Microfluidic chip and sample detection method

Abstract

The present disclosure provides a microfluidic chip and a sample detection method, the microfluidic chip comprising: a connection chip comprising a rapid detection unit interface, at least one storage unit interface, a fluid control unit interface, a waste liquid chamber, a fluid driving unit interface, and one or more chip microfluids, the chip microfluids being used to form a fluid flow path; a fluid control unit connected with the fluid control unit interface, used to control the flow path of the fluid in the chip microfluids; a fluid driving unit connected with the fluid driving unit interface, used to provide positive pressure or negative pressure to drive the fluid flow; a storage unit connected with the storage unit interface, used to store at least one reagent; and a rapid detection unit connected with the rapid detection unit interface, used to detect the sample.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biological detection processing, and in particular to a microfluidic chip and a sample detection method. BACKGROUND

[0002] Biosecurity refers to measures and techniques for preventing, controlling and mitigating biological threats, thereby reducing their potential harm to human and animal health, ecosystems and socio-economic stability. In recent years, frequent large-scale infection events of pathogens have seriously harmed human and animal health, hindered economic development, and continuously challenged biosecurity. Rapid and accurate pathogen identification is crucial for guiding early intervention and timely treatment strategies.

[0003] Currently, some microfluidic-based point-of-care testing devices have achieved complete molecular diagnosis. However, these microfluidic testing devices are limited by high cost, long detection time, complex operation process, large volume, and power supply, which limits their application in resource-limited environments for rapid on-site diagnosis of pathogens. SUMMARY

[0004] In view of the above problems, the present disclosure provides a microfluidic chip and a sample detection method, which at least solve one of the above problems.

[0005] According to a first aspect of the present disclosure, a microfluidic chip is provided, comprising: a sample rapid processing unit 100, a rapid detection unit 500, the sample rapid processing unit 100 comprising a connection chip 101, a fluid control unit 200, a fluid driving unit 300, a storage unit 400;

[0006] The connection chip 101 comprises a rapid detection unit interface 110, at least one storage unit interface 120, a fluid control unit interface 130, a waste liquid chamber 140, a fluid driving unit interface 150, and one or more chip microfluidic channels 160, the chip microfluidic channels 160 being used to form a fluid flow path;

[0007] The fluid control unit 200 is connected with the fluid control unit interface 130, and is used to control the flow path of the fluid in the chip microfluidic channel 160;

[0008] The fluid driving unit 300 is connected with the fluid driving unit interface 150, and is used to provide positive pressure or negative pressure to drive the fluid flow;

[0009] The storage unit 400 is connected with the storage unit interface 120, and is used to store at least one reagent;

[0010] The rapid detection unit 500 is connected with the rapid detection unit interface 110, and is used to detect the sample.

[0011] According to the embodiment of the present disclosure, a plurality of cylindrical protrusions 131 are arranged on the fluid control unit interface 130.

[0012] The rapid detection unit interface 110 is connected to the rapid detection unit 500 by interference fit, the chip microfluidic channel 160 forms a liquid flow passage through the connecting pipe, the storage unit interface 120 is connected to the storage unit 400 by snap connection, the plurality of cylindrical protrusions 131 correspond to a plurality of connecting through holes 231 on the fluid control unit 200, and the fluid control unit 200 is connected to the connecting chip 101 by interference fit.

[0013] According to the embodiment of the present disclosure, the fluid control unit 200 comprises a fixing member 210, a rotating member 220, a connecting member 230, and a sealing member 240.

[0014] A plurality of positioning protrusions 211 are arranged on the upper surface of the fixing member 210, and the positioning protrusions 211 are used for angle positioning of the rotating member 220; the fixing member 210 is provided with internal threads, the connecting member 230 is provided with external threads, the internal threads of the fixing member 210 and the external threads of the connecting member 230 are connected by thread cooperation, and the rotating member 220 and the sealing member 240 are pressed for sealing by thread pre-tightening force.

[0015] The rotating member 220 is rotated by a fixed angle to make the built-in microfluidic channel 221 communicate with different chip microfluidic channels 160 in the connecting chip 101 to form a liquid passage, thereby controlling the flow of fluid.

[0016] The connecting member 230 is provided with a plurality of connecting through holes 231 at the bottom.

[0017] According to the embodiment of the present disclosure, the fluid driving unit 300 comprises a sealing top cover 310, a moving piston 320, and a reaction chamber 330.

[0018] The moving piston 320 is used to apply negative pressure and positive pressure by sliding up and down to drive fluid to flow into and out of the reaction chamber 330.

[0019] The reaction chamber 330 is provided with a first through hole 331, and the fluid driving unit 300 is connected to the connecting chip 101 by interference fit or adhesive bonding through the first through hole 331.

[0020] According to the embodiment of the present disclosure, the storage unit 400 comprises at least one chamber, and the at least one chamber comprises at least one of an elution reagent storage chamber 410, a washing reagent II storage chamber 420, a washing reagent I storage chamber 430, and a lysis solution storage chamber 440.

[0021] According to the embodiment of the present disclosure, the rapid detection unit 500 comprises at least one pressing seal 510, a cover plate 520, a base plate 530, and a sealing film 540.

[0022] The pressing seal 510 is used to open when the sample flows in.

[0023] The upper surface of the cover plate 520 is provided with at least one pressing seal slot 521 and a buffer chamber 522.

[0024] The base plate 530 comprises a detection chamber 531, a base plate microfluidic channel 532, a connecting hole 533, and an air hole 534. The detection chamber 531 is used to pre-store amplification detection reagents, and the detection chamber 531 is closed using a PCR sealing film. During the sample inflow process, the air in the detection chamber 531 and the base plate microfluidic channel 532 is discharged through the air hole 534.

[0025] According to the first aspect of the present disclosure, a sample detection method is provided, comprising:

[0026] S10, controlling the chip microfluidic channel 160 to communicate the storage unit 400 and the fluid driving unit 300 through the fluid control unit 200, so that the reagent and the sample are mixed and reacted under the action of the fluid driving unit 300;

[0027] S20, switching the chip microfluidic channel 160 to communicate the waste liquid chamber 140 through the fluid control unit 200, so as to discharge the waste liquid;

[0028] S30, repeating the operations of S10-S20 to sequentially react the sample with different reagents and discharge the corresponding waste liquid;

[0029] S40, controlling the chip microfluidic channel 160 to communicate the rapid detection unit 500 through the fluid control unit 200, so that the processed sample enters the rapid detection unit 500 for detection;

[0030] S50, placing the microfluidic chip in a detection device for nucleic acid amplification detection.

[0031] According to the embodiment of the present disclosure, the S10 comprises:

[0032] The sample is loaded into the lysis solution storage chamber 440, the rotating member 220 is adjusted to make the lysis solution storage chamber 440 communicate with the reaction chamber 330, the piston 320 is moved to drive the lysis solution to mix with the sample, so that the cells are lysed to release nucleic acid, and the magnetic beads adsorb the nucleic acid.

[0033] The S20 comprises: applying an external magnet to adsorb the magnetic beads, adjusting the rotating member 220 to make the reaction chamber 330 communicate with the waste liquid chamber 140, and moving the piston 320 to discharge the lysis waste liquid.

[0034] According to the embodiment of the present disclosure, the S30 comprises:

[0035] Remove the external magnet, adjust the rotating member 220 to make the reaction chamber 330 communicate with the washing reagent I storage chamber 430, drive the washing reagent I to mix with the magnetic beads by the moving piston 320, and wash the impurities;

[0036] Apply the external magnet to adsorb the magnetic beads, adjust the rotating member 220 to make the reaction chamber 330 communicate with the waste liquid chamber 140, and discharge the first washing waste liquid by the moving piston 320;

[0037] Remove the external magnet, adjust the rotating member 220 to make the reaction chamber 330 communicate with the washing reagent II storage chamber 420, drive the washing reagent II to mix with the magnetic beads by the moving piston 320, and wash the impurities again;

[0038] Apply the external magnet to adsorb the magnetic beads, adjust the rotating member 220 to make the reaction chamber 330 communicate with the waste liquid chamber 140, and discharge the second washing waste liquid by the moving piston 320.

[0039] According to the embodiment of the present disclosure, the S40 comprises:

[0040] Remove the external magnet, adjust the rotating member 220 to make the reaction chamber 330 communicate with the elution reagent storage chamber 410, drive the elution reagent to mix with the magnetic beads by the moving piston 320, and release the nucleic acid from the magnetic beads;

[0041] Apply the external magnet to adsorb the magnetic beads, adjust the rotating member 220 to make the reaction chamber 330 communicate with the rapid detection unit 500, press the sealing member 510 to open, and drive the purified nucleic acid to flow into the detection chamber 531 by the moving piston 320.

[0042] According to the image processing method, device, equipment, medium and product provided by the embodiment of the present disclosure, the display and utilization of the height prediction result, that is, the height information, realize accurate and efficient feature extraction of a large range of multi-scale targets, improve the feature representation under the premise of minimizing the occupation of computing resources, and improve the accuracy of downstream tasks such as target detection and bird's eye view segmentation.

[0043] The embodiment of the present disclosure has at least the following advantages:

[0044] 1) The present disclosure stores reagents by physical separation, realizes the flow of complex fluid in the pathogen detection process through the cooperation of the fluid control unit and the fluid driving unit, meets the needs of pathogen nucleic acid extraction and amplification detection reaction, can effectively improve the reaction efficiency, and shorten the reaction time;

[0045] 2) The microfluidic chip disclosed in the present disclosure has high integration degree, simple operation steps, does not depend on professional technicians, can perform rapid detection of pathogens in an environment with limited resources, realizes one-stop "sample in and result out", and effectively avoids aerosol pollution;

[0046] 3) The detachable microfluidic chip designed in the present disclosure can be combined with the sample processing unit and the rapid detection unit to perform complete pathogen detection, and can also be used alone to perform rapid nucleic acid extraction or rapid detection, thereby meeting the needs of various application scenarios;

[0047] 4) The present disclosure greatly supplements the resources for pathogen detection in limited environments, effectively solves the problems of high cost, complex operation, long detection time and power limitation of on-site rapid detection equipment. BRIEF DESCRIPTION OF DRAWINGS

[0048] The above and other objects, features and advantages of the present disclosure will become more apparent from the following description of embodiments of the present disclosure with reference to the accompanying drawings, in which:

[0049] Figure 1 The result diagram of the microfluidic chip according to the embodiment of the present disclosure is schematically shown;

[0050] Figure 2 The sample processing unit schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0051] Figure 3 The sample processing unit explosion schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0052] Figure 4 The connection chip microfluidic channel and connection interface schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0053] Figure 5 The fluid control unit explosion schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0054] Figure 6 The fluid control unit cross-sectional explosion schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0055] Figure 7 The rotating part groove schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0056] Figure 8 The fluid driving unit schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0057] Figure 9 The reagent storage unit schematic diagram according to the embodiment of the present disclosure is schematically shown;

[0058] Figure 10A schematic diagram of a rapid detection unit according to an embodiment of the present disclosure is shown schematically;

[0059] Figure 11 A schematic diagram of a microfluidic channel and an amplification detection chamber distribution of a rapid detection unit according to an embodiment of the present disclosure is shown schematically;

[0060] Figure 12 A schematic diagram of a pressing seal structure according to an embodiment of the present disclosure is shown schematically;

[0061] Figure 13 A flowchart of a sample detection method according to an embodiment of the present disclosure is shown schematically;

[0062] Figure 14 A schematic diagram of a nucleic acid extraction principle of embodiment one according to an embodiment of the present disclosure is shown schematically;

[0063] Figure 15 Optimization results of magnetic bead dosage of embodiment one according to an embodiment of the present disclosure are shown schematically;

[0064] Figure 16 Optimization results of proteinase K dosage of embodiment one according to an embodiment of the present disclosure are shown schematically

[0065] Figure 17 Comparison of simulation sample nucleic acid extraction results of embodiment one according to an embodiment of the present disclosure is shown schematically

[0066] Figure 18 A schematic diagram of a matched miniaturized device according to an embodiment of the present disclosure is shown schematically;

[0067] Figure 19 A schematic diagram of detection results of embodiment two according to an embodiment of the present disclosure is shown schematically;

[0068] Figure 20 A schematic diagram of sample nucleic acid extraction and qPCR detection results 1 of embodiment three according to an embodiment of the present disclosure is shown schematically;

[0069] Figure 21 A schematic diagram of sample nucleic acid extraction and qPCR detection results 2 of embodiment three according to an embodiment of the present disclosure is shown schematically;

[0070] 100 - sample rapid processing unit; 101 - connecting chip; 110 - rapid detection unit interface; 120 - storage unit interface; 121 - elution solution storage chamber interface; 122 - washing solution II solution storage chamber interface; 123 - washing solution I storage chamber interface; 124 - lysis solution storage chamber interface; 130 - fluid control unit interface; 131 - cylindrical protrusion; 140 - waste liquid chamber; 150 - fluid driving unit interface; 160 - chip microfluidic channel; 200 - fluid control unit; 210 - fixing member; 211 - positioning protrusion; 220 - rotating member; 221 - built-in microfluidic channel; 230 - connecting member; 231 - connecting through hole; 240 - sealing member; 300 - fluid driving unit; 310 - sealing top cover; 320 - moving piston; 330 - reaction chamber; 331 - first through hole; 400 - storage unit; 410 - elution reagent storage chamber; 420 - washing reagent II storage chamber; 430 - washing reagent I storage chamber; 440 - lysis solution storage chamber; 500 - rapid detection unit; 510 - pressing sealing member; 520 - cover plate; 530 - base plate; 540 - sealing film; 521 - pressing sealing member clamping groove; 522 - buffer chamber; 531 - detection chamber; 532 - base plate microfluidic channel; 533 - connecting hole; 534 - air hole. DETAILED DESCRIPTION

[0071] Hereinafter, embodiments of the present disclosure will be described with reference to the accompanying drawings. It should be understood, however, that the description is merely exemplary and is not intended to limit the scope of the present disclosure. In the following detailed description of the embodiments, numerous specific details are set forth in order to provide a thorough understanding of the embodiments of the present disclosure. However, it would be apparent to those skilled in the art that the embodiments can be practiced without these specific details. In other instances, well-known structures and techniques have been described in detail in order to avoid obscuring the concepts of the present disclosure.

[0072] The terms used herein are merely used to describe specific embodiments and are not intended to limit the present disclosure. The terms "include", "comprise" and the like used herein indicate the presence of the described features, steps, operations, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, or components.

[0073] All terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art unless otherwise defined. It should be noted that the terms used herein should be interpreted as having a meaning consistent with the context of the specification, and should not be interpreted in an idealized or overly formal manner.

[0074] In the case of using expressions similar to "at least one of A, B, and C", etc., it generally should be interpreted to include any of them, to exclude any of them, or to include some of them. For example, "at least one of A, B, and C" should be interpreted to include only A, only B, only C, both A and B, both A and C, both B and C, or all of A, B, and C, etc.

[0075] Figures 1-3 A structural diagram of a microfluidic chip according to an embodiment of the disclosure is schematically shown.

[0076] As shown in Figures 1-3 , the microfluidic chip provided by the embodiment of the disclosure includes a sample rapid processing unit 100 and a rapid detection unit 500. The sample rapid processing unit 100 includes a connection chip 101, a fluid control unit 200, a fluid driving unit 300, and a storage unit 400. The connection chip 101 includes a rapid detection unit interface 110, at least one storage unit interface 120, a fluid control unit interface 130, a waste liquid chamber 140, and a fluid driving unit interface 150.

[0077] Figure 4 A structural diagram of a microfluidic chip according to an embodiment of the disclosure is schematically shown.

[0078] As shown in Figure 4 , the connection chip 101 provided by the embodiment of the disclosure includes a rapid detection unit interface 110, a storage unit interface 120, an elution solution storage chamber interface 121, a washing solution II solution storage chamber interface 122, a washing solution I storage chamber interface 123, a lysis solution storage chamber interface 124, a fluid control unit interface 130, a waste liquid chamber 140, a fluid driving unit interface 150, and one or more chip microfluidic channels 160 for forming a fluid flow path. The fluid control unit 200 is connected with the fluid control unit interface 130, and is used to control the flow path of the fluid in the chip microfluidic channel 160. The fluid driving unit 300 is connected with the fluid driving unit interface 150, and is used to provide positive pressure or negative pressure to drive the fluid flow. The storage unit 400 is connected with the storage unit interface 120, and is used to store at least one reagent. The rapid detection unit 500 is connected with the rapid detection unit interface 110, and is used to detect the sample.

[0079] In some embodiments, a plurality of cylindrical protrusions 131 are arranged on the fluid control unit interface 130. The rapid detection unit interface 110 is connected to the rapid detection unit 500 by interference fit, the chip microfluidic channel 160 forms a liquid flow passage through the connecting pipe, the storage unit interface 120 is connected to the storage unit 400 by snap connection, the plurality of cylindrical protrusions 131 correspond to a plurality of connecting through holes 231 on the fluid control unit 200, and the fluid control unit 200 is connected to the connecting chip 101 by interference fit.

[0080] Figures 5-7 A structural diagram of a microfluidic chip according to an embodiment of the present disclosure is schematically shown.

[0081] As shown in Figures 5-7 the fluid control unit 200 provided by the embodiment of the present disclosure includes a fixing member 210, a rotating member 220, a connecting member 230, and a sealing member 240. A plurality of positioning protrusions 211 are arranged on the upper surface of the fixing member 210, and the positioning protrusions 211 are used for positioning the rotating angle of the rotating member 220. The fixing member 210 is provided with internal threads, the connecting member 230 is provided with external threads, the internal threads of the fixing member 210 and the external threads of the connecting member 230 are connected by thread cooperation, and the rotating member 220 and the sealing member 240 are pressed tightly by the thread pre-tightening force for sealing. The rotating member 220 is rotated by a fixed angle to make the built-in microfluidic channel 221 communicate with different chip microfluidic channels 160 in the connecting chip 101 to form a liquid passage, thereby controlling the flow of fluid. The connecting member 230 is provided with a plurality of connecting through holes 231 at the bottom.

[0082] Figure 8 A structural diagram of a microfluidic chip according to an embodiment of the present disclosure is schematically shown.

[0083] As shown in Figure 8 the fluid driving unit 300 provided by the embodiment of the present disclosure includes a sealing top cover 310, a moving piston 320, and a reaction chamber 330. The moving piston 320 is used to apply negative pressure and positive pressure by sliding up and down to drive fluid to flow into and out of the reaction chamber 330. The reaction chamber 330 is provided with a first through hole 331, and the fluid driving unit 300 is connected to the connecting chip 101 by interference fit or adhesive bonding through the first through hole 331.

[0084] Figure 9 A structural diagram of a microfluidic chip according to an embodiment of the present disclosure is schematically shown.

[0085] As shown in Figure 9 the storage unit 400 provided by the embodiment of the present disclosure includes at least one chamber, and the at least one chamber includes at least one of an elution reagent storage chamber 410, a washing reagent II storage chamber 420, a washing reagent I storage chamber 430, and a lysis solution storage chamber 440.

[0086] Figures 10-12 A structural diagram of a microfluidic chip according to an embodiment of the present disclosure is schematically shown.

[0087] As shown in Figures 10-12 The rapid detection unit 500 provided by the embodiment of the present disclosure includes at least one pressing seal 510, a cover plate 520, a substrate 530, and a sealing film 540. The pressing seal 510 is used to open when the sample flows in. The upper surface of the cover plate 520 is provided with at least one pressing seal slot 521 and a buffer chamber 522. The substrate 530 includes a detection chamber 531, a substrate microfluidic channel 532, a connecting hole 533, and an air hole 534. The detection chamber 531 is used to pre-store amplification detection reagents, and the detection chamber 531 is closed using a PCR sealing film. During the sample flowing in process, the air in the detection chamber 531 and the substrate microfluidic channel 532 is discharged through the air hole 534.

[0088] Figure 13 A flowchart of a sample detection method according to an embodiment of the present disclosure is schematically shown.

[0089] As shown in Figure 13 The sample detection method can be applied to the microfluidic chip described above, and the method includes operations S10-S50.

[0090] In operation S10, the chip microfluidic channel 160 is connected to the storage unit 400 and the fluid driving unit 300 by the fluid control unit 200, so that the reagent and the sample are mixed and reacted under the action of the fluid driving unit 300.

[0091] In operation S20, the chip microfluidic channel 160 is connected to the waste liquid chamber 140 by the fluid control unit 200, and the waste liquid is discharged.

[0092] In operation S30, operations S10-S20 are repeated to sequentially react the sample with different reagents and discharge the corresponding waste liquid.

[0093] In operation S40, the chip microfluidic channel 160 is connected to the rapid detection unit 500 by the fluid control unit 200, so that the processed sample enters the rapid detection unit 500 for detection.

[0094] In operation S50, the microfluidic chip is placed in a detection device for nucleic acid amplification detection.

[0095] The embodiment of the present disclosure also provides a sample detection method, which includes operations S1-S9.

[0096] In operation S1, the sample is loaded into the storage unit 400.

[0097] In operation S2, the fluid control unit 200 is adjusted to make the reaction chamber 330 communicate with the waste liquid chamber 140, and the lysed waste liquid is discharged by moving the piston 320;

[0098] In operation S3, the external magnet is removed, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the washing reagent I storage chamber 430, and the washing reagent I is mixed with the magnetic beads by moving the piston 320 to wash the impurities;

[0099] In operation S4, the external magnet is applied to adsorb the magnetic beads, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the waste liquid chamber 140, and the first washing waste liquid is discharged by moving the piston 320;

[0100] In operation S5, the external magnet is removed, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the washing reagent II storage chamber 420, and the washing reagent II is mixed with the magnetic beads by moving the piston 320 to wash the impurities again;

[0101] In operation S6, the external magnet is applied to adsorb the magnetic beads, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the waste liquid chamber 140, and the second washing waste liquid is discharged by moving the piston 320;

[0102] In operation S7, the external magnet is removed, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the elution reagent storage chamber 410, and the elution reagent is mixed with the magnetic beads by moving the piston 320 to release the nucleic acid from the magnetic beads;

[0103] In operation S8, the external magnet is applied to adsorb the magnetic beads, the rotating member 220 is adjusted to make the reaction chamber 330 communicate with the rapid detection unit 500, the sealing member 510 is pressed to open, and the purified nucleic acid is driven to flow into the detection chamber 531 by moving the piston 320;

[0104] In operation S9, the microfluidic chip is placed in a detection device for nucleic acid amplification detection.

[0105] Embodiment one

[0106] Figure 14 The principle diagram of nucleic acid extraction according to the embodiment of the present disclosure is schematically shown.

[0107] As Figure 14 shown, the microfluidic chip provided by the embodiment of the present disclosure is used for rapid nucleic acid extraction of a sample, and the specific operation steps are as follows:

[0108] The 3D printing photosensitive resin is used to process the connection chip 101, the fluid control unit 200, the fluid driving unit 300, and the reagent storage unit 400, and the components are sequentially connected, and the nucleic acid extraction reagent is stored in the corresponding chamber of the storage unit;

[0109] S11 adds the simulated sample containing plasmid into the lysis solution storage chamber, adjusts the rotating member to connect the micro flow channel between the lysis solution storage chamber and the reaction chamber to form a liquid passage, moves the piston in the reaction chamber to mix the sample and the lysis solution uniformly, the cells are lysed to release nucleic acid, and the magnetic beads in the lysis solution specifically adsorb the nucleic acid; S12 applies an external magnet to adsorb the magnetic beads, adjusts the rotating member to connect the reaction chamber and the waste liquid chamber, and removes the lysis waste liquid by moving the piston; S13 removes the external magnet, adjusts the rotating member to connect the reaction chamber and the washing reagent I storage chamber, and moves the piston in the reaction chamber to mix the magnetic beads and the washing reagent I uniformly to sufficiently wash the impurities; S14 applies an external magnet to adsorb the magnetic beads, adjusts the rotating member to connect the reaction chamber and the waste liquid chamber, and removes the first washing waste liquid by moving the piston; S15 removes the external magnet, adjusts the rotating member to connect the reaction chamber and the washing reagent II storage chamber, and moves the piston in the reaction chamber to mix the magnetic beads and the washing reagent II uniformly to wash the impurities again; S16 applies an external magnet to adsorb the magnetic beads, adjusts the rotating member to connect the reaction chamber and the waste liquid chamber, and removes the second washing waste liquid by moving the piston; S17 removes the external magnet, adjusts the rotating member to connect the reaction chamber and the elution reagent storage chamber, and moves the piston in the reaction chamber to mix the magnetic beads and the elution reagent uniformly, so that the magnetic beads release the nucleic acid; and S18 applies an external magnet to adsorb the magnetic beads, adjusts the rotating member to connect the reaction chamber and the micro flow channel, and controls the purified nucleic acid substance to flow out from the rapid detection unit interface 110 and be collected by moving the piston.

[0110] The embodiments of the present disclosure verify the use of one or more of an avian influenza virus H9N2 plasmid simulated sample and a chicken tissue sample, the genetic sequence of which and the probe primer information of the PCR amplification system are shown in Tables 1 and 2.

[0111] First, the use amount of magnetic beads and proteinase K in the lysis solution is optimized by using the detachable handheld rapid sample processing and detection microfluidic chip. The use amount of magnetic beads in the lysis solution is gradually increased from 5 μL to 30 μL at a volume gradient of 5 μL, other components in the lysis solution remain unchanged, the same volume of chicken tissue sample is used for nucleic acid extraction on the chip, the concentration and purity of the extracted nucleic acid are tested by a spectrophotometer, and the quantity is quantified by PCR amplification. As shown in Figure 15 As the use amount of magnetic beads increases, the concentration of the extracted nucleic acid gradually increases, the purity of the nucleic acid first increases and then decreases, the purity of the nucleic acid is the highest when the use amount of magnetic beads is 20 μL, and the PCR amplification effect is the best according to the relative fluorescence intensity of the PCR quantification, so the use amount of magnetic beads in the lysis solution is 20 μL. Then, the use amount of proteinase K in the lysis solution is optimized by using the same method. As shown in Figure 16As shown, with the increase of the amount of proteinase K, the concentration of the extracted nucleic acid first decreases and then increases, and the purity of the nucleic acid first increases and then decreases. When the amount of proteinase K is 20 μL, the purity of the nucleic acid is the highest, and the PCR quantitative relative fluorescence intensity proves that the PCR amplification effect is the best at this time. Therefore, the amount of proteinase K in the lysis solution is 20 μL.

[0112] The simulated samples containing different concentrations of plasmids are subjected to nucleic acid extraction using the microfluidic chip of the present disclosure, and the whole extraction process is completed within 5 minutes. Then, the purified nucleic acid obtained by extraction is subjected to PCR quantification, and the quantification results are compared with the results of the purified nucleic acid obtained by the standard process of the A42352 nucleic acid extraction kit and the original sample, and the results are as follows Figure 17 As shown, the fluorescence intensity of the purified nucleic acid obtained by extraction using the microfluidic chip of the present disclosure and the relative fluorescence intensity of the purified nucleic acid obtained by the standard process of the commercial kit and the original sample have a small gap, which proves that the microfluidic chip proposed in the present disclosure has high efficiency in the process of rapid nucleic acid extraction of samples.

[0113] Example Two

[0114] Figure 18 The schematic diagram of the matched miniaturized device according to the embodiment of the present disclosure is schematically shown. Figure 19 The schematic diagram of the detection result according to the embodiment of the present disclosure is schematically shown.

[0115] The microfluidic chip provided in the embodiment of the present disclosure is used for PCR detection, and the specific operation steps are as follows:

[0116] The PCR detection is performed using the rapid detection unit alone. The rapid detection unit 500 component is machined by using a machined polycarbonate material, and then the sealing is completed by using a hot-press bonding method. The pressing seal 510 is machined by using a silica gel reverse mold method, and the two pressing seals are assembled in the rapid detection unit. The PCR reaction reagent is added to the detection chamber 531, and the chamber is closed by using a PCR sealing film after the detection sample is added. Finally, the detection unit is placed in the matched miniaturized device for PCR detection. The results of the PCR detection using the simulated sample containing plasmids are as follows Figure 19 As shown, the whole process takes 13 min, and when the plasmid concentration is 10 3 ~10 0 copies / μL, a relatively strong relative fluorescence intensity is presented.

[0117] Example Three

[0118] Figure 20 The schematic diagram of the sample nucleic acid extraction and qPCR detection result 1 according to the embodiment of the present disclosure is schematically shown. Figure 21A schematic diagram of sample nucleic acid extraction and qPCR detection results 2 according to an embodiment of the present disclosure is shown.

[0119] As shown in Figure 19 The microfluidic chip provided by the embodiment of the present disclosure is used for sample rapid nucleic acid extraction and qPCR detection, and the specific operation steps are as follows:

[0120] The 3D printing photosensitive resin is used to process the connection chip 101, the fluid control unit 200, the fluid driving unit 300, and the reagent storage unit 400, the polycarbonate material is used to process the rapid detection unit 500 components, and then the sealing is completed by the method of hot pressing bonding. The pressing seal 510 is processed in the form of silicone inverse mold, and the components are connected in sequence. The nucleic acid extraction reagent and the RT-qPCR reagent are pre-stored in the corresponding chamber.

[0121] S31 loads the sample into the lysis solution storage chamber, adjusts the rotating part to connect the microfluidic channel between the lysis solution storage chamber and the reaction chamber to form a liquid passage, moves the piston in the reaction chamber to mix the sample and the lysis solution uniformly, and releases the nucleic acid by cell lysis. The magnetic beads in the lysis solution specifically adsorb the nucleic acid; S32 external magnet, adsorb magnetic beads, adjust the rotating part to make the reaction chamber and the waste liquid chamber communicate, and remove the lysis waste liquid by moving the piston; S33 remove the external magnet, adjust the rotating part to make the reaction chamber and the washing reagent I storage chamber communicate, and mix the magnetic beads and the washing reagent I uniformly by moving the piston in the reaction chamber, and fully wash the impurities; S34 external magnet, adsorb magnetic beads, adjust the rotating part to make the reaction chamber and the waste liquid chamber communicate, and remove the first washing waste liquid by moving the piston; S35 remove the external magnet, adjust the rotating part to make the reaction chamber and the washing reagent II storage chamber communicate, and mix the magnetic beads and the washing reagent II uniformly by moving the piston in the reaction chamber, and wash the impurities again; S36 external magnet, adsorb magnetic beads, adjust the rotating part to make the reaction chamber and the waste liquid chamber communicate, and remove the second washing waste liquid by moving the piston; S37 remove the external magnet, adjust the rotating part to make the reaction chamber and the elution reagent storage chamber communicate, and mix the magnetic beads and the elution reagent uniformly by moving the piston in the reaction chamber, and release the nucleic acid by the magnetic beads; S38 external magnet, adsorb magnetic beads, adjust the rotating part to make the reaction chamber and the rapid detection unit communicate, and open the pressing seal of the rapid detection unit by moving the piston. The purified nucleic acid substance flows into the amplification detection chamber; S39 places the microfluidic chip in the matching miniaturized equipment for amplification detection.

[0122] The performance of the rapid nucleic acid extraction and RT-qPCR detection of the present disclosure is verified by using a simulated sample containing two different plasmids. The plasmid concentration gradient is 10 30.5 copies / μL, nucleic acid extraction and qPCR detection were performed according to the above procedure, and the whole process was completed within 18 minutes. The detection results of the two plasmids are shown in FIGS. 10 and 11, respectively. The real-time fluorescence curve was complete and the inflection point was clear when the sample plasmid concentration was as low as 0.5 copies / μL, proving the feasibility of the present disclosure for sample nucleic acid extraction and qPCR detection. Figure 20 、 Figure 21 The real-time fluorescence curve was complete and the inflection point was clear when the sample plasmid concentration was as low as 0.5 copies / μL, proving the feasibility of the present disclosure for sample nucleic acid extraction and qPCR detection.

[0123] As shown in Table 1, the synthetic sequence of H9N2 avian influenza virus is shown in Table 2, the probe primer information of H9N2 avian influenza virus PCR reaction system is shown.

[0124] Table 1

[0125] Table 2

[0126]

[0127] The above describes embodiments of the present disclosure. However, these embodiments are merely for illustrative purposes, and are not intended to limit the scope of the present disclosure. Although each embodiment is described above separately, this does not mean that the measures in each embodiment cannot be used advantageously in combination. The scope of the present disclosure is defined by the appended claims and their equivalents. Without departing from the scope of the present disclosure, those skilled in the art can make various substitutions and modifications, which should all fall within the scope of the present disclosure.

Claims

1. A microfluidic chip, characterized in that, include: The sample rapid processing unit (100) and the rapid detection unit (500) include a connection chip (101), a fluid control unit (200), a fluid drive unit (300), and a storage unit (400). The connection chip (101) includes a fast detection unit interface (110), at least one storage unit interface (120), a fluid control unit interface (130), a waste liquid chamber (140), a fluid drive unit interface (150), and one or more chip microchannels (160), the chip microchannels (160) being used to form fluid flow paths; The fluid control unit (200) is connected to the fluid control unit interface (130) and is used to control the flow path of the fluid in the chip microchannel (160); The fluid drive unit (300) is connected to the fluid drive unit interface (150) and is used to provide positive or negative pressure to drive the fluid flow; The storage unit (400) is connected to the storage unit interface (120) and is used to store at least one reagent; The rapid detection unit (500) is connected to the rapid detection unit interface (110) and is used to detect samples; The fluid control unit (200) includes a fixing member (210), a rotating member (220), a connecting member (230), and a sealing member (240); The upper surface of the fixing member (210) is provided with a plurality of positioning protrusions (211), which are used for positioning the rotation angle of the rotating member (220); the fixing member (210) is provided with an internal thread, and the connecting member (230) is provided with an external thread. The internal thread of the fixing member (210) and the external thread of the connecting member (230) are connected by a threaded engagement, and the rotating member (220) and the sealing member (240) are pressed together by a thread preload for sealing; The rotating component (220) rotates at a fixed angle to connect the built-in microchannel (221) with different chip microchannels (160) in the connecting chip (101) to form a liquid passage, thereby controlling the flow of fluid; The bottom of the connector (230) is provided with multiple connecting through holes (231); The fluid drive unit (300) includes a sealed top cover (310), a movable piston (320), and a reaction chamber (330). The movable piston (320) is used to apply negative and positive pressure by sliding up and down to drive fluid into and out of the reaction chamber (330). A first through hole (331) is provided on the reaction chamber (330), and the fluid drive unit (300) is connected to the connecting chip (101) through the first through hole (331) by interference fit or adhesive bonding. The storage unit (400) includes at least one chamber, which includes at least one of the following: an elution reagent storage chamber (410), a washing reagent II storage chamber (420), a washing reagent I storage chamber (430), and a lysis solution storage chamber (440).

2. The microfluidic chip according to claim 1, characterized in that, The fluid control unit interface (130) is provided with a plurality of cylindrical protrusions (131). The fast detection unit interface (110) is connected to the fast detection unit (500) by an interference fit. The chip microchannel (160) forms a liquid flow path through a connecting tube. The storage unit interface (120) is connected to the storage unit (400) by a snap-fit ​​connection. The multiple cylindrical protrusions (131) correspond to the multiple connecting through holes (231) on the fluid control unit (200). The fluid control unit (200) is connected to the connecting chip (101) by an interference fit.

3. The microfluidic chip according to claim 1, characterized in that, The rapid detection unit (500) includes at least one press-to-seal element (510), a cover plate (520), a base plate (530), and a sealing film (540). The press-fit seal (510) is used to open when the sample flows in; The upper surface of the cover plate (520) is provided with at least one press-sealing groove (521) and a buffer chamber (522); The substrate (530) includes a detection chamber (531), a substrate microchannel (532), a connection hole (533), and an air hole (534). The detection chamber (531) is used to pre-store amplification detection reagents and is sealed with a PCR sealing film. During the sample inflow process, the air in the detection chamber (531) and the substrate microchannel (532) is discharged through the air hole (534).

4. A sample detection method based on any one of claims 1 to 3, characterized in that, include: S10. The microchannel (160) of the chip is controlled by the fluid control unit (200) to connect the storage unit (400) and the fluid driving unit (300), so that the reagent and the sample are mixed and reacted under the action of the fluid driving unit (300); S20. The fluid control unit (200) switches the chip microchannel (160) to connect to the waste liquid chamber (140) to discharge the waste liquid. S30. Repeat operations S10-S20 to react the sample with different reagents in sequence and discharge the corresponding waste liquid; S40. The microchannel (160) of the control chip is connected to the rapid detection unit (500) through the fluid control unit (200) so that the processed sample enters the rapid detection unit (500) for detection. S50. Place the microfluidic chip in the detection device for nucleic acid amplification and detection.

5. The sample detection method according to claim 4, characterized in that, S10 includes: The sample is loaded into the lysis solution storage chamber (440), and the rotating part (220) is adjusted to connect the lysis solution storage chamber (440) with the reaction chamber (330). The lysis solution is driven to mix with the sample by moving the piston (320), so that the cells lyse and release nucleic acid, and the magnetic beads adsorb the nucleic acid. The S20 includes: applying an external magnet to attract magnetic beads, adjusting the rotating part (220) to connect the reaction chamber (330) with the waste liquid chamber (140), and discharging the pyrolysis waste liquid through the moving piston (320).

6. The sample detection method according to claim 4, characterized in that, S30 includes: Remove the external magnet, adjust the rotating part (220) to connect the reaction chamber (330) with the washing reagent I storage chamber (430), and drive the washing reagent I to mix with the magnetic beads through the moving piston (320) to wash away impurities; An external magnet is applied to attract magnetic beads, and the rotating part (220) is adjusted to connect the reaction chamber (330) with the waste liquid chamber (140), and the first washing waste liquid is discharged through the moving piston (320); Remove the external magnet, adjust the rotating part (220) to connect the reaction chamber (330) with the washing reagent II storage chamber (420), and drive the washing reagent II to mix with the magnetic beads through the moving piston (320) to wash the impurities again; An external magnet is applied to attract magnetic beads, and the rotating component (220) is adjusted to connect the reaction chamber (330) with the waste liquid chamber (140), and the second washing waste liquid is discharged through the moving piston (320).

7. The sample detection method according to claim 4, characterized in that, S40 includes: Remove the external magnet, adjust the rotating part (220) to connect the reaction chamber (330) with the elution reagent storage chamber (410), and drive the elution reagent to mix with the magnetic beads by moving the piston (320) so that the magnetic beads release nucleic acid; An external magnet is applied to attract magnetic beads. The rotating component (220) is adjusted to connect the reaction chamber (330) with the rapid detection unit (500). The sealing component (510) is pressed to open, and the purified nucleic acid is driven to flow into the detection chamber (531) by the moving piston (320).

Images