CN109647553B - Multi-index disease joint detection microfluidic device - Google Patents
Multi-index disease joint detection microfluidic device Download PDFInfo
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- CN109647553B CN109647553B CN201811635744.5A CN201811635744A CN109647553B CN 109647553 B CN109647553 B CN 109647553B CN 201811635744 A CN201811635744 A CN 201811635744A CN 109647553 B CN109647553 B CN 109647553B
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- 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
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- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention discloses a multi-index disease joint detection microfluidic device, which comprises: the device comprises a microfluidic reaction chip, a microfluidic storage chip, a direct current motor driving module, a reaction chip fluid control module, a storage and release fluid control module, a temperature control module and a CCD detection module. Through the mutual matching of the reaction chip fluid control module, the storage and release fluid control module, the temperature control module and the microfluidic chip, the mixing of a sample and a sample diluent is respectively completed, the capture of an antibody coated on the surface of the microsphere to a specific antigen of a target cell, the cleaning of a cell antigen nonspecifically adsorbed on the surface of the microsphere, the capture of a target cell antigen specifically adsorbed on the surface of the microsphere to an enzyme-labeled antibody, the mixing of an exciting liquid A, B and the chemiluminescence process of an enzyme-catalyzed exciting liquid specifically adsorbed on the surface of the microsphere are completed, and a CCD detection module is matched to collect and detect chemiluminescence signals on the surface of the microsphere, so that the automatic operation of the capture and detection processes of a plurality of index antigens in a serum sample is realized.
Description
Technical Field
The invention relates to the field of detection and diagnosis of life medicine, in particular to a multi-index disease joint detection microfluidic chip and a detection device based on enzyme-linked immunosorbent assay and chemiluminescence principle. The microfluidic detection device can be used for simultaneously detecting a plurality of disease markers, such as five indexes for detecting prenatal and postnatal care: levels of five IgG (immunoglobulin) antibodies in serum, such as Toxoplasma gondii (TOX), Rubella (RUB), giant Cell (CMV), herpes (HSV-1), herpes (HSV-2), and the like.
Background
Enzyme-linked immunosorbent assay (ELISA), the principle of which is to demonstrate the presence of a specific protein through a color change or luminescence reaction with an enzyme, by the specificity of the bond between an antigen (i.e., protein) and an antibody, has been widely used in various disease diagnoses based on protein detection. Highly specific antibody reactions and result amplification processes have been widely used in immunoassays, environmental analyses, and biotechnology research. In conventional protocols, the ELISA detection process first generates an antibody specific for the expressed protein targeting the foreign transgene, which is referred to as the primary antibody (or detection antibody). In the detection process, the humoral protein to be detected is first placed on a membrane that prevents the binding of non-specific antibodies. Primary antibodies are added to detect and capture specific proteins. A secondary antibody (or enzyme-labeled antibody) is then added. The secondary antibody is linked to an enzyme and is specific for the primary antibody. Specific linkage generated by the secondary antibody and the primary antibody carries the enzyme to the site of the protein, followed by addition of the enzyme substrate. Over a period of time, the enzyme catalyzes a reaction of the substrate that involves a color change or luminescence. Finally, if the remaining color change or a particular product remains at a particular location on the membrane, the presence of a particular protein is confirmed.
In the traditional ELISA detection, a large-scale biomedical diagnostic instrument is often required, the price is high, the detection process is complex, the detection cost is high, the detection time is long, the efficiency is low, and the clinical popularization of the diagnostic method is not facilitated. Particularly, the traditional ELISA detection method can only complete single index detection at one time, and the detection efficiency is low; current biomedical detection methods require faster analysis and measurement procedures. The platform of the test sample and the test solution is reduced, and the automatic, integrated, high-throughput, multi-index, low-cost and portable Point-of-Care test (POI-of-Care test) simultaneous combined detection can be realized.
Disclosure of Invention
The invention aims to design an integrated, portable and widely-applied multi-index disease combined detection micro-fluidic chip automatic detection system based on enzyme-linked immunosorbent assay and chemiluminescence principles. The invention can be applied to various disease diagnoses based on protein detection, takes the detection of five indexes of prepotency as an implementation example, and realizes the automatic combined detection of the five indexes of prepotency (toxoplasma, rubella, giant cells, herpes-I and herpes-II).
In order to achieve the above object, the present invention adopts a technical solution of a multi-index disease joint detection microfluidic device, which is characterized in that the detection device comprises: the device comprises a microfluidic reaction chip, a microfluidic storage chip, a direct current motor driving module, a reaction chip fluid control module, a storage and release fluid control module, a temperature control module and a CCD detection module. The method comprises the steps of respectively completing the mixing of a sample and a sample diluent through the mutual matching of a reaction chip fluid control module, a storage and release fluid control module, a temperature control module and a microfluidic chip, capturing a target cell specific antigen by an antibody coated on the surface of a microsphere, cleaning a cell antigen nonspecifically adsorbed on the surface of the microsphere, capturing an enzyme-labeled antibody by a target cell antigen specifically adsorbed on the surface of the microsphere, mixing an excitation liquid A, B, catalyzing an excitation liquid chemiluminescence process by enzyme of microsphere surface specific adsorption, and collecting and detecting chemiluminescence signals on the surface of the microsphere by matching with a CCD (charge coupled device) detection module, so that the integrated operation of capturing and detecting processes of a plurality of index antigens in a serum sample is realized. Multi-index antigens such as: five indexes of prepotency include TOX (Toxoplasma gondii), RUB (rubella), CMV (giant cell), HSV-1 (herpes 1), HSV-2 (herpes 2);
the microfluidic storage chip 1 is composed of a storage chip top layer sealing soft film 2, a storage chip upper cover plate, a storage chip liquid storage layer 4, a storage chip lower cover plate 5 and a storage chip bottom layer sealing soft film 6, wherein the storage chip top layer sealing soft film 2, the storage chip upper cover plate, the storage chip liquid storage layer 4, the storage chip lower cover plate 5 and the storage chip bottom layer sealing soft film 6 are sequentially connected from top to bottom. The storage chip liquid storage layer 4 is provided with a sample solution storage cavity 7, a sample diluent storage cavity 8, a cleaning solution storage cavity I9, an enzyme-labeled antibody solution storage cavity 10, a cleaning solution storage cavity II 11, an exciting liquid A storage cavity 12, an exciting liquid B storage cavity 13, a related reagent release channel 14 and a related cut-off air channel 15; a sample solution storage cavity 7, a sample diluent storage cavity 8, a cleaning solution storage cavity I9, an enzyme-labeled antibody solution storage cavity 10, a cleaning solution storage cavity II 11, an exciting liquid A storage cavity 12 and an exciting liquid B storage cavity 13 are dispersedly arranged on the storage chip liquid storage layer 4; the sample solution storage cavity 7, the sample diluent storage cavity 8, the first cleaning solution storage cavity 9, the enzyme-labeled antibody solution storage cavity 10, the second cleaning solution storage cavity 11, the exciting liquid A storage cavity 12 and the exciting liquid B storage cavity 13 are all connected with the cut-off air channel 15 through a reagent release channel 14;
the microfluidic reaction chip 16 consists of a reaction chip upper cover plate 17, a reaction chip main body layer 18, microspheres 19, a waste liquid storage layer 20 and a reaction chip lower cover plate 21; the upper cover plate 17 of the reaction chip, the main body layer 18 of the reaction chip, the waste liquid storage layer 20 and the lower cover plate 21 of the reaction chip are connected up and down in sequence; the microspheres 19 are arranged in the reaction chip main body layer 18; the upper cover plate 17 of the reaction chip comprises three pins 22 for assembling the microfluidic storage chip 1 and seven air holes, wherein the seven air holes are respectively a sample inlet, a first cleaning solution inlet 25, an enzyme-labeled antibody solution inlet 26, a second cleaning solution inlet 27, an exciting solution inlet, an injector vent hole 29 and an air suction pump vent hole 30. The reaction chip main body layer 18 comprises a sample dilute mixing channel 31, a cleaning solution channel I, an enzyme-labeled antibody solution channel, a cleaning solution channel II, an exciting solution mixing channel 35, a biochemical reaction channel 36, five microsphere fixing grooves 37, an injector vent hole 29, an air suction pump vent hole 30 and a waste liquid cavity inlet 38. The waste liquid storage layer comprises a waste liquid cavity and absorbent paper 40;
the first reaction chip fluid control system device is formed by connecting an air pump connecting hole to an air pump through an air pump connecting hose 42, the other end of the air pump is connected with an air pump electromagnetic valve, and the air pump is fixed on a bottom plate 46 through an air pump bracket 45;
the second reaction chip fluid control system device is formed by connecting an injector connecting hole to a small injector 49 through an injector connecting hose 48, and the tail part of the small injector 49 is connected with a micro stepping motor 50; the small injector 49 and the micro stepping motor 50 are respectively fixed on the bottom plate 46 by an injector bracket 51 and a micro stepping motor bracket 52;
the direct current motor driving module comprises a direct current motor 53, a screw 54, a sliding block, an upper touch switch 56, a lower touch switch, a first guide rail 58 and a second guide rail 59. When the module works, the direct current motor 53 drives the screw rod 54 to rotate, so that the sliding block on the screw rod 54 is driven to move up and down, the sliding block is positioned through the upper touch switch 56 and the lower touch switch, and the sliding block is guided through the first guide rail 58 and the second guide rail 59;
the storage and release fluid control module comprises a pressing block 60, a rubber column, a vent needle tube, a vent column hose 64 and a solenoid valve group 65. The pressing block 60 is fixed on the sliding block of the direct current motor driving module, the pressing block 60 moves downwards under the action of the direct current motor driving module, and fourteen ventilation needle tubes are inserted into the storage chip top layer sealing soft film 2. Each vent needle tube is communicated with the corresponding vent column, a vent column hose 64 is hermetically assembled on each vent column, each vent column hose 64 is correspondingly connected with one of fourteen inlets of two electromagnetic valve groups 65, the electromagnetic valve groups 65 are fixed on an electromagnetic valve group seat 66, and the electromagnetic valve group seat 66 is fixed on the bottom plate 46;
the temperature control module is embedded in the chip composite function base 67 and comprises a heating resistance film 68, a heating aluminum block 69 and a thermistor 70; the heating resistance film 68 is adhered to the lower surface of the heating aluminum block 69, the thermistor 70 is fixed in the groove in the middle of the heating aluminum block 69, and the heating aluminum block 69 is tightly attached to the right lower part of the biochemical reaction channel 36 of the microfluidic reaction chip 16; the position of the composite function base 67 and the bottom plate 46 is determined by mechanical fixation;
the CCD detection module comprises a CCD chemiluminescence intensity detector 71 which is positioned right above the biochemical reaction channel 36 in the microfluidic reaction chip 16.
A sample dilute mixing channel 31 and an exciting liquid mixing channel 35 are arranged in a reaction chip main body layer 18 of the microfluidic reaction chip 16; the two mixing channels are composed of three parts, namely a mixing channel premixing cavity 72, a Z-shaped narrow channel 73 and a mixing channel final mixing cavity 74, wherein liquid inlets and outlets of the mixing channel premixing cavity 72 and the mixing channel final mixing cavity 74 are in a horn shape, and the two cavities are connected through the Z-shaped narrow channel 73; when the mixing device works, two liquids sequentially enter the mixing channel premixing cavity 72, enter the mixing channel final mixing cavity 74 through the Z-shaped narrow channel 73 under the driving of the injector 49, and return to the mixing channel premixing cavity 72 through the Z-shaped narrow channel 73, so that the two liquids are mixed under the reciprocating motion.
The reaction chip main body layer 18 of the microfluidic reaction chip 16 is provided with a biochemical reaction channel 36 and five microsphere fixing grooves 37; the microsphere fixing groove 37 is a hemispherical groove in the biochemical reaction channel 36, and the microspheres 19 are placed in the microsphere fixing groove 37; the distance from the center of the cross section of the microsphere fixing groove 37 to the two vertical surfaces of the biochemical reaction channel 36 is equal; the inlet of the biochemical reaction channel 36 is horn-shaped, and the whole biochemical reaction channel is Z-shaped, but the biochemical reaction channel can also be L-shaped or straight line section-shaped; connected to the end of the biochemical reaction channel is a waste chamber inlet 38.
The waste liquid storage layer 20 of the microfluidic reaction chip 16 is adhered below the reaction chip main body layer 18, various reacted samples, reagents and cleaning liquid are collectively called waste liquid, and the waste liquid enters the waste liquid cavity through the waste liquid cavity inlet 38 and is absorbed by absorbent paper 40; the waste chamber volume communicates with the syringe 49 through the syringe vent 29 and with the suction pump through the suction pump vent 30.
A sample dilute mixing channel 31, a cleaning solution channel I, an enzyme-labeled antibody solution channel, a cleaning solution channel II and an exciting liquid mixing channel 35 are arranged in a reaction chip main body layer 18 of the microfluidic reaction chip 16; the sample diluent enters the biochemical reaction channel 36 to react with the microspheres 19, and then a plurality of cleaning solutions alternately pass through the first cleaning solution channel and the second cleaning solution channel to wash the biochemical reaction channel 36.
The storage chip liquid storage layer 4 of the microfluidic storage chip 1 is provided with seven storage cavities, and each storage cavity is connected with an upstream passage 79, a reagent release channel 14 and a cut-off air channel 15; after the injector 49 is moved to give negative pressure, the upstream passage 79 of the storage chamber which needs to release liquid is communicated with the atmosphere under the condition that other through holes are not ventilated, so that the liquid in the chamber is released outwards, the upstream passage 79 is not ventilated after a certain amount is released, the cut-off air channel 15 is communicated with the atmosphere, and the injector 49 is continuously given negative pressure to cut off the liquid in the reagent release channel 14, so that the function of releasing the liquid in a quantitative volume is completed.
Fourteen through holes are arranged on the upper cover plate of the storage chip of the microfluidic storage chip 1, namely upstream through holes 75 and cut-off air holes 76 of seven reagent storage cavities; a top sealing soft film 2 of the storage chip is adhered above the upper cover plate of the storage chip, and the material of the top sealing soft film can be rubber or other soft materials meeting the requirement; the lower cover plate 5 of the storage chip is provided with five liquid releasing holes 77 which respectively correspond to the inlets of the five channels of the reaction chip main body layer 18; a storage chip bottom layer sealing soft film 6 is adhered to the lower part of the storage chip lower cover plate 5, and five cross-shaped openings are formed in the storage chip bottom layer sealing soft film 6; one cross opening corresponds to a through hole on the lower cover plate 5 of the storage chip, and the cross opening can be opened under the pressure action of the pressing block 60 and the rubber column, so that the microfluidic storage chip 1 is communicated with the microfluidic reaction chip 16.
In the direct current motor driving module, the position of the pressing block 60 when lifted is determined by the upper touch switch 56, and the position of the pressing block 60 when extruded is determined by the lower touch switch; when the pressing block 60 is extruded, the microfluidic reaction chip 16 and the microfluidic storage chip 1 are hermetically combined through the storage chip bottom layer sealing soft film 6, and meanwhile, the communication process of the microfluidic storage chip 1 and the microfluidic reaction chip 16 is completed; fourteen venting needle tubes pierce the sealing soft film 2 on the top layer of the storage chip and are correspondingly inserted into the upstream through hole 75 or the cutoff air hole 76, so that the venting needle tubes are connected with corresponding channels of fourteen venting columns above the pressing block.
Fourteen ventilation columns above the pressing block are in one-to-one corresponding sealing connection with two groups of electromagnetic valve groups 65 with seven inlets through fourteen ventilation column hoses 64; the single chip microcomputer controls the opening and closing of one electromagnetic valve in the electromagnetic valve group 65, so that one ventilation column is communicated with the atmosphere, and further one upstream passage 79 is communicated with the atmosphere.
After the micro-fluidic storage chip 1 is communicated with the micro-fluidic reaction chip 16 and the micro-fluidic storage chip 1 is communicated with the electromagnetic valve group 65, the micro stepping motor 50 is controlled by the microprocessor to rotate, and the tail part of the small injector 49 is driven by the thread structure to realize the control of fluid in the device, so that the mixing of two kinds of liquid and the directional flow of various kinds of liquid are finished; the thread structure converts a larger angle of rotation of the micro stepper motor 50 into a smaller displacement of the rear of the small syringe 49, making the control of the displacement of the syringe 49 more accurate.
In the temperature control module, the heating aluminum block 69 is tightly attached to the lower surface of the microfluidic reaction chip 16 and is positioned right below the biochemical reaction channel 36, the heating resistance film 68 is adhered to the lower surface of the heating aluminum block 69, the heating resistance film 68 and the heating aluminum block are physically adhered by adopting a pressure-sensitive double-sided adhesive, or are chemically adhered by adopting an organic solvent or thermal adhesion, the thermistor 70 is fixed in a groove in the middle of the aluminum block, the microprocessor heats the aluminum block by controlling the heating resistance film 68, the temperature control of the aluminum block is realized by matching with the thermistor 70, and the aluminum block further realizes the reaction temperature control of reagents in the liquid storage chip by virtue of a heat conduction effect.
Compared with the prior art, the invention has the following beneficial effects
1. The invention uses an automatic and integrated working mode to complete the operation steps of capturing and detecting five target antigens in a sample through the connection and the matching among a microfluidic reaction chip, a microfluidic storage chip, a direct current motor driving module, a reaction chip fluid control module, a storage and release fluid control module, a temperature control module and a CCD detection module, and realizes the combined detection of multi-index diseases in a serum sample in a production line manner.
2. The invention provides a multi-liquid mixing mode applied to a micro-fluidic chip platform, which utilizes the structures of a mixing channel premixing cavity, a Z-shaped narrow channel and a mixing channel final mixing cavity to complete the full mixing of various microfluids under the action of a fluid driving device.
3. The invention fixes the micro-beads in the micro-fluidic chip, and automatically realizes the allocation and transportation of different reagents, thereby completing the processes of capturing a plurality of target cell antigens by the antibody coated on the surface of the micro-beads, cleaning the cell antigens non-specifically adsorbed on the surface of the micro-beads, capturing the enzyme-labeled antibody by the target cell antigens specifically adsorbed on the surface of the micro-beads and the like in a short time.
4. The invention designs a microfluidic storage chip with a reagent storage function, and the quantitative sequential release of the reagent volume in the microfluidic storage chip can be realized by matching with other devices in the invention; the microfluidic reaction chip provided by the invention is provided with the waste liquid cavity, and can be used for hermetically storing various waste liquids generated in the detection process, so that the on-site rapid detection (POCT) can be conveniently realized.
5. The invention has the advantages of small volume, simple operation, high automation degree, high detection sensitivity, simple structure, low cost and the like, can expand and supplement the existing detection mode, and can realize the high-efficiency combined detection of multi-index diseases by using microspheres coated by different antibodies.
Drawings
Fig. 1 is a schematic structural diagram of a multi-index disease joint detection microfluidic device.
Fig. 2.1 shows the structure of each layer of the microfluidic storage chip of the multi-index disease joint detection microfluidic device.
Fig. 2.2 shows the top view angle structures of the liquid storage layer of the storage chip of the multi-index disease joint detection microfluidic device.
Fig. 2.3 shows the bottom view angle structures of the liquid storage layer of the storage chip of the multi-index disease joint detection microfluidic device.
Fig. 3.1 shows the structure of each layer of the microfluidic reaction chip of the multi-index disease joint detection microfluidic device.
Fig. 3.2 is a main body layer structure of a microfluidic reaction chip of the multi-index disease joint detection microfluidic device.
Fig. 4 is a block diagram of a direct current motor driving module of the multi-index disease joint detection microfluidic device.
Fig. 5 is a diagram of a temperature control module of the multi-index disease joint detection microfluidic device. In the figure:
1. microfluidic storage chip 2 and storage chip top layer sealing soft film
4. Storage chip liquid storage layer 5, storage chip lower cover plate 6 and storage chip bottom layer sealing soft film
7. A sample solution storage cavity 8, a sample diluent storage cavity 9 and a cleaning solution storage cavity I
10. An enzyme-labeled antibody solution storage cavity 11, a cleaning solution storage cavity II 12 and an exciting liquid A storage cavity
13. Exciting liquid B storage cavity 14, reagent release channel 15 and cut-off air channel
16. Microfluidic reaction chip 17, upper cover plate 18 of reaction chip, and reaction chip main body layer
19. Microsphere 20, waste liquid storage layer 21 and reaction chip lower cover plate
22. Pin bolt
25. A first cleaning liquid inlet 26, an enzyme-labeled antibody solution inlet 27 and a second cleaning liquid inlet
An exciting liquid inlet 29, an injector vent 30, and an air pump vent
31. Sample dilute mixing channel
35. Exciting liquid mixing channel 36 and biochemical reaction channel
37. Microsphere fixing groove 38 and waste liquid cavity inlet
40. Water absorption paper 42 and air pump connecting hose
45. Air pump support
46. Bottom plate 48, syringe connection hose
49. Syringe 50, micro stepping motor 51, syringe support
52. Miniature stepping motor support 53, direct current motor 54, screw rod
56. Upper touch switch
58. Guide rail 159, guide rail 260, press block
64. Air vent column hose 65, solenoid valve group 66 and solenoid valve group seat
67. Composite functional base 68, heating resistance film 69, heating aluminum block
70. Thermistor 71, CCD chemiluminescence intensity detector 72 and mixing channel premixing cavity
73. Z-shaped narrow channel 74, mixing channel final mixing cavity 75 and upstream through hole
76. Shut-off vent 77, liquid release vent
79. Upstream passage 80, pin hole
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to fig. 1 to 5 in the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. The following description of an exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. 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.
The relative arrangement of the components and steps, the numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless specifically stated otherwise.
Meanwhile, it should be understood that the sizes of the respective portions shown in the drawings are not drawn in an actual proportional relationship for the convenience of description.
Techniques, methods, and apparatus known to those of ordinary skill in the relevant art may not be discussed in detail but are intended to be part of the specification where appropriate.
In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
The operation process of one embodiment of the invention is that the complete detection flow of the device is as follows, firstly, the upper layer and the lower layer of plastic sealing films of the microfluidic storage chip 1 are torn off, and a sample is extracted by a syringe and injected into the sample solution storage cavity 7 of the microfluidic storage chip 1. Removing the plastic sealing film on the top of the microfluidic reaction chip 16, placing the microfluidic reaction chip 16 in the chip composite function base 67, and positioning the two chips by the pin 22 of the microfluidic storage chip 1 and the pin hole 80 on the microfluidic reaction chip 16; a direct current motor 53 of the direct current motor driving module drives a sliding block to move from the position of an upper touch switch 56 to the position of a lower touch switch, so that a pressing block 60 tightly presses the microfluidic reaction chip 16 and the microfluidic storage chip 1 by using a rubber column; while the pressure block 60 is pressed, the vent needle tubes on the pressure block are inserted into the corresponding reagent release channel 14 and the cut-off air channel 15 of the microfluidic storage chip 1; the microprocessor controls the heating resistor film 68 to cooperate with the thermistor 70 to heat the three aluminum blocks so that the biochemical reaction channel 36 is kept at a constant temperature.
Opening the electromagnetic valve corresponding to the upstream through hole 75 of the sample solution storage cavity 7, closing all the other electromagnetic valves, utilizing the tail part of the small injector 49 and the micro stepping motor 50 to drive the injector to move for a corresponding distance, releasing a certain amount of sample solution, closing the reagent release channel 14 of the sample diluent storage cavity 8, opening the air intercepting channel 15 of the sample diluent storage cavity 8, enabling the sample and the diluent to jointly enter the sample diluent mixing channel 31 of the microfluidic reaction chip 16 by the corresponding distance, enabling the reagent to enter the mixing channel final mixing cavity 74 from the corresponding Z-shaped narrow channel 73 under the driving of the injector 49, and returning to the mixing channel premixing cavity 72 from the Z-shaped narrow channel 73, and completing the mixing of the sample and the cleaning solution under the reciprocating motion of the two liquids. After mixing, the reagent is driven to the biochemical reaction channel 36 by the injector, and the mixed reagent reacts with the microspheres in the five microsphere fixing grooves 37 in the biochemical reaction channel 36 for a certain time. During the reaction, the small injector 49 makes a back-and-forth micro-motion to drive the reagent in the biochemical reaction channel 36 to make a micro-reciprocating motion, so that the reaction reagent and the microspheres make a relative motion, thereby accelerating the reaction. And after the set time is reached, opening the air pump, and simultaneously opening an electromagnetic valve connected with the air pump to pump the reagent in the biochemical reaction channel to the waste liquid cavity.
Opening the reagent release channel 14 of the first cleaning solution storage cavity 9, moving the small injector for a corresponding distance to release a corresponding volume of sample diluent, for example, 250ul, closing the reagent release channel 14 of the first cleaning solution storage cavity 9, opening the first cleaning solution storage cavity 9 to block the air channel 15, moving the small injector for a corresponding distance to enable the cleaning solution to enter the cleaning solution channel of the microfluidic reaction chip 16 and enter the biochemical reaction channel 36 again, cleaning the biochemical reaction channel 36 for a period of time by the cleaning solution, opening the air pump, and simultaneously opening the electromagnetic valve connected with the air pump to pump the reagent in the biochemical reaction channel to the waste liquid cavity; the cleaning process is repeated many times.
Opening a reagent release channel 14 of an enzyme-labeled antibody solution storage cavity 10, moving a small injector for a corresponding distance to release an enzyme-labeled antibody solution storage cavity with a corresponding volume, for example, 200ul, closing the reagent release channel 14 of the enzyme-labeled antibody solution storage cavity 10, opening an intercepting air channel 15 of the enzyme-labeled antibody solution storage cavity 10, moving the small injector for a corresponding distance to enable washing liquor to enter an enzyme-labeled antibody solution channel of a microfluidic reaction chip 16 and then enter a biochemical reaction channel 36 for a corresponding reaction time, and performing back-and-forth micro-motion by using a small injector 49 during the reaction to drive a reagent in the biochemical reaction channel 36 to perform micro-reciprocating motion, so that the reaction reagent and microspheres perform relative motion, thereby accelerating the reaction. And after the set time is reached, opening the air pump, and simultaneously opening an electromagnetic valve connected with the air pump to pump the reagent in the biochemical reaction channel to the waste liquid cavity.
Opening the reagent release channel 14 of the second cleaning solution storage cavity 11, moving the small injector for a corresponding distance to release a corresponding volume of cleaning solution, for example, 250ul, closing the reagent release channel 14 of the second cleaning solution storage cavity 11, opening the air intercepting channel 15 of the second cleaning solution storage cavity 11, moving the small injector for a corresponding distance to enable the cleaning solution to enter the second cleaning solution channel of the microfluidic reaction chip 16 and then enter the biochemical reaction channel 36, cleaning the biochemical reaction channel 36 for a period of time by the cleaning solution, opening the air pump, and simultaneously opening the electromagnetic valve connected with the air pump to pump the reagent in the biochemical reaction channel to the waste liquid cavity; the cleaning process is repeated many times.
The small syringe moves corresponding distance to release corresponding volume of the exciting liquid A, for example, 125ul by opening the reagent release channel 14 of the exciting liquid A storage chamber 12, and the small syringe moves corresponding distance to release corresponding volume of the exciting liquid A, for example, 125ul by opening the reagent release channel 14 of the exciting liquid A storage chamber 12 and opening the reagent release channel 14 of the exciting liquid B storage chamber 13. Closing the reagent release channel 14 of the excitation liquid B storage cavity 13, opening the cut-off air channel 15 of the excitation liquid B storage cavity 13, driving the reagent to the excitation liquid mixing channel 35 by the small-sized injector, completing the mixing of the excitation liquid by the movement of the reagent in the mixing channel premixing cavity 72 and the mixing channel final mixing cavity 74, driving the reagent to the biochemical reaction channel 36 after the mixing is completed, exciting the excitation liquid by the reaction product on the microspheres to generate chemiluminescence, and finally acquiring and detecting the chemiluminescence signals on the surfaces of the microspheres 19 in the five liquid storage cavities by the CCD chemiluminescence intensity detector 71.
Therefore, the automatic and integrated combined detection process of the multi-index disease combined detection microfluidic device for the five indexes of prepotency is realized.
It will be understood by those skilled in the art that all or part of the steps for implementing the above embodiments may be implemented by hardware, or may be implemented by a program instructing relevant hardware, where the program may be stored in a computer-readable storage medium, and the above-mentioned storage medium may be a read-only memory, a magnetic disk or an optical disk, etc.
The description of the present invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the invention in the form disclosed. Many modifications and variations will be apparent to practitioners skilled in this art. The embodiment was chosen and described in order to best explain the principles of the invention and the practical application, and to enable others of ordinary skill in the art to understand the invention for various embodiments with various modifications as are suited to the particular use contemplated.
Claims (10)
1. The multi-index disease joint detection microfluidic device is characterized by comprising: the device comprises a microfluidic reaction chip, a microfluidic storage chip, a direct current motor driving module, a reaction chip fluid control module, a storage release fluid control module, a temperature control module and a CCD (charge coupled device) detection module; the method comprises the following steps of respectively completing the mixing of a sample and a sample diluent through the mutual matching of a reaction chip fluid control module, a storage and release fluid control module, a temperature control module and a microfluidic chip, capturing a target cell specific antigen by an antibody coated on the surface of a microsphere, cleaning a cell antigen nonspecifically adsorbed on the surface of the microsphere, capturing an enzyme-labeled antibody by a target cell antigen specifically adsorbed on the surface of the microsphere, mixing an excitation liquid A, B, and catalyzing an excitation liquid chemiluminescence process through the specific adsorption of the enzyme-labeled antibody by the target cell antigen on the surface of the microsphere, and collecting and detecting chemiluminescence signals on the surface of the microsphere by matching with a CCD (charge coupled device) detection module, so that the integrated operation of capturing and detecting processes of a plurality of;
the microfluidic storage chip (1) consists of a storage chip top layer sealing soft film (2), a storage chip upper cover plate (3), a storage chip liquid storage layer (4), a storage chip lower cover plate (5) and a storage chip bottom layer sealing soft film (6), wherein the storage chip top layer sealing soft film (2), the storage chip upper cover plate (3), the storage chip liquid storage layer (4), the storage chip lower cover plate (5) and the storage chip bottom layer sealing soft film (6) are sequentially connected from top to bottom; a sample solution storage cavity (7), a sample diluent storage cavity (8), a cleaning solution storage cavity I (9), an enzyme-labeled antibody solution storage cavity (10), a cleaning solution storage cavity II (11), an exciting liquid A storage cavity (12), an exciting liquid B storage cavity (13), a related reagent release channel (14) and a cut-off air channel (15) are arranged on the storage chip liquid storage layer (4); a sample solution storage cavity (7), a sample diluent storage cavity (8), a cleaning solution storage cavity I (9), an enzyme-labeled antibody solution storage cavity (10), a cleaning solution storage cavity II (11), an exciting liquid A storage cavity (12) and an exciting liquid B storage cavity (13) are dispersedly arranged on a storage chip liquid storage layer (4); a sample solution storage cavity (7), a sample diluent storage cavity (8), a cleaning solution storage cavity I (9), an enzyme-labeled antibody solution storage cavity (10), a cleaning solution storage cavity II (11), an exciting liquid A storage cavity (12) and an exciting liquid B storage cavity (13) are all connected with a cut-off air channel (15) through a reagent release channel (14);
the microfluidic reaction chip (16) consists of a reaction chip upper cover plate (17), a reaction chip main body layer (18), microspheres (19), a waste liquid storage layer (20) and a reaction chip lower cover plate (21); the reaction chip upper cover plate (17), the reaction chip main body layer (18), the waste liquid storage layer (20) and the reaction chip lower cover plate (21) are connected in sequence from top to bottom; the microspheres (19) are arranged in the reaction chip main body layer (18); the reaction chip upper cover plate (17) comprises three pins (22) for assembling the microfluidic storage chip (1) and seven air holes (23), wherein the seven air holes (23) are respectively a sample inlet (24), a cleaning solution inlet I (25), an enzyme-labeled antibody solution inlet (26), a cleaning solution inlet II (27), an exciting solution inlet (28), an injector air hole (29) and an air suction pump air hole (30); the reaction chip main body layer (18) comprises a sample dilute mixing channel (31), a cleaning solution channel I (32), an enzyme-labeled antibody solution channel (33), a cleaning solution channel II (34), an exciting solution mixing channel (35), a biochemical reaction channel (36), five microsphere fixing grooves (37), an injector vent hole (29), an air suction pump vent hole (30) and a waste liquid cavity inlet (38); the waste liquid storage layer (20) comprises a waste liquid cavity (39) and absorbent paper (40);
the reaction chip fluid control system device I is formed by connecting an air pump connecting hole (41) to an air pump (43) through an air pump connecting hose (42), the other end of the air pump is connected with an air pump electromagnetic valve (44), and the air pump (43) is fixed on a bottom plate (46) through an air pump bracket (45);
the second reaction chip fluid control system device is formed by connecting an injector connecting hole (47) to a small injector (49) through an injector connecting hose (48), and the tail part of the small injector (49) is connected with a micro stepping motor (50); the small injector (49) and the micro stepping motor (50) are respectively fixed on the bottom plate (46) by an injector bracket (51) and a micro stepping motor bracket (52);
the direct current motor driving module comprises a direct current motor (53), a screw rod (54), a sliding block (55), an upper touch switch (56), a lower touch switch (57), a first guide rail (58) and a second guide rail (59); when the module works, the direct current motor (53) drives the screw rod (54) to rotate, so that the sliding block (55) on the screw rod (54) is driven to move up and down, the sliding block (55) is positioned through the upper touch switch (56) and the lower touch switch (57), and the sliding block (55) is guided through the first guide rail (58) and the second guide rail (59);
the storage and release fluid control module comprises a pressing block (60), a rubber column (61), a ventilation needle tube (62), a ventilation column (63), a ventilation column hose (64) and an electromagnetic valve group (65); the pressing block (60) is fixed on a sliding block (55) of the direct current motor driving module, the pressing block (60) moves downwards under the action of the direct current motor driving module, and fourteen ventilation needle tubes (62) are inserted into the storage chip top layer sealing soft film (2); each vent needle tube (62) is communicated with the corresponding vent column (63), each vent column (63) is hermetically provided with a vent column hose (64), each vent column hose (64) is correspondingly connected with one of fourteen inlets of two electromagnetic valve groups (65), the electromagnetic valve groups (65) are fixed on an electromagnetic valve group seat (66), and the electromagnetic valve group seat (66) is fixed on the bottom plate (46);
the temperature control module is embedded into the chip composite function base (67) and comprises a heating resistance film (68), a heating aluminum block (69) and a thermistor (70); the heating resistance film (68) is adhered to the lower surface of the heating aluminum block (69), the thermistor (70) is fixed in a groove in the middle of the heating aluminum block (69), and the heating aluminum block (69) is tightly attached to the right lower part of the biochemical reaction channel (36) of the microfluidic reaction chip (16); the position of the composite functional base (67) and the bottom plate (46) is determined by mechanical fixation;
the CCD detection module comprises a CCD chemiluminescence intensity detector (71) which is positioned right above a biochemical reaction channel (36) in the microfluidic reaction chip (16).
2. The multi-index disease joint detection microfluidic device of claim 1, wherein: a sample dilute mixing channel (31) and an exciting liquid mixing channel (35) are arranged in a reaction chip main body layer (18) of the microfluidic reaction chip (16); the two mixing channels are composed of three parts, namely a mixing channel premixing cavity (72), a Z-shaped narrow channel (73) and a mixing channel final mixing cavity (74), the liquid inlets and outlets of the mixing channel premixing cavity (72) and the mixing channel final mixing cavity (74) are in a horn shape, and the two cavities are connected through the Z-shaped narrow channel (73); when the mixing device works, two kinds of liquid sequentially enter the mixing channel premixing cavity (72), enter the mixing channel final mixing cavity (74) through the Z-shaped narrow channel (73) under the driving of the injector (49), and return to the mixing channel premixing cavity (72) through the Z-shaped narrow channel (73), and the two kinds of liquid are mixed under the reciprocating motion.
3. The multi-index disease joint detection microfluidic device of claim 1, wherein: a biochemical reaction channel (36) and five microsphere fixing grooves (37) are arranged in a reaction chip main body layer (18) of the microfluidic reaction chip (16); the microsphere fixing groove (37) is a hemispherical groove in the biochemical reaction channel (36), and the microspheres (19) are placed in the microsphere fixing groove (37); the distances from the circle center of the cross section of the microsphere fixing groove (37) to two vertical surfaces of the biochemical reaction channel (36) are equal; the inlet of the biochemical reaction channel (36) is horn-shaped, and the whole biochemical reaction channel is Z-shaped, but the biochemical reaction channel can also be L-shaped or straight line section-shaped; the tail end of the biochemical reaction channel is connected with a waste liquid cavity inlet (38);
a waste liquid storage layer (20) of the microfluidic reaction chip (16) is adhered below a reaction chip main body layer (18), various reacted samples, reagents and cleaning liquid are collectively called waste liquid, and the waste liquid enters a waste liquid cavity (39) through a waste liquid cavity inlet (38) and is absorbed by absorbent paper (40); the cavity of the waste liquid cavity (39) is communicated with an injector (49) through an injector vent hole (29) and is communicated with an air suction pump (43) through an air suction pump vent hole (30).
4. The multi-index disease joint detection microfluidic device of claim 1, wherein: a sample dilute mixing channel (31), a cleaning solution channel I (32), an enzyme-labeled antibody solution channel (33), a cleaning solution channel II (34) and an exciting liquid mixing channel (35) are arranged in a reaction chip main body layer (18) of the microfluidic reaction chip (16); the sample diluent enters the biochemical reaction channel (36) to react with the microspheres (19), and then a plurality of cleaning solutions alternately pass through the first cleaning solution channel (32) and the second cleaning solution channel (34) to flush the biochemical reaction channel (36).
5. The multi-index disease joint detection microfluidic device of claim 1, wherein: the memory chip reservoir layer (4) of the microfluidic memory chip (1) has seven storage chambers according to claim 1, each of which is connected to an upstream channel (79), a reagent release channel (14), a trapped air channel (15); after the injector (49) moves to give negative pressure, an upstream passage (79) of the storage cavity which needs to release liquid is communicated with the atmosphere under the condition of no ventilation, so that the liquid in the cavity is released outwards, the upstream passage (79) is not ventilated after a certain amount is released, the cut-off air channel (15) is communicated with the atmosphere, and the injector (49) continues to give negative pressure to cut off the liquid in the reagent release channel (14), so that the function of 'quantitative volume liquid release' is completed.
6. The multi-index disease joint detection microfluidic device of claim 1, wherein: the upper cover plate (3) of the storage chip of the microfluidic storage chip (1) is provided with fourteen through holes which are respectively an upstream through hole (75) and a cut-off air hole (76) of seven reagent storage cavities; a top sealing soft film (2) of the memory chip is adhered above the upper cover plate (3) of the memory chip, and the material of the top sealing soft film can be rubber or other soft materials meeting the requirement; the lower cover plate (5) of the storage chip is provided with five liquid release holes (77) which respectively correspond to the inlets of five reagent release channels (14) of the reaction chip main body layer (18); a memory chip bottom layer sealing soft film (6) is adhered to the lower part of the memory chip lower cover plate (5), and five cross-shaped openings (78) are formed in the memory chip bottom layer sealing soft film (6); a cross opening (78) corresponds to a through hole on a cover plate (5) at the lower part of the storage chip, and the cross opening (78) can be opened under the pressure action of a pressing block (60) and a rubber column (61), so that the microfluidic storage chip (1) is communicated with the microfluidic reaction chip (16).
7. The multi-index disease joint detection microfluidic device of claim 1, wherein: in the direct current motor driving module, the position of the pressing block (60) when lifted is determined by an upper touch switch (56), and the position of the pressing block (60) when extruded is determined by a lower touch switch (57); when the pressing block (60) is extruded, the microfluidic reaction chip (16) and the microfluidic storage chip (1) are hermetically combined through the storage chip bottom layer sealing soft film (6), and meanwhile, the communication process of the microfluidic storage chip (1) and the microfluidic reaction chip (16) is completed; fourteen ventilation needle tubes (62) pierce the sealing soft film (2) on the top layer of the storage chip and are correspondingly inserted into the upstream through hole (75) or the cutoff air hole (76), so that the ventilation needle tubes (62) are connected with corresponding channels of fourteen ventilation columns (63) above the pressing block;
fourteen ventilation columns (63) above the pressing block are in one-to-one corresponding sealing connection with two groups of electromagnetic valve groups (65) respectively provided with seven inlets through fourteen ventilation column hoses (64); the single chip microcomputer controls the opening and closing of a certain electromagnetic valve in the electromagnetic valve group (65) so that a certain vent column (63) is communicated with the atmosphere, and further a certain upstream passage (79) is communicated with the atmosphere.
8. The multi-index disease joint detection microfluidic device of claim 1, wherein: after the micro-fluidic storage chip (1) is communicated with the micro-fluidic reaction chip (16) and the micro-fluidic storage chip (1) of the electromagnetic valve group (65), the micro stepping motor (50) is controlled by the microprocessor to rotate, and the tail part of the small injector (49) is driven by the thread structure to realize the control of fluid in the device, so that the mixing of two kinds of liquid and the directional flow of various kinds of liquid are finished; the thread structure enables a larger rotating angle of the micro stepping motor (50) to be converted into smaller displacement of the tail part of the small injector (49), and the displacement of the injector (49) is controlled more accurately.
9. The multi-index disease joint detection microfluidic device of claim 1, wherein: in the temperature control module, a heating aluminum block (69) is tightly attached to the lower surface of a microfluidic reaction chip (16) and located right below a biochemical reaction channel (36), a heating resistance film (68) is adhered to the lower surface of the heating aluminum block (69), the heating resistance film (68) and the heating aluminum block are physically adhered by adopting a pressure-sensitive double faced adhesive tape or chemically adhered by adopting an organic solvent or thermal adhesion, a thermistor (70) is fixed in a groove in the middle of the aluminum block, a microprocessor heats the aluminum block by controlling the heating resistance film (68), the temperature control of the aluminum block is realized by matching with the thermistor (70), and the aluminum block further realizes the reaction temperature control of reagents in the liquid storage chip by virtue of a heat conduction effect.
10. The multi-index disease joint detection microfluidic device of claim 1, wherein: firstly tearing off an upper layer and a lower layer of plastic sealing films of the microfluidic storage chip (1), and extracting a sample by using an injector to inject the sample into a sample solution storage cavity (7) of the microfluidic storage chip (1); removing the plastic sealing film on the top of the microfluidic reaction chip (16), placing the microfluidic reaction chip (16) in a chip composite function base (67), and positioning the two chips by pins (22) of the microfluidic storage chip (1) and pin holes (80) on the microfluidic reaction chip (16); a direct current motor (53) of the direct current motor driving module drives a sliding block to move from the position of an upper touch switch (56) to the position of a lower touch switch (57), so that a pressing block (60) compresses the microfluidic reaction chip (16) and the microfluidic storage chip (1) by using a rubber column (61); while the compression is carried out, the vent needle tube (62) on the pressing block (60) is inserted into the corresponding reagent release channel (14) and the cut-off air channel (15) of the microfluidic storage chip (1); the microprocessor controls the heating resistance film (68) to be matched with the thermistor (70) to heat the three aluminum blocks, so that the biochemical reaction channel (36) is kept at a constant temperature;
opening the electromagnetic valve corresponding to the upstream through hole (75) of the sample solution storage cavity (7), closing all the other electromagnetic valves, utilizing the tail part of the small injector (49) and the micro stepping motor (50) to drive the injector to move for a corresponding distance to release a certain amount of sample solution, closing the reagent release channel (14) of the sample diluent storage cavity (8), opening the air intercepting channel (15) of the sample diluent storage cavity (8), and enabling the sample and the diluent to jointly enter the sample diluent mixing channel (31) of the microfluidic reaction chip (16) by the corresponding distance of the movement of the small injector, under the drive of the injector (49), the reagent enters the mixing channel final mixing cavity (74) from the corresponding Z-shaped narrow channel (73) and returns to the mixing channel premixing cavity (72) from the Z-shaped narrow channel (73), and the two liquids complete the mixing of the sample and the cleaning liquid under the reciprocating motion; after mixing, driving the reagent to the biochemical reaction channel (36) by the injector, and reacting the mixed reagent with the microspheres in the five microsphere fixing grooves (37) in the biochemical reaction channel (36) for a certain time; during the reaction, the small injector (49) is used for carrying out back and forth micro-motion to drive the reagent in the biochemical reaction channel (36) to carry out micro-reciprocating motion, and the reaction reagent and the microspheres generate relative motion, so that the reaction is accelerated; after the set time is reached, opening the air pump (43), and simultaneously opening an electromagnetic valve connected with the air pump (43) to pump the reagent in the biochemical reaction channel to the waste liquid cavity (39);
opening a reagent release channel (14) of a first cleaning solution storage cavity (9), enabling a small-sized injector to move for a corresponding distance to release 250ul of sample diluent with a corresponding volume, closing the reagent release channel (14) of the first cleaning solution storage cavity (9), opening a first cleaning solution storage cavity (9) to cut off an air channel (15), enabling the cleaning solution to enter a first cleaning solution channel (32) of a microfluidic reaction chip (16) and then enter a biochemical reaction channel (36) by the corresponding distance, after the cleaning solution is cleaned in the biochemical reaction channel (36) for a period of time, opening an air suction pump (43), and simultaneously opening an electromagnetic valve connected with the air suction pump (43) to pump the reagent in the biochemical reaction channel to a waste liquid cavity (39); the cleaning process is repeated for a plurality of times;
opening a reagent release channel (14) of an enzyme-labeled antibody solution storage cavity (10), moving a small injector by a corresponding distance to release 200ul enzyme-labeled antibody solution storage cavities with corresponding volumes, closing the reagent release channel (14) of the enzyme-labeled antibody solution storage cavity (10), opening an intercepting air channel (15) of the enzyme-labeled antibody solution storage cavity (10), moving the small injector by a corresponding distance to enable washing liquor to enter an enzyme-labeled antibody solution channel (33) of a microfluidic reaction chip (16) and then enter a biochemical reaction channel (36) for a corresponding reaction time, and performing back-and-forth micro motion by using the small injector (49) during the reaction to drive a reagent in the biochemical reaction channel (36) to perform micro-reciprocating motion, so that the reaction reagent and microspheres generate relative motion, and the reaction is accelerated; after the set time is reached, opening the air pump (43), and simultaneously opening an electromagnetic valve connected with the air pump (43) to pump the reagent in the biochemical reaction channel to the waste liquid cavity (39);
opening a reagent release channel (14) of a second cleaning solution storage cavity (11), enabling the small-sized injector to move for a corresponding distance to release 250ul of cleaning solution with a corresponding volume, closing the reagent release channel (14) of the second cleaning solution storage cavity (11), opening a second air intercepting channel (15) of the second cleaning solution storage cavity (11), enabling the cleaning solution to enter a second cleaning solution channel (34) of the microfluidic reaction chip (16) and then enter a biochemical reaction channel (36) by the corresponding distance, after the cleaning solution is cleaned in the biochemical reaction channel (36) for a period of time, opening an air suction pump (43), and simultaneously opening an electromagnetic valve connected with the air suction pump (43) to pump the reagent in the biochemical reaction channel to a waste liquid cavity (39); the cleaning process is repeated for a plurality of times;
opening the reagent release channel (14) of the storage chamber (12) of the exciting liquid A and moving the mini-injector for corresponding distances to release the corresponding volume of exciting liquid A125ul, closing the reagent release channel (14) of the storage chamber (12) of the exciting liquid A and opening the reagent release channel (14) of the storage chamber (13) of the exciting liquid B and moving the mini-injector for corresponding distances to release the corresponding volume of exciting liquid B125 ul; closing a reagent release channel (14) of a storage cavity (13) of the exciting liquid B, opening an intercepting air channel (15) of the storage cavity (13) of the exciting liquid B, driving a reagent to an exciting liquid mixing channel (35) by a small-sized injector, completing the mixing of the exciting liquid by the movement of the reagent in a mixing channel premixing cavity (72) and a mixing channel final mixing cavity (74), driving the reagent to a biochemical reaction channel (36) after the mixing is completed, exciting the exciting liquid by reaction products on microspheres to generate chemiluminescence, and finally acquiring and detecting chemiluminescence signals on the surfaces of the microspheres (19) in five liquid storage cavities by a CCD chemiluminescence intensity detector (71);
therefore, the detection of multiple disease indexes of the multi-index disease combined detection microfluidic device is realized.
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