to the surface of a flat chip, microbeads have a larger effective surface area, allowing for more effective reagent fixation and higher reaction efficiency. Furthermore, fixing reagents onto microbeads separates reagent fixation from chip encapsulation into independent operations, facilitating better quality control and making chip assembly and application more flexible. The second method involves directly immobilizing reagents within the microfluidic chip. There are two immobilization methods: one involves dehydrating the reagent through methods such as lyophilization, vacuum drying, or porous media adsorption drying, then adding the solid reagent into the reaction chamber; during the experiment, it is reconstituted with buffer solution before subsequent reactions. The other method involves patterning and immobilizing the reaction reagent on a reaction substrate within the microfluidic chip using non-contact spraying, contact spotting, micro-contact printing, or dip pen nanofabrication, such as glass, quartz, silicon, polymer materials, or metal electrodes. In use, the molecule to be detected reacts with the formatted and immobilized reagent, generating optical, electrical, or other directly measurable signals.

[0294] In some embodiments of the present invention, the digital microfluidic chip is pre-packaged with reagents, including at least one of the reagents required for nucleic acid extraction and the reagents required for library construction.

[0295] In some embodiments of the present invention, the pre-sealed reagent may have an independent packaging film layer that can be broken during use to release the pre-sealed reagent into the digital microfluidic chip. The packaging film layer may be broken by means of heating or mechanical puncture, for example.

[0296] In some embodiments of the present invention, the digital microfluidic chip is pre-loaded with a filling oil with a specified melting point. The melting point of the filling oil may be less than or greater than 0°C. Preferably, the melting point of the filling oil is greater than 0°C, for example, greater than 10°C, or greater than 25°C, or greater than 35°C.

[0297] Unless otherwise stated, in this specification, the melting point of a substance refers to the temperature at which the substance changes from a solid state to a liquid state under standard atmospheric pressure (atm).

[0298] Specifically, in the specification of this invention, the terms "filler oil" and "filler liquid" are used interchangeably, referring to substances that can completely or partially fill the gaps between DMF chips, are substantially insoluble in droplets, have no significant impact on droplet control, are compatible with chip materials and coatings, and have no significant impact on the droplet reaction process within the chip. It can be one or more substances; for example, a digital microfluidic chip may be filled only with a first filler liquid, or may contain both a first filler liquid and a second filler liquid, with the second filler liquid surrounding or intersecting with the first filler liquid. The filler liquid can fill the entire gap or part of the gaps between the DMF chips; alternatively, samples or reagents and the filler liquid can be placed in specific reservoirs on the DMF chip, such that droplets dispensed from the reservoirs by electrowetting (or dielectrophoresis) are coated with a very thin layer of filler liquid.

[0299] Specifically, in the specification of this invention, the term "reaction procedure" refers to the process of reacting and / or measuring one or more substances according to a specified sequence and conditions (e.g., degree of mixing, temperature, magnetic field strength, electric field strength, time, mass ratio, etc.). The terms "no significant effect" or "no significant effect" can be used interchangeably, meaning that the influence of newly introduced factors (such as materials, processes, formulations, temperature, etc.) on the experimental results is within the allowable error range.

[0300] Specifically, the filling fluid (including the first and second filling fluids) can directly change between liquid and solid states during the experiment by altering experimental conditions (primarily temperature). The melting point of the filling fluid is typically higher than the melting point of the specified droplets in the digital microfluidic chip. For example, the melting points of the first and second filling fluids may be greater than 0 degrees Celsius, or greater than 15 degrees Celsius, or greater than 25 degrees Celsius, or greater than 35 degrees Celsius. The first and second filling fluids may contain low-viscosity oils, such as one or more silicone oils, more specifically, one or more fluorosilicone oils; they may also contain one or more mineral oils, such as one or more paraffin oils; or they may contain one or more hydrocarbon compounds, such as one or more aromatic hydrocarbons, and / or one or more alkanes. During droplet operation (at temperatures typically below 100 degrees Celsius), the kinematic viscosity is typically less than 1000 cSt (centiStokes), or less than 100 cSt, or less than 50 cSt, or less than 20 cSt, or less than 10 cSt, or less than 5 cSt.

[0301] Specifically, the first and second filling solutions may contain small amounts of one or more surfactants that are miscible with them. These surfactants may include nonionic surfactants such as Triton X-15 (Octylphenol ethoxylate, HLB value 4.9) and Span 20 (Sorbitan monostearte, chemical formula C...). 18 H 34 O6 (HLB value 8.6), Span 40 (Sorbitan monopalmitate, chemical formula C60) 22 H 42 O6 (HLB value 6.7), Span 60 (Sorbitanmonostearte, chemical formula C60) 24 H 46 O6 (HLB value 4.7), Span 65 (Sorbitan tristearate, chemical formula C60) 60 H 114 O8 (HLB value 2.1), Span 80 (Sorbitan monoleate, chemical formula C) 24 H 44O6 (HLB value 4.3), Span 83 (Sorbitan sesquioleate, chemical formula C60) 66 H 126 O 16 (HLB value 3.7), Span 85 (Sorbitantrioleate, chemical formula C... 60 H 108 O8 (HLB value 1.8) and fluorinated surfactants.

[0302] In this invention, the mass ratio of surfactant to first filling liquid is usually no more than 10%, for example less than 1%, or less than 0.1%, and the HLB value of surfactant is usually less than 15, for example less than 10, or less than 5.

[0303] It should be noted that whether or not a surfactant is added to the first filling solution does not limit the addition of surfactants miscible with the reaction droplets. For example, appropriate amounts of Tween 20 (polyoxyethylene-20-sorbitan monolauraate) (HLB = 16.7), Tween 40 (polyoxyethylene-40-sorbitan monolauraate) (HLB = 15.6), Tween 60 (polyoxyethylene-60-sorbitan monolaurate) (HLB = 14.9), Tween 65 (polyoxyethylene-65-sorbitan monolaurate) (HLB = 10.5), Tween 80 (polyoxyethylene-80-sorbitan monolaurate) (HLB = 15.0), Tween 85 (polyoxyethylene-85-sorbitan monolaurate) (HLB = 11.0), or Triton X-100 (octylphenol) are acceptable. ethoxylate (HLB=13.5) and other similar substances are often added to reaction droplets to reduce the aggregation of magnetic beads in the droplets and reduce the adsorption of protein molecules in the droplets on the chip surface when no external magnetic field is applied.

[0304] Specifically, in many applications, such as nucleic acid extraction, sequencing library construction, and immunoassay, magnetic beads are included in the reaction droplets. Magnetic bead cleaning is a common step in the reaction process. Ethanol is a commonly used component in magnetic bead cleaning. Therefore, in related applications, it is necessary to select a first filling solution that is insoluble or has low miscibility with ethanol at room temperature.

[0305] Specifically, in the specification of this invention, the terms "dye" and "staining agent" are used interchangeably, referring to substances added to the filling liquid that have no significant impact on experimental conditions and results, and can be used to help better observe experimental phenomena and effects; it can be a fluorescent dye (a substance that absorbs a certain wavelength of light and emits another wavelength of light with a longer wavelength than the absorbed light) or a non-fluorescent dye. The dye used in this invention is miscible with the filling oil system (including any surfactants that may be present therein), but substantially insoluble with the reaction droplets, thus ensuring that the first filling liquid and / or the second filling liquid contain one or more surfactants. As a non-limiting example, one or more dyes (staining agents) and filling oil are uniformly mixed so that the color of the mixed filling liquid is suitable for observation but different from the color of the liquid in the chip, and then injected into the digital microfluidic chip. In this way, when the droplets are removed from the chip, it is easy to observe whether there are components of the filling liquid in the sampler. The mass ratio of the dye to the first filling liquid is typically less than 20%, for example, less than 10%, or less than 5%, or less than 1%.

[0306] It should be noted that whether or not a dye is added to the filling solution does not limit the addition of dyes miscible with the reaction droplets. Of course, for ease of experimental observation, the colors of the dyes that may be added to the droplets and the colors of the dyes that may be added to the filling solution should be avoided to the greatest extent possible.

[0307] It should be noted that, in addition to possible surfactants or dyes, other components may be added to the filler solution as needed.

[0308] Specifically, in the specification of this invention, the term "substantially insoluble" generally means that after mixing, the two substances exist more continuously in their original physical state (liquid or solid). For example, when a small amount of oil is mixed with water, the oil exists mainly on the surface of the water or as droplets in the water after mixing. Quantitatively speaking, essentially insoluble means that the total mass (or volume) of a substance existing in a continuous state (micrometer scale or larger) after mixing accounts for not less than 70%, or not less than 80%, or not less than 90%, or not less than 95%, or not less than 98%, or not less than 99% of the mass (or volume) of the species before mixing.

[0309] Unless otherwise stated, the terms "miscible" and "mutually soluble" are used interchangeably in this invention, referring to the homogeneous mixing of one substance and another, where the original substances no longer exist continuously in the mixture in their pre-mixing state; that is, both substances coexist in any microscopic region (e.g., micrometers or smaller). Unless specifically indicated, no chemical reaction occurs between the two (or more) miscible substances in this disclosure.

[0310] The digital microfluidic chip of the present invention includes a chip body and at least one nucleic acid extraction and library construction unit. The nucleic acid extraction and library construction unit is disposed in the chip body and includes a connected nucleic acid extraction module and a library construction module, so that the nucleic acid extraction and library construction processes can be continuously completed on the same digital microfluidic chip without manual or external intervention. The constructed library can be transferred to a sequencer for direct sequencing.

[0311] Example 2

[0312] This invention also provides an integrated sequencing system. Using this integrated sequencing system, raw samples can be lysed, nucleic acid molecules captured, washed, and eluted on a microfluidic chip. The extracted nucleic acid samples can then be used to construct libraries (such as enzyme fragmentation, end repair, linker addition, concentration amplification, etc.) on the same chip. The constructed libraries can be directly extracted and sequenced using a sequencer.

[0313] Please see Figure 5 The diagram shows the modular structure of the integrated sequencing system 400 of the present invention, including a nucleic acid extraction and library construction module 401, a library transfer module 402, and a sequencing module 403. The nucleic acid extraction and library construction module 401 includes a digital microfluidic chip as described above, used for nucleic acid extraction and library construction. The sequencing module 403 is used to sequence the library constructed by the nucleic acid extraction and library construction module 401. The library transfer module 402 is used to transfer the library constructed by the nucleic acid extraction and library construction module 401 to the sequencing module for sequencing.

[0314] As an example, the nucleic acid extraction and library construction module 401 includes a control motherboard, which includes a CPU and firmware running on it. When using a DMF chip for nucleic acid extraction and library construction, the operations performed on the samples mainly include droplet manipulation, temperature control, magnetic bead manipulation, and quantitative library measurement.

[0315] As an example, the nucleic acid extraction and library construction module 401 includes a droplet control module, which provides an electrical signal to the digital microfluidic chip to operate the liquid or droplets in the digital microfluidic chip.

[0316] As an example, the droplet control module is used to provide a voltage control signal to the droplet control electrode 2021a in the digital microfluidic chip, wherein the droplet control module typically has multiple outputs, with a maximum number of 1,000,000, 100,000, 10,000, or 1,000.

[0317] As an example, the voltage output of the droplet control module can be unipolar or bipolar, with a voltage amplitude less than 1000 volts, or less than 300 volts, or less than 100 volts, or less than 60 volts, or less than 30 volts. The voltage frequency can be an AC or DC signal less than 10 MHz, or less than 1 MHz, or less than 100 kHz, or less than 20 kHz, or less than 5 kHz, or less than 1 kHz. The voltage waveform can be a square wave, sine wave, sawtooth wave, pulse width modulation signal, etc.

[0318] As an example, the voltage control module is programmed by a microprocessor or computer on a circuit board via SPI (Serial Peripheral Interface), I2C (Inter-Integrated Circuit), USB (Universal Serial Bus), parallel port, Ethernet, Wi-Fi, or Bluetooth to control the sequence, duration, amplitude, and frequency of the output signals. A spring-loaded electrical contact pin or connector pad can be used to transmit multiple high-voltage control signals to the droplet control electrode 2021a in the digital microfluidic chip.

[0319] As an example, the nucleic acid extraction and library construction module 401 further includes at least one magnet control module for controlling the movement of one or more magnets to drive the magnetic beads in the digital microfluidic chip to move, thereby realizing magnetic bead operation, and each magnet has at least two degrees of freedom of motion.

[0320] Specifically, in the specification of this invention, "magnetic bead operation" or "magnetic bead control" can be used interchangeably to refer to one or more of the following operations:

[0321] (1) Focusing: Magnetic beads are focused within droplets, troughs, or areas between droplets (troughs) on the DMF chip. The size of the focused beads is less than 10 mm, or less than 5 mm, or less than 2 mm. It should be noted that focused beads typically have clear boundaries, but may also be quite blurred. The focused beads account for more than 30%, or more than 50%, or more than 70%, or more than 80%, or more than 90% of all individual beads.

[0322] (2) Immobilize: The magnetic bead is essentially confined to a designated location on the DMF chip within the droplet, reservoir, and filling liquid, during which operations such as droplet control can be performed. For example, in one embodiment, the immobilized magnetic bead is essentially confined to a location within the droplet to allow for droplet separation, resulting in a droplet that is essentially free of the magnetic bead and another droplet (remaining liquid) containing the majority of the magnetic bead.

[0323] (3) Transport: Moving a magnetic bead from one location to another on a DMF chip, including but not limited to moving a magnetic bead from one droplet or tank to another droplet or tank.

[0324] (4) Disperse: By removing (or otherwise controlling) the magnetic field, the aggregated magnetic beads are dispersed in the droplet or liquid tank. This can be done simultaneously with the droplet operation.

[0325] Specifically, in this invention's specification, the terms "magnet" and "magnetic body" are used interchangeably to refer to a magnetic object with a certain shape, including samarium cobalt magnets, neodymium iron boron magnets, ferrite magnets, AlNiCo magnets, and iron-chromium-cobalt magnets. Shapes include cylindrical, toroidal, disc-shaped, conical, pyramidal, and other irregular shapes. Magnets include permanent and non-permanent magnets. Permanent magnets are inherently magnetic, while non-permanent magnets, such as electromagnets, only exhibit magnetism under certain conditions (e.g., the passage of an electric current).

[0326] As an example, the magnet includes a focusing magnet, which is a magnet (permanent magnet or electromagnet) with a specific shape such that the magnetic field on one side is stronger than the magnetic field on the opposite side. Examples include, but are not limited to, conical magnets or pyramidal magnets, wherein the magnetic field at the tip of the conical magnet (or pyramidal magnet) is stronger than the magnetic field of its base.

[0327] For example, please refer to Figure 6 The diagram shows a structural schematic of the magnet control module 500 in one embodiment. The magnet control module 500 can simultaneously control two magnets 501, thereby synchronously manipulating the magnetic beads in the two nucleic acid extraction and library construction units of the digital microfluidic chip. During the nucleic acid extraction process, the magnets 501 are used to gather the magnetic beads that capture nucleic acid molecules and move them to the washing tank for cleaning, and then to the elution tank to elute the nucleic acid molecules, so that the eluent contains purified nucleic acid molecules. The magnets can also purify nucleic acid molecules at different steps during library construction.

[0328] In other embodiments, when a digital microfluidic chip includes one, three or other numbers of the nucleic acid extraction and library construction units 300, the number of magnets 501 installed in the magnet control module 500 can be adjusted accordingly.

[0329] Specifically, in the digital microfluidic chip, the gap 103 may be surrounded by air or filling fluid. For example, please refer to [link to example]. Figures 7-11 This demonstrates the process of using a magnet to manipulate a magnetic bead in a DMF device, moving the bead from one reservoir to another through a medium (air or filling liquid):

[0330] (1) Please refer to Figure 7 The DMF device includes a first liquid reservoir 601 and a second liquid reservoir 602. The first liquid reservoir 601 contains a first liquid 701, and the second liquid reservoir 602 contains a second liquid 702. Magnetic beads 800 are uniformly distributed in the first liquid reservoir 601. It should be noted that the DMF device structure shown here is for illustrative purposes only and does not represent all possibilities.

[0331] (2) Please refer to Figure 8 Magnet 501 is placed in close contact with the DMF device at the position of the first liquid storage tank 601. Under the action of the magnetic field generated by the magnet, the magnetic beads 800 are gathered at the bottom of the first liquid storage tank 601.

[0332] (3) Please refer to Figure 9 and Figure 10 By moving the magnet 501, the magnetic bead 800 is moved to the second liquid storage tank 602.

[0333] (4) Please refer to Figure 11 The magnet 501 is then removed from the DMF device, and the magnetic beads 800 are resuspended and dispersed in the second liquid reservoir 602. This may require droplet movement or other methods to assist in the suspension and dispersion of the magnetic beads in the liquid within the second liquid reservoir 602.

[0334] It should be noted that phrases such as "the magnet is moved (or brought in, transported) to approach" or "the magnet is moved closer" used in the specification of this invention are intended to refer to the relative positions of the magnet and the DMF device. The magnetic force generated by the magnet has a significant effect on the magnetic beads on the device. Conversely, phrases such as "the magnet is moved away" or "the magnet is moved out" are intended to indicate that the magnet has no effect or a negligible effect on the magnetic beads on the device.

[0335] Specifically, depending on the specific measurement, the moving speed of the magnet can usually be controlled by a motor. Different applications require different magnet speeds, typically ranging from 0.1 to 100 mm / sec, or 0.5 to 20 mm / sec, or 1 to 10 mm / sec.

[0336] In the above embodiments, the magnet contacts the base plate 101 and moves along the bottom of the DMF device. In other embodiments, the magnet may also be located on top of the DMF device, or a pair of magnets, one on top and the other on the bottom, for manipulating magnetic beads in the DMF device.

[0337] As an example, the nucleic acid extraction and library construction module 401 further includes at least one temperature control module, which is used to heat or cool a designated area of ​​the digital microfluidic chip to a designated temperature to assist nucleic acid lysis, elution, PCR amplification and other required reactions.

[0338] For example, please refer to Figure 12 The diagram shows a structural schematic of the temperature control module 900 in one embodiment, which includes multiple temperature control units, such as a first temperature control unit 901, a second temperature control unit 902, a third temperature control unit 903, a fourth temperature control unit 904, a fifth temperature control unit 905, a sixth temperature control unit 906, and a seventh temperature control unit 907 arranged side by side and spaced apart. Each temperature control unit can be controlled independently.

[0339] For example, please refer to Figure 13 and Figure 14 ,in, Figure 13 The display shows the temperature control module 900 being placed... Figure 2 The diagram below the digital microfluidic chip is shown. Figure 14 The display shows the temperature control module 900 being placed... Figure 4 The diagram below shows the digital microfluidic chip.

[0340] It should be noted that the movement range of the magnet is limited when the temperature control module 900 is close to the DMF device. Since not all functional modules are needed simultaneously during the experiment, different modules can approach or move away from the DMF chip at different times, allowing other modules more room to move when needed. Therefore, in one embodiment, the instrument is designed such that when temperature control is not required, one or more temperature control units are moved away from the location near or in contact with the DMF device, allowing the magnet to move to a desired position close to the DMF device. In another embodiment, when the magnet is not in use, it can be moved to one end of the DMF device to allow the temperature control module to move to a position close to or in contact with the DMF device. In yet another embodiment, the magnet can be held in one or more spaces between the temperature control modules, allowing the temperature control modules to be moved closer to or in contact with the DMF device.

[0341] As an example, the nucleic acid extraction and library construction module 401 also includes a library quantification module (not shown), which includes an excitation light source and an optical detection module. The excitation light source is used to project excitation light onto the nucleic acid concentration measurement area of ​​the digital microfluidic chip, and the optical detection module is used to perform optical measurement on at least one site of the digital microfluidic chip.

[0342] Specifically, quantitative analysis and quality assessment of sequencing libraries are crucial for obtaining high-quality nucleic acid sequencing data. This is because: (1) if the copy number of the sequencing library is too low, insufficient sequencing data will not be obtained; (2) if the copy number of the sequencing library is too high, low-quality sequencing data will be generated, and sequencing may even fail. Therefore, to control the amount of sequencing library loaded on the sequencer and ensure the quality of sequencing data, quantitative analysis of the gene sequencing library is required before sequencing.

[0343] Specifically, there are different methods to achieve quantitative analysis of documents:

[0344] (1) Ultraviolet Spectrophotometry. Nucleic acids, including DNA and RNA, are composed of ribose, phosphate groups, and bases. Because bases contain aromatic ring structures, they absorb light in the ultraviolet band. This characteristic can be used for library quantification, and the corresponding method is called ultraviolet spectrophotometry. The ultraviolet absorption peak of nucleic acids is at 260 nm. Simultaneously, the purity of nucleic acids can be estimated by calculating the values ​​of A260 / A280 and A260 / A230. Absorption at 280 nm typically originates from proteins, while absorption at 230 nm typically originates from sugars and phenols. Ultraviolet spectrophotometry cannot distinguish between DNA and RNA because the structures with ultraviolet absorption are the bases of nucleic acids, and both DNA and RNA contain bases. Therefore, when a sample contains both DNA and RNA, the measured concentration will be higher than the actual DNA concentration.

[0345] (2) Fluorescence intensity method. This method uses target-specific dyes that specifically bind to different types of nucleic acid strands and emit fluorescence when excited by a light source of a specific wavelength. Fluorescent dyes bound to nucleic acids can amplify the signal by tens to hundreds of times compared to free nucleic acid dyes, thus distinguishing them from the background. In the measurement process, the DNA sample to be tested is first mixed with the fluorescent dye, which binds to the DNA to form a fluorescent complex. Then, this fluorescent complex is excited with light of a corresponding wavelength, causing it to emit a fluorescent signal. The concentration of DNA can be calculated by measuring the intensity of this fluorescent signal. The fluorescence intensity method is currently the most widely used method for sequencing library quantification. For example, the fluorescent dyes PicoGreen and SYBR Green I are two methods used to measure the concentration of double-stranded DNA in solution. When the liquid is irradiated (excited) with light at approximately 480 nm, it will fluoresce at approximately 520 nm. Within a certain concentration range (0–20 μg / ml), the fluorescence intensity is directly proportional to the concentration of double-stranded DNA in the liquid. First, measure the fluorescence value of a solution with a known nucleic acid concentration, then measure the fluorescence value of an unknown library. The concentration of DNA in the library can be calculated based on the fluorescence intensity.

[0346] (3) Quantitative PCR. Quantitative PCR combines fluorescence detection technology with PCR technology. A fluorescent group is added to the PCR reaction system. The change in fluorescence signal during PCR temperature cycling directly reflects the change in the concentration of the nucleic acid to be tested in the reaction system. The concentration of unknown samples can be calculated using a standard curve with known concentrations, thus allowing the measurement of library concentration. Because its specific primers only quantify libraries with complete adapter ligation at both ends, it can eliminate interference from unsequential libraries with no adapter ligation at either end, resulting in high accuracy.

[0347] Specifically, in this invention's specification, the terms "testing," "detection," and "measurement" are used interchangeably to refer to the process of acquiring physical quantities (e.g., position, charge, temperature, concentration, pH value, brightness, fluorescence, etc.). Typically, at least one sensor (or detector) is used to acquire the physical quantity and convert it into a signal or information that can be recognized by a human or instrument. Other components may be present between the analyte and the sensor, such as lenses, mirrors, and filters used in optical measurements, and resistors, capacitors, and transistors used in electrical measurements. Furthermore, other auxiliary devices or components are often used to make measurement possible or easier. For example, light sources such as lasers or laser diodes are used to excite particles from their electronic ground state to an electronic excited state. When excited-state particles return to their ground state, they sometimes emit fluorescence, and measuring the intensity of this fluorescence can be used to measure the concentration of a certain particle in a liquid sample. Optical sensors include CCDs, photodiodes, and photomultiplier tubes (PMTs), while electrical sensors include operational amplifiers, analog-to-digital converters, thermocouples, and thermistors.

[0348] Specifically, measurements can be performed simultaneously or sequentially on multiple parameters in multiple samples. For example, while measuring the fluorescence of a particle in a droplet using a photodiode, the droplet's position can also be obtained simultaneously by capacitance measurement. Sensors or detectors are typically connected to a central processing unit (CPU) or computer, which runs corresponding software that analyzes the measured signals and usually converts them into information that can be understood by the user or other instruments. For example, the concentration of a particle can be inferred by measuring and analyzing its fluorescence intensity in a liquid. As non-limiting examples, optical measurements include laser-induced fluorescence measurement, infrared spectroscopy, Raman spectroscopy, chemiluminescence measurement, surface plasmon resonance measurement, absorption spectroscopy, etc.; electrical measurements include amperometry, voltammetry, photoelectrochemistry, coulometric analysis, capacitance measurement, and AC impedance measurement, etc.

[0349] As an example, the document transfer module 402 uses a pump to power the transfer of liquids. A pump is a device that transports fluids (including liquids and gases) or pressurizes fluids. It transfers mechanical energy or other external energy to the fluid, increasing its energy. Pumps can be used to transport liquids such as water, oil, acids and alkalis, emulsions, suspensions, and liquid metals, as well as liquid-gas mixtures and liquids containing suspended solids. There are many types of pumps, including gear pumps, centrifugal pumps, screw pumps, reciprocating pumps, piston pumps, hydraulic plunger pumps, pneumatic diaphragm pumps, and so on. In this invention, a peristaltic pump is preferred, primarily because it facilitates non-contact liquid control, but the use of other types of pumps to achieve similar functions of powering liquid transfer is not excluded. A peristaltic pump mainly consists of three parts: a driver, a pump head, and a hose. The fluid is isolated in the hose, which can be quickly replaced. Advantages of peristaltic pumps include reversible fluid flow, dry operation capability, and low maintenance costs. Controlled by the program on the motherboard, the peristaltic pump can be used to perform functions such as venting the sequencing chip, introducing sequencing reagents into the sequencing chip, and transferring libraries from the DMF chip to the sequencing chip.

[0350] As an example, the library transfer module 402 also includes a flowmeter for quantitatively monitoring the transferred liquid. Flowmeters, typically including liquid flowmeters and gas flowmeters, are devices used to measure the flow rate of fluids in a pipeline. There are many types of flowmeters, including differential pressure flowmeters, rotor flowmeters, throttling flowmeters, slot flowmeters, volumetric flowmeters, electromagnetic flowmeters, and ultrasonic flowmeters. This invention preferentially uses a non-contact ultrasonic flowmeter, which has several advantages, including not contacting the liquid being measured and not affecting the fluid being measured. However, this invention does not exclude the use of other types of flowmeters.

[0351] As an example, the library transfer module 402 also includes a demister disposed in the liquid transfer path. It should be noted that bubble formation in liquids is easy, and bubbles in microfluidic chips often affect experimental procedures and analytical results; therefore, debubbling is a crucial step in liquid handling. This invention selects a demister in the liquid path during transfer. This demister is a device with a liquid inlet, a liquid outlet, and a gas inlet and outlet, allowing any gas that may be present in the liquid entering through the liquid inlet to be discharged through the gas inlet and outlet, while the liquid exits through the liquid outlet. One implementation method is to use a microporous membrane (e.g., a polytetrafluoroethylene membrane) within the device along the liquid inlet to liquid outlet path, allowing bubbles or gas in the liquid to be discharged through this membrane, while the liquid continues to flow along the original liquid path.

[0352] It should be noted that in biological assays, it is often necessary to transfer liquids from one functional module to another. In the system described in this invention, the constructed library needs to be transferred from the DMF library preparation chip to the sequencing chip. To avoid cross-contamination between different sample assays, the reuse of liquid transfer circuits should be minimized. Therefore, the tubes, valves, connectors, etc., used in the liquid transfer module of this invention are all disposable, and reusable parts or modules such as pumps and flow meters do not directly contact the transferred liquid.

[0353] As an example, the sequencing module 403 includes a nanopore sequencing module, a single-molecule fluorescence sequencing module, or an NGS sequencing module, wherein the nanopore sequencing module employs a biological nanopore or a solid nanopore.

[0354] In one embodiment, a nanopore chip (or nanopore sequencing chip) is used together with a nanopore sequencer to sequence nucleic acid molecules.

[0355] Specifically, the basic working principle of nanopore sequencing includes: within a cavity filled with electrolyte, an insulating, impermeable membrane with nanoscale pores divides the cavity into two chambers. When a voltage is applied to the electrolyte chamber, charged ions and small molecules can pass through the pores, forming a stable and detectable ionic current. By controlling the size and surface properties of the nanopores, the applied voltage, and the solution conditions, different types of biomolecules can be detected.

[0356] Since the four bases that make up DNA / RNA, A, G, C, T / U, have different molecular structures and sizes, single-stranded DNA (ssDNA) is rapidly cleaved into deoxyribonucleotide molecules one by one by exonucleases. When a single base passes through a nanoscale pore under the drive of an electric field, the differences in the chemical properties of the different bases cause different amplitudes of current changes when passing through the nanopore, thus obtaining the sequence information of the DNA being measured.

[0357] Specifically, there are two main types of nanopores currently used for gene sequencing: biological nanopores and solid nanopores. Among them, biological nanopores are nanodevices made by self-assembling proteins or peptides, which have specific pore structures, biological activities and the ability to insert into lipid bilayer membranes, and can be chemically or biologically modified; the most common ones include: (1) α-hemolysin (αHL) nanopores, which are composed of 293 amino acid peptides and can be inserted into pure lipid bilayer membranes to form mushroom-shaped heptamers and assemble into transmembrane channels. αHL heptamer nanopores are mainly composed of three parts: a cap region (Cap, with an inlet cis end diameter of 2.6 nm), a rim region (Rim, with a diameter of 1.4 nm), and a stem region (Stem, with an inlet trans end diameter of 2.2 nm). αHL nanopores are permanently open and never close, resistant to strong acids and alkalis, and relatively stable under high temperature and high voltage; (2) MspA (Mycobacterium smegmatisporin A) nanopores are conical, octamer pore proteins, with a short and narrow contraction region of about 1.2nm × 0.6nm. Compared with the 5nm long α-hemolysin protein pore, it is more conducive to the determination of single bases. Gundlach first reported the technique of connecting nucleic acid ends to nucleic acid molecular clips and using MspA nanopores to identify four single bases, which can slow down the DNA crossing speed and improve the detection sensitivity of DNA single bases. Solid nanopore chips are mainly made by using ion etching technology, electron etching technology, focused electron beam (FEB) or ion beam (FIB) to create tiny pores on insulating materials such as silicon nitride, silicon dioxide and graphene. Currently, the fabrication of solid-state nanopores involves first creating a suspended film 30–500 nm thick using conventional microfabrication techniques, and then drilling pores of 2–100 nm on the surface of the silicon or other material film using ion beams or electron beams. Since the nanopore diameters required for DNA detection are typically 1–2 nm, further shrinkage techniques such as precipitation shrinkage, ion beam radiation, and electron beam radiation can be employed to reduce the size of the nanopores, thereby achieving even smaller target nanopore sizes.

[0358] Compared to biological nanopores, solid-state nanopores offer advantages such as tunable size, good chemical stability, and easy expansion. However, the insufficient detection resolution of traditional solid-state nanopores limits their application in biomolecule detection. Two-dimensional materials such as graphene, molybdenum disulfide, and tungsten disulfide, with their atomic-layer thickness, can improve the detection resolution of solid-state nanopore sensors. Among them, graphene, being the thinnest two-dimensional material discovered to date, possesses excellent mechanical properties at a single atomic layer thickness, allowing it to be fabricated into suspended nanopore sensors, significantly enhancing the detection resolution of solid-state nanopores.

[0359] Specifically, nanopore sequencing mainly involves the following steps:

[0360] (1) Unwinding: unwinding double-stranded DNA into single strands (this step is not required for RNA sequencing);

[0361] (2) A single-stranded DNA molecule passes through a pore protein, and there is a protein molecule in the pore that acts as a converter;

[0362] (3) When a single DNA molecule remains in the pore, it causes a change in current. Different bases cause different changes in current.

[0363] (4) Based on the spectrum of current change over time, the base sequence is obtained by applying a pattern recognition algorithm.

[0364] Specifically, there are two main methods for nanopore sequencing of RNA. The first is direct sequencing of RNA molecules, which requires special library preparation by ligating primers to the 3' end of the natural RNA without reverse transcription. The second method requires synthesizing a cDNA strand to obtain an RNA-cDNA hybrid double strand, followed by ligation of an adapter. The first method is suitable for small sample sizes and is faster, while the second produces a more stable library for longer sequencing processes, thus offering higher yields. In both methods, only the RNA strand passes through the nanopore. Studies have shown that nanopore sequencing devices typically have lower average accuracy for direct RNA sequencing compared to gene sequencing (currently around 85%).

[0365] For example, please refer to Figure 15 and Figure 16 The diagram shows a schematic representation of the integrated sequencing system of the present invention in one embodiment, wherein... Figure 15 This is shown as a top-front-side view of the device. Figure 16 This is a rear view of the device.

[0366] Specifically, such as Figure 15 As shown, the spring probe 1001 serves as an interface for droplet control between the instrument and the DMF device, providing a droplet control voltage to the electrowetting electrode on the DMF device. The touchscreen 1002 provides a user-friendly graphical interface for inputting commands and displaying help, experimental status, and measurement results. A sliding tray 1003 is used for loading and unloading the DMF device. When pushed in, the spring probe 1001 contacts the bottom substrate of the DMF device 1004.

[0367] Specifically, such as Figure 16 As shown, vent 1005 (typically equipped with an internal fan) is used to dissipate heat generated during instrument operation. USB port 1006 and Ethernet port 1007 optionally provide a means for application software running on a computer to communicate with the instrument (e.g., send commands or receive data). AC power port 1008 is used for external power supply.

[0368] The integrated sequencing system of this invention can be used to process or measure target analytes in sample solutions, which may include, but are not limited to, bodily fluids (including but not limited to blood, serum, saliva, urine, etc.), purified samples (e.g., purified DNA, RNA, proteins, cells, etc.), environmental samples (including but not limited to water, air, agricultural samples, etc.), and biological warfare agent samples. Bodily fluids can be derived from any organism. In some embodiments, the bodily solution may be derived from mammalian bodily fluids, such as those from humans.

[0369] Specifically, in the specification of this invention, the terms "analyte" and "analyte" are used interchangeably, referring to the substance or chemical component to be analyzed or tested. An "analyte" can be organic or inorganic. It can refer to biomolecules (such as proteins, lipids, cytokines, hormones, carbohydrates, etc.), viruses (such as herpesviruses, retroviruses, adenoviruses, lentiviruses), intact cells (including prokaryotic and eukaryotic cells), environmental pollutants (including toxins, pesticides, etc.), drug molecules (such as antibiotics, therapeutic drugs, and drugs of abuse), cell nuclei, spores, etc.

[0370] It should be noted that some or all of the instrument functional modules in this invention, such as the temperature control module, droplet control module, magnet control module, optical detection module, liquid transfer module, etc., can be automatically controlled by a program (software or firmware) running on a microprocessor or computer.

[0371] The integrated sequencing system of this invention automates the entire process of nucleic acid extraction, library construction, and sequencing, reducing reagent consumption and thus lowering library construction and sequencing costs and time. This integrated sequencing system also features smaller size and lower power consumption, requires less manual operation, and has low requirements for the operating environment (such as temperature, humidity, and ventilation), making it suitable for use in environments outside the laboratory. It is applicable to various settings including hospitals of all sizes, community health clinics, disease control centers, environmental monitoring, customs, and forensic identification.

[0372] Example 3

[0373] This invention also provides an integrated sequencing method, comprising the following steps:

[0374] Nucleic acid extraction and library construction steps: Nucleic acid extraction and library construction are performed on the digital microfluidic chip described in any of the above embodiments;

[0375] Library transfer step: Transfer the library constructed in the digital microfluidic chip to the sequencing module;

[0376] Sequencing steps: Sequencing is performed on the sequencing module.

[0377] Specifically, the integrated sequencing methods described above generate a series of "reactions," namely physical and chemical reactions between the sample and reagents. Physical reactions refer to changes in the state or form of matter without altering its properties. Examples include lyophilized reagents dissolving in a solvent (such as deionized water), cleaning magnetic beads by removing adsorbed impurities from them in a washing solution, uniformly mixing two miscible liquids (or a solid and a liquid), and cooling a filling solution from a liquid to a solid state. Chemical reactions, on the other hand, refer to the process by which atoms in a molecule rearrange and recombine to form new molecules. Essentially, this involves the breaking of old chemical bonds and the formation of new ones. Examples include PCR reactions, DNA enzymatic digestion, DNA fragment and end repair, adding A tails, and adding linkers.

[0378] For example, please refer to Figure 17 The flowchart shown is an embodiment of the integrated sequencing method of the present invention, including the following steps:

[0379] S1: Set the temperature control module to a specified temperature to melt the filling oil in the digital microfluidic chip;

[0380] S2: Add the sample to be tested into the designated liquid tank of the digital microfluidic chip;

[0381] S3: Add the required reagent (if not pre-sealed) to the designated liquid tank of the digital microfluidic chip;

[0382] S4: Nucleic acid extraction and library construction are performed on the digital microfluidic chip;

[0383] S5: Transfer the library constructed in the digital microfluidic chip to the sequencing module;

[0384] S6: Start sequencing;

[0385] S7: Analyze sequencing data;

[0386] S8: Deliver the analysis results to the user.

[0387] As an example, the above process is implemented based on the integrated sequencing system of the present invention. Except for loading the sample into the DMF chip, everything else is automatically implemented by the instrument according to the specified program. If the reagent has been pre-packaged on the chip, step S3 can be omitted. In step S8, the analysis results are given to the user in the form of electronic documents, direct display on the screen, etc.

[0388] As an example, in the nucleic acid extraction and library construction step, the nucleic acid extraction is performed using the magnetic bead method, including the following steps:

[0389] (1) Add the sample to be tested, lysis buffer and magnetic beads to the lysis tank of the nucleic acid extraction module, use the lysis buffer to release the nucleic acid molecules in the sample to be tested, and use the magnetic beads to adsorb the nucleic acid molecules;

[0390] (2) A magnet is used to gather the magnetic beads with nucleic acid molecules adsorbed in the lysis cell from below the digital microfluidic chip, and the magnetic beads are moved along the bottom plate of the digital microfluidic chip to the nucleic acid cleaning tank of the nucleic acid extraction module for cleaning to remove the impurities adsorbed on the magnetic beads.

[0391] (3) A magnet is used to move the cleaned magnetic beads into the nucleic acid elution tank of the nucleic acid extraction module, and the nucleic acid elution tank is filled with elution solution to elute the nucleic acid molecules adsorbed on the magnetic beads into the solution.

[0392] As an example, the library construction includes one or more of the following steps:

[0393] (1) Fragmenting nucleic acid molecules using enzyme reactions;

[0394] (2) Using enzyme reactions to repair the ends of nucleic acid molecular fragments;

[0395] (3) Adding an A tail to the end of a nucleic acid molecule fragment using an enzyme reaction;

[0396] (4) Adding adapters to nucleic acid molecular fragments using enzyme reactions;

[0397] (5) PCR amplification;

[0398] (6) Nucleic acid molecules were purified using the magnetic bead method;

[0399] (7) Quantitative analysis of the library.

[0400] As an example, at least a portion of the reagents are added to the digital microfluidic chip via a pre-sealing method. This pre-sealing method includes placing the reagents to be pre-sealed at a designated location on the digital microfluidic chip, and then solidifying the filling oil in the digital microfluidic chip by lowering the temperature to fix the position of the pre-sealed reagents within the chip. When nucleic acid extraction and library construction are performed on the digital microfluidic chip, the chip is first heated to a designated temperature to change the filling oil in the chip from a solid state to a liquid state, allowing the pre-sealed reagents to move within the digital microfluidic chip.

[0401] As an example, multiple nucleic acid extraction and library construction units in the digital microfluidic chip are operated synchronously.

[0402] The following is based on Figure 2The following example illustrates the usage process of a digital microfluidic chip that integrates magnetic bead-based nucleic acid extraction and nanopore sequencing library preparation. For details involving heating, please refer to [reference needed]. Figure 13 :

[0403] (1) Load a specified amount of filling oil and all the required reagents into the digital microfluidic chip;

[0404] (2) Load the sample into the lysis tank 2011 which has been loaded with lysis buffer and magnetic beads, and use the first temperature control unit 901, the second temperature control unit 902 and the third temperature control unit 903 to heat the area where the lysis tank 2011 is located to a specified temperature. After a specified time, place the magnet close to the bottom of the DMF chip below the lysis tank 2011 and move it within the range of the lysis tank 2011 along a specified path and speed. The purpose is to gather the magnetic beads (which have adsorbed nucleic acid molecules) in the lysis tank 2011.

[0405] (3) Move the magnetic beads to the first nucleic acid cleaning tank 2012a and move them within the range of the first nucleic acid cleaning tank 2012a to clean any possible debris from the magnetic beads.

[0406] (4) Optionally, the magnetic beads are moved to the second nucleic acid cleaning tank 2012b and moved within the range of the second nucleic acid cleaning tank 2012b to further clean any possible debris on the magnetic beads;

[0407] (5) Move the magnetic beads to the nucleic acid elution tank 2013 and move the magnet away from the DMF substrate. Optionally, use the third temperature control unit 903 and the fourth temperature control unit 904 to heat the nucleic acid elution tank 2013 to a specified temperature. After a specified time, move the magnet back close to the DMF chip below the nucleic acid elution tank 2013 and move the magnet within the area of ​​the nucleic acid elution tank 2013 along a specified trajectory and speed to collect the magnetic beads, and then move the magnetic beads to a specified position on the chip to discard them.

[0408] (6) Using the electrowetting effect of the droplet control electrode assembly 2021, a specified amount of eluent (containing purified nucleic acid molecules) is taken out from the nucleic acid elution tank 2013 and moved to the first liquid addition hole 20201 and the second liquid addition hole 20202 respectively, so that the eluent is mixed with the fragmentation enzyme and buffer at that location, and the mixed droplet is moved to the designated location, and the temperature control module 624 is used to heat the location to the specified temperature required for the fragmentation reaction;

[0409] (7) After a specified time, the fragmentation termination buffer droplet at the liquid addition hole 1223 is moved to a designated location and mixed with the above-mentioned mixed droplet. The fourth temperature control unit 904 is used to heat the location to a designated temperature to terminate the fragmentation reaction.

[0410] (8) After a specified time, the previously mixed droplets are mixed with the magnetic bead solution at the fourth liquid filling hole 20204;

[0411] (9) After a specified time, the magnetic beads in the mixed droplets from the previous step are gathered by a magnet and then moved sequentially to the first magnetic bead cleaning tank 2022a and the second magnetic bead cleaning tank 2022b for cleaning.

[0412] (10) Use a magnet to move the cleaned magnetic beads to the fifth liquid filling hole 20205 for elution, and remove the eluted magnetic beads.

[0413] (11) Move the elution droplet and mix it sequentially with the sequencing connector at the sixth well 20206 and the sequencing buffer at the seventh well 20207;

[0414] (12) Move the mixed droplets from the previous step to the library collection storage tank 2023 and mix them with the sequencing buffer and other reagents therein; at this time, the liquid in the library collection storage tank 2023 can be taken out for nanopore sequencing; in order to further optimize the sequencing steps, the library collection storage tank 2023 can also be loaded or pre-sealed with reagents required for the operation of the nanopore sequencer, such as conduction buffer and other reagents.

[0415] Optionally, in order to control the amount of sequencing library loaded on the sequencer and ensure the quality of sequencing data, the quantitative detection reagent loading and storage tank 2024 is loaded with and stores quantitative detection reagents, and the concentration of the constructed library is measured using the corresponding detection module (library quantification module) on the integrated sequencing system 400.

[0416] Thus, nucleic acid extraction and library construction were completed on the same digital microfluidic chip, and the constructed library was transferred to a sequencer for direct sequencing. After the user adds samples and reagents, the instrument can automatically complete nucleic acid extraction and library construction. Reagents can also be pre-packaged; the user only needs to add the sample to be tested to the chip, and the system can automatically complete nucleic acid extraction and library construction without manual intervention.

[0417] Next, let's take... Figure 3 The following example illustrates the usage process of a digital microfluidic chip integrating magnetic bead-based nucleic acid extraction and NGS sequencing library preparation. For details involving heating, please refer to [reference needed]. Figure 14 :

[0418] (1) Load the specified amount of filler oil and all required reagents into the DMF;

[0419] (2) Load the sample into the lysis cell 3011, which has been loaded with lysis buffer and magnetic beads. Optionally, the lysis cell 3011 is heated to a specified temperature using the first temperature control unit 901 and the second temperature control unit 902. After a specified time, a magnet is placed close to the bottom of the DMF chip below the lysis cell 3011 and moved along a specified path and speed within the area of ​​the lysis cell 3011 to gather the magnetic beads (which have adsorbed nucleic acid molecules) in the lysis cell 3011.

[0420] (3) Move the magnetic beads to the first nucleic acid cleaning tank 3012a and move them within the range of the first nucleic acid cleaning tank 3012a to clean any possible debris from the magnetic beads.

[0421] (4) Optionally, the magnetic beads are moved to the second nucleic acid cleaning tank 3012b and moved within the range of the second nucleic acid cleaning tank 3012b to further clean any possible impurities on the magnetic beads;

[0422] (5) Move the magnetic beads to the nucleic acid elution tank 3013 and move the magnet away from the DMF substrate. Optionally, use the fourth temperature control unit 904 and the fifth temperature control unit 905 to heat the nucleic acid elution tank 3013 to a specified temperature. After a specified time, move the magnet back close to the DMF chip below the nucleic acid elution tank 3013 and move the magnet within the area of ​​the nucleic acid elution tank 3013 along a specified trajectory and speed to collect the magnetic beads, and then move the magnetic beads to a specified position on the chip to discard them.

[0423] (6) Using the electrowetting effect of the droplet control electrode assembly 2021, a specified amount of eluent (containing purified nucleic acid molecules) is taken out from the nucleic acid elution tank 3013, moved to the first liquid addition hole 30201 and mixed with the fragmentation reagent there, and the fourth temperature control unit 906 is used to control the temperature at that point to the specified temperature required for the fragmentation reaction.

[0424] (7) After a specified time, the above liquid is moved to the third liquid filling hole 30203 and mixed with the ER / AT reagent there, and the fourth temperature control unit 907 is used to control the temperature there to the specified temperature.

[0425] (8) After a specified time, mix the previously mixed droplets with the connector solution at the second liquid filling hole 30202 and the ligase reagent at the fourth liquid filling hole 30204;

[0426] (9) After a specified time, the droplet is moved to the fifth liquid filling hole 30205 and mixed with the magnetic bead solution there;

[0427] (10) After a specified time, the magnetic beads in the droplets are gathered by a magnet and moved to the first magnetic bead cleaning tank 3022a and the second magnetic bead cleaning tank 3022b for magnetic bead cleaning.

[0428] (11) Move the cleaned magnetic beads to the sixth liquid filling hole 30206 for elution, and remove the eluted magnetic beads;

[0429] (12) Move the elution droplet and mix it with the PCR solution in the eighth well 30208;

[0430] (13) The first temperature control unit 901, the second temperature control unit 902 and the third temperature control unit 903 are used to heat the corresponding area of ​​the chip to the temperature required for PCR reaction. The droplets are moved back and forth in the corresponding temperature area a specified number of times to perform PCR amplification.

[0431] (14) Mix the droplet after the PCR reaction with the magnetic bead solution at the ninth liquid filling well 30209;

[0432] (15) Use a magnet to gather the magnetic beads in the droplets and move them to the seventh liquid filling hole 30207 for elution. Remove the eluted magnetic beads. The elution solution is then a library that can be used for sequencing.

[0433] Optionally, in order to control the amount of sequencing library loaded on the sequencer and ensure the quality of sequencing data, the quantitative detection reagent loading and storage slot 3024 is loaded with and stores quantitative detection reagents, and the concentration of the constructed library is measured using the corresponding detection module (library quantification module) on the integrated sequencing system 400.

[0434] The above process completes nucleic acid extraction and library construction on the same digital microfluidic chip, and then transfers the constructed library to the sequencer for direct sequencing. The entire process is automated and reduces reagent consumption, thereby reducing the cost of library construction and sequencing, and shortening the time required for library construction and sequencing.

[0435] In summary, the digital microfluidic chip, integrated sequencing system, and method of this invention include a chip body and at least one nucleic acid extraction and library construction unit. The nucleic acid extraction and library construction unit is disposed within the chip body and includes a connected nucleic acid extraction module and a library construction module, enabling continuous completion of nucleic acid extraction and library construction processes on the same digital microfluidic chip without manual or external intervention. The constructed library can be directly transferred to a sequencer for sequencing. This invention automates the entire process of nucleic acid extraction, library construction, and sequencing, reducing reagent consumption and thus lowering library construction and sequencing costs and shortening sequencing time. The integrated sequencing system of this invention achieves smaller size and lower power consumption, requires less manual operation, and has low requirements for the operating environment (such as temperature, humidity, and ventilation), allowing it to be used in environments outside the laboratory. It is suitable for applications in hospitals of all sizes, community health clinics, disease control centers, environmental monitoring, customs, forensic identification, and other scenarios. Therefore, this invention effectively overcomes the various shortcomings of existing technologies and has high industrial application value.

[0436] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. A digital microfluidics chip, characterized by, The chip body comprises a bottom plate and an upper cover plate, and the upper cover plate is located above the bottom plate and forms a gap between the bottom plate to provide space for the movement of liquid or droplets in the digital microfluidic chip; The bottom plate comprises a bottom plate body layer and a first dielectric layer located on the bottom plate body layer, at least a part of the surface of the first dielectric layer is hydrophobic; the upper cover plate comprises a cover plate body layer and a second dielectric layer located below the cover plate body layer, at least a part of the surface of the second dielectric layer is hydrophobic. The nucleic acid extraction module comprises a lysis tank for sample lysis and nucleic acid capture, at least one nucleic acid washing tank for nucleic acid washing, and a nucleic acid elution tank for nucleic acid elution, the lysis tank, the nucleic acid washing tank and the nucleic acid elution tank are arranged in different regions of the upper cover plate and are all in communication with the gap, and the top of the lysis tank, the nucleic acid washing tank and the nucleic acid elution tank is respectively provided with a corresponding liquid addition hole.

2. The digital microfluidics chip of claim 1, wherein: The library construction module comprises a droplet control electrode assembly, a magnetic bead washing tank and a library collection storage tank, the droplet control electrode assembly is used for controlling droplets or liquid in the digital microfluidic chip, the droplet control electrode assembly comprises a plurality of droplet control electrodes arranged between the bottom plate body layer and the first dielectric layer and spaced according to a preset path, and a ground electrode layer arranged between the cover plate body layer and the second dielectric layer, the preset path starts from the nucleic acid elution tank and comprises a plurality of branch paths, the end region of the branch path is provided with a liquid addition hole penetrating through the upper cover plate to communicate with the gap, and the magnetic bead washing tank and the library collection storage tank are arranged in different regions of the upper cover plate and are all in communication with the gap, and the top of the magnetic bead washing tank and the library collection storage tank is respectively provided with a corresponding liquid addition hole.

3. The digital microfluidics chip of claim 1, wherein: The library construction module further comprises a quantitative detection reagent loading storage tank, the quantitative detection reagent loading storage tank is located at the end of one of the branch paths and penetrates through the upper cover plate to communicate with the gap, and the top of the quantitative detection reagent loading storage tank is provided with a corresponding liquid addition hole.

4. The digital microfluidics chip of claim 1, wherein: The number of the nucleic acid extraction and library construction units is single or multiple, when the number of the nucleic acid extraction and library construction units is multiple, the multiple nucleic acid extraction and library construction units are arranged side by side and spaced in the chip body.

5. The digital microfluidics chip of claim 4, wherein: The gap between adjacent two nucleic acid extraction and library construction units is in communication, or a barrier is arranged in the gap between adjacent two nucleic acid extraction and library construction units.

6. The digital microfluidics chip of claim 1, wherein: The library constructed by the library construction module is used for nanopore sequencing or second-generation sequencing.

7. The digital microfluidics chip of claim 6, wherein: At least a part of the bottom plate is optically transparent.

8. The digital microfluidics chip of claim 1, wherein: ​ 9. The digital microfluidics chip of claim 1, wherein: ​ 10. The digital microfluidics chip of claim 9, wherein: The optical transmittance of the optically transparent portion of the bottom plate is greater than 50% in the visible light range, or greater than 70%, or greater than 80%, or greater than 90%.

11. The digital microfluidics chip of claim 1, wherein: The digital microfluidic chip is pre-loaded with a filling oil with a specified melting point.

12. The digital microfluidics chip of claim 11, wherein: The melting point of the filling oil is greater than 0℃, or greater than 10℃, or greater than 25℃, or greater than 35℃.

13. The digital microfluidics chip of claim 1, wherein: The digital microfluidic chip is pre-sealed with reagents, and the pre-sealed reagents include at least one of reagents required for nucleic acid extraction and reagents required for library construction.

14. The digital microfluidics chip of claim 13, wherein: The pre-sealed reagents have an independent packaging film layer that can be broken to release the pre-sealed reagents in the digital microfluidic chip during use.

15. The digital microfluidics chip of claim 14, wherein: The breaking mode of the packaging film layer includes heating or mechanical puncture.

16. An integrated sequencing system, comprising: Comprising: a nucleic acid extraction and library construction module comprising the digital microfluidic chip according to any one of claims 1-15, for performing nucleic acid extraction and library construction; a sequencing module for sequencing the library constructed by the nucleic acid extraction and library construction module; a library transfer module for transferring the library constructed by the nucleic acid extraction and library construction module to the sequencing module for sequencing.

17. The integrated sequencing system of claim 16, wherein: The nucleic acid extraction and library construction module comprises a droplet control module for providing an electrical signal to the digital microfluidic chip to operate the liquid or droplets in the digital microfluidic chip.

18. The integrated sequencing system of claim 16, wherein: The nucleic acid extraction and library construction module comprises at least one magnet control module for controlling the movement of one or more magnets to drive the movement of magnetic beads in the digital microfluidic chip, and each magnet has at least two degrees of freedom.

19. The integrated sequencing system of claim 18, wherein: The magnet includes a focused magnet.

20. The integrated sequencing system of claim 16, wherein: The nucleic acid extraction and library construction module comprises at least one temperature control module for heating or cooling a specified region of the digital microfluidic chip to a specified temperature.

21. The integrated sequencing system of claim 16, wherein: The nucleic acid extraction and library construction module further comprises a library quantification module, which comprises an excitation light source and an optical detection module, the excitation light source is used to project excitation light to the nucleic acid concentration measurement region of the digital microfluidic chip, and the optical detection module is used to perform optical measurement on at least one site of the digital microfluidic chip.

22. The integrated sequencing system of claim 16, wherein: The library transfer module comprises a peristaltic pump for providing power for the transfer of liquid.

23. The integrated sequencing system of claim 16, wherein: The library transfer module comprises a flow meter for quantitative monitoring of the transferred liquid.

24. The integrated sequencing system of claim 23, wherein: The flow meter includes an ultrasonic flow meter that is not in contact with the liquid.

25. The integrated sequencing system of claim 16, wherein: The library transfer module comprises a deaerator arranged in the liquid transfer path.

26. The integrated sequencing system of claim 16, wherein: The sequencing module comprises a nanopore sequencing module, a single molecule fluorescent sequencing module, or an NGS sequencing module, and the nanopore sequencing module uses a biological nanopore or a solid-state nanopore.

27. An integrated sequencing method, comprising: Comprising the following steps: a nucleic acid extraction and library construction step: completing nucleic acid extraction and library construction on the digital microfluidic chip according to any one of claims 1-15; a library transfer step: transferring the library constructed in the digital microfluidic chip to a sequencing module; a sequencing step: completing sequencing on the sequencing module.

28. The integrated sequencing method of claim 27, wherein: In the nucleic acid extraction and library construction step, the temperature control module is first set to a specified temperature to melt the filling oil in the digital microfluidic chip, and the sample to be tested is added to the designated reservoir of the digital microfluidic chip. If the required reagents are not pre-sealed in the digital microfluidic chip, they are also added to the designated reservoir of the digital microfluidic chip.

29. The integrated sequencing method of claim 27, wherein, The following steps are also included: Analyzing the sequencing data; Transferring the analysis results to the user.

30. The integrated sequencing method of claim 27, wherein, In the nucleic acid extraction and library construction step, the nucleic acid extraction is performed using a magnetic bead method, including the following steps: In the lysis tank of the nucleic acid extraction module, the sample to be tested, lysis solution, and magnetic beads are added. The lysis solution is used to release the nucleic acid molecules in the sample to be tested, and the magnetic beads are used to adsorb the nucleic acid molecules. The magnetic beads adsorbed with nucleic acid molecules in the lysis tank are gathered from below the digital microfluidic chip using a magnet, and are moved along the bottom plate of the digital microfluidic chip to the nucleic acid washing tank of the nucleic acid extraction module for washing to remove impurities adsorbed on the magnetic beads. The washed magnetic beads are moved to the nucleic acid elution tank of the nucleic acid extraction module using a magnet, and the nucleic acid elution tank is added with an elution solution to elute the nucleic acid molecules adsorbed on the magnetic beads into the solution.

31. The integrated sequencing method of claim 27, wherein, The library construction includes one or more of the following steps: Fragmenting the nucleic acid molecules using an enzyme reaction; End repair of the nucleic acid molecule fragments using an enzyme reaction; Adding an A tail to the ends of the nucleic acid molecule fragments using an enzyme reaction; Adding adapters to the nucleic acid molecule fragments using an enzyme reaction; PCR amplification; Purifying the nucleic acid molecules using a magnetic bead method; Library quantification.

32. The integrated sequencing method of claim 27, wherein: At least a portion of the reagents are added to the digital microfluidic chip by pre-sealing, which includes placing the reagents to be pre-sealed at a designated position in the digital microfluidic chip, and fixing the position of the reagents to be pre-sealed in the digital microfluidic chip by lowering the temperature to solidify the filling oil in the digital microfluidic chip. When performing nucleic acid extraction and library construction in the digital microfluidic chip, the digital microfluidic chip is first heated to a specified temperature to change the filling oil in the digital microfluidic chip from a solid state to a liquid state, so that the pre-sealed reagents can be moved in the digital microfluidic chip.

33. The integrated sequencing method of claim 27, wherein: The multiple nucleic acid extraction and library construction units in the digital microfluidic chip are operated synchronously.

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