TW201928055A - One-step isolation, concentration and/or purification of nucleic acids using ATPS-embedded microfluidic device - Google Patents

Abstract

This invention relates to a method for the isolation, concentration and/or purification of nucleic acids by an aqueous two-phase system (ATPS) embedded microfluidic device. In one embodiment, the present invention provides an apparatus, a method, a system and a kit for the isolation, concentration and/or purification of nucleic acids from biological or non-biological samples.

Description

Isolate, concentrate and / or purify nucleic acids in one step using microfluidic devices embedded in ATPS

Related applications

This application claims priority from US Provisional Application Serial No. 62 / 599,001, filed on December 14, 2017, which is incorporated by reference in its entirety.

Throughout this application, various publications are cited. The entire disclosures of these publications are incorporated by reference into this application to more fully describe the state of the art to which this invention belongs.

The present invention relates to a method for separating, concentrating, and / or purifying nucleic acids by a microfluidic device embedded with a two-liquid phase system (ATPS) component. In one embodiment, the invention provides instruments, methods, systems, and kits for separating, concentrating, and / or purifying nucleic acids from biological or non-biological samples.

Methods for isolating and purifying nucleic acids (such as DNA and RNA) from complex matrices (such as blood, tissue samples, bacterial cell culture fluids, and forensic samples) in genetic research, diagnosis of nucleic acid probes, forensic DNA testing, and others that require amplification and processing Or very important in the field of analyzing nucleic acids.

Techniques for extracting and purifying nucleic acids from mammalian cells have emerged many years ago (N. Blin, DW Stafford (1976), A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 3 (9): 2303 -8). Generally, the isolation and purification process involves two main steps: lysing the target cell or cell population, and purifying nucleic acids from organelles (such as the nucleus) and cellular components (such as proteins and cells or tissue debris). Lysing cells and lysing DNA can be performed mechanically or chemically. Chemical lysing agents such as detergents are commonly used to disrupt the lipid bilayer of cells, thereby releasing the cell contents into the lysis solution. Kothari and Taylor (1972) describe a method for nucleic acid isolation in which oligodeoxyribonucleotides are coupled to cellulose and one or more organic solvents are used at various stages of the method. All of the above methods require several purification cycles to isolate pure nucleic acids. In addition, the substrates used to capture nucleic acids in these methods are generally only suitable for capturing nucleic acids exceeding a certain size, such as 1000 bp.

For cell-derived DNA, purification usually involves gentle lysis of the cell, lysis of the DNA, and the use of chemical methods to release the DNA from other cellular material or contaminants such as proteins, RNA, and other substances. Conventionally, multiple procedures are required to perform the entire purification process, including cell lysis, binding of nucleic acids to a solid substrate, washing steps, and elution steps.

With the modern advancement of benchtop solid-phase extraction methods, nucleic acid extraction has become simpler and faster. Commercial kits such as those provided by Qiagen ^® and Invitrogen ^® allow extraction and purification of nucleic acids in 20-30 minutes. However, it still requires benchtop centrifuges, basic laboratory equipment such as pipettes and consumables, which means that extraction and purification are largely confined to basic-equipment laboratories and rely on power. In addition, because conventional nucleic acid extraction processes require multiple consecutive steps to be performed manually from time to time, experimental steps (including assays and molecular diagnostics) that require amplification or analysis of nucleic acids from a particular sample can be very time consuming and laborious. The need for significant human interference has limited the development of POC. Therefore, there is a need for a method for preparing nucleic acids that is convenient and has minimal manipulation interference.

Microfluidics is a discipline that studies the behavior of fluids in microchannels and has been applied in various fields. Microfluidics is a relatively new technology in the field of biomedical research; for example, it is applied to the real-time detection of Ca ²⁺ channel activity in single cells (Li and Li, 2010, Expert Review of Clinical Pharmacology, 3: 267 -80) and miniature laboratory (LOC) or point-of-care (POC) equipment to extract and amplify DNA from bacteria (Oblath et al., 2013, Lab Chip, 13 (7)). By aggregating multiple steps within a microfluidic device, you can increase sensitivity and efficiently perform sample processing and biological material analysis, while minimizing reagent consumption and risk of contamination. However, most microfluidic devices require external forces (such as electricity and vacuum) to operate, limiting their use outside the laboratory environment.

To solve the above problems, some embodiments of the present invention provide a microfluidic device embedded with a two-liquid phase system (ATPS) component, and use the device to prepare samples in research and / or clinical applications. In one embodiment, ATPS contains two different liquid phases, the ratio of which can be easily controlled. Biomolecules suspended in ATPS, based on their physico-chemical properties (such as hydrophilicity), are partitioned into one of two phases, resulting in separation and in situ concentration.

In some embodiments, the invention also provides a fast, sensitive, and specific nucleic acid purification system that combines the steps of sample collection, removal of contaminants, and purification of nucleic acids in a microfluidic device. In some embodiments, the present invention does not require any external force, complex instrumentation or electronic hardware to operate, and therefore provides a fast and economical method for nucleic acid isolation, concentration, and purification.

In one embodiment, the invention provides a microfluidic device comprising a porous matrix embedded in a two-liquid phase system component (also known as an ATPS component). Such microfluidic devices are sometimes referred to herein as ATPS-embedded microfluidic devices. When the ATPS component is hydrated (or rehydrated), the resulting solution undergoes spontaneous phase separation into a first phase and a second phase.

In one embodiment, the invention also provides instruments, methods, systems, and kits for separating, concentrating, and / or purifying nucleic acids from biological or non-biological materials using the ATPS-embedded microfluidic devices described herein.

In one embodiment, the apparatus, methods, systems, and kits of the present invention enable cell lysis and nucleic acid purification in a single microfluidic device in a fast, efficient, and convenient manner.

In one embodiment, the invention is capable of isolating, concentrating, and / or purifying nucleic acids that are present in low abundance in a small amount of sample, resulting in an isolated nucleic acid solution with a high concentration.

In one embodiment, the present invention enables nucleic acids to be obtained from a sample in a higher yield or purity compared to related methods in the art.

The two-liquid phase system (ATPS) is a mixed-phase solution capable of spontaneous phase separation. Some embodiments of the invention provide a microfluidic device comprising a porous matrix (sometimes referred to as an ATPS-embedded microfluidic device) embedded with ATPS components. The invention further provides instruments, methods, systems, and kits for separating, concentrating, and / or purifying nucleic acids from biological or non-biological materials using the microfluidic devices described herein.

The ATPS used in this application can have a variety of compositions. For example, ATPS can be formed from two polymers or polymers and salts in water with different properties. Various methods can be used to prepare ATPS. For example, ATPS can be prepared by mixing two solutions with certain components. These two solutions are sometimes referred to herein as a phase solution or a first phase solution and a second phase solution. In some embodiments, one of the phase solutions may be a salt-rich solution and the other phase solution may be a polymer-rich solution. In some embodiments, one phase solution may contain a first polymer and the other phase solution may contain a second polymer. When the two phase solutions are mixed, the resulting ATPS spontaneously undergoes phase separation, that is, the two phases separate from each other to form different layers. These phases are sometimes referred to herein as the first and second phases.

ATPS can also be formed by the simultaneous or sequential addition of certain compounds (sometimes collectively or separately referred to herein as ATPS components) to an aqueous solution. ATPS can also be formed when the ATPS component is embedded in a porous matrix and an aqueous solution is allowed to flow through this matrix. When an aqueous solution flows through this matrix, the embedded ATPS components dissolve in the aqueous solution and form ATPS. Since these phases have different physicochemical properties and can flow through the porous matrix at different rates, it is possible to separate molecules in a mixture by their properties and further concentrate the separated molecules in one of the two phases of ATPS And reduce equipment and human intervention required.

Microfluidics technology allows processing of very small volumes of materials. In order to improve current nucleic acid extraction methods in terms of sensitivity, efficiency, and ease of use, the present invention combines and improves ATPS and microfluidic technologies. Some embodiments of the invention allow quick and easy extraction, concentration and purification of nucleic acids. Essentially, when a nucleic acid-containing sample containing an aqueous solution is applied to a microfluidic device containing a porous matrix embedded with an ATPS component, the ATPS component dissolves in the aqueous matrix as it flows through the porous matrix and forms ATPS. Nucleic acids are concentrated in one of the two phases of ATPS. Therefore, extraction, concentration, and purification of nucleic acids are achieved. By modifying the ATPS and microfluidic matrix, the present invention provides a new ATPS-embedded microfluidic device that significantly accelerates the isolation of nucleic acids from various types of crude samples and produces target nucleic acids with high purity and yield.

In one embodiment, the invention provides apparatus, methods, systems, and kits for separating, concentrating, and / or purifying nucleic acids from biological or non-biological materials using the microfluidic devices described herein.

In one embodiment, the apparatus, methods, systems, and kits of the present invention enable cell lysis and nucleic acid purification in a single microfluidic device in a fast, efficient, and convenient manner.

In one embodiment, the method for isolating, concentrating, and / or purifying nucleic acids from a sample includes the following steps:
a) obtaining a sample containing the target nucleic acid; and
b) contacting the sample with a porous matrix of a microfluidic device, wherein the porous matrix is embedded to form an ATPS two-liquid phase system (ATPS) component comprising a first phase and a second phase,
The target nucleic acid can enter and pass through the porous matrix, resulting in the separation of the target nucleic acid.

In one embodiment, ATPS is formed by mixing a first phase solution and a second phase solution. The two phases of the resulting ATPS can then be separated. In another embodiment, the components of the first phase solution and the second phase solution are embedded in a porous matrix. In some embodiments, components of different types of phase solutions are embedded in the same area of the porous matrix. In other embodiments, components of different types of phase solutions are embedded in different regions of the porous matrix. FIG. 4 shows some embodiments of the device of the present invention, which includes a plurality of porous matrix layers, wherein the components of the phase solution are embedded in different regions of the porous matrix separately or one after the other, or simultaneously in a mixture.

In one embodiment, the method further comprises the step of eluting the target nucleic acid from the porous matrix of the microfluidic device.

In one embodiment, the instrument, method, system, or kit of the present invention is capable of separating, concentrating, and / or purifying nucleic acids present in low abundance in a small amount of sample, thereby producing an isolated nucleic acid solution with a high concentration .

In one embodiment, the instrument, method, system, or kit of the present invention is capable of obtaining nucleic acids from a sample in a higher yield or purity than related methods in the art.

In one embodiment, the invention is used to separate nucleic acids from cellular material, components or contaminants. Isolated nucleic acids are concentrated and free of contaminants, making them suitable for a variety of molecular biology procedures in research or clinical laboratories.

The microfluidic device of the present invention can be made of any material compatible with ATPS and microfluidic devices.

In one embodiment, a microfluidic device includes a porous matrix containing a capillary that allows another material to pass through. In various embodiments, the porous matrix is easily passed by a flowing material, such as a solvent, solute, or solution, in response to capillary forces, gravity, electrostatic forces, positive pressure, and the like. In one embodiment, the porous matrix is a porous material, including but not limited to the following materials and combinations thereof: inorganic powders, such as silica, magnesium sulfate, alumina, etc .; natural polymer materials, especially cellulose materials, Fiber paper (such as filter paper, chromatography paper, glass fiber paper or other suitable fiber-containing paper); synthetic or modified naturally occurring polymers such as nitrocellulose, cellulose acetate, poly (vinyl chloride), polyacrylamide , Cross-linked dextran, agarose, polyacrylate, etc., they can be used alone or in combination with other materials; ion exchange resins, ceramic materials and so on.

In one embodiment, the microfluidic device of the present invention includes a porous matrix composed of one, two, three or more layers of porous material. In one embodiment, the microfluidic device of the present invention further comprises one or more non-porous materials.

In one embodiment, a fiber-containing paper or other porous material or a combination thereof forms a laminate. These stacks can be obtained by stacking layers (or sheets) of material and cutting the resulting stack to a suitable size. The stack may be formed of two or more (or more) porous materials. In one embodiment, the stack is formed of four layers of porous material. In one embodiment, the porous material is glass fiber paper. A stack formed from a fiber-containing paper is sometimes referred to herein as a paper stack. In one embodiment, the stack is rectangular with a size of 0.5 cm x 4 cm.

The diameter of the capillary in the porous matrix depends on the length and size of the nucleic acid to be extracted. In one embodiment, the average diameter of the capillaries in the porous matrix is about 0.015 μm to 12 μm. In another embodiment, the average diameter of the capillary is about 1 μm to 4 μm.

In one embodiment, the microfluidic device of the invention is used to isolate nucleic acids of small size (about 1000 base pairs (bp) or less). In one embodiment, a paper with a capillary having a diameter of 1-2 μm is used to prepare the porous matrix of the present invention for the isolation of nucleic acids below 500 bp.

It should be understood that those skilled in the art will be able to implement the description of the present invention according to the length, size, and type of the nucleic acid to be separated, to select an appropriate size for the porous material or porous matrix stack applied in the present invention.

In one embodiment, one or more portions of the porous matrix of the microfluidic device are embedded in a two-phase system (ATPS) component containing components of the first phase solution and / or the second phase solution. In one embodiment, ATPS is present as a mixed phase solution, which can be phase separated, whereby the two phases form different layers. In one embodiment, the components of the first phase solution and the second phase solution are mixed before embedding in the porous matrix. In one embodiment, the components of the first phase solution and the second phase solution of ATPS are embedded in the porous matrix, respectively. In one embodiment, the components of the first phase solution are embedded in the porous matrix before the sample is added, and the second phase solution is applied to the microfluidic device with the sample. In another embodiment, the components of the second phase solution are embedded in the porous matrix before the sample is added, while the first phase solution is applied to the microfluidic device along with the sample.

The ATPS component can be embedded in a porous matrix in a variety of ways. Some methods are described below. In one embodiment, ATPS is dehydrated on a porous substrate. In one embodiment, the porous matrix is inserted into ATPS and then dried in air. In one embodiment, the porous matrix is inserted into pre-mixed ATPS and then dried by hot air. In one embodiment, the porous matrix is inserted into pre-mixed ATPS and then lyophilized. In one embodiment, the porous matrix is inserted into pre-mixed ATPS and then dried by supercritical drying. In one embodiment, the pre-mixed ATPS component is sprayed onto a porous substrate and then dried by any of the drying methods described above or other methods. In one embodiment, different types or concentrations of ATPS components are sprayed onto the same or different areas of the porous substrate and then dried. In one embodiment, in the case of using a multilayer porous substrate, different types or concentrations of ATPS components are sprayed onto different layers of the porous substrate, respectively, and then dried by the drying method mentioned above or other methods. In one embodiment, different types or concentrations of ATPS components are sprayed on different areas of the porous substrate, respectively, and then dried.

In one embodiment, the first and / or second phase solution comprises a polymer. In one embodiment, the polymer includes, but is not limited to, a polyalkylene glycol, such as a hydrophobically modified polyalkylene glycol, a poly (alkylene oxide) polymer, a poly (alkylene oxide) copolymer, such as Hydrophobically modified poly (oxyalkylene) copolymer, polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl caprolactam, polyvinyl methyl ether, alkoxylated surfactant, alkoxylated starch, alkoxylated Cellulose, alkyl hydroxyalkyl cellulose, silicone-modified polyether and poly-N-isopropylacrylamide and copolymers thereof. In one embodiment, the first polymer includes, but is not limited to, polyethylene glycol, polypropylene glycol, or dextran. In another embodiment, the second polymer is a polyelectrolyte, such as a polycation or a polyanion. Polyelectrolytes dissociate in aqueous solution and carry one or more charges depending on the pH. Examples of polyelectrolytes include, but are not limited to, natural polyelectrolytes such as gum (polygalacturonic acid), alginate (alginic acid), carboxymethyl cellulose, and polypeptides, and synthetic polyelectrolytes such as polyacrylic acid, polystyrene sulfonate Acid salts and their salts.

In one embodiment, the polymer concentration in the first or second phase solution is from about 0.01% to about 90% (w / w) (based on the total weight of the aqueous solution). In various embodiments, the polymer solution is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, and about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w , Or about 1% w / w polymer solution. In some embodiments, the polymer solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, and about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20%% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w About 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, About 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, or about 50% w / w polymer solution. In one embodiment, the polymer concentration is about 2% w / w to 25% w / w. In another implementation, the polymer concentration is about 3% w / w to 15% w / w.

In one embodiment, the first and / or second phase solution comprises a salt, thereby forming a salt solution. In one embodiment, the salt includes, but is not limited to, a lyophilic salt, a chaotropic salt, and an inorganic salt. The inorganic salt cation includes: linear or branched trimethylammonium, triethylammonium, tripropylammonium, Butylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium, the inorganic salt anions include: phosphate, sulfate, nitrate, chloride or bicarbonate. In another embodiment, the salt is selected from sodium chloride, sodium phosphate, disodium phosphate, sodium sulfate, sodium citrate, sodium acetate, potassium chloride, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium sulfate, Potassium citrate, ammonium sulfate and combinations thereof. Other salts, such as ammonium acetate, can also be used. In one embodiment, the first phase solution and / or the second phase solution comprises two or more salts.

In one embodiment, the total salt concentration is from 0.001 mM to 100 mM. Those skilled in the art will understand that the amount of salt required to form a two liquid phase system will be affected by the molecular weight, concentration, and physical state of the polymer.

In various embodiments, the salt solution is selected from a salt solution of about 0.001% to 90% w / w. In various embodiments, the salt solution is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, and about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w About 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, Or about 1% w / w of a salt solution. In some embodiments, the salt solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, and about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w About 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, About 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35 % w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, about 50% w / w of salt solution. In one embodiment, the concentration of the salt solution is about 2% w / w to 20% w / w. In another embodiment, the concentration of the salt solution is about 5% w / w to 15% w / w.

In one embodiment, the first and / or second phase solution comprises a water-immiscible solvent. In some embodiments, the solvent comprises a non-polar organic solvent. In some embodiments, the solvent comprises an oil. In some embodiments, the solvent is selected from the group consisting of pentane, cyclopentane, benzene, 1,4-dioxane, diethyl ether, dichloromethane, chloroform, toluene, and hexane.

In various embodiments, the non-polar solution is selected from a non-polar solution of about 0.001% to 90% w / w. In various embodiments, the non-polar solution is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, and about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w , Or about 1% w / w non-polar solution. In some embodiments, the non-polar solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, and about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w About 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, About 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42 % w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, and a non-polar solution of about 50% w / w. In one embodiment, the concentration of the non-polar solution is about 0.1% w / w to 20% w / w. In another embodiment, the concentration of the non-polar solution is about 1% w / w to 10% w / w.

In one embodiment, the first and / or second phase solution comprises a micellar solution. In some embodiments, the micellar solution comprises a non-ionic surfactant. In some embodiments, the micellar solution comprises a detergent. In some embodiments, the micellar solution comprises Triton-X. In some embodiments, the micellar solution comprises a detergent similar to Triton-X, such as Igepal CA-630 and Nonidet P-40. In some embodiments, the micellar solution consists essentially of Triton-X.

In various embodiments, the non-ionic surfactant solution is selected from about 0.001% to 90% w / w of non-ionic surfactant solutions. In various embodiments, the non-ionic surfactant solution is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, about 0.25% w / w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6 % w / w, about 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, or about 1% w / w of a non-ionic surfactant solution. In some embodiments, the non-ionic surfactant solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13 % w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w, about 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, about 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w About 42% w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, A non-ionic surfactant solution of about 49% w / w, and about 50% w / w. In one embodiment, the concentration of the non-ionic surfactant solution is about 0.1% w / w to 30% w / w. In another embodiment, the concentration of the non-ionic surfactant solution is about 1% w / w to 15% w / w.

In one embodiment, the first phase solution comprises a micellar solution and the second phase solution comprises a polymer. In one embodiment, the second phase solution comprises a micellar solution and the first phase solution comprises a polymer. In one embodiment, the first phase solution comprises a micellar solution and the second phase solution comprises a salt. In one embodiment, the second phase solution comprises a micellar solution and the first phase solution comprises a salt. In one embodiment, the micellar solution is a Triton-X solution. In one embodiment, the first phase solution comprises a first polymer and the second phase solution comprises a second polymer. In one embodiment, the first / second polymer is selected from polyethylene glycol and dextran. In one embodiment, the first phase solution comprises a polymer and the second phase solution comprises a salt. In one embodiment, the second phase solution comprises a polymer and the first phase solution comprises a salt. In some embodiments, the first phase solution comprises polyethylene glycol and the second phase solution comprises potassium phosphate. In some embodiments, the second phase solution comprises polyethylene glycol and the first phase solution comprises potassium phosphate. In one embodiment, the first phase solution comprises a salt and the second phase solution comprises a salt. In one embodiment, the first phase solution comprises a lyophilic salt and the second phase solution comprises a chaotropic salt. In some embodiments, the second phase solution comprises a lyophilic salt and the first phase solution comprises a chaotropic salt.

In one embodiment, the first phase solution is a polyelectrolyte or salt solution, and the second phase solution is a non-polar solution. In another embodiment, the first phase solution and the second phase solution have opposite charges. The size of the charge difference and the choice of the first and second phase solutions can be adjusted according to the length, size and type of the nucleic acid to be extracted and / or purified.

In various embodiments, the polyelectrolyte solution is selected from a polyelectrolyte solution of about 0.001% to 90% w / w. In various embodiments, the polyelectrolyte is selected from about 0.01% w / w, about 0.05% w / w, about 0.1% w / w, about 0.15% w / w, about 0.2% w / w, and about 0.25% w / w. w, about 0.3% w / w, about 0.35% w / w, about 0.4% w / w, about 0.45% w / w, about 0.5% w / w, about 0.55% w / w, about 0.6% w / w About 0.65% w / w, about 0.7% w / w, about 0.75% w / w, about 0.8% w / w, about 0.85% w / w, about 0.9% w / w, about 0.95% w / w, Or about 1% w / w polyelectrolyte solution. In some embodiments, the polyelectrolyte solution is selected from about 1% w / w, about 2% w / w, about 3% w / w, about 4% w / w, about 5% w / w, and about 6% w / w, about 7% w / w, about 8% w / w, about 9% w / w, about 10% w / w, about 11% w / w, about 12% w / w, about 13% w / w, about 14% w / w, about 15% w / w, about 16% w / w, about 17% w / w, about 18% w / w, about 19% w / w, about 20% w / w About 21% w / w, about 22% w / w, about 23% w / w, about 24% w / w, about 25% w / w, about 26% w / w, about 27% w / w, About 28% w / w, about 29% w / w, about 30% w / w, about 31% w / w, about 32% w / w, about 33% w / w, about 34% w / w, about 35% w / w, about 36% w / w, about 37% w / w, about 38% w / w, about 39% w / w, about 40% w / w, about 41% w / w, about 42 % w / w, about 43% w / w, about 44% w / w, about 45% w / w, about 46% w / w, about 47% w / w, about 48% w / w, about 49% w / w, and about 50% w / w polyelectrolyte solution. In one embodiment, the concentration of the polyelectrolyte solution is about 0.1% w / w to 20% w / w. In another embodiment, the concentration of the polyelectrolyte solution is about 1% w / w to 10% w / w.

In one embodiment, the ratio of the first phase solution to the second phase solution is in the range of 1: 1 to 1: 1000. In some embodiments, the ratio of the first phase solution to the second phase solution is selected from about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about Ratios of 1: 7, about 1: 8, about 1: 9, and about 1:10. In some embodiments, the ratio of the first component to the second component is selected from about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, and about 1:70. In some embodiments, the ratio of the first component to the second component is selected from about 1: 200, about 1: 300, about 1: 400, about 1: 100, about 1:90, and about 1: 90.1 : 500, about 1: 600, about 1: 700, about 1: 800, about 1: 900, and about 1: 1000.

In one embodiment, the ratio of the second phase solution to the first phase solution is selected from about 1: 1, about 1: 2, about 1: 3, about 1: 4, about 1: 5, about 1: 6, about The ratio of 1: 7, about 1: 8, about 1: 9, and about 1:10. In some embodiments, the ratio of the second component to the first component is selected from about 1:20, about 1:30, about 1:40, about 1:50, about 1:60, about 1:70, about 1:80, about 1:90, about 1: 100. In some embodiments, the ratio of the second component to the first component is selected from about 1: 200, about 1: 300, about 1: 400, about 1: 500, about 1: 600, about 1: 700, about The ratio of 1: 800, about 1: 900, and about 1: 1000.

In one embodiment, the first phase solution is a polymer solution having a concentration of 1-25 (w / w)%, and the second phase solution is a salt solution having a concentration of 1-40% (w / w).

In one embodiment, the first phase solution is a polymer solution comprising polyethylene glycol (PEG) selected from PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG1400, and PEG1500. In one embodiment, the concentration of PEG is 1-25% (w / w).

In one embodiment, the second phase solution is a salt solution comprising one or more salts selected from the group consisting of potassium dihydrogen phosphate, dipotassium hydrogen phosphate, potassium sulfate, sodium sulfate, potassium chloride, and sodium chloride. In one embodiment, the total concentration of one or more salts does not exceed 10% (w / w).

In one embodiment, the sample is a nucleic acid-containing sample obtained from a non-biological source, such as soil, water, food, and beverages. In another embodiment, the sample is a nucleic acid-containing biological material.

The nucleic acid can be any nucleic acid, such as a human nucleic acid, a bacterial nucleic acid, or a viral nucleic acid. In some embodiments, the nucleic acid can be DNA, RNA, or DNA products of RNA, single- or double-stranded, or any other form, obtained from reverse transcription or other means.

In one embodiment, the nucleic acid can be from one or more cells, tissues or body fluids, such as blood, urine, semen, lymph fluid, cerebrospinal fluid or amniotic fluid, or other biological samples, such as cultured cells, oral swabs, mouthwash , Feces, tissue sections, biopsies, and archeological samples such as bone or mummy tissue.

In various embodiments, the biological material may be derived from any source including, but not limited to, eukaryotes, plants, animals, vertebrates, fish, mammals, humans, non-humans, bacteria, microorganisms, viruses, biological sources, serum, Plasma, blood, urine, semen, lymph fluid, cerebrospinal fluid, amniotic fluid, biopsy, needle biopsy, cancer, tumor, tissue, cells, cell lysate, crude cell lysate, tissue lysate, tissue culture cells, oral swabs , Mouthwash, feces, mummy tissue, forensic source, autopsy, archeological resources, infection, nosocomial infection, production source, pharmaceutical preparation, biomolecule product, protein preparation, lipid preparation, carbohydrate preparation, inanimate object, air, soil, Sap, metals, fossils, excavated materials, water, food, beverages, and / or other land or field materials and sources. The sample may also contain a mixture of materials from one source or different sources.

In one embodiment, the invention can isolate nucleic acids of multiple base pair (bp) size lengths. In one embodiment, the present invention is used to isolate 1000 bp or less nucleic acid, and the microfluidic device comprises a porous matrix that embeds ATPS components by dehydrating ATPS onto the porous matrix, wherein the ATPS comprises % (w / w) polymer, or in the range of 2-6% (w / w), or in the range of 3-5% (w / w). In another embodiment, the invention is used to isolate 200 bp or smaller nucleic acids. In another embodiment, the invention is used to isolate 250 bp or smaller nucleic acids. In another embodiment, the invention is used to isolate a 300 bp or smaller nucleic acid. In one embodiment, a microfluidic device comprises a porous matrix that embeds ATPS components by dehydrating ATPS to the porous matrix, wherein the ATPS comprises 12% (w / w) PEG 1000, 1% (w / w ) SDS, 3% (w / w) Triton-X114 and 18% (w / w) Na ₂ SO ₄ , pH is about 7. In another embodiment, the present invention is used to isolate 500 bp or less nucleic acid, and the ATPS component used in the microfluidic device contains 12% (w / w) PEG 600, 1% (w / w) SDS, 4% (w / w) Triton-X114 and 14% (w / w) Na ₂ SO ₄ , pH is about 7. In another embodiment, the present invention is used to isolate 1000 bp or smaller nucleic acids, and the ATPS component used in the microfluidic device contains 4% (w / w) PEG 400, 1% (w / w) SDS, 2% (w / w) Triton-X114 and 10% (w / w) Na ₂ SO ₄ , pH is about 7.

In one embodiment, the present invention can simultaneously isolate and concentrate nucleic acids from a relatively large volume of sample to a very small volume (eg, 1 μl) to reduce the sample volume for downstream processing. In another embodiment, the present invention can concentrate low-abundance nucleic acids present in a sample, for example, 1 picogram per milliliter (pg / ml), to be concentrated 2 to 1000 times.

In one embodiment, the present invention can isolate nucleic acids from a sample in high yield and purity. In one embodiment, the present invention can isolate nucleic acids from a sample in a yield and purity compatible with downstream applications. In one embodiment, the resulting isolated nucleic acid can be used directly for downstream processing, including PCR amplification, restriction digestion, and different sequencing methods. In another embodiment, a microfluidic device is used for purification or purification of PCR products.

Some embodiments do not require external driving forces (such as electricity or vacuum) to operate, thus allowing easy isolation and purification of nucleic acids at any time and anywhere. However, an external driving force may be used in some embodiments of the invention.

In one embodiment, the present invention provides a device for rapid separation of nucleic acids from a nucleic acid-containing material, the device comprising a porous matrix embedded in a two-liquid phase system (ATPS component), wherein the ATPS includes a first phase solution and a second Phase solution.

In one embodiment of the device of the invention, the first phase solution is selected from the group consisting of a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution. In one embodiment, the second phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution.

In one embodiment of the apparatus of the present invention, the ratio of the first phase solution to the second phase solution is 1: 1 to 1: 1000.

In one embodiment of the device of the invention, the first phase solution and the second phase solution are premixed into a mixed phase solution before being embedded into the porous matrix. In another embodiment, the first phase solution and the second phase solution are embedded in the porous matrix, respectively. In yet another embodiment, the first phase solution and the second phase solution are embedded on the porous matrix at the same or different locations.

In one embodiment of the device of the invention, the porous matrix is made of one or more porous materials containing a plurality of capillaries. In one embodiment, the average diameter of the capillary is about 0.015 μm to 12 μm.

In one embodiment of the device of the invention, the nucleic acid to be isolated has a size of about 1000 bp or less.

In one embodiment of the device of the invention, the nucleic acid-containing material is from a biological or non-biological material.

In one embodiment of the device of the invention, the nucleic acid is present in the nucleic acid-containing material at a concentration of 1 pg / ml or higher. In one embodiment, the device is used to concentrate nucleic acids 2 to 1000 times. In another embodiment, the device can isolate the target nucleic acid in 2 to 5 minutes.

In one embodiment, the invention provides a method for rapidly isolating and concentrating nucleic acids from nucleic acid-containing material using the devices described herein.

In one embodiment of the method, the first phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution.

In one embodiment of the method, the second phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution.

In one embodiment of the method, the ratio of the first phase solution to the second phase solution is 1: 1 to 1: 1000.

In one embodiment of the method, the first phase solution and the second phase solution are premixed into a mixed phase solution before being embedded in the porous matrix. In another embodiment, the first phase solution and the second phase solution are embedded in the porous matrix, respectively. In another embodiment, the first phase solution and the second phase solution are embedded on the porous matrix at the same or different locations.

In one embodiment of the method, the porous matrix is made of one or more porous materials containing a plurality of capillaries. In one embodiment, the average diameter of the capillary is about 0.015 μm to 12 μm.

In one embodiment of the method, the target nucleic acid has a size of 1000 bp or less.

In one embodiment of the method, the sample is from a biological or non-biological material.

In one embodiment of the method, the target nucleic acid in the sample has a concentration of 1 pg / ml or higher. In one embodiment, the target nucleic acid is concentrated 2 to 1000 times after isolation. In another embodiment, the method can isolate the target nucleic acid in 2 to 5 minutes.

In one embodiment, the present invention provides a microfluidic device for rapid separation of nucleic acids from a material containing these nucleic acids, the device comprising a porous matrix embedded with components of a dual liquid phase system (ATPS), wherein the Hydration of the porous matrix produces the ATPS containing the first phase solution and the second phase solution.

In one embodiment, the first phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution.

In one embodiment, the second phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution.

In one embodiment, the volume ratio of the first phase solution and the second phase solution is from 1: 1 to 1: 1000.

In one embodiment, the ATPS components are mixed in a solution and then dehydrated onto a porous substrate. In one embodiment, the components forming the first phase solution and the components forming the second phase solution are dissolved in separate solutions and then dehydrated to the porous substrate separately.

In one embodiment, the components forming the first phase solution and the components forming the second phase solution are embedded in the porous matrix at the same or different positions.

In one embodiment, the porous matrix comprises one or more porous materials, and the average pore diameter of the porous matrix is about 0.015 μm to 12 μm.

In one embodiment, the microfluidic device of the present invention is capable of separating nucleic acids of about 1000 bp or less.

In one embodiment, the microfluidic device of the present invention is capable of separating nucleic acids present in the starting material at a concentration of 1 pg / ml or higher.

In one embodiment, the microfluidic device of the present invention concentrates nucleic acids 2 to 1000 times.

In one embodiment, the microfluidic device of the present invention can separate a target nucleic acid in 2 to 5 minutes.

In one embodiment, the first phase solution is a polymer solution having a concentration of 1-25% (w / w), and the second phase solution is a salt solution having a concentration of 1-40% (w / w).

In one embodiment, the first phase solution comprises a polymer selected from PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400 With PEG 1500, the polymer concentration is 1-25% (w / w).

In one embodiment, the second phase solution comprises one or more salts selected from the group consisting of sodium chloride, sodium phosphate, disodium phosphate, sodium sulfate, sodium citrate, sodium acetate, potassium chloride, potassium dihydrogen phosphate, Dipotassium hydrogen phosphate, potassium sulfate, sodium sulfate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate, and ammonium acetate, wherein the concentration of the salt is not more than 10% (w / w).

In one embodiment, the present invention provides a method for isolating and concentrating a target nucleic acid from a sample, including the following steps:
a) obtaining a sample solution containing the target nucleic acid; and
b) contacting the solution with a microfluidic device comprising a porous matrix embedded with components of a dual liquid phase system (ATPS), wherein when the solution flows through the porous matrix, a solution comprising a first phase solution and a second In phase solution ATPS, the target nucleic acid can pass through the pores of the porous matrix and is preferentially distributed to one phase solution, resulting in separation of the target nucleic acid in the first phase solution or the second phase solution.

In one embodiment, the invention provides a method, further comprising the step of eluting the target nucleic acid from the porous matrix of the microfluidic device.

In one embodiment, the present invention provides a method, wherein the first phase solution is a polymer solution, a salt solution, a non-polar solution, a micellar solution, or a polyelectrolyte solution.

In one embodiment, the present invention provides a method, wherein the second phase solution is a polymer solution, a salt solution, a non-polar solution, a micellar solution, or a polyelectrolyte solution.

In one embodiment, the present invention provides a method, wherein the volume ratio of the first phase solution and the second phase solution is from 1: 1 to 1: 1000.

In one embodiment, the present invention provides a method wherein the porous matrix comprises one or more layers of porous material, and wherein the average pore diameter of the porous matrix is about 0.015 μm to 12 μm.

In one embodiment, the invention provides a method wherein the target nucleic acid has a size of about 1000 bp or less.

In one embodiment, the invention provides a method wherein the target nucleic acid in solution has a concentration of 1 pg / ml or higher.

In one embodiment, the present invention provides a method wherein the target nuclear acid is concentrated 2 to 1000 times.

In one embodiment, the present invention provides a method that can isolate a target nucleic acid in 2 to 5 minutes.

The invention will be better understood by reference to the following examples. However, those skilled in the art will readily understand that the examples provided are for illustrative purposes only and are not meant to limit the scope of the invention, which is defined by the following claims.

Throughout this application, it should be noted that the transitional term "including", which is synonymous with "including,""containing," or "characterized by," is inclusive or open-ended, and does not exclude other, unrecited elements or method steps.
Examples
Example 1 Simultaneous separation and concentration of plasmid DNA of C. trachomatis cells in a PBS solution using a two-liquid phase system

Different amounts of Chlamydia trachomatis (CT) cells were incorporated into 1 × PBS solution to a final concentration of 2 × 10, 2 × 10 ² , 2 × 10 ³ , 2 × 10 ⁴ and 2 × 10 ⁵ cells / ml. Two-liquid phase system (ATPS) components were added to 1 ml of PBS solution mixed with CT cells of different concentrations, so that the final concentration of the ATPS component in the resulting solution was 12% (w / w) PEG 1000, 1% ( w / w) SDS, 3% (w / w) Triton-X114 and 18% (w / w) Na ₂ SO ₄ , pH is about 7. The added ATPS component forms a mixed phase solution in PBS solution. The mixed phase solution was vortexed for 2 minutes and then rotated for 15 seconds for phase separation. The top phase was pipetted into a PCR tube for terminal PCR.

In the terminal PCR, the 201 bp fragment was amplified using the following primers: 5'-TAGTAACTGCCACTTCATCA-3 '(SEQ ID NO: 1) and 5'-TTCCCCTTTATATCTCGTTGC-3' (SEQ ID NO: 2), in which the secreted plasmid of C. trachomatis . The final PCR reaction was prepared as follows: 10 ul TAQ2X standard buffer, 0.2 μM primer, and 5 μl DNA template per 20 μl reaction. The PCR amplification process was performed with a denaturation step at 94 ° C for 4 minutes, followed by 30 cycles of amplification using a miniPCR ^™ thermal cycler. Each cycle consisted of 1 minute at 94 ° C, 1 minute at 48 ° C, 1 minute at 68 ° C, and final elongation at 68 ° C for 5 minutes. Gel electrophoresis followed by terminal PCR (using Thermo Scientific ™ GeneRuler low-range DNA ladder, that is, type 25 to 700bp as a label).

According to the manufacturer's instructions, the results were compared with the results of DNA isolation using a QIAamp DNA Blood Mini Kit (Qiagen) on a sample containing 2 × 10 ⁵ cells / ml and a total volume of 1 ml. As shown in Figure 2, the detection limit of this method reaches 20 cells / ml. Based on a comparison of the band intensities in agarose gel electrophoresis depicted in Figure 2, the results indicate that the yield of purified nucleic acid obtained by our method is comparable to that obtained by the QIAamp DNA Blood Mini Kit (Qiagen).

Example 2 Simultaneous separation and concentration of Chlamydia trachomatis cell plasmid DNA incorporated in a PBS solution using a porous matrix embedded in a two-liquid phase system

Add the prepared ATPS component to the solution so that the final concentration of the component is 12% (w / w) PEG 1000, 1% (w / w) SDS, 3% (w / w) Triton-X114 and 18 % (w / w) Na ₂ SO ₄ . The resulting solution was mixed and then dehydrated to one end of a paper stack formed of four layers of fiberglass paper (as shown in the lower right of FIG. 4). Add the paper stack embedded with ATPS components to a premixed suspension of 4 × 10 ⁵ CT cells in 1 × PBS, bring the bottom of the paper stack into contact with the suspension, and draw the sample for about 2 minutes until the solution reaches the paper stack the top of. As shown in Figure 3, DNA from the lysed cells was drawn to the top of the paper stack. Nucleic acid was extracted from the top of the paper stack by pipetting the solution from the top of the paper stack into a PCR tube, followed by terminal PCR, followed by gel electrophoresis as described in Example 1. As shown in FIG. 3, using the extracted DNA as a template, a fragment of the C. trachomatis concealing plasmid was successfully amplified.

Example 3 Isolation and concentration of Chlamydia trachomatis cell plasmid DNA in serum using a porous paper matrix embedded in a two-liquid phase system

Under sterile conditions, blood was collected from fasting patients by venipuncture. After the blood was coagulated in a test tube for 1 hour at room temperature, the test tube was centrifuged at 1600 g for 15 minutes at 4 ° C. The serum was immediately separated and 3 ml of serum was centrifuged on a Beckman centrifuge AN (Beckman Coulter, Inc., USA) at 15000 rpm for 15 minutes at 4 ° C. Discard the supernatant. The obtained pellet was gently resuspended in a 0.5 mL SPG buffer using a micropipette (pH 7.2; 250 mM sucrose, 10 mM sodium phosphate, 5 mM L-glutamic acid). Add the prepared ATPS components to the pretreated serum samples so that their final concentration is 12% (w / w) PEG 1000, 1% (w / w) SDS, 3% (w / w) Triton-X114 And 18% (w / w) Na ₂ SO ₄ , the pH is about 7. The ATPS sample mixture was incubated for 10 minutes at room temperature. A paper stack formed of 4 layers of fiberglass paper was then inserted into the mixture so that the top of the paper stack was not submerged in the mixture, and the sample was drawn for about 3 minutes until the solution reached the top. The solution was extracted from the top of the paper stack by aspirating the solution with a pipette. The nucleic acid concentrated in the extraction solution was then subjected to terminal PCR, and then subjected to gel electrophoresis as described in Example 1. Based on the comparison of the band intensities in agarose gel electrophoresis, the yield of the purified nucleic acid obtained was comparable to that obtained by the QIAamp DNA Blood Mini Kit (Qiagen).

Example 4 Simultaneous isolation and concentration of Chlamydia trachomatis cell plasmid DNA in saliva using a porous paper matrix embedded in a two-liquid phase system

Fasting patients collect saliva samples themselves using dry polypropylene containers without preservatives. Dissolve the prepared ATPS components in solution so that their final concentration is 12% (w / w) PEG 1000, 1% (w / w) SDS, 3% (w / w) Triton-X114 and 18% (w / w) Na ₂ SO ₄ . The resulting solution was mixed and dehydrated to the end of a paper stack formed from 4 layers of fiberglass paper as described in Example 3. The paper stack with the ATPS component embedded was inserted into a 1 ml saliva sample so that the top of the paper stack was not immersed in the sample, and the sample was drawn for about 3 minutes until the solution reached the top. The solution was extracted from the top by a pipette for terminal PCR, and then subjected to gel electrophoresis as described in Example 1. Based on the comparison of the band intensities in agarose gel electrophoresis, the yield of the purified nucleic acid obtained was comparable to that obtained by the QIAamp DNA Blood Mini Kit (Qiagen).

Example 5 Simultaneous separation and concentration of Chlamydia trachomatis cell plasmid DNA in urine using a porous paper matrix embedded in a two-liquid phase system

Fasting patients collect their own urine samples in a dry polypropylene container without preservatives. A 3.5 ml urine sample was mixed with 560 μl buffer AVL from QIAamp Viral RNA Mini Kit (Qiagen), incubated for 10 minutes, and then run on a Beckman centrifuge AN (Beckman Coulter, Inc., USA) at 5000 rpm at 4 Centrifuge at ° C for 5 minutes. The obtained pellet was gently resuspended in a 0.5 mL PBS using a micropipette. Add the prepared ATPS components to the suspension so that their final concentration is 12% (w / w) PEG 1000, 1% (w / w) SDS, 3% (w / w) Triton-X114 and 18% (w / w) Na ₂ SO ₄ with a pH of about 7. The ATPS sample mixture was incubated for an additional 10 minutes at room temperature. The paper stack described in Example 3 was then inserted into the mixture so that the top of the paper stack was not immersed in the mixture, and the sample was drawn for about 3 minutes until the solution reached the top. The solution was extracted from the top by a pipette for terminal PCR, and then subjected to electrophoretic analysis as described in Example 1. Based on the comparison of the band intensities in agarose gel electrophoresis, the yield of the purified nucleic acid obtained was comparable to that obtained by the QIAamp DNA Blood Mini Kit (Qiagen).

In summary, the results of the above examples show that the present invention can isolate and purify DNA from Chlamydia trachomatis cells present in various samples. The DNA products obtained by the present invention can be amplified and analyzed by high-sensitivity electrophoresis.

Figure 1 shows a picture of phase separation induced by adding polymer and salt to water. In the test tube shown in Figures A1 and B1, the two phases are mixed to form a mixed phase, and then the salt-rich phase and polymer-rich phase gradually separate to form different layers, as shown in the test tubes of A2 and B2. The salt-rich phase in this example settles at the bottom of the test tube, while the polymer-rich phase is above it. By changing the ratio of polymer to salt, the volume ratio of the two phases can be changed so that the target molecule is concentrated in the smaller volume phase. For example, the ratio of the top phase to the bottom phase is 1: 1 in Figure A2, and the ratio of 9: 1 is depicted in Figure B2. Compared with the bottom of Figure A2, the bottom phase (salt-rich phase) in Figure B2 shows a darker color, and it can be seen that the target molecule (in this example, gold nanoparticles) is in the bottom The target molecule is concentrated in the bottom phase to obtain a higher concentration in the bottom phase.

FIG 2 shows a schematic ATPS using Qiagen ^® or a nucleic acid preparation kit (QIAamp DNA Mini Kit) extracting DNA from Chlamydia trachomatis (CT) cells, and the resulting electropherogram amplified DNA extracted from various cell product CT. Thermo Scientific ™ Gene Ruler low-range DNA ladders, ready-to-use 25 to 700 bp, are used as markers in the bottom image. It can be observed that the ATPS can successfully extract and amplify DNA from only 20 cells with a concentration of 20 cells / ml.

Figure 3 shows a schematic diagram of extracting DNA from Chlamydia trachomatis cells and analyzing the extracted and amplified DNA by electrophoresis using a microfluidic device according to an embodiment of the present invention. As the marker, the same DNA ladder as described above was used.

Figure 4 shows some embodiments of the microfluidic device of the invention, where the ATPS component is embedded in the porous matrix in different ways.

Claims (23)

A microfluidic device for rapidly separating a nucleic acid from a nucleic acid-containing material, the device comprising a porous matrix embedded with components of a dual liquid phase system (ATPS), wherein hydration of the porous matrix produces the ATPS containing a first phase solution and a second phase solution. The apparatus of claim 1, wherein the first phase solution is selected from a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution. The apparatus of claim 1, wherein said second phase solution is selected from the group consisting of a polymer solution, a salt solution, a non-polar solution, a micellar solution, and a polyelectrolyte solution. The device according to claim 1, wherein the volume ratio of the first phase solution to the second phase solution is 1: 1 to 1: 1000. The apparatus of claim 1, wherein the components are mixed in a solution and then dehydrated onto a porous substrate; or the components forming the first phase solution and the components forming the second phase solution are dissolved in separate solutions And then dehydrate them separately onto a porous substrate. The apparatus of claim 5, wherein the component forming the first phase solution and the component forming the second phase solution are embedded in the porous substrate at the same or different positions. The apparatus of claim 1, wherein the porous matrix comprises one or more porous materials, and the average diameter of the pores of the porous matrix is about 0.015 μm to 12 μm. The device of claim 1, wherein the size of said nucleic acid is about 1000 bp or less. The apparatus of claim 1, wherein said nucleic acid is present in the material at a concentration of 1 pg / ml or higher. The device of claim 1, wherein said device concentrates the nucleic acid 2 to 1000 times. The device of claim 1, wherein said device is capable of separating the target nucleic acid in 2 to 5 minutes. The apparatus of claim 1, wherein the first phase solution is a polymer solution having a concentration of 1-25% (w / w), and the second phase solution is a salt solution having a concentration of 1-40% (w / w) . The device of claim 1, wherein the first phase solution comprises a polymer selected from the group consisting of PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, PEG 1000, PEG 1100, PEG 1200, PEG 1300, PEG 1400, and PEG 1500, wherein the concentration of the polymer is 1-25% (w / w); and The second phase solution contains one or more salts selected from the group consisting of sodium chloride, sodium phosphate, disodium phosphate, sodium sulfate, sodium citrate, sodium acetate, potassium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. , Potassium sulfate, sodium sulfate, potassium citrate, ammonium sulfate, sodium citrate, sodium acetate, and ammonium acetate, wherein the concentration of the salt does not exceed 10% (w / w). A method for isolating and concentrating a target nucleic acid from a sample, including the following steps: a) obtaining a sample solution containing the target nucleic acid; and b) contacting the solution with a microfluidic device comprising a porous matrix embedded with components of a two-liquid phase system (ATPS), wherein the first phase is formed as the solution flows through the porous matrix Solution and second phase solution, Wherein the target nucleic acid can pass through the pores of the porous matrix and is preferentially distributed to a phase solution, resulting in separation of the target nucleic acid in the first phase solution or the second phase solution. The method of claim 14, further comprising the step of eluting the target nucleic acid from the porous matrix of the microfluidic device. The method of claim 14, wherein the first phase solution is a polymer solution, a salt solution, a non-polar solution, a micellar solution, or a polyelectrolyte solution. The method of claim 14, wherein the second phase solution is a polymer solution, a salt solution, a non-polar solution, a micellar solution, or a polyelectrolyte solution. The method of claim 14, wherein the volume ratio of the first phase solution to the second phase solution is from 1: 1 to 1: 1000. The method of claim 14, wherein the porous matrix comprises one or more porous materials, and wherein the average pore diameter of the porous matrix is about 0.015 μm to 12 μm. The method of claim 14, wherein the size of the target nucleic acid is about 1000 bp or less. The method of claim 14, wherein the target nucleic acid in the solution has a concentration of 1 pg / ml or higher. The method of claim 14, wherein the target nucleic acid is concentrated 2 to 1000 times. The method of claim 14, wherein the method can isolate the target nucleic acid in 2 to 5 minutes.