Handheld microfluidic sample processing device and method of use thereof

Abstract

The present invention provides a handheld microfluidic sample processing device and a method for using the same. The device comprises a fixture upper plate, an air path connection plate, a joint sealing plate, a chip sealing plate, a microfluidic chip, and a fixture lower plate, which are stacked in sequence from top to bottom. The upper plate is provided with a hollowed-out area for connecting to a second positioning hole of the fixture lower plate. The air path connection plate is provided with a plurality of closely spaced air path connection ports, which connect to a first positioning hole of the fixture lower plate. The joint sealing plate is provided with a joint connection port corresponding to the air path connection port of the air path connection plate, which fixes a third positioning hole of the chip sealing plate. The chip sealing plate is provided with a liquid reservoir corresponding to the joint connection port of the joint sealing plate. The microfluidic chip comprises a channel layer and a substrate layer. The lower plate is provided with a chip positioning area for placing the microfluidic chip. The present invention reduces hardware costs and can meet multiple functional operations.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics technology, and in particular relates to a handheld microfluidics sample processing device and a method for using the same. Background Art

[0002] Microfluidics refers to the science and technology involved in systems that use microchannels (tens to hundreds of microns in size) to process or manipulate tiny fluids. It is an emerging interdisciplinary discipline involving chemistry, fluid physics, microelectronics, new materials, biology, and biomedical engineering. It integrates the basic units involved in sample analysis in biology, chemistry, medicine, and other fields, including preparation, reaction, separation, and detection, onto a micron-scale chip, automating the entire analysis process. Because of their miniaturization and integration, microfluidic devices are often referred to as microfluidic chips, also known as labs on a chip.

[0003] Microfluidics technology offers the advantages of miniaturization, high throughput, low sample volume, and minimal reagent consumption, giving it broad application prospects across diverse fields. In the field of in vitro diagnostics, it demonstrates its unique strengths in diverse IVD sub-sectors, including biochemical analysis, immunodiagnosis, and molecular diagnostics, and holds significant potential as a replacement for traditional IVD testing methods. Its advantages in cell separation can significantly improve the detection rate and purity of circulating tumor cells, providing crucial support for the treatment of related cancers. In the field of drug screening, stem cell microarrays, for example, can overcome the limitations of existing in vitro stem cell research by precisely controlling various factors in the stem cell microenvironment in real time, closely mimicking the complex environment in which stem cells grow and differentiate. For example, a single-cell library preparation system (ZL202110977986.8) developed by Beijing Xunyin Biotechnology Co., Ltd. is used for cell labeling and library construction in single-cell sequencing, while a microfluidic chip and its droplet generation device (ZL201710364655.0) developed by Guangdong Yongnuo Medical Technology Co., Ltd. is used for droplet generation in digital PCR. The application of its microfluidic technology requires matching microfluidic chips and microfluidic control systems. The currently developed microfluidic operating systems have single functions and can only be used with a single chip for fixed projects. Different equipment is needed to carry out different projects. The hardware cost is high, the operation is complicated, and the convenience is poor. Summary of the Invention

[0004] To solve the above problems, the present invention provides a handheld microfluidic sample processing device and its use method to achieve the purpose of matching with multiple microfluidic chips, completing multiple projects with one device, reducing hardware costs, and meeting multiple functional operations.

[0005] In order to achieve the above purpose, the technical solution is as follows:

[0006] A handheld microfluidic sample processing device comprises a fixture upper plate, an air path connection plate, a joint sealing plate, a chip sealing plate, a microfluidic chip, and a fixture lower plate, which are stacked in sequence from top to bottom;

[0007] The upper plate of the fixture is provided with a hollow area for passing the gas path connection port of the gas path connection plate, and is used to connect to the second positioning hole of the lower plate of the fixture;

[0008] The gas connection plate is provided with a plurality of closely spaced gas connection ports for connecting to an external gas source, and is used to connect to the first positioning hole of the lower plate of the fixture;

[0009] The joint sealing plate is provided with a joint connection port corresponding to the gas path connection port of the gas path connection plate, and is used to fix the third positioning hole of the chip sealing plate;

[0010] The chip sealing plate is provided with a liquid storage tank corresponding to the joint connection port of the joint sealing plate, and is used for clamping the hook of the third positioning hole of the joint sealing plate;

[0011] The microfluidic chip includes a channel layer and a substrate layer, wherein the channel layer is tightly sealed to the chip sealing plate above and below, and the substrate layer is tightly attached to the bottom of the channel layer;

[0012] The lower plate of the fixture is provided with a chip positioning area for placing the microfluidic chip, a first positioning pin for connecting the air path connecting plate, and a second positioning pin for connecting the upper plate of the fixture. The lower plate of the fixture and the upper plate of the fixture are connected by a stainless steel buckle.

[0013] Furthermore, spring beads are provided around the hollow area of ​​the upper plate of the fixture for forming point contact with the gas path connection plate. The second positioning holes are provided at the four corners of the upper plate of the fixture. A buckle is provided on each side of the upper plate of the fixture. The thickness of the upper plate of the fixture is less than or equal to 5 mm.

[0014] Furthermore, the gas path connecting plate also includes a connecting plate body, the connecting plate body is made of stainless steel or aluminum plate, and the gas path connecting ports are closely arranged in the middle position of the connecting plate body; the first positioning holes are arranged at the four corners of the connecting plate body.

[0015] Furthermore, the air circuit connection port is a pneumatic quick-connect connector, and the pneumatic quick-connect connector is threadedly connected to the air inlet on the connecting plate body.

[0016] Furthermore, a third positioning hole is provided on the joint sealing plate, and the third positioning hole is arranged at the four corners of the joint sealing plate; the joint sealing plate has one or more groups of joint connection ports, which are arranged corresponding to the liquid storage tank of the chip sealing plate; the material of the joint sealing plate is one of silicone, fluororubber or rubber.

[0017] Furthermore, the liquid storage tank of the chip sealing plate is a cylinder, and the liquid storage tank is arranged in one or more groups; the storage volume of the liquid storage tank is various, and can store 10μL-2000μL of reaction liquid; the liquid outlet at the bottom of the liquid storage tank is provided with an arc, and the liquid outlet of the liquid storage tank is a conical card hole, and the top of the liquid storage tank is tightly sealed with the joint sealing plate up and down.

[0018] Furthermore, the channel layer of the microfluidic chip is located above the substrate layer and is tightly attached to the substrate layer; one or more reaction units are arranged on the channel layer of the microfluidic chip; the reaction units include channels and openings, the openings of the reaction units correspond to the positions of the liquid storage tanks of the chip sealing plate above and below, and the channels of each reaction unit are connected to the corresponding openings; the size of the openings is 1-6 mm.

[0019] Furthermore, the channel is the core reaction area, and its characteristic size is 10um-500um, which is processed by soft lithography precision method; the microfluidic chip material is one of polydimethylsiloxane or polycarbonate; the bonding method of the channel layer and the substrate layer is reversible bonding or irreversible bonding, and the bonding method is one of plasma bonding, chemical reagent bonding or biological tape bonding.

[0020] Furthermore, the lower plate of the fixture is a rectangular groove structure with an opening on one side, and a chip positioning area is set at the inner bottom of the lower plate of the fixture; the first positioning pins are set at the four corners of the inner bottom of the lower plate of the fixture, and the second positioning pins are set at the four corners of the top of the lower plate of the fixture; buckle grooves are provided on both sides of the lower plate of the fixture corresponding to the buckle positions of the upper plate of the fixture, and the thickness of the lower plate of the fixture does not exceed 5 mm; an observation port is provided in the middle of the lower plate of the fixture.

[0021] A method for using a handheld microfluidic sample processing device comprises the following steps:

[0022] Make or select a suitable microfluidic chip according to the experimental purpose;

[0023] Assembling the microfluidic chip with the chip sealing plate, adding microparticles, solutions or chemicals into the liquid reservoir of the chip sealing plate, and then assembling the microfluidic sample processing device as a whole;

[0024] Connect the external gas source through the gas connection plate and set the gas source channels and the gas source pressure parameters of each channel according to the experimental purpose;

[0025] Run the microfluidic sample processing device and select an appropriate run time to obtain the target amount of mixture.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The device of the present invention has a small size, simple structure, low manufacturing cost, and simple operation. The modules can be assembled in sequence and are easy to carry. Secondly, the device can be matched with microfluidic chips with different functional structures. The corresponding microfluidic chip can be selected according to the purpose. After assembly, an external gas source with corresponding channel and pressure can be connected to realize the separation, mixing and encapsulation of different particles, solutions and chemicals in the chip, providing a physical basis for subsequent biochemical reactions, and can be used for cell labeling in single-cell sequencing, droplet generation in digital PCR, preparation of gel microbeads, etc. In addition, the device of the present invention can adjust the gas access pressure and reaction liquid dosage according to different projects to optimize experimental conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the overall structure of the device of the present invention;

[0029] Figure 2 It is a schematic diagram of the structural decomposition of the device of the present invention;

[0030] Figure 3 This is a schematic structural diagram of the upper plate of the fixture of the device of the present invention;

[0031] Figure 4 This is a schematic structural diagram of the gas path connecting plate of the device of the present invention;

[0032] Figure 5 This is a schematic structural diagram of the joint sealing plate of the device of the present invention;

[0033] Figure 6 This is a schematic structural diagram of the chip sealing plate of the device of the present invention;

[0034] Figure 7 A side view of a chip sealing plate of the device of the present invention;

[0035] Figure 8 Schematic diagram of the structure of chip A in the device of the present invention;

[0036] Figure 9 Schematic diagram of the structure of chip B in the device of the present invention;

[0037] Figure 10 This is a schematic structural diagram of the lower plate of the fixture of the device of the present invention;

[0038] Figure 11 Generate a microscope image of the PCR droplet of Example 2 of the present invention;

[0039] Figure 12 This is a microscope image of the hydrogel microbeads of Example 3 of the present invention;

[0040] Figure 13 This is a graph showing the quality inspection results of the single-cell sequencing library of the control group in Example 4 of the present invention;

[0041] Figure 14 This is a graph showing the quality inspection results of the single-cell sequencing library of the test group of Example 4 of the present invention;

[0042] Figure 15 This is a graph showing the analysis results of single-cell sequencing data for the control group of Example 4 of the present invention;

[0043] Figure 16 This is a graph showing the analysis results of single-cell sequencing data from the test group of Example 4 of the present invention.

[0044] Figure markings: 1. Upper plate of the fixture; 101. Buckle; 102. Spring bead; 103. Second positioning hole; 2. Gas path connecting plate; 201. Gas path connecting port; 202. First positioning hole; 3. Joint sealing plate; 301. Joint connecting port; 302. Third positioning hole; 4. Chip sealing plate; 401. Liquid storage tank; 402. Hook; 403. Liquid outlet; 5. Microfluidic chip; 501. Channel layer; 502. Opening; 503. Substrate layer; 504. Reaction unit; 505. Channel; 6. Lower plate of the fixture; 601. Chip positioning area; 602. First positioning pin; 603. Second positioning pin; 604. Buckle groove; 605. Observation port. DETAILED DESCRIPTION

[0045] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0046] In the description of the present invention, it should be noted that the terms "center", "upper", "lower", "left", "right", "vertical", "horizontal", "inner", "outer", etc., indicating directions or positional relationships, are based on the directions or positional relationships shown in the accompanying drawings, and are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the devices or components referred to must have a specific direction, be constructed and operated in a specific direction, and therefore should not be understood as limiting the present invention; the terms "first", "second", and "third" are only used for descriptive purposes and should not be understood as indicating or implying relative importance. In addition, unless otherwise clearly specified and limited, the terms "installed", "connected", and "connected" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection, an indirect connection through an intermediate medium, or it can be internal communication between two components. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0047] Example 1

[0048] A handheld microfluidic sample processing device, such as Figure 1-2 As shown, it includes a fixture upper plate 1, an air path connection plate 2, a joint sealing plate 3, a chip sealing plate 4, a microfluidic chip 5, and a fixture lower plate 6 stacked in sequence from top to bottom;

[0049] like Figure 3 As shown, the upper plate 1 of the fixture is provided with a hollow area for passing the gas connection port 201 of the gas connection plate 2 and a second positioning hole 103 for connecting to the lower plate 6 of the fixture;

[0050] like Figure 4 As shown, the gas connection plate 2 is provided with a plurality of closely spaced gas connection ports 201 for connecting to an external gas source, and a first positioning hole 202 for connecting to the lower plate 6 of the fixture;

[0051] like Figure 5 As shown, the joint sealing plate 3 is provided with closely spaced joint connection ports 301 corresponding to the gas path connection ports 201 of the gas path connection plate 2, and the joint connection ports 301 connect the upper gas path connection plate 2 and the lower chip sealing plate 4; the joint sealing plate 3 is also provided with a third positioning hole 302 for fixing the chip sealing plate 4; in particular, the material of the joint sealing plate 3 includes but is not limited to silicone, fluororubber or rubber;

[0052] like Figure 6-7 As shown, the chip sealing plate 4 is provided with a liquid storage tank 401 corresponding to the joint connection port 301 of the joint sealing plate, and a hook 402 for clamping the positioning hole of the joint sealing plate;

[0053] The microfluidic chip 5 includes a channel layer 501 and a substrate layer 503. The channel layer 501 is tightly sealed to the chip sealing plate 4 from top to bottom, and the substrate layer 503 is tightly attached to the bottom of the channel layer 501.

[0054] like Figure 10 As shown, the lower plate 6 of the fixture is provided with a chip positioning area 601 for placing the microfluidic chip 5, a first positioning pin 602 for connecting the air path connecting plate 2, and a second positioning pin 603 for connecting the upper plate 1 of the fixture. The lower plate 6 of the fixture is connected to the upper plate 1 of the fixture by a stainless steel buckle.

[0055] In this embodiment, spring beads are provided around the hollow area of ​​the upper plate 1 of the clamp for forming point contact with the air path connecting plate 2, which are used to form point contact with the lower air path connecting plate 2 to avoid poor contact caused by processing flatness; second positioning holes 103 are provided at the four corners of the upper plate 1 of the clamp; a buckle 101 is provided on each side of the upper plate 1 of the clamp, and the buckle 101 is preferably a stainless steel buckle 101, which is used to tightly buckle with the lower plate 6 of the clamp to form an overall seal of the device; the thickness of the upper plate 1 of the clamp does not exceed 5 mm.

[0056] In this embodiment, the gas connection plate 2 further includes a connection plate body, which is made of stainless steel or aluminum plate and has a thickness of 5-10 mm. The gas connection ports 201 are closely spaced in the middle of the connection plate body. The first positioning holes 202 are located at the four corners of the connection plate body.

[0057] In this embodiment, the air connection port 201 is a pneumatic quick-connect connector, which is threadedly connected to the air inlet on the connecting plate body, and the external air source supplies the required pressure to the device through the pneumatic quick-connect connector.

[0058] In this embodiment, the liquid storage tank 401 of the chip sealing plate 4 is a cylinder, and the liquid storage tank 401 is provided in one or more groups. The storage volume of the liquid storage tank 401 is various and can store reaction liquids ranging from 10 μL to 2000 μL. The liquid outlet 403 at the bottom of the liquid storage tank 401 is provided with an arc to effectively avoid liquid residue. The liquid outlet 403 of the liquid storage tank 401 is a conical card hole, and the top of the liquid storage tank 401 is tightly attached to the joint sealing plate 3 up and down to ensure sealing.

[0059] In this embodiment, the channel layer 501 is located above the substrate layer 503 and is tightly attached to the substrate layer 503; the channel layer 501 is provided with one or more groups of reaction units 504; each reaction unit 504 includes a channel 505 and an opening 502, and the opening 502 of the reaction unit corresponds to the position of the liquid storage tank of the chip sealing plate up and down, and the channel 505 of each reaction unit is connected to the corresponding opening 502, and the opening 502 corresponds to the position of the liquid storage tank 401 of the chip sealing plate 4 up and down, and the size of the opening 502 is 1-6 mm, which is convenient for connecting with the conical structure liquid outlet 403 of the liquid storage tank 401 of the chip sealing plate 4.

[0060] In this embodiment, the channel 505 is the core reaction area, and its characteristic size is 10um-500um, and it is precisely processed by methods including but not limited to soft lithography; the microfluidic chip 5 is a material including but not limited to polydimethylsiloxane or polycarbonate; the channel layer 501 and the substrate layer 503 are bonded in a reversible or irreversible manner, and the bonding method includes but is not limited to plasma bonding, chemical reagent bonding or biological tape bonding.

[0061] In particular, such as Figure 8-9 As shown, the microfluidic chip 5 is a chip A for digital PCR or gel bead preparation, or a chip B for single-cell sequencing cell labeling, wherein chip A consists of 4 groups of reaction units 504, each group contains 3 openings 502, which are an oil phase pool, a reaction liquid pool, and a collection pool in sequence; chip B consists of 4 groups of reaction units 504, each group contains 4 openings 502, which are an oil phase pool, a cell suspension pool, a beads pool, and a collection pool in sequence.

[0062] In this embodiment, the lower plate 6 of the clamp is a rectangular groove structure with an opening on one side, and a chip positioning area 601 is set at the inner bottom of the lower plate 6 of the clamp; the first positioning pins 602 are set on the four corners of the inner bottom of the lower plate 6 of the clamp, and the second positioning pins 603 are set on the four corners of the top of the outer wall of the lower plate 6 of the clamp; buckle grooves 604 are provided on both sides of the lower plate 6 corresponding to the buckle 101 positions of the upper plate 1 of the clamp, the thickness of the lower plate 6 does not exceed 5 mm, and an observation port 605 is provided in the middle of the lower plate 6 to provide an observation window for the core area of ​​the microfluidic chip 5.

[0063] The present invention also discloses an assembly method of a handheld microfluidic sample processing device, which specifically includes the following steps:

[0064] Align the liquid outlet 403 of the liquid reservoir 401 of the chip sealing plate 4 with the opening 502 of the microfluidic chip 5 and seal tightly;

[0065] Align the joint connection port 301 of the joint sealing plate 3 with the liquid storage tank 401 of the chip sealing plate 4, and engage the hook 402 of the chip sealing plate 4 with the positioning hole of the joint sealing plate 3;

[0066] Place the microfluidic chip 5 on the chip positioning area 601 of the lower plate 6 of the fixture;

[0067] With the help of the first positioning pin 602 of the lower plate 6 of the fixture, gently insert the first positioning hole 202 of the gas circuit connecting plate 2 into the first positioning pin 602 and fit it into the joint sealing plate;

[0068] The upper plate 1 of the fixture is fitted onto the second positioning pins 603 of the lower plate 6 of the fixture through the second positioning holes 103 , and the microfluidic sample processing device is sealed by the buckle 101 .

[0069] The present invention also discloses a method for using a handheld microfluidic sample processing device, comprising the following steps:

[0070] Make or select a suitable microfluidic chip 5 according to the experimental purpose;

[0071] Assemble the microfluidic chip 5 and the chip sealing plate 4, add particles, solutions or chemicals into the liquid reservoir 401 of the chip sealing plate 4, and then assemble the microfluidic sample processing device as a whole;

[0072] Connect the external gas source through the gas connection plate 2, and set the gas source channels and the gas source pressure parameters of each channel according to the experimental purpose;

[0073] Run the microfluidic sample processing device and select an appropriate run time to obtain the target amount of mixture.

[0074] Example 2

[0075] This example is a specific example of the application of digital PCR detection. The specific operation steps are as follows:

[0076] (1) Reagents: Digital PCR premix, droplet generation oil, and analytical oil were purchased from Biorad. PBMC DNA samples were homemade. HER2 CNV primers and probes were purchased from Sangon Biotech (Shanghai) Co., Ltd. DNase / RNase-free water was purchased from Thermo Fisher Scientific.

[0077] (2) Instruments: The droplet generation device used was the handheld microfluidic sample processing device of the present invention. The digital PCR chip was a homemade PDMS chip. The external gas source had an accuracy of 1 mPa and a range of 20 mbar–2000 mbar. The PCR instrument (Mastercycler X50) was purchased from Eppendorf, and the droplet analyzer (QX200) and QuantaSoft software were purchased from Biorad.

[0078] (3) Prepare the digital PCR system: 2 μl of PBMC DNA, 15 μl of digital PCR premix, 1 μl of HER2CNV primer, 0.8 μl of probe, and add nuclease-free water to 30 μl, then mix thoroughly.

[0079] (4) Detection: Align the liquid outlet 403 of the liquid reservoir 401 of the chip sealing plate 4 with the opening 502 of the microfluidic chip 5 and fit them tightly together. Add the digital PCR reaction system through the liquid reservoir 401 and seal it with the joint sealing plate 3. Here, the joint sealing plate 3 is preferably a silicone sealing plate. Continue to assemble the gas path connecting plate 2, the upper plate 1 of the fixture and the lower plate 6 of the fixture. After fastening the device with the buckle 101, connect the external gas source and operate at an air pressure of 250 mbar for the oil phase and 235 mbar for the water phase for 6 minutes to generate about 30,000 droplets.

[0080] (5) After the droplets are generated, Figure 11 As shown, the clamp was opened, and the droplets were aspirated from the collection wells into a 96-well plate for PCR, and PCR amplification was performed by denaturation at 95°C for 10 min, 40 cycles of 95°C for 20 s, 60°C for 1 min, and continuous storage at 4°C.

[0081] (6) Reading data: After the PCR is completed, take out the 96-well plate and place it in the droplet reader fixture for assembly, then place it in the droplet reader, turn on the digital PCR reader, load the digital PCR droplet analysis oil, and perform detection and data analysis.

[0082] Example 3

[0083] This example is a specific example of preparing gel microbeads. The specific steps are as follows:

[0084] (1) Reagents: Acrylamide, methylenebisacrylamide, ammonium persulfate, Tris-HCl, EDTA, NaCl, and TEMED were purchased from Merck. Fluoride solution 7500 (Novec HFE-7500) was purchased from 3M. Surfactant (008-FluoroSurfactant-10G) was purchased from RNA Biotech. Acrydite-modified DNA primers were purchased from Sangon Biotech (Shanghai) Co., Ltd.

[0085] (2) Instruments and equipment: The equipment uses the handheld microfluidic sample processing device of the present invention, the microbead generation chip is a homemade PDMS chip, the main channel 505 is 60 μm wide, and the external gas source accuracy is 1 mPa;

[0086] The dispersed phase is composed of Tris-HCl pH 7.6, EDTA, NaCl, acrylamide, methylene bisacrylamide, ammonium persulfate and acrydite-modified DNA primers; the continuous phase is composed of Fluoride 7500, TEMED and a surfactant.

[0087] (3) Device assembly: Align the liquid outlet 403 of the liquid reservoir 401 of the chip sealing plate 4 with the opening 502 of the microfluidic chip 5 and fit them tightly together. Add the dispersed phase and continuous phase mixtures to the liquid reservoir 401 respectively, cover with the joint sealing plate 3 to seal, continue to assemble the gas path connecting plate 2 and the clamp, fasten them, connect the external gas source, generate droplets into the centrifuge tube at a flow rate of 450 μL / h for the dispersed phase and 1000 μL / h for the continuous phase, and react at 65°C for 12 h to polymerize the gel beads;

[0088] (4) After the gel beads are generated, Figure 12 As shown, open the clamp and aspirate the gel beads from the collection well into a centrifuge tube for subsequent quality control and other applications.

[0089] The handheld microfluidic sample processing device of the present invention selects the corresponding microfluidic chip according to the purpose. After assembly, it is connected to an external gas source with corresponding channels and pressures to achieve the separation, mixing and encapsulation of different particles, solutions and chemicals in the chip, providing a physical basis for subsequent biochemical reactions.

[0090] Example 4

[0091] This embodiment is applied in single-cell sequencing detection.

[0092] In this example, the single-cell RNA-Seq assay from 10X Genomics was used to compare the performance of the Chromium single-cell marker on the market, with the Chromium single-cell marker serving as the control group.

[0093] (1) Test reagents:

[0094]

[0095]

[0096] (2) Experimental equipment:

[0097]

[0098] (3) Cell suspension preparation and single cell labeling

[0099] Peripheral blood was drawn to obtain fresh PBMC cells, and the cell suspension was diluted to 1*10^6 cells / ml; cells were labeled using a Chromium single cell labeling instrument and the device of the present invention, respectively, to obtain cell labeled products.

[0100] (4) Library preparation

[0101] The cell-labeled products were reverse transcribed, purified, and library prepared. The prepared library was detected for fragment distribution using the Agilent 2100 chip bioanalysis system.

[0102] (5) Sequencing and data analysis

[0103] Sequencing was performed using an Illumina NovaSeq 6000 sequencer, and data such as the number of cells effectively captured by the encoded beads, the number of molecular tag UMIs, and the number of captured genes were analyzed. The results are as follows:

[0104]

[0105]

[0106] Conclusion: The test results are as follows Figure 13-16 From the experimental analysis data, it can be seen that the microfluidic sample processing device of the present invention can achieve the same effect as 10X Genomics' products in capturing cells, capturing mRNA molecules and analyzing them in single-cell transcriptome sequencing applications.

[0107] The device of the present invention is small in size, simple in structure, low in manufacturing cost, and easy to operate. The modules can be assembled sequentially, making it portable. Furthermore, the device can be used with microfluidic chips of varying functional structures. The corresponding microfluidic chip is selected based on the intended purpose. After assembly, an external gas source with the appropriate channels and pressure is connected to the chip, enabling the separation, mixing, and encapsulation of various particles, solutions, and chemicals within the chip, providing a physical foundation for subsequent biochemical reactions. Furthermore, the device of the present invention can adjust the gas connection pressure and reaction solution volume according to the project, optimizing experimental conditions.

[0108] The above description is merely a preferred embodiment of the present invention and does not limit the present invention in any way. Any person skilled in the art who, without departing from the scope of the present invention, makes any equivalent substitution, modification, or other changes to the technical solution and technical content disclosed in the present invention shall be deemed to be within the scope of the present invention and still fall within the scope of protection of the present invention.

Claims

1. A handheld microfluidic sample processing device, characterized in that: It comprises a fixture upper plate (1), an air path connection plate (2), a joint sealing plate (3), a chip sealing plate (4), a microfluidic chip (5), and a fixture lower plate (6) which are stacked in sequence from top to bottom; The clamp upper plate (1) is provided with a hollow area for passing the gas path connection port (201) of the gas path connection plate (2), and a second positioning hole (103) for connecting to the clamp lower plate (6); The gas path connection plate (2) is provided with a plurality of closely spaced gas path connection ports (201) for connection to an external gas source, and a first positioning hole (202) for connection to the clamp lower plate (6); The joint sealing plate (3) is provided with a joint connection port (301) corresponding to the gas path connection port (201) of the gas path connection plate (2), and a third positioning hole (302) for fixing the chip sealing plate (4); The chip sealing plate (4) is provided with a liquid storage tank (401) corresponding to the joint connection port (301) of the joint sealing plate (3), and a hook (402) for clamping the third positioning hole (302) of the joint sealing plate (3); The microfluidic chip (5) comprises a channel (505) layer (501) and a substrate layer (503), wherein the channel (505) layer (501) is tightly sealed with the chip sealing plate (4) above and below, and the substrate layer (503) is tightly attached to the bottom of the channel (505) layer (501); The clamp lower plate (6) is provided with a chip positioning area (601) for placing the microfluidic chip (5), a first positioning pin (602) for connecting to the gas path connecting plate (2), and a second positioning pin (603) for connecting to the clamp upper plate (1). The clamp lower plate (6) and the clamp upper plate (1) are connected by a stainless steel buckle (101).

2. A handheld microfluidic sample processing device according to claim 1, characterized in that: Spring top beads (102) for forming point contact with the air path connecting plate (2) are provided around the hollow area of ​​the clamp upper plate (1), and the second positioning holes (103) are provided at the four corners of the clamp upper plate (1); the thickness of the clamp upper plate (1) is less than or equal to 5 mm.

3. The handheld microfluidic sample processing device according to claim 1, wherein: The gas path connecting plate (2) comprises a connecting plate body, the connecting plate body is made of stainless steel or aluminum plate, and the thickness of the connecting plate body is 5-10 mm; the gas path connecting ports (201) are closely arranged in the middle position of the connecting plate body; and the first positioning holes (202) are arranged at the four corners of the connecting plate body.

4. A handheld microfluidic sample processing device according to claim 3, characterized in that: The air circuit connection port (201) is a pneumatic quick-connect connector, which is threadedly connected to the air inlet on the connecting plate body.

5. The handheld microfluidic sample processing device according to claim 1, wherein: The third positioning holes (302) on the joint sealing plate (3) are arranged at the four corners of the joint sealing plate (3); the joint sealing plate (3) has one or more joint connection ports (301) arranged corresponding to the liquid storage tank (401) of the chip sealing plate (4); the material of the joint sealing plate (3) is one of silicone, fluororubber or rubber.

6. The handheld microfluidic sample processing device according to claim 1, wherein: The liquid storage tank (401) of the chip sealing plate (4) is cylindrical, and one or more liquid storage tanks (401) are provided. The liquid outlet (403) of the liquid storage tank (401) is a conical card hole, which forms a seal with the microfluidic chip (5); the top of the liquid storage tank (401) is tightly attached to the joint sealing plate (3) up and down.

7. The handheld microfluidic sample processing device according to claim 1, wherein: The channel (505) layer (501) of the microfluidic chip (5) is located above the substrate layer (503) and is tightly attached to the substrate layer (503); one or more reaction units (504) are arranged on the channel (505) layer (501) of the microfluidic chip (5); the reaction units (504) include channels (505) and openings (502); the openings (502) of the reaction units (504) correspond to the positions of the liquid storage tank (401) of the chip sealing plate (4) in the upper and lower directions, and the channels (505) of each reaction unit (504) are connected to the corresponding openings (502); the size of the openings (502) is 1-6 mm.

8. The handheld microfluidic sample processing device according to claim 7, wherein: The channel (505) is a core reaction area and is processed by a soft photolithography precision method; the material of the microfluidic chip (5) is one of polydimethylsiloxane or polycarbonate; the bonding method of the channel (505) layer (501) and the substrate layer (503) is reversible bonding or irreversible bonding, and the bonding method is one of plasma bonding, chemical reagent bonding or biological tape bonding.

9. The handheld microfluidic sample processing device according to claim 1, wherein: The lower plate (6) of the clamp is a rectangular groove structure with an opening on one side. A chip positioning area (601) is provided at the inner bottom of the lower plate (6); the first positioning pins (602) are provided at the four corners of the inner bottom of the lower plate (6), and the second positioning pins (603) are provided at the four corners of the top of the lower plate (6); buckle grooves (604) are provided on both sides of the lower plate (6) corresponding to the buckle (101) positions of the upper plate (1) of the clamp, and the thickness of the lower plate (6) does not exceed 5 mm; an observation port (605) is provided in the middle of the lower plate (6).

10. A method for using a handheld microfluidic sample processing device, for use with the handheld microfluidic sample processing device according to any one of claims 1 to 9, comprising the following steps: Make or select a suitable microfluidic chip according to the experimental purpose (5); Assembling the microfluidic chip (5) and the chip sealing plate (4), adding microparticles, solutions or chemicals into the liquid storage tank (401) of the chip sealing plate (4), and then assembling the microfluidic sample processing device as a whole; Connect the external gas source through the gas connection plate (2), and set the gas source channels and the gas source pressure parameters of each channel according to the experimental purpose; Run the microfluidic sample processing device and select an appropriate run time to obtain the target amount of mixture.

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