CN218910385U - Single-cell nucleic acid analysis system based on digital microfluidic - Google Patents

Abstract

The utility model discloses a single-cell nucleic acid analysis system based on digital microfluidics. The system comprises a digital microfluidic device, wherein the digital microfluidic device comprises a bottom plate and an upper cover plate, a plurality of electrowetting electrodes for controlling movement of liquid drops are arranged on the bottom plate, some electrodes form a plurality of paths for realizing DNA amplification, and at least one part of the bottom plate is optically transparent and used for light excitation and measurement; the upper cover plate is provided with a liquid storage tank for storing and reacting the reagent. The system is used for single-cell nucleic acid analysis, and can reduce errors of manual operation, reduce cross contamination and improve detection speed.

Description

Single-cell nucleic acid analysis system based on digital microfluidic

Technical Field

The utility model relates to the technical field of biological analysis, in particular to a single-cell nucleic acid analysis system based on digital micro-flow control.

Background

Single cell nucleic acid analysis often requires multiple steps, which can be integrated into the same device, omitting the transfer of fluids between different steps has many advantages, such as reduced cross contamination, safer operation, increased detection sensitivity, reduced detection time, etc. The utility model provides a nucleic acid detection method based on digital microfluidic integration of multiple steps.

There are a number of instruments for biochemical treatment and analysis. When using these instruments, a significant proportion of the sample or reagent required for the biochemical reaction does not participate in the reaction or measurement and is therefore wasted. In digital microfluidic (Digital Microfluidics, DMF) systems, the dead volume (the volume of liquid added to the system but not involved in the reaction and detection) can be significantly reduced, sometimes even to zero. This means that the amount of sample or reagent required for the experiment is the amount used for the measurement. This not only greatly reduces the cost of using the sample and reagent, since the smaller reaction volume can shorten the mixing time of the reagent and sample, and thus the time required for detection and analysis.

In contrast to the large number of manual processing steps required in many current biochemical analysis systems, digital microfluidic-based systems (instruments, devices, methods, etc.) can provide a high degree of integration and automation. This can greatly reduce possible human error and can greatly improve the reliability and data quality of detection.

In a droplet-based digital microfluidic system, the liquid is manipulated in a two-dimensional space in a discrete format (droplets), which can be individually manipulated, which is why it is called digital microfluidic. In DMF devices, the travel path of the droplet may be defined at run-time and may be dynamically altered. In contrast to typical pressure-driven (by external pumps) or electrically-driven (by high pressure) channel-based microfluidic, DMF devices require only low voltages to control the droplets therein. The driving force in digital microfluidic is based on electrostatic effects, such as electrowetting or dielectrophoresis, which are generally not correlated with the biological sample to be detected, so that the design of the digital microfluidic system and specific detection items are independent, and therefore the digital microfluidic system has better universality.

In recent years, digital microfluidic technology has attracted a lot of attention because of its ability to handle individual droplets and the ease of miniaturization, integration, and automation. The digital micro-fluidic technology reduces the usage amount of reagents, simplifies experimental steps, shortens detection time and has great superiority.

The polymerase chain reaction (Polymerase Chain Reaction, or PCR) fundamentally changes the scientific field, and as a well-established method, PCR requires repeated heating and cooling cycles of a reaction system containing specific DNA primers, dntps (deoxynucleotide triphosphates), thermostable DNA polymerase (e.g., taq DNA polymerase), etc., to double the number of target DNA molecules per temperature cycle for possible target DNA molecules, resulting in exponential accumulation of target sequences. This technique amplifies trace amounts of DNA or RNA in a sample to a level that can be measured and analyzed. PCR techniques have been applied in many different fields including viral load testing, food borne pathogen quantification, clinical diagnosis, drug resistance analysis and forensic science. Using PCR techniques, doctors and researchers can determine the source of a viral infection by analyzing a single cell. A number of infectious organisms can now be detected using PCR, such as COVID-19, HIV-1, hepatitis B, hepatitis C, SARS virus, west Nile Virus, mycobacterium tuberculosis, and the like.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, an object of the present utility model is to provide a single-cell nucleic acid analysis system based on digital microfluidic for implementing nucleic acid analysis of single-cell sorting and quantitative PCR. The utility model utilizes the digital microfluidic chip to quantitatively divide liquid, move liquid drops, mix, focus and move magnetic beads, integrates the cell sorting and nucleic acid detection processes on the chip, can obviously improve the nucleic acid analysis and detection efficiency, and reduces the manual labor intensity and errors caused by manual operation.

The technical scheme of the utility model is specifically introduced as follows.

The utility model provides a single-cell nucleic acid analysis system based on digital microfluidics, which comprises a bottom plate and an upper cover plate, wherein a plurality of electrowetting electrodes for controlling liquid drop movement are arranged on the bottom plate, each electrowetting electrode comprises a first electrode for cell sorting, a second electrode for separating DNA (deoxyribonucleic acid) and a third electrode for DNA amplification, and a plurality of third electrodes are arranged in an array to form a plurality of DNA amplification paths for realizing DNA amplification; the upper cover plate is provided with a liquid storage tank, wherein the liquid storage tank comprises a first liquid storage tank for adding a cell sample solution to be analyzed, a second liquid storage tank for storing sample waste liquid which does not contain cells to be tested after separation, a third liquid storage tank for adding a lysis solution containing magnetic beads, a fourth liquid storage tank for adding a magnetic bead cleaning solution and a fifth liquid storage tank for adding a DNA eluent;

the first liquid storage tank is respectively connected with the second liquid storage tank and the third liquid storage tank through the first electrode; the output end of the third liquid storage tank is connected with the input end of the fourth liquid storage tank, the output end of the fourth liquid storage tank is connected with the input end of the fifth liquid storage tank, and the fifth liquid storage tank is connected with a sample adding hole for adding a DNA amplification reagent through a second electrode and a DNA amplification path formed by a third electrode; the middle position of the DNA amplification reagent is provided with a plurality of fixed PCR temperature areas, optical detection points for light excitation and measurement are arranged between the fixed PCR temperature areas, the optical detection points and the sample adding holes are arranged on the bottom plate, the peripheral area of the bottom plate where the optical detection points are arranged is optically transparent, and the bottom of the bottom plate is provided with a magnet for gathering and driving the magnetic beads to move.

In the utility model, the electrowetting electrode is connected with the voltage control module.

In the utility model, the bottom plate comprises a substrate, an electrowetting electrode and a dielectric layer, wherein the electrowetting electrode is deposited on the substrate; the upper cover plate includes a substrate, a ground electrode, and a dielectric layer, the ground electrode being deposited on the substrate.

In the utility model, two or three fixed PCR temperature areas are provided, and the fixed PCR temperature areas are connected with a temperature control module.

In the present utility model, the magnet is connected to the motor.

Compared with the prior art, the utility model has the beneficial effects that:

the system is used for single-cell nucleic acid analysis and detection, and can reduce errors of manual operation, reduce cross contamination, improve detection speed and the like.

Drawings

Fig. 1 shows the layout of electrowetting electrodes on the bottom plate of a digital microfluidic DMF device.

Fig. 2 shows a schematic diagram of a digital microfluidic DMF chip for cell sorting, lysis, and nucleic acid detection.

Fig. 3 is a functional schematic combining fig. 1 and 2.

Fig. 4A-4E show side views of a portion of a digital microfluidic DMF device containing a liquid and magnetic beads, and a process for manipulating the magnetic beads with a magnet.

Detailed Description

A description will be given below of the structure and operation of various embodiments of the present utility model, as well as detailed features and advantages, with reference to the accompanying drawings. It should be noted that the present utility model is not limited to the particular embodiments described herein. These embodiments are presented herein for illustrative purposes only and other embodiments will be apparent to those skilled in the relevant arts.

For the purposes of this disclosure, the word "comprising" and variations thereof, such as "comprises", should be understood to mean "including but not limited to".

Throughout this description of the utility model, "one embodiment" or "an aspect" or the like means that the embodiment or aspect includes some particular feature, structure, and characteristic that may be included in one embodiment, but not necessarily all embodiments. Furthermore, the features, structures, and characteristics of the utility model may be combined in any suitable manner in one or more embodiments.

For the purposes of this disclosure, the term "microfluidic" refers to a device or system capable of manipulating at least one liquid having a cross-sectional dimension of a few microns to about a few millimeters. While the term "digital microfluidic (digital microfluidics)" refers to a device or system that can manipulate one or a droplet based on electrowetting or dielectrophoresis effects.

For purposes of this disclosure, "DMF chip," "DMF device," and "DMF device" may be used interchangeably, including a first substrate (bottom plate) having a first substrate surface and a second substrate (top plate) having a second substrate surface, the second substrate being spaced from the first substrate by a distance to define a gap (space between the first and second substrate surfaces), wherein the distance is sufficient to accommodate a droplet disposed 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. For grounding purposes at least one electrode is provided on the second substrate surface, at least a part of which electrode is covered by a layer of dielectric and at least a part of which dielectric layer is hydrophobic. It also includes DMF device introduced in the utility model:

for the purposes of this disclosure, the term "drop" refers to a quantity of liquid (a mixture of one or more) separated from other parts by air or other gases, other (generally non-fusing) liquids, and solid surfaces (e.g., the inner surface of a DMF device), etc. The volume of the "droplets" is very large, typically ranging from a few picoliters (picoliters) to hundreds of microliters (microlite). The "droplets" may have any shape, including spherical, hemispherical, flat rounded, irregular, etc.

For the purposes of this disclosure, the term "reservoir" or "tank" is used to indicate the portion of the DMF device that may be used to store, hold and supply liquid, whether fully enclosed or partially enclosed. The reservoir may be associated with a fluid path that allows liquid to be introduced into the gap of the DMF devices for droplet operations, or from between the DMF devices into the reservoir for liquid storage or temporary preservation.

For the purposes of this disclosure, the terms "filler liquid" and "filler oil" are used interchangeably to refer to a liquid that may completely or partially fill the gap of a DMF device, is substantially immiscible with liquid droplets, and does not substantially affect the ability of the DMF device to electrowetting operation. The filler fluid may comprise a low viscosity oil, such as silicone oil (fluorosilicone oil), fluorosilicone oil (fluorosilicone oil), mineral oil (mineral oil), liquid paraffin (Paraffin liquid), and the like. The kinematic viscosity (kinematic viscosity) of the filler liquid is typically less than 100 cSt (centrstokes), or 50 cSt, or 20 cSt,10cSt,5cSt,2cSt, or 1cSt.

In this disclosure, unless otherwise indicated, the term "less than" generally means "equal to or less than," and "greater than" generally means "equal to or greater than.

For purposes of this disclosure, the term "droplet operations" may include droplet dispensing (from a container or continuous fluid flow), moving, combining and mixing, dividing (symmetrical or asymmetrical), shaping (forming a specified shape), suspending and distributing particles (within a liquid or droplet), and the like.

The present utility model provides devices and methods for processing or measuring target analytes in a sample solution. As will be appreciated by those skilled in the art, the sample solution may include, but is not limited to, body 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, among others. The bodily fluid may be from any organism, in some embodiments, the solid solution may be a bodily fluid from a mammal, such as a bodily fluid from a human.

For the purposes of this disclosure, the terms "analyte" and "analyte" are used interchangeably to refer to a substance or chemical component to be measured in an assay or test. The "analyte" may be an organic or inorganic substance. It may refer to biomolecules (e.g., proteins, lipids, cytokines, hormones, carbohydrates, etc.), viruses (e.g., herpes viruses, retroviruses, adenoviruses, lentiviruses), intact cells (including prokaryotic and eukaryotic cells), environmental contaminants (including toxins, pesticides, etc.), drug molecules (e.g., antibiotics, therapeutic drugs, and drug abuse), nuclei, spores, etc.

For purposes of this disclosure, the term "reagent" refers to any material used to react with, dilute, mordant, suspend, emulsify, encapsulate, interact with, and add to a sample.

For the purposes of this disclosure, the terms "lyophilized reagent" and "lyophilized reagent" are used interchangeably to refer to reagents prepared using a lyophilization process, typically for reagents containing active substances that are not resistant to high temperatures. Biological reagent preservation is an important link in medical diagnosis. From the viewpoint of reagent preservation, many kits for Point-of-Care Testing (or POCT) require preservation at room temperature, which requires that the test reagents must be dehydrated and exist in a solid form. When in use, the buffer solution is used for redissolution to carry out subsequent reactions. For example, the liquid reagent is dropped into liquid nitrogen, solidified into pellets in a very short time, and then the solid pellets are placed into a pre-chilled freeze dryer, a freeze-drying curve is designed, and freeze-drying is completed. The freeze-dried ball form can keep the activity of enzyme to the maximum extent, and the freeze-dried balls have loose network structure and are quickly redissolved. The freeze-dried solid pellets can be stored and transported at normal temperature after being packaged, so that the transportation cost and the preservation time are greatly reduced. These outstanding advantages of the freeze-dried bead technology are not only suitable for the POCT process production flow of nucleic acid reagents, but also suitable for other production processes requiring preservation, ensuring active substances, biological products stored and transported at normal temperature, and the like. This new technology has been rapidly applied in the past few years.

For the purposes of this disclosure, the term "lysate (or lysis buffer)" refers to an agent that can detach nucleic acids in a sample from its original host (e.g., cell, virus, etc.). Protease can also be added into the lysate; the use of proteases to digest proteins into small fragments facilitates separation of nucleic acids from proteins, and also facilitates subsequent purification operations and the obtaining of purer nucleic acids. The effect of the salt, in addition to providing a suitable cleavage environment (e.g., tris), includes inhibiting the destruction of nucleic acids by nucleases in the sample during cleavage (e.g., EDTA), maintaining the stability of the nucleic acid structure (e.g., naCl), and the like.

In the present utility model, the term "particle" is used to refer to an entity on the order of micrometers or nanometers, which may be natural or artificial, such as cells, subcellular components, liposomes (liposome), viruses, nanospheres, and microspheres, or smaller entities such as biological macromolecules, proteins, DNA, and RNA, which may also refer to liquid beads that are immiscible with a suspending medium, which may also refer to small bubbles in a liquid, and the like. The (linear) size of the "particles" may be from a few nanometers to hundreds of micrometers.

For the purposes of this disclosure, the term "immobilized" is used to indicate that the magnetic beads are substantially confined to a specified location in the droplet, reservoir, and fill fluid on the DMF device. For example, in one embodiment, the immobilized magnetic beads are substantially confined to a location in the droplet to allow the droplet separation operation to be performed, thereby producing another droplet that is substantially free of magnetic beads and one droplet (remaining liquid) that has a majority of magnetic beads.

For the purposes of this disclosure, "amplification" refers to a process by which the number or concentration of analytes to be measured can be increased. Non-limiting examples include polymerase chain reaction (Polymerase Chain Reaction or PCR) and variants thereof (e.g., quantitative competitive PCR, immuno PCR, reverse transcription PCR, etc.), strand displacement amplification (StrandDisplacement 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), melt-enzyme amplification (Helicase-dependent amplification or HAD), and the like.

For the purposes of this disclosure, the terms "quantitative PCR" and "fluorescent quantitative PCR" and "real-time fluorescent quantitative PCR" are used interchangeably to refer to a method of adding a fluorescent group into a PCR reaction system, measuring the fluorescent intensity of the reaction system at each PCR temperature cycle, and finally quantitatively analyzing an unknown template by a standard curve. The common quantitative PCR method is 1) SYBRGreen fluorescent dye method, in the PCR reaction system, excessive SYBR fluorescent dye is added, after the SYBR fluorescent dye is nonspecifically doped into DNA double chains, fluorescent signals are emitted, and SYBR dye molecules which are not doped into the chains cannot emit any fluorescent signals, so that the increase of the fluorescent signals and the increase of PCR products are completely synchronous. SYBR binds only to double stranded DNA and thus can determine whether the PCR reaction is specific by a lysis curve; 2) The Taqman fluorescent probe method is characterized in that after a Taqman probe marked with fluorescein is mixed with template DNA, the Taqman probe complementarily paired with the template DNA is cut off during PCR amplification, the fluorescein is dissociated in a reaction system and emits fluorescence under specific light excitation, the amplified target gene fragment grows exponentially along with the increase of the circulation times, ct value is obtained by detecting the corresponding fluorescent signal intensity which changes along with the amplification in real time, and meanwhile, the copy number of the target gene of a sample to be detected can be obtained by using a plurality of standard products with known template concentration as a reference; 3) The molecular beacon (molecular beacon) method is a stem-loop double-labeled oligonucleotide probe which forms a hairpin structure of about 8 bases at the 5 and 3 terminal, and the nucleic acid sequences at the two ends are in complementary pairing, so that a fluorescent group is closely adjacent to a quenching group and does not generate fluorescence. After the PCR product is generated, in the annealing process, the middle part of the molecular beacon is paired with a specific DNA sequence, and the fluorescent gene and the quenching gene are separated to generate fluorescence.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a series of DNA sequences found in the genome of prokaryotes such as bacteria and archaebacteria. These sequences are derived from DNA fragments of viruses previously infected with these prokaryotes and are used by these prokaryotes in subsequent infections to detect and destroy viral-like DNA.

Diagnostic techniques based on CRISPR are a new but very promising approach due to their rapid nucleic acid detection capability and single base specificity, the rationale behind which is the trans-splicing activity of CRISPR-Cas proteases. CRISPR and CRISPR-Cas adaptive immune systems contain programmable endonucleases, which can be used for CRISPR-based diagnostics. Some Cas enzymes, such as Cas12a, can detect the target DNA. While some RNase enzymes, such as Cas13a, that direct a single effector RNA can be reprogrammed with CRISPR RNA (crRNA) so that the target DNA can be detected. For example, by using isothermal pre-amplification in combination with Cas13, sholock (specific high sensitivity enzyme reporter unlock) can be used to detect a single molecule of RNA or DNA.

For the purposes of this 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 generally deposited, formed, coated, or otherwise disposed on another structure.

For the purposes of this disclosure, the term "ground" (as used for a "ground electrode" or "ground voltage") refers to the voltage of the respective electrode being zero or sufficiently close to zero. All other voltage values, although the amplitude is typically less than 300 volts, should be high enough to enable an adequate observation of the electrowetting effect.

It should be noted that when a covered dielectric layer is arranged, the space between adjacent electrodes in the same layer is typically filled with a dielectric material. These spaces may 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.

As used herein, the term "contact angle" refers to the angle formed when a liquid-vapor interface contacts a solid surface. Inside the pure liquid, each molecule is pulled equally in every direction by the adjacent liquid molecules, resulting in zero net force. However, the molecules of the liquid surface do not have adjacent molecules in all directions to provide a balanced net force, they are pulled inward by the adjacent molecules, creating an internal pressure, with the result that the liquid surface area contracts to maintain its lowest surface free energy. This surface-contracting intermolecular force, i.e. liquid-gas interfacial energy gamma _LG Referred to as surface tension, which determines the shape of the droplet. Other external forces, such as gravity, can also deform the droplet. Thus, the contact angle is determined by both the surface tension and the external force (typically gravity). The contact angle is also a characteristic parameter of a solid-liquid system in a specific environment. The hydrophobic surface has liquid repellent properties, while the hydrophilic surface has liquid attractive properties. For purposes of this disclosure, a "hydrophobic surface" has a contact angle greater than 90 °, while a hydrophilic surface has a contact angle less than 90 °.

For the purposes of this disclosure, it will be understood that when any form of liquid (e.g., a droplet or a continuous body, possibly in motion or stationary) is described as "on", "at" or "over" an electrode, array, matrix, and surface, the liquid may be in direct contact with the electrode, array, matrix, and surface, or may be in contact with one or more layers or films interposed between the liquid and the electrode, array, matrix, and surface.

For the purposes of this disclosure, it will be understood that when a given component, such as a layer, region, and substrate, is referred to as being "disposed on", "in" and "at" another component, the given component can be directly on the other component or, alternatively, intervening components (e.g., one or more buffer layers, interlayers, and electrodes) may also be present. It will also be understood that the terms "disposed on" and "formed on" may be used interchangeably to describe how a given component is positioned or disposed relative to another component. Thus, the terms "disposed on" and "formed on" are not intended to introduce any limitations on the particular method of material transport, deposition, and fabrication.

For the purposes of this disclosure, the terms "printed wiring board (Printed Circuit Board, or PCB)" or "printed wiring board" are used interchangeably to refer to a circuit board without soldered components consisting essentially of:

1. line and Pattern (Pattern): the material used for the traces is typically copper, which provides a conductive path between electronic components, and large copper surfaces are typically designed to serve as ground and power planes. The lines are made simultaneously with the drawing.

2. Dielectric layer (Dielectric layer): for maintaining insulation between the wires and the layers, also called the substrate.

3. Hole (or Via): the via holes can conduct more than two layers of lines, the larger via hole is used as a part plug-in, and the non-via holes are commonly used for surface mounting and positioning.

4. Solder Mask ink (solver Mask): not all copper surfaces need to be plated with tin, so that a layer of substances (usually epoxy resin) for isolating the copper surfaces from tin is printed in a non-tin-plated area, and short circuits among non-tin-plated circuits are avoided. According to different processes, green oil, red oil and blue oil are classified.

5. Screen (Silk screen): the main function of the device is to mark the names and position frames of all parts on the circuit board, so that the device is convenient to maintain and identify after assembly.

For the purposes of this disclosure, the terms "detecting", "detecting" and "measuring" are used interchangeably to refer to the process of acquiring a physical quantity (e.g., location, charge level, temperature, concentration, pH, brightness, fluorescence, etc.). Typically, at least one sensor (or detector) will be used to acquire a physical quantity and convert it into a signal or information recognizable by the person or instrument. Other components can be arranged between the object to be measured and the sensor, such as a lens, a reflector, a filter and the like used in optical measurement, and a resistor, a capacitor, a triode and the like used in electrical measurement. Moreover, in order to make the measurement possible or easy, other auxiliary devices or means are often used in the measurement. For example, a light source such as a laser or laser diode is used to excite particles from an electron ground state to an electron excited state, and the excited state particles sometimes emit fluorescence when returning to the ground state, and measuring the fluorescence intensity here can be used to measure the concentration of a certain particle in a liquid sample. The optical sensors include CCD, photodiode, photomultiplier, etc., and the electrical sensors include operational amplifier, A/D converter, thermocouple, thermistor, etc.

The measurements may be performed simultaneously or in a sequence for a plurality of parameters in a plurality of samples. For example, while measuring the fluorescence of a certain particle in a droplet with a photodiode, the position of its droplet can also be obtained simultaneously by capacitance measurement. The sensor or detector is typically connected to a central processing unit (Central Processing Unit, or CPU) or computer (computer) on which corresponding software is run to analyze the measured signals and convert them into information that can be read by humans or other instruments. For example, using measurement and analysis of the fluorescence intensity of a particle in a liquid can be used to infer the concentration of the particle. As non-limiting examples, optical measurements include laser-induced fluorescence measurements (laserinduced fluorescence measurement), infrared spectra (infrared spectroscopy), raman spectra (Raman spectroscopy), chemiluminescence measurements (chemiluminescence measurement), surface plasmon resonance measurements (surface plasmon resonance measurement), absorption spectra (absorption spectroscopy), and the like; electrical measurements include amperometric (amperometric), voltammetric (voltment), photoelectrochemical (photoelectrochemical), coulometric (coulometric), capacitive (capacitive measurement), and ac impedance (and AC impedance measurement), among others.

In nucleic acid detection, fluorescence measurement (or fluorescence detection) is a method that is commonly used to detect the presence or absence of a certain nucleic acid to be detected in a sample to be detected, or the concentration of the nucleic acid to be detected. This method is mainly two parts, 1) light excitation-irradiation of the sample to be measured with a light source of suitable wavelength, such as LED (Light Emitting Diode) and Laser (Laser), are two light sources commonly used today, and band pass filters are used. 2) Light measurement-measuring the intensity of fluorescence of a specified wavelength range emitted by a sample to be measured using a light sensor. By utilizing fluorescent probes with different wavelengths and measuring the fluorescent intensity in the corresponding wavelength range, a plurality of different nucleic acids can be detected simultaneously in the same reaction system; although being used for fluorescent quantitative PCR, the method is also applicable to isothermal amplification real-time fluorescence method, CRISPR detection and the like.

In one aspect, the utility model provides a DMF device generally comprising a bottom plate and an upper cover plate. The base plate comprises a substrate provided with a plurality of electrowetting electrodes for controlling the droplet or liquid. At least a portion of the base plate is optically transparent for optical excitation and measurement. The upper cover plate includes a surface substantially parallel to the bottom plate, an access port for loading and unloading of samples and reagents, and a reservoir for reagent storage and reaction. At least a portion of the surface is electrically conductive. In another aspect, some of the electrodes on the bottom plate form multiple paths for performing amplification and the like. In another aspect, some of the electrowetting electrode regions on the bottom substrate contain a lyophilization reagent, including but not limited to a polymerase (particularly a thermostable enzyme such as Taq polymerase and variants thereof), deoxyribonucleotide triphosphates (dNTPs; typically 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 guide RNAs that bind to a particular target molecule, and the like.

In another aspect, a DMF device includes a device housing that provides some protection for a bottom plate and a top plate that are bonded together and provides an interface for control signals of an instrument.

The utility model further provides a method of using the DMF device of the utility model. In one aspect, the utility model provides a method of measuring a plurality of target nucleic acids in a sample comprising a) adding the sample to a DMF device, b) performing specified steps to lyse cells in the sample, extract nucleic acids in the sample, purify the nucleic acids, amplify and measure nucleic acids in the sample.

In one aspect, the method provides for adding a sample to a DMF device of the present utility model and performing an assay operation, comprising: a) Mixing the sample with a lysis buffer; b) Adding a binding buffer and capture magnetic beads to the sample; c) Mixing magnetic beads with a sample; d) Optionally washing the magnetic beads; e) Eluting nucleic acids from the magnetic beads; f) Adding an amplification reagent to the eluted sample; g) Amplification (isothermal amplification and PCR); h) Fluorescence measurement, and the like.

In another aspect, the utility model provides a method for washing magnetic beads by moving the magnetic beads in a fill liquid of a DMF device. The magnetic beads for extracting the biological nucleic acid are usually superparamagnetic microspheres with small diameters (nano-scale to micron-scale), can be rapidly aggregated in a magnetic field, can be uniformly dispersed in liquid after the magnetic field is removed, and are not easy to settle. The extraction of nucleic acids can be achieved by coating the beads with a corresponding coating (e.g., silica-based, amino, hydroxyl, etc.). Under the action of the lysate, the nucleic acid (DNA or RNA) is released, and the surface modified magnetic beads and the nucleic acid are specifically combined to form a magnetic bead-nucleic acid complex. Nucleic acid-magnetic bead complexes typically have non-specifically adsorbed impurities that may interfere with the next step of detection and thus need to be removed. A general method for removing impurities from a magnetic bead-nucleic acid complex is to use a washing solution, and the present utility model provides a method for removing impurities from a magnetic bead-nucleic acid complex by moving a magnetic bead in an immiscible (immiscable) path using a magnet.

The following is a detailed description of embodiments of the present utility model for processing biological samples, and for convenience of description, the corresponding figures (fig. 1 to 4) will be referred to as needed. It should be noted that these examples are intended to aid in the description and are not intended to limit the intent and spirit of the utility model.

The principles of the present disclosure are further illustrated and described herein, together with the accompanying drawings and detailed description, and enable one skilled in the relevant art to make and use the corresponding apparatus, DMF devices, and methods described.

For purposes of this disclosure, some or all of the functional modules (e.g., temperature control modules, optical measurement modules, etc.) herein may be automatically controlled. Existing programs (software or firmware ) running on a microprocessor or computer are typically used to implement the automatic control.

Fig. 1 shows a corresponding electrowetting electrode layout of DMF device 100 for single-cell analysis, wherein the small squares represent droplet-driving electrodes, which are also different in size depending on their specific function, e.g. a first electrode 160 for cell sorting is smallest, a second electrode 170 for separating droplets from the elution tank is larger, and third electrodes 181-188 for DNA amplification are largest.

Fig. 2 is a top view of an upper cover plate of the DMF device 100 of the present utility model, wherein a first liquid storage tank 211 may be used for adding a cell sample solution to be analyzed, a second liquid storage tank 212 may be used for sorting sample waste liquid without cells to be analyzed, a third liquid storage tank 213 may be used for sample lysis and nucleic acid capture, a fourth liquid storage tank 214 may be used for magnetic bead washing, and a fifth liquid storage tank 215 may be used for DNA elution, and each liquid storage tank is designed with a hole for adding (or removing) a corresponding sample or reagent (or waste liquid). 221 is a well for adding a DNA amplification reagent.

Fig. 3 is a functional schematic combining fig. 1 and 2. The fixed PCR temperature zones 231 and 232, the plurality of optical detection spots 241, and the cell sorting spot 251 are shown in fig. 3. In one embodiment, both optical excitation and detection are accomplished by the bottom plate of the DMF device. Thus, this portion of the DMF device backplane needs to be optically transparent (including the substrate, electrodes and dielectric layers). In the present utility model, the optical measurement is performed in the visible range, typically between 400 and 700 nm. Higher light transmittance means higher system detection sensitivity. The transmittance of the backplane optical measurement portion of DMF devices is typically greater than 70%, or greater than 80%, or greater than 90%.

FIGS. 4A-4E show side views of a portion of a DMF chip for magnetic bead control of the present utility model, generally designated 100, as a preferred embodiment for achieving magnetic bead control. Fig. 4A shows different reservoirs of liquid 401 and liquid 402 on a DMF device. The backplane 300 includes a drop control electrode 302 and a dielectric layer 303 deposited on a substrate 301. The upper cover plate 200 includes a ground electrode 202 and a dielectric layer 203 on a deposition substrate 201. It should be noted that the DMF device structure shown here is for the purpose of illustrating the bead manipulation and is by no means representative of all possibilities. In some embodiments, DMF devices may be implemented in a variety of different ways. For example, 1) the control electrodes may have different shapes, such as rectangular, square, trapezoidal, pentagonal, hexagonal, and irregular shapes, and may be arranged in a straight line or other shape; 2) The control electrodes may be in different layers (typically electrically isolated from each other); 3) The dielectric layer may also have two or more layers, and the materials used include Parylene C (Parylene C), silicon nitride, silicon dioxide, and the like; one of the layers may be a hydrophobic material such as Teflon, cytop, fluoroPel, and the like.

The substrate may be any non-conductive material or conductive material coated with a non-conductive layer, so long as it has sufficient mechanical strength to maintain its shape within the desired system operating and storage conditions. In terms of light transmission, it may be transparent, translucent, and opaque. The transparent substrate may be made of various transparent materials such as glass, quartz, plastic, transparent ceramic, transparent printed wiring board, etc. The electrodes may be made of any electrically conductive material, such as metals, alloys, and conductive polymers. It may be made of one material or a mixture of different materials. The transparent electrode on the DMF device may be made of a transparent conductive material (e.g., indium Tin Oxide (ITO), aluminum doped zinc oxide (AZO), transparent conductive polymer (polyacetylene, polyaniline, etc.), or a transparent nanomaterial, etc.

The voltage control module is used to provide a voltage control signal to the drop control electrode 302. It typically has multiple outputs, with a maximum number of 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. The voltage frequency is less than 10 MHz, or less than 1 MHz, or less than 100 KHz, or less than 20 KHz, or less than 5 KHz, or less than 1 KHz of alternating current signals, or direct current signals. The waveform of the voltage may be a square wave, sine wave, saw tooth, pulse width modulated signal, etc. The voltage control module is typically programmed with the sequence, duration, amplitude, and frequency of the output signals by a microprocessor or computer on a circuit board through SPI (Serial Peripheral Interface), I2C (Inter-Integrated Circuit), USB (Universal Serial Bus), parallel port (parallel port), ethernet, wi-Fi, bluetooth, or the like. Spring probes (springloaded electrical contact pin) or interface boards (connector pads) can be used to deliver multiple high voltage control signals to electrodes on the DMF device.

In fig. 4A, the magnetic beads 500 are uniformly distributed in the reservoir 401 of the DMF device. In fig. 4B, magnet 600 is placed in close proximity to DMF device at reservoir 401. Under the magnetic field generated by the magnet, the magnetic beads 500 are collected at the bottom of the reservoir 401. As shown in fig. 4C and 4D, the magnet moves the beads to the reservoir 402. In fig. 4E, the magnet is removed from the DMF device and the magnetic beads are resuspended and dispersed in reservoir 402. The magnetic beads are suspended and dispersed in the liquid in the reservoir 402, and may require droplet motion or other means to assist.

The reservoir gap in DMF devices may be surrounded by air or a filling liquid. Figures 4A-4E illustrate the process of the beads being moved from the interior of one container through a medium (air or fill liquid) into another reservoir. As shown, the device is hydrophobic to the droplets. The droplets on the electrodes may be manipulated (moved, split, combined, mixed, etc.) by the electrowetting effect.

As used herein, phrases such as "magnet is moved (or brought in, transported) to be proximate", "magnet is moved proximate" are intended to refer to the relative positions of the magnet and DMF device. The magnetic force generated by the magnet has obvious influence on the magnetic beads on the device. Conversely, phrases such as "magnet removed", "magnet removed" are intended to mean that the magnet has no or insignificant effect on the magnetic beads on the device.

The speed of movement of the magnet may typically be controlled by a motor, depending on the measurement, and the magnet speed required for different applications may vary, typically ranging from 0.1 to 100mm/sec, or from 0.5 to 20mm/sec, or from 1 to 10mm/sec.

In the above embodiment, the magnet is in contact with the base substrate and moves along the bottom of the DMF device. In some embodiments, the magnet may also be located on top of the DMF device; or a pair of magnets, one on top and the other on the bottom, for manipulating the magnetic beads in the DMF device.

The utility model further shows examples of magnetic bead-based nucleic acid extraction and quantitative PCR analysis using the nucleic acid analysis system.

In step S501, a cell sample solution is added to the first reservoir 211, a lysis solution and a bead solution are added to the third reservoir 213, a bead washing solution is added to the fourth reservoir 214, an eluent is added to the fifth reservoir 215, and a DNA amplification reagent is added to the sample addition well 221.

In step S502, droplets are generated from the first liquid reservoir 211, and based on the result of the cell sorting point 251 on the chip, droplets containing the test cells are moved to the first liquid reservoir 213, and droplets not containing the test cells are moved to the second liquid reservoir 212.

In step S503, the magnet is moved upward, and the DMF apparatus is abutted against the third reservoir 213. The magnet is moved along a predetermined trajectory within the third reservoir 213 to assist the capture of the DNA molecules by the magnetic beads within the third reservoir 213.

In step S504, the magnet is moved from the third reservoir 213 to the fourth reservoir 214 against the DMF device, which carries the magnetic beads to the fourth reservoir 214. The magnet is moved along a predetermined trajectory within the fourth reservoir 214 to clean the magnetic beads of impurities that may be adsorbed.

In step S505, the magnet is moved from the fourth reservoir 214 to the fifth reservoir 215, which carries the magnetic beads to the fifth reservoir 215. The magnet was removed from the DMF apparatus so that the beads would be dispersed into the eluent. Moving the magnet, moving the magnet back to be close to the fifth reservoir 215 of the DMF device, and moving the magnet back and forth within the range of the fifth reservoir 215 to re-collect the magnetic beads. The magnet was moved to a designated position against the DMF apparatus and the beads were discarded.

In step S506, 8 droplets are separated from the eluate in the fifth reservoir 215 and moved along paths 181-188 to the 8 wells 221, respectively, where DNA amplification reagents are mixed.

In step S507, 8 droplets are moved back and forth along paths 181 to 188 between the fixed PCR temperature zones 231 and 232 and fluorescence measurements are made through the optical detection spot 241 in each movement cycle.

In step S508, an analysis report is generated based on the obtained quantitative PCR data.

It should be mentioned here that the reagents may be encapsulated in DMF devices, and the user only needs to load the sample. This makes the device easier to handle and reduces the chance of contamination (or cross-contamination) of the test.

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

While the preferred embodiment of the utility model has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the utility model.

The above embodiments are merely illustrative of the principles of the present utility model and its effects, and are not intended to limit the scope of the utility model. Various modifications and alterations may be made to this utility model by those skilled in the art without departing from the spirit and scope of this utility model. Accordingly, all modifications and alterations may be made by anyone having ordinary skill in the art without departing from the spirit and technical teachings of the utility model.

Claims (5)

1. A single-cell nucleic acid analysis system based on digital micro-fluidic is characterized by comprising a bottom plate, an upper cover plate and a bottom plate

The device is provided with a plurality of electrowetting electrodes for controlling the movement of liquid drops, each electrowetting electrode comprises a first electrode for cell sorting, a second electrode for separating DNA and a third electrode for amplifying DNA, and the plurality of third electrodes are arranged in an array to form a plurality of DNA amplification paths for realizing DNA amplification; the upper cover plate is provided with a liquid storage tank, wherein the liquid storage tank comprises a first liquid storage tank for adding a cell sample solution to be analyzed, a second liquid storage tank for storing sample waste liquid which does not contain cells to be tested after separation, a third liquid storage tank for adding a lysis solution containing magnetic beads, a fourth liquid storage tank for adding a magnetic bead cleaning solution and a fifth liquid storage tank for adding a DNA eluent;

the first liquid storage tank is respectively connected with the second liquid storage tank and the third liquid storage tank through the first electrode; the output end of the third liquid storage tank is connected with the input end of the fourth liquid storage tank, the output end of the fourth liquid storage tank is connected with the input end of the fifth liquid storage tank, and the fifth liquid storage tank is connected with a sample adding hole for adding a DNA amplification reagent through a second electrode and a DNA amplification path formed by a third electrode; the middle position of the DNA amplification reagent is provided with a plurality of fixed PCR temperature areas, optical detection points for light excitation and measurement are arranged between the fixed PCR temperature areas, the optical detection points and the sample adding holes are arranged on the bottom plate, the peripheral area of the bottom plate where the optical detection points are arranged is optically transparent, and the bottom of the bottom plate is provided with a magnet for gathering and driving the magnetic beads to move.

2. The digital microfluidic based single cell nucleic acid analysis system according to claim 1 wherein the electrowetting electrode is connected to a voltage control module.

3. The digital microfluidic based single cell nucleic acid analysis system according to claim 1 wherein the base plate comprises a substrate, an electrowetting electrode and a dielectric layer, the electrowetting electrode being deposited on the substrate; the upper cover plate includes a substrate, a ground electrode, and a dielectric layer, the ground electrode being deposited on the substrate.

4. The digital microfluidic based single cell nucleic acid analysis system according to claim 1, wherein the number of the fixed PCR temperature zones is two or three, and the fixed PCR temperature zones are connected with the temperature control module.

5. The digital microfluidic based single cell nucleic acid analysis system according to claim 1 wherein the magnet is coupled to the motor.

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