WO2016086374A1 - 一种模拟细胞体内环境的微流体器件及其应用 - Google Patents

一种模拟细胞体内环境的微流体器件及其应用 Download PDF

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Publication number
WO2016086374A1
WO2016086374A1 PCT/CN2014/092950 CN2014092950W WO2016086374A1 WO 2016086374 A1 WO2016086374 A1 WO 2016086374A1 CN 2014092950 W CN2014092950 W CN 2014092950W WO 2016086374 A1 WO2016086374 A1 WO 2016086374A1
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Prior art keywords
microfluidic
micro
microfluidic device
slope
slope structure
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PCT/CN2014/092950
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English (en)
French (fr)
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蒋春盛
董毅
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士捷医疗设备(武汉)有限公司
蒋春盛
董毅
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Application filed by 士捷医疗设备(武汉)有限公司, 蒋春盛, 董毅 filed Critical 士捷医疗设备(武汉)有限公司
Priority to PCT/CN2014/092950 priority Critical patent/WO2016086374A1/zh
Priority to CN201480001474.6A priority patent/CN106170301A/zh
Publication of WO2016086374A1 publication Critical patent/WO2016086374A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/44—Antibodies bound to carriers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/40—Concentrating samples

Definitions

  • the present invention relates to microfluidic technology, and more particularly to a microfluidic device that mimics the in vivo environment of cells and its use in detecting low abundance cells, low abundance proteins, and low abundance nucleic acid biomarkers, as well as in cell release, Application of DNA, mDNA, RNA, and second-generation sequencing analysis.
  • Microfluidic technology refers to the technology of controlling, operating and detecting complex fluids under microscopic dimensions.
  • fluids such as sample DNA preparation, liquid chromatography, PCR reaction, and electrophoresis.
  • the operations are all carried out in a liquid phase environment. If the steps of sample preparation, biochemical reaction, and results detection are integrated on the biochip, the amount of fluid used in the experiment will be reduced from milliliters, micro-upgrade to nanoliter or skin upgrade.
  • a powerful microfluidic device is very important. From a biomedical point of view, microfluidic devices can save a lot of reagents, increase productivity, and improve the effectiveness of the analysis.
  • the direct consequence of the limitation of the shear stress is that the body fluid, such as a blood sample, has been separated from the human body. It must flow through the microfluidic device at a very low flow rate, resulting in prolonged enrichment of the target cells, low efficiency; and in this process is susceptible to degrading enzymes in the wash solution, as well as biological, chemical, and physical effects, Causes damage to biomarkers.
  • the present invention proposes a novel microfluidic device that mimics the in vivo environment of cells and uses thereof.
  • a microfluidic device that mimics the in vivo environment of a cell, comprising a liquid inlet, a liquid outlet, and at least one microfluidic chamber; the microfluidic chamber having an inlet channel and an outlet channel, wherein the inlet channel is in communication a liquid inlet, the outlet passage communicating with the liquid outlet; and the microfluidic chamber
  • a plurality of microstructure elements are disposed therein, and at least some of the microstructure elements have a micro-slope structure.
  • the microstructured elements are randomly arranged within the microfluidic cavity.
  • each of the microstructure elements has the same or a different micro-slope structure.
  • the maximum height of the micro-slope structure is at least 10 microns lower than the height of the microfluidic cavity.
  • microfluidic chambers For example, only one of the microfluidic chambers is provided.
  • the micro-slope structure has a first slope.
  • the first bevel faces the direction of liquid flow inside the microfluidic cavity.
  • the micro-slope structure has a second bevel that faces away from the direction of liquid flow within the microfluidic cavity.
  • the inclination of the second slope is smaller than the inclination of the first slope.
  • the inclination of the second slope is greater than or equal to the inclination of the first slope.
  • At least a portion of the cross-section of the micro-slope structure has an arcuate closed shape.
  • a plurality of studs are also disposed on the micro-slope structure.
  • the angle between the micro-slope structure and the direction of liquid flow inside the microfluidic cavity is 30 to 150 degrees.
  • the cross-section of the micro-slope structure has a long side and a short side, wherein the long side is perpendicular to the direction of liquid flow inside the microfluidic chamber.
  • the corners of the micro-slope structure are smoothly arranged.
  • the inner wall of the microfluidic cavity is provided with a carbohydrate matrix coating.
  • the microfluidic cavity is transparently disposed above.
  • the micro-slope structure is disposed at the top and bottom of the inner wall of the microfluidic cavity.
  • each of the microfluidic cavities is of a width design.
  • each of the microfluidic cavities has a structure that is small and large.
  • the microfluidic device includes a number of microfluidic chambers.
  • each of the microfluidic chambers is arranged in series.
  • each of the microfluidic cavities is arranged in parallel.
  • the inlet passage and the outlet passage include a plurality of linear passages, a polygonal passage, an arcuate passage, a spiral passage, or a combination thereof.
  • the microfluidic device is useful for detecting low abundance cells, low abundance proteins, low abundance nucleic acid biomarkers.
  • the microfluidic device is used in cell release, DNA, mDNA, RNA, second generation sequencing analysis.
  • the invention has the beneficial effects that: by adopting the design of the micro-slope structure, the shear stress is small, so that the cells in the microfluidic device can withstand higher flow rates, which can greatly shorten the sample processing time and improve the separation efficiency; more importantly, It protects the originality and integrity of biomarkers such as cells, proteins, and nucleic acids.
  • Figure 1A is a schematic illustration of one embodiment of a microfluidic chamber of the present invention.
  • FIGS. 1B to 1E are schematic views of respective embodiments of a microfluidic cavity of the present invention.
  • FIG. 2A is a schematic view of an embodiment of a microfluidic device of the present invention.
  • FIGS. 2B and 2C are schematic views of still another embodiment of the microfluidic device of the present invention.
  • 2D is a schematic view of still another embodiment of the microfluidic cavity of the present invention.
  • Figure 5B is a schematic illustration of an embodiment of a 360 degree micro-slope structure of the present invention.
  • FIGS. 6A to 6C are respectively schematic views showing still another embodiment of the micro-slope structure of the present invention.
  • FIG. 7A to 7D are schematic views respectively showing a cross section of the micro-slope structure
  • Figure 8 is a schematic illustration of one embodiment of a spiral flow path of a microfluidic device of the present invention.
  • 9A-9D are schematic illustrations of one embodiment of a capture antibody or fragment thereof, respectively.
  • a microfluidic device that mimics the in vivo environment of a cell, which is efficient, gentle, and facilitates cell protection, identification, and release, the microfluidic device including a liquid inlet, a liquid outlet, and at least one microfluidic cavity;
  • the microfluidic chamber has an inlet passage and an outlet passage, wherein the inlet passage communicates with the liquid inlet, the outlet passage communicates with the liquid outlet; and a plurality of microstructure elements are disposed in the microfluidic chamber, At least a portion of the microstructure elements have a micro-slope structure.
  • the microfluidic device is used to separate and identify circulating tumor cells and other non-cellular biomarkers from whole blood and other body fluids.
  • the inner wall of the microfluidic cavity is provided with a carbohydrate matrix coating.
  • all surfaces within the microfluidic cavity including microstructured element surfaces, micro-slope surfaces, and the like, are provided with a carbohydrate matrix overlay.
  • the microfluidic device is capable of mimicking the in vivo environment of cells, is highly efficient and gentle, is conducive to cell protection, recognition and release, enhances and promotes the affinity of cells and antibodies, increases the contact time and area of cells and antibodies, and can be used for cancer research, various Research on the clinical diagnosis of cancer, as well as research on personal diagnosis of tumor-targeted therapy. Because of its small shear stress, the cells can withstand higher flow rates, shortening sample processing time and improving separation efficiency; thus achieving rapid sample processing, reducing biomarker damage, and protecting the originality and integrity of biomarkers.
  • the microfluidic device is used to extract rare biomarkers from blood and body fluids.
  • Blood Including red blood cells, white blood cells, platelets, etc.; body fluids include blood, urine, saliva, etc.
  • Rare biomarkers include rare cells such as circulating tumor cells, as well as proteins, ribonucleic acids, and deoxyribonucleic acids.
  • the microfluidic device includes one or more microfluidic chambers, or the microfluidic device is comprised of one or more microfluidic chambers.
  • a large number of micro-slope structures are placed in each microfluidic cavity. For example, the shape of these micro-slope structures is optimized by computer simulation to greatly reduce the shear stress while promoting cell tumbling on the surface of the microfluidic device.
  • the micro-slope structure captures the target while maintaining the biological function and physical structure of the cell.
  • the surface of the microfluidic device is chemically coated via a carbohydrate matrix to minimize non-target characteristic adsorption to avoid interference from non-target cells, proteins, nucleic acids, and the like.
  • a surface of a carbohydrate is immobilized with a capture ligand, and the capture ligand has a specific antibody against a target cell surface antigen; capture of the target cell is achieved by the specific antigen-antibody reaction.
  • the micro-slope structure also has two important functions: one is to reduce the interference of light waves in optical detection, and the other is to keep the cells intact and help to re-enrich the target cells and target cells.
  • the microfluidic chamber as shown in FIG. 1A, has an inlet channel 101 and an outlet channel 102, and a fluid in the microfluidic device, or liquid, enters the microfluidic chamber from the inlet channel 101 through a number of similar After the micro-slope structure of the ski slope, it flows out from the outlet passage 102.
  • the microfluidic device can be made of materials such as glass, plastic, metal, silicon, silicon oxide, and the like.
  • the microfluidic device is a plastic device whose surface is treated with oxygen plasma or ultraviolet light to produce an active binding site, i.e., having an activated surface.
  • the surface of the plastic device is changed from hydrophobic to hydrophilic, facilitating antibodies or other capture coatings on the plastic surface.
  • the surface of the microfluidic cavity such as the activated surface, is provided with a carbohydrate-based matrix to avoid the effects of non-specific blood cells, proteins and nucleotides as much as possible.
  • a cell-binding ligand such as antibody 901 corresponding to a cell surface antigen, is immobilized on a carbohydrate matrix 903 via a hydrophilic spacer 902, such as a PEG having a functional group at both ends, and the carbohydrate matrix 903 is located in the plastic device. 904 surface.
  • Monoclonal antibodies can be immobilized in different shapes. As shown in Figure 9A, all antibodies are immobilized on the surface; as shown in Figures 9B to 9D, antibody fragment 905, immunoglobulin G906, etc. can be immobilized, better targeted binding sites or lower for blood cells. Non-specific binding. For easy storage and replacement The body cover is protected with a stabilizer.
  • the model microfluidic device shown in Figure 1 has a microfluidic chamber.
  • the microfluidic cavity has an inlet and an outlet before and after, for example, for the ingress or egress of a blood sample.
  • the blood sample flows out of the outlet and eventually flows to waste collection.
  • the microfluidic cavity is provided with four successively arranged micro-slope structures.
  • a real microfluidic device would contain hundreds to tens of thousands of micro-slope structures.
  • the highest height of the micro-slope structure is at least 10-20 microns lower than the height of the microfluidic cavity.
  • each micro-slope structure in the entire microfluidic cavity has a uniform shape as shown in FIG. 1B or as shown in FIG.
  • each micro-slope structure in the entire microfluidic cavity is as shown in FIG. 1D or as shown in FIG. 1E.
  • the display has a constantly changing shape.
  • the microfluidic device can be assembled with a flat top layer to form a structure as shown in Figure 1B or as shown in Figure 1D.
  • the top layer of the flat sheet must be optically transparent, and the material may be a special transparent adhesive tape or a plastic or glass sheet.
  • the microfluidic device can also be assembled with a matching microfluidic device to form the structure shown in Figure 1C or as shown in Figure 1E.
  • One of the innovations of the microfluidic device of this embodiment is that the blood flowing therein and the target cells therein flow down from the flow of several micro-slope structures and are captured by the microfluidic device during this flow.
  • the top 103 of the inner wall of the microfluidic cavity is a flat plate structure, and the bottom portion 104 is provided with the micro-slope structure.
  • the top 103 of the inner wall of the microfluidic cavity is a flat plate structure, and the bottom portion 104 is provided with the micro-slope structure.
  • the top 103 and the bottom 104 of the inner wall of the microfluidic cavity are provided with the micro-slope structure.
  • the top portion 103 and the bottom portion 104 of the inner wall of the microfluidic chamber are provided with the micro-slope structure.
  • the microfluidic cavity is transparently disposed above; for example, as shown in any of Figures 1B-1E, the top 103 of the inner wall of the microfluidic cavity is transparently disposed. And/or, the microfluidic cavity is disposed transparently below, for example, as shown in any of FIGS. 1B-1E, the bottom 104 of the inner wall of the microfluidic cavity is transparently disposed. In this way, it is advantageous to collect optical signals.
  • the microfluidic device includes a number of microfluidic chambers.
  • a microfluidic device as shown in FIG. 2A, includes a liquid inlet 201, a liquid outlet 202, and four microfluidic chambers 203; the liquid inlet 201 communicates with each microfluidic chamber inlet through a liquid inlet passage 204, respectively.
  • Channel 101 for example, as shown in Figure 2A, each of the microfluidic cavities is of a width design.
  • each of the microfluidic cavities has a structure that is small in front and large in size.
  • FIG. 2A the liquid inlet 201
  • a liquid outlet 202 includes a liquid outlet 202, and four microfluidic chambers 203; the liquid inlet 201 communicates with each microfluidic chamber inlet through a liquid inlet passage 204, respectively.
  • Channel 101 for example, as shown in Figure 2A, each of the microfluidic cavities is of a width design.
  • each of the microfluidic cavities has a
  • the microfluidic device includes six microfluidic chambers 203, each of which is a microfluidic chamber.
  • the outlet passages 102 communicate with the liquid outlets 202 through the liquid outlet passages 205, respectively, wherein the microfluidic chambers have a structure of small front and large rear, that is, the inlet port is small, and the outlet port is large, thus contributing to The fluid diffuses after entering the microfluidic cavity.
  • the microfluidic device includes seven microfluidic chambers 203 having a structure of front large and small, and the liquid outlet passage 205 has a structure of front small and large, thus contributing to the fluid.
  • each of the microfluidic chambers is disposed in parallel.
  • each of the microfluidic chambers is disposed in series.
  • the microfluidic cavity contains a plurality of identical micro-slope structures as shown in Figure 2D.
  • the microstructure elements are randomly arranged within the microfluidic cavity, for example, each of the microstructure elements has the same structure, the position and orientation of which are randomly set within the microfluidic cavity; and, as shown in FIG. 5A, at least two The microstructure elements have distinct structures, the position and orientation of each of the microstructure elements are randomly set within the microfluidic cavity, and so on.
  • the microstructure elements are regularly arranged within the microfluidic cavity; for example, the microstructure elements are arranged in rows or arrays within the microfluidic cavity; as another embodiment, as shown in Figure 2D, each of the microstructure elements Having the same structure, the positions of each of the microstructure elements are regularly arranged in the microfluidic cavity and the directions are consistent; for example, as shown in FIG. 3 or FIG. 4, at least two microstructure elements have different structures, each of which The locations of the microstructure elements are generally regularly arranged within the microfluidic cavity and the orientation is generally uniform. As another example, as shown in FIG. 5A, each of the microstructure elements has the same structure or a different structure, and the microstructure elements are randomly disposed within the microfluidic cavity.
  • each of the microstructure elements has the same or different micro-slope structure.
  • at least two microstructure elements have distinct micro-slope structures; for example, several microstructure elements have different two or more micro-slope structures.
  • the maximum height of the micro-slope structure is at least 10 microns lower than the height of the microfluidic cavity, that is, the highest micro-slope structure, one end of which is fixed to one side of the microfluidic cavity The other end is at least 10 microns from the other side of the microfluidic cavity.
  • the maximum height of the micro-slope structure is at least 12 microns lower than the height of the microfluidic cavity; as another example, the maximum height of the micro-slope structure is at least 20 microns lower than the height of the microfluidic cavity.
  • capturing objects based on the target Set the specific height of the micro-slope structure.
  • microfluidic chambers only one of the microfluidic chambers is provided, an inlet passage communicating with the inlet port, and an outlet passage communicating with the outlet port; and wherein the microfluidic chamber is provided with a plurality of microstructure elements, at least a portion of the microstructure Among the elements, there is a micro-slope structure.
  • microfluidic device designs Due to the diversity and variability of circulating tumor cells, it is necessary for microfluidic device designs to include a diverse number of such microstructure elements that have the same or different functional units, for example, having the same or different micro-slope structures.
  • the micro-slope structure is aligned in the tangential direction to the flow direction, diversification can be achieved by banding.
  • the microfluidic device is provided with four sets of micro-slope structure strips.
  • the micro-slope structure strips are uniform within the group and repeat the same curvature and size.
  • the micro-slope structure belts are non-uniform between groups and have different curvatures and scales. The purpose of this design is to better enrich the diverse circulating tumor cells or other rare cells. It should be noted that FIG.
  • microfluidic device 3 is only a schematic diagram, and the true microfluidic device contains a large number of sets of micro-slope structure bands to maximize the enrichment efficiency.
  • the microfluidic device randomly lays out each micro-slope structure.
  • micro-slope structures of different curvatures and sizes are randomly arranged in the microfluidic cavity by computer.
  • fluid such as blood flow
  • the blood flow flows through the sides of the functional unit while the blood flow also flows up and down.
  • Target cells can be captured by all surface areas. This is not found in previous microfluidic devices.
  • the micro-slope structures may be arranged along the direction of blood flow, may be orthogonal to the direction of blood flow, or may intersect the direction of blood flow at any angle.
  • a 360 degree micro-slope structure is also provided.
  • a micro-slope structure along the direction of blood flow and an orthogonal blood flow direction is used in combination with a 360-degree micro-slope structure; wherein the 360-degree micro-slope structure is as shown in FIG. 5B.
  • the 360-degree micro-slope structure also has the function of diversion and shunt while promoting cell tumbling.
  • the 360-degree micro-slope structure can be arranged not only at the bottom of the microfluidic cavity, but also at different angles on the slope of other micro-slope structures.
  • the micro-slope structure has a first slope.
  • the first bevel faces the direction of liquid flow inside the microfluidic cavity.
  • the micro-slope structure has a second bevel that faces away from the direction of liquid flow within the microfluidic cavity.
  • the inclination of the second inclined surface is smaller than the inclination of the first inclined surface; or the inclination of the second inclined surface is greater than or equal to the inclination of the first inclined surface.
  • the first slope or the second slope is an inclined plane having a certain inclination angle, for example, 10 degrees to 80 degrees, for example, the inclination angle of the first slope or the second slope is 20 degrees to 65 degrees, and, for example, The inclination angle of the first slope or the second slope is 10 degrees, 15 degrees, 20 degrees, 25 degrees, 30 degrees, 40 degrees, 45 degrees, 52 degrees, 60 degrees, 65 degrees, 72 degrees or 80 degrees.
  • the first slope or the second slope has a curved section, a parabolic section or a section formed by a combination of an arc and a parabola, for example, a first slope or a second slope having an arcuate section, which is a cylinder Part of the cylindrical surface, and so on.
  • the first bevel or the second bevel is concave or convex.
  • the first slope 301 faces the liquid flow direction inside the microfluidic chamber, that is, toward the inlet passage 101, which is concavely disposed; the second slope 302 faces the liquid flow inside the microfluidic chamber.
  • the direction, ie towards the outlet channel 102, is concave.
  • the first inclined surface 401 is concavely disposed, and the second inclined surface 402 is concavely disposed.
  • the first inclined surface 501 is convexly disposed, and the second inclined surface 502 is concavely disposed.
  • the first inclined surface and the second inclined surface are both concavely disposed. In this way, it is advantageous to reduce the shear stress.
  • a plurality of studs 503 are also disposed on the micro-slope structure to facilitate capturing an object, such as a target cell.
  • a plurality of 360 degree micro-slope structures 504 are disposed inside the microfluidic cavity.
  • at least one micro-slope structure 505 is disposed inside the microfluidic cavity, and a side facing the liquid inside the microfluidic cavity is non-beveled.
  • the cross-section of the micro-slope structure has a long side and a short side, wherein, as shown in FIG. 3, the long side is in a direction of liquid flow inside the microfluidic chamber. vertical.
  • the corners of the micro-slope structure are smoothly arranged.
  • the corners of the micro-slope structure are smoothly disposed, which is another example of a 360-degree micro-slope structure. In this way, it is advantageous to reduce the shear stress and thereby increase the flow rate to increase the processing efficiency.
  • the corners of the micro-slope structure as shown in FIG. 6A are smoothly set to the corners of the micro-slope structure as shown in FIG. 6B, or smoothly set to the corners of the micro-slope structure as shown in FIG. 6C.
  • the cross-section of the micro-slope structure has an arcuate closed shape.
  • the cross-section of the micro-slope structure of this embodiment has an arcuate closed shape.
  • the cross-section of the micro-slope structure of this embodiment has an arcuate closed shape. This It is beneficial to reduce the shear stress.
  • the cross section of the 360 degree micro-slope structure is circular.
  • the inlet passage and the outlet passage include a plurality of linear passages, a polygonal passage, an arcuate passage, a spiral passage or a combination thereof; for example, the inlet passage and the inlet Between the outlet passages, a line-shaped passage or an arc-shaped passage is included; for example, the inlet passage and the outlet passage include a combined passage having at least a linear passage, at least one polygonal passage, and at least one arc a channel, and/or at least one spiral channel.
  • a plurality of spiral channels 802 are disposed in the microfluidic cavity 801, and fluid flows in each of the spiral channels 802.
  • Circulating tumor cells and other biomarkers for example, smooth micro-slope surface promotes cell-antibody contact and reduces shear stress; for example, fluid route design in spiral channels uses Archimedes spirals, isometric spirals , logarithmic spiral or other spiral implementation.
  • a preferred embodiment is a microfluidic device comprising a liquid inlet, a liquid outlet, and at least one microfluidic chamber; the microfluidic chamber having an inlet channel and an outlet channel, wherein the inlet channel is in communication The liquid inlet, the outlet channel communicates with the liquid outlet; and the microfluidic cavity is provided with a plurality of microstructure elements, at least part of the microstructure elements having a micro-slope structure; and, the micro The structural elements are randomly arranged in the microfluidic cavity, and the angle between the arrangement direction of the micro-slope structure in the microfluidic cavity and the direction of liquid flow may be 0-180 degrees.
  • the angle between the micro-slope structure and the direction of liquid flow inside the microfluidic cavity is 30 to 150 degrees.
  • the angle between the micro-slope structure and the direction of liquid flow inside the microfluidic chamber is 45 to 135 degrees. In this way, it is advantageous to reduce the shear stress.
  • a further preferred embodiment is a microfluidic device comprising a liquid inlet, a liquid inlet, a liquid outlet, a liquid outlet, and a plurality of microfluidic chambers, wherein the microfluidic chamber is transparently disposed, each The microfluidic cavity is arranged in parallel; the microfluidic cavity has an inlet channel and an outlet channel, wherein the inlet port communicates with the inlet channel of each microfluidic cavity through the inlet channel, and the outlet channel of each microfluidic cavity
  • the liquid outlets are respectively connected through the liquid outlet passages; each of the microfluidic chambers has a structure of a small front and a large rear, and the liquid inlet passage has a structure of a front large and a small, and the liquid outlet passage has a structure of a small front and a large front; a plurality of microstructure elements are disposed in the microfluidic cavity, and at least a portion of the microstructure elements have a micro-slope structure; And, the microstructure elements are
  • the slope of the slope is a curved surface, not a plane, and the inclination gradually increases from 0 to 90 degrees, and then decreases to 0 degrees.
  • the angle between the slope unit and the liquid flow inside the microfluidic chamber is between 0 and 180 degrees.
  • a preferred embodiment for a micro-slope structure is a micro-slope structure for use in a microfluidic device that is randomly disposed within a microfluidic cavity in a microfluidic device, the micro-slope structure being provided with at least one slope, The slope of the slope is 10 to 80 degrees, and the angle between the at least one slope of the micro-slope structure and the liquid flow direction inside the microfluidic chamber is 30 to 150 degrees, and the corner of the micro-slope structure is smoothly arranged.
  • the surface of the micro-slope structure is provided with a carbohydrate matrix cover layer.
  • shear stress experienced by cells in microfluidic devices is an important limiting factor in the design of rare cell enriched microfluidic devices.
  • Shear stress is directly related to flow rate. When the shear stress exceeds 10-11 per square centimeter of dyne (dynes/cm 2 or dyn/cm 2 ), the cells begin to rupture. The diagnostic value of ruptured cells will be greatly reduced.
  • a direct consequence of the shear stress limitation is that the blood sample must flow through the microfluidic device at a very low flow rate. The enrichment process of the target cells is forced to prolong.
  • the micro-slope structure design largely overcomes this limitation.
  • the following four functional units are compared: cylindrical, round table (cone with rounded top), Gaussian clock and micro-slope structure.
  • the maximum shear stress test is carried out with a flow rate of 40 ⁇ L/min. The results are shown in Table 1 below.
  • the shear stress caused by the three functional units is much higher than that of the micro-slope structural functional unit.
  • Another limitation for the design of cell-enriched microfluidic devices is the presence of a solid-liquid interfacial layer, which is the surface interface layer of the blood-microfluidic device.
  • the blood flow rate in this interface layer is extremely low, close to zero.
  • the thickness of the interface layer is close to or greater than the target cell, the target cell is difficult to contact the surface of the microfluidic device.
  • Target cell capture efficiency is greatly reduced.
  • the thickness of this interface layer can be greatly reduced.
  • 7A to 7D compare the thickness of the interface layer of the four functional unit design shapes; wherein the conclusion of the simulation experiment is also applicable to the column shape; when the fluid flows from top to bottom, the four functional units have the same cross-cut.
  • FIGS. 7A to 7D show the interface layer, and the degree of magnification is different, so that the shapes of FIGS. 7A to 7D are different, wherein FIG. 7A is a circle having a radius of 20 ⁇ m, FIG. 7B It is an ellipse of 25 x 16 microns, Figure 7C is an ellipse of 40 x 10 microns, and Figure 7D is a specially selected shape of the waterdrop shape, thus optimizing the design of the 360 degree micro-slope.
  • the highest round or cylindrical design has the thickest interface layer, for example 11.2 microns.
  • This thickness corresponds to the average diameter of blood ring tumor cells.
  • Microfluidic devices using this design can only have very low target cell capture efficiencies.
  • the elliptical design can be progressively optimized to reduce the interfacial layer thickness as shown in Figures 7B and 7C.
  • the optimized shape design has the lowest interface layer thickness as shown in Fig. 7D, which is an elliptical variant.
  • the thickness of the interface layer is shown in Table 2 below. It can be seen from Table 2 that the selected shape has the best cell-micro Fluid device surface contact efficiency.
  • the microfluidic device of any of the above embodiments is useful for detecting low abundance cells, low abundance proteins, and low abundance nucleic acid biomarkers.
  • a carbohydrate on a surface in contact with a fluid Matrix overlays, buffers, and test parameters can be implemented with reference to existing assays and adjusted for actual testing.
  • microfluidic device of any of the above embodiments is used in cell release, DNA, mDNA (mitochondrial DNA), RNA, and second generation sequencing analysis.
  • a small portion of the inner surface of a fluid chamber or fluid channel holds an antigen or antibody and enriches the low abundance protein while enriching the cells. Since the amount of 7-20 mL blood sample usually used for CTC enrichment is more than 100 times that of the general protein assay, it is possible to enrich 100 times or more of low-abundance protein. CTC and low abundance proteins are simultaneously enriched. The combined information of the two is also more clinically valuable.
  • the release and collection of captured CTCs is achieved by a combination of mechanical, chemical and biological forces.
  • the design of microfluidic devices not only uses CTCs containing trap size and physical adsorption to capture CTC, but also has some chemical and biological mechanisms when CTC is released after capture.
  • pH and chemical adjustment can significantly weaken the binding between the capture molecules on the surface of the chip and the target molecules on the cell surface.
  • a carbohydrate matrix coating on the plastic surface can dissolve in the enzyme solution, releasing the captured CTC. Crosslinking of the matrix with the capture molecule can be enzymatically cleaved or chemically cleaved to release the CTC.
  • the release and collection of captured CTCs can be achieved by reverse flow direction and rinsing with a release buffer that combines optimized chemical and biological factors.
  • the embodiment of the present invention further includes the microfluidic device having the micro-slope structure formed by combining the technical features of the above embodiments, and detecting low-abundance cells, low-abundance proteins, and low-abundance nucleic acids. Biomarker applications, as well as applications in cell release, DNA, mDNA, RNA, and second-generation sequencing.
  • the microfluidic device can be used for cancer research, clinical diagnosis of various cancers, and personal diagnosis of tumor targeted therapy.
  • the shear stress is small, so that the cells in the microfluidic device can withstand higher flow rates, which can greatly shorten the sample processing time and improve the separation efficiency; more importantly, the realization is achieved. Rapid sample processing reduces biomarker damage and protects the originality and integrity of biomarkers such as cells, proteins and nucleic acids.

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Abstract

一种模拟细胞体内环境的微流体器件及其应用，包括进液口、出液口、以及至少一微流体腔；微流体腔具有入口通道与出口通道，入口通道连通进液口，出口通道连通出液口；微流体腔内设置众多微结构元素，其中具有微斜坡结构。该微流体器件能够模拟细胞体内环境，高效、柔和，利于细胞保护、识别和释放，增强并促进细胞和抗体的亲和作用，增加细胞和抗体的接触时间和面积，可用于癌症研究，各种癌症的临床诊断的研究，以及个人诊断肿瘤靶向治疗的研究。因其切变应力小，使细胞能够承受较高的流速，可以缩短样品处理时间，提高分离效率；从而实现了快速样品处理，减少生物标记的损伤，保护了生物标记的原始性和完整性。

Description

一种模拟细胞体内环境的微流体器件及其应用 【技术领域】
本发明涉及微流体技术，尤其涉及的是，一种模拟细胞体内环境的微流体器件及其在检测低丰度细胞、低丰度蛋白以及低丰度核酸生物标记的应用，以及在细胞释放、DNA、mDNA、RNA、二代测序分析的应用。
【背景技术】
微流体技术是指在微观尺寸下控制、操作和检测复杂流体的技术，在生物、化学等科学实验中，经常需要对流体进行操作，如样品DNA的制备、液相色谱、PCR反应、电泳检测等操作都是在液相环境中进行。如果要将样品制备、生化反应、结果检测等步骤集成到生物芯片上，则实验所用流体的量就从毫升、微升级降至纳升或皮升级，这时功能强大的微流体装置就非常重要，从生物医学的角度看，微流体器件可大量节省试剂的用量，提高生产率、改善分析的有效性。
但是，现有的微流体器件，当切变应力较大时，细胞开始破裂，从而导致信息处理价值将大大降低；这样，切变应力局限的直接后果是对已经脱离人体的体液，例如血样，必须以很低的流速流经微流体器件，从而导致目标细胞的富集过程被迫延长，效率低；并且在此过程中容易受到洗液中的降解酶作用，以及生物、化学、物理作用，造成生物标记的损伤。
【发明内容】
为了至少部分解决以上问题，本发明提出了一种新的模拟细胞体内环境的微流体器件及其应用。
例如，一种模拟细胞体内环境的微流体器件，其包括进液口、出液口、以及至少一微流体腔；所述微流体腔具有入口通道与出口通道，其中，所述入口通道连通所述进液口，所述出口通道连通所述出液口；并且，所述微流体腔 内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构。
例如，所述微结构元素在微流体腔内随机布局。
例如，各所述微结构元素具有相同或相异的微斜坡结构。
例如，所述微流体腔中，所述微斜坡结构的最大高度比所述微流体腔的高度至少低10微米。
例如，仅设置一所述微流体腔。
例如，所述微斜坡结构具有第一斜面。
例如，所述第一斜面朝向所述微流体腔内部的液体流动方向。
例如，所述微斜坡结构具有第二斜面，其背向所述微流体腔内部的液体流动方向。
例如，所述第二斜面的倾斜度小于所述第一斜面的倾斜度。
例如，所述第二斜面的倾斜度大于或等于所述第一斜面的倾斜度。
例如，至少部分所述微斜坡结构的横切面具有弧形的封闭形状。
例如，所述微斜坡结构上还设置若干凸柱。
例如，所述微斜坡结构与所述微流体腔内部的液体流动方向的夹角为30至150度。
例如，所述微斜坡结构的横切面具有长边与短边，其中，所述长边与所述微流体腔内部的液体流动方向相垂直。
例如，所述微斜坡结构的角部平滑设置。
例如，所述微流体腔内壁设置碳水化合物基质覆盖层。
例如，所述微流体腔上方透明设置。
例如，所述微流体腔内壁的顶部与底部均设置所述微斜坡结构。
例如，各所述微流体腔等宽度设计。
例如，各所述微流体腔具有前小后大的结构。
例如，所述微流体器件包括若干微流体腔。
例如，各所述微流体腔串联设置。
例如，各所述微流体腔并联设置。
例如，所述微流体腔中，所述入口通道与所述出口通道之间包括若干直线形通道、折线形通道、弧线形通道、螺旋形通道或其组合。
又如，所述微流体器件在检测低丰度细胞、低丰度蛋白、低丰度核酸生物标记的应用。
又如，所述微流体器件在细胞释放、DNA、mDNA、RNA、二代测序分析的应用。
本发明的有益效果是：通过采用微斜坡结构的设计，切变应力小，从而使得微流体器件中的细胞能够承受较高的流速，这样可以大大缩短样品处理时间，提高分离效率；更重要的是保护了细胞，蛋白，和核酸等生物标记的原始性和完整性。
【附图说明】
图1A为本发明微流体腔的一个实施例的示意图；
图1B至图1E分别为本发明微流体腔的各个实施例的示意图；
图2A为本发明微流体器件的一个实施例的示意图；
图2B与图2C分别为本发明微流体器件的又一实施例的示意图；
图2D为本发明微流体腔的又一实施例的示意图；
图3、图4、图5A分别为本发明微流体腔的又一实施例的示意图；
图5B为本发明的一个360度微斜坡结构的实施例的示意图；
图6A至图6C分别为本发明微斜坡结构的又一实施例的示意图；
图7A至图7D分别为微斜坡结构的横切面的示意图；
图8为本发明微流体器件的螺旋形流向路线的一个实施例的示意图；
图9A至图9D分别为捕获抗体或其片段的一个实施例的示意图。
【具体实施方式】
为了便于理解本发明，下面结合附图和具体实施例，对本发明进行更详细的说明。需要说明的是，当元件被表述“固定于”另一个元件，它可以直接在另一个元件上、或者其间可以存在一个或多个居中的元件。当一个元件被表述“连接”另一个元件，它可以是直接连接到另一个元件、或者其间可以存在一个或多个居中的元件。本说明书所使用的术语“垂直的”、“水平的”、“左”、“右”以及类似的表述只是为了说明的目的。
除非另有定义，本说明书所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本说明书中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是用于限制本发明。本说明书所使用的术语“和/或”包括一个或多个相关的所列项目的任意的和所有的组合。
例如，一种模拟细胞体内环境的微流体器件，其高效、柔和(gentle)，利于细胞的保护、识别和释放，该微流体器件包括进液口、出液口、以及至少一微流体腔；所述微流体腔具有入口通道与出口通道，其中，所述入口通道连通所述进液口，所述出口通道连通所述出液口；并且，所述微流体腔内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构。例如，所述微流体器件用于从全血和其它体液中，分离和鉴定循环肿瘤细胞和其它非细胞的生物标记物。例如，所述微流体腔内壁设置碳水化合物基质覆盖层。这样，有利于捕获目标对象。例如，所述微流体腔内所有表面，包括微结构元素表面、微斜坡表面等，均设置碳水化合物基质覆盖层。该微流体器件能够模拟细胞体内环境，高效、柔和，利于细胞保护、识别和释放，增强并促进细胞和抗体的亲和作用，增加细胞和抗体的接触时间和面积，可用于癌症研究，各种癌症的临床诊断的研究，以及个人诊断肿瘤靶向治疗的研究。因其切变应力小，使细胞能够承受较高的流速，可以缩短样品处理时间，提高分离效率；从而实现了快速样品处理，减少生物标记的损伤，保护了生物标记的原始性和完整性。
其中，所述微流体器件用于从血液和体液中富集提取稀有生物标记。血液 包括红血球、白血球、血小板等；体液包括血液，尿液，唾液等。稀有生物标记包括稀有细胞如循环肿瘤细胞，以及蛋白，核糖核酸，和脱氧核糖核酸等。该微流体器件包括一个或多个微流体腔，或者，该微流体器件由一个或多个微流体腔组成。每个微流体腔内布有大量微斜坡结构。例如，这些微斜坡结构的外形经由计算机模拟优化而成，在促进细胞在微流体器件表面翻滚的同时，大大降低切变应力。微斜坡结构在捕获目标的同时又能保持细胞生物功能和物理结构健全。该微流体器件表面经由碳水化合物基质化学覆盖，将非目标的特征性吸附降低到最小，以避免从非目标的细胞，蛋白，核酸等的干扰。例如，碳水化合物的表面固定有捕捉配体，捕捉配体具有针对目标细胞表面抗原的特效性抗体；目标细胞的捕获通过该特效性抗原-抗体反应来实现。微斜坡结构外形同时还具有以下两个重要功能：一是减低光学检测中的光波干涉，二是保持细胞完整，帮助释放目标细胞和目标细胞的再富集。
例如，所述微流体腔如图1A所示，其具有入口通道101与出口通道102,微流体器件中的流体，或称为液体，从入口通道101进入所述微流体腔，经过若干个类似于滑雪坡的微斜坡结构后，从出口通道102流出。
例如，该微流体器件可由玻璃、塑料、金属、硅、氧化硅等材料制成。例如，微流体器件为塑料装置，其微流体腔的表面采用氧气等离子体或紫外线处理，以产生活性结合位，即具有活化的表面。通过处理或改变，将塑料装置表面从疏水性变成亲水性，有利于在塑料表面上的抗体或其他捕获覆盖层。其中，微流体腔的表面，例如活化的表面，设置基于碳水化合物的基质，尽可能避免非特异性血细胞、蛋白与核苷酸的影响。细胞结合配体，例如对应于细胞表面抗原的抗体901，通过一个亲水的间隔区902，例如两端都有功能基团的PEG，固定在碳水化合物基质903上，碳水化合物基质903位于塑料装置904表面。单克隆抗体可通过不同的形状被固定。如图9A所示，全部抗体都被固定在表面上；如图9B至9D所示，抗体的片段905、免疫球蛋白G906等能够被固定，更好的定向结合位或者对于血细胞的更低的非特异性结合。为便于储存备用，抗 体覆盖层采用稳定剂进行保护。
图1显示的模型微流体器件有一个微流体腔。微流体腔前后有入口和出口，例如，用于血样的进或出。血样从出口流出后最终流向废物收集。微流体腔内布有四个相继排列的微斜坡结构。例如，一个真实的微流体器件将含有数百至数万个微斜坡结构。例如，微斜坡结构的最高处高度比微流体腔高度低至少10-20微米。例如，整个微流体腔内的各微斜坡结构如图1B所示或如图1C所示具有均一的外形；又如，整个微流体腔内的各微斜坡结构如图1D所示或如图1E所示具有不断变化的外形。例如，该微流体器件可以与一个平板顶层组装在一起而形成如图1B所示或如图1D所示的结构。例如，该平板顶层必须光学透明，其材料可以是专用透明胶带，也可以是塑料或者玻璃片。该微流体器件也可以与一个相匹配的微流体器件组装而形成图1C所示或如图1E所示的结构。该实施例的微流体器件的一个创新点是使其中流动的血液和其中的目标细胞从若干微斜坡结构流上流下，并在这个流动过程中被微流体器件捕获。
例如，如图1B所示，所述微流体腔内壁的顶部103为平板结构，底部104设置所述微斜坡结构。又如，如图1D所示，所述微流体腔内壁的顶部103为平板结构，底部104设置所述微斜坡结构。例如，如图1C所示，所述微流体腔内壁的顶部103与底部104均设置所述微斜坡结构。又如，如图1E所示，所述微流体腔内壁的顶部103与底部104均设置所述微斜坡结构。例如，所述微流体腔上方透明设置；例如，如图1B至图1E任一所示，所述微流体腔内壁的顶部103透明设置。和/或，所述微流体腔下方透明设置，例如，如图1B至图1E任一所示，所述微流体腔内壁的底部104透明设置。这样，有利于采集光学信号。
例如，所述微流体器件包括若干微流体腔。例如，一种微流体器件如图2A所示，其包括进液口201、出液口202，以及四个微流体腔203；进液口201通过进液通道204分别连通各微流体腔的入口通道101；例如，如图2A所示，各所述微流体腔等宽度设计。例如，如图2B所示，各所述微流体腔具有前小后大的结构。又如，如图2B所示，微流体器件包括六个微流体腔203，各微流体腔 的出口通道102分别通过出液通道205连通出液口202，其中，各微流体腔具有前小后大的结构，即进液口处较小，出液口处较大，这样，有助于流体进入微流体腔后分散开。又如，如图2C所示，微流体器件包括七个微流体腔203，进液通道204具有前大后小的结构，出液通道205具有前小后大的结构，这样，有助于流体进入进液通道后，逐渐被各微流体腔分流；流体从出液通道流出后，逐渐合流到出液口。例如，如图2A至图2C所示，各所述微流体腔并联设置。又如，各所述微流体腔串联设置。例如，所述微流体腔内含有多个相同的微斜坡结构如图2D所示。
例如，所述微结构元素在微流体腔内随机布局，例如，各所述微结构元素具有相同结构，其位置和方向在微流体腔内随机设置；又如，如图5A所示，至少两个微结构元素具有相异结构，各所述微结构元素的位置和方向在微流体腔内随机设置，以此类推。又如，所述微结构元素在微流体腔内规则排列；例如，所述微结构元素在微流体腔内按行排列或者阵列排列；又如，如图2D所示，各所述微结构元素具有相同结构，各所述微结构元素的位置在微流体腔内规则排列且方向保持一致；又如，如图3或图4所示，至少两个微结构元素具有相异结构，各所述微结构元素的位置在微流体腔内总体规则排列且方向总体保持一致。又如，如图5A所示，各所述微结构元素具有相同结构或相异结构，所述微结构元素在微流体腔内随机设置。
例如，如图2D、图3、图4或图5A所示，各所述微结构元素具有相同或相异的微斜坡结构。例如，至少两个微结构元素具有相异的微斜坡结构；又如，若干微结构元素具有相异的两种或多种微斜坡结构。
例如，所述微流体腔中，所述微斜坡结构的最大高度比所述微流体腔的高度至少低10微米，也就是说，最高的微斜坡结构，其一端固定于微流体腔中的一面，另一端距离微流体腔中的另一面的距离不小于10微米。例如，所述微斜坡结构的最大高度比所述微流体腔的高度至少低12微米；又如，所述微斜坡结构的最大高度比所述微流体腔的高度至少低20微米。又如，根据目标捕获对象 设置微斜坡结构具体的高度。
例如，仅设置一所述微流体腔，其入口通道连通所述进液口，出口通道连通所述出液口；并且，所述微流体腔内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构。
由于循环肿瘤细胞的多样性和多变性，微流体器件设计有必要包含多样化的所述微结构元素，其具有相同或相异的功能单元，例如，其具有相同或相异的微斜坡结构。当微斜坡结构在与流向相切方向排列时，多样化可以通过带状化来实现。例如，如图3所示，微流体器件设置四组微斜坡结构带。各微斜坡结构带的组内均一，重复相同的曲度和尺寸。微斜坡结构带的组间非均一，拥有不同的曲度和尺度。这样设计的目的是为了更好的富集变化多样的循环肿瘤细胞或其他稀有细胞。需要说明的是，图3仅仅作为一个示意图，真正的微流体器件含有大量组数的微斜坡结构带以最大化富集效率。又如，微流体器件随机布局各微斜坡结构。如图4所示，不同曲度和尺寸的微斜坡结构经计算机随机排布在微流体腔内。在这两种设计中，流体，例如血流，流过功能单元的侧面的同时，血流还有上下方向的流动。目标细胞可以被所有的表面区域抓获。这是以往微流体器件中没有的。
例如，微斜坡结构可以沿着血流方向排列，可以与血流方向正交，也可以以任何角度与血流方向相交。又如，还设置360度微斜坡结构。例如，如图5A所示，沿血流方向和正交血流方向的微斜坡结构与360度微斜坡结构并用的设计；其中，360度微斜坡结构如图5B所示。360度微斜坡结构在促进细胞翻滚的同时还具有导流和分流的作用。其中，如图5A所示，360度微斜坡结构不仅可以排列在微流体腔的底部，也可以不同角度排列在其他微斜坡结构的坡面上。
例如，所述微斜坡结构具有第一斜面。例如，所述第一斜面朝向所述微流体腔内部的液体流动方向。例如，所述微斜坡结构具有第二斜面，其背向所述微流体腔内部的液体流动方向。例如，所述第二斜面的倾斜度小于所述第一斜面的倾斜度；或者，所述第二斜面的倾斜度大于或等于所述第一斜面的倾斜度。 例如，第一斜面或第二斜面为倾斜的平面，其具有一定的倾斜角度，例如10度至80度，例如，第一斜面或第二斜面的倾斜角度为20度至65度，又如，第一斜面或第二斜面的倾斜角度为10度、15度、20度、25度、30度、40度、45度、52度、60度、65度、72度或80度。又如，第一斜面或第二斜面具有弧形截面、抛物线形截面或者弧形与抛物线形相组合所形成的截面，例如，具有弧形截面的第一斜面或第二斜面，其为一圆柱体的部分圆柱面，以此类推。又如，第一斜面或第二斜面内凹或者外凸设置。例如，如图3所示，第一斜面301朝向所述微流体腔内部的液体流动方向，即朝向入口通道101，其内凹设置；第二斜面302背向所述微流体腔内部的液体流动方向，即朝向出口通道102，其内凹设置。又如，如图4所示，第一斜面401内凹设置，第二斜面402内凹设置。又如，如图5A所示，第一斜面501外凸设置，第二斜面502内凹设置。又如，如图6A所示，第一斜面与第二斜面均内凹设置。这样，有利于降低切变应力。
例如，如图5A所示，所述微斜坡结构上还设置若干凸柱503，这样，有助于捕获对象，例如目标细胞。又如，如图5A所示，所述微流体腔内部还设置若干360度微斜坡结构504。又如，如图5A所示，所述微流体腔内部还设置至少一个微斜坡结构505，其朝向所述微流体腔内部的液体流向的一面为非斜面。
例如，如图3或图6B所示，所述微斜坡结构的横切面具有长边与短边，其中，如图3所示，所述长边与所述微流体腔内部的液体流动方向相垂直。
为了保证微斜坡结构捕获到的目标细胞不会遭到破坏例如，如图6B所示，所述微斜坡结构的角部平滑设置。又如，如图6C所示，所述微斜坡结构的角部平滑设置，这是360度微斜坡结构的又一个例子。这样，有利于降低切变应力，从而提升流速，以增加处理效率。又如，将如图6A所示的微斜坡结构的角部平滑设置为如图6B所示的微斜坡结构的角部，或者平滑设置为如图6C所示的微斜坡结构的角部。例如，至少部分所述微斜坡结构的横切面具有弧形的封闭形状。例如，如图6B所示，该实施例的微斜坡结构的横切面具有弧形的封闭形状。又如，如图6C所示，该实施例的微斜坡结构的横切面具有弧形的封闭形状。这 样，有利于降低切变应力。其中，360度微斜坡结构的横切面为圆形。
例如，所述微流体腔中，所述入口通道与所述出口通道之间包括若干直线形通道、折线形通道、弧线形通道、螺旋形通道或其组合；例如，所述入口通道与所述出口通道之间包括折线形通道或者弧线形通道；又如，所述入口通道与所述出口通道之间包括组合通道，其具有至少一直线形通道、至少一折线形通道、至少一弧线形通道、和/或至少一螺旋形通道。例如，如图8所示，所述微流体腔801中设置若干螺旋形通道802，流体在各螺旋形通道802中流动，这样，特别适用于设计利用密度和抗原-抗体亲和力来分离血细胞、捕获循环肿瘤细胞及其他生物标记；又如，平滑的微斜坡结构表面促进细胞-抗体接触，减低切变应力；例如，螺旋形通道中的流体路线设计采用阿基米德螺线、等角螺线、对数螺线或其他螺线实现。
一个较佳的实施例是，一种微流体器件，其包括进液口、出液口、以及至少一微流体腔；所述微流体腔具有入口通道与出口通道，其中，所述入口通道连通所述进液口，所述出口通道连通所述出液口；并且，所述微流体腔内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构；并且，所述微结构元素在微流体腔内随机布局，微斜坡结构在微流体腔内的排布方向与液体流动方向的夹角可以是0-180度。例如，所述微斜坡结构与所述微流体腔内部的液体流动方向的夹角为30至150度。又如，所述微斜坡结构与所述微流体腔内部的液体流动方向的夹角为45至135度。这样，有利于降低切变应力。
又一个较佳的实施例是，一种微流体器件，其包括进液口、进液通道、出液口、出液通道以及若干微流体腔，其中，所述微流体腔上方透明设置，各所述微流体腔并联设置；所述微流体腔具有入口通道与出口通道，其中，所述进液口通过所述进液通道分别连通各微流体腔的入口通道，各微流体腔的出口通道分别通过所述出液通道连通所述出液口；各微流体腔具有前小后大的结构，进液通道具有前大后小的结构，出液通道具有前小后大的结构；并且，所述微流体腔内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构； 并且，所述微结构元素在微流体腔内随机布局，至少两个微结构元素具有相异的微斜坡结构，所述微斜坡结构的横切面具有弧形的封闭形状，所述微斜坡结构至少一斜面与所述微流体腔内部的液体流动方向的夹角为30至150度，所述微斜坡结构的角部平滑设置，所述微流体腔内壁设置碳水化合物基质覆盖层，所述微流体腔上方透明设置。例如，斜坡的坡面是弧面，不是平面，倾斜度由0度到90度逐渐增加，再减少到0度。又如，斜坡单元与微流体腔内部的液体流向夹角在0度至180度之间。
对于微斜坡结构的一个较佳实施例是，一种应用于微流体器件中的微斜坡结构，其在微流体器件中的微流体腔内随机设置，所述微斜坡结构设置至少一斜面，所述斜面的倾斜度为10至80度，所述微斜坡结构至少一斜面与所述微流体腔内部的液体流动方向的夹角为30至150度，所述微斜坡结构的角部平滑设置，所述微斜坡结构的表面设置碳水化合物基质覆盖层。
需要说明的是，细胞在微流体器件中所经历的切变应力是稀有细胞富集微流体器件设计中一个重要的限制因素。切变应力与流速直接相关。当切变应力超过10-11每平方厘米达因(dynes/cm2或dyn/cm2)，细胞开始破裂。破裂细胞的医疗诊断价值将大大降低。切变应力局限的直接后果是血样必须以很低的流速流经微流体器件。目标细胞的富集过程被迫延长。微斜坡结构设计在很大程度上克服了这个局限。下面对比四种功能单元：圆柱、圆台(具有圆形顶部的圆锥)、高斯钟和微斜坡结构，采用流速为40μL/min，进行最大切变应力测试，结果如下表1所示，可见，前三种功能单元所导致的切变应力都远远高于微斜坡结构功能单元。
Figure PCTCN2014092950-appb-000001
表1
由表1可见，上述四种微斜坡结构设计中循环肿瘤细胞经历的切变应力水 平和切变应力最高的位置，从圆柱到圆台再到高斯钟，切变应力的变化很小。而微斜坡结构设计中的切变应力只有其他三种设计的1/3或更低。由于切变应力与流速成正比，在同等条件下，微斜坡结构微流体器件可以使用3倍于其他三种设计的流速而不破坏细胞的完整性。
另一个用于细胞富集微流体器件设计的局限是固体-液体界面层的存在，这个固-液界面层也就是血液-微流体器件表面界面层。在这个界面层内血液流速极低，近于零。当界面层厚度接近或者大于目标细胞时，目标细胞很难与微流体器件表面接触。目标细胞捕获效率大大降低。经由设计优化，这个界面层的厚度可以大大减低。图7A至图7D比较了四种功能单元设计外形的界面层厚度；其中，该模拟实验的结论对柱形同样适用；当流体由上而下流动时，这四种功能单元有相同的横切面积，或称为横截面积，但界面层厚度大大不同。模拟中横切面积是相同的，截图时的图7A至图7D为显示界面层，放大程度不同，使得图7A至图7D看上去大小不同，其中，图7A是半径20微米的圆，图7B是25×16微米的椭圆，图7C是40×10微米的椭圆，图7D是面积相当的水珠形特选形状，这样，优化了360度微斜坡的设计。最高的圆形设计或圆柱形设计有最厚的界面层，例如11.2微米。这个厚度相当于血环肿瘤细胞的平均直径。使用这个设计的微流体器件只能有很低的目标细胞捕获效率。例如，椭圆形设计可以被逐步优化以减低界面层厚度如图7B和7C所示。优化后的特选形状设计具有最低的界面层厚度如图7D所示，为椭圆形的变体，界面层厚度如下表2所示，由表2可见，特选形状具有最佳的细胞-微流体器件表面接触效率。
结构设计 360°微斜坡结构的前面 360°微斜坡结构的侧面
A.圆形(半径为20μm) 11.2 3.2
B.椭圆形(16x25μm) 8.7 4.0
C.椭圆形(10x40μm) 4.8 4.7
D.特选形状 3.3 3.0
表2
又如，上述任一实施例所述微流体器件在检测低丰度细胞、低丰度蛋白、低丰度核酸生物标记的应用。例如，其中与流体向接触的表面上的碳水化合物 基质覆盖层、缓冲液以及试验参数，可以参考现有的检测方法实现，以及根据实际试验而调整。
又如，上述任一实施例所述微流体器件在细胞释放、DNA、mDNA(mitochondrial DNA，线粒体DNA)、RNA、二代测序分析的应用。
例如，流体腔或者流体通道内表面小部分区域固定抗原或抗体，在富集细胞的同时富集低丰度蛋白。由于CTC富集通常使用的7-20mL血样量是一般蛋白化验用量的100倍以上，这样，可以富集100倍以上的低丰度蛋白。CTC和低丰度蛋白同时富集。两者综合的信息也更加有临床价值。
例如，捕获所得到的CTC(循环肿瘤细胞)的释放与采集，是通过机械力、化学力与生物力的结合来实现的。在实现机制上，微流体器件的设计不仅采用包含陷阱大小和物理吸附的物理力来捕获CTC，当CTC在捕获后被释放时，还有一些化学与生物机制共同作用。例如，释放缓冲液时，进行pH和化学调节，能够显著削弱在芯片表面的捕获分子与细胞表面上的目标分子之间的结合。又如，塑料表面上的碳水化合物基质覆盖层能够在酶液中溶解，释放出捕获的CTC。基质与捕获分子的交联可以被酶促裂解或化学裂解，从而释放出CTC。这样，通过反向流动方向，并用结合优化的化学和生物因素的释放缓冲液进行冲洗，即可实现捕获所得到的CTC的释放与采集。
进一步地，本发明的实施例还包括，上述各实施例的各技术特征，相互组合形成的具有微斜坡结构的微流体器件及其在检测低丰度细胞、低丰度蛋白以及低丰度核酸生物标记的应用，以及在细胞释放、DNA、mDNA、RNA、二代测序分析的应用。该微流体器件可用于癌症研究，各种癌症的临床诊断的研究，以及个人诊断肿瘤靶向治疗的研究。采用本发明各实施例所述微流体器件，切变应力小，从而使得微流体器件中的细胞能够承受较高的流速，这样可以大大缩短样品处理时间，提高分离效率；更重要的是实现了快速样品处理，减少生物标记的损伤，保护了细胞、蛋白和核酸等生物标记的原始性和完整性。
需要说明的是，本发明的说明书及其附图中给出了本发明的较佳的实施例， 但是，本发明可以通过许多不同的形式来实现，并不限于本说明书所描述的实施例，这些实施例不作为对本发明内容的额外限制，提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。并且，上述各技术特征继续相互组合，形成未在上面列举的各种实施例，均视为本发明说明书记载的范围；进一步地，对本领域普通技术人员来说，可以根据上述说明加以改进或变换，而所有这些改进和变换都应属于本发明所附权利要求的保护范围。

Claims (10)

  1. 一种模拟细胞体内环境的微流体器件，其特征在于，包括进液口、出液口、以及至少一微流体腔；所述微流体腔具有入口通道与出口通道，其中，所述入口通道连通所述进液口，所述出口通道连通所述出液口；并且，所述微流体腔内设置众多微结构元素，至少部分所述微结构元素中，具有微斜坡结构。
  2. 根据权利要求1所述微流体器件，其特征在于，各所述微结构元素具有相同或相异的微斜坡结构。
  3. 根据权利要求1所述微流体器件，其特征在于，所述微斜坡结构具有第一斜面，其朝向所述微流体腔内部的液体流动方向。
  4. 根据权利要求3所述微流体器件，其特征在于，所述微斜坡结构具有第二斜面，其背向所述微流体腔内部的液体流动方向。
  5. 根据权利要求1所述微流体器件，其特征在于，至少部分所述微斜坡结构的横切面具有弧形的封闭形状。
  6. 根据权利要求5所述微流体器件，其特征在于，所述微斜坡结构上还设置若干凸柱。
  7. 根据权利要求1所述微流体器件，其特征在于，所述微斜坡结构与所述微流体腔内部的液体流动方向的夹角为30至150度。
  8. 根据权利要求1所述微流体器件，其特征在于，所述微流体腔内壁的顶部与底部均设置所述微斜坡结构。
  9. 根据权利要求1所述微流体器件，其特征在于，所述微流体腔中，所述入口通道与所述出口通道之间包括若干直线形通道、折线形通道、弧线形通道、螺旋形通道或其组合。
  10. 权利要求1至9任一所述微流体器件在检测低丰度细胞、低丰度蛋白、低丰度核酸生物标记的应用，以及在细胞释放、DNA、mDNA、RNA、二代测序分析的应用。
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