CN112763701A - Microfluidic detection chip and microfluidic detection method - Google Patents

Abstract

The invention relates to a microfluidic detection chip and a microfluidic detection method, wherein the microfluidic detection chip comprises at least one detection unit, and the detection unit comprises a sample adding cavity, an immune combination pool, an immune detection pool, an immune cleaning solution pool, an immune buffer solution pool, a nucleic acid detection pool and a waste solution pool. The microfluidic detection chip disclosed by the invention takes a microfluidic technology as a carrier, combines an immunological detection method and a molecular biological detection method for detecting tumor markers into a closed microfluidic chip, realizes that a small amount of samples can simultaneously complete detection and typing of different tumor markers, obtains a test result based on specific immune and molecular detection items, can provide more detailed, comprehensive and multidimensional data for clinical judgment, simplifies the requirements of a process and instrument equipment, shortens the detection period, reduces cross contamination, and also improves the effectiveness and accuracy of the detection result.

Description

Microfluidic detection chip and microfluidic detection method

Technical Field

The invention relates to the technical field of microfluidics, in particular to a microfluidic detection chip and a microfluidic detection method.

Background

The immunological detection method for detecting tumor marker is to take protein marker in body fluid as antigen and produce specific binding with antibody labeled with enzyme, isotope, biotin, avidin, fluorescein, etc. to locate and detect, and is used in enzyme-linked immunoassay, chemiluminescence immunoassay, radioimmunoassay, etc. conventionally. Although many protein markers have been discovered, not all cancers have protein markers, nor are all elevated protein markers due to cancer, and some non-cancer conditions may also result in elevated protein markers.

In addition, molecular biological detection methods (liquid biopsy) have been continuously applied to the course management of cancer patients, and in recent years, the application potential thereof has been actively developed, which can compensate for some of the drawbacks of immunological detection methods. In the liquid biopsy, tumor-related products in a body fluid sample such as blood, urine and the like of a patient are collected, including free Circulating Tumor Cells (CTC), circulating tumor DNA (ctDNA), exosomes and the like in the blood, and then a molecular biology detection method of a tumor target gene is combined to realize high-throughput, high-specificity and high-sensitivity detection results, so that detection and analysis of free DNA (cfDNA) in plasma and other body fluids successfully identify various types of cancer-related molecules, including gene mutation, copy number variation, translocation, methylation variation, gene expression variation, virus DNA and the like.

The tumor detection means, either an immunological detection method (protein marker detection) or a molecular biological detection method (liquid biopsy), have respective advantages and strong complementarity, and are essentially diagnostic means for different test target objects at different time periods. The detection results of the two are combined, so that more comprehensive and comprehensive dynamic data and indexes aiming at patients at different tumor stages on the immune level and the molecular level can be obtained, and a better reference basis can be provided for a clinician in actual diagnosis. However, the conventional method for respectively carrying out the immunological detection method and the molecular biological detection method not only has complex flow, but also needs to respectively and independently use various instruments and equipment, has a large amount of complicated manual operation, has long detection period, and has great risk of cross contamination in the detection process due to sample transfer among the equipment, thereby influencing the detection result.

Disclosure of Invention

In view of the above, it is necessary to provide a microfluidic detection chip capable of performing both an immunological detection method and a molecular biological detection method.

A microfluidic detection chip comprises at least one detection unit, wherein the detection unit comprises a sample adding cavity, an immune combination pool, an immune detection pool, an immune cleaning solution pool, an immune buffer solution pool, a nucleic acid detection pool and a waste solution pool;

the sample adding cavity is used for inputting and storing sample liquid; the immunological binding pool is connected with the sample adding cavity and is used for pre-loading a capture carrier coated with a capture antibody or a capture antigen; the immune detection pool is connected with the immune combination pool and is used for pre-loading a labeled antibody; the immune cleaning solution pool is connected with the immune detection pool and is used for pre-loading a cleaning buffer solution capable of washing off the labeled antibody; the immune buffer liquid pool is connected with the immune detection pool and is used for pre-loading a detection buffer liquid; the waste liquid pool is connected with the immunoassay pool for accommodating waste liquid; the nucleic acid detection pool is connected with the sample adding cavity and is used for pre-loading a PCR reaction reagent capable of amplifying a target sequence;

the micro-fluidic detection chip is provided with a rotation center, the immune combination pool, the immune detection pool and the waste liquid pool are sequentially distributed from the proximal end to the distal end of the rotation center, the immune cleaning liquid pool and the immune buffer liquid pool are closer to the rotation center relative to the immune detection pool, the nucleic acid detection pool is closer to the rotation center relative to the waste liquid pool, and valves are arranged between the immune combination pool, the immune detection pool and the waste liquid pool.

When the microfluidic detection chip is used, the sample liquid can be added into the sample adding cavity, and then the sample liquid is released to the immunological binding pool to be incubated with the capture carrier, so that the antigen to be detected is captured by the capture carrier. And then releasing the capture carrier and the sample liquid to an immunoassay pool to incubate with the labeled antibody so as to form a labeled antibody-antigen to be detected-capture carrier complex, discharging the incubated liquid into a waste liquid pool, and reserving the capture carrier in the immunoassay pool. And then releasing the cleaning buffer solution of the immune cleaning solution tank to the immune detection tank for cleaning, then discharging the cleaning solution into a waste solution tank, and reserving the capture carrier in the immune detection tank, so that the labeled antibody, impurities and the like which are not bound to the capture carrier are washed into the waste solution tank. And then releasing the detection buffer solution in the immune buffer solution pool to the immune detection pool, and detecting the marker in the immune detection pool to finish the immunological detection. Meanwhile, partial sample liquid in the sample adding cavity can be released to a nucleic acid detection pool, and then PCR amplification and detection are carried out, so that the molecular biological detection can be completed. The microfluidic detection chip disclosed by the invention takes a microfluidic technology as a carrier, combines an immunological detection method (protein marker detection) and a molecular biological detection method (liquid biopsy) for detecting tumor markers into a closed microfluidic chip, realizes that a small amount of samples can simultaneously complete the detection and typing of tumor markers of different categories, obtains a test result based on immune and molecular detection items, can provide more detailed, comprehensive and multidimensional data for clinical judgment, simplifies the requirements of processes and instruments, shortens the detection period, reduces cross contamination, and also improves the effectiveness and accuracy of the detection result.

In one embodiment, the sample application cavity comprises a first sample application pool and a second sample application pool, the immunological binding pool is connected with the first sample application pool, and the nucleic acid detection pool is connected with the second sample application pool.

In one embodiment, the detection unit further comprises a flow splitting cell, the flow splitting cell is connected with the immunological binding cell, the immunological detection cell is connected with the immunological binding cell through the flow splitting cell, and the nucleic acid detection cell is connected with the sample adding cavity through the flow splitting cell and the immunological binding cell; the flow distribution pool is far away from the rotation center relative to the immunological combination pool, and is closer to the rotation center relative to the immunological detection pool and the nucleic acid detection pool, and valves are arranged between the flow distribution pool and the immunological detection pool and the nucleic acid detection pool.

In one embodiment, the detection unit further comprises a sample lysis cell for pre-loading with an enzyme capable of releasing nucleic acid and a lysate cell for pre-loading with a sample lysate; the sample cracking cell is respectively connected with the sample adding cavity and the cracking liquid cell, the nucleic acid detecting cell is connected with the sample cracking cell, the sample cracking cell is farther away from the rotation center relative to the sample adding cavity, and is closer to the rotation center relative to the nucleic acid detecting cell, and the cracking liquid cell is closer to the rotation center relative to the sample cracking cell.

In one embodiment, the detection unit further comprises a nucleic acid enrichment pool, a nucleic acid cleaning solution pool and a nucleic acid eluent pool, wherein the nucleic acid enrichment pool is used for pre-loading nucleic acid carriers capable of binding nucleic acids, the nucleic acid cleaning solution pool is used for pre-loading cleaning solutions capable of washing off non-nucleic acid impurities, and the nucleic acid eluent pool is used for pre-loading eluent capable of separating nucleic acids from the nucleic acid carriers; the nucleic acid enrichment pool is respectively connected with the sample cracking pool, the nucleic acid cleaning solution pool and the nucleic acid eluent pool, the nucleic acid detection pool and the waste solution pool are respectively connected with the nucleic acid enrichment pool, and the nucleic acid enrichment pool is farther away from the rotation center relative to the sample cracking pool, the nucleic acid cleaning solution pool and the nucleic acid eluent pool and is closer to the rotation center relative to the nucleic acid detection pool and the waste solution pool.

In one embodiment, the number of the nucleic acid detection pools is multiple, the detection unit further comprises a liquid separation channel and a second siphon channel, the liquid separation channel is connected with the nucleic acid enrichment pool through the second siphon channel and extends from the connection end around the rotation center, the multiple nucleic acid detection pools are distributed around the rotation center outside the liquid separation channel, and each nucleic acid detection pool is respectively connected with the liquid separation channel.

In one embodiment, the number of the nucleic acid washing solution reservoirs is at least two, wherein at least one nucleic acid washing solution reservoir is used for pre-filling a washing solution containing an alcohol-based washing agent, and at least one nucleic acid washing solution reservoir is used for pre-filling a washing solution capable of washing the alcohol-based washing agent.

In one embodiment, the detection unit further includes a sedimentation basin, a separation basin, and a first siphon channel, the separation basin is closer to the rotation center than the sedimentation basin, the sedimentation basin is connected to the sample-adding cavity, the separation basin is connected to the sedimentation basin, and the immunological binding basin is connected to the separation basin through the first siphon channel.

In one embodiment, the microfluidic detection chip comprises a valve control layer, a detection core layer and a centrifugal layer, which are sequentially stacked, the valve is disposed on the valve control layer, the sample adding cavity, the immunological binding pool, the immunological detection pool, the immunological cleaning solution pool, the immunological buffer solution pool, the nucleic acid detection pool and the waste solution pool are disposed on the detection core layer, and the sedimentation pool and the separation pool are disposed on the centrifugal layer.

The invention also provides a microfluidic detection method, which adopts the microfluidic detection chip and comprises the following steps:

adding a sample liquid to the sample adding cavity;

releasing the sample fluid to the immunological binding cell for incubation with the capture carrier;

releasing the capture carrier and the sample fluid into the immunoassay reservoir to incubate with the labeled antibody, and then draining the incubated fluid into the waste reservoir and retaining the capture carrier in the immunoassay reservoir;

releasing the washing buffer solution of the immune washing solution pool to the immune detection pool for washing, then discharging the washing buffer solution into the waste solution pool and retaining the capture carrier in the immune detection pool;

releasing the detection buffer solution of the immunoassay buffer solution pool to the immunoassay pool, and then detecting the labeled antibody in the immunoassay pool; and/or

And releasing the sample liquid to the nucleic acid detection pool, and then carrying out PCR amplification and detection.

Drawings

FIG. 1 is a schematic structural diagram of a microfluidic detection chip according to an embodiment;

FIG. 2 is a schematic diagram of a partial structure of a microfluidic detection chip according to an embodiment;

FIG. 3 is a schematic structural diagram of a microfluidic detection chip according to another embodiment;

fig. 4 is an exploded view of a microfluidic detection chip according to an embodiment.

Detailed Description

In order that the invention may be more fully understood, a more particular description of the invention will now be rendered by reference to specific embodiments thereof that are illustrated in the appended drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items. Furthermore, the terms "first", "second" and "first" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. In the present invention, unless otherwise expressly stated or limited, the terms "mounted," "connected," "secured," and the like are to be construed broadly and can, for example, be fixedly connected, detachably connected, or integrally formed; can be mechanically or electrically connected; they may be directly connected or indirectly connected through intervening media, or they may be connected internally or in any other suitable relationship, unless expressly stated otherwise. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.

Microfluidic technology refers to the science and technology involved in systems that process or manipulate tiny fluids (in micro-or even nano-liter volumes) using micro-channels (tens to hundreds of microns), and is an emerging cross-technology involved in chemical, fluid physical, microelectronic, new materials, biological, and biomedical engineering. The system has the following 5 advantages: integration and automation. The microfluidic technology can concentrate a plurality of steps of sample detection on a small chip, and integrates the operation steps through the matching and combination of the size and the curvature of a flow channel, a micro valve and a cavity design, so that the whole detection integration is miniaturized and automated. ② high flux. The microfluidic can be designed into a multi-channel, so that a sample to be detected can be simultaneously distributed to a plurality of reaction units through a micro-channel network, and the reaction units are mutually isolated, so that the reactions are not mutually interfered, and the detection of a plurality of items can be parallelly carried out on the same sample as required. Compared with the conventional one-by-one item detection, the method greatly shortens the detection time, improves the detection efficiency and has the characteristic of high flux. Consumption of detection reagent is low. Due to the miniaturization of integrated detection, the reaction unit cavity on the microfluidic chip is very small, although the concentration of a reagent formula can be improved in a certain proportion, the use amount of the reagent is far lower than that of a conventional reagent, and the consumption amount of the reagent is greatly reduced. Fourthly, the sample size requirement is low. Since the detection is done on small chips only, the sample size to be detected is very small, often in the order of microliters. In addition, the whole blood can be directly used for detection, and the detection is more convenient for the people with small blood volume and difficult vein collection, such as infants, old people and disabled people. Meanwhile, for rare and precious samples, the detection of multiple indexes of the samples becomes possible. Less pollution. The integration function of the microfluidic chip enables all the operations which need to be manually completed in a laboratory to be automatically completed on the chip, and the pollution of the sample to the environment is reduced to the minimum degree.

As shown in fig. 1, the microfluidic detection chip 200 according to an embodiment of the present invention includes at least one detection unit, and the detection unit includes a sample adding cavity 11, an immunological binding pool 21, an immunological detection pool 31, an immunological cleaning solution pool 41, an immunological buffer solution pool 51, a nucleic acid detection pool 61, and a waste solution pool 71.

The sample adding cavity 11 is used for inputting and storing sample liquid. The immunological binding pool 21 is connected to the sample application chamber 11, and the immunological binding pool 21 is used for pre-loading a capture carrier coated with a capture antibody or a capture antigen. The immunoassay pool 31 is connected with the immunological binding pool 21, and the immunoassay pool 31 is pre-filled with labeled antibody. The immune cleaning solution pool 41 is connected with the immune detection pool 31, and the immune cleaning solution pool 41 is used for pre-loading a cleaning buffer solution capable of washing off the labeled antibody. The immune buffer pool 51 is connected with the immune detection pool 31, and the immune buffer pool 51 is used for pre-loading detection buffer. The waste liquid tank 71 is connected to the immunoassay tank 31 for receiving waste liquid. The nucleic acid detection cell 61 is connected with the sample adding cavity 11, and the nucleic acid detection cell 61 is used for pre-loading a PCR reaction reagent capable of amplifying a target sequence.

It is understood that the middle portion of the microfluidic detection chip 200 is a rotation mounting portion having a rotation center, which is a rotation center in the centrifugal operation. The immune combination pool 21, the immune detection pool 31 and the waste liquid pool 71 are sequentially distributed from the near center end to the far center end of the rotating center, the immune cleaning liquid pool 41 and the immune buffer liquid pool 51 are closer to the rotating center relative to the immune detection pool 31, the nucleic acid detection pool 61 is closer to the rotating center relative to the waste liquid pool 71, and valves are arranged among the immune combination pool 21, the immune detection pool 31, the immune cleaning liquid pool 41, the immune buffer liquid pool 51 and the waste liquid pool 71.

When the microfluidic detection chip 200 of the present invention is used, the sample solution can be added to the sample adding cavity 11, and then the sample solution is released to the immunological binding pool 21 to be incubated with the capture carrier, so that the antigen to be detected is captured by the capture carrier. The capture carrier and the sample liquid are then released to the immunoassay detection cell 31 to be incubated with the labeled antibody, thereby forming a labeled antibody-antigen to be detected-capture carrier complex, and the incubated liquid is discharged to the waste liquid cell 71 and the capture carrier is left in the immunoassay detection cell 31. Then, the washing buffer solution in the immune washing solution tank 41 is released to the immunoassay detection tank 31 to be washed, and then the washing solution is discharged into the waste solution tank 71 and the capture carrier is left in the immunoassay detection tank 31, so that the labeled antibody and impurities, etc. that are not bound to the capture carrier are washed into the waste solution tank 71. Then, the detection buffer solution in the immune buffer solution pool 51 is released to the immune detection pool 31, and the marker in the immune detection pool 31 is detected, so that the immunological detection can be completed. Meanwhile, part of the sample solution in the sample adding cavity 11 can be released to the nucleic acid detection cell 61, and then PCR amplification and detection are performed, so that molecular biological detection can be completed. The microfluidic detection chip 200 of the invention uses the microfluidic technology as a carrier, combines an immunological detection method (protein marker detection) and a molecular biological detection method (liquid biopsy) for detecting tumor markers into a closed microfluidic chip, realizes that a small amount of samples simultaneously complete the detection and typing of tumor markers of different categories, obtains a test result based on immune and molecular detection items, can provide more detailed, comprehensive and multidimensional data for clinical judgment, simplifies the requirements of processes and instruments, shortens the detection period, reduces cross contamination, and also improves the validity and accuracy of the detection result.

It will be appreciated that, according to the immunoassay principle, the type of the carrier may be different depending on the detection object in the sample (detection antibody or detection antigen), i.e., if the detection object is an antibody, the capture antigen is coated on the capture carrier, and if the detection object is an antigen, the capture antibody is coated on the capture carrier.

In a specific example, the detection unit further includes a flow splitting cell 22, the flow splitting cell 22 is connected to the immunological binding cell 21, the immunological detection cell 31 is connected to the immunological binding cell 21 through the flow splitting cell 22, and the nucleic acid detection cell 61 is connected to the sample application cavity 11 through the flow splitting cell 22 and the immunological binding cell 21. The flow splitting cell 22 is farther from the rotation center relative to the immunological binding cell 21 and closer to the rotation center relative to the immunological detection cell 31 and the nucleic acid detection cell 61, and valves are arranged between the flow splitting cell 22 and the immunological detection cell 21 and the nucleic acid detection cell 61. Thus, the sample solution after incubation with the capture carrier can be divided into two parts by the flow dividing cell 22 for immunological detection and molecular biological detection, respectively. The inner diameter of the middle part of the flow distribution cell 22 is reduced, so that the cavity is divided into two areas with the same size, and the liquid is conveniently divided into two parts equally for immunological detection and molecular biological detection respectively. Optionally, the detection unit further comprises an overflow tank connected to the diversion tank 22, and a distance between the overflow tank and the rotation center is equal to a distance between the diversion tank 22 and the rotation center, for accommodating excess liquid. It can be understood that the immunoassay pool 31 may be pre-loaded with different labeled antibodies according to different detection targets, and different detectors and optical systems of different detection devices are selected to achieve the requirement of detecting multiple antibody targets.

Optionally, the capture antibody in the immunological binding pool 21 is a specific antibody against a tumor cell surface antigen. Because the content of CTC (circulating tumor cells) in a human blood circulation system is extremely low, and each milliliter of whole blood of a tumor metastasis patient only contains 1-10 CTC, the detection of the CTC is realized, and the sorting and enrichment of the CTC is a key step. The CTC is captured and enriched by utilizing the specific combination of the specific antibody and the tumor cell surface antigen in the immune combination pool 21, and the CTC sorting and enriching characteristics of high sensitivity, rapidness and high cell activity are realized. CTCs are classified into epithelial cell phenotype, mesenchymal cell phenotype, and mixed epithelial-mesenchymal cell phenotype, identifying epithelial markers, identifying mesenchymal markers, and identifying epithelial mesenchymal markers. The epithelial marker is expressed on normal epithelial cells and epithelial tumors, is absent on interstitial leukocytes, and can distinguish cancer cells from normal blood cells. Optionally, the cell surface marker for positive enrichment of epithelial phenotype CTCs is epithelial cell adhesion molecule (EpCAM) or a cytokeratin family member (i.e., CK8, CK18, and CK19) for detecting CTCs in a cancer patient having an epithelial phenotype. In addition to detecting the captured CTC cells, tumor markers, including hormones, enzymes, etc., released by tumor cells in plasma can be detected as products of tumor cell metabolism, as shown in the table below. The subsequent application can be based on the result of the tumor marker before treatment as a baseline and as a reference standard for the later detection of curative effect and relapse monitoring, if the originally increased marker is reduced after treatment, the treatment is effective, otherwise, if the marker is not reduced or even increased, the treatment is possibly ineffective.

In a specific example, as shown in fig. 2, the detection unit further includes a sedimentation basin 12, a separation basin 13, and a first siphon channel 14, which are disposed at different layers, the separation basin 13 is closer to the rotation center relative to the sedimentation basin 12, the sedimentation basin 12 is connected to the sample-adding cavity 11, the separation basin 13 is connected to the sedimentation basin 12, and the immunological binding basin 21 is connected to the separation basin 13 through the first siphon channel 14. Thus, when the sample fluid is a sample to be pretreated, such as blood, the sample fluid can be layered by high-speed centrifugation, red blood cells, white blood cells, platelets, impurities, and the like in the sample fluid can be precipitated into the precipitation tank 12, serum can fill the separation tank 13, and then the centrifugation speed is reduced or the centrifugation is stopped, and the serum can enter the immunological binding tank 21 along the siphon channel 14 to prepare for subsequent tests. Preferably, the separation tank 13 and the settling tank 12 are arranged in succession in a radial direction away from the centre of rotation. It is understood that the settling tank 12, the separation tank 13, the first siphon channel 14, etc. may be disposed on the same layer as other chambers, without being limited thereto.

Optionally, the detection unit further comprises a residual sample cell 15 communicated with the separation cell 13, and a distance between the residual sample cell 15 and the rotation center is greater than or equal to a distance between the separation cell 13 and the rotation center, so as to accommodate the redundant sample liquid. As the sedimentation basin 12 and the separation basin 13 are gradually filled with the sample liquid, the excess sample liquid enters the adjacent excess sample basin 15. Optionally, the detection unit further comprises an indication cell 16 in communication with the separation cell 13, the indication cell 16 being filled to indicate that the added sample liquid can meet the detection requirement, which helps to guide the addition of the sample liquid.

In one specific example, the detection unit further comprises a sample lysis cell 62 and a lysate cell 63, the sample lysis cell 62 being pre-loaded with an enzyme capable of releasing nucleic acids, and the lysate cell 63 being pre-loaded with a sample lysate. The sample lysis cell 62 is connected with the sample adding cavity 11 and the lysis solution cell 63 respectively, the nucleic acid detection cell 61 is connected with the sample lysis cell 62, the sample lysis cell 62 is farther away from the rotation center relative to the sample adding cavity 11 and is closer to the rotation center relative to the nucleic acid detection cell 61, and the lysis solution cell 63 is closer to the rotation center relative to the sample lysis cell 62. It can be understood that the sample lysis solution contains components such as a reagent and a buffer solution for lysing cells, bacteria or viruses and denaturing proteins, and the enzyme in the sample lysis cell 62 may be lysozyme or protease (proteinase K, plant protease, pronase, or the like) and the like for promoting the cell or virus shell to be ruptured and degraded to release nucleic acid, so that the microfluidic detection chip 200 does not need to perform additional lysis pretreatment on the sample solution when performing molecular biological detection, and thus can be applied to a wider range of sample types and simplify manual operation steps.

In a specific example, the detection unit further comprises a nucleic acid enrichment pool 64, a nucleic acid washing solution pool 65 and a nucleic acid eluent pool 66, wherein the nucleic acid enrichment pool 64 is pre-filled with nucleic acid carriers capable of binding nucleic acids, the nucleic acid washing solution pool 65 is pre-filled with washing solutions capable of washing off non-nucleic acid impurities, and the nucleic acid eluent pool 66 is pre-filled with eluent capable of separating nucleic acids from nucleic acid carriers. The nucleic acid enrichment pool 64 is respectively connected with the sample cracking pool 62, the nucleic acid cleaning solution pool 65 and the nucleic acid eluent pool 66, the nucleic acid detection pool 61 and the waste solution pool 71 are respectively connected with the nucleic acid enrichment pool 64, and the nucleic acid enrichment pool 64 is farther away from the rotation center relative to the sample cracking pool 62, the nucleic acid cleaning solution pool 65 and the nucleic acid eluent pool 66 and is closer to the rotation center relative to the nucleic acid detection pool 61 and the waste solution pool 71. Thus, the sample solution is released to the nucleic acid enrichment pool 64 after the sample lysis pool 62 is subjected to nucleic acid lysis treatment, the nucleic acid carrier in the nucleic acid enrichment pool 64 can adsorb and enrich nucleic acid components, then unbound non-nucleic acid impurities can be washed to the waste solution pool 71 by the washing solution in the nucleic acid washing solution pool 65, then the nucleic acid can be separated from the nucleic acid carrier by the washing solution in the nucleic acid eluent pool 66 and washed to the nucleic acid detection pool 61, so that the subsequent better PCR amplification and detection can be carried out. It is understood that valves are provided between the nucleic acid enrichment cell 64, the sample lysis cell 62, the nucleic acid wash solution cell 65, the nucleic acid eluent cell 66 and the waste solution cell 71 to control the flow of the liquid.

In a specific example, the number of the nucleic acid washing solution reservoirs 65 is at least two, wherein at least one nucleic acid washing solution reservoir 65 is pre-filled with a washing solution containing an alcohol-based washing agent, and at least one nucleic acid washing solution reservoir 65 is pre-filled with a washing solution capable of washing off the alcohol-based washing agent. Thus, firstly, the impurities such as cell debris, virus debris, lipid, protein debris and the like can be washed away by the cleaning solution containing the alcohol cleaning agent, and then the residual alcohol cleaning agent is washed away so as to avoid the influence of the substances such as the alcohol cleaning agent and the like on the PCR amplification effect and improve the detection accuracy. In the embodiment shown in FIG. 1, the number of the nucleic acid washing solution reservoirs 65 is three, wherein two nucleic acid washing solution reservoirs 65 are pre-filled with a washing solution containing an alcohol-based washing agent, and the other nucleic acid washing solution reservoir 65 is pre-filled with a washing solution capable of washing off the alcohol-based washing agent. It is understood that the specific number of the nucleic acid washing liquid reservoir 65 and the type of the washing liquid to be pre-filled are not limited thereto, and may be adjusted as necessary.

In a specific example, the number of the nucleic acid detecting cells 61 is plural, the detecting unit further includes a liquid separating channel 67, the liquid separating channel 67 is connected to the nucleic acid enriching cell 64 and extends from the connecting end around the rotation center, the plural nucleic acid detecting cells 61 are distributed around the rotation center outside the liquid separating channel 67, and the respective nucleic acid detecting cells 61 are connected to the liquid separating channel 67, respectively. In this way, multiple parallel PCR amplifications and detections can be performed simultaneously to improve detection accuracy and detection efficiency, and for example, PCR reagents for different target sequences can be pre-loaded in multiple nucleic acid detection cells 61, so as to obtain detection results of multiple targets at one time. It is understood that the nucleic acid detecting pool 61 may be pre-embedded with freeze-dried microspheres or air-dried powder of PCR reaction reagents, including primer/probe combination for specific target sequence, reverse transcriptase required for amplification, Taq DNA polymerase, etc. Optionally, the detection unit further comprises a second siphon channel 68, and the separating channel 67 is connected with the nucleic acid enrichment pool 64 through the second siphon channel 68. Optionally, the detection unit further comprises a residual liquid pool, and the residual liquid pool is connected with the end of the liquid separation flow channel 67, namely the end far away from the nucleic acid enrichment pool 64, so as to accommodate the redundant liquid.

As shown in fig. 3, in the microfluidic detection chip 200 of another embodiment, the sample application cavity 11 includes a first sample application cell 111 and a second sample application cell 112, the immunological binding cell 21 is connected to the first sample application cell 111, and the nucleic acid detection cell 61 is connected to the second sample application cell 112. Thus, the first sample addition cell 111 and the second sample addition cell 112 are provided separately from each other, and the first sample addition cell 111 can be used for detection of a blood sample, and the second sample addition cell 112 can be used for a non-blood sample such as a throat swab, a nose swab, and an alveolar lavage fluid. It can be understood that the first sample adding well 111 and the second sample adding well 112 have corresponding sample adding holes respectively.

In one specific example, as shown in fig. 4, the microfluidic detection chip 200 includes a valve control layer 210, a detection core layer 220, and a centrifugal layer 230, which are sequentially stacked. The valves are disposed on the valve control layer 210, and can be configured to provide specific regions for interaction with the valves, thereby completing all reaction steps of the chip in sequence in steps and time. The sample adding cavity 11, the immunological combination pool 21, the immunological detection pool 31, the immunological cleaning liquid pool 41, the immunological buffer liquid pool 51, the nucleic acid detection pool 61 and the waste liquid pool 71 are arranged on the detection core layer 220, and mainly complete the processes of mixing, separating, reacting and detecting the detection reagent and the sample. The sedimentation tank 12 and the separation tank 13 are arranged on the centrifugal layer 230, and mainly complete the pretreatment of the sample. Further, the diversion cell 22, the sample lysis cell 62, the lysis solution cell 63, the nucleic acid enrichment cell 64, the wash solution cell 65, and the eluent cell 66 are also disposed on the detection core layer 220, and the first siphon channel 14, the remaining sample cell 15, and the indicator cell 16 are disposed on the centrifugation layer 230. In addition, as shown in FIG. 2, the centrifugation layer 230 is further provided with a sample addition hole 203 communicating with the sample addition chamber 11, and an air hole 204 connected to each chamber. Optionally, a filter element is disposed on the air hole 204, so that aerosol possibly generated by biological reaction in the chip can be blocked before entering an external environment, thereby preventing biological pollution.

In a specific example, the centrifugal layer 230 includes a siphon channel layer 231, a separation storage layer 232, and an excess buffer layer 233, which are sequentially stacked. The excess buffer layer 233 is provided with an excess sample well 15 to provide a storage space for excess samples. The separation reservoir 232 is provided with a sedimentation tank 12, a separation tank 13 and an indication tank 16, so that the blood sample can be separated into serum and blood cell sediment by high-speed centrifugation. The siphon channel layer 231 is provided with a first siphon channel 14 for introducing the separated serum from the separation cell 13 into the immuno-binding cell 21 of the detection core layer 220 via the first siphon channel 14, and the siphon channel layer 231 may also be provided with a micro channel for connecting the sample adding cavity 11 and the sedimentation cell 12.

In one specific example, the detection core layer 220 includes a fluid channel layer 221, a chamber storage layer 222, and a gas channel layer 223, which are sequentially stacked. As shown in fig. 1, the gas channel layer 223 is provided with a gas flow channel 205 for connecting each chamber and the gas hole 204, so as to guide the air flow of each chamber and uniformly join to the gas hole 204. The chamber storage layer 222 mainly provides storage and reaction spaces for samples and reagents, for example, the sample adding cavity 11, the immunological binding pool 21, the immunological detection pool 31, the immunological cleaning liquid pool 41, the immunological buffer liquid pool 51, the nucleic acid detection pool 61, the waste liquid pool 71, and the like are all disposed in the chamber storage layer 222. The fluid channel layer 221 is provided with corresponding micro channels for transferring and flowing the sample and each reaction reagent in each chamber, and can complete each link of detection by matching with the valve control layer 210.

In one particular example, valvular layer 210 includes an exchange control layer 211, a flexible valve layer 212, and a reagent seal layer 213, disposed in sequence in a stack. The reagent sealing layer 213 may be an aluminum foil, which can seal the connection between the bottom of each chamber and the channel, so that the liquid can be stably stored in each chamber before use, and when in use, the reagent sealing layer 213 is burst by an external puncturing mechanism, so as to realize liquid export, i.e., the reagent sealing layer 213 may be used as a valve between the immune cleaning liquid pool 41, the immune buffer liquid pool 51, the lysis liquid pool 63, the nucleic acid cleaning liquid pool 65, the nucleic acid eluent pool 66 and the chambers to which they are correspondingly connected. Or the liquid reagent can be stored in a thin capsule form and then preset in the corresponding chamber, and the subsequent external equipment punctures the thin capsule according to the requirement to enable the reagent to flow into the flow channel. The flexible valve layer 212 is matched with an external device positioning mechanism, can accurately control the communication and the closing of each chamber and the channel at fixed points and at fixed time, and provides an interface for the liquid flow after the chambers are punctured, namely the flexible valve layer 212 can be used as a valve among the diversion pool 22, the immunodetection pool 31, the sample cracking pool 62, the waste liquid pool 71 and the nucleic acid enrichment pool 64. The interaction control layer 211 is the bottom layer of the chip package, and provides an accurate point for the supporting device to interact with the flexible valve layer 212.

In a specific example, the microfluidic detection chip 200 further includes a top layer 240 and an isolation layer 250, the top layer 240 covers the centrifugal layer 230, and serves as the topmost layer of the chip package, and through holes communicated with the air holes are correspondingly formed in the top layer, and are inlets and outlets through which air pressure in the chip interacts with external air pressure; the isolation layer 250 is provided between the detection core layer 220 and the centrifugal layer 230 to isolate the chambers of the centrifugal layer 230 and the detection core layer 220 from each other, and above and below which the liquid can flow without interfering with each other.

Optionally, the material of the microfluidic detection chip 200 is one or more of glass, silicon wafer, quartz and common polymer materials, and the polymer materials are one or more of Polydimethylsiloxane (PDMS), polyurethane, epoxy resin, polymethyl methacrylate (PMMA), Polycarbonate (PC), Cyclic Olefin Copolymer (COC), Polystyrene (PS), Polyethylene (PE), polypropylene (PP), fluoroplastic and silicone rubber.

Optionally, the processing manner of the microfluidic detection chip 200 includes, but is not limited to, CNC, laser engraving, soft lithography, 3D printing, and injection molding to form a mold. Optionally, the capture carrier is an immunomagnetic bead coated with a capture antibody, the nucleic acid carrier is a magnetic bead capable of adsorbing nucleic acid, and the label bound to the labeled antibody is a fluorescent label.

Optionally, the microfluidic chip 200 is substantially circular, and is suitable for various detection samples such as whole blood, plasma, saliva, and the like. Of course, in other embodiments, the microfluidic detection chip 200 may also have other shapes, such as rectangular, polygonal, and so on. The number of detection cells on the microfluidic detection chip 200 can be one, two, three, five, seven, and so on.

The microfluidic detection method of an embodiment of the present invention employs the above microfluidic detection chip 200, and includes the following steps:

adding a sample liquid to the sample adding cavity 11;

releasing the sample fluid into the immuno-binding cell 21 for incubation with the capture carrier;

releasing the capture carrier and the sample solution to the immunoassay tank 31 to incubate with the labeled antibody, then discharging the incubated solution to the waste solution tank 71 and retaining the capture carrier in the immunoassay tank 31;

releasing the cleaning buffer solution in the immune cleaning solution tank 41 to the immune detection tank 31 for cleaning, then discharging the cleaning buffer solution into a waste solution tank 71 and retaining the capture carrier in the immune detection tank 31;

releasing the detection buffer solution in the immunoassay buffer solution pool 51 to the immunoassay detection pool 31, and then detecting the labeled antibody in the immunoassay detection pool 31; and/or

The sample solution is discharged to the nucleic acid detecting cell 61, and then PCR amplification and detection are performed.

The microfluidic detection method disclosed by the invention takes a microfluidic technology as a carrier, combines an immunological detection method (protein marker detection) and a molecular biological detection method (liquid biopsy) for detecting tumor markers into a closed microfluidic chip, realizes that a small amount of samples can simultaneously complete the detection and typing of different tumor markers, obtains a targeted test result based on immune and molecular detection items, and provides a more detailed, comprehensive and multidimensional basis for clinical judgment. It can be understood that the microfluidic detection method using the microfluidic detection chip 200 can be used not only for early screening and diagnosis of tumors, but also for detection of samples from various sources such as environment and food, and the types of detectable molecules are not limited to disease-related molecules, and practically any antigen antibody can be detected by the method, i.e., the application thereof has universality.

In one specific example, the method for detecting CTC immune indicators and molecular indicators in a blood sample using the microfluidic detection chip 200 as shown in fig. 1 is as follows:

the sample liquid is injected into the sample adding cavity 11, the sample liquid flows into the sedimentation tank 12 along the flow path, and the separation tank 13 and the indication tank 16 communicated with the separation tank are filled successively along with the gradual increase of the sample amount. The indication that the well 16 is full indicates that the amount of sample added is sufficient for the detection, and the excess sample will fill the remaining well 15 if any. Two-step gradient centrifugation can be adopted, wherein the first step is intermediate speed centrifugation at about 5000-6000 rpm to ensure the separation of blood plasma and blood cells, and the second step is high speed centrifugation at 15000-20000 rpm to ensure the removal of residual blood cells. Under the condition of high-speed centrifugation, red blood cells, white blood cells, platelets and the like can be precipitated into the sedimentation tank 12, and serum can fill part of the sedimentation tank 12 near the axial region of the chip and the separation tank 13. Then, by turning to low speed centrifugation or stopping centrifugation, the serum will enter the immunological binding pool 21 along the first siphon channel 14 in preparation for subsequent testing. In the process, the valves are all in a closed state.

After the serum enters the immune combination pool 21, the oscillation chip is rotated clockwise and anticlockwise for 5-10 s, and then incubation is carried out for 5-10 min, so that the antigen (CTC cells) in the serum is specifically combined with the magnetic beads coated by the antibody. And (4) centrifuging at a medium speed, allowing the antigen-antibody-magnetic bead complex mixed solution formed by incubation to enter the shunting pool 22 from the immunological binding pool 21, and finally equally dividing into two parts. And opening a valve between the immunological combination pool 21 and the immunological detection pool 31, centrifuging at a low speed, and allowing a part of the antigen-antibody-magnetic bead complex mixed solution in the diversion pool 22 to flow into the immunological detection pool 31. And when the mixed solution completely flows in, closing a valve between the immunological combination pool 21 and the immunological detection pool 31, rotating the oscillation chip clockwise and anticlockwise for 5-10 s to ensure that the antigen-antibody-magnetic bead compound is specifically combined with the fluorescence labeling antibody in the immunological detection pool 31, and incubating for 5-10 min. And enriching and fixing the incubated magnetic beads at the bottom of the chip by using a magnet, opening a valve between the immunoassay pool 31 and the waste liquid pool 71, centrifuging at a low speed, and discharging the waste liquid into the waste liquid pool 71. The bottom of the immune cleaning solution tank 41 is punctured by a puncture valve, and then centrifuged at a low speed, and the cleaning buffer solution flows into the immunoassay detection tank 31. And (3) when the washing buffer solution completely flows in, removing the magnet at the bottom of the chip, and rotating the oscillation chip clockwise and anticlockwise for 15-30 s, so that the washing buffer solution can fully dissolve the unbound magnetic beads and the fluorescence labeled antibody. After the immunoassay pool 31 is fully cleaned, the cleaned magnetic beads are enriched and fixed by using a magnet, a valve between the immunoassay pool 31 and the waste liquid pool 71 is opened, and the immunoassay pool is centrifuged at medium speed to discharge the cleaning waste liquid into the waste liquid pool 71. Then the bottom of the immune buffer solution pool 51 is punctured by the puncture valve, other valves are closed, the low-speed centrifugation is carried out, and the detection buffer solution flows into the immune detection pool 31. And (3) completely flowing the buffer solution to be detected, removing the magnet at the bottom of the chip, and rotating the oscillation chip clockwise and anticlockwise for 5-10 s so that the detection buffer solution can fully disperse the magnetic beads. When the magnetic beads are sufficiently dispersed, the antibody-antigen-fluorescent labeled antibody specifically bound thereto is also sufficiently dispersed, and fluorescence detection is possible.

Then, the valve between the flow splitting cell 22 and the sample lysis cell 62 is opened, the centrifugation is performed at a low speed, and the antigen-antibody-magnetic bead complex mixture in the flow splitting cell 22 flows into the sample lysis cell 62. And after the mixed liquid completely flows into the sample cracking cell 62, closing a valve between the shunt cell 22 and the sample cracking cell 62, and centrifuging at a low speed after the bottom of the cracking liquid cell 63 is punctured by the puncture valve, so that the sample cracking liquid enters the sample cracking cell 62. Rotating the oscillation chip clockwise and anticlockwise for 5-10 s to enable the antigen-antibody-magnetic bead compound to be fully mixed with the sample lysate and release nucleic acid substances inside the cells. A magnet is arranged at the bottom of the chip to enrich and fix the immunomagnetic beads, a valve between the sample lysis pool 62 and the nucleic acid enrichment pool 64 is opened, low-speed centrifugation is carried out, and the lysis mixed liquid in the sample lysis pool 62 flows into the nucleic acid enrichment pool 64. And after the mixed liquid completely flows into the nucleic acid enrichment pool 64, closing a valve between the sample cracking pool 62 and the nucleic acid enrichment pool 64, incubating for 10min, and rotating the oscillation chip 10s clockwise and anticlockwise every 2min in the period so that the mixed liquid and the nucleic acid magnetic beads pre-embedded in the nucleic acid enrichment pool 64 fully react. Then, a magnet is arranged at the bottom of the chip to enrich and fix nucleic acid magnetic beads, a valve between the nucleic acid enrichment pool 64 and the waste liquid pool 71 is opened, and the waste liquid is discharged into the waste liquid pool 71 through medium-speed centrifugation.

The bottom of a nucleic acid washing liquid tank 65 is pierced by a piercing valve, centrifuged at a low speed, and a washing liquid containing an alcohol-based washing agent is introduced into the nucleic acid enrichment tank 64. And after the cleaning solution completely flows into the nucleic acid enrichment pool 64, removing the magnet, and rotating the oscillation chip clockwise and anticlockwise for 15-30 s, so that the cleaning solution is fully contacted with the magnetic beads capturing the nucleic acids for washing. And arranging a magnet at the bottom of the chip to enrich and fix nucleic acid magnetic beads, opening a valve between the nucleic acid enrichment pool 64 and the waste liquid pool 71, and centrifuging at a medium speed to discharge the cleaning waste liquid into the waste liquid pool 71. The bottom of the other nucleic acid washing solution pool 65 is punctured by the puncture valve, and the previous step is repeated, so that the washing solution and the nucleic acid magnetic beads are washed for the second time. After the bottom of the other nucleic acid washing solution tank 65 is pierced by the piercing valve, low-speed centrifugation is performed, and the washing solution for washing off the alcohol-based washing solution flows into the nucleic acid enrichment tank 64, and the previous step is repeated to wash and dissolve the alcohol residues in the previous washing solution. The bottom of the nucleic acid eluent pool 66 is punctured by a puncture valve, and then the elution liquid flows into the nucleic acid enrichment pool 64 after low-speed centrifugation. And (3) completely flowing the to-be-eluted liquid into the nucleic acid enrichment pool 64, removing the magnet, rotating the oscillation chip clockwise and anticlockwise for 10-15 s to ensure that the elution liquid is fully contacted with the nucleic acid magnetic beads and then is incubated for 5min, and rotating the oscillation chip clockwise and anticlockwise for 5-8 s every 2min during incubation to ensure that the nucleic acid is thoroughly eluted and dissolved from the surfaces of the magnetic beads. And fixing the nucleic acid magnetic beads again, stopping or turning to low-speed centrifugation after medium-speed centrifugation, and allowing the eluent containing the nucleic acid to enter the liquid separation flow channel 67 through the second siphon channel 68. All the eluent flows into and fills the full liquid separating channel 67 through the second siphon channel 68, and is centrifuged at medium or high speed, so that the eluent in the liquid separating channel 67 enters each nucleic acid detecting pool 61 (13 in fig. 1) and is mixed with the nucleic acid detecting reagent embedded in the nucleic acid detecting pool 61 in advance. Then, a real-time fluorescence quantitative PCR process is carried out, and the nucleic acid amplification and signal detection are completed by the equipment.

Optionally, the low-speed centrifugation is about 1000 to 3000rpm, the medium-speed centrifugation is about 5000 to 6000rpm, and the high-speed centrifugation is about 15000 to 20000rpm, but not limited thereto. Optionally, the parameters for rotating the oscillating chip clockwise and counterclockwise are as follows: the oscillation angle is about 90-180 degrees, the oscillation frequency is about 1-2 times/s, and the oscillation time is selected according to needs.

cfDNA molecules are a mixture of DNA released by different tissues of the human body, a portion of which is derived from tumor cells. The amount of ctDNA released by tumor cells is a critical factor affecting the accuracy of the liquid biopsy. cfDNA concentrations are quite low compared to blood cell counts, and blood cell contamination in plasma samples can greatly dilute ctDNA from tumor sources, thus affecting downstream analysis. It is therefore preferred to minimize cell lysis during blood collection and storage, particularly during blood collection, when preparing the sample fluid, and to avoid hemolysis. The blood sample should be treated for separation of plasma and blood cells within 6 hours after collection, and a time exceeding 6 hours may result in release of a large amount of blood cell DNA. The chip is used for extracting a small amount of peripheral blood (about a few milliliters) from a patient body to be used as a sample, the process is a non-invasive sample acquisition mode which is to be measured immediately after extraction and has no side effect, the same patient can be repeatedly acquired, and tissue slices do not need to be acquired through modes such as operation or puncture on the patient. Of course, whole blood samples loaded with blood collection tubes of the following specification types can also be used: firstly, the loading capacity is more than or equal to 5mL, an EDTA anticoagulant blood collection tube is used, and the detection point is reached within 4 h; and secondly, the loading capacity is more than or equal to 5mL, a PAXgene ccfDNA blood collection tube or a Streck BCT blood collection tube is used for transportation at 6-28 ℃, and the blood is conveyed to a detection point within 3 days. The blood collection tube used above is an anticoagulated blood collection tube (short-term transport and storage) or a blood collection tube for exclusive cfDNA analysis (medium-term transport and storage), and contains an additive for stabilizing blood cells, and the type and specification thereof are not limited to those listed above. In order to meet the test accuracy, the blood sampling tube needs to be gently rotated and uniformly mixed after the blood sampling is finished, and the blood sampling tube is filled as far as possible, so that hemolysis caused by vibration in the transportation process is reduced.

In one embodiment, the method of detection using the microfluidic detection chip 200 shown in FIG. 3 is substantially the same as the previous steps, except that the blood sample is added to the first sample reservoir 111, and then subjected to steps such as centrifugal sedimentation to perform immunological detection, for example, detection of anti-pathogen antibodies produced in human body, and the sample with relatively simple other components can be added to the second sample reservoir 112 to perform molecular biological detection, for example, detection of pathogen nucleic acid.

Compared with the traditional detection method, the microfluidic detection method provided by the invention can complete the risk detection of a large number of cancers at one time based on the microfluidic detection chip 200, and the immunodetection of the detection index coverage protein marker and the molecular detection of the tumor target gene provide more complete and comprehensive guidance information for clinical judgment. The required sample is convenient to extract, only 5mL of venous blood needs to be extracted, and compared with invasive methods for obtaining tumor specimens through operations and tissue biopsy or image examination such as MRI, PET-CT and the like, the detection method is noninvasive and risk-free and does not need to worry about radiation. Except the sample application, all other processes are all accomplished in the chip, need not to possess medical staff of professional knowledge and also can easily accomplish the detection and obtain the result, and the chip structure is the enclosure space, reduces cross contamination, and the result accuracy is higher, and the interference is littleer, and the reaction process can not pollute external environment yet. The detection process that the patient participated in consumes very little time, and once draws blood can accomplish immune and many item detection results of molecule, greatly improves detection efficiency and can have the detection cost of more having the price/performance ratio. The detection method brings revolutionary breakthrough to cancer disease tracking and medication guidance, detects the level of a related marker of a circulating tumor cell and the variation condition and the change of content of a tumor cell genetic gene, and can provide the type and the dosage of a medicine suitable for individual conditions by matching with analysis of physiology or drug resistance, comprehensively evaluate the prognosis and monitor the disease condition of a patient, and update a suitable treatment scheme in time. The chip can simultaneously analyze a large number of biological indexes in a short time, quickly and accurately acquire biological information in a sample, has the efficiency which is thousands of times that of the traditional detection means, and is compatible with almost all requirements of immune and molecular detection.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims (10)

1. A microfluidic detection chip is characterized by comprising at least one detection unit, wherein the detection unit comprises a sample adding cavity, an immune combination pool, an immune detection pool, an immune cleaning liquid pool, an immune buffer liquid pool, a nucleic acid detection pool and a waste liquid pool;

the sample adding cavity is used for inputting and storing sample liquid; the immunological binding pool is connected with the sample adding cavity and is used for pre-loading a capture carrier coated with a capture antibody or a capture antigen; the immune detection pool is connected with the immune combination pool and is used for pre-loading a labeled antibody; the immune cleaning solution pool is connected with the immune detection pool and is used for pre-loading a cleaning buffer solution capable of washing off the labeled antibody; the immune buffer liquid pool is connected with the immune detection pool and is used for pre-loading a detection buffer liquid; the waste liquid pool is connected with the immunoassay pool for accommodating waste liquid; the nucleic acid detection pool is connected with the sample adding cavity and is used for pre-loading a PCR reaction reagent capable of amplifying a target sequence;

the micro-fluidic detection chip is provided with a rotation center, the immune combination pool, the immune detection pool and the waste liquid pool are sequentially distributed from the proximal end to the distal end of the rotation center, the immune cleaning liquid pool and the immune buffer liquid pool are closer to the rotation center relative to the immune detection pool, the nucleic acid detection pool is closer to the rotation center relative to the waste liquid pool, and valves are arranged between the immune combination pool, the immune detection pool and the waste liquid pool.

2. The microfluidic detection chip according to claim 1, wherein the sample application cavity comprises a first sample application cell and a second sample application cell, the immunological binding cell is connected to the first sample application cell, and the nucleic acid detection cell is connected to the second sample application cell.

3. The microfluidic detection chip according to claim 1, wherein the detection unit further comprises a flow splitting cell, the flow splitting cell is connected to the immunological binding cell, the immunological detection cell is connected to the immunological binding cell via the flow splitting cell, and the nucleic acid detection cell is connected to the sample application cavity via the flow splitting cell and the immunological binding cell; the flow distribution pool is far away from the rotation center relative to the immunological combination pool, and is closer to the rotation center relative to the immunological detection pool and the nucleic acid detection pool, and valves are arranged between the flow distribution pool and the immunological detection pool and the nucleic acid detection pool.

4. The microfluidic detection chip according to claim 1, wherein the detection unit further comprises a sample lysis cell and a lysate cell, the sample lysis cell is pre-filled with an enzyme capable of releasing nucleic acid, and the lysate cell is pre-filled with a sample lysate; the sample cracking cell is respectively connected with the sample adding cavity and the cracking liquid cell, the nucleic acid detecting cell is connected with the sample cracking cell, the sample cracking cell is farther away from the rotation center relative to the sample adding cavity, and is closer to the rotation center relative to the nucleic acid detecting cell, and the cracking liquid cell is closer to the rotation center relative to the sample cracking cell.

5. The microfluidic detection chip according to claim 4, wherein the detection unit further comprises a nucleic acid enrichment pool, a nucleic acid washing liquid pool and a nucleic acid eluent pool, wherein the nucleic acid enrichment pool is pre-filled with nucleic acid carriers capable of binding nucleic acids, the nucleic acid washing liquid pool is pre-filled with washing liquid capable of washing off non-nucleic acid impurities, and the nucleic acid eluent pool is pre-filled with eluent capable of separating nucleic acids from the nucleic acid carriers; the nucleic acid enrichment pool is respectively connected with the sample cracking pool, the nucleic acid cleaning solution pool and the nucleic acid eluent pool, the nucleic acid detection pool and the waste solution pool are respectively connected with the nucleic acid enrichment pool, and the nucleic acid enrichment pool is farther away from the rotation center relative to the sample cracking pool, the nucleic acid cleaning solution pool and the nucleic acid eluent pool and is closer to the rotation center relative to the nucleic acid detection pool and the waste solution pool.

6. The microfluidic detection chip according to claim 5, wherein the number of the nucleic acid detection cells is multiple, the detection unit further comprises a liquid separation channel and a second siphon channel, the liquid separation channel is connected to the nucleic acid enrichment cell through the second siphon channel and extends from the connection end around the rotation center, the multiple nucleic acid detection cells are distributed around the rotation center outside the liquid separation channel, and each nucleic acid detection cell is connected to the liquid separation channel.

7. The microfluidic detection chip according to claim 5, wherein the number of the nucleic acid cleaning solution reservoirs is at least two, at least one of the nucleic acid cleaning solution reservoirs is pre-filled with a cleaning solution containing an alcohol cleaning agent, and at least one of the nucleic acid cleaning solution reservoirs is pre-filled with a cleaning solution capable of washing off the alcohol cleaning agent.

8. The microfluidic detection chip according to any one of claims 1 to 7, wherein the detection unit further comprises a sedimentation basin, a separation basin, and a first siphon channel, the separation basin is closer to the rotation center than the sedimentation basin, the sedimentation basin is connected to the sample-adding cavity, the separation basin is connected to the sedimentation basin, and the immunological binding basin is connected to the separation basin through the first siphon channel.

9. The microfluidic detection chip according to claim 8, wherein the microfluidic detection chip comprises a valve control layer, a detection core layer and a centrifugation layer, the valve is disposed on the valve control layer, the sample adding cavity, the immuno-binding pool, the immuno-detection pool, the immuno-cleaning liquid pool, the immuno-buffer liquid pool, the nucleic acid detection pool and the waste liquid pool are disposed on the detection core layer, and the sedimentation pool and the separation pool are disposed on the centrifugation layer.

10. A microfluidic detection method is characterized in that the microfluidic detection chip of any one of claims 1 to 9 is adopted, and the microfluidic detection method comprises the following steps:

adding a sample liquid to the sample adding cavity;

releasing the sample fluid to the immunological binding cell for incubation with the capture carrier;

releasing the capture carrier and the sample fluid into the immunoassay reservoir to incubate with the labeled antibody, and then draining the incubated fluid into the waste reservoir and retaining the capture carrier in the immunoassay reservoir;

releasing the washing buffer solution of the immune washing solution pool to the immune detection pool for washing, then discharging the washing buffer solution into the waste solution pool and retaining the capture carrier in the immune detection pool;

releasing the detection buffer solution of the immunoassay buffer solution pool to the immunoassay pool, and then detecting the labeled antibody in the immunoassay pool; and/or

And releasing the sample liquid to the nucleic acid detection pool, and then carrying out PCR amplification and detection.

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