Method for removing a droplet from a digital microfluidic chip

Abstract

The present application provides a method for effectively removing a droplet from a digital microfluidic chip filled with a filler fluid. Specifically, by selecting a filler fluid with a melting point higher than that of the droplet, moving the droplet to a designated area in the chip, and lowering the temperature of the designated area to below the melting point of the filler fluid in the designated area and above the melting point of the droplet in the designated area, the filler fluid in the designated area becomes solid while the droplet remains liquid. Thus, when the droplet is removed from the digital microfluidic chip, the amount of filler fluid that can be mixed in is minimized, even to zero. This can simplify the subsequent storage, handling, and use of the removed droplet.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biomedical technology, and in particular to a method for removing droplets from a digital microfluidic chip. BACKGROUND

[0002] There are many instruments for biomedical detection and analysis, and in the use of these instruments, a large proportion of the sample or reagent required for the reaction is wasted without participating in the reaction or measurement. In the digital microfluidic (DMF) system, the dead volume (the volume of liquid added to the system but not participating in the reaction and detection) can be significantly reduced, sometimes even to zero, which 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, because smaller reaction volumes can shorten the time for mixing and reaction of reagents and samples, and the time required for detection and analysis can also be greatly shortened. Moreover, many current biomedical analysis systems require a large number of manual processing steps when used, while digital microfluidic-based systems (including DMF chips and instruments) can provide high integration and automation, greatly reducing the possibility of human error and thus improving the reliability and data quality of the experiment.

[0003] In a digital microfluidic system based on droplet control, liquid is manipulated in a discrete format (droplets) in two-dimensional space, and each droplet can be independently controlled, which is why the technology is called digital microfluidic. In a digital microfluidic device, the motion path of the droplets can be determined at the time of the experiment and can be dynamically changed. Compared with the usual pressure-driven (by vacuum pump) or centrifugal force-driven (by rotating centrifugal device) pipe microfluidics, digital microfluidic devices only require low voltage to control the droplets therein. The driving force in digital microfluidics is based on the electric field effect, such as electrowetting or dielectrophoresis, which is usually not related to the biological sample to be detected, which makes the design of the digital microfluidic system independent of the specific detection project, and thus has better universality.

[0004] In recent years, digital microfluidic technology has attracted widespread attention due to its ability to handle individual droplets, as well as its advantages of easy miniaturization, integration and automation. Digital microfluidic technology reduces the amount of reagent used, simplifies the experimental steps, and shortens the detection time, and has great advantages in medical diagnosis, gene sequencing, DNA synthesis, drug development, and other fields.

[0005] The following is a brief introduction to the technical field that may be involved in the present application.

[0006] Polymerase Chain Reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments, a special DNA replication outside the organism, its biggest feature is to greatly increase the trace amount of DNA. PCR requires repeated temperature cycling of the reaction system, which contains specific DNA primers, dNTP (deoxy-ribonucleoside triphosphate), heat-stable DNA polymerase (such as Taq enzyme) and other components. Each temperature cycle will double the number of target DNA molecules (theoretically, but in practice it will be a little smaller), resulting in exponential growth of the number of target sequences. The technology amplifies the trace amount of DNA or RNA in the sample to a level that can be measured and analyzed. PCR technology has been applied in many different fields, including viral load testing, foodborne pathogen quantification, clinical diagnosis, drug resistance analysis and forensic science. Using PCR technology, doctors and researchers can determine the source of viral infection by analyzing a single cell. Many infectious organisms can now be detected using PCR, such as COVID-19, HIV, hepatitis B, hepatitis C, SARS virus, West Nile virus, Mycobacterium tuberculosis, etc.

[0007] Gene Sequencing, also known as DNA Sequencing, is a new type of genetic testing method that can analyze and determine part or all of the gene sequence from biological samples (such as blood, saliva, etc.). Gene sequencing technology can lock in individual disease genes and prevent and treat in advance. Currently, gene sequencing-related products and technologies have evolved from laboratory research to clinical use, which is of great significance.

[0008] The current main sequencing technologies are: 1) Sanger et al. invented the end-termination sequencing method in 1977, which is also commonly known as first-generation sequencing; 2) second-generation sequencing technology of sequencing by synthesis; 3) third-generation sequencing technology that does not require PCR amplification to achieve individual sequencing of each DNA molecule; 4) fourth-generation sequencing technology - without biological or chemical processing of DNA or RNA, real-time sequencing by inferring base composition from "electrical signal" changes generated by ssDNA (single strand DNA) or RNA template molecules through nanopores.

[0009] Next Generation Sequencing (NGS), also known as High Throughput Sequencing (HTS), has been widely accepted due to its low cost, high accuracy (more than 99%) and the ability to sequence hundreds of thousands to millions of DNA molecules simultaneously in a short time. The first step in NGS is library construction. DNA library, strictly speaking, should be called cDNA library, which refers to the collection of cDNA obtained by reverse transcription of mRNA from all expressible gene fragments in the genome of an organism. In NGS, the library usually refers to the collection of DNA fragments with similar lengths and adaptors at the 3' and 5' ends corresponding to the same sample. Multiple libraries (each with a unique adaptor) are usually pooled together for sequencing on a sequencer. Library construction is the process of processing the DNA to be sequenced into the format required for sequencing. This formatting process may include sample fragmentation, end repair, A-tailing, adaptor ligation, concentration amplification, purification (such as magnetic bead cleaning), etc. Library construction is a very important step in DNA sequencing, as it directly affects the quality of sequencing. If the library construction is not good, the sequencing quality cannot be good.

[0010] For the illumina sequencing platform, which has the largest market share, DNA libraries are mainly divided into: DNA small fragment library, DNA large fragment library, exon library, PCR-Free library and single cell library, etc. The following is a brief introduction to some library construction processes.

[0011] The typical DNA small fragment library construction process includes: 1) Fragmentation - breaking the DNA molecules to be sequenced into fragments of a certain length (usually 150-500 bp) using ultrasonic or enzyme digestion; 2) End repair (ER) - repairing the ends of the broken genome; 3) A-tailing - adding A to the 3' end of the repaired fragments and phosphorylating the 5' end; 4) Adaptor ligation - ligating adaptors to the repaired fragments; 5) Magnetic bead purification; 6) DNA elution; 7) PCR amplification of the adaptor-ligated DNA fragments; 8) Magnetic bead purification; 9) DNA elution; 10) Library collection.

[0012] The construction of the DNA large fragment library mainly includes the following steps: 1) breaking the genome to be sequenced; 2) end repair and biotin labeling of the broken genome; 3) circularization of the labeled genome; 4) breaking the circularized genome; 5) capturing the fragmented genome by magnetic beads; 6) end repair plus A and adapter addition to the captured genome; 7) PCR amplification of the adapter-ligated genome; and 8) library collection.

[0013] The construction of the exon library mainly includes the following steps: 1) ultrasonic breaking of qualified genomic DNA into fragments of about 200-300 bp; 2) end repair (ER) of the broken genomic DNA fragments, 3' end A addition and 5' end phosphorylation (AT); 3) adapter ligation to the repaired fragments; 4) linear amplification (LM-PCR) of the adapter-ligated DNA fragments to prepare a hybridization library; 5) probe hybridization capture of the constructed library; 6) PCR amplification of the captured library; and 7) library collection.

[0014] As can be seen from the above simple description, the steps of the prior art sequencing library construction are relatively cumbersome and the cost is also relatively high. The inventors have found through a large number of experiments that the construction of a library on a digital microfluidic chip can automate the entire process and greatly reduce the amount of reagents used, thereby reducing the cost. In order to prevent sample evaporation and cross contamination during the construction of a library or other reactions (such as DNA synthesis, etc.) on a digital microfluidic chip, a filling liquid that is not miscible with the reaction liquid, such as silicone oil, fluorosilicone oil, mineral oil, etc., is usually added in the chip. When the library is collected, a certain amount of filling liquid may be collected together with the library, which will affect the subsequent concentration measurement, sequencing, etc. to some extent. The library and the filling liquid can be separated by centrifugation, etc., but these steps will increase the cost of the entire sequencing, and are also prone to cause cross contamination. Therefore, accurate collection of the library obtained through a long library construction process is of great significance. SUMMARY

[0015] In view of the above-mentioned disadvantages of the prior art, the purpose of the present application is to provide a method for removing a droplet from a digital microfluidic chip, so as to solve the problems of cumbersome steps and high cost in the prior art sequencing library construction. In the present application, a filling liquid with a higher melting point than that of a specified droplet is filled in the digital microfluidic chip, the droplet is moved to a specified area in the chip, and the temperature of the specified area is lowered to below the melting point of the filling liquid in the specified area but above the melting point of the droplet in the specified area, so that the filling liquid in the specified area becomes solid while the droplet remains liquid. In this way, when the droplet is removed from the digital microfluidic chip, the amount of filling liquid that may be mixed can be minimized, even to zero.

[0016] The method of the present application is based on a digital microfluidic chip, which generally comprises a bottom plate and a top cover plate. The bottom plate comprises a substrate provided with a plurality of droplet control electrodes (the substrate is not conductive), and a medium layer covering at least part of the electrodes, which has at least partial hydrophobic properties. The top cover plate comprises a surface substantially parallel to the bottom plate, which is provided with at least one electrode, and a medium layer covering at least part of the electrode, which has at least partial hydrophobic properties. There are also access ports (sample loading and sampling holes) for loading and unloading samples and reagents, and optional liquid tanks for reagent storage and reaction. In another aspect, some electrodes on the bottom plate form a plurality of paths for NGS library construction reactions, PCR amplification and the like required by experiments. In another aspect, the digital microfluidic chip optionally comprises a device housing, which provides protection for the bottom plate and the top cover plate bonded together, and provides an interface for the control signals of the instrument.

[0017] The present application uses an instrument for droplet operation, temperature control, magnetic control and the like on a digital microfluidic chip. Through software control, the instrument can implement a complete experimental process, such as NGS library construction reaction, PCR amplification, DNA synthesis and the like, on the digital microfluidic chip, without the need for manual intervention. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figures 1A-1B A schematic diagram showing droplet operation and temperature control in a digital microfluidic chip.

[0019] Figures 2A-2D A schematic diagram showing effective droplet removal from a digital microfluidic chip through temperature control.

[0020] Figures 3A-3B An exemplary structural schematic diagram of a digital microfluidic chip.

[0021] Figures 4A-4D An exemplary structural schematic diagram of a digital microfluidic instrument.

[0022] Figures 5A-5E A schematic diagram showing effective droplet removal from a digital microfluidic device through instrument temperature control.

[0023] Figure 6 An exemplary schematic diagram showing the completion of automated NGS library construction by a digital microfluidic system (including a chip and an instrument).

[0024] Figure 7 An auxiliary explanatory schematic diagram of the Yang-Laplace equation. DETAILED DESCRIPTION

[0025] For the purposes of the present disclosure, the word "comprise" and variations such as "comprises" and "comprising", shall not be understood as "consist only of", "consists only of" or "consists entirely of".

[0026] Throughout the description of this patent, "one embodiment", "one example", or "one aspect" means that a described embodiment or aspect includes some, but not necessarily all, of the features, structures, or characteristics described. Moreover, the patent discloses features, structures, or characteristics in any combination that can be applied to one or more embodiments.

[0027] Throughout the description of this patent, the term "Dielectrophoresis" refers to an effect that a neutral particle experiences a force in a non-uniform electric field. When a particle suspended in a liquid medium is subjected to a non-uniform electric field, it can move to a region with a stronger electric field (positive dielectrophoresis) or a region with a weaker electric field (negative dielectrophoresis). Unlike electrophoresis, the particles subjected to dielectrophoresis can be neutral particles without electric charges, and the dielectrophoresis force is not sensitive to the polarity of the electric field. Both direct current and alternating current can be used to generate the dielectrophoresis effect. All particles will experience a certain degree of dielectrophoresis in a non-uniform electric field, and the strength of the dielectrophoresis 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 the convenience of operation, the frequency of the electric field is often used as a parameter for adjusting the control of different particles in the liquid. See the patent "Dielectrophoresis based apparatuses and methods for the manipulation of particles in liquids" (International Patent No. WO2014 / 036915 Al) for a detailed description.

[0028] For the purpose of this disclosure, the term "electrowetting" is used to refer to the effect that the contact angle of a liquid with a solid surface changes with the applied electric field. It should be noted that when the applied voltage or electric field is alternating, both "electrowetting" effect and "dielectrophoresis" effect exist, and when the frequency of the voltage or electric field increases, the relative proportion of the "dielectrophoresis" effect usually increases accordingly. In this invention, "electrowetting" effect and "dielectrophoresis" effect are not strictly distinguished, and both refer to the mechanism of using electric field to operate liquid droplets.

[0029] For the purposes of the present disclosure, the term "microfluidics" refers to a device, system or method that has the ability to manipulate a fluid having at least one cross-sectional dimension in the range of a few microns to about a few millimeters. As a subfield of microfluidics, "digital microfluidics" refers to a device, system or method that can manipulate one or more individual droplets using some control mechanism, such as electrowetting or dielectrophoresis effects.

[0030] For the purposes of the present disclosure, the terms "digital microfluidic chip", "digital microfluidic device", "digital microfluidic apparatus", "DMF chip", "DMF device", and "DMF apparatus" are used interchangeably and include a first substrate (bottom plate) having a first substrate surface and a second substrate (top cover plate) having a second substrate surface, the second substrate being spaced apart from the first substrate by a distance sufficient to accommodate a droplet disposed in the gap (the space between the first and second substrate surfaces). A plurality of droplet control electrodes are disposed on the bottom plate and at least some of the electrodes are covered by a layer of dielectric and at least a portion of the dielectric layer is hydrophobic. For grounding purposes, at least one electrode is disposed on the top cover plate surface, at least a portion of the electrode is covered by a layer of dielectric and at least a portion of the dielectric layer is hydrophobic. It is noted that the digital microfluidic apparatus of the present disclosure can operate on individual droplets (e.g., create, move, merge, split, etc.) using electrical signals, although electrowetting and dielectrophoresis are the two common droplet control mechanisms, the present disclosure does not limit the possibility of using other control mechanisms, such as electrophoresis, electro-osmosis, opto-electrowetting, or combinations thereof, etc.

[0031] For the purposes of the present disclosure, the term "droplet" refers to a quantity of liquid (one or a mixture of several) partitioned from other portions by air or other gas, other (typically immiscible) liquids, and solid surfaces (e.g., internal surfaces of a DMF apparatus), etc. The volume of a "droplet" can range widely, typically from a few picoliters to a few hundred microliters. A "droplet" can have any shape, including spherical, semi-spherical, flattened circular, irregular, etc.

[0032] For the purpose of the present disclosure, the term "reservoir" or "liquid reservoir" is used to indicate a portion on a DMF device that can be used to store, hold, and supply liquid, which can be fully enclosed or partially enclosed. The reservoir can be associated with a fluid path that allows liquid to be introduced into the DMF device gap for droplet operations, or from the DMF device gap into the reservoir for liquid storage or temporary holding.

[0033] For the purpose of the present disclosure, the term "reaction" refers to both physical and chemical reactions between samples and reagents. A physical reaction refers to a change in the state or form of existence of a substance without a change in the nature of the substance itself; for example, dissolving a lyophilized reagent in a solvent (e.g., deionized water), washing magnetic beads to remove adsorbed impurities from the magnetic beads in a washing solution, uniformly mixing two miscible liquids (or a solid and a liquid), cooling a filled liquid from a liquid state to a solid state, etc. are all physical reactions. A chemical reaction refers to the process of rearranging atoms in a molecule to form a new molecule, which is essentially a process of breaking old chemical bonds and forming new chemical bonds; for example, PCR reactions, DNA enzyme digestion reactions, DNA fragment and DNA end repair, A addition, linker addition, etc. are all chemical reactions.

[0034] For the purpose of the present disclosure, the term "reaction procedure" refers to the process of reacting and / or measuring one or more substances in a specified order and under specified conditions (e.g., degree of mixing, temperature, magnetic field strength, electric field strength, time, mass ratio, etc.).

[0035] For the purpose of the present disclosure, the term "reaction substance" refers to all substances involved in the experiment added to the chip, including samples and reagents. Samples and reagents are usually added to the chip in liquid form, but can also be added in solid form, such as lyophilized reagents. In addition, reaction substances can be used immediately after being added, or can be pre-packaged in the chip for use at a later time; for example, the chip can be pre-packaged with specified reagents (which can be liquid reagents or lyophilized reagents) when the chip is being manufactured, the chip is transported to the place where the user needs it, and the user adds samples to the chip with pre-packaged reagents to start the corresponding experiment.

[0036] For the purpose of the present disclosure, the terms "lyophilized reagent" and "lyophilized reagent" can be used interchangeably to refer to a reagent prepared using a lyophilization method, which is usually used for reagents containing active substances that are not resistant to high temperatures. Biological reagent preservation is an important part of medical diagnosis. From the perspective of reagent preservation, many nucleic acid point-of-care testing (POCT) reagent cartridges need to be stored at room temperature, which requires that the detection reagent must be dehydrated to exist in solid form. When used, the subsequent reaction is carried out by reconstituting with a buffer. For example, liquid reagents are dropped into liquid nitrogen and solidified into small spherical shapes in a very short time, and then the solid small balls are placed into a pre-cooled lyophilizer, a lyophilization curve is designed, and freeze-drying is completed. The lyophilized ball form can maximize the activity of the enzyme, and the lyophilized small ball has a loose network structure and is quickly reconstituted. The lyophilized solid small ball, after packaging, can be stored and transported at room temperature, greatly reducing the transportation cost and preservation time. These outstanding advantages of lyophilized bead technology are not only suitable for nucleic acid reagent POCT process production process, but also suitable for other biological products that require preservation, ensure the activity of the active substance, and the production process of room temperature storage and transportation. In the past few years, this new technology has been rapidly applied.

[0037] For the purpose of the present disclosure, the term "surfactant" refers to a substance that can reduce the surface tension of a target liquid, has a fixed hydrophilic and lipophilic group, and can be oriented on the surface of the liquid. The molecular structure of the surfactant has two properties, one end is a hydrophilic group, and the other end is a hydrophobic group; the hydrophilic group is often a polar group, such as carboxylic acid, sulfonic acid, sulfuric acid, amino, or amine group, and salt, hydroxyl, amide group, ether bond, etc. can also be used as a polar hydrophilic group; and the hydrophobic group is often a non-polar hydrocarbon chain, such as a hydrocarbon chain of 8 or more carbon atoms. Surfactants are classified into ionic surfactants (including cationic surfactants and anionic surfactants), non-ionic surfactants, zwitterionic surfactants, combination surfactants, etc.

[0038] In order for the surfactant to exhibit unique interfacial activity, there must be a certain balance between the hydrophobic group and the hydrophilic group. The hydrophilic-lipophilic balance (Hydrophile-Lipophile Balance), abbreviated as HLB value, represents the hydrophilic and hydrophobic properties of the surfactant.

[0039] The non-ionic surfactant is a surfactant with ether group as the main hydrophilic group in the molecule which does not dissociate in aqueous solution, and does not exist in the form of ions in the solution, and the surface activity is embodied by neutral molecules, so it has good stability, is not easily affected by strong electrolytes, is not easily affected by acid and alkali, can be easily mixed with other types of surfactants, has good solubility in various solvents, does not strongly adsorb on solid surfaces, and therefore the surfactant used in the application is usually a non-ionic surfactant. The HLB value of the non-ionic surfactant ranges from 0 to 20, the HLB value of a paraffin molecule composed of only hydrophobic carbon and hydrogen groups is 0, the HLB value of polyethylene glycol (PEG) composed of only hydrophilic oxyethylene groups is 20, and the HLB value of a surfactant with both carbon and hydrogen chains and oxyethylene chains is between the two. The higher the HLB value, the stronger the hydrophilicity, and the lower the HLB value, the stronger the lipophilicity; the surfactant with high lipophilicity or hydrophilicity is easily dissolved in oil or water, so the amount of positive adsorption at the solution interface is less, and the effect of reducing surface tension is weaker. The HLB value can be used as a reference for selecting surfactants.

[0040] For the purpose of the present disclosure, the terms "oil" and "wax" are not specifically distinguished, and refer to neutral, non-polar chemical substances that are liquid at the temperature required for the experiment (usually less than 100 degrees Celsius).

[0041] For the purpose of the present disclosure, the terms "filler liquid" and "filler oil" can be used interchangeably, and refer to substances that can completely or partially fill the gap of a DMF device, are substantially insoluble in droplets, have no obvious effect on droplet control, are compatible with chip materials and coatings, have no obvious effect on the droplet reaction process in the chip; it can be one or more, such as a digital microfluidic chip only filled with a first filler liquid, or containing a first filler liquid and a second filler liquid at the same time, the second filler liquid surrounding the first filler liquid, or in contact with the first filler liquid. The filler liquid can fill the entire gap or part of the gap of the DMF device; the sample or reagent and the filler liquid can also be placed in a specific reservoir on the DMF device, so that the droplet dispensed from the reservoir by electrowetting (or dielectrophoresis) is wrapped in a thin layer of filler liquid.

[0042] The fill liquids (including the first fill liquid and the second fill liquid) are liquid at the conditions (primarily temperature) required for the experiment. The fill liquids have a higher melting point than the melting point of the designated droplet in the digital microfluidic chip. For example, the first fill liquid and the second fill liquid have a melting point of about -10 to 60 degrees Celsius, such as about 0 to 40 degrees Celsius, and more specifically, 5 to 25 degrees Celsius. The first fill liquid and the second fill liquid can include a lower viscosity oil, such as one or more silicone oils, and more specifically, such as one or more fluorosilicone oils; one or more mineral oils, such as one or more paraffin oils; one or more hydrocarbons, such as one or more aromatic hydrocarbons, and / or one or more alkanes. The kinematic viscosity at the temperature at which the droplet is operated (typically less than 100 degrees Celsius) 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.

[0043] The first fill liquid and the second fill liquid can include a small amount of one or more surfactants that are soluble therewith, including non-ionic surfactants, such as Triton X-15 (Octylphenol ethoxylate, HLB value of 4.9), Span 20 (Sorbitan monostearte, chemical formula of C18H34O6, HLB value of 8.6), Span 40 (Sorbitan monopalmitate, chemical formula of C22H42O6, HLB value of 6.7), Span 60 (Sorbitan monostearte, chemical formula of C24H46O6, HLB value of 4.7), Span 65 (Sorbitan tristearate, chemical formula of C60H114O8, HLB value of 2.1), Span 80 (Sorbitan monoleate, chemical formula of C24H44O6, HLB value of 4.3), Span 83 (Sorbitan sesquioleate, chemical formula of C66H126O16, HLB value of 3.7), Span 85 (Sorbitan trioleate, chemical formula of C60H108O8, HLB value of 1.8), and fluorinated surfactants.

[0044] In the present application, the mass ratio of the surfactant to the first filling liquid is generally no more than 10%, such as less than or equal to 1%, or less than or equal to 0.1%, and the HLB value of the surfactant is generally less than 15, such as less than 10, or less than 5.

[0045] It should be noted that whether the surfactant is added to the first filling liquid does not limit the addition of a surfactant compatible with the droplet in the reaction droplet. For example, an appropriate amount of Tween 20 (polyoxyethylene-20-sorbitan monolaurate) (HLB = 16.7), Tween 40 (polyoxyethylene-40-sorbitan monolaurate) (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 ethoxylate) (HLB = 13.5) and the like are often added to the reaction droplet to reduce the aggregation of magnetic beads in the droplet in the absence of an external magnetic field, reduce the adsorption of protein molecules in the droplet on the surface of the chip, etc.

[0046] In many applications, such as immunoassay, nucleic acid analysis, NGS library construction, etc., magnetic beads will be included in the reaction droplet, and magnetic bead washing is a common step in the reaction process, and ethanol is a commonly used component in magnetic bead washing. Therefore, in related applications, a first filling liquid that is not mutually soluble or has relatively weak mutual solubility with ethanol at room temperature needs to be selected.

[0047] For the purposes of the present disclosure, the terms "dye" and "stain" are used interchangeably to refer to a substance that is added to the fill fluid that has no significant effect on the experimental conditions and results, and that can be used to aid in better visualization of experimental phenomena and effects; it can be a fluorescent dye (a substance that absorbs light of one wavelength and emits light of a different wavelength that is longer than the absorbed light), or it can be a non-fluorescent dye. The dye used in the present invention is miscible with the fill fluid system (including any surfactants that can be mixed therein), but is substantially immiscible with the reaction droplet, even if the first fill fluid and / or the second fill fluid contain one or more surfactants. As a non-limiting example, one or more dyes (stains) are mixed homogeneously with the fill fluid, such that the resulting fill fluid has a color (e.g., yellow, red, green, etc.) that is suitable for observation but is different from the color of the fluid in the chip, and then the fill fluid is injected into the digital microfluidic chip, and it is easy to observe whether the fill fluid components are present in the sampler when the droplet is removed from the chip. The mass ratio of the dye to the first fill fluid is typically less than 20%, such as less than 10%, or less than 5%, or less than 1%.

[0048] It is noted that the presence of a dye in the fill fluid does not limit the presence of a dye in the reaction droplet that is miscible with the droplet. Of course, to facilitate observation of the experiment, the color of the dye that can be present in the droplet should be different from the color of the dye that can be present in the fill fluid.

[0049] It is noted that the fill fluid can contain other components in addition to the possible surfactants or dyes.

[0050] For the purposes of the present disclosure, the term "substantially immiscible" between two substances generally means that after mixing, the two substances exist more in their pre-mixed physical state (liquid or solid) in a continuous phase. For example, when a small amount of oil is mixed in water, the oil exists primarily on the surface of the water, or in the water as droplets; from a quantitative perspective, substantially immiscible means that the total mass (or volume) of the substance that exists in a continuous phase (on the order of microns or larger) after mixing is 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 substance prior to mixing.

[0051] Unless otherwise specified, the terms "miscible" and "mutually miscible" are used interchangeably in the present disclosure to mean that one substance and another substance can be mixed homogeneously, such that the original substances no longer exist in a continuous phase in the mixture, i.e., both substances are present in any microscopic region (e.g., on the order of microns or smaller). Unless specifically indicated, mutually miscible substances in the present disclosure do not undergo a chemical reaction with each other.

[0052] Unless otherwise indicated, in the present disclosure, the melting point of a substance refers to the melting point of the substance at one standard atmosphere (atm).

[0053] Unless otherwise indicated, in the present disclosure, the term "less than" generally means "equal to or less than," and "greater than" generally means "equal to or greater than."

[0054] For the purposes of the present disclosure, the terms "droplet driving," "droplet control," and "droplet manipulation" can be used interchangeably and can include droplet generation (from a reservoir or continuous liquid), movement, merging and mixing, splitting (symmetric or asymmetric), shaping (into a specified shape), suspending and dispersing particles (within a liquid or droplet), etc.

[0055] Unless otherwise indicated, in the present disclosure, the term "chip" is an abbreviation for "digital microfluidic chip."

[0056] The present disclosure presents instruments, devices, and methods for processing or measuring a target analyte in a sample solution. As will be appreciated by those skilled in the art, the sample solution can include, but is not limited to, a bodily fluid (including, but not limited to, blood, serum, saliva, urine, etc.), a purified sample (e.g., purified DNA, RNA, protein, cells, etc.), an environmental sample (including, but not limited to, water, air, agricultural samples, etc.), and a biological warfare agent sample, etc. The bodily fluid can be from any organism. In some embodiments, the bodily fluid can be from a mammal, e.g., a human.

[0057] For the purposes of the present disclosure, the terms "no significant effect" or "no significant effects" can be used interchangeably to mean that the effect of a newly introduced factor (such as a material, process, formulation, temperature, etc.) on the experimental results is within the range of allowable error.

[0058] For the purposes of the present disclosure, the terms "analyte" and "analyte to be analyzed" can be used interchangeably to mean the substance or chemical component to be measured or tested in an analysis or test. The "analyte" can be an organic or inorganic substance. It can refer to a biomolecule (such as a protein, lipid, cytokine, hormone, carbohydrate, etc.), a virus (such as a herpes virus, retrovirus, adenovirus, lentivirus), an intact cell (including prokaryotic and eukaryotic cells), an environmental contaminant (including toxins, pesticides, etc.), a pharmaceutical molecule (such as antibiotics, pharmaceuticals, and drug abuse, and drugs), a nucleus, a spore, etc.

[0059] For the purposes of the present disclosure, 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.

[0060] For the purposes of the present disclosure, the terms "magnet" and "magnetic body" are used interchangeably to refer to a body having a shape and magnetic properties, including samarium cobalt magnets, neodymium iron boron magnets, ferrite magnets, alnico magnets, and ferroxcube magnets. Shapes include cylindrical, toroidal, disc, conical, pyramidal, and other irregular shapes. Magnets include permanent magnets, which are always magnetic, and non-permanent magnets, such as electromagnets, which are only magnetic under certain conditions (e.g., when current is passing through them).

[0061] For the purposes of the present disclosure, the term "focusing magnet" is used to refer to a magnet (permanent or electromagnet) having a shape such that the magnetic field is stronger on one side than on the opposite side. Examples include, but are not limited to, conical magnets or pyramidal magnets. The magnetic field at the tip of a conical magnet (or pyramidal magnet) is stronger than the magnetic field at its base.

[0062] In the present disclosure, the term "particle" is used to refer to entities of the order of microns or nanometers, which can be natural or artificially made, such as cells, subcellular components, liposomes, viruses, nanospheres, and microspheres, or smaller entities such as biological macromolecules, proteins, DNA, and RNA. A "particle" can also refer to liquid droplets that are not miscible with the suspending medium, and can also refer to small gas bubbles in a liquid. The (linear) size of a "particle" can range from a few nanometers to a few hundred microns.

[0063] In the present disclosure, the term "bead" can be any bead or particle that reacts with a solution. The bead can be of any different shape, such as spherical, egg-shaped, cubic, disc-shaped, or irregular. The bead can be inside a droplet, on the inner surface of a DMF device, in the filling liquid of a DMF device, in a liquid bath, etc. The bead can be made of a variety of materials, such as resins, polymers, glass, nanomaterials, etc., and can be of any size, such as microbeads and nanobeads. The bead can be magnetically responsive, in which case at least one or some of its components is made of a magnetically responsive material, while the remaining material can include polymeric material, a coating, or a group linked to a detection reagent, etc. Examples of beads include quantum dots, polyethylene microbeads, silica microbeads, fluorescent microspheres or nanospheres, magnetic microbeads, magnetic nanobeads, flow cytometry microbeads, etc.

[0064] For the purposes of the present disclosure, the term "magnetic bead" refers to a bead comprising a magnetically responsive material. Examples of magnetically responsive materials include ferromagnetic materials, paramagnetic materials, superparamagnetic materials, ferrimagnetic materials, and the like. Examples of paramagnetic materials include metals such as nickel, iron, and cobalt, as well as metal oxides such as Fe3O4, Cr2O3, NiO, Mn2O3, and the like. The magnetically responsive material can substantially constitute the entirety of the magnetic bead, a portion of the magnetic bead, or a component of the magnetic bead. The remainder of the magnetic bead can include polymeric material and coating portions that allow for attachment of a target particle. Magnetic beads can be used in a variety of assays in which the magnetic beads are typically used to bind one or more target species, such as an analyte or a contaminant, in a mixture. Assays often require an efficient magnetic bead washing process to reduce the amount of one or more target species in the mixture that are not bound to the surface of the magnetic bead, thereby avoiding interference with the assay results.

[0065] Verification experiments based on digital microfluidics demonstrated that magnetic beads coated with antihuman serum albumin antibodies can be used to isolate human serum albumin. Verification experiments for extracting DNA from a whole blood sample using magnetic beads were also performed on a digital microfluidics platform. DNA extraction on a digital microfluidics platform was achieved similarly to traditional methods, which typically require a pre-treatment step that includes cell lysis.

[0066] For the purposes of the present disclosure, "magnetic bead manipulation" or "magnetic bead control" can be used interchangeably to refer to one of the following operations or a combination thereof:

[0067] 1. Focusing - gathering magnetic beads in a droplet on a DMF device, in a reservoir, between droplets (reservoirs), and the like. The size of the focused magnetic beads is less than 10 mm, or less than 5 mm, or less than 2 mm. It should be noted that focused magnetic beads typically have a well-defined boundary, but can also be rather diffuse. The proportion of magnetic beads that are focused is more than 30%, or more than 50%, or more than 70%, or more than 80%, or more than 90% of all the particular magnetic beads.

[0068] 2. Immobilizing - substantially confining magnetic beads to a designated location in a droplet, reservoir, and fill solution on a DMF device, during which time operations such as droplet control can be performed. For example, in one embodiment, immobilized magnetic beads are substantially confined to a location in a droplet to allow for performing operations for droplet splitting, resulting in one droplet that is substantially free of magnetic beads and another droplet (the remaining fluid) that contains a majority of the magnetic beads.

[0069] 3. Transport - moving magnetic beads from one location to another on a DMF device, including but not limited to moving magnetic beads from one droplet or well to another droplet or well.

[0070] 4. Disperse - allowing aggregated magnetic beads to disperse in a droplet or well by removing (or otherwise controlling) a magnetic field, which can be done simultaneously with droplet operations.

[0071] For the purposes of the present disclosure, "amplification" refers to a process that can increase the number or concentration of an analyte to be detected. Non-limiting examples include Polymerase Chain Reaction (PCR) and its variants (e.g., quantitative competitive PCR, immuno-PCR, reverse-transcriptase PCR, etc.), Strand Displacement Amplification (SDA), Nucleic Acid Sequence Based amplification (NASBA), Loop-mediated isothermal amplification (LAMP), Helicase-dependent amplification (HAD), etc.

[0072] For the purposes of the present disclosure, the terms "layer" and "film" are used interchangeably to refer to a structure of a body that is generally, but not necessarily, planar or substantially planar, and is typically deposited, formed, coated, or otherwise placed on another structure.

[0073] For the purposes of the present disclosure, the terms "dielectric layer", "dielectric film", and "dielectric layer" are used interchangeably to refer to a thin film made of a dielectric material on a chip. A dielectric material is an insulating material that can be electrically polarized, and materials for making a dielectric layer include, but are not limited to, Teflon, Cytop, Parylene C, silicon nitride, silicon oxide, etc. Methods for making include spin coating, dip coating, spray coating, sputtering, PECVD (Plasma Enhanced Chemical Vapor Deposition), etc.

[0074] For the purposes of the present disclosure, the term "Ground" (as used in "ground electrode" or "ground voltage") means that the voltage of the respective electrode is zero or sufficiently close to zero so that the electrode (when a ground voltage is applied) does not have a noticeable electro wetting effect on the area of the DMF chip to which it corresponds. All other voltage values, although typically less than 300 volts in magnitude, should be sufficiently high so that electro wetting effects can be observed adequately.

[0075] It should be noted that when a covered dielectric layer is arranged, the spaces between adjacent electrodes in the same layer are typically filled with dielectric material. These spaces can be left empty, or filled with a gas such as air, nitrogen, helium, and argon. All electrodes in the same layer and electrodes at different layers are preferably electrically insulated.

[0076] As used herein, the term "contact angle" denotes the angle formed when a liquid-vapor interface contacts a solid surface. When the three phases - liquid, solid, and vapor (which can be a mixture of ambient atmosphere and equilibrium concentration of the liquid) reach thermodynamic equilibrium, the shape of the liquid-gas interface is determined by the Young-Laplace equation,

[0077] γ SG -γ SL -γ LG coSθ c = 0

[0078] where γ SG represents the solid-gas interface energy, γ SL represents the solid-liquid interface energy, and γ LG represents the liquid-gas interface energy (i.e. surface tension), and θ represents the contact angle at equilibrium. Referring to Figure 7 , which shows the quantities in the Young-Laplace equation. It should be noted that this equation also applies if the vapor phase is replaced by another immiscible liquid phase.

[0079] Within a pure liquid, each molecule is pulled equally in every direction by neighboring liquid molecules, resulting in a net force of zero. However, molecules at the surface of a liquid do not have a net force in all directions balanced by neighboring molecules, they are pulled inward by neighboring molecules, resulting in an internal pressure, which in turn causes the surface area of the liquid to shrink to maintain its lowest surface free energy. This intermolecular force that causes the surface to shrink is called the liquid-gas interface energy γ LG , which is called the surface tension, determines the shape of a liquid drop. Other external forces, such as gravity, also deform the drop. Thus, the contact angle is determined by both the surface tension and the external forces (typically gravity). The contact angle is also a characteristic parameter of a certain solid-liquid system under a particular environment.

[0080] A hydrophobic surface has the property of repelling liquids, while a hydrophilic surface has the property of attracting liquids. For the purposes of the present disclosure, a "hydrophobic surface" has a contact angle greater than 90°, while a "hydrophilic surface" has a contact angle less than 90°.

[0081] For the purposes of the present disclosure, it is understood that when a liquid in any form (such as a droplet or a continuum, possibly in motion or at rest) is described as being "on," "at," or "over" an electrode, array, matrix, and surface, the liquid can be in direct contact with the electrode, array, matrix, and surface, or can be in contact with one or more layers or films interposed between the liquid and the electrode, array, matrix, and surface.

[0082] For the purposes of the present disclosure, it is understood that when a given component such as a layer, region, and substrate is referred to as being disposed on or formed on another component, the given component can be directly on the other component, or alternatively, an intervening component (such as one or more buffer layers, interlayers, and electrodes) can also be present. It is also understood that the terms "disposed on" and "formed on" can be used interchangeably to describe how a given component is positioned or located relative to another component. Accordingly, the terms "disposed on" and "formed on" are not intended to introduce any limitations on the particular methods of material transport, deposition, and fabrication.

[0083] For the purposes of the present disclosure, the terms "printed circuit board" (or PCB) or "printed wiring board" can be used interchangeably to refer to a circuit board without soldered components, which is primarily composed of the following parts.

[0084] 1. Circuitry and pattern: The material used for the circuitry is usually copper, and the circuitry can provide a conductive path between electronic components. In addition, a large copper surface is usually designed as a ground and power layer. The circuitry and pattern are made at the same time.

[0085] 2. Dielectric layer: This is used to maintain insulation between the circuitry and the layers, and is also called the base material.

[0086] 3. Through hole or via: A through hole allows the circuitry on two or more layers to be connected to each other, while a larger through hole is used for component insertion. In addition, a non-through hole is usually used for surface mount positioning.

[0087] 4. Solder mask: Not all copper surfaces need to be tinned with components, so a layer of material that prevents tinning of the copper surface (usually epoxy) is printed in the non-tinning area to prevent short circuits between non-tinning lines. Depending on the process, it is divided into green oil, red oil, and blue oil.

[0088] 5. Silk screen: This is an optional component. Its main function is to mark the name and location of each component on the circuit board, which is convenient for maintenance and identification after assembly.

[0089] For the purpose of this disclosure, the terms "Testing", "Detection" and "Measurement" are used interchangeably to refer to the process of obtaining a physical quantity (e.g., position, charge, temperature, concentration, pH, brightness, fluorescence, etc.). In general, at least one sensor (or detector) is used to obtain the physical quantity and convert it into a signal or information that can be recognized by a person or an instrument. There can be other components between the object to be measured and the sensor, such as lenses, mirrors, filters used in optical measurements, and resistors, capacitors, transistors used in electrical measurements. Moreover, other auxiliary devices or components are often used to make the measurement possible or easier. For example, a light source such as a laser or a laser diode is used to excite a particle from an electronic ground state to an electronic excited state, and the fluorescence emitted when the excited state particle returns to the ground state can be used to measure the concentration of a certain particle in a liquid sample. Optical sensors include CCDs, photodiodes, photomultiplier tubes, and electrical sensors include operational amplifiers, analog-to-digital converters, thermocouples, and thermistors.

[0090] The following is a detailed description of the embodiments of the application for processing biological samples, i.e., the method of removing droplets from a digital microfluidic chip. For the convenience of illustration, the corresponding drawings ( Figures 1A-6 ) will be mentioned when necessary. It should be noted that the purpose of these examples is to help illustrate, not to limit the intent and spirit of the invention.

[0091] The accompanying drawings and detailed description herein serve to further illustrate and describe the principles of the present disclosure, and enable one skilled in the relevant art to make and use the corresponding instruments, DMF devices, and methods described.

[0092] For the purpose of this disclosure, some and all functional modules (e.g., temperature control module, droplet control module, etc.) herein can be automatically controlled. Programs (software or firmware) running on a microprocessor or computer are usually used to achieve automatic control.

[0093] Figures 1A-1BA top view of a DMF device (generally labeled 100, i.e., a digital microfluidic chip) is shown for droplet manipulation and temperature control, including droplet actuation electrodes (typically fabricated on a bottom plate) 112, four sets of droplet actuation electrodes 131-134, which can be used to perform parallel manipulations on multiple droplets 201. Figure 1B Four temperature control modules 301-304, which are independent of the DMF device, are also shown in the figure, which can be used to control different regions of the DMF device 100 to different temperatures.

[0094] Figures 2A-2D A side view of a portion of the DMF device 100 (i.e., a digital microfluidic chip) is shown as a preferred embodiment for efficient liquid removal from the DMF device. The bottom plate 110 includes droplet actuation electrodes 112 and a dielectric layer 113 deposited on a substrate 111. The top plate 150 includes ground electrodes 152 and a dielectric layer 153 deposited on a substrate 151; two liquid reservoirs 154 and 155 are fabricated on the top plate, in which the liquid reservoir 154 is enclosed from the outside, and the liquid reservoir 155 is an open (or open) liquid reservoir, through which liquid, such as a first filling liquid, can be added or removed from the DMF device 100.

[0095] By controlling the droplet actuation electrodes 102 in a specified order, the droplet 201 can be moved from Figure 2A to the position of the liquid reservoir 155 in Figure 2B . Temperature control of the DMF device 100 in the specified region of the liquid reservoir 155 causes the filling liquid 250 to become solid in the specified region, but the droplet 201 remains liquid, as shown in Figure 2C ; at this time, the shape of the droplet can become irregular due to being surrounded by the solid filling oil.

[0096] Figure 2DFigure 21 shows a schematic diagram of the process of piercing the solidified fill oil in the reservoir 155 with the sampler 211 and removing the droplet 201 from the bottom of the DMF device. The original location of the droplet on the chip is now empty (of air or other ambient gas) in the designated area 202. The description here is for the case where the solidified fill oil has a lower density than the droplet. It should be noted that the present application does not exclude the case where the solidified fill oil has a higher density than the droplet. In this case, the droplet will be on top of the solidified fill oil. The sampler should be designed so that it does not insert into the solidified fill oil when removing the droplet, so that the droplet is not contaminated with fill oil. The sampler here refers to a device that sucks liquid into it by controlling the pressure of the gas in it (so that the tip that is in contact with the liquid has a negative pressure). Non-limiting examples include: 1) pipette (also called pipet) - can be divided into air-displacement pipette and positive-displacement pipette according to the principle; 2) dropper (including disposable dropper); 3) ear bulb (also called ear syringe) - a tool made of rubber that can be used to quantitatively extract liquid from a pipette; 4) syringe - a set of devices that use a needle to extract or inject gas or liquid; 5) vacuum pump (requires a small tube that can be inserted into the chip).

[0097] It should be noted that the DMF device structure shown here is for the purpose of illustrating the process of droplet removal and does not represent all possibilities. In some embodiments, the DMF device can be implemented in various different ways. For example, 1) the control electrodes can have different shapes, such as rectangular, square, trapezoidal, pentagonal, hexagonal, and irregular shapes, and can be arranged in a straight line or other shapes; 2) the control electrodes can be in different layers (usually electrically insulated from each other), as described in the patent "Electrowetting Based Digital Microfluidics" (International Patent No. (WO 2008 / 147568); 3) the dielectric layer can also have two or more layers, and the materials used include Parylene C, silicon nitride, silicon dioxide, tantalum oxide, etc.; one of the layers can be a hydrophobic material, such as Teflon, Cytop, and FluoroPel, etc.

[0098] The substrate can be any non-conductive material or conductive material coated with a non-conductive layer, as long as it has sufficient mechanical strength to maintain its shape under the required system operating and storage conditions, it can be transparent, translucent and opaque, such as glass, quartz, plastic (such as polycarbonate (PC), cyclic olefin copolymer (COC), acrylic, etc.), ceramic, PCB, silicon wafer, etc. The electrode can be made of any conductive material, such as metal, alloy and conductive polymer, it can be made of one material or a mixture of different materials, it can be transparent, translucent and opaque. The transparent electrode on the DMF device can be made of transparent conductive material, such as indium tin oxide (ITO), aluminum-doped zinc oxide (AZO), transparent conductive polymer (polyacetylene, polyaniline, etc.), or transparent nanomaterial, etc.

[0099] The voltage control module is used to provide voltage control signals to the droplet control electrodes. It usually has multiple outputs, with a maximum number of 1000000, or 100000, or 10000, or 1000. The voltage output can be unipolar (only positive voltage or only negative voltage) or bipolar (both positive and negative voltage); when the voltage output is bipolar, it can be symmetric or asymmetric; the voltage amplitude is less than 1000 volts, or less than 500 volts, or less than 300 volts, or less than 100 volts, or less than 60 volts, or less than 30 volts. The voltage frequency is less than 10 MHz (megahertz), or less than 1 MHz, or less than 100 KHz (kilohertz), or less than 20 KHz, or less than 5 KHz, or less than 1 KHz AC signal, or DC signal. The waveform of the voltage can be square wave, sine wave, sawtooth, pulse width modulation signal, etc. The voltage control module is usually programmed by a microprocessor or computer on the circuit board through SPI (Serial Peripheral Interface), I2C (Inter-Integrated Circuit), USB (Universal Serial Bus), parallel port (parallel port), Ethernet (Ethernet), Wi-Fi or Bluetooth (Bluetooth) to program the sequence, duration, amplitude, and frequency of the output signal. Spring probe (Spring loaded electrical contact pin, also called Pogo pin) or connector pad can be used to deliver multiple high-voltage control signals to the electrodes on the DMF device.

[0100] Figure 3A and 3Bis the perspective view and top view of the digital microfluidic chip based on the present application. Only the upper cover plate part can be seen here. The material of the upper cover plate is PC, which is made by injection molding process. The hole diameter of the sample loading and taking hole 156 on the digital microfluidic chip is about 3mm. Using this digital microfluidic chip, hybridization capture library construction can be performed on 4 samples simultaneously.

[0101] Figures 4A-4D A preferred embodiment of the control instrument used in the present application is shown. Figure 4A An embodiment of the temperature control module used in the instrument of the present application is shown. The temperature control modules 311 to 316 can be independently temperature controlled, and can be used to control the temperature of the corresponding regions of the digital microfluidic chip to any temperature in the range of -10 to 100 degrees Celsius. Figure 4B is the top-front-side view of the device, and 4C is the top view, Figure 4D is the rear view. The spring probes 350 can be used to implement the interface for droplet control of the instrument and the digital microfluidic chip 100, and to provide voltage signals to the droplet control electrodes on the digital microfluidic chip. The magnet control module 360 can control the position of the magnets above, and can be used to control the magnetic beads in the digital microfluidic chip (e.g. aggregation, fixation, movement, etc.), thereby achieving the capture, enrichment, washing, elution, etc. of nucleic acid molecules on the chip. The magnets on the magnet control module can be aggregation magnets. The touch screen 401 is used to provide a graphical interface operable by the user, for the user to input commands and display help, experimental status, and measurement results, etc. The slidable tray 402 is used to load and unload the digital microfluidic chip, and when pushed in, the spring probes 400 come into contact with the bottom substrate of the digital microfluidic chip. Figure 4D In the above, the air vent 404 is used to discharge heat generated during operation of the instrument (usually equipped with a fan inside); the USB port 405 provides a way for the application software running on the computer to communicate with the instrument (such as sending commands or receiving data); and an AC power port 406 is also provided.

[0102] Figures 5A-5E An example of effective droplet removal from the digital microfluidic chip by temperature control is shown. Figures 5A-5D A digital microfluidic chip 100 placed in the control instrument is shown. The temperature control modules 311 to 316 in the instrument are in contact with the bottom of the digital microfluidic chip 100, and are used to control the temperature of different regions of the digital microfluidic chip. Figure 5A A state is shown in which the chip has been filled with filling oil, but there is no droplet in the sample hole 156. Figure 5B A state is shown in which the chip has been filled with filling oil, and there is a droplet in the sample hole 156. Figure 5CThe temperature module 313 is shown to have reduced the temperature of the designated area 180 to near 0 degrees Celsius, causing the fill oil to solidify, but the droplet in the sample well 156 remains liquid; Figure 5D The sample well 156 is shown to have been sampled by the sampler to extract the droplet through the solidified fill oil; Figure 5E The sampler is shown to have extracted the liquid 201 from one of the sample wells 156 in the chip, with the top 501 and bottom 502 of the droplet 201 in the sampler being air, indicating that the only component in the sampler is the extracted liquid, without the fill oil mixed in.

[0103] Figure 6 The steps S601-S616 of the method 600 show an example of the application for automated library construction on a digital microfluidic chip for NGS, and extraction of the library after the construction is completed.

[0104] In step S601, a digital microfluidic chip is placed in the instrument, and a specified volume of fill oil is added to the digital microfluidic chip through the fill oil well;

[0105] In step S602, reagents required for library construction, including fragmentation reagents (optional), ER and AT reagents, adapter reagents, two types of magnetic bead reagents, PCR reagents, and samples, are added to the digital microfluidic chip through the corresponding sample wells in specified volumes;

[0106] In step S603, the sample droplet and the fragmentation reagent droplet on the digital microfluidic chip are moved from the corresponding sample wells to a designated location on the chip for merging and mixing, and the merged droplet is moved to a designated area on the chip, which has the fill oil (i.e., the first fill liquid). The designated area is controlled to a specified temperature and maintained for a specified period of time to complete the fragmentation of the sample DNA (usually by enzymatic cleavage);

[0107] In step S604, the fragmented sample droplet and the ER and AT reagent droplet on the digital microfluidic chip are moved to a designated location on the chip for merging and mixing, and the merged droplet is moved to a designated area on the chip. The designated area is controlled to a specified temperature and maintained for a specified period of time to complete the ER and AT reaction;

[0108] In step S605, the droplet after the ER and AT reaction and the adapter reagent droplet on the digital microfluidic chip are moved to a designated location on the chip for merging and mixing, and the merged droplet is moved to a designated area on the chip. The designated area is controlled to a specified temperature and maintained for a specified period of time to complete the adapter ligation reaction;

[0109] In step S606, the droplet after the completed joint connection reaction and the magnetic bead droplet on the chip are moved to a designated position on the chip to be mixed and combined, after a designated time, the magnet on the instrument is used to gather the magnetic beads in the mixed droplet in the digital microfluidic chip and transfer them to the first washing liquid tank on the digital microfluidic chip according to a designated route, and then the magnetic beads are moved in the range of the washing liquid tank according to a designated trajectory and speed to realize the washing of the magnetic beads;

[0110] In step S607, the magnet of the instrument is used to transfer the magnetic beads washed in step S606 to the second washing liquid tank on the chip according to a designated route, and then the magnetic beads are moved in the range of the washing liquid tank according to a designated trajectory and speed to realize the re-washing of the magnetic beads;

[0111] In step S608, the magnet of the instrument is used to transfer the magnetic beads washed in step S607 to the first elution tank on the chip according to a designated route, and the magnetic control of the instrument on the chip is stopped (for example, the magnet is moved away from the chip), at this time, the magnetic beads are dispersed in the elution liquid from the aggregated state, thereby realizing the elution of the adsorbed DNA on the magnetic beads;

[0112] In step S609, the magnetic beads in the first elution tank are re-gathered by using the magnetic force control of the instrument, and are moved to a designated position on the chip according to a designated path to be discarded;

[0113] In step S610, a droplet of a designated amount of elution liquid in the first elution tank and a droplet of PCR reagent on the chip are moved to a designated position on the chip to be mixed and combined, and the mixed droplet is moved back and forth on the chip in a designated temperature area to realize the amplification of the PCR concentration;

[0114] In step S611, the droplet after the PCR amplification in step S610 and the second magnetic bead droplet on the chip are moved to a designated position on the chip to be mixed and combined, after a designated time, the magnet on the instrument is used to gather the magnetic beads in the mixed droplet on the chip and transfer them to the third washing liquid tank on the chip according to a designated route, and then the magnetic beads are moved in the range of the washing liquid tank according to a designated trajectory and speed to realize the washing of the magnetic beads;

[0115] In step S612, the magnet of the instrument is used to transfer the magnetic beads washed in step S611 to the fourth washing liquid tank on the chip according to a designated route, and then the magnetic beads are moved in the range of the washing liquid tank according to a designated trajectory and speed to realize the re-washing of the magnetic beads;

[0116] In step S613, the magnetic beads in the second elution tank are moved to a designated position on the chip by the magnetic force control of the instrument, and the magnetic force control of the instrument is stopped (e.g., the magnet is moved away from the chip), at which time the magnetic beads are dispersed in the elution liquid from the aggregated state, thereby achieving elution of the adsorbed DNA on the magnetic beads;

[0117] In step S614, the magnetic beads in the second elution tank are re-aggregated by the magnetic force control of the instrument, and are moved to a designated position on the chip for discarding by a designated path;

[0118] In step S615, the temperature control module of the instrument is used to control the temperature of the second elution tank region of the chip, and the temperature is lowered to below the melting point of the filling oil but above the melting point of the droplet, and is maintained for a specified time, so that the filling oil in the region becomes solid.

[0119] In step S616, the droplet in the specified region of the chip is taken out by using the sampler to pass through (pierce) the solid filling oil at the second elution tank of the chip, thereby obtaining a library that can be used for sequencing on a machine.

[0120] It should be noted that the above examples and advantages are for illustrative purposes only and are by no means exhaustive.

[0121] Although the preferred embodiments of the present application have been shown and described, it should be understood that various changes can be made without departing from the spirit and scope of the present application.

[0122] The above embodiments only illustrate the principles and effects of the present application, and are not intended to limit the present application. Any person skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes made by those skilled in the art without departing from the spirit and technical ideas disclosed by the present application should be covered by the claims of the present application.

Claims

1. A method for removing droplets from a digital microfluidic chip, characterized in that, Including steps: a) Add the first filling liquid to the digital microfluidic chip; b) Add one or more reactive substances to the digital microfluidic chip; c) Execute the specified reaction process on the digital microfluidic chip; d) Move a designated droplet from the digital microfluidic chip to a designated area on the digital microfluidic chip, wherein the designated area contains the first filling liquid; e) Control the temperature of the designated area to be lower than the melting point of the first filling liquid in the designated area, but higher than the melting point of the droplets in the designated area, and maintain it for a designated time so that the first filling liquid in the designated area becomes solid, while the droplets in the designated area remain liquid. f) Use a sampling tool to remove the droplet from the designated area.

2. The method according to claim 1, characterized in that, The melting point of the first filling liquid is -10 to 60 degrees Celsius.

3. The method according to claim 2, characterized in that, The melting point of the first filling liquid is 0 to 40 degrees Celsius.

4. The method according to claim 3, characterized in that, The melting point of the first filling liquid is 5 to 25 degrees Celsius.

5. The method according to claim 1, characterized in that, The first filling liquid contains one or more silicone oils.

6. The method according to claim 5, characterized in that, The silicone oil contains one or more fluorosilicone oils.

7. The method according to claim 1, characterized in that, The first filling fluid contains one or more mineral oils.

8. The method according to claim 7, characterized in that, The mineral oil contains one or more paraffin oils.

9. The method according to claim 1, characterized in that, The first filling liquid contains one or more hydrocarbon compounds.

10. The method according to claim 9, characterized in that, The hydrocarbon compound contains one or more alkanes and / or one or more aromatic hydrocarbons.

11. The method according to claim 1, characterized in that, The first filling solution is insoluble in ethanol at room temperature.

12. The method according to claim 1, characterized in that, The first filling liquid contains one or more surfactants.

13. The method according to claim 12, characterized in that, The mass ratio of surfactant to first filling liquid is no more than 10%.

14. The method according to claim 13, characterized in that, The mass ratio of surfactant to first filling liquid is no more than 1%.

15. The method according to claim 14, characterized in that, The mass ratio of surfactant to first filling liquid is no more than 0.1%.

16. The method according to claim 12, characterized in that, Surfactants include nonionic surfactants.

17. The method according to claim 16, characterized in that, Nonionic surfactants have an HLB value of less than 15.

18. The method according to claim 17, characterized in that, Nonionic surfactants have an HLB value of less than 10.

19. The method according to claim 18, characterized in that, Nonionic surfactants have an HLB value of less than 5.

20. The method according to claim 1, characterized in that, The first filling solution contains one or more staining agents.

21. The method according to claim 1, characterized in that, Droplet control is achieved through electrowetting or dielectrophoresis.

22. The method according to claim 1, characterized in that, The digital microfluidic chip is filled with a second filling liquid, which surrounds or intersects with the first filling liquid.

23. The method according to claim 22, characterized in that, The second filling fluid contains one or more surfactants.

24. The method according to claim 22, characterized in that, The second filling solution contains one or more staining agents.

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