WO2023040012A1 - 集成核酸分析***和测量样本中的目标核酸的方法 - Google Patents

集成核酸分析***和测量样本中的目标核酸的方法 Download PDF

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WO2023040012A1
WO2023040012A1 PCT/CN2021/127976 CN2021127976W WO2023040012A1 WO 2023040012 A1 WO2023040012 A1 WO 2023040012A1 CN 2021127976 W CN2021127976 W CN 2021127976W WO 2023040012 A1 WO2023040012 A1 WO 2023040012A1
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nucleic acid
dmf device
storage tank
magnet
magnetic beads
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PCT/CN2021/127976
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English (en)
French (fr)
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吴传勇
周丽
汪志芳
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上海衡芯生物科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/36—Apparatus for enzymology or microbiology including condition or time responsive control, e.g. automatically controlled fermentors
    • C12M1/38—Temperature-responsive control
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the invention relates to the technical field of biological detection, in particular to an integrated nucleic acid analysis system and a method for measuring at least one target nucleic acid in a sample.
  • the present invention utilizes a digital microfluidics device (Digital Microfluidics, DMF device for short) to realize the integration of magnetic bead method extraction, purification, amplification and measurement of nucleic acid.
  • a digital microfluidics device Digital Microfluidics, DMF device for short
  • Magnetic beads are commonly used in sample processing, such as extraction, separation and purification of analytes.
  • the target analyte is chemically specifically bound to the surface of the magnetic beads, and the separation or purification can be achieved by using a pipette or flowing the liquid through the magnetic beads held by the magnet to remove unwanted particles or liquid.
  • magnets magnetic beads can also be focused and moved away from their original liquid environment to remove unwanted particles or liquid.
  • Magnetic beads are often used as carriers for nucleic acids, antigen antibodies, catalysts, and proteins, and are widely used in DNA isolation, mRNA purification, protein purification, cell separation, immunoassay, biomolecular capture, etc.
  • the liquid is manipulated in a two-dimensional space in a discrete format (droplet), and the droplets can be manipulated individually, which is why it is called digital microfluidics.
  • digital microfluidics In a DMF device, the running path of a droplet can be defined at runtime and can be changed dynamically. Compared with the usual pressure-driven (via external pump) or electrically-driven (via high pressure) channel-based microfluidics, the DMF device requires only low voltage to control the droplets in it.
  • the driving force in digital microfluidics is based on electrostatic effects, such as electrowetting or dielectrophoresis. This electrostatic effect is usually not related to the biological samples to be tested, which makes the design of digital microfluidics systems independent of specific detection items. Therefore, it has better versatility.
  • PCR Polymerase Chain Reaction
  • dNTP deoxy-ribonucleoside triphosphate, deoxyribonucleoside triphosphate
  • thermostable DNA polymerase such as Taq DNA polymerase
  • 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 technology, doctors and researchers can determine the source of a viral infection by analyzing a single cell. Many infectious organisms such as COVID-19, HIV-1, Hepatitis B, Hepatitis C, SARS virus, West Nile virus, Mycobacterium tuberculosis, etc. can now be detected using PCR.
  • nucleic acid amplification can also be achieved isothermally.
  • isothermal amplification methods such as SDA (Strand Displacement Amplification, strand displacement amplification), NASBA (Nucleic Acid Sequence Based Amplification, nucleic acid sequence-based amplification), TMA (transcription-mediated amplification), RCA (rolling circle Amplification), RPA (Recombinase Polymerase Amplification, recombinase polymerase amplification), LAMP (Loop-Mediated Isothermal Amplification, loop-mediated amplification), and HDA (Helicase-Dependent Amplification, helicase amplification), can DNA or RNA amplification is performed at a specified temperature.
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats, Clustered Regularly Interspaced Short Palindromic Repeats
  • prokaryotes such as bacteria and archaea. These sequences were derived from fragments of DNA from viruses that had previously infected these prokaryotes and were used by these prokaryotes to detect and destroy the DNA of similar viruses during subsequent infections.
  • Cas CRISPR associated system, CRISPR associated system
  • Cas protein and gRNA guide RNA, guide RNA
  • Cas protein and gRNA can recognize and cut specific nucleic acid molecules complementary to this gRNA sequence, and some Cas proteins will further cut other nucleic acid molecules around it, including reporter molecule (reporter molecule). This is the basis of CRISPR detection.
  • CRISPR-based diagnostic technology is a new but very promising approach due to its rapid nucleic acid detection capability and single-base specificity.
  • a system that can realize sample input and result output has many advantages, for example, it can be used for on-site diagnosis, requires less training for operators, ensures the safety of operators, and reduces cross-contamination.
  • the present invention proposes an improvement scheme.
  • the present invention provides an integrated nucleic acid analysis system capable of performing magnetic bead-based nucleic acid extraction and purification, thermal amplification (e.g., isothermal amplification and PCR), and fluorescence measurement, the integrated nucleic acid analysis system comprising at least one DMF device , for detecting at least one sample, the DMF device includes: a base plate with a first surface, the base plate is provided with an electrowetting electrode for droplet or liquid control, the first surface is a flat surface, at least a part of the base plate is optically transparent; an upper cover plate comprising a second surface parallel to the first surface and cooperating with the first surface to form a gap for liquid manipulation, the gap comprising a nucleic acid extraction The first liquid storage tank and the third liquid storage tank for nucleic acid elution, the upper cover plate is provided with holes for sample or reagent loading or unloading; for performing a series of operations on the sample in the DMF device Instruments for operations including magnetic bead extraction, purification,
  • a base plate comprising a substrate provided with a plurality of electrowetting electrodes for controlling droplets or liquids, at least a portion of the base plate being optically transparent (including where possible electrodes and dielectric layers) for optical excitation and measurement;
  • the upper cover plate comprises a surface substantially parallel to the bottom plate, an inlet and outlet (hole) for loading and unloading of samples and reagents, and a liquid storage tank for reagent storage and reaction, and at least a part of the bottom plate surface is conductive; on the other hand , some electrodes on the bottom plate form multiple paths for amplification etc.
  • some of the electrowetting electrode areas on the bottom substrate contain lyophilized reagents, including but not limited to polymerases (particularly thermostable enzymes such as Taq polymerase and variants thereof), deoxyribonucleotide triphosphates (dNTPs), ; usually a mixture of dCTP, dTTP, dGTP, and dATP), PCR primers, labeled probes, reverse transcriptase (if the target nucleic acid is RNA), exonuclease, Cas protease, one or more Combined guide RNA, etc.
  • polymerases particularly thermostable enzymes such as Taq polymerase and variants thereof
  • dNTPs deoxyribonucleotide triphosphates
  • PCR primers usually a mixture of dCTP, dTTP, dGTP, and dATP
  • labeled probes labeled probes
  • reverse transcriptase if the target nucleic acid is RNA
  • DMF devices usually include a device housing, which provides certain protection for the bonded bottom plate and upper cover plate, and provides an interface for the control signal of the instrument.
  • the device housing includes a liquid reagent cartridge that includes: a) a plurality of liquid capsules containing sample processing or detection reagents and fill fluid, b) connecting the capsules to the upper cover plate The fluid channel of the filling hole, and c) the liquid inlet.
  • the DMF device further includes a second liquid storage tank for nucleic acid washing and/or a fourth liquid storage tank for magnetic bead washing.
  • the gap of the DMF device has a plurality of different height values, and when the gap height of the DMF device changes from one value to another value, the change corresponding to the slope of the second surface is continuous.
  • the light transmittance of the transparent bottom plate of the DMF device is greater than 30% in the visible light range, such as greater than 50%, 70% or even greater than 90%.
  • the first liquid storage tank is placed with sample lysate and magnetic bead solution
  • the second liquid storage tank is placed with magnetic bead washing solution
  • the third liquid storage tank is placed with nucleic acid eluent .
  • the gap of the DMF device is filled with liquid, such as mineral oil.
  • the instruments include:
  • At least one module for loading and unloading DMF devices is connected to the DMF device;
  • At least one voltage control module connected to the DMF device, for providing electrical signals to the DMF device, to operate the liquid or droplets in the DMF device; for providing electrical signals to the DMF device to operate the DMF device liquid or droplets in
  • each magnet has at least two degrees of freedom of movement, that is, it can move in two directions;
  • At least one temperature control module used to control the designated area of the DMF device to a designated temperature
  • At least one light source used for optical excitation during nucleic acid detection in the DMF device
  • At least one optical detection module for fluorescence measurement reaction used for optical measurement of at least one site on the DMF device.
  • the integrated nucleic acid analysis system also includes a touch screen display, which provides a graphical interface for the user to perform experiments and display the experimental status, and/or includes a central processing unit that performs unified operation control on the aforementioned modules, both of which are compatible with The optical detection module is electrically connected.
  • the magnets are focusing magnets.
  • the present invention also provides a method for measuring at least one target nucleic acid in a sample, the method comprising adding the sample to the DMF device of the integrated nucleic acid analysis system described in any of the above schemes, and then performing a detection operation on the sample , the detection operation comprises the following steps: 1) mixing the sample with a lysate to lyse the cells in the sample; 2) adding a magnetic bead solution to the sample; 3) mixing the magnetic bead solution—making the magnetic beads and Sample mixing; 4) washing the magnetic beads; 5) extracting the nucleic acid in the sample, purifying the nucleic acid, and eluting the nucleic acid from the magnetic beads to obtain the target nucleic acid; 6) for the eluted obtained Adding an amplification reagent to the target nucleic acid to thermally amplify the nucleic acid; 7) measuring the fluorescence intensity from the target nucleic acid.
  • the step of washing the magnetic beads is as follows: a) using the magnet in the instrument, the magnetic beads are moved from the first liquid storage tank, through the filling solution, to the second liquid storage tank on the DMF device, and The magnetic beads move within the range of the second liquid storage tank according to a specified trajectory; c) passing the magnetic beads through the filling solution and moving to the third liquid storage tank.
  • steps a) and c) further include: step b) moving the magnetic beads from the second storage tank through the filling solution to the fourth storage tank, and transferring the magnetic beads to the fourth storage tank Move according to the specified trajectory within the scope of the four liquid storage tanks.
  • the magnetic beads used for biological nucleic acid extraction are usually superparamagnetic microspheres with small diameters (nanoscale to micronscale), which can quickly aggregate in a magnetic field, and can be uniformly dispersed in liquid after the magnetic field is removed, and are not easy to settle. Nucleic acid extraction can be realized by coating the magnetic beads accordingly (such as silicon base, amino group, hydroxyl group, etc.).
  • nucleic acid DNA or RNA
  • the surface-modified magnetic beads specifically bind to the nucleic acid to form a magnetic bead-nucleic acid complex.
  • Nucleic acid-magnetic bead complexes usually have non-specifically adsorbed impurities, which may affect the next step of detection and thus need to be removed.
  • the usual removal method is to use a washing solution.
  • the present invention provides a method for removing impurities on the magnetic bead-nucleic acid complex by using a magnet to move the magnetic beads in an immiscible (immiscible) according to a specified trajectory.
  • the steps of cleaning the magnetic beads are as follows: using the magnet in the instrument, the magnetic beads are directly moved from the first liquid storage tank through the filling solution to the third liquid storage tank on the DMF device.
  • the implementation steps of eluting the nucleic acid from the magnetic beads are as follows: a) moving the magnet to a designated position with a certain distance from the DMF device, so that the magnetic beads are dispersed in the eluent; Magnet is in the direction parallel with DMF device, moves in the specified way in the scope of the 3rd fluid storage tank, to help the nucleic acid on the elution magnetic bead; c) magnet is moved to the 3rd fluid storage tank place and is close to DMF device; D) will The magnet moves along the specified path at the third liquid storage tank to gather the magnetic beads therein; e) moves the magnet close to the DMF device to the designated discarding position of the magnetic beads; f) moves the magnet away from the position of the DMF device.
  • thermally amplifying the target nucleic acid and measuring the fluorescence intensity from the target nucleic acid is performed as follows: a) using an electrowetting electrode to dispense one or more solutions containing the nucleic acid from a third reservoir; droplet; b) moving the droplet along the designated electrowetting electrode path for nucleic acid amplification and optical measurement. All steps of the detection operation are preferably performed automatically by the instrument.
  • the thermal amplification is PCR amplification and/or isothermal amplification.
  • the PCR amplification is realized by moving the reaction liquid droplet between different temperature regions on the DMF device and moving according to a specified trajectory, and the temperature of the specified region remains constant during the PCR reaction.
  • optical measurements are performed by mixing a portion of the complex with different reagents.
  • the integrated nucleic acid analysis system provided by the present invention has the advantages of high sensitivity, multiple detection indicators and short detection time, which can reduce manual operation errors, reduce cross-contamination, and improve detection speed.
  • FIG. 1A-1E show an exemplary partial side-view structure of a DMF device containing liquid and magnetic beads, and the process of manipulating the magnetic beads with a magnet.
  • FIG. 2A shows an exemplary top view of a DMF device with reservoirs, samples, and reagent loading wells.
  • FIG. 2B shows a top view of the DMF device of FIG. 2A.
  • FIG. 3 is a schematic diagram showing an exemplary layout of electrowetting electrodes on the bottom plate of the DMF device illustrated in FIG. 2A .
  • Figure 4 is shown as a combined view of Figure 2A and Figure 3 and provides a schematic of thermal amplification and optical measurements.
  • 5A-5C show schematic diagrams of different designs of the upper lid portion for the gap height variation of the DMF device.
  • FIG. 6A-6D show exemplary schematic diagrams of different functional modules of the integrated nucleic acid analysis system provided by the present invention, wherein, FIG. 6A shows a temperature control module; FIG. 6B shows a magnet control module that can control 2 magnets at the same time; Figure 6C shows the optical measurement module; Figure 6D is a top view of 4 temperature control modules and 2 magnets (part of the magnet control module).
  • FIGS. 7A and 7B are schematic diagrams showing an exemplary structure of the integrated nucleic acid analysis system provided by the present invention.
  • Fig. 8 shows an exemplary flowchart of magnetic bead-based nucleic acid extraction and quantitative PCR detection implemented using the integrated nucleic acid analysis system provided by the present invention.
  • FIG. 9 shows a top view of another example of the integrated nucleic acid analysis system provided by the present invention.
  • FIG. 10 shows an exemplary flowchart for implementing magnetic bead-based nucleic acid extraction and CRISPR detection using the integrated nucleic acid analysis system of FIG. 9 .
  • Embodiments of the present invention are described below through specific examples, and 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 implementation modes, and various modifications or changes can be made to the details in this specification based on different viewpoints and applications without departing from the spirit of the present invention.
  • the cross-sectional view showing the device structure will not be partially enlarged according to the general scale, and the schematic diagram is only an example, which should not limit the protection scope of the present invention.
  • the three-dimensional space dimensions of length, width and depth should be included in actual production.
  • spatial relation terms such as “below”, “below”, “below”, “below”, “above”, “on” etc. may be used herein to describe an element or element shown in the drawings.
  • a layer is referred to as being “between” two layers, it can be the only layer between the two layers, or one or more intervening layers may also be present.
  • structures described as having a first feature "on top of" a second feature may include embodiments where the first and second features are formed in direct contact, as well as additional features formed between the first and second features. Embodiments between the second feature such that the first and second features may not be in direct contact.
  • one embodiment or “an aspect”, etc. means that the embodiment or aspect includes some specific features, structures and characteristics, which may be included in an embodiment, but not necessarily included in the All examples. Furthermore, the features, structures, and characteristics disclosed in this patent may be implemented in any suitable combination in one or more embodiments.
  • microfluidics refers to devices or systems capable of manipulating liquids with at least one cross-sectional dimension ranging from a few micrometers to about a few millimeters.
  • digital microfluidics refers to devices or systems that can manipulate one or more droplets based on electrowetting or dielectrophoretic effects.
  • DMF chip for the purposes of this disclosure, “DMF chip,” “DMF device,” and “DMF device” are used interchangeably to include a first substrate (the bottom plate) having a first substrate surface and a second substrate (the upper substrate) having a second substrate surface. cover plate), the second substrate is spaced from the first substrate by a distance to define a gap (the space between the first and second substrate surfaces), wherein the distance is sufficient to accommodate a droplet placed in the gap.
  • a plurality of first liquid control electrodes are disposed on the first substrate surface, 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.
  • at least one electrode is provided on the second substrate 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.
  • the term "droplet” refers to a droplet that is separated from other parts by air or other gases, other (usually non-miscible) liquids, and solid surfaces (such as the interior surfaces of DMF devices), etc. A certain amount of liquid (one or a mixture of several) that comes out.
  • the volume of “droplets” ranges from a few picoliters (picoliters) to hundreds of microliters (microliters).
  • the “droplet” can have any shape, including spherical, hemispherical, flat circular, irregular, etc.
  • the terms “reservoir” or “reservoir” are used to indicate a portion of a DMF device that can be used to store, hold and supply liquid, either fully enclosed or partially enclosed.
  • the reservoir may be associated with a fluid path that allows liquid to be introduced into the DMF device gap for droplet manipulation, or from between the DMF devices into the reservoir for liquid storage or temporary preservation.
  • filling liquid and “filler oil” are used interchangeably to refer to a liquid that is substantially immiscible with liquid droplets that can fully or partially fill the interstices of a DMF device.
  • the filling fluid may contain low viscosity oil, such as silicone oil, mineral oil, paraffin liquid, fluorosilicone oil and the like.
  • the kinematic viscosity of the filling fluid is usually less than 100cSt (centiStokes), or 50cSt, or 20cSt, 10cSt, 5cSt, or 2cSt.
  • the fill fluid may contain a small amount of surfactant soluble in the fill fluid, typically in a volume ratio of 0.00001% to about 1%, or 0.0001% to about 0.1%, or 0.001% to about 0.01% by volume of surfactant to fill fluid In the range.
  • the filling fluid can fill the entire gap or part of the gap of the DMF device. It is also possible to place the sample or reagent and the filling liquid in a specific reservoir on the DMF device, so that the liquid droplets dispensed from the reservoir using spot wetting are coated with a thin layer of filling liquid.
  • the term “less than” generally means “equal to or less than”, and the term “greater than” generally means “equal to or greater than”.
  • droplet manipulation may include droplet dispensing (from a container or continuous fluid stream), movement, merging and mixing, splitting (symmetrical or asymmetrical), shaping (forming into a specified shape), levitation And distribution of particles (in liquid or droplet), etc.
  • sample solutions 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, etc. Bodily fluids can come from any living organism.
  • the solid solution may be a bodily fluid from a mammal, such as a bodily fluid from a human.
  • analyte and “analyte” are used interchangeably to refer to a substance or chemical constituent to be measured in an analysis or test.
  • An “analyte” can be an organic or inorganic substance. 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.), pharmaceutical molecules (such as antibiotics, therapeutic drugs and drugs of abuse, and drugs), cell nuclei, spores, etc.
  • 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
  • the term "antigen” is a toxin or other foreign substance capable of inducing an immune response (production of antibodies) in a host organism.
  • the term "antibody” is a large Y-shaped protein molecule produced primarily by plasma cells that is used by the immune system to neutralize pathogens, such as pathogenic bacteria or viruses.
  • An antibody specifically binds to its corresponding antigen.
  • the antibody can be labeled with another molecule (eg, a fluorescent tag or an enzyme) to facilitate detection or quantitative measurement of the antibody.
  • reagent and “detection reagent” are used interchangeably to refer to a reagent used to react with a sample, dilute a sample, mix a sample, suspend a sample, emulsify a sample, encapsulate a sample, Sample interactions and any material added to the sample.
  • lyophilized reagent and “lyophilized reagent” are used interchangeably to refer to reagents prepared using lyophilization methods, typically for reagents containing active substances that are not resistant to high temperatures. Preservation of biological reagents is an important part of medical diagnosis. From the perspective of reagent storage, many nucleic acid point-of-care testing (POCT) reagent cartridges need to be stored at room temperature, which requires that the detection reagents must be dehydrated and exist in solid form. When in use, reconstitute with buffer for subsequent reactions.
  • POCT nucleic acid point-of-care testing
  • freeze-dried spheres can maintain the activity of the enzyme to the greatest extent, and the lyophilized spheres have a loose network structure and can be reconstituted quickly. Freeze-dried solid pellets can be stored and transported at room temperature after packaging, which greatly reduces transportation costs and preservation time.
  • magnet and “magnet” are used interchangeably to refer to a shaped, magnetic object, including samarium cobalt magnets, neodymium iron boron magnets, ferrite magnets, AlNiCo magnets, and FeCrCo magnets. Shapes include cylinders, donuts, disks, cones, pyramids and other irregular shapes. Magnets include permanent magnets and non-permanent magnets. Permanent magnets themselves are always magnetic, while non-permanent magnets, such as electromagnets, only appear magnetic under certain conditions (such as current passing through).
  • focusing magnet is used to refer to a magnet (permanent or electromagnet) that has a specific shape such that the magnetic field on one side is stronger than that on the opposite side. Examples include, but are not limited to, conical or pyramidal magnets. The magnetic field at the tip of a conical magnet (or pyramidal magnet) is stronger than that of its base.
  • the term "particle” is used to refer to micro- or nano-sized entities, which may be natural or artificial, such as cells, subcellular components, liposomes ), viruses, nanospheres, and microspheres, or smaller entities such as biomacromolecules, proteins, DNA, and RNA, etc. Bubbles etc.
  • the (linear) size of the "particles” can range from a few nanometers to hundreds of microns.
  • the term "bead” may be any bead or particle that reacts with a solution.
  • Beads can be any of a variety of shapes, such as spherical, egg-shaped, cubic, disc-shaped, or irregular.
  • the beads can be inside the droplet, on the inner surface of the DMF device, in the fill fluid of the DMF device, in a tank, and the like.
  • Beads can be made from a wide variety of materials such as resins, polymers, glass, nanomaterials, etc., and can be of any size such as microbeads and nanobeads.
  • Beads may be magnetically responsive, in which case at least one or part of their components consist of a magnetically responsive material, while the remaining material may contain polymeric materials, coatings, or groups to which detection reagents are attached, etc.
  • examples of beads include quantum dots, polyethylene microbeads, silica microbeads, fluorescent microspheres or nanospheres, magnetic microbeads, magnetic nanobeads, flow cytometric beads, and the like.
  • magnetic bead refers to a bead comprising a magnetically responsive material.
  • magnetically responsive materials include ferromagnetic materials, paramagnetic materials, supermagnetic materials, and ferrimagnetic materials, among others.
  • paramagnetic materials include metals such as nickel, iron, and cobalt, and metal oxides such as Fe 3 O 4 , Cr 2 O 3 , NiO, Mn 2 O 3 .
  • the magnetically responsive material may constitute substantially all of the magnetic beads, a portion of the magnetic beads, or a certain component of the magnetic beads.
  • the remainder of the magnetic bead may include a polymer material and a coating portion to allow attachment of the target particle.
  • Magnetic beads can be used in a variety of assays, where magnetic beads are typically used to bind one or more target species, such as analytes or contaminants, in a mixture. Assays often require an efficient bead washing process to reduce the amount of one or more species in a bead-containing droplet that may come into contact with the bead surface.
  • immobilized is used to indicate that the magnetic beads are substantially confined to a given location in the droplet, reservoir, and fill fluid on the DMF device.
  • immobilized magnetic beads are substantially constrained in position within the droplet to allow the operation of droplet separation to produce another droplet that is substantially free of magnetic beads and has a majority of magnetic beads.
  • One droplet residual liquid
  • magnetic bead washing and “magnetic bead washing” can be used interchangeably with respect to washing magnetic beads and washing, and are used to indicate the use of liquid to reduce certain (or several) substances on magnetic beads. amount or concentration. The reduction in the amount or concentration of a substance may be partial or complete.
  • the substance can be any of a variety of substances, such as components of the sample, contaminants, and excess reagents.
  • a “magnetic bead operation” may consist of one or a combination of the following operations:
  • Aggregation - Magnetic beads are aggregated within the droplet, within the trough, and in the area between the droplet (sink) and the droplet (sink), etc. on the DMF device.
  • the size of the magnetic beads after aggregation is less than 10 mm, or less than 5 mm, or less than 2 mm. It should be noted that aggregated beads usually have sharp boundaries, but can also be rather fuzzy.
  • the aggregated magnetic beads account for more than 30%, or more than 50%, or more than 70%, or more than 80%, or more than 90% of all the specific magnetic beads.
  • Movement - Moving magnetic beads from one location to another on a DMF device including but not limited to moving magnetic beads from one droplet or reservoir to another.
  • the aggregated magnetic beads are dispersed in the droplet or tank, which can be performed simultaneously with the droplet operation.
  • amplification refers to a process by which the amount or concentration of an analyte being measured can be increased.
  • Non-limiting examples include Polymerase Chain Reaction (Polymerase Chain Reaction or PCR) and its variants (such as quantitative competitive PCR, immune PCR, reverse transcription PCR, etc.), strand displacement amplification (Strand Displacement Amplification or SDA), nucleic acid sequence-based Amplification (Nucleic Acid Sequence Based amplification or NASBA), loop-mediated isothermal amplification (Loop-mediated isothermal amplification or LAMP), helicase-dependent amplification (Helicase-dependent amplification or HAD), etc.
  • layer and “film” are used interchangeably to refer to a structure of a body that is usually, but not necessarily, planar or substantially planar, and typically deposited , formed, coated or otherwise placed on another structure.
  • ground means that the voltage of the corresponding electrode is zero or sufficiently close to zero. All other voltage values, although typically less than 300 volts in magnitude, should be high enough to enable the electrowetting effect to be fully observed.
  • the space between adjacent electrodes in the same layer is usually filled with a dielectric material. These spaces can also be empty, or filled with gases such as air, nitrogen, helium and argon. All electrodes in the same layer and electrodes at different layers are preferably electrically insulated.
  • contact angle means the angle formed when a liquid-vapor interface contacts a solid surface.
  • the shape of the liquid-gas interface is determined by the Young-Laplace equation at the thermodynamic equilibrium of the three phases - liquid, solid and gas (which can be the ambient atmosphere and a mixture vapor at equilibrium concentration of the liquid).
  • ⁇ SG represents the solid-gas interface energy
  • ⁇ SL represents the solid-liquid interface energy
  • ⁇ LG represents the liquid-gas interface energy (ie surface tension)
  • ⁇ represents the contact angle at equilibrium.
  • each molecule is pulled equally in every direction by adjacent liquid molecules, resulting in a net force of zero.
  • molecules at the surface of a liquid are not in all directions free from the net forces provided by their neighbors to provide balance, they are pulled inwards by their neighbors, creating internal pressure, with the result that the liquid surface area shrinks to maintain its minimum surface free able.
  • This intermolecular force that shrinks the surface that is, the liquid-gas interfacial energy ⁇ LG
  • surface tension is called surface tension, and it determines the shape of the droplet.
  • Other external forces, such as gravity can also deform the droplets. Therefore, the contact angle is determined by both surface tension and an external force (usually gravity). The contact angle is also a characteristic parameter of a certain solid-liquid system in a specific environment.
  • Hydrophobic surfaces have the property of repelling liquids, while hydrophilic surfaces have the properties of attracting liquids.
  • a "hydrophobic surface” has a contact angle greater than 90°, while a hydrophilic surface has a contact angle less than 90°.
  • liquids in any form are described as being “on,” “at” or on electrodes, arrays, matrices, and surfaces
  • the liquid may be in direct contact with the electrodes, array, matrix, and surface, or may be in contact with one or more layers or films interposed between the liquid and the electrodes, array, matrix, and surface.
  • PCB printed Circuit Board
  • PCB printed Circuit Board
  • Circuit and pattern The material used in the circuit is usually copper.
  • the circuit can provide a conduction path between electronic components.
  • a large copper surface is usually designed as a grounding and power supply layer. Lines and drawings are made at the same time.
  • Dielectric layer It is used to maintain the insulation between lines and layers, also called substrate.
  • the through hole can make the lines of more than two levels conduct with each other, the larger through hole is used as a component plug-in, and the non-conductive hole is usually used as a surface mount mounting position.
  • solder Mask Not all copper surfaces need to be tinned, so the non-tinned area will be printed with a layer of material (usually epoxy resin) that isolates the copper surface from eating tin to avoid non-stained parts. There is a short circuit between the tinned lines. According to different processes, it is divided into green oil, red oil and blue oil.
  • Silk screen This is a non-essential component. Its main function is to mark the name and position box of each part on the circuit board, which is convenient for maintenance and identification after assembly.
  • testing is used interchangeably to obtain a physical quantity (e.g., position, charge, temperature, concentration, pH value, brightness, fluorescence, etc.).
  • a sensor or detector
  • the terms “testing”, “detection” and “measurement” are used interchangeably to obtain a physical quantity (e.g., position, charge, temperature, concentration, pH value, brightness, fluorescence, etc.).
  • at least one sensor or detector
  • There may be other components between the object to be tested and the sensor such as lenses, mirrors, filters, etc. used in optical measurement, and resistors, capacitors, transistors, etc. used in electrical measurement.
  • other auxiliary devices or devices are often used in the measurement.
  • a light source such as a laser or a laser diode is used to excite particles from an electronic ground state to an electronically excited state.
  • the excited state particles return to the ground state, they sometimes emit fluorescence. Measuring the fluorescence intensity at this time can be used to measure the concentration of certain particles.
  • Optical sensors include CCDs, photodiodes, photomultiplier tubes, etc.
  • electrical sensors include operational amplifiers, analog-to-digital converters, thermocouples, and thermistors.
  • Measurements can be made simultaneously or sequentially for multiple parameters in multiple samples. For example, while a photodiode is used to measure the fluorescence of a particle in a droplet, its position can also be obtained simultaneously by capacitive measurements. Sensors or detectors are usually connected to a central processing unit (Central Processing Unit, or CPU) or a computer (computer), and the corresponding software runs on the central processing unit or computer to analyze the measured signal and usually convert it to Transform into information that can be read by humans or other devices. For example, the measurement and analysis of the fluorescence intensity of a particle in a liquid can be used to infer the concentration of the particle.
  • CPU Central Processing Unit
  • computer computer
  • optical measurements include laser induced fluorescence measurement, infrared spectroscopy, Raman spectroscopy, chemiluminescence measurement, surface plasmon resonance measurement plasmon resonance measurement), absorption spectroscopy (absorption spectroscopy), etc.; electrical measurements include amperometry, voltammetry, photoelectrochemistry, coulometry, capacitance measurement ( capacitance measurement), and AC impedance measurement (and AC impedance measurement), etc.
  • FIG. 1A to FIG. 10 The following is a specific description of the embodiment of processing biological samples in the present invention, and for the convenience of explanation, the corresponding drawings (FIG. 1A to FIG. 10) will be referred to when necessary. It should be noted that the purpose of these examples is to aid in illustration and not to limit the spirit and spirit of the invention.
  • some and all of the functional modules herein may be automatically controlled.
  • a program (software or firmware) running on a microprocessor or computer is usually used to implement automatic control.
  • FIG. 1A-1E show side views of a portion of a DMF device, generally designated 100, in an integrated nucleic acid analysis system of the present invention, as a preferred embodiment for achieving magnetic bead control.
  • Figure 1A shows different reservoirs for liquid 401 and liquid 402 on a DMF device.
  • Base plate 300 comprises a droplet control electrode 302 and a dielectric layer 303 deposited on a substrate 301 .
  • the upper cover 200 includes a ground electrode 202 and a dielectric layer 203 on a deposition substrate 201 .
  • the DMF device structures shown here are for the purpose of illustrating magnetic bead manipulation and by no means represent all possibilities. In some embodiments, DMF devices can be implemented in various ways.
  • control electrodes can have different shapes, such as rectangle, square, trapezoid, pentagon, hexagon, and irregular shapes, and can be arranged and combined 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 WO 2008/147568 entitled “Electrowetting Based Digital Microfluidics” ("Digital Microfluidics Based on Electrowetting", inventor Wu Chuanyong); 3)
  • the electrical layer can also have two or more layers, and the materials used include parylene C (Parylene C), silicon nitride, silicon dioxide, tantalum oxide, etc.; one of them can be a hydrophobic material, such as iron fluoride Dragon (Teflon), Cytop, and FluoroPel, etc.
  • 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 hold its shape within the desired system operating and storage conditions. In terms of light transmission ability, it can be transparent, translucent and opaque.
  • the transparent substrate can be made of various transparent materials, such as glass, quartz, plastic, transparent ceramics, transparent printed circuit boards, and the like.
  • Electrodes can be made of any conductive material, such as metals, alloys, and conductive polymers. It can be made of one material or a mixture of different materials.
  • Transparent electrodes on DMF devices 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 wait.
  • transparent conductive materials such as indium tin oxide (ITO), aluminum-doped zinc oxide (AZO), transparent conductive polymers (polyacetylene, polyaniline, etc.), or transparent nanomaterials wait.
  • the voltage control module is used to provide voltage control signals to the droplet control electrodes. It usually has multiple outputs, the maximum number is 1000000, or 100000, or 10000, or 1000.
  • the voltage output may be unipolar or bipolar, with a voltage amplitude of less than 1000 volts, or less than 300 volts, or less than 100 volts, or less than 60 volts, or less than 30 volts.
  • AC signal or DC signal with voltage frequency less than 10MHz (megahertz), or less than 1MHz, or less than 100KHz (kilohertz), or less than 20KHz, or less than 5KHz, or less than 1KHz.
  • the waveform of the voltage can be square wave, sine wave, sawtooth, pulse width modulated signal, etc.
  • the voltage control module is usually controlled by the 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), etc., program the sequence, duration, amplitude, and frequency of the output signal.
  • SPI Serial Peripheral Interface
  • I2C Inter-Integrated Circuit
  • USB Universal Serial Bus
  • parallel port parallel port
  • Ethernet Ethernet
  • Wi-Fi Wi-Fi
  • Bluetooth Bluetooth
  • FIG. 1A magnetic beads 500 are evenly distributed in the reservoir 401 of the DMF device.
  • the magnet 600 is placed close to the DMF device at the position of the reservoir 401 . Under the action of the magnetic field generated by the magnet, the magnetic beads 500 are gathered at the bottom of the liquid storage tank 401 . The magnet moves the beads to the reservoir 402 as shown in FIGS. 1C and 1D .
  • FIG. 1E the magnet is removed from the DMF device and the beads are resuspended and dispersed in reservoir 402. Suspending and dispersing magnetic beads in the liquid in reservoir 402 may require droplet motion or other means to assist.
  • the reservoir gap in DMF devices may be surrounded by air or fill fluid.
  • Figures 1A–1E illustrate the process of magnetic beads being moved from the inside of one container through the medium (air or filling liquid) to another reservoir. As shown, the device is hydrophobic to the droplets. Droplets on the electrodes can be manipulated (move, split, merge, mix, etc.) through the electrowetting effect.
  • phrases such as “the magnet is moved (or brought, transported) to approach”, “the magnet is moved closer” as used herein are intended to refer to the relative positions of the magnet and the DMF device.
  • the magnetic force generated by the magnet has a clear effect on the magnetic beads on the device.
  • phrases such as “the magnet moves out of the way”, “the magnet moves out of the way” are intended to mean that the magnet has no and negligible effect on the magnetic beads on the device.
  • the moving speed of the magnet can usually be controlled by a motor, and the magnet speed required by different applications can be different, usually ranging from 0.1 to 100mm/sec, or 0.5 to 20mm/sec, or 1 to 10mm/sec.
  • the magnet is in contact with the base substrate and moves along the bottom of the DMF device.
  • a magnet can also be located on the top of the DMF device; or a pair of magnets, one on the top and one on the bottom, are used to manipulate the magnetic beads in the DMF device.
  • FIG. 2A shows a top view of a DMF device 200 of the present invention, generally indicated at 200, capable of running and analyzing two samples simultaneously.
  • Both the bottom plate and the top cover have two areas with the same function, which can be used to process two samples respectively.
  • the liquid storage tank 204 can be used for sample lysis and nucleic acid capture, and the liquid filling holes 205 and 206 are used for loading samples, lysate, and magnetic bead solutions, etc., for nucleic acid extraction.
  • Reservoirs 207 and 209 are used for nucleic acid washing.
  • Liquid filling holes 208 and 210 are used for loading magnetic bead washing liquid.
  • the liquid storage tank 211 is used for nucleic acid elution, and the liquid filling hole 213 is used for loading eluent.
  • the liquid filling hole 215 is used for loading reagents for nucleic acid amplification and detection.
  • Filling well 214 can be used to load reagents for negative or positive controls.
  • a simplified detection operation procedure comprise: 1) magnet is contacted with DMF device at the position of 204, by moving magnet by specified trajectory in the scope of liquid storage tank 204, will have The magnetic beads that have captured the nucleic acid gather at the bottom of the liquid storage tank; 2) use a magnet to move the focused magnetic beads to the liquid storage tank 207, and wash the magnetic beads in the liquid storage tank; 3) put the focused magnetic beads Move to the liquid storage tank 209, and do cleaning again in the liquid storage tank; 4) move the cleaned magnetic beads to the liquid storage tank 211, and carry out nucleic acid elution; 5) use electrowetting electrode Separate one or more droplets of the eluate containing nucleic acid from the elution tank, and move to the right side, and mix with the PCR reagent loaded through the liquid addition hole 215; 6) Perform thermal amplification and optical measurement.
  • Fig. 2B is a top view of the physical design of the upper cover plate of the DMF device based on the present invention.
  • the material of the upper cover is polycarbonate (Polycarbonate), which is made by injection molding.
  • the AL and BL liquid tanks are used for the magnetic bead extraction of the nucleic acid of the two samples (A and B) respectively;
  • the AE1 and AE2 liquid tanks are used for the magnetic bead cleaning of the sample A
  • the BE1 and BE2 liquid tanks are used for the magnetic bead cleaning of the sample B ;
  • the AE and BE liquid tanks are used for nucleic acid elution of A and B samples respectively.
  • A1-A4 holes are used to add reagents for detection sample A
  • B1-B4 holes are used for adding reagents for detection sample B
  • OIL holes are used to add filling liquid to DMF devices.
  • Figure 3 shows a corresponding electrowetting electrode layout for the DMF device shown in Figure 2A.
  • the optional electrode 311 can be used to assist in mixing the sample with the lysis buffer and magnetic beads, etc.
  • the optional electrodes 312 and 313 are used to assist in the washing of the magnetic beads
  • the electrodes 314 and 315 are used to separate the droplets from the elution tank.
  • the electrodes 321 to 324 are used to move (including repeatedly moving back and forth) the liquid droplets to achieve amplification and/or measurement.
  • FIG. 4 is a functional schematic diagram combining FIG. 2A and FIG. 3 .
  • the temperature control areas 611 and 612 and the optical detection point 620 are shown in FIG. 4 .
  • both optical excitation and detection are accomplished through the backplane of the DMF device. Therefore, this part of the DMF device backplane needs to be optically transparent (including the substrate, electrodes and dielectric layers).
  • optical measurements are performed in the visible range, typically at wavelengths between 350-750nm. Higher light transmittance means higher detection sensitivity of the system.
  • the transmittance of the optical measurement portion of the bottom plate of the DMF device is generally greater than 30%, or greater than 50%, or greater than 70%, or greater than 90%.
  • FIGS 5A-5C show the side views of the part of the DMF device where the gap height changes from H1 to a smaller value H2, wherein Figures 5A and 5B are two common designs, and Figure 5C is the design proposed by the present invention.
  • the gap height changes from H1 in region 251 to H2 in region 252 at a single point 253; in FIG. H2, the corresponding slope (slope) is a constant (constant).
  • the slope of a curve at a point is the slope of the tangent at that point.
  • the slope is zero at regions 251 and 252 .
  • the slope is infinite at point 253 .
  • the slope is constant throughout region 254, which can be calculated from the gap height difference and region length. It is equal to (H1-H2) divided by the length of the region 254, eg if H1 is 2.5mm, H2 is 0.5mm, and the length of the region 254 is 10mm, the absolute value of the slope is 0.2.
  • FIG. 5C is presented in the present invention, where the gap height varies over region 255 from H1 in region 251 to H2 in region 252 .
  • region 255 the (absolute value of) slope continuously increases from zero to a finite value and then decreases continuously to zero.
  • Figure 5A the slope changes from zero to infinity at 253, and then back to zero; in Figure 5B, the slope changes directly from zero to a non-zero value, and then directly back to zero.
  • the slope of the lower surface of the upper cover plate at the gap change of the DMF device in Figures 5A and 5B will undergo a non-continuous (non-continuous) change, while in Figure 5C, the change of the corresponding slope is continuous (continuous) ).
  • Figure 5C proposes a design that makes it easier for a droplet to transition from one gap height to another. In particular, this makes it easier to remove droplets from the tank.
  • Figure 6A illustrates one embodiment of a temperature control module.
  • the temperature control modules 611 and 612 are used for nucleic acid amplification.
  • the temperature control module 613 is optionally used to assist nucleic acid elution, and the temperature control module 614 is optionally used to assist nucleic acid extraction.
  • Figure 6B is a magnet control module 630, 631 among the figure is 2 magnets for manipulating the magnetic beads in the DMF device,
  • Figure 6C shows the design of the optical detection module,
  • Figure 6D shows the temperature control modules 611 to 614, magnet 630 and a top view of the optical detection module 620.
  • the instrument is designed such that when temperature control is not required, one or more temperature control elements are moved away from close to or in contact with the DMF device, thereby allowing the magnet to move to a desired position proximate to the DMF device .
  • magnet 630 when the magnet 630 is not in use, it can be moved to one end of the DMF device to allow the temperature control modules 613 and 614 to be moved close to or in contact with the DMF device. In yet another embodiment, magnet 630 may be held in one or more spaces between temperature control modules 613 and 614 such that temperature control modules 613 and 614 may be moved close to or in contact with the DMF device.
  • the magnet and the heating block mentioned above are independent devices.
  • the two devices can be integrated together, that is, one device can be used to control the temperature of the DMF device, and can also generate magnetism for the DMF device. magnetic beads for control.
  • Using this integrated module saves space and simplifies instrument design. Since the relevant design is relatively simple, no specific example description will be given here.
  • Figures 7A and 7B show a preferred embodiment of the integrated nucleic acid analysis system.
  • Figure 7A is a top-front-side view of the device and
  • Figure 7B is a rear view.
  • 700 is a spring probe, which can be used to realize the interface between the instrument and the droplet control of the DMF device, and provide droplet control voltage to the electrowetting electrode on the DMF device.
  • 701 is a touch screen, which is used to provide a user-operable graphical interface for the user to input commands and display help, experiment status, and measurement results.
  • 702 is a slidable tray for loading and unloading DMF devices. When pushed in, the spring probes 700 make contact with the bottom substrate of the DMF device.
  • 703 is a DMF device. In FIG.
  • 7B , 704 is a vent (usually equipped with a fan inside) for externally discharging the heat generated when the instrument is in operation.
  • 705 is a USB port and 706 is an Ethernet port. They optionally provide a means for application software running on a computer to communicate with the instrument (eg, send commands or receive data).
  • 707 is an AC power port.
  • Figure 8 shows an example of magnetic bead-based nucleic acid extraction and quantitative PCR analysis using the nucleic acid analysis system.
  • step S801 add the lysate and the magnetic bead solution in the liquid storage tank 204, add the magnetic bead cleaning solution in the liquid storage tank 207 and 209, add the eluent in the liquid storage tank 211, and add the eluent in the liquid storage tank 215.
  • step S802 the magnet is moved to stick to the DMF device at the liquid storage tank 204 .
  • the magnet is moved within the range of the liquid storage tank 204 according to a specified trajectory, to help the magnetic beads in the liquid storage tank to capture nucleic acid.
  • step S803 moving the magnet against the DMF device from the reservoir 204 to the reservoir 207 , which will carry the magnetic beads to the reservoir 207 .
  • the magnet is moved within the range of the liquid storage tank 207 according to a specified trajectory, so as to clean possible impurities on the magnetic beads.
  • step S804 the magnet is moved from the reservoir 207 to the reservoir 209 , which will carry the magnetic beads to the reservoir 209 .
  • the magnet is moved within the scope of the liquid storage tank 209 according to the specified trajectory, and further cleaning of the magnetic beads is performed.
  • step S805 the magnet is moved from the reservoir 209 to the reservoir 211 , which will carry the magnetic beads to the reservoir 211 .
  • step S806 4 liquid droplets are separated from the liquid storage tank 211 and moved to the liquid storage tank 215 along the paths 321 to 324 to mix with the quantitative PCR reagents there.
  • step S807 the four droplets are moved back and forth along the paths 321 to 324 in the PCR temperature zones 611 and 612 , and fluorescence measurement is performed at the detection point 620 in each movement cycle.
  • step S808 an analysis report is generated according to the obtained quantitative PCR data.
  • Fig. 9 shows a top view of a DMF device and an instrument module in another example of the present invention
  • the DMF device can run and analyze four samples simultaneously.
  • the liquid storage tank 901 can be used for sample lysis and nucleic acid capture, the liquid storage tanks 902 and 903 are used for nucleic acid cleaning, the liquid storage tank 904 is used for nucleic acid elution, the liquid storage tank 905 is used for loading PCR reagents, and the liquid storage tank 906 is used for loading Cas reagent.
  • 911 is an electrowetting electrode, which is used to operate the liquid in the DMF device, and there is a solid (air-dried) gRNA reagent on each small square in 912, which is used to test (can be different) in the reactant Nucleic acid molecules are detected.
  • 921, 922, and 923 are three independent temperature control modules in the instrument, which can control the corresponding chip area at different temperatures.
  • Figure 10 shows an example of magnetic bead-based nucleic acid extraction and CRISPR detection using the integrated nucleic acid analysis system provided in Figure 9 .
  • step S1001 add the lysate and the magnetic bead solution in the liquid storage tank 901, add the magnetic bead cleaning solution in the liquid storage tank 902 and 903, add the eluent in the liquid storage tank 904, and add the eluent in the liquid storage tank 905.
  • step S1002 the magnet is moved upwards, and the DMF device is attached to the liquid storage tank 901 .
  • the magnet is moved within the range of the liquid storage tank 901 according to a predetermined track, so as to help the magnetic beads in the liquid storage tank to capture nucleic acid molecules.
  • step S1003 moving the magnet against the DMF device from the reservoir 901 to the reservoir 902 , which will carry the magnetic beads to the reservoir 902 .
  • the magnet is moved according to a predetermined trajectory in the range of the liquid storage tank 902, to clean possible foreign matter adsorbed on the magnetic beads.
  • step S1004 the magnet is moved from the reservoir 902 to the reservoir 903 , which will carry the magnetic beads to the reservoir 903 .
  • the magnet is moved in the range of the liquid storage tank 903 according to a predetermined trajectory for further cleaning of the magnetic beads.
  • step S1005 the magnet is moved from the reservoir 903 to the reservoir 904 , which will carry the magnetic beads to the reservoir 904 .
  • step S1006 one droplet is separated from the eluate in the storage tank 904, and moved to the storage tank 905 to dissolve the freeze-dried PCR reagent stored there. Then the droplet is moved back and forth between the temperature zones 921 and 922 for PCR amplification.
  • step S1007 the droplets amplified by PCR are divided into two parts, one part is moved to areas such as 904 or 905 and discarded, and the other part is mixed with the Cas reagent in 906.
  • step S1008 the mixed droplet in S1007 is moved to the gRNA spotting place 912, and mixed with the gRNA there. At this time, Cas and gRNA are combined. After finding the DNA molecule corresponding to the gRNA, the nucleic acid in the droplet is analyzed. Molecule (including reporter molecule) shearing. Fluorescence measurements were taken on the reaction droplets at specified time intervals for the specified time.
  • step S1009 an analysis report is generated according to the obtained fluorescence data.
  • the reagents can be encapsulated on the DMF device and the user only needs to load the sample. This makes the device easier to handle and reduces the chance of the test being contaminated (or cross-contaminated).

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Abstract

一种集成核酸分析***和测量样本中的至少一个目标核酸的方法。集成核酸分析***包括至少一个DMF器件（100），DMF器件（100）包括底板（300）、盖板（200）和用于对DMF器件（100）中的样本执行包括核酸的磁珠（500）提取、纯化、热扩增和光学测量等一系列操作的仪器。其中，核酸提取和纯化基于磁珠法，利用仪器将磁珠（500）在数字微流控芯片上移动到不同功能区域，例如裂解槽、清洗槽、洗脱槽等来实现。该***将样本处理和检测集成在一个数字微流控芯片上，整个检测操作过程由相应的仪器自动完成，以此来减少人工操作的失误、降低交叉污染、提高检测速度。

Description

集成核酸分析***和测量样本中的目标核酸的方法 技术领域
本发明涉及生物检测技术领域，具体涉及一种集成核酸分析***和测量样本中的至少一个目标核酸的方法。本发明利用数字微流控器件(Digital Microfluidics，简称DMF器件)实现核酸的磁珠法提取、纯化、扩增及测量的集成。
背景技术
生物样本在进行检测分析前，通常需要经过一系列的机械、热、电、磁、光或化学处理步骤。在现代生命科学和医学诊断应用中，磁珠常用于样本处理，例如分析物的提取、分离和纯化。在提取过程中，目标分析物通过化学特异性结合在磁珠表面，使用移液管或让液体流过被磁铁固定的磁珠，去除不需要的颗粒或液体，可以实现分离或纯化。利用磁铁，磁珠也可以被聚焦，并从其原始的液体环境移开，以去除不需要的颗粒或液体。磁珠经常被用作核酸、抗原抗体、催化剂和蛋白质的载体，并广泛应用于DNA分离、mRNA纯化、蛋白质纯化、细胞分离、免疫测定、生物分子捕获等。
用于生化处理和分析的仪器有很多。当使用这些仪器时，生化反应所需样本或试剂有很大比例都不参与反应或测量，因而被浪费。在数字微流控(DMF)体系中，死体积(加入体系但不参与反应和检测的液体体积)可以显著减少，有时甚至可以减少到零。这意味着实验所需的样本或试剂的量就是测量的量。这不仅大大降低了样本和试剂的使用成本，因为较小的反应体积可以缩短试剂和样本的混合时间，检测分析所需的时间也因此可以大大缩短。
当前许多生化分析***中需要大量手动处理步骤。与此相比，基于数字微流控的***(仪器、装置和方法等)可以提供高度的集成度和自动化，这可以大大减少可能的人为错误，并可以大大提高检测的可靠性和数据质量。
在基于液滴的数字微流控***中，液体以离散的格式(液滴)在二维空间中***控，液滴可以单独的***控，这就是被称为数字微流控的原因。在DMF器件中，液滴的运行路径可以在运行时定义，并可以动态更改。与通常的压力驱动(通过外部泵)或电动驱动(通过高压)基于通道的微流控相比，DMF器件仅需要低电压即可控制其中的液滴。数字微流控中的驱动力基于静电效应，例如电润湿或介电泳，这个静电效应和待检的生物样本通常没有相关性，这使得数字微流控***的设计和具体的检测项目独立，因而具有比较好的通用性。
近年来，数字微流控技术由于具有处理单个液滴的能力，以及容易做到小型化、集成化和自动化等优点，吸引了广泛的关注。数字微流控技术减少了试剂的使用量、简化了实验步 骤、缩短了检测时间，具有很大的优越性。
聚合酶链反应(Polymerase Chain Reaction,或PCR)从根本上改变了科学领域，作为一种成熟的方法，PCR需要对反应体系重复加热和制冷的循环，这个反应体系包含有特定DNA引物、dNTP(deoxy-ribonucleoside triphosphate，脱氧核糖核苷三磷酸)、热稳定DNA聚合酶(如Taq DNA聚合酶)等，以对可能有的目标DNA分子每个温度循环都会使目标DNA分子的数量增加一倍，从而导致目标序列的指数积累。该技术将样本中的微量DNA或RNA扩增到可以测量和分析的水平。PCR技术已应用于许多不同领域，包括病毒载量测试，食源性病原体定量，临床诊断，耐药性分析和法医科学。利用PCR技术，医生和研究人员可以通过分析一个单细胞来确定病毒感染的来源。现在可以使用PCR检测到许多传染性生物，例如COVID-19，HIV-1，乙型肝炎，丙型肝炎，SARS病毒，西尼罗河病毒，结核分枝杆菌等。
除了PCR外，核酸扩增也可以等温实现。等温扩增方法有很多，例如SDA(Strand Displacement Amplification，链置换扩增)，NASBA(Nucleic Acid Sequence Based Amplification，基于核酸序列的扩增)，TMA(转录介导的扩增)，RCA(滚环扩增)，RPA(Recombinase Polymerase Amplification，重组酶聚合酶扩增)，LAMP(Loop-Mediated Isothermal Amplification，环介导的扩增)，和HDA(Helicase-Dependent Amplification，解链酶扩增)，可以在一个指定的温度下进行DNA或RNA扩增。
CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats，成簇的规律间隔的短回文重复序列)是在如细菌和古细菌等原核生物基因组中发现的一系列DNA序列。这些序列来自先前感染过这些原核生物的病毒的DNA片段，并被这些原核生物在随后的感染中用来检测和摧毁类似病毒的DNA。
Cas(CRISPR associated system,CRISPR关联***)是一种核酸内切酶。Cas蛋白和gRNA(guide RNA,向导RNA)，可以识别并且切割特定与此gRNA序列互补的核酸分子，有些Cas蛋白会进一步切割其周围其他的核酸分子，其中也包括reporter molecule(报道分子)，这就是CRISPR检测的基础。基于CRISPR的诊断技术由于其快速的核酸检测能力和单碱基特异性而成为一种新的但非常有前途的方法。
在医疗诊断中，能实现样本进、结果出的***具有许多优点，例如可用于现场诊断，较少的操作人员培训，保障操作人员的安全，降低交叉污染等。在核酸诊断领域，尽管有相应的***存在，它们普通存在检测时间长、灵敏度低和检测指标少等问题。为此，本发明提出了一种改善方案。在本发明中，我们提出了一种具有高灵敏度、多检测指标和检测时间短的集成核酸分析***以及基于此的测量样本中的至少一个目标核酸的方法。
发明内容
一方面，本发明提供了能够执行基于磁珠的核酸提取和纯化、热扩增(例如等温扩增和PCR)、以及荧光测量的集成核酸分析***，所述集成核酸分析***包括至少一个DMF器件，用于检测至少一个样本，DMF器件包括：具有第一表面的底板，所述底板上设置有用于液滴或液体控制的电润湿电极，所述第一表面为平坦表面，底板的至少一部分是光学透明的；上盖板，所述上盖板包括平行于所述第一表面并与第一表面配合以形成用于液体操作的间隙的第二表面，所述间隙包括用于核酸提取的第一储液槽和用于核酸洗脱的第三储液槽，所述上盖板设置有用于样本或试剂加载或卸载的孔；用于对所述DMF器件中的样本执行一系列操作的仪器，所述操作包括核酸的磁珠提取、纯化、热扩增和光学测量。底板上包括设置有多个电润湿电极的衬底，用于控制液滴或液体，底板的至少一部分是光学透明的(包括该处可能的电极和电介质层)，用于光激发和测量；上盖板包括基本平行于底板的表面，用于样本及试剂的装卸载的出入口(孔)，以及用于试剂存储及反应的储液槽，底板表面的至少一部分是导电的；在另一方面，底板上的一些电极构成多个路径，用于实现扩增等。且作为示例，底部基板上的一些电润湿电极区域包含冻干试剂，包括但不限于聚合酶(特别是热稳定酶，例如Taq聚合酶及其变种)、脱氧核糖核苷酸三磷酸(dNTP；通常是dCTP，dTTP，dGTP，和dATP的混合物)、PCR引物、标记探针、逆转录酶(如果目标核酸为RNA)、核酸外切酶，Cas蛋白酶、一种或多种与特定靶分子结合的guide RNA等。
DMF器件通常包括器件外壳，为粘合在一起的底板和上盖板提供一定的保护，并为仪器的控制信号提供接口。比如在一示例中，器件外壳包括液体试剂卡盒，该液体试剂卡盒包括：a)多个包含样本处理或检测试剂及填充液的液体胶囊，b)将所述胶囊连接到上盖板的加液孔的流体通道,和c)液体入口。
作为示例，所述DMF器件还包括用于核酸清洗的第二储液槽和/或用于磁珠洗涤的第四储液槽。
作为示例，所述DMF器件的间隙具有多个不同的高度值，且所述DMF器件的间隙高度从一个值改变为另一值时，对应第二表面斜率的变化是连续的。
作为示例，所述DMF器件的底板透明处的光透射率在可见光范围内大于30％，比如大于50％、70％乃至大于90％。
作为示例，所述第一储液槽置放有样本裂解液和磁珠溶液，所述第二储液槽置放有磁珠洗涤液，所述第三储液槽置放有核酸洗脱液。
作为示例，所述DMF器件的间隙内有填充液，比如填充有矿物油。
作为示例，所述仪器包括：
a)至少一个用于加载及卸载DMF器件的模块，比如装卸载托盘，与所述DMF器件相连接；
b)至少一个电压控制模块，与所述DMF器件相连接，用于向DMF器件提供电信号，以操作所述DMF器件中的液体或液滴；用于向DMF器件提供电信号以操作DMF器件中的液体或液滴
c)至少一个磁铁控制模块，位于所述DMF器件的上部或下部，用于控制一个或多个磁铁，每个磁铁具有至少两个运动自由度，即可以在两个方向上运动；
d)至少一个温度控制模块，用于将DMF器件的指定区域控制到指定的温度；
e)至少一个光源，用于所述DMF器件中核酸检测时的光激发；
f)至少一个用于荧光测量反应的光学检测模块，用于对所述DMF器件上的至少一个位点进行光学测量。
作为示例，所述集成核酸分析***还包括触摸屏显示器，其提供用于用户执行实验和实验状态显示的图形界面，和/或包括对前述各模块进行统一操作控制的中央处理单元，两者均与所述光学检测模块电连接。
作为示例，所述磁铁是聚焦磁铁。
本发明还提供了一种测量样本中的至少一个目标核酸的方法，该方法包括将样本添加到如上述任一方案所述的集成核酸分析***的DMF器件中，然后对所述样本执行检测操作，所述检测操作包括以下步骤：1)将所述样本与裂解液混合以裂解样本中的细胞；2)向样本中加入磁珠溶液；3)混合所述磁珠溶液一是使磁珠和样本混合；4)清洗所述磁珠；5)提取样本中的核酸，纯化所述核酸，从所述磁珠上洗脱所述核酸以得到目标核酸；6)对洗脱后得到的所述目标核酸添加扩增试剂以对核酸进行热扩增；7)测量来自目标核酸的荧光强度。
在一示例中，清洗所述磁珠的步骤如下：a)利用仪器中的磁铁，在DMF器件上将磁珠从第一储液槽、经过填充液，移动到第二储液槽，并将磁珠在第二储液槽范围内按指定轨迹移动；c)将磁珠经过填充液，移动到第三储液槽。
在进一步的示例中，所述步骤a)和c)之间还包括：步骤b)将磁珠从第二储液槽、经过填充液，移动到第四储液槽，并将磁珠在第四储液槽范围内按指定轨迹移动。用于生物核酸提取的磁珠通常是直径很小(纳米级至微米级)的超顺磁微球，在磁场中能迅速聚集，磁场去除后又能在液体中均匀分散，而且不容易沉降。通过对磁珠做相应的包被(如硅基，氨基，羟基等)，可以实现对核酸的提取。细胞或组织在裂解液的作用下，其中的核酸(DNA 或RNA)被释放出来，经过表面修饰的磁珠与核酸进行特异性结合，形成磁珠-核酸复合物。核酸-磁珠复合物通常会有非特异性吸附的杂质，这些杂质可能会影响下一步的检测，因而需要去除。通常的去除方法是使用洗涤液,本发明提供了利用磁铁将磁珠在非混溶(immiscible)里按指定轨迹移动，从而实现去除磁珠-核酸复合物上杂质的方法。
在一示例中，清洗所述磁珠的实现步骤如下：利用仪器中的磁铁，在DMF器件上将磁珠从第一储液槽、经过填充液，直接移动到第三储液槽。
在一示例中，从所述磁珠上洗脱所述核酸的实现步骤如下：a)将磁铁移至距离DMF器件一定距离的指定位置，从而让磁珠在洗脱液中分散；b)将磁铁在和DMF器件平行的方向，在第三储液槽范围按指定方式移动，以帮助洗脱磁珠上的核酸；c)将磁铁移至第三储液槽处贴近DMF器件；d)将磁铁在第三储液槽处按指定路径移动以聚集其中的磁珠；e)将磁铁贴近DMF器件移至指定的磁珠废弃位置；f)将磁铁从DMF器件的位置移开。
作为示例，对所述目标核酸进行热扩增和测量来自目标核酸的荧光强度按如下方式进行：a)利用电润湿电极从第三储液槽分出一个或多个包含所述核酸的液滴；b)沿着指定的电润湿电极路径移动液滴以进行核酸扩增及光学测量。所述检测操作的所有步骤均优选由所述仪器自动完成。
在一具体示例中，所述热扩增为PCR扩增和/或等温扩增。在进一步的示例中，所述PCR扩增是通过将反应液滴在所述DMF器件上的不同温度区域之间，并按指定轨迹移动实现的，在PCR反应期间指定区域的温度保持不变。
作为示例，通过将一部分的复合物与不同的试剂混合来进行光学测量。
相较于现有技术，本发明提供的集成核酸分析***具有高灵敏度、多检测指标和检测时间短等优点，可以减少人工操作的失误、降低交叉污染、提高检测速度。
为使本发明的技术方案和优点更加突出，接下来将结合附图做进一步说明。
附图说明
图1A-1E显示为含有液体和磁珠的DMF器件的示例性局部侧视结构图，以及利用磁铁操作磁珠的过程。
图2A显示为具有储液槽、样本和试剂加载孔的DMF器件的示例性俯视结构图。
图2B显示为图2A的DMF器件的实物俯视图。
图3显示为图2A示例的DMF器件的底板上的电润湿电极的示例性布局示意图。
图4显示为图2A和图3的组合视图，并提供了热扩增和光学测量的示意图。
图5A-5C显示为DMF器件的间隙高度变化的上盖板部分的不同设计示意图。
图6A-6D显示为本发明提供的集成核酸分析***的不同功能模块的示例性示意图，其中，图6A展示了一个温度控制模块；图6B展示了一个可以同时控制2个磁铁的磁铁控制模块；图6C展示了光学测量模块；图6D是4个温度控制模块和2个磁铁(磁铁控制模块的一部分)的俯视图。
图7A和7B显示为本发明提供的集成核酸分析***的例示性结构示意图。
图8显示为使用本发明提供的集成核酸分析***实施的基于磁珠的核酸提取和定量PCR检测的示例性流程图。
图9显示为本发明提供的集成核酸分析***于另一示例中的俯视图。
图10显示为利用图9的集成核酸分析***实施基于磁珠核酸提取和CRISPR检测的示例性流程图。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。如在详述本发明实施例时，为便于说明，表示器件结构的剖面图会不依一般比例作局部放大，而且所述示意图只是示例，其在此不应限制本发明保护的范围。此外，在实际制作中应包含长度、宽度及深度的三维空间尺寸。
为了方便描述，此处可能使用诸如“之下”、“下方”、“低于”、“下面”、“上方”、“上”等的空间关系词语来描述附图中所示的一个元件或特征与其他元件或特征的关系。将理解到，这些空间关系词语意图包含使用中或操作中的器件的、除了附图中描绘的方向之外的其他方向。此外，当一层被称为在两层“之间”时，它可以是所述两层之间仅有的层，或者也可以存在一个或多个介于其间的层。
在本申请的上下文中，所描述的第一特征在第二特征“之上”的结构可以包括第一和第二特征形成为直接接触的实施例，也可以包括另外的特征形成在第一和第二特征之间的实施例，这样第一和第二特征可能不是直接接触。
需要说明的是,本实施例中所提供的图示仅以示意方式说明本发明的基本构想，遂图式中仅显示与本发明中有关的组件而非按照实际实施时的组件数目、形状及尺寸绘制，其实际实施时各组件的型态、数量及比例可为一种随意的改变，且其组件布局型态也可能更为复杂。 为使图示尽量简洁，各附图中并未对所有的结构全部标示。
为了本公开的目的，单词“包括”及其变种，例如“包含”，应当被理解为“包括但不限于”。
在本专利的整个说明中，“一个实施例”或“一个方面”等是指该实施例或方面包括一些特定的特征、结构和特性，可以包括在一个实施例中，但不一定会包括在所有的实施例。此外，本专利中公开的特征、结构和特性可以以任何合适的组合方式，应用在一个或多个实施例中。
为了本公开的目的，术语“微流控(microfluidics)”是指能对至少一个横截面尺寸为几微米至约几毫米的液体具有操纵能力的装置或***。而术语“数字微流控(digital microfluidics)”指的是基于电润湿或介电泳效应，可以对一个或多个液滴进行操控的装置或***。
为了本公开的目的，“DMF芯片”、“DMF装置”和“DMF器件”可以互换使用，包括具有第一基底表面的第一基底(底板)和具有第二基底表面的第二基底(上盖板)，该第二基底与第一基底间隔一定距离，以限定一个间隙(第一和第二基板表面之间的空间)，其中该距离足以容纳置于间隙中的液滴。多个第一液体控制电极设置在第一基板表面上，并且至少一些电极被一层电介质覆盖，并且电介质层的至少一部分是疏水的。出于接地的目的，在第二衬底表面上至少设置一个电极，该电极的至少一部分被一层电介质覆盖，并且该电介质层的至少一部分是疏水的。
出于本公开的目的，术语“液滴(droplet)”指的是和其他部分由空气或其他气体、其他(通常指相互不融合的)液体和固体表面(例如DMF器件的内表面)等分离开来的一定量的液体(一种或几种的混合)。“液滴”的体积范围很大,一般从几皮升(picoliter，皮升)到几百微升(microliter)。“液滴”可以有任意的形状，包括球形、半球形、扁状的圆形、不规则形等。
为了本公开的目的，术语“储液槽(reservoir)”或“液槽”用于指示DMF器件上的可以被用来存储、保持和供应液体的部分，可以是全封闭或部分封闭的。储液槽可以与流体路径相关联，该流体路径允许液体被引入DMF器件间隙进行液滴操作，或从DMF器件间进入储液槽进行液体存储或临时保存。
为了本公开的目的，术语“填充液(filler liquid)”和“填充油(filler oil)”可以互换使用，指的是可以全部充满或部分充满DMF器件间隙的、与液滴基本上不混溶、而且基本不影响DMF器件电润湿操作的能力的液体。填充液可包含低粘度油，例如硅油(silicone  oil)，矿物油(mineral oil)，液体石蜡(paraffin liquid)，氟硅油(fluorosilicone oil)等。填充液的运动粘度(kinematic viscosity)通常小于100cSt(centiStokes)，或50cSt，或20cSt，10cSt，5cSt，或2cSt。填充液可包含少量可溶于所述填充液的表面活性剂，表面活性剂与填充液的体积比通常在0.00001％至约1％，或0.0001％至约0.1％，或0.001％至约0.01％的范围内。填充液可以填充DMF器件的整个间隙或部分间隙。也可以将样本或试剂和填充液置放在DMF器件上特定的储液槽中，使得从该储液槽中利用点润湿分配出的液滴包裹有一层薄薄的填充液。
除非另有说明，本公开中，术语“小于”通常表示“等于或小于”，“大于”公开通常表示“等于或大于”。
为了本公开的目的，术语“液滴操作”可以包括液滴分配(从容器或连续的流体流)、移动、合并及混合、分割(对称或不对称)、成形(形成指定的形状)、悬浮及分布颗粒(在液体或液滴内)等。
本发明提出了用于处理或测量样本溶液中的目标分析物的集成核酸分析***和测量样本中的至少一个目标核酸的方法。如本领域技术人员将理解的，样本溶液可以包括但不限于体液(包括但不限于血液，血清，唾液，尿液等)，纯化的样本(例如纯化的DNA，RNA，蛋白质，细胞等)，环境样本(包括但不限于水，空气，农业样本等)和生物战剂样本等。体液可以来自任何生物。在一些实施方案中，实体溶液可以是来自哺乳动物的体液，例如来自人的体液。
出于本公开的目的，术语“分析物(analyte)”和“待分析物”可以互换使用，指的是分析或测试中的待测物质或化学成分。“分析物”可以是有机或无机物质。它可以指生物分子(如蛋白质、脂质、细胞因子、激素、碳水化合物等)，病毒(如疱疹病毒、逆转录病毒、腺病毒、慢病毒)，完整细胞(包括原核和真核细胞)、环境污染物(包括毒素、杀虫剂等)、药物分子(如抗生素、治效药物和药物滥用、及毒品)，细胞核，孢子，等等。
为了本公开的目的，术语“抗原(antigen)”是能够在宿主生物体中诱导免疫应答(产生抗体)的毒素或其他异物。
为了本公开的目的，术语“抗体(antibody)”是主要由浆细胞产生的大的Y形蛋白分子，其被免疫***用来中和病原体，例如病原性细菌或病毒。抗体与其相应抗原特异性结合。可将抗体用另一分子(例如，荧光标签或酶)进行标记，以促进抗体的检测或定量测量。
出于本公开的目的，术语“试剂(reagent)”和“检测试剂”可以互换使用，指的是用于与样本反应、稀释样本、混合样本、悬浮样本、乳化样本、包封样本、与样本相互作用和 添加到样本中的任何材料。
出于本公开的目的，术语“冻干试剂”和“冻干型试剂”可以互换使用，指的是使用冻干方法制备的试剂，通常用于含有不耐高温的活性物质的试剂。生物试剂保存是医疗诊断的重要环节。从试剂保存的角度，很多核酸即时检测(Point-of-Care Testing,或POCT)的试剂卡盒需要在室温下保存，这就要求检测试剂必须经过脱水处理，以固体形态存在。使用时，用缓冲液复溶进行后续反应。例如，液体试剂滴到液氮中，在极短的时间内固化成小球状，然后将固态小球放入预冷好的冻干机，设计好冻干曲线，完成冷冻干燥。冻干球形式能够最大程度地保持酶的活性，且冻干小球有疏松网状结构，复溶迅速。冻干的固态小球，经过封装后，可以常温储存运输，极大降低了运输成本及保鲜保存时间。冻干珠技术的这些突出优点，不仅适合核酸试剂POCT工艺生产流程，而且适合其他要求保鲜，保证活性物质，常温储存运输的生物制品等产品的生产工艺。在过去几年里，这项新技术快速得到了应用。
为了本公开的目的，术语“磁铁(magnet)”和“磁体”可以互换使用，用于指具有一定形状的、有磁性的物体，包括钐钴磁铁、钕铁硼磁铁、铁氧体磁铁、铝镍钴磁铁、及铁铬钴磁铁。形状包括圆柱形、圆环形、圆片形、圆锥形、金字塔形和其他不规则形状。磁铁包括永久磁铁和非永久磁铁，永久磁铁自身一直有磁性，而非永久性磁铁，例如电磁铁，只有在一定条件下(例如有电流通过)才会出现磁性。
为了本公开的目的，术语“聚焦磁铁(focusing magnet)”用于指具有特定形状的磁铁(永久磁铁或电磁铁)，使得一侧的磁场强于相反侧的磁场。示例包括但不限于圆锥形磁铁或金字塔形磁铁。圆锥形磁铁(或金字塔形磁铁)尖端的磁场强于其基体的磁场。
在本发明中，术语“颗粒(particle)”被用来指微米或纳米量级的实体，这些实体可以是天然的，也可以是人工制作的，例如细胞、亚细胞成分、脂质体(liposome)、病毒、纳米球，和微米球，或更小的如生物大分子、蛋白质、DNA及RNA等实体，它也可指与悬浮介质不相融合的液珠，它还可指液体中的小气泡等。“颗粒”的(线性)大小可以从几纳米到几百微米。
本发明中，术语“珠(bead)”可以是任何与溶液反应的珠子或粒子。珠子可以是任意不同的形状，如球形、鸡蛋形、立方形、圆盘形或者不规则形。珠子可以在液滴的里面、DMF器件的内表面上、DMF器件的填充液里、液槽里等。珠子可以由各种各样的材料制成，如树脂、聚合物、玻璃、纳米材料等，并且可以有任意的尺寸如微珠和纳米珠。珠子可以有磁响应性，在这种情况下，至少其一种或部分成分由磁响应材料构成，同时剩下的材料可能含聚合材料、涂层或链接有检测试剂的基团等。关于珠子的实例包括量子点、聚乙烯微珠、二氧 化硅微珠、荧光微球或纳米球、磁微珠、磁纳米珠，流式细胞微珠等。
为了本公开的目的，术语“磁珠(magnetic bead)”指的是包含磁响应材料珠子。磁响应材料的示例包括铁磁材料、顺磁性材料、超磁性材料、以及亚铁磁性材料等。顺磁性材料的示例包括诸如镍、铁和钴的金属，以及诸如Fe 3O 4,Cr 2O 3,NiO,Mn 2O 3等金属氧化物。磁响应材料可以基本上构成磁珠的全部、磁珠的一部分、或磁珠的某一种成分。磁珠的其余部分可以包括允许附着目标颗粒的聚合物材料和涂层部分。磁珠可用于各种测定中，其中磁珠通常被用来结合混合物中的一种或多种目标物质，例如分析物或污染物。测定通常需要有效的磁珠清洗过程，以减少含磁珠的液滴中可能与磁珠表面接触一种或多种物质的量。
基于数字微流控的验证实验表明涂有人抗血清白蛋白抗体(antihuman serum albumin antibodies)的磁珠，可用于分离人血清白蛋白(human serum albumin)。利用磁珠从全血样本中提取DNA的验证实验也在数字微流控平台上实现。在数字微流控平台上实现DNA提取和传统的方法类似，通常需要包含一步实现细胞裂解的预处理。
为了本公开的目的，术语“固定”用于指示磁珠在DMF器件上基本上被限制在液滴、储液槽和填充液中的某个指定位置。例如，在一个实施方案中，固定的磁珠基本上被限制在液滴中的位置，以允许执行液滴分离的操作，从而产生基本上没有磁珠的另一液滴和具有大部分磁珠的一个液滴(剩余液体)。
为了本公开的目的，相对于洗涤磁珠、洗涤，术语“磁珠洗涤”和“磁珠清洗”可以互换使用，用于表示利用液体来减少磁珠上某种(或几种)物质的数量或浓度。物质量或浓度的降低可以是部分的或完全的。该物质可以是多种物质中的任何一种，例如样本的成分、污染物和过量的试剂。
出于本公开的目的，“磁珠操作”可以由以下操作或其组合之一组成：
1.聚集–在DMF器件上的液滴内、液槽内和液滴(液槽)和液滴(液槽)之间的区域等将磁珠聚集。聚集后磁珠的尺寸小于10毫米，或小于5毫米，或小于2毫米。应该指出的是，聚集磁珠通常具有清晰的边界，但也可能相当模糊。被聚集的磁珠占所有特定磁珠的比例超过30％，或超过50％，或超过70％，或超过80％，或超过90％。
2.固定–将磁珠在DMF器件上的指定位置在指定的时间内固定不动。在此期间可以执行如液滴控制等操作。
3.移动–在DMF器件上将磁珠从一个位置移动到另一个位置，包括但不限于将磁珠从一个液滴或液槽移动到另一液滴或液槽。
4.打散-通过去除(或其他控制)磁场，让聚集的磁珠在液滴或液槽中分散开，这可以 和液滴操作同时进行。
出于本公开的目的，“扩增(amplification)”指的是可以增加待测分析物的数量或浓度的过程。非限制的例子包括聚合酶链反应(Polymerase Chain Reaction或PCR)及其变种(如定量竞争PCR、免疫PCR、逆转录PCR等)，链置换扩增(Strand Displacement Amplification或SDA)，基于核酸序列的扩增(Nucleic Acid Sequence Based amplification或NASBA)，环介导等温扩增(Loop-mediated isothermal amplification或LAMP)，解链酶扩增(Helicase-dependent amplification或HAD)等。
出于本公开的目的，术语“层(layer)”和“膜(film)”可以互换使用，用来指主体的结构，该结构通常但不必须是平面或基本上平面的，而且通常沉积、形成、涂覆或其他方式放置在另一结构上。
出于本公开的目的，术语“接地(ground)”(如用于“接地电极”或“接地电压”)指的是相应的电极的电压是零或足够接近于零。所有其他电压值，尽管幅度通常小于300伏，应当足够高，以使得能够充分观察到电润湿效应。
应当指出，当布置覆盖的电介质层时，同一层中相邻电极之间的空间通常填充有介电材料。这些空间也可以空着，或填充有诸如空气、氮气、氦气和氩气等气体。同一层中的所有电极和不同层处的电极优选的是电绝缘的。
如本文所用，术语“接触角”表示在液体-蒸汽界面接触到固体表面时形成的角度。在三相-液相、固相和气相(可以是环境大气和液体平衡浓度的混合物蒸汽)达到热力学平衡时，液-气界面的形状由Young-Laplace方程确定。
γ SG-γ SL-γ LGcosθ c＝0，
其中γ SG表示固气界面能，γ SL表示固液界面能，γ LG表示液气界面能(即表面张力)，θ表示平衡态时的接触角。以下是液滴的示意图，显示了Young-Laplace方程中的量。应当指出，如果气相被另一种不混溶的液相代替，则该方程式也适用。
Figure PCTCN2021127976-appb-000001
在纯液体内部，每个分子在每个方向上都被相邻的液体分子均等地拉动，导致净力为零。然而，液体表面的分子不是在所有方向上不具有相邻分子提供平衡的净力，它们被相邻的分 子向内拉，从而产生内部压力，其结果就是液体表面积收缩以保持其最低的表面自由能。这种使表面收缩的分子间力即液-气界面能γ LG，称为表面张力，它决定了液滴的形状。其他外力，如重力，也会使液滴变形。因此，接触角由表面张力和外力(通常为重力)共同决定。接触角也是某种固液***在特定环境下的一个特征参数。
疏水性表面具有排斥液体的特性，而亲水性表面具有吸引液体的特性。为了本公开的目的，“疏水表面”具有大于90°的接触角，而亲水表面具有小于90°的接触角。
出于本公开的目的，可以理解，当任何形式(如液滴或连续体，可能是在运动或静止的)的液体被描述为在电极、阵列、矩阵和表面“上”、“处”或“之上”时，该液体可能与电极、阵列、矩阵和表面直接接触，或可能与***液体和电极、阵列、矩阵、及表面之间的一个或多个层或膜相接触。
出于本公开的目的，可以理解，当诸如层、区域和基底的给定组件被称为置于或形成在另一组件“上”、“中”和“处”时，该给定组件可以直接位于该另一组件上，或备选地，也可以存在中间组件(例如一个或更多个缓冲层、夹层和电极)。还可以理解，术语“置于...上”和“形成在...上”可以互换使用，用来描述给定组件如何相对于另一组件进行定位或安置。因此，术语“置于...上”和“形成在...上”并不意在对材料传输、沉积和制造的特定方法引入任何限制。
出于本公开的目的，术语“印刷线路板(Printed Circuit Board，或PCB)”或“印制线路板”可以互换使用，指的是没有焊接元器件的电路板，主要由以下部分组成：
1、线路与图面(Pattern)：线路所用的材料通常是铜，线路可以为电子元器件之间提供导通的途径，另外通常设计大铜面作为接地及电源层。线路与图面是同时做出的。
2、介电层(Dielectric layer)：用来保持线路及各层之间的绝缘性，也叫基材。
3、孔(Through hole,或Via)：导通孔可使两层次以上的线路彼此导通，较大的导通孔则做为零件插件用，另外有非导通孔通常用来作为表面贴装定位。
4、防焊油墨(Solder Mask)：并非全部的铜面都要吃锡上零件，因此非吃锡的区域，会印一层隔绝铜面吃锡的物质(通常为环氧树脂)，避免非吃锡的线路间短路。根据不同的工艺，分为绿油、红油、蓝油。
5、丝印(Silk screen)：此为非必要之构成，主要的功能是在电路板上标注各零件的名称、位置框，方便组装后维修及辨识用。
出于本公开的目的，术语“检测(testing)”、“探测(detection)”和“测量(measurement)”可以互换使用，用来获取物理量(例如，位置、带电量、温度、浓度、pH 值、亮度、荧光等)的过程。在通常情况下，至少有一个传感器(或探测器)会被用来获取物理量，并将其转换成人或仪器可以识别的信号或信息。待测物和传感器之间可以有其他元器件，比如光学测量中使用的透镜、反光镜、滤光片等，和电学测量中的电阻、电容、三极管等。而且，为了使得测量成为可能或容易些，测量中常会用到其他的辅助装置或器件。例如，诸如激光或激光二极管等光源被用来将粒子从电子基态激发到电子激发态，激发态粒子回到基态时有时会发射荧光，而测量这时的荧光强度就可以用来测量液体样本中某种粒子的浓度。光学方面的传感器有CCD、光电二极管、光电倍增管等，在电学方面有运算放大器、模数转换器、热电偶、热敏电阻等。
测量可以对多个样本中的多个参量同时或按一定的顺序进行。例如，在用光电二极管测量液滴中某种粒子荧光的同时，其液滴的位置也可以由电容测量来同时获得。传感器或探测器通常会跟中央处理器(Central Processing Unit,或CPU)或电脑(computer)连接起来，中央处理器或电脑上运行有相应的软件，对所测量的信号进行分析，并通常将其转化成人或其他仪器可以读懂的信息。例如，利用对液体中某粒子荧光强度的测量和分析可以用来推断该粒子的浓度。作为非限制性示例，光学测量包括激光诱导的荧光测量(laser induced fluorescence measurement)、红外光谱(infrared spectroscopy)、拉曼光谱(Raman spectroscopy)、化学发光测量(chemiluminescence measurement)、表面等离子共振测量(surface plasmon resonance measurement)、吸收光谱(absorption spectroscopy)等；电学测量包括电流分析法(amperometry)、伏安测量法(voltammetry)、光电化学测量法(photoelectrochemistry)、库仑分析法(coulometry)、电容测量法(capacitance measurement)、以及交流阻抗测量法(and AC impedance measurement)，等。
以下是对本发明中的处理生物样本的实施方案的具体描述，为了便于说明，相应的附图(图1A至图10)会在需要的时候提到。应该说明的是，这些例子的目的是为了帮助说明，而不是为了限制发明的意愿和精神。
本文中作为辅助的附图和具体描述一起，进一步展示和诠释本专利公开的原理，并使相关领域的技术人员能够制造和使用相应的仪器、DMF器件、以及描述的方法。
出于本公开的目的，这里的一些和所有功能模块(例如磁铁控制模块，温度控制模块，光学测量模块等)都可以自动控制。在微处理器或计算机上运行的程序(软件或固件，software or firmware)通常被用来实现自动控制。
图1A-1E展出了本发明的集成核酸分析***中的DMF器件的一部分的侧视图，总体上标为100，作为实现磁珠控制的优选实施例。图1A示出了液体401和液体402在DMF器件上 不同的储液槽。底板300包含沉积在衬底301上的液滴控制电极302和介电层303。上盖板200包含了沉积衬底201上的接地电极202和介电层203。应该指出的是，这里示出的DMF器件结构出于说明磁珠操纵的目的，绝不代表所有的可能性。在一些实施例中，DMF器件可以以各种不同的方式实现。例如，1)控制电极可以具有不同的形状，例如长方形、正方形、梯形、五边形、六边形、和不规则形状，并且可以按直线或其他形状排列组合；2)控制电极可以在不同的层(通常是相互电绝缘的)，如其标题为“Electrowetting Based Digital Microfluidics”(“基于电润湿的数字微流控”，发明人吴传勇)的专利WO 2008/147568中所述的；3)介电层也可以具有两层或更多层，所用材料的包括聚对二甲苯C(Parylene C)，氮化硅，二氧化硅，钽氧化物等；其中一层可以是疏水材料，例如铁氟龙(Teflon)、Cytop、和FluoroPel等。
衬底可以是任何非导电材料或涂覆有非导电层的导电材料，只要它具有足够的机械强度,使其形状保持在所需的***操作和存储条件内即可。就透光能力而言，它可以是透明的、半透明的和不透明的。透明基板可以由多种透明材料制成，例如玻璃、石英、塑料、透明陶瓷、透明印刷线路板等。电极可以由任何导电材料制成，例如金属、合金和导电聚合物。它可以由一种材料或不同材料的混合物制成。DMF器件上的透明电极可以由透明导电材料(例如铟锡氧化物(ITO)，掺杂铝的氧化锌(AZO)，透明导电聚合物(聚乙炔，聚苯胺等)，或透明纳米材料制成等。
电压控制模块用于向液滴控制电极提供电压控制信号。它通常具有多个输出，最大数量为1000000，或100000，或10000，或1000。电压输出可以是单极性或双极性的，电压幅度小于1000伏，或小于300伏，或小于100伏，或小于60伏，或小于30伏。电压频率小于10MHz(兆赫兹)，或小于1MHz，或小于100KHz(千赫兹)，或小于20KHz，或小于5KHz，或小于1KHz的交流信号，或直流信号。电压的波形可以是方波，正弦波，锯齿形，脉宽调制信号等。电压控制模块通常由电路板上的微处理器或计算机通过SPI(Serial Peripheral Interface)，I2C(Inter-Integrated Circuit)，USB(Universal Serial Bus)，并行端口(parallel port)，以太网(Ethernet)，Wi-Fi，或蓝牙(Bluetooth)等，对输出信号的顺序、持续时间、幅度、及频率进行编程。弹簧探针(spring loaded electrical contact pin)或接口板(connector pad)可用来将多个高压控制信号传递到DMF器件上的电极。
在图1A中，磁珠500均匀地分布在DMF器件的储液槽401中。在图1B中，将磁铁600在储液槽401的位置紧贴DMF器件。在磁铁产生的磁场作用下，磁珠500被聚集在储液槽401的底部。如图1C和1D所示，磁铁将磁珠移到储液槽402处。在图1E中，将磁铁从DMF器件 移开，磁珠重新悬浮和分散在储液槽402中。磁珠悬浮和分散在储液槽402中的液体中，可能需要液滴运动或其他方式来协助。
DMF器件中储液槽间隙中可能会被空气或填充液包围。图1A–1E展示了磁珠从一个容器内部通过介质(空气或填充液)被移动到另一个储液槽中的过程。如图所示，该装置对于液滴是疏水的。电极上的液滴可以通过电润湿效应***作(移动、分割、合并、混合等)。
本文所使用的诸如“磁铁被移动(或带入、运输)以接近”，“磁铁被移近”之类的短语旨在指代磁铁和DMF器件的相对位置。磁铁产生的磁力对装置上的磁珠有明显的影响。相反地，诸如“磁铁移开”，“磁铁移出”之类的短语旨在表示磁铁对装置上的磁珠的没有影响和影响微不足道。
取决于不同的测定，磁铁的移动速度通常可以由马达控制，不同的应用需要的磁铁速度可以不同，通常范围是0.1至100mm/sec，或0.5至20mm/sec，或1至10mm/sec。
在上述实施例中，磁铁与底板衬底接触并沿着DMF器件的底部移动。在一些实施例中，磁铁也可以位于DMF器件的顶部；或者一对磁铁，一个在顶部，另一个在底部，用于操纵DMF器件中的磁珠。
图2A示出了本发明中的DMF器件200的俯视图，该DMF器件总体上以200表示，可以同时运行和分析两个样本。底板或上盖板都有2个功能一样的区域，可以分别用于处理2个的样本。储液槽204可用于样本裂解和核酸捕获，加液孔205和206用于装载样本，裂解液，和磁珠溶液等，用于核酸提取。储液槽207和209用于核酸清洗。加液孔208和210用于装载磁珠清洗液。储液槽211用于核酸洗脱，加液孔213用于装载洗脱液。加液孔215用于装载用于核酸扩增及检测的试剂。加液孔214可用于加载用于阴性或阳性对照的试剂。
为了进一步的阐述，此处给出的是一个简化的检测操作程序，包括：1)将磁铁在204的位置与DMF器件接触，通过在储液槽204范围内按指定轨迹移动磁铁，将带有捕获了核酸的磁珠聚集在储液槽的底部；2)利用磁铁将聚焦的磁珠移至储液槽207中，并在该储液槽内进行磁珠清洗；3)将聚焦的磁珠移至储液槽209中，并在该储液槽内做再一次的清洗；4)将清洗后的磁珠移至储液槽211中，并进行核酸洗脱；5)利用电润湿电极将含有核酸的洗脱液从洗脱槽分出一个或多个液滴，并移动到右侧，与通过加液孔215加载的PCR试剂混合；6)执行热扩增和光学测量。
图2B是基于本发明的DMF器件的上盖板的实物设计俯视图。上盖板的材料是聚碳酸酯(Polycarbonate)，利用注塑工艺制作。其中AL和BL液槽分别用于2个样本(A和B)核酸的磁珠提取；AE1和AE2液槽用于样本A的磁珠清洗，BE1和BE2液槽用于样本B的磁珠清洗； 其中AE和BE液槽分别用于A、B样本的核酸洗脱。A1-A4孔用于加检测样本A的试剂，B1-B4孔用于加检测样本B的试剂；OIL孔用于对DMF器件加填充液。
图3示出了用于图2A所示的DMF器件的相应的电润湿电极布局。可选电极311可以用于辅助样本与裂解缓冲液和磁珠等的混合，可选电极312和313用于辅助磁珠的洗涤，电极314和315用于将液滴从洗脱槽分出，并移动到扩增反应区域，电极321至324用于移动(包括来回重复移动)液滴，以实现扩增及或测量。
图4是结合图2A和图3的功能示意图。图4中给出了温控区域611和612以及光学检测点620。在一个实施例中，光学激发和检测均通过DMF器件的底板完成。因此，DMF器件底板的该部分需要是光学透明的(包括衬底、电极和介质层)。在本发明中，光学测量是在可见光范围内完成的，波长通常在350-750nm之间。更高的光透过率意味着更高的***检测灵敏度。DMF器件的底板光学测量部分的透射率通常大于30％，或者大于50％，或者大于70％，或者大于90％。
在DMF器件中，为了便于液滴分样或液滴控制，有时会需要在不同的功能位置将DMF器件的间隙设计为不同的高度。图5A-5C示出了DMF器件间隙高度从H1变化到较小值H2的部分的侧视图，其中图5A和5B是两种常见的设计，图5C是本发明提出的设计。在图5A中，间隙高度在单个点253处从区域251中的H1变为区域252中的H2；在图5B中，间隙高度在区域254上从区域251中的H1线性变化为区域252中的H2，对应的斜率(slope)是一个常数(constant)。
用数学术语来说，曲线某点处的斜率是该点处切线(tangent)的斜率。在图5A-5C中，斜率在区域251和252处为零。在图5A中，斜率在点253处是无穷大的。在图5B中，斜率在整个区域254中是一个常数，这个常数可以通过间隙高度差和区域长度来计算得到。它等于(H1-H2)除以区域254的长度，例如，如果H1为2.5mm，H2为0.5mm，区域254的长度为10mm，则斜率的绝对值为0.2。
在本发明中提出了图5C，其中间隙高度在区域255上从区域251中的H1变化到区域252中的H2。在区域255中，斜率(的绝对值)连续地从零增加到一个有限值，然后再连续的减小到零。作为对比，在图5A中，斜率从零在253处变化到无穷大，然后又变回到零；在图5B中，斜率从零直接变化为一个非零的值，然后又直接变回到零。换句话说，图5A和5B中DMF器件间隙变化处上盖板的下表面的斜率会经历一个非连续(non-continuous)的变化，而在图5C中，相应斜率的变化是连续的(continuous)。在不进一步纠结液体行为的数学细节的情况下，图5C提出了一种设计，可使得液滴更容易从一个间隙高度过渡到另一个间隙高 度。特别地，这使得从液槽中取出液滴更容易。
图6A展示了温度控制模块的一个实施方式。温度控制模块611和612用于核酸的扩增。温度控制模块613可选地用于辅助核酸洗脱，而温度控制模块614可选地用于辅助核酸提取。图6B是磁铁控制模块630，图中的631是2个用于操控DMF器件中磁珠的磁铁，图6C展示了光学检测模块的设计，图6D给出了温度控制模块611至614，磁铁630和光学检测模块620的俯视图。
从图6A至6D可以看出，当温度控制模块(尤其是613和614)在物理距离上靠近DMF器件时，磁铁的运动范围受到限制。由于在实验过程中并非同时需要所有功能模块，因此可以将某些模块移开，以便其他模块在需要时可以有更多空间移动。因此，在一个实施例中，该仪器被设计成使得当不需要温度控制时，一个或多个温度控制元件从靠近或接触DMF器件的地方移开，从而让磁铁移动到贴近DMF器件的预期位置。在另一个实施例中，当不使用磁铁630时，可以将其移动到DMF器件的一端，以允许温度控制模块613和614移动到靠近或接触DMF器件的位置。在又一个实施例中，磁铁630可以被保持在温度控制模块613和614之间的一个或多个空间中，使得温控模块613和614可以被移动靠近或接触DMF器件。
前面所述的磁铁和加热块是相互独立的器件，在实际应用中，两个器件可以集成在一起，也就是一个器件既可以用来对DMF器件做温度控制，又可以产生磁性对DMF器件中的磁珠进行控制。使用这种集成的模块可以节省空间，简化仪器设计。由于相关的设计比较简单，这里不再给出具体的实例描述。
图7A和7B展示了集成核酸分析***的一个优选实施例。图7A是该装置的顶-前-侧视图，图7B是后视图。700是弹簧探针，可用来实现仪器和DMF器件的液滴控制的接口，向DMF器件上的电润湿电极提供液滴控制电压。701是一个触摸屏，用来提供用户可操作的图形界面，供用户输入命令并显示帮助、实验状态、及测量结果等。702是可滑动的托盘，用于加载及卸载DMF器件。当推入时，弹簧探针700与DMF器件的底部基板接触。703是DMF器件。图7B中，704是用于对外排放仪器工作时产生的热量的通风口(内部通常装有具有风扇)。705是USB端口，而706是以太网端口。它们可选的为在计算机上运行的应用软件提供与仪器进行通讯(如发送命令或接收数据)的方式。707是交流电源端口。
图8展示了利用核酸分析***做基于磁珠核酸提取和定量PCR分析的示例。
在步骤S801中，在储液槽204中加裂解液和磁珠溶液，在储液槽207和209中加磁珠清洗液，在储液槽211中加洗脱液,在储液槽215中加定量PCR试剂。将样本加入储液槽204。
在步骤S802中，移动磁铁，在储液槽204处贴紧DMF器件。将磁铁在储液槽204范围内 按指定轨迹移动，帮助储液槽内的磁珠进行核酸捕获。
在步骤S803中，将磁铁贴紧DMF器件从储液槽204移动到储液槽207，这会将磁珠携带至储液槽207。将磁铁在储液槽207范围内按指定轨迹移动，以清洗磁珠上可能的杂物。
在步骤S804中，将磁铁从储液槽207移动到储液槽209，这会将磁珠携带至储液槽209。将磁铁在储液槽209范围内按指定轨迹移动，做进一步的磁珠清洗。
在步骤S805中，将磁铁从储液槽209移动到储液槽211，这会将磁珠携带至储液槽211。将磁铁从DMF器件处移开，这样磁珠会分散到洗脱液中。移动磁铁，将磁铁移回至贴紧DMF器件的储液槽211处，将磁铁在储液槽211范围内按指定轨迹移动，以重新聚集磁珠。将磁铁贴紧DMF器件移至一个指定的位置，丢弃磁珠。
在步骤S806中，从储液槽211中分4个液滴，分别沿着路径321至324移至储液槽215处和那里的定量PCR试剂混合。
在步骤S807中，将4个液滴沿着路径321至324在PCR温度区611和612件来回移动，并在每个移动循环中在检测点620做荧光测量。
在步骤S808中，根据获得定量PCR数据，产生分析报告。
图9示出了本发明中的另一个示例中的DMF器件及仪器模块的俯视图，该DMF器件可以同时运行和分析四个样本。储液槽901可用于样本裂解和核酸捕获，储液槽902和903用于核酸清洗，储液槽904用于核酸洗脱，储液槽905用于装载PCR试剂，储液槽906用于装载Cas试剂。911为电润湿电极，用于对DMF器件中的液体进行操作，912中的每一个小方块上有固态(风干)的gRNA试剂，用于对反应物中的(可以是不同的)待测核酸分子进行检测。921，922，和923分别为仪器中三个可以独立控温的模块，可以将对应芯片的区域控制在不同的温度。
图10展示了利用图9提供的集成核酸分析***做基于磁珠核酸提取和CRISPR检测的示例。
在步骤S1001中，在储液槽901中加裂解液和磁珠溶液，在储液槽902和903中加磁珠清洗液，在储液槽904中加洗脱液,在储液槽905中加PCR试剂，在储液槽906中加Cas试剂。将样本加入储液槽901。
在步骤S1002中，向上移动磁铁，在储液槽901处贴紧DMF器件。将磁铁在储液槽901范围按预定的轨迹移动，帮助储液槽内的磁珠进行核酸分子的捕获。
在步骤S1003中，将磁铁贴紧DMF器件从储液槽901移动到储液槽902，这会将磁珠携带至储液槽902。将磁铁在储液槽902范围按预定的轨迹移动，以清洗磁珠上可能吸附的杂 物。
在步骤S1004中，将磁铁从储液槽902移动到储液槽903，这会将磁珠携带至储液槽903。将磁铁在储液槽903范围按预定轨迹移动，做进一步磁珠清洗。
在步骤S1005中，将磁铁从储液槽903移动到储液槽904，这会将磁珠携带至储液槽904。将磁铁从DMF器件处移开，这样磁珠会分散到洗脱液中。移动磁铁，将磁铁移回至贴紧DMF器件的储液槽904处，将磁铁在储液槽904范围内来回移动，以重新聚集磁珠。将磁铁贴紧DMF器件移至一个指定的位置，丢弃磁珠。
在步骤S1006中，从储液槽904里的洗脱液中分出1个液滴，移至储液槽905将存放在那里的冻干PCR试剂溶解。然后将该液滴移在温度区域921和922之间来回移动，做PCR扩增。
在步骤S1007中，将PCR扩增后的液滴分成2部分，一部分移至904或905等区域丢弃，另一部分和906中的Cas试剂混合。
在步骤S1008中，将S1007中混合后的液滴移至gRNA点样处912，与那里的gRNA混合，此时Cas和gRNA结合，找到和gRNA相对应的DNA分子后，对液滴中的核酸分子(包括reporter molecule)剪切。对反应液滴在指定的时间内按指定的时间间隔进行荧光测量。
在步骤S1009中，根据获得的荧光数据，产生分析报告。
这里应该提到的是，试剂可以封装在DMF器件上，用户只需要加载样本即可。这使装置更易于操作，并减少了测试被污染(或交叉污染)的机会。
应当指出，上述示例和上述优点是出于说明的目的，绝不是穷举的。
尽管已经示出和描述了本发明的优选实施例，但是应当理解，可以在不脱离本发明的精神和范围的情况下进行各种改变。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (22)

  1. 一种集成核酸分析***，其特征在于，所述集成核酸分析***包括至少一个DMF器件，用于检测至少一个样本，所述DMF器件包括：
    具有第一表面的底板，所述底板上设置有用于液滴或液体控制的电润湿电极，所述第一表面为平坦表面，底板的至少一部分是光学透明的；
    上盖板，所述上盖板包括平行于所述第一表面并与第一表面配合以形成用于液体操作的间隙的第二表面，所述间隙包括用于核酸提取的第一储液槽和用于核酸洗脱的第三储液槽，所述上盖板设置有用于样本或试剂加载或卸载的孔；
    用于对所述DMF器件中的样本执行一系列操作的仪器，所述操作包括核酸的磁珠提取、纯化、热扩增和光学测量。
  2. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件还包括用于核酸清洗的第二储液槽和/或用于磁珠洗涤的第四储液槽。
  3. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件的所述底板还设置有用于核酸浓度扩增的电润湿电极路径。
  4. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件的间隙具有多个不同的高度值，且所述DMF器件的间隙高度从一个值改变为另一值时，对应第二表面斜率的变化是连续的。
  5. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件的底板透明处的光透射率在可见光范围内大于30％。
  6. 根据权利要求5所述的集成核酸分析***，其特征在于，所述透射率大于90％。
  7. 根据权利要求2所述的集成核酸分析***，其特征在于，所述第一储液槽置放有样本裂解液和磁珠溶液，所述第二储液槽置放有磁珠洗涤液，所述第三储液槽置放有核酸洗脱液。
  8. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件的间隙内有填充液。
  9. 根据权利要求1所述的集成核酸分析***，其特征在于，所述DMF器件预装有用于检测的冻干试剂。
  10. 根据权利要求1所述的集成核酸分析***，其特征在于，所述仪器包括：
    a)至少一个用于加载及卸载DMF器件的模块，与所述DMF器件相连接；
    b)至少一个电压控制模块，与所述DMF器件相连接，用于向DMF器件提供电信号，以操作所述DMF器件中的液体或液滴；
    c)至少一个磁铁控制模块，位于所述DMF器件的上部或下部，用于控制一个或多个磁铁，每个磁铁具有至少两个运动自由度；
    d)至少一个温度控制模块，用于将DMF器件的指定区域控制到指定的温度；
    e)至少一个光源，用于所述DMF器件中核酸检测时的光激发；
    f)至少一个光学检测模块，用于对所述DMF器件上的至少一个位点进行光学测量。
  11. 根据权利要求10所述的集成核酸分析***，其特征在于，所述集成核酸分析***还包括触摸屏显示器和中央处理单元中的一个或两个，与所述光学检测模块电连接。
  12. 根据权利要求10所述的集成核酸分析***，其特征在于，所述磁铁是聚焦磁铁。
  13. 一种测量样本中的至少一个目标核酸的方法，其特征在于，包括将样本添加到如权利要求1-12中任一项所述的集成核酸分析***的DMF器件中，然后对所述样本执行检测操作，以测量目标核酸，其中，所述检测操作包括以下步骤：
    1)将所述样本与裂解液混合；
    2)加入磁珠溶液；
    3)混合所述磁珠溶液；
    4)清洗所述磁珠；
    5)从所述磁珠上洗脱所述核酸以得到目标核酸；
    6)对所述目标核酸进行热扩增；
    7)测量来自目标核酸的荧光强度。
  14. 根据权利要求13所述的方法，其特征在于，清洗所述磁珠的步骤如下：
    a)利用仪器中的磁铁，在DMF器件上将磁珠从第一储液槽、经过填充液，移动到第二储液槽，并将磁珠在第二储液槽范围内按指定轨迹移动；
    c)将磁珠经过填充液，移动到第三储液槽。
  15. 根据权利要求14所述的方法，其特征在于，所述步骤a)和c)之间还包括：步骤b)将磁珠从第二储液槽、经过填充液，移动到第四储液槽，并将磁珠在第四储液槽范围内按指定轨迹移动。
  16. 根据权利要求13所述的方法，其特征在于，清洗所述磁珠的实现步骤如下：利用仪器中的磁铁，在DMF器件上将磁珠从第一储液槽、经过填充液，直接移动到第三储液槽。
  17. 根据权利要求13所述的方法，其特征在于，从所述磁珠上洗脱所述核酸的实现步骤如下：
    a)将磁铁移至距离DMF器件一定距离的指定位置，从而让磁珠在洗脱液中分散；
    b)将磁铁在和DMF器件平行的方向，在第三储液槽范围按指定方式移动，以帮助洗脱磁珠上的核酸；
    c)将磁铁移至第三储液槽处贴近DMF器件；
    d)将磁铁在第三储液槽处按指定路径移动以聚集其中的磁珠；
    e)将磁铁贴近DMF器件移至指定的磁珠废弃位置；
    f)将磁铁从DMF器件的位置移开。
  18. 根据权利要求13所述的方法，其特征在于，对所述目标核酸进行热扩增和测量来自目标核酸的荧光强度按如下方式进行：
    a)利用电润湿电极从第三储液槽分出一个或多个包含所述核酸的液滴；
    b)沿着指定的电润湿电极路径移动液滴以进行核酸扩增及光学测量。
  19. 根据权利要求13所述的方法，其特征在于，所述检测操作的所有步骤由所述仪器自动完成。
  20. 根据权利要求13所述的方法，其特征在于，所述热扩增为PCR扩增和/或等温扩增。
  21. 根据权利要求20所述的方法，其特征在于，所述PCR扩增是通过将反应液滴在所述DMF器件上的不同温度区域之间，并按指定轨迹移动实现的，在PCR反应期间指定区域的温度保持不变。
  22. 根据权利要求18所述的方法，其特征在于，通过将一部分的复合物与不同的试剂混合来进行光学测量。
PCT/CN2021/127976 2021-09-17 2021-11-01 集成核酸分析***和测量样本中的目标核酸的方法 WO2023040012A1 (zh)

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