CN110568201B - Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip - Google Patents

Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip Download PDF

Info

Publication number
CN110568201B
CN110568201B CN201910864179.8A CN201910864179A CN110568201B CN 110568201 B CN110568201 B CN 110568201B CN 201910864179 A CN201910864179 A CN 201910864179A CN 110568201 B CN110568201 B CN 110568201B
Authority
CN
China
Prior art keywords
liquid
pool
detection
channel
centrifugal
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201910864179.8A
Other languages
Chinese (zh)
Other versions
CN110568201A (en
Inventor
廖晓玲
徐文峰
王溢
徐鹤丹
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Chongqing University of Science and Technology
Original Assignee
Chongqing University of Science and Technology
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Chongqing University of Science and Technology filed Critical Chongqing University of Science and Technology
Priority to CN201910864179.8A priority Critical patent/CN110568201B/en
Publication of CN110568201A publication Critical patent/CN110568201A/en
Application granted granted Critical
Publication of CN110568201B publication Critical patent/CN110568201B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/5302—Apparatus specially adapted for immunological test procedures
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6887—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids from muscle, cartilage or connective tissue

Abstract

The invention provides a use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip, the chip mainly comprises a chip body, a sample addition area, a centrifugal channel and a detection channel, and during detection, a bidirectional pneumatic valve is connected, a pressure valve of the bidirectional pneumatic valve is opened, and an interface valve of the chip is opened; the air pressure instantly pushes away a sealing pad wrapping a movable sealing plug pad tightly attached to the control plug, and an upper fluid channel and a lower fluid channel are opened; simultaneously, instantly pressing down the centrifugal liquid closing plate to the bottom surface of the plug inner cavity of the control plug, and pressing down a sealing gasket of the centrifugal liquid closing plate to seal a sample inlet of the detection channel; keeping the pneumatic valve in a pneumatic gas opening state and standing for 3-30min, and after the liquid flows over the liquid fixing pool, allowing the redundant liquid to flow into the residual liquid pool through the residual liquid guide pipe to finish the constant volume of the detection sample. The method overcomes the defects that the prior method aims at the detection of large sample amount, greatly reduces accessory parts of detection equipment, simplifies the operation and saves the detection cost.

Description

Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip
Technical Field
The invention belongs to the technical field of biomedical detection, relates to a use method of a microfluidic detection chip for centrifugally separating and processing a liquid sample to be detected in an early stage, and particularly relates to a use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip.
Background
The immunoassay is an analysis method for detecting a trace amount of a substance in a biological sample by utilizing specific binding of an antigen and an antibody. With the improvement of living standard of people, more and more attention is paid to body health, which promotes the development of medical industry, and the immunoassay technology also plays an important role in clinical detection. The fluorescence immunochromatography technology is a novel immunoassay method which is based on the specific immunoreaction of antigen and antibody and is combined with immunoassay technology and chromatographic analysis technology. The method comprises the steps of taking a microporous membrane as a solid phase carrier, taking an antibody as a detection line, coating an anti-antibody as a quality control line on the microporous membrane, fixing a fluorescence-labeled antibody on a connecting pad, adding a sample to be detected, and combining an antigen in the sample and the antibody on the membrane through capillary siphoning or percolation to form a detection strip. The method has the characteristics of strong specificity, wide detection range, simple operation, quick detection and the like. Test paper strips represented by colloidal gold and quantum dots are widely applied to the fields of food, medical treatment, environment and the like.
At present, with the development of detection technology and medical science, multi-factor cooperative detection and comprehensive diagnosis become the judgment basis of medical diagnosis. When one index is detected independently for diagnosis, the diagnosis result is often not accurate enough. For example, during the activity of inflammatory diseases such as autoimmune diseases (e.g., lupus erythematosus, reactive arthritis, and inflammatory bowel disease), if Procalcitonin (PCT) is measured alone, no significant difference from the normal level is found; however, if C-reactive protein (CRP) is measured, the CRP content is significantly higher than that of normal persons. Therefore, a combination of these two criteria is required to determine the presence of an inflammatory response in a patient. Similarly, in medical diagnosis, there is an increasing need for index detection of multiple markers simultaneously. Therefore, rapid, multi-sample high-throughput quantitative detection is essential. However, the high flux simultaneously requires the completion of the rapid quantitative liquid feeding of a high number of samples, which is difficult. In order to solve the problem, a complex mechanical automatic sample sucking needle is generally adopted for completion, so that an auxiliary mechanism of the detection equipment is complicated, and the contradiction between the complication and the miniaturization of the rapid detection equipment is caused. The micro-fluidic technology is applied more and more widely in the actual diagnosis of realizing high-flux and rapid target detection.
The invention aims to solve the technical problem that a whole blood sample needs to be detected by fast liquid separation and fast constant volume test solution or a body fluid or liquid sample needs to be centrifugally separated and processed by utilizing a micro-fluidic chip technology. In view of the defects of the prior detection technology, the invention provides a using method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects in the prior art and provides a use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip.
In order to solve the technical problems, the invention adopts the following technical scheme:
a method for using an automatic sample-separating constant-volume immunofluorescence quantitative rapid detection microfluidic chip comprises a chip body, a sample-adding region, a centrifugal channel and a detection channel,
a sample adding area is processed at the circle center of the chip body; a cylindrical hollow round pit is processed at the center of the sample adding area, and a cylindrical control plug is processed in the hollow round pit in a matching way; the plane of the bottom end of the processed control plug is h height away from the plane of the bottom of the round pit, and a liquid fixing pool is formed in the h height area of the plane of the bottom of the round pit away from the bottom of the control plug; the liquid-fixing pools are divided into more than 2 by h height liquid-fixing pool dividing walls;
processing a residual liquid pool with a concentric circle center and a diameter larger than that of the round pit and annularly winding the residual liquid pool around the fixed liquid pool at a certain thickness part of the periphery of the round pit of the fixed liquid pool, wherein the height of the residual liquid pool is consistent with that of the fixed liquid pool h, and the upper plane is flush with the upper plane of the fixed liquid pool; the residual liquid pool is divided into more than 2 by the partition wall of the residual liquid pool; the residual liquid pool partition wall and the constant liquid pool partition wall are in one-to-one correspondence on the same diameter line of the circle center of the circular pit, the separated residual liquid pool and the constant liquid pool are in one-to-one correspondence in a sector area with the circle center of the circular pit and the same circle center included angle, and the residual liquid pool and the constant liquid pool which are respectively corresponding are communicated through 1 residual liquid conduit pipe at the same plane height at the top of the pool; ensuring that each liquid determination pool corresponds to an independent residual liquid pool; each residual liquid pool is communicated with 1 independent residual liquid pool vent pipe, or is communicated with 1 or more residual liquid pool vent pipes connected with a plurality of residual liquid pools, so as to ensure that the redundant liquid of the liquid determining pool flows into the residual liquid pools; the volumes of the liquid fixing pools 10 of the chips with the same specification are consistent, or the volumes of the liquid fixing pools on the same diameter which is symmetrically distributed on the circumference are consistent; the bottom surface of each liquid fixing pool is provided with 1 detection channel sample inlet with a hydrophobic sealing rubber ring; a detection channel with one end below the liquid fixing pool is arranged below the sample inlet of each detection channel;
the detection channels are uniformly distributed on the circumference of the round pit and move along the direction of the diameter line, and the other end of each detection channel is opened on the side face of the disc-shaped chip body; 2-4 openings are processed on the bottom surface of the chip body of the detection channel to be used as detection windows; the detection test strip can be inserted, installed and fixed in the detection channel from the opening of the detection channel; each detection channel is provided with a channel mark on the chip body;
processing a hollow cylinder with the same circle center and the diameter larger than that of the round pit from the half height of the round pit to the top; the hollow cylinder is completely communicated with the round pit; the annular hollow cylinder is communicated with the round pit to form a concave table, and the annular concave table is used as a liquid adding pool for detecting a sample stock solution;
the liquid adding pool is divided into two specifications of adding sample stock solution into the partition wall and adding no sample stock solution into the partition wall, and each specification forms a series of small specifications according to the number of the liquid fixing pools; the sample stock solution adding partition wall processed in the liquid adding pool corresponds to the partition wall of the liquid fixing pool from top to bottom, and the liquid adding pool is uniformly divided into more than 2 parts which correspond to the liquid fixing pool from top to bottom; the liquid adding pool is arranged with 1 pair of n corresponding to the liquid fixing pool, wherein n is a natural number more than or equal to 1; the volume capacity of each liquid adding pool is more than 2 times of the volume capacity of each liquid fixing pool, so that sufficient centrifuged test solution can flow to the liquid fixing pools and redundant liquid can flow into the residual liquid pool, and the constant volume of the test solution detected by each liquid fixing pool is ensured to be consistent;
1 centrifugal channel is processed on the outer circular wall of each liquid adding pool, the height of the bottom plane of an opening of each centrifugal channel is consistent with that of the bottom plane of the liquid adding pool, and liquid adding scale marks are processed on the upper plane of the opening of each centrifugal channel; the centrifugal channel is provided with an upward inclination outwards along the center of the circular pit; each centrifugal channel is uniformly distributed on the circumference of a round pit of the sample adding area and is symmetrical one by one on the same diameter line of the circle center of the round pit; the inclination angles of the centrifugal channels of the chip bodies 1 with different specifications are different specifications from 1 degree to 15 degrees according to the viscosity of the detection liquid; in order to ensure centrifugal balance, the inclination angles of the centrifugal channels of the same tablet core body are consistent, or the inclination angles of the centrifugal channels with the same diameter and symmetry are consistent; the outer end of the centrifugal channel, namely the highest inclined position, is provided with a section of horizontal centrifugal channel for collecting centrifugal sediments, the top of the horizontal centrifugal channel is provided with an opening, and a sealed cleaning cover is processed;
the cylindrical control plug is internally hollow and is provided with a plug inner cavity, and the top surface of the control plug is provided with an interface valve which is communicated with the plug inner cavity and is connected with a bidirectional pneumatic valve; the top surface of the control plug is provided with an interface valve which is communicated with the cavity in the plug and is connected with a bidirectional pneumatic valve; a through hole which traverses the side wall of the control plug is processed on the outer wall of the control plug at the position facing the centrifugal channel port of the liquid adding pool, and a movable bolt is arranged in the through hole; the movable plug is connected with sealing gaskets at the inner end and the outer end of the side wall of the control plug to form a movable sealing plug gasket; the two ends of the movable bolt are connected with sealing gaskets to ensure that the test solution cannot flow into the cavity in the plug;
a control plug is arranged on the outer wall below the bottom surface facing the liquid adding pool, and a groove is processed above each liquid fixing pool to form an upper fluid channel and a lower fluid channel; or the outer wall below the middle of the movable sealing plug cushion is processed to reduce the diameter of the outer wall of the control plug corresponding to the upper part of each fixed liquid pool to the bottom end of the side surface of the control plug, a gap is generated between the outer wall of the control plug and the inner wall of the round pit of each fixed liquid pool to form an upper fluid channel and a lower fluid channel, and the upper fluid channel and the lower fluid channel correspond to the sample stock solution adding area and the partition wall of each fixed liquid pool which are consistent up and down; a control plug partition wall is also processed on the outer wall of the control plug; ensuring that the test solutions are not mixed when flowing down;
a circular centrifugate closing plate matched with the diameter of the cavity in the plug is arranged at the bottom surface of the cavity in the plug of the control plug; the bottom surface of the centrifugate closing plate is provided with movable bolts or sealing needles with the same number as the fixed liquid pool at the position corresponding to the sample inlet of the fixed liquid pool detection channel under the control plug; the movable plug penetrates through the bottom surface of the cavity in the plug, and the lower end of the movable plug is provided with a sealing gasket; when the centrifugate closing plate is abutted against the bottom surface of the cavity in the control plug, a sealing gasket or a sealing needle on the centrifugate closing plate can seal the sample inlet of the detection channel; a sealing limiting block is processed and installed on the inner wall of the lower part of the cavity in the control plug, and the centrifugal liquid sealing plate is limited to move up and down only within a height range of 1 mm-5 mm;
the using method comprises the following steps:
(1) preparing liquid adding; according to the requirements for detecting different projects, chips with different specifications are selected, and the parameters of the chips with different specifications are as follows: the number of the centrifugal channels and the detection channels, the inclination angle of the centrifugal channels, the capacity of the liquid fixing pool and the number of the detection windows of the detection channels; selecting a detection test strip of each detection channel according to the detection items, and inserting the detection test strip into the detection channel for fixing; fixing the chip on the centrifugal detection all-in-one machine through a centrifugal locking hole;
connecting a bidirectional pneumatic valve to an interface valve of a control plug, starting the bidirectional pneumatic valve, and opening a pneumatic valve air suction valve; the cavity in the plug of the control plug generates negative pressure, so that the movable plug of the movable sealing plug gasket slides inwards to drive the sealing gasket connected with the end head to be tightened and wrapped inwards to be tightly attached to the outer wall of the control plug, an upper fluid channel and a lower fluid channel are blocked, and the communication between the liquid adding pool and the liquid fixing pool is closed; at the moment, the centrifugate sealing plate 11 in the cavity in the plug of the control plug is tightly attached to the lower part of the sealing limiting block due to the upward sliding of the movable plug, and drives the sealing gasket of the centrifugate sealing plate to upwards leave the sample inlet of the detection channel, or drives the sealing needle of the centrifugate sealing plate to upwards leave the sample inlet of the detection channel with the hydrophobic sealing rubber ring; closing an interface valve of the chip and disconnecting the bidirectional pneumatic valve;
different detection sample stock solutions are symmetrically added into a liquid adding pool with the specification of the sample stock solution adding partition wall according to the channel mark; if the liquid adding pool and the liquid fixing pool are 1 to 1, only the constant volume is finished; if the liquid adding pool and the liquid fixing pool are 1 to 2 or more, different liquid adding pools and liquid fixing pools are required to complete sample separation and volume fixing;
or adding the same detection sample stock solution into a liquid adding pool without adding the sample stock solution into the specification of the partition wall to complete sample separation and constant volume work; the liquid adding amount is just to the liquid adding scale line at the top end of the centrifugal channel opening and does not exceed the liquid adding scale line;
(2) centrifuging; selecting 5000 r/min-15000 r/min of revolution according to the test solution conditions, and starting a centrifuge; detecting a section of a sample stock solution which is horizontal along the highest position of the centrifugal channel and flows upwards along the centrifugal channel under the action of centrifugal force, precipitating precipitates, and keeping the pneumatic valve in a suction opening state for standing for 3-30min after the centrifugation is finished; the supernatant liquid flows back into the liquid adding pool under the action of the gravity of the inclined angle of the centrifugal channel, and the centrifugal precipitate is left at the section of the centrifugal channel with the highest level;
(3) fixing the volume of the test solution; connecting a bidirectional pneumatic valve, opening a pneumatic valve of the bidirectional pneumatic valve, and opening an interface valve of the chip; the air pressure instantly pushes away a sealing pad wrapping a movable sealing plug pad tightly attached to the control plug, and an upper fluid channel and a lower fluid channel are opened; simultaneously, instantly pressing down the centrifugal liquid sealing plate to the bottom surface of the plug inner cavity of the control plug, pressing down a sealing gasket of the centrifugal liquid sealing plate to seal a detection channel sample inlet, or driving a sealing needle of the centrifugal liquid sealing plate to press down to seal a detection channel sample inlet with a hydrophobic sealing rubber ring; keeping the pneumatic valve in a pneumatic pressure gas opening state and standing for 3-30min, allowing the centrifuged supernatant in the liquid adding pool to flow into the liquid fixing pool through the upper and lower fluid channels on the outer wall of the control plug, and allowing the redundant liquid to flow into the residual liquid pool through the residual liquid guide pipe after the liquid flows over the liquid fixing pool to finish the constant volume of the detection sample;
(4) testing; opening an air suction valve of the bidirectional pneumatic valve, and controlling a centrifugate sealing plate in a cavity in a plug of the plug to cling to the lower part of the sealing limiting block due to upward sliding of the movable bolt under the action of negative pressure so as to drive a sealing gasket of the centrifugate sealing plate to upwards leave a sample inlet of the detection channel or drive a sealing needle of the centrifugate sealing plate to upwards leave the sample inlet of the detection channel with a hydrophobic sealing rubber ring; the liquid to be tested with the fixed volume in the liquid fixing pool flows into the test strip in the detection channel through the sample inlet of the detection channel; opening a fluorescence detection system, and detecting fluorescence intensity through a detection window at the bottom of the chip;
(5) cleaning; after the detection is finished, the interface valve of the chip is closed, the bidirectional pneumatic valve is closed, the connection of the bidirectional pneumatic valve is disconnected, the centrifugal locking hole 8 is loosened to take the chip out of the centrifugal detection all-in-one machine, the detection test strip is taken out, the cleaning cover of the centrifugal channel is opened, the control plug is pulled out, and the chip is cleaned for later use.
As a preferred technical scheme, the capacity of each liquid determination pool is precisely calibrated through the height of a residual liquid guide pipe after processing.
As a preferred technical scheme, the cross sections of the hollow round pits and the hollow cylinders are T-shaped.
Preferably, the control plug is removable from the well of the sample addition zone for cleaning.
As a preferred technical scheme, the chip body is fixed on a centrifuge or a centrifugal detection all-in-one machine through a centrifugal locking hole.
As a preferable technical scheme, the height of the partition wall of the liquid determination pool is equal to the height of the plane of the bottom end of the control plug from the bottom plane of the hollow round pit.
As a preferable technical solution, the chip body is disc-shaped.
As a preferable technical scheme, the centrifugal channel and the detection channel are both buried in the chip body.
As a preferred technical scheme, the whole chip is prepared in a layered mode and is connected with glue or bonded together.
As an optimal technical scheme, the chip is provided with different centrifugal channels and detection channels according to the number of the centrifugal channels and the detection channels, the inclination angle of the centrifugal channels, the capacity of the liquid fixing pool and the number of detection windows of the detection channels to form chips with different specification parameters.
Has the advantages that:
1) the invention provides a use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip, which can separate sample liquid, can rapidly, uniformly and automatically distribute the sample liquid and perform constant volume sample addition, overcomes the defect that most of the sample liquid needs to be distributed and quantitatively added by a mechanical arm and a sample injection needle aiming at large sample amount detection at present, greatly reduces accessory parts of detection equipment, simplifies operation and saves detection cost.
2) The application method of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip provided by the invention can be used for centrifugal separation of sample stock solution and multi-channel multi-component marker detection.
3) The invention provides a use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip, which can be used in cooperation with a dry immunofluorescence analyzer, can adopt a targeted quantum dot detection test strip to simultaneously detect C-reactive protein, cardiac troponin, glycosylated hemoglobin, urine microalbumin, prostate specific antigen, alpha fetoprotein and the like, has low detection cost and convenient use, and is easy to popularize, and relates to detection and diagnosis of the protein and general investigation and screening work of related diseases.
Drawings
In order to more clearly illustrate the detailed description of the invention or the technical solutions in the prior art, the drawings that are needed in the detailed description of the invention or the prior art will be briefly described below. Throughout the drawings, like elements or portions are generally identified by like reference numerals. In the drawings, elements or portions are not necessarily drawn to scale.
Fig. 1 is a schematic view of a chip top view structure of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in an embodiment of the present invention.
Fig. 2 is a schematic diagram of a cross-sectional structure of a main view of a sample addition area of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention.
Fig. 3 is a schematic diagram of a main sectional view of a sample injection plug of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention.
Fig. 4 is a schematic structural diagram of a sealing plate for an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in an embodiment of the present invention in a front view.
Fig. 5 is a schematic structural diagram of a sealing plate for an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in an embodiment of the present invention in a front view.
Fig. 6 is a schematic structural diagram of the main view of the upper and lower fluid channels of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention.
Fig. 7 is a schematic structural diagram of the main view of the upper and lower fluid channels of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention.
FIG. 8 is a schematic top view of the sample application region of the movable sealing plug pad of the automatic sample separation and volume determination immunofluorescence quantitative rapid detection microfluidic chip according to the embodiment of the present invention.
FIG. 9 is a schematic top view of the sample application region of the movable sealing plug pad of the automatic sample separation and volume determination immunofluorescence quantitative rapid detection microfluidic chip according to the embodiment of the present invention.
Fig. 10 is a schematic top view of the sample application region of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention with the control plug removed.
Fig. 11 is a schematic top view of the sample application regions of the microfluidic chip without control plugs and the channels of the microfluidic chip for automatic sample separation, volume determination, immunofluorescence and rapid detection in the embodiment of the present invention.
Fig. 12 is a schematic diagram of a cross-sectional front view of a sample addition region of a micro-fluidic chip with detection test strips and a schematic diagram of chip layering processing, wherein the micro-fluidic chip is used for automatic sample separation and volume determination immunofluorescence quantitative rapid detection in an embodiment of the invention.
Fig. 13 is a schematic view of the structure of the bottom of the chip of the automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip in the embodiment of the present invention looking down on the residual liquid pool.
FIG. 14 is a graph showing the results of detection of C-reactive protein in the examples of the present invention.
FIG. 15 is a graph showing the results of glycated hemoglobin assay in the example of the present invention.
FIG. 16 is a graph showing the results of urine microalbumin assay in accordance with the present invention.
FIG. 17 is a graph showing the results of detection of prostate specific antigen in the examples of the present invention.
FIG. 18 is a graph showing the results of detection of alpha-fetoprotein in the examples of the present invention.
In the figure: 1. a chip body; 2. a centrifugal channel; 3. cleaning the cover; 4. detecting a window; 5. a detection channel; 6. a sample adding area; 7. marking a channel; 8. a centrifugal locking hole; 9. a residual liquid pool 10, a liquid fixing pool; 11. a centrate containment plate; 12. a liquid adding pool; 13. a control plug; 14. a port valve; 15. a plug inner cavity; 16. a movable sealing plug pad; 17. a movable bolt; 18. a gasket; 19. upper and lower fluid passages; 20. a seal limiting block; 21. a raffinate conduit; 22. detecting a channel sample inlet; 23. a dividing wall; 23-1, adding the sample stock solution into a partition wall; 23-2, controlling the plug separating wall; 23-3, a liquid determination pool partition wall; 23-4. a residual liquid pool partition wall; 24. sealing the needle; 25. a residual liquid pool vent pipe; 26. detecting the test strip; 27. and (5) processing a boundary line in a layered manner.
Detailed Description
The technical solutions in the embodiments of the present invention will be described clearly and completely below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
It should be noted that all directional indicators (such as upper, lower, left, right, front and rear … …) in the embodiment of the present invention are only used to explain the relative position relationship between the components, the movement situation, etc. in a specific posture (as shown in the drawing), and if the specific posture is changed, the directional indicator is changed accordingly.
In addition, the descriptions related to "first", "second", etc. in the present invention are only for descriptive purposes and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one such feature. In the description of the present invention, "a plurality" means at least two, e.g., two, three, etc., unless specifically limited otherwise.
The invention will now be further described with reference to the accompanying drawings.
Example 1: automatic sample separation constant volume immunofluorescence quantitative rapid detection micro-fluidic chip
In the embodiment of the invention, referring to fig. 1-13, an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip comprises a chip body 1, a sample adding region 6, a centrifugal channel 2 and a detection channel 5, and is characterized in that,
a sample adding region 6 is processed at the circle center of the disc-shaped chip body 1; a cylindrical hollow round pit is processed at the center of the sample adding area 6, and a cylindrical control plug 13 is processed in the hollow round pit in a matching way; the distance between the bottom end plane of the processed control plug 13 and the bottom plane of the round pit is h, and a liquid fixing pool 10 is formed in the area, h, between the bottom plane of the round pit and the bottom of the control plug 13; the liquid-fixing pool 10 is divided into more than 2 by h height liquid-fixing pool dividing walls (23-3);
processing a residual liquid pool 9 with a diameter which is concentric and larger than the diameter of the round pit and annularly wound around the fixed liquid pool 10 at a certain thickness part of the periphery of the round pit of the fixed liquid pool 10, wherein the height of the residual liquid pool 9 is consistent with that of the fixed liquid pool 10h, and the upper plane is flush with the upper plane of the fixed liquid pool 10; the residual liquid pool 9 is divided into more than 2 by the separation wall 23-4 of the residual liquid pool; the residual liquid pool partition wall 23-4 and the fixed liquid pool partition wall 23-3 are in one-to-one correspondence on the same diameter line of the circle center of the circular pit, the separated residual liquid pool 9 and the fixed liquid pool 10 are in one-to-one correspondence in a sector area with the circle center of the circular pit and the same circle center included angle, and the respectively corresponding residual liquid pool 9 and the fixed liquid pool 10 are communicated through 1 residual liquid conduit 21 at the same plane height at the top of the pools; each liquid fixing pool 10 is ensured to correspond to an independent residual liquid pool 9; each residual liquid pool 9 is communicated with 1 independent residual liquid pool vent pipe 25, or is communicated with 1 or more residual liquid pool vent pipes 25 connected with a plurality of residual liquid pools 9, so as to ensure that the redundant liquid of the liquid fixing pool 10 flows into the residual liquid pools 9; the capacity of each liquid determination pool 10 is precisely calibrated through the height of a residual liquid guide pipe 21 after processing; the volumes of the liquid fixing pools 10 of the chips with the same specification are consistent, or the volumes of the liquid fixing pools 10 on the same diameter which is symmetrically distributed on the circumference are consistent; the bottom surface of each liquid fixing pool 10 is provided with 1 detection channel sample inlet 22 with a hydrophobic sealing rubber ring; a detection channel 5 with an end under the liquid fixing pool 10 is arranged under the sample inlet 22 of each detection channel;
the detection channels 5 are uniformly distributed on the circumference of the round pit and move along the direction of the diameter line, and the other end of each detection channel is opened on the side surface of the disc-shaped chip body 1; the detection channel 5 is provided with 2-4 openings as detection windows 4 on the bottom surface of the chip body 1; the test strip 26 can be inserted, installed and fixed in the detection channel 5 from the opening of the detection channel 5; each detection channel 5 is provided with a channel mark 7 on the chip body 1;
processing a hollow cylinder with the same circle center and the diameter larger than that of the round pit from the half height of the round pit to the top; the hollow cylinder and the round pit are completely communicated, and the cross sections of the hollow round pit and the hollow cylinder are T-shaped; the annular hollow cylinder is communicated with the round pit to form a concave table, and the annular concave table is used as a liquid adding pool 12 for detecting the stock solution of the sample;
the liquid adding pool 12 is divided into 2 specifications of a partition wall 23-1 with sample stock solution and a partition wall (23-1) without sample stock solution, and each specification forms a series of small specifications according to the number of the liquid determining pools 10; the sample stock solution adding partition wall 23-1 processed in the solution adding pool 12 corresponds to the upper part and the lower part of the solution fixing pool partition wall 23-3 of the solution fixing pool 10, and the solution adding pool 12 is uniformly divided into more than 2 parts which correspond to the upper part and the lower part of the solution fixing pool 10; 1 to 1, 1 to 2, 1 to 3 and more than are arranged correspondingly, and 1 liquid adding pool 12 corresponds to 1 or 2 or more than 3 liquid fixing pools 10 below; the volume capacity of each liquid adding pool 12 is more than 2 times of the volume capacity of each liquid fixing pool 10, so that sufficient centrifuged test liquid can flow to fill each liquid fixing pool 10, redundant liquid can flow into the residual liquid pool 9, and the constant volume of the detection test liquid in each liquid fixing pool 10 is consistent;
1 centrifugal channel 2 is processed on the outer circular wall of each liquid adding pool 12, the height of the bottom plane of an opening of each centrifugal channel 2 is consistent with that of the bottom plane of the liquid adding pool 12, and liquid adding scale marks are processed on the upper plane of the opening of each centrifugal channel 2; the centrifugal channel 2 has an upward inclination outwards along the center of the circular pit; each centrifugal channel 2 is uniformly distributed on the circumference of a round pit of the sample adding area 6 and is symmetrical one by one on the same diameter line of the circle center of the round pit; the inclination angles of the centrifugal channels 2 of the chip bodies 1 with different specifications are different from 1 degree to 15 degrees according to the viscosity of the detected liquid by the centrifugal channels 2; in order to ensure centrifugal balance, the inclination angles of the centrifugal channels 2 of the same tablet core body 1 are consistent, or the inclination angles of the centrifugal channels 2 with symmetrical diameters are consistent; the outer end of the centrifugal channel 2, namely the highest inclined position, is provided with a horizontal centrifugal channel 2 which is used for collecting centrifugal sediments, the top of the horizontal centrifugal channel 2 is provided with an opening, and a sealed cleaning cover 3 is processed; the cleaning cover 3 is used for conveniently cleaning centrifugal sediments; the design of the centrifugation channel 2 is to facilitate liquid-solid separation and reflux centrifugation of the supernatant.
The cylindrical control plug 13 is internally hollow and is provided with a plug inner cavity 15, and the top surface of the control plug 13 is provided with a port valve 14 which is communicated with the plug inner cavity 15 and is connected with a bidirectional pneumatic valve; a port valve 14 which is communicated with a cavity 15 in the plug and is connected with a bidirectional pneumatic valve is processed on the top surface of the control plug 13; a through hole which traverses the side wall of the control plug 13 is processed on the outer wall of the control plug 13 at the position facing the centrifugal channel 2 of the liquid adding pool 12, and a movable bolt 17 is arranged in the through hole; the movable plug 17 is connected with sealing gaskets 18 at the inner end and the outer end of the side wall of the control plug 13 to form a movable sealing plug gasket 16; the two ends of the movable bolt 17 are connected with sealing gaskets 18 to ensure that the test solution cannot flow into the plug inner cavity 15;
the control plug 13 is provided with a groove corresponding to the upper part of each liquid fixing pool 10 on the outer wall below the bottom surface facing the liquid adding pool 12, so as to form an upper fluid channel 19 and a lower fluid channel 19; or the outer wall below the middle of the movable sealing plug pad 16 is processed to reduce the diameter of the outer wall of the control plug 13 from the upper part corresponding to each fixed liquid pool 10 to the bottom end of the side surface of the control plug 13, a gap is generated between the outer wall of the control plug 13 and the inner wall of the circular pit of the fixed liquid pool 10 to form an upper fluid channel 19 and a lower fluid channel 19, and the upper fluid channel and the lower fluid channel correspond to the sample stock solution adding area 12 and the partition wall 23 of the fixed liquid pool 10 which are consistent up and down; a control plug partition wall 23-2 is also processed on the outer wall of the control plug 13; ensuring that the test solutions are not mixed when flowing down;
a circular centrifugate closing plate 11 matched with the diameter of the plug inner cavity 15 is arranged at the bottom surface of the plug inner cavity 15 of the control plug 13; the bottom surface of the centrifugate closing plate 11 is provided with movable bolts 17 or sealing needles 24 with the same number as the fixed liquid pool 10 at the position corresponding to the detection channel sample inlet 22 of the fixed liquid pool 10 under the control plug 13; the movable bolt 17 penetrates through the bottom surface of the plug inner cavity 15, and the lower end of the movable bolt is provided with a sealing gasket 18; when the centrifugate closing plate 11 abuts against the bottom surface of the cavity 15 in the plug of the control plug 13, the sealing gasket 18 or the sealing needle 24 on the centrifugate closing plate can seal the sample inlet 22 of the detection channel; a sealing limiting block 20 is processed and installed on the inner wall of the lower part of the plug inner cavity 15 of the control plug 13, and the centrifugal liquid sealing plate 11 is limited to move up and down only within a height range of 1 mm-5 mm;
the chip body 1 with different specifications has the following parameters: the number of the centrifugal channels 2 and the detection channels 5, the inclination angle of the centrifugal channels 2, the capacity of the liquid fixing pool 10 and the number of the detection windows of the detection channels 5;
the centrifugal channel 2 and the detection channel 5 are both embedded in the chip body 1; the chip is prepared in a layered way and is glued or bonded together; the chip body 1 is fixed on a centrifugal machine or a centrifugal detection all-in-one machine through a centrifugal locking hole 8; the control plug 13 can be pulled out from the round pit of the sample adding area 6 for cleaning; the detection channel sample inlet 22 with the hydrophobic sealing rubber ring is convenient for the sealing needle 24 of the centrifugate sealing plate 11 to seal.
Example 2: use method of automatic sample separation constant volume rapid detection micro-fluidic chip
The method for detecting by using the automatic sample separation constant volume rapid detection microfluidic chip in the embodiment 1 specifically comprises the following steps:
(1) liquid adding preparation; selecting a chip body 1, wherein the chip body 1 is provided with 4 centrifugal channels 2 and detection channels 5, the inclination angle of the centrifugal channels 2 is 45 degrees, and the number of detection windows of the detection channels 5 is 4; selecting the detection test strips 26 of the detection channels 5 according to the detection items, giving corresponding label marks according to the items (C reactive protein, cardiac troponin, glycosylated hemoglobin, urine microalbumin, prostate specific antigen, alpha fetoprotein) detected by the detection test strips 26 for each detection item, and inserting the detection test strips 26 into the detection channels 5 for fixing; fixing the chip body 1 on the centrifugal detection all-in-one machine through a centrifugal locking hole 8;
connecting a bidirectional pneumatic valve to an interface valve 14 of a control plug 13, starting the bidirectional pneumatic valve, and opening a pneumatic valve air suction valve; a plug inner cavity 15 of the control plug 13 generates negative pressure, so that a movable plug 17 of a movable sealing plug gasket 16 slides inwards to drive a sealing gasket 18 connected with the end head to tighten and wrap inwards to be tightly attached to the outer wall of the control plug 13, an upper fluid channel 19 and a lower fluid channel 19 are blocked, and the communication between the liquid adding pool 12 and the liquid fixing pool 10 is closed; at this time, the centrifugate sealing plate 11 in the plug inner cavity 15 of the control plug 13 slides upwards to cling to the lower part of the sealing limiting block 20 due to the movable plug 17, so as to drive the sealing gasket 18 of the centrifugate sealing plate 11 to leave upwards the detection channel sample inlet 22, or drive the sealing needle 24 of the centrifugate sealing plate 11 to leave upwards the detection channel sample inlet 22 with the hydrophobic sealing rubber ring; the interface valve 14 of the chip body 1 is closed, and the bidirectional pneumatic valve connection is disconnected;
different detection sample stock solutions are symmetrically added into a liquid adding pool 12 with the specification of a sample stock solution adding partition wall (23-1) according to a channel mark 7; if the liquid adding pool 12 and the liquid fixing pool 10 are 1 to 1, only the constant volume is finished; if the liquid adding pool 12 and the liquid fixing pool 10 are 1 to 2 or more, different liquid adding pools 12 and liquid fixing pools 10 are required to finish sample separation and constant volume work;
or the same detection sample stock solution is added into a liquid adding pool 12 without the sample stock solution added into the partition wall (23-1) to complete the sample separation and volume fixing work; the liquid adding amount is just to the liquid adding scale line at the top end of the opening of the centrifugal channel 2 and does not exceed the liquid adding scale line;
(2) centrifuging; selecting 5000 r/min-15000 r/min of revolution according to the test solution conditions, and starting a centrifuge; the detection sample stock solution flows upwards to the section of the centrifugal channel 2 with the highest level along the centrifugal channel 2 under the action of centrifugal force, precipitates, and keeps the pneumatic valve in an air suction opening state for 3-30min after the centrifugation is finished; the supernatant liquid flows back into the liquid adding pool 12 under the action of the gravity of the inclined angle of the centrifugal channel 2, and the centrifugal precipitate is left at the section of the centrifugal channel 2 with the highest level;
(3) fixing the volume of the test solution; connecting a bidirectional pneumatic valve, opening a pneumatic valve of the bidirectional pneumatic valve, and opening an interface valve 14 of the chip body 1; the air pressure pushes away the sealing gasket 18 of the movable sealing plug 16 which wraps and clings to the control plug 13 instantly, and the upper and lower fluid channels 19 are opened; simultaneously, instantly pressing the centrifugate sealing plate 11 down to the bottom surface of the plug inner cavity 15 of the control plug 13, pressing a sealing gasket 18 of the centrifugate sealing plate 11 down to seal a detection channel sample inlet 22, or driving a sealing needle 24 of the centrifugate sealing plate 11 down to seal a detection channel sample inlet 22 with a hydrophobic sealing rubber ring; keeping the pneumatic valve and the pneumatic valve in an open state and standing for 3-30min, allowing the centrifuged supernatant in the liquid adding pool 12 to flow into the liquid fixing pool 10 through the upper and lower fluid channels 19 on the outer wall of the control plug 13, allowing the liquid to flow into the liquid fixing pool 10, and allowing the redundant liquid to flow into the residual liquid pool 9 through the residual liquid guide pipe 21 after the liquid flows over the liquid fixing pool 10, thereby completing the constant volume of the detection sample;
(4) testing; opening an air suction valve of the bidirectional pneumatic valve, and controlling the centrifugate sealing plate 11 in the plug inner cavity 15 of the plug 13 to cling to the lower part of the sealing limiting block 20 due to the upward sliding of the movable bolt 17 under the action of negative pressure, so as to drive the sealing gasket 18 of the centrifugate sealing plate 11 to leave the sample inlet 22 of the detection channel upwards, or drive the sealing needle 24 of the centrifugate sealing plate 11 to leave the sample inlet 22 of the detection channel with the hydrophobic sealing rubber ring upwards; the liquid to be tested which has fixed volume in the liquid fixing pool 10 flows into the test strip 26 in the test channel 5 through the test channel sample inlet 22; opening a fluorescence detection system, and detecting fluorescence intensity through a detection window 4 at the bottom of the chip body 1;
(5) cleaning; after the detection is finished, the interface valve 14 of the chip body 1 is closed, the bidirectional pneumatic valve is closed, the connection of the bidirectional pneumatic valve is disconnected, the centrifugal locking hole 8 is loosened to take the chip body 1 out of the centrifugal detection all-in-one machine, the detection test paper strip 26 is taken out, the cleaning cover 3 of the centrifugal channel 2 is opened, the control plug 13 is pulled out, and the chip body 1 is cleaned for standby.
Referring to fig. 14, the fitting equation of the linear correlation between the detection results of C-reactive protein and the hospital measured values in the embodiment of the present invention is y-0.996 x-0.0635, and the correlation coefficient R2-0.9971, that is, the correlation coefficient R-0.9985. The result of the C-reactive protein detection is not obviously different from the hospital detection result.
Referring to fig. 15, the fitting equation of the linear correlation between the glycated hemoglobin measurement and the hospital measurement in the embodiment of the present invention is y-1.0157 x-0.0712, and the correlation coefficient R2-0.9983, i.e., the correlation coefficient R-0.9991. The result of the glycated hemoglobin test is not obviously different from the result of the hospital test.
Referring to fig. 16, the fitting equation of the linear correlation between the urine microalbumin test results and the hospital measurement values in the embodiment of the present invention is y-1.1037 x +0.0712, and the correlation coefficient R2-0.9951, i.e. the correlation coefficient R-0.9975. The result of the urine microalbumin test is not obviously different from the hospital test result.
Referring to fig. 17, the fitting equation of the linear correlation between the prostate specific antigen detection results and the hospital measurement values in the embodiment of the present invention is y-1.0154 x +0.3315, and the correlation coefficient R2-0.9933, i.e., the correlation coefficient R-0.9967. The result of prostate specific antigen detection is not obviously different from the hospital test result.
Referring to fig. 18, the fitting equation of the linear correlation between the detection result of the alphafetoprotein and the hospital measurement value in the embodiment of the present invention is y-1.0034 x-0.083, and the correlation coefficient R2-0.9960, that is, the correlation coefficient R-0.9980. The results of the alpha fetoprotein test are not obviously different from the results of the hospital test.
The experimental detection result of the target marker determined in the embodiment is fitted with the linear correlation of the hospital measured value, and the correlation coefficients all reach r more than or equal to 0.9900, thereby meeting the requirement that the correlation r is more than 0.9750. And T test Sig (both sides) all reach P ≧ 0.21, i.e., P >0.05 with no significant difference. The quantitative determination effect of the experiment is better.
The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

Claims (10)

1. A use method of an automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip comprises a chip body (1), a sample adding region (6), a centrifugal channel (2) and a detection channel (5),
a sample adding region (6) is processed at the center of the chip body (1); a cylindrical hollow round pit is processed at the center of the sample adding area (6), and a cylindrical control plug (13) is processed in the hollow round pit in a matching way; the bottom end plane of the processed control plug (13) has a certain height from the bottom plane of the hollow round pit to form a liquid fixing pool (10); the liquid determination pool (10) is divided into more than 2 by liquid determination pool dividing walls (23-3);
processing a residual liquid pool (9) which is concentric and has a diameter larger than that of the round pit and annularly surrounds the fixed liquid pool (10) at a certain thickness part of the periphery of the round pit of the fixed liquid pool (10), wherein the height of the residual liquid pool (9) is consistent with that of the fixed liquid pool (10), and the upper plane is flush with that of the fixed liquid pool (10); the residual liquid pool (9) is divided into more than 2 by the partition wall (23-4) of the residual liquid pool; the residual liquid pool partition walls (23-4) and the fixed liquid pool partition walls (23-3) are in one-to-one correspondence on the same diameter line of the circle center of the circular pit, the separated residual liquid pools (9) and the fixed liquid pools (10) are in one-to-one correspondence in a sector area with the circle center of the circular pit and the same circle center included angle, and the respectively corresponding residual liquid pools (9) and the fixed liquid pools (10) are communicated through 1 residual liquid conduit (21) at the same plane height at the top of the pools; each liquid fixing pool (10) is exactly corresponding to an independent residual liquid pool (9); each residual liquid pool (9) is communicated with 1 independent residual liquid pool vent pipe (25), or is communicated with 1 or more residual liquid pool vent pipes (25) connected with a plurality of residual liquid pools (9) so as to ensure that the redundant liquid of the liquid fixing pool (10) flows into the residual liquid pool (9); the volumes of the liquid fixing pools (10) of the chips with the same specification are consistent, or the volumes of the liquid fixing pools (10) on the same diameter which is symmetrically distributed on the circumference are consistent; the bottom surface of each liquid fixing pool (10) is provided with 1 detection channel sample inlet (22) with a hydrophobic sealing rubber ring; a detection channel (5) with one end arranged below the liquid fixing pool (10) is arranged below the sample inlet (22) of each detection channel;
the detection channels (5) are uniformly distributed on the circumference of the round pit and move along the direction of the diameter line, and the other end of each detection channel is opened on the side surface of the chip body (1); 2-4 openings are processed on the bottom surface of the chip body (1) of the detection channel (5) to be used as detection windows (4); the detection test strip (26) can be inserted, installed and fixed in the detection channel (5) from the opening of the detection channel (5); each detection channel (5) is provided with a channel mark (7) on the chip body (1);
processing a hollow cylinder with the same circle center and the diameter larger than that of the round pit from the half height of the round pit to the top; the hollow cylinder is completely communicated with the round pit; the annular hollow cylinder is communicated with the round pit to form a concave table, and the annular concave table is used as a liquid adding pool (12) for detecting the stock solution of the sample;
the liquid adding pool (12) is divided into two specifications of a partition wall (23-1) with sample stock solution and a partition wall (23-1) without sample stock solution, and each specification forms a series of small specifications according to the number of the liquid fixing pools (10); the sample stock solution adding partition wall (23-1) processed in the solution adding pool (12) is vertically corresponding to the solution fixing pool partition wall (23-3) of the solution fixing pool (10), and the solution adding pool (12) is uniformly divided into more than 2 parts which are vertically corresponding to the solution fixing pool (10); the liquid adding pool (12) is provided with 1 pair of n corresponding to the liquid fixing pool (10), wherein n is a natural number more than or equal to 1; the volume capacity of each liquid adding pool (12) is more than 2 times of the volume capacity of each liquid fixing pool (10) and is used for ensuring that enough centrifuged test solution flows over each liquid fixing pool (10) and redundant liquid flows into the residual liquid pool (9) and ensuring that the constant volume of the detection test solution of each liquid fixing pool (10) is consistent;
1 centrifugal channel (2) is processed on the outer circular wall of each liquid adding pool (12), the height of the bottom plane of an opening of each centrifugal channel (2) is consistent with that of the bottom plane of the liquid adding pool (12), and liquid adding scale marks are processed on the upper plane of the opening of each centrifugal channel (2); the centrifugal channel (2) is provided with an upward inclination outward along the center of the round pit; each centrifugal channel (2) is uniformly distributed on the circumference of a round pit of the sample adding region (6) and is symmetrical one by one on the same diameter line of the circle center of the round pit; the inclination angle of the centrifugal channel (2) of the chip body (1) has different specifications of 1-15 degrees according to the viscosity of the detection liquid and the centrifugal channel (2); the inclination angles of the centrifugal channels (2) of the same tablet core body (1) are consistent, or the inclination angles of the centrifugal channels (2) with the same diameter and symmetry are consistent, so as to ensure centrifugal balance; the outer end of the centrifugal channel (2), namely the highest inclined position, is provided with a horizontal centrifugal channel (2) for collecting centrifugal sediments, the top of the horizontal centrifugal channel (2) is opened, and a sealed cleaning cover (3) is processed;
the cylindrical control plug (13) is internally hollow and is provided with a plug inner cavity (15), and the top surface of the control plug (13) is provided with a connector valve (14) which is communicated with the plug inner cavity (15) and is connected with a bidirectional pneumatic valve; a through hole which traverses the side wall of the control plug (13) is processed on the outer wall of the control plug (13) at the position facing the centrifugal channel (2) of the liquid adding pool (12), and a movable bolt (17) is arranged in the through hole; the movable plug (17) is connected with sealing gaskets (18) at the inner end and the outer end of the side wall of the control plug (13) to form a movable sealing plug gasket (16); the two ends of the movable bolt (17) are connected with sealing gaskets (18) to ensure that the test solution cannot flow into the inner cavity (15) of the plug;
a control plug (13) is arranged on the outer wall below the bottom surface opposite to the liquid adding pool (12), and a groove is formed above each liquid fixing pool (10) to form an upper fluid channel and a lower fluid channel (19); or the outer wall below the middle of the movable sealing plug pad (16) is processed to reduce the diameter of the outer wall of the control plug (13) from the upper part corresponding to each fixed liquid pool (10) to the bottom end of the side surface of the control plug (13), a gap is generated between the outer wall of the control plug (13) and the inner wall of a circular pit of the fixed liquid pool (10) to form an upper fluid channel (19) and a lower fluid channel (19), and the upper fluid channel and the lower fluid channel correspond to a sample stock solution adding area (12) and a partition wall (23) of the fixed liquid pool (10) which are consistent up and down; a control plug partition wall (23-2) is also processed on the outer wall of the control plug (13); ensuring that the test solutions are not mixed when flowing down;
a round centrifugate closing plate (11) which is matched with the diameter of the plug inner cavity (15) is arranged at the bottom surface of the plug inner cavity (15) of the control plug (13); the bottom surface of the centrifugate closing plate (11) is provided with movable plugs (17) or sealing needles (24) which have the same number as that of the fixed liquid pool (10) at the position corresponding to the sample inlet (22) of the detection channel of the fixed liquid pool (10) under the control plug (13); the movable bolt (17) penetrates through the bottom surface of the plug inner cavity (15), and the lower end of the movable bolt is provided with a sealing gasket (18); when the centrifugate closing plate (11) abuts against the bottom surface of the plug inner cavity (15) of the control plug (13), a sealing gasket (18) or a sealing needle (24) on the centrifugate closing plate can seal a detection channel sample inlet (22); a sealing limiting block (20) is processed and installed on the inner wall of the lower part of the plug inner cavity (15) of the control plug (13) to limit the centrifugal liquid sealing plate (11) to move up and down only within a height range of 1 mm-5 mm;
the using method comprises the following steps:
(1) liquid adding preparation; according to the requirements for detecting different projects, chips with different specifications are selected, and the parameters of the chips with different specifications are as follows: the number of the centrifugal channels (2) and the detection channels (5), the inclination angle of the centrifugal channels (2), the capacity of the liquid fixing pool (10) and the number of detection windows of the detection channels (5); selecting the detection test strip (26) of each detection channel (5) according to the detection items, and inserting the detection test strip (26) into the detection channel (5) for fixing; fixing the chip (1) on the centrifugal detection all-in-one machine through a centrifugal locking hole (8);
connecting a bidirectional pneumatic valve to an interface valve (14) of a control plug (13), starting the bidirectional pneumatic valve, and opening an air suction valve of the pneumatic valve; a plug inner cavity (15) of the control plug (13) generates negative pressure, so that a movable plug (17) of a movable sealing plug gasket (16) slides inwards to drive a sealing gasket (18) connected with a terminal to tighten and wrap inwards and cling to the outer wall of the control plug (13), an upper fluid channel and a lower fluid channel (19) are blocked, and the communication between the liquid adding pool (12) and the liquid fixing pool (10) is sealed; at the moment, the centrifugate sealing plate (11) in the plug inner cavity (15) of the control plug (13) is tightly attached to the lower part of the sealing limiting block (20) due to the upward sliding of the movable plug (17), and the sealing gasket (18) of the centrifugate sealing plate (11) is driven to upwards leave the sample inlet (22) of the detection channel, or the sealing needle (24) of the centrifugate sealing plate (11) is driven to upwards leave the sample inlet (22) of the detection channel with the hydrophobic sealing rubber ring; closing an interface valve (14) of the chip (1) and disconnecting the bidirectional pneumatic valve;
different detection sample stock solutions are symmetrically added into a liquid adding pool (12) with the specification of a partition wall (23-1) according to a channel mark (7); if the liquid adding pool (12) and the liquid fixing pool (10) are 1 to 1, only constant volume is completed; if the liquid adding pool (12) and the liquid fixing pool (10) are 1 to 2 or more, different liquid adding pools (12) and liquid fixing pools (10) are required to complete sample separation and volume fixing;
or the same detection sample stock solution is added into a liquid adding pool (12) without the sample stock solution added into the partition wall (23-1) to complete the sample separation and constant volume work; the liquid adding amount is just to the liquid adding scale mark at the top end of the opening of the centrifugal channel (2) and does not exceed the liquid adding scale mark;
(2) centrifuging; selecting 5000 r/min-15000 r/min of revolution according to the test solution conditions, and starting a centrifuge; the detection sample stock solution flows upwards to the section of the highest position of the centrifugal channel (2) which is horizontal along the centrifugal channel (2) under the action of centrifugal force, precipitates, and keeps the pneumatic valve in a suction opening state for standing for 3-30min after the centrifugation is finished; the supernatant liquid flows back into the liquid adding pool (12) under the action of the gravity of the inclined angle of the centrifugal channel (2), and the centrifugal precipitate is left at the section of the highest position of the centrifugal channel (2) which is horizontal;
(3) fixing the volume of the test solution; connecting a bidirectional pneumatic valve, opening a pneumatic valve of the bidirectional pneumatic valve, and opening an interface valve (14) of the chip (1); the air pressure pushes away a sealing gasket (18) of a movable sealing plug gasket (16) which wraps and is tightly attached to the control plug (13) instantly, and an upper fluid channel and a lower fluid channel (19) are opened; simultaneously instantly pressing down the centrifugal liquid closing plate (11) to the bottom surface of the plug inner cavity (15) of the control plug (13), pressing down a sealing gasket (18) of the centrifugal liquid closing plate (11) to seal a detection channel sample inlet (22), or driving a sealing needle (24) of the centrifugal liquid closing plate (11) to press down to seal a detection channel sample inlet (22) with a hydrophobic sealing rubber ring; keeping the pneumatic valve and the pneumatic valve in an open state and standing for 3-30min, allowing the centrifuged supernatant in the liquid adding pool (12) to flow into the liquid determining pool (10) through the upper and lower fluid channels (19) on the outer wall of the control plug (13), allowing the liquid to flow into the liquid determining pool (10), and allowing the redundant liquid to flow into the residual liquid pool (9) through the residual liquid guide pipe (21) after the liquid flows over the liquid determining pool (10) to finish the constant volume of the detection sample;
(4) testing; opening an air suction valve of the bidirectional pneumatic valve, and controlling a centrifugate sealing plate (11) in a plug inner cavity (15) of a plug (13) to cling to a sealing limiting block (20) due to upward sliding of a movable bolt (17) under the action of negative pressure so as to drive a sealing gasket (18) of the centrifugate sealing plate (11) to upwards leave a detection channel sample inlet (22) or drive a sealing needle (24) of the centrifugate sealing plate (11) to upwards leave the detection channel sample inlet (22) with a hydrophobic sealing rubber ring; the liquid to be tested which has the fixed volume in the liquid fixing pool (10) flows into a test strip (26) in the test channel (5) through a sample inlet (22) of the test channel; opening a fluorescence detection system, and detecting fluorescence intensity through a detection window (4) at the bottom of the chip (1);
(5) cleaning; after the detection is finished, the interface valve (14) of the chip (1) is closed, the bidirectional pneumatic valve is closed, the connection of the bidirectional pneumatic valve is disconnected, the centrifugal locking hole (8) is loosened to take the chip (1) out of the centrifugal detection all-in-one machine, the detection test paper strip (26) is taken out, the cleaning cover (3) of the centrifugal channel (2) is opened, the control plug (13) is pulled out, and the chip (1) is cleaned for later use.
2. The micro-fluidic chip for the automatic sample separation and volume determination immunofluorescence quantitative rapid detection according to claim 1, wherein the volume of each liquid determination pool (10) is precisely calibrated through the height of a residual liquid guide pipe (21) after processing.
3. The micro-fluidic chip for automatic sample separation and volume constant immunofluorescence quantitative rapid detection according to claim 1, wherein the cross section of the hollow round pit and the hollow cylinder is T-shaped.
4. The micro-fluidic chip for the automatic sample separation and volume measurement immunofluorescence quantitative rapid detection according to claim 1, wherein the control plug (13) can be pulled out and cleaned from a round pit of the sample adding region (6).
5. The micro-fluidic chip for the automatic sample separation, volume measurement, immunofluorescence quantitative and rapid detection according to claim 1, wherein the chip body (1) is fixed on a centrifuge or a centrifugal detection all-in-one machine through a centrifugal locking hole (8).
6. The micro-fluidic chip for the automatic sample separation and volume determination immunofluorescence quantitative rapid detection according to claim 1, wherein the height of the liquid determination pool partition wall (23-3) is equal to the height of the bottom end plane of the control plug (13) from the bottom plane of the hollow round pit.
7. The micro-fluidic chip for the automatic sample separation, volume measurement, immunofluorescence quantitative and rapid detection according to claim 1, wherein the chip body (1) is disc-shaped.
8. The micro-fluidic chip for the automatic sample separation and volume constant immunofluorescence quantitative rapid detection according to claim 1, wherein the centrifugal channel (2) and the detection channel (5) are both embedded in the chip body (1).
9. The micro-fluidic chip for automatic sample and volume separation and immunofluorescence quantitative rapid detection according to claim 1, wherein the whole chip is prepared in layers, and is connected by glue or bonded together.
10. The micro-fluidic chip for the automatic sample separation and volume constant immunofluorescence quantitative rapid detection according to claim 1, wherein the chip is provided with different numbers of the centrifugal channels (2) and the detection channels (5), inclination angles of the centrifugal channels (2), capacities of the liquid fixing pools (10), and numbers of detection windows of the detection channels (5) according to the chip body (1) to form chips with different specification parameters.
CN201910864179.8A 2019-09-12 2019-09-12 Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip Active CN110568201B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910864179.8A CN110568201B (en) 2019-09-12 2019-09-12 Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910864179.8A CN110568201B (en) 2019-09-12 2019-09-12 Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip

Publications (2)

Publication Number Publication Date
CN110568201A CN110568201A (en) 2019-12-13
CN110568201B true CN110568201B (en) 2022-05-24

Family

ID=68779716

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910864179.8A Active CN110568201B (en) 2019-09-12 2019-09-12 Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip

Country Status (1)

Country Link
CN (1) CN110568201B (en)

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1579191A2 (en) * 2002-12-19 2005-09-28 Bayer HealthCare LLC Method and apparatus for separation of particles in a microfluidic device
CN1880329A (en) * 2005-06-13 2006-12-20 中国科学院电子学研究所 Reversible packaging micro fluent chip for separating and purifying biological sample
CN101642725A (en) * 2008-07-16 2010-02-10 奥索临床诊断有限公司 Use of fluid aspiration/dispensing tip as a microcentrifuge tube
CN102175840A (en) * 2010-12-30 2011-09-07 北京大学 Whole blood centrifugal separation chip and preparation method thereof
EP2439262A1 (en) * 2007-08-22 2012-04-11 Samsung Electronics Co., Ltd. Centrifugal force-based microfluidic device for blood chemistry analysis
CN104111324A (en) * 2013-04-15 2014-10-22 贝克顿·迪金森公司 Blood sampling transfer device, blood separation and testing system, and blood sampling transfer system
CN106268994A (en) * 2016-09-22 2017-01-04 博奥生物集团有限公司 A kind of centrifugal type microfludic chip
WO2017066485A1 (en) * 2015-10-13 2017-04-20 Landers James P Devices and methods for extraction, separation and thermocycling
CN107102131A (en) * 2017-07-03 2017-08-29 沈阳微流控生物科技有限公司 A kind of full-automatic micro-fluidic chip fluorescence immunoassay detecting system and its detection method
CN108025320A (en) * 2015-07-17 2018-05-11 统计诊断与创新有限公司 The method and apparatus that sample is separated and collected
CN108704684A (en) * 2018-09-04 2018-10-26 重庆科技学院 A kind of application method of detection multi-layer micro-fluidic chips
CN108760686A (en) * 2018-08-07 2018-11-06 李浩元 Scattered light urbidmetry detects micro-fluidic chip and the biochemical immunity machine using the chip
CN108745426A (en) * 2018-04-24 2018-11-06 齐齐哈尔医学院 A kind of micro-fluidic chip and its preparation method and application for the detection of Alzheimer disease Complicated with Depression blood-related proteins
CN109030369A (en) * 2018-09-08 2018-12-18 重庆科技学院 Multichannel micro-fluidic chip associated with a kind of and cuvette
CN109211870A (en) * 2018-11-17 2019-01-15 郑州亲和力科技有限公司 A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection cTnI
CN109738632A (en) * 2019-01-09 2019-05-10 南京岚煜生物科技有限公司 Multi objective micro-fluidic chip and its application method

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2329276A4 (en) * 2008-10-01 2012-07-04 Samsung Electronics Co Ltd Centrifugal-based microfluid apparatus, method of fabricationg the same, and method of testing samples using the microfluidic apparatus

Patent Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1579191A2 (en) * 2002-12-19 2005-09-28 Bayer HealthCare LLC Method and apparatus for separation of particles in a microfluidic device
CN1880329A (en) * 2005-06-13 2006-12-20 中国科学院电子学研究所 Reversible packaging micro fluent chip for separating and purifying biological sample
EP2439262A1 (en) * 2007-08-22 2012-04-11 Samsung Electronics Co., Ltd. Centrifugal force-based microfluidic device for blood chemistry analysis
CN101642725A (en) * 2008-07-16 2010-02-10 奥索临床诊断有限公司 Use of fluid aspiration/dispensing tip as a microcentrifuge tube
CN102175840A (en) * 2010-12-30 2011-09-07 北京大学 Whole blood centrifugal separation chip and preparation method thereof
CN104111324A (en) * 2013-04-15 2014-10-22 贝克顿·迪金森公司 Blood sampling transfer device, blood separation and testing system, and blood sampling transfer system
CN108025320A (en) * 2015-07-17 2018-05-11 统计诊断与创新有限公司 The method and apparatus that sample is separated and collected
WO2017066485A1 (en) * 2015-10-13 2017-04-20 Landers James P Devices and methods for extraction, separation and thermocycling
CN106268994A (en) * 2016-09-22 2017-01-04 博奥生物集团有限公司 A kind of centrifugal type microfludic chip
CN107102131A (en) * 2017-07-03 2017-08-29 沈阳微流控生物科技有限公司 A kind of full-automatic micro-fluidic chip fluorescence immunoassay detecting system and its detection method
CN108745426A (en) * 2018-04-24 2018-11-06 齐齐哈尔医学院 A kind of micro-fluidic chip and its preparation method and application for the detection of Alzheimer disease Complicated with Depression blood-related proteins
CN108760686A (en) * 2018-08-07 2018-11-06 李浩元 Scattered light urbidmetry detects micro-fluidic chip and the biochemical immunity machine using the chip
CN108704684A (en) * 2018-09-04 2018-10-26 重庆科技学院 A kind of application method of detection multi-layer micro-fluidic chips
CN109030369A (en) * 2018-09-08 2018-12-18 重庆科技学院 Multichannel micro-fluidic chip associated with a kind of and cuvette
CN109211870A (en) * 2018-11-17 2019-01-15 郑州亲和力科技有限公司 A kind of micro-fluidic fluorescence immunoassay chip of rapid quantitative detection cTnI
CN109738632A (en) * 2019-01-09 2019-05-10 南京岚煜生物科技有限公司 Multi objective micro-fluidic chip and its application method

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
"Volumetric measurements by image segmentation on centrifugal microfluidic platforms in motion";Kazarine A 等;《Lab on a Chip》;20141231;第20卷(第10期);2733-2739 *
微流控芯片电泳分离血清中小而密低密度脂蛋白的研究;汪骅等;《分析化学》;20081115;第36卷(第11期);1531-1534 *

Also Published As

Publication number Publication date
CN110568201A (en) 2019-12-13

Similar Documents

Publication Publication Date Title
CN110568200B (en) Combined diagnosis paper-based micro-fluidic chip and detection method
US20180272331A1 (en) Device for preparing and dispensing biological sample
CN106662573B (en) For determining the container of urinary precipitation in analyte and/or chemico-physical parameter and determining urine in urine；And the method for using the container carrying out full urinalysis
JPH03170060A (en) Tester of molecular sample in organic fluid and method of testing prescribed molecule in blood
CN102841210A (en) Retinol detection kit and preparation method thereof
KR20190106128A (en) Concentration analyzing system of analytes in biological sample using diagnosis kit capable of identifying concentration of analyte and portable terminal
CN110568201B (en) Use method of automatic sample separation constant volume immunofluorescence quantitative rapid detection microfluidic chip
CN110568202B (en) Automatic sample separation constant volume immunofluorescence quantitative rapid detection micro-fluidic chip
CN110568199B (en) Multi-channel fluorescence immunochromatography detection microfluidic chip
EP4006553A1 (en) Blood group antigen testing component
KR102451165B1 (en) The pressure-type sampling device
KR102520865B1 (en) The pressure-type sampling device
CN217410812U (en) Multi-channel thromboelastogram detection chip based on micro-fluidic
CN110568203B (en) Application method of multi-channel fluorescence immunochromatographic microfluidic chip
CN109212185B (en) One-step fast detection kit for pepsinogen I
CN206146947U (en) Guang chalone C and joint detect reagent box of beta 2 microglobulin sxemiquantitative gold mark
CN112280665A (en) Gene detection equipment integrating extraction, amplification and detection
CN112226352A (en) Multifunctional gene detection device
CN208399514U (en) A kind of micro-fluidic immunoassay device
CN109212183B (en) One-step fecal hemoglobin rapid detection kit
CN105716905A (en) Sample collecting and detecting method
CN210045262U (en) Cardiovascular emergency detection micro-fluidic chip
CN2485656Y (en) Plasma separating and analyzing reagnet box
CN217377883U (en) Molecular diagnosis test card
CN212433188U (en) Reagent card for detecting dog blood type and cat blood type by chromatography

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant