CN108956558B - Microfluidic chip and immunofluorescence analyzer - Google Patents
Microfluidic chip and immunofluorescence analyzer Download PDFInfo
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- CN108956558B CN108956558B CN201810509672.3A CN201810509672A CN108956558B CN 108956558 B CN108956558 B CN 108956558B CN 201810509672 A CN201810509672 A CN 201810509672A CN 108956558 B CN108956558 B CN 108956558B
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/533—Production of labelled immunochemicals with fluorescent label
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Abstract
The application discloses a microfluidic chip and an immunofluorescence analyzer, wherein the microfluidic chip comprises: a substrate on which a protrusion is provided; and the cover plate is provided with grooves which correspond to and are matched with the positions of the protrusions, and the protrusions on the substrate can be separated from the grooves on the cover plate, so that the substrate of the microfluidic chip is detachably connected with the cover plate. Through the mode, the substrate and the cover plate of the microfluidic chip can be detachably connected, so that technical support is provided for recycling of the microfluidic chip.
Description
Technical Field
The application relates to the technical field of medical equipment, in particular to a microfluidic chip and an immunofluorescence analyzer.
Background
Microfluidic chip technology is a technology for processing micro-scale (10 -9 L-10 -18 L) the reaction system of the sample is widely applied to cell screening, immunodetection, cell detection analysis and the like at present; the traditional microfluidic chip integrates functional components such as a micro pipeline, a micro pump, a micro valve, a micro liquid storage device, a micro detection element and the like on a chip material like an integrated circuit by micro processing technology according to the micro structural characteristics of a micro pipeline network so as to finish sample processing and detection.
The inventor of the application finds that most of the existing microfluidic chips cannot be reused in a long-term research process, the processing difficulty is high, the processing cost is generally 50-200 yuan/piece, the detection cost of detection items related to hospitals is about 30 yuan, and the cost of the microfluidic chips is far more than the cost range which can be born by hospitals.
Disclosure of Invention
The application mainly solves the technical problem of providing a microfluidic chip and an immunofluorescence analyzer, which can lead a substrate and a cover plate of the microfluidic chip to be detachably connected, thereby providing technical support for the reutilization of the microfluidic chip.
In order to solve the technical problems, the application adopts a technical scheme that: there is provided a microfluidic chip including: a substrate on which a protrusion is provided; and the cover plate is provided with grooves which correspond to and are matched with the positions of the protrusions, and the protrusions on the substrate can be separated from the grooves on the cover plate, so that the substrate of the microfluidic chip is detachably connected with the cover plate.
In order to solve the technical problems, the application adopts another technical scheme that: there is provided an immunofluorescence analyzer comprising the microfluidic chip of any of the embodiments described above.
The beneficial effects of the application are as follows: different from the situation of the prior art, the substrate provided by the application is provided with the bulge, the cover plate is provided with the groove which corresponds to the bulge in position and is matched with the bulge, when the cover plate is covered on the substrate, the bulge on the substrate is embedded into the groove on the cover plate to enable the substrate and the cover plate to be positioned, and the bulge on the substrate can be separated from the groove on the cover plate, so that the substrate of the microfluidic chip is detachably connected with the cover plate, and further technical support is provided for recycling of the microfluidic chip.
In one application scenario, after the microfluidic chip is used, the substrate and the cover plate are detached and separated, and the substrate and the cover plate are placed in a cleaning solution containing active ingredients such as sodium hypochlorite for cleaning, and the substrate and the cover plate are dried for repeated use next time after the cleaning is completed.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for the description of the embodiments will be briefly described below, and it is apparent that the drawings in the following description are only some embodiments of the present application, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art. Wherein:
FIG. 1 is a schematic structural diagram of an embodiment of a microfluidic chip according to the present application;
FIG. 2 is a schematic diagram of an embodiment of the substrate of FIG. 1;
FIG. 3 is a schematic view of an embodiment of the cover plate in FIG. 1;
FIG. 4 is a schematic view of another embodiment of the substrate of FIG. 1;
FIG. 5 is a schematic structural diagram of another embodiment of a microfluidic chip according to the present application;
FIG. 6 is a schematic diagram of an embodiment of the substrate of FIG. 5;
FIG. 7 is a schematic diagram of an embodiment of the cover plate of FIG. 5;
FIG. 8 is a schematic diagram showing the structure of an embodiment of the immunofluorescence analyzer of the present application.
Detailed Description
The following description of the embodiments of the present application will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present application, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.
At present, most of the existing microfluidic chips cannot be reused, the processing difficulty is high, the processing cost is generally 50-200 yuan/piece, the detection cost of detection items related to hospitals is about 30 yuan, and the cost of the microfluidic chips far exceeds the cost range born by hospitals, so that the development of the reusable microfluidic chips is particularly important.
Referring to fig. 1 to 3, fig. 1 is a schematic structural diagram of an embodiment of a microfluidic chip according to the present application, fig. 2 is a schematic structural diagram of an embodiment of a substrate in fig. 1, and fig. 3 is a schematic structural diagram of an embodiment of a cover plate in fig. 1. The microfluidic chip 1 provided by the application comprises a substrate 10 and a cover plate 12; in one application scenario, the material of the microfluidic chip 1 is surface-modified Polydimethylsiloxane (PDMS), where the manner of surface modification of PDMS includes any one of plasma, surfactant, ultraviolet irradiation, and ozone treatment, which is not limited in the present application. PDMS itself is a strongly hydrophobic material on which the microfluidic channel 102 is constructed, and if no surface modification is performed, the surface modification of the PDMS material is necessary because the strong hydrophobicity of the PDMS surface results in a large flow resistance of the polar liquid like an aqueous solution in the microfluidic channel 102 after the entire assembly is completed, i.e., after the cover plate 12 is covered on the substrate 10. By modifying the PDMS surface, the inert PDMS surface can be activated, the interaction of the interface is enhanced, and the sample can flow on the surface more easily. In other application scenarios, the material of the microfluidic chip 1 may be other, for example, silicon, glass, quartz, plastic, etc., which is not limited in the present application.
Specifically, the substrate 10 is provided with a bump 100; the number of the protrusions 100 may be 1, 2, 3, etc., and when the number of the protrusions 100 is even, the protrusions 100 may be symmetrically or asymmetrically arranged in pairs, which is not limited in the present application; for ease of assembly, in this embodiment, the cross-section of the protrusion 100 is circular, and in other embodiments, the cross-section of the protrusion 100 may be other (e.g., triangular, rectangular, etc.), which is not limited by the present application. In an application scenario, the substrate 10 is provided with the micro-fluidic channel 102, the micro-fluidic channel 102 is a channel formed on one side surface of the substrate 10 by using a photolithography process, a region occupied by the micro-fluidic channel 102 on the substrate 10 is defined as a functional region 104, a region on the substrate 10 except for the micro-fluidic channel 102 is defined as a non-functional region 106, and in order not to affect the implementation of the functions of the functional region 104 on the substrate 10, the bump 100 may be disposed on the non-functional region 106 of the substrate 10.
Specifically, the cover plate 12 covers the microfluidic channel 102 on the substrate 10, and is provided with a groove 120 corresponding to and matching the position of the protrusion 100, when the cover plate 12 is covered on the substrate 10, the protrusion 100 on the substrate 10 is embedded into the groove 120 on the cover plate 12 to position the substrate 10 and the cover plate 12, and the protrusion 100 on the substrate 10 can be separated from the groove 120 on the cover plate 12, so that the substrate 10 of the microfluidic chip 1 is detachably connected with the cover plate 12. The groove 120 may or may not extend through the cover plate 12; when the groove 120 does not penetrate the cover plate 12, the height h1 of the protrusion 100 may be equal to or slightly lower than the depth d1 of the groove 120; when the groove 120 penetrates the cover plate 12, the height of the protrusion 100 may be higher than or equal to or lower than the depth of the groove 120, which is not limited in the present application.
Of course, in other embodiments, grooves may be formed on the substrate 10, and protrusions corresponding to and matching the positions of the grooves may be formed on the cover 12, which is not limited in the present application. The microfluidic chip 1 provided in the above embodiment is simple in structure and detachable; after the microfluidic chip 1 is used, the substrate 10 and the cover plate 12 are separated and then put into an ultrasonic cleaning device filled with a cleaning solution of active ingredients such as sodium hypochlorite for cleaning, and the cleaning is dried and then reused next time. Through the mode, the microfluidic chip 1 can be recycled, and the aim of reducing the detection cost is achieved.
In one embodiment, referring to fig. 1-3, the microfluidic channel 102 disposed on the substrate 10 includes a sample injection region 108, and a through hole 122 is disposed on the cover 12 corresponding to the sample injection region 108, where the through hole 122 communicates with the sample injection region 108, so that a sample enters the sample injection region 108 through the through hole 122. In one application scenario, the contact surfaces of the sample injection region 108 and the through hole 122 are a first plane a and a second plane B, respectively, where the first plane a covers the second plane B, so that the sample flows into the sample injection region 108 through the through hole 122; the cross-section of the through-hole 122 may be circular, square, oval, etc., and the through-hole 122 may be an equal-diameter or an unequal-diameter hole (e.g., the diameter of the cross-section of the through-hole 122 gradually decreases or gradually increases, etc., in a direction toward the substrate 10); the cross-section of the sample injection region 108 may be square, circular, oval, pentagonal, etc., and the sample injection region 108 may be provided with one or more outlets C at the edges (e.g., at the corners of a pentagon when the sample injection region 108 is pentagon) in order to allow the sample within the sample injection region 108 to enter the subsequent detection region.
In another embodiment, the microfluidic channel 102 on the substrate 10 is further formed with a sample filtering section 101, a capturing section 103, and a sample collecting section 105 connected to each other in this order after the sample injection section 108.
Specifically, the first end C of the sample injection zone 108 is connected to the first end D of the sample filtration zone 101. The sample filtering area 101 is used for filtering red blood cells and other impurities in a sample, the sample filtering area 101 is provided with a plurality of protruding columns E (e.g. 5, 10, 50, etc.) extending from the surface of the substrate 10, the radius of the protruding columns E is 15um-20um (e.g. 15um, 18um, 20um, etc.), and the interval between two adjacent protruding columns E is 5um-10um (e.g. 5um, 8um, 10um, etc.). The radius of the pillars E and the distance between two adjacent pillars E in the sample filtering region 101 can be adjusted according to specific requirements, which is not limited in the present application.
Specifically, the bottom of the capture area 103 is provided with a plurality of recesses F (e.g., 5, 10, 50, etc.) arranged in a microarray, the recesses F being for capturing the micro particulate matter in the sample entering the capture area 103, the space of the recesses F being sized to accommodate only one or two micro particulate matter. In one application scenario, the microparticulate material can be magnetic beads, microbeads (e.g., polystyrene microspheres, etc.), or cells, etc. When the micro-particle material is a magnetic bead or a micro-bead, the magnetic bead or the micro-bead may be coated with other materials such as an antibody, which is not limited in the present application. To ensure that the flow of the micro particulate matter in the sample is not impeded, the minimum distance between the bottom of the micro flow channel 102 on the substrate 10 and the cover plate 12 is a first distance d2, the first distance d2 being larger than the diameter of the micro particulate matter. In another application scenario, the shape of the cross-section of the recess F includes circular, triangular, rectangular, diamond, etc. When the cross section of the recess F is circular, the diameter of the circular shape is greater than or equal to the diameter of the micro particulate matter and less than or equal to twice the diameter of the micro particulate matter. In one embodiment, the diameter of the circle is 10um-30um, e.g., 10um, 20um, 30um, etc.
In particular, the sample collection area 105 is used to collect samples that do not flow into the capture area 103.
In one application scenario, the sample is a fluorescent-labeled magnetic bead sample which is pre-bound with a specific protein in whole blood/serum, and after entering the sample injection region 108 through the through hole 122, the magnetic bead sample flows to the sample filtering region 101 under the driving of capillary force; the magnetic bead sample passing through the sample filtering area 101 enters the capturing area 103 under the driving of capillary force; an external magnetic field is applied to the capture area 103, and the magnetic bead samples fall into a plurality of concave portions F of the capture area 103 under the magnetic force of the external magnetic field, and no more than two magnetic bead samples are in one concave portion F. After applying the external magnetic field for a certain time, the external magnetic field is removed, and the rest of the sample that does not fall into the plurality of recesses F of the capture zone 103 enters the sample collection zone 105 under the drive of capillary force. The sample in the microfluidic chip 1 provided by the embodiment flows under the drive of capillary action, and an external electromechanical driving component is not needed, so that the operation is simple, and the practicability is strong. Through reasonable light path design, a single concave part F of the capturing area 103 is irradiated by laser with specific wavelength, whether fluorescence exists in the currently irradiated concave part F or not is observed through a fluorescence microscope, all concave parts F in the capturing area 103 are traversed, and whether the sample contains the designated protein or not is further judged. The method for detecting the fluorescence of the capture area 103 and the magnetic bead sample can realize quantitative detection of single magnetic beads, and has higher sensitivity compared with the detection of the traditional chemiluminescence method. When the material of the microfluidic chip 1 is transparent, the laser can directly irradiate the capture area 103 of the microfluidic chip 1; when the material of the microfluidic chip 1 is a non-transparent material, a via hole (not shown) may be formed at a position of the cover plate 12 corresponding to the capture area 103, and laser light may be irradiated to the capture area 103 of the microfluidic chip 1 through the via hole.
In another embodiment, referring to fig. 4, fig. 4 is a schematic structural diagram of another embodiment of a substrate. The substrate 20 includes a sample injection zone 108, a sample filtration zone 101, a sample reaction zone 200, a capture zone 103, and a sample collection zone 105 in communication with one another; in this embodiment, the structures of the sample injection region 108, the sample filtering region 101, the capturing region 103 and the sample collection region 105 are the same as those in the above embodiment, and will not be described again. The sample reaction region 200 is pre-embedded with a fluorescent-labeled first antibody, and the recess (not labeled) of the capture region 103 is pre-embedded with a magnetic-bead-labeled second antibody, wherein the first antibody may bind to a specific protein in the sample, the second antibody may bind to a specific protein in the sample, or the second antibody may bind to the first antibody.
In one application scenario, the sample corresponding to the substrate 20 provided in the above embodiment is a whole blood/serum sample. After the whole blood/serum sample enters the sample injection region 108 through the through hole 122, the whole blood/serum sample flows to the sample filtering region 101 under the driving of capillary force; the whole blood/serum sample passing through the sample filtering area 101 enters the sample reaction area 200 under the driving of capillary force, and the appointed protein in the whole blood/serum sample reacts with the fluorescent-labeled first antibody pre-embedded in the sample reaction area 200; the reacted whole blood/serum sample enters the capture area 103, an external magnetic field is applied to the capture area 103, and the magnetic bead-labeled secondary antibodies are embedded in the plurality of recesses of the capture area 103 in advance to further react with the reacted sample. After applying the external magnetic field for a certain time, the external magnetic field is removed, and the rest of the sample that does not fall into the plurality of recesses of the capture zone 103 enters the sample collection zone 105 under the drive of capillary force. Through reasonable light path design, a single concave part of the capturing area 103 is irradiated by laser with specific wavelength, whether fluorescence exists in the currently irradiated concave part or not is observed through a fluorescence microscope, all concave parts in the capturing area 103 are traversed, and whether the sample contains the designated protein or not is further judged. When the material of the microfluidic chip 1 is transparent, the laser can directly irradiate the capture area 103 of the microfluidic chip 1; when the material of the microfluidic chip 1 is a non-transparent material, a via hole may be formed at a position of the cover plate 12 corresponding to the capture region 103, and laser irradiates the capture region 103 of the microfluidic chip 1 through the via hole.
To control the flow rate of a sample (a magnetic bead sample, or a whole blood sample, or a serum sample, etc.) within the microfluidic channel 102, the microfluidic channel 102 further includes a serpentine bend, the flow rate of the sample being inversely proportional to the length of the serpentine bend; as shown in fig. 2, the first serpentine bend 107 is located between the sample injection zone 108 and the sample filtration zone 101 and/or the second serpentine bend 109 is located between the sample filtration zone 101 and the capture zone 103; as shown in fig. 4, the first serpentine bend 107 is located between the sample injection zone 108 and the sample filtration zone 101, and/or the third serpentine bend 202 is located between the sample filtration zone 101 and the sample reaction zone 200, and/or the fourth serpentine bend 204 is located between the sample reaction zone 200 and the capture zone 103.
At present, a traditional microfluidic chip can only detect a single item, when detecting a plurality of items of the same sample, the sample to be detected needs to be divided into a plurality of samples, and then the allocated samples are respectively detected by the corresponding items on different microfluidic chips. Therefore, it is important to develop a microfluidic chip capable of performing multi-item joint inspection.
Referring to fig. 5, fig. 5 is a schematic structural diagram of another embodiment of a microfluidic chip according to the present application, and fig. 6 is a schematic structural diagram of an embodiment of a substrate in fig. 5. In this embodiment, the microfluidic chip 3 is a detachable structure, and the design of the detachable structure is the same as that in the above embodiment, and will not be described here again.
In this embodiment, the microfluidic channel 300 on the substrate 30 includes a sample diversion area 302, where the sample diversion area 302 includes a main channel 3000 and a plurality of branch channels 3002 (e.g., 2, 4, 9, etc.), and first ends G of the main channel 3000 for letting out the sample are connected to first ends H of the plurality of branch channels 3002 for letting in the sample, respectively; wherein the sum of the cross-sectional areas of the plurality of branch flow channels 3002 is the cross-sectional area of the trunk flow channel 3000.
In one application scenario, the number N of the branch channels 3002, the radius R1 of the branch channels 3002, and the radius R2 of the trunk channel 3000 satisfy the following relationships: r2=r1×n 1/2 . In one embodiment, the radius of the trunk flow path is 200um-500um, e.g., 200um, 300um, 400um, 500um, etc.
In yet another application scenario, the number N of the branch flow channels 3002, the length L1 of the branch flow channels 3002, and the length L2 of the trunk flow channel 3000 satisfy the following relationships: l2=l1×n 1/2 . In one embodiment, the length L2 of the trunk flow path 3000 is proportional to the number N of the branch flow paths 3002; the length L2 of the trunk flow path 3000 is 3cm to 8cm, for example, 3cm, 5cmc, 8cm, etc.
For example, when the number of the branch flow passages 3002 is 4, the sum of the cross-sectional areas of the 4 branch flow passages 3002 is equal to the cross-sectional area of the trunk flow passage 3000; the radius R2 of the trunk flow path 3000 is 2 times the radius R1 of the branch flow path 3002; meanwhile, in order to ensure that the sample flowing through the main channel 3000 is sufficiently spread over the branch channel 3002, the length of the branch channel 3002 is as short as possible, and the length L2 of the main channel 3000 is 2 times the length L1 of the branch channel 3002. Note that, the lengths of the trunk flow channel 3000 and the branch flow channel 3002 include, but are not limited to, straight lengths, and the trunk flow channel 3000 and the branch flow channel 3002 may be designed to be curved, so as to increase the sample storage capacity. In addition, in order to divide the sample into the branch flow channels 3002 as uniformly as possible, the flow rate of the sample may be reduced by reducing the flow rate of the sample, for example, designing a serpentine bend (not shown) on the trunk flow channel 3000, and reducing the flow rate of the sample by the curved structure and length of the serpentine bend.
In one embodiment, the microfluidic channel 300 located on the substrate 30 in this embodiment further includes a sample injection region 304, and the sample injection region 304 is configured to connect one end of the sample flowing out with the second end I of the trunk flow channel 3000 for flowing in the sample. Through holes 320 are formed in the cover plate 32 at positions corresponding to the sample injection regions 304, and the through holes 320 are in communication with the sample injection regions 304, so that samples can enter the sample injection regions 304 through the through holes 320.
In another embodiment, the sample injected from the sample injection region 304 is a whole blood/serum sample, and in order to avoid that red blood cells or impurities therein affect the detection items corresponding to the respective branch flow channels 3002, the microfluidic channel 300 further includes a sample filtering region 306 located between the sample injection region 304 and the trunk flow channel 3000, and the structure of the sample filtering region 306 is the same as that of the above-mentioned embodiment and will not be repeated here.
In another embodiment, the microfluidic channel 300 on the substrate 30 further includes a plurality of sample reaction regions 308, a capturing region 301 and a sample collecting region 303, which are sequentially connected and arranged side by side, wherein the second end J of each bypass flow channel 3002 for flowing out the sample is respectively connected to one end K of each corresponding sample reaction region 308 for flowing in the sample. Each of the branch flow channels 3002 corresponds to the sample reaction area 308 and the capture area 301, and the plurality of branch flow channels 3002 may share the same sample collection area 303, or each of the branch flow channels 3002 corresponds to one of the sample collection areas 303, which is not limited in the present application. Each sample reaction zone 308 is pre-embedded with a fluorescent-labeled first antibody and each capture zone 301 is pre-embedded with a magnetic bead labeled second antibody, wherein the first antibody may bind to a designated protein in the sample, the second antibody may bind to a designated protein in the sample or the second antibody may bind to the first antibody. With a reasonable light path design, a single recess of the capture zone 301 is illuminated with a laser of a specific wavelength and the presence or absence of fluorescence in the currently illuminated recess is observed by a fluorescence microscope. When the material of the microfluidic chip 3 is transparent, the laser can directly irradiate the capture area 301 of the microfluidic chip 3; when the material of the microfluidic chip 3 is a non-transparent material, a via hole 322 may be formed at a position of the cover plate 32 corresponding to the capturing area 301 of each branch flow channel 3002, and the laser irradiates the capturing area 301 of the microfluidic chip 3 through the via hole 322.
Referring to fig. 8, fig. 8 is a schematic structural diagram of an immunofluorescence analyzer according to an embodiment of the application. The immunofluorescence analyzer 4 provided by the present application comprises a microfluidic chip (not shown in fig. 8) in any of the embodiments described above. In one application scenario, as shown in fig. 8, the immunofluorescence analyzer 4 includes an inlet 40, and the microfluidic chip is placed on a detection platform (not shown) of the immunofluorescence analyzer 4 through the inlet 40. In use, taking the microfluidic chip 1 shown in fig. 1 as an example, firstly, the substrate 10 and the cover plate 12 of the microfluidic chip 1 are assembled, and sample injection is completed; then, the microfluidic chip 1 is placed on the detection platform of the fluorescence analyzer 4 through the inlet and outlet 40; after the detection is completed, the microfluidic chip 1 is taken out from the inlet and outlet 40, and the substrate 10 and the cover plate 12 of the microfluidic chip 1 are detached and separated outside the immunofluorescence analyzer 4.
The microfluidic chip provided by the application is further described below in terms of a few specific application scenarios.
Embodiment one: procalcitonin detection;
procalcitonin (PCT) is a protein whose levels in plasma are elevated when severely infected by bacteria, fungi, parasites, sepsis and multiple organ failure. Whereas PCT does not rise upon autoimmune, allergic and viral infections. Locally limited bacterial infections, mild infections and chronic inflammation do not lead to an increase. PCT reflects the activity level of the systemic inflammatory response, and tests against PCT are clinically significant. For PCT detection, the double-antibody sandwich immunoluminescence method is widely applied, and the principle is that a double monoclonal antibody is applied, wherein one antibody is a calcitonin antibody (first antibody) and is combined with magnetic beads and the calcitonin part of PCT molecules; the other is an anti-calcein antibody (secondary antibody), and an anti-calcein moiety that labels fluorescein and PCT molecules. The two antibodies are combined with PCT molecules to form a sandwich complex, and then the sandwich complex emits fluorescence under the excitation of laser with specific wavelength, and the corresponding PCT value can be judged according to the fluorescence intensity.
The detection of PCT can be performed by using the microfluidic chip 1 shown in fig. 1 to screen the coated sample, and the specific operation process is as follows:
A. magnetic beads with the diameter of 5um and the primary antibody are added into a reaction cup, and the mixture is incubated and coated for 20-30min at a proper temperature. Placing the incubated sample into a magnetic field, adsorbing the magnetic beads on the wall of a reaction cup under the action of the magnetic field, and cleaning the magnetic beads for 2-3 times by using a cleaning solution to remove the first antibodies which are not coated on the magnetic beads, so as to obtain the magnetic beads which are coated by the first antibodies theoretically;
B. and adding a sample into the magnetic bead reaction cup coated with the first antibody, and incubating and coating for 20-30min at a proper temperature. Placing the incubated sample into a magnetic field, adsorbing the magnetic beads on the wall of a reaction cup under the action of the magnetic field, and cleaning the magnetic beads for 2-3 times by using a cleaning solution to remove the sample which is not coated on the magnetic beads, so as to obtain a first antibody and a magnetic bead with a good sample coating in theory;
C. adding the fluorescent-labeled secondary antibody into the magnetic bead reaction cup with the primary antibody and the sample coated, and incubating and coating for 20-30min at a proper temperature. Placing the incubated sample into a magnetic field, adsorbing the magnetic beads on the wall of a reaction cup under the action of the magnetic field, and cleaning the reaction cup for 2-3 times by using a cleaning solution to remove the second antibody which is not coated on the magnetic beads, so as to obtain a magnetic bead-first antibody-sample-fluorescent second antibody sample with a theoretical sandwich structure;
D. adding a certain buffer solution into a theoretical magnetic bead-primary antibody-sample-fluorescent secondary antibody sample to obtain a sample which can be detected by the microfluidic chip 1;
E. the substrate 10 and the cover plate 12 of the microfluidic chip 1 are assembled, a sampling tube is used for taking 10-15uL of samples, the samples are added into a sample injection area 108 of the microfluidic chip 1, and after waiting for 5-10min, the samples flow to a capturing area 103 under the driving of capillary action;
F. applying an external magnetic field to the capture zone 103, waiting for 5-10min, removing the external magnetic field, and allowing the redundant sample to flow into the sample collection zone 105 through the capture zone 103;
G. the laser with specific wavelength irradiates a single concave part F on the micro-fluidic chip 1 in sequence, detects whether a fluorescent signal is collected, judges whether the signal is positive or not according to the existence and intensity of the fluorescent signal, and if the signal is collected, the signal is positive, otherwise the signal is negative;
H. the substrate 10 and the cover plate 12 of the microfluidic chip 1 are separated, the microfluidic chip 1 is cleaned by ultrasonic waves by using cleaning liquid containing hypochlorous acid according to the components, and the microfluidic chip 1 is put into a drying oven to be dried for reuse.
Example two, brain natriuretic peptide and troponin combined detection;
the clinical significance of Brain Natriuretic Peptide (BNP) and troponin I (cTnI) for aiding in the diagnosis of cardiac function was examined in combination. In general, the detection of Brain Natriuretic Peptide (BNP) and troponin I (cTnI) is performed in two separate steps, which increase the cost of the detection on the one hand and make the detection more cumbersome and complicated on the other hand. For the detection of Brain Natriuretic Peptide (BNP) and troponin I (cTnI), the double-antibody sandwich immunofluorescence method is adopted in the embodiment, the principle is that a double monoclonal antibody is adopted, wherein the first antibody is brain natriuretic peptide primary antibody, the first antibody is combined with Brain Natriuretic Peptide (BNP) molecules, and the surface of the primary antibody is modified by fluorescein; the second antibody is brain natriuretic peptide secondary antibody, which is combined with magnetic beads and another site of Brain Natriuretic Peptide (BNP) molecule. The detection principle of troponin I (cTnI) is consistent with that of Brain Natriuretic Peptide (BNP), and will not be described in detail herein.
The microfluidic chip 3 supporting multi-item joint inspection shown in fig. 5 is adopted to improve the detection speed, simplify the operation steps, and the specific operation process is as follows:
A. embedding antibodies of corresponding Brain Natriuretic Peptide (BNP) and troponin I (cTnI) in a sample reaction area and a capture area corresponding to a first branch flow channel and a second branch flow channel of the multi-item joint detection microfluidic chip 3;
B. the substrate 30 and the cover plate 32 of the microfluidic chip 3 are assembled, a sampling tube is used for taking 10-15uL of whole blood/serum sample, the sample is added into a sample injection area 304 of the microfluidic chip 3, the sample contains Brain Natriuretic Peptide (BNP) and troponin I (cTnI) antigens, and after waiting for 5-6min, the sample flows through a sample filtering area 306 under the drive of capillary action;
C. the sample passing through the sample filtering area 306 flows into the sample reaction area 308 corresponding to the first branch flow channel and the second branch flow channel under the driving of capillary action, and Brain Natriuretic Peptide (BNP) or troponin I (cTnI) in the sample reacts with antibodies (primary antibodies) embedded in the corresponding channels in an immune manner;
an antigen (brain natriuretic peptide (BNP) or troponin i (cTn i)) that binds to the primary antibody is driven by capillary force to flow through the capture zone 301, and the antigen of the primary antibody binds to the secondary antibody on the surface of the magnetic beads pre-embedded in the capture zone 301, forming a sandwich structure of magnetic bead-secondary antibody-antigen-primary antibody;
applying an external magnetic field to the capture zone 301 and allowing unbound excess sample to flow into the sample collection zone 303;
D. the magnetic bead samples of a plurality of concave parts in the capturing areas 301 of the different branch flow channels are sequentially irradiated by laser with specific wavelength, whether fluorescence signals are collected or not is detected, negative and positive can be judged according to the existence and strength of the fluorescence signals, if the fluorescence signals are collected, the negative is positive, otherwise the negative is negative;
E. the substrate 30 and the cover plate 32 of the microfluidic chip 3 are separated, the microfluidic chip 3 is cleaned by ultrasonic waves by using cleaning liquid containing hypochlorous acid according to the components, and the microfluidic chip 3 is put into a drying oven to be dried for reuse.
The foregoing description is only of embodiments of the present application, and is not intended to limit the scope of the application, and all equivalent structures or equivalent processes using the descriptions and the drawings of the present application or directly or indirectly applied to other related technical fields are included in the scope of the present application.
Claims (10)
1. A microfluidic chip, the microfluidic chip comprising:
a substrate on which a protrusion is provided;
the cover plate is provided with grooves which correspond to and are matched with the protrusions in position, the protrusions on the base plate can be separated from the grooves on the cover plate, and therefore the base plate of the microfluidic chip is detachably connected with the cover plate;
the substrate is further provided with a micro-flow channel, the micro-flow channel comprises a sample diversion area, the sample diversion area comprises a trunk flow channel and a plurality of branch flow channels, and first ends of the trunk flow channels for enabling samples to flow out are respectively connected with first ends of the branch flow channels for enabling the samples to flow in; wherein the sum of the cross sectional areas of the plurality of branch flow passages is the cross sectional area of the trunk flow passage; the trunk flow channel and the branch flow channel are curves and are used for increasing the storage capacity of a sample;
the number N of the branch flow passages, the length L1 of the branch flow passages and the length L2 of the trunk flow passages meet the following relation: l2=l1×n 1/2 The method comprises the steps of carrying out a first treatment on the surface of the The length of the trunk flow passage is 3cm-8 cm;
wherein the microfluidic channel further comprises a sample injection zone, a sample filtration zone and a capture zone, the main channel comprises a serpentine bend for reducing the flow rate of the sample; wherein a first serpentine bend is provided between the sample injection zone and the sample filtration zone, and/or a second serpentine bend is provided between the sample filtration zone and the capture zone.
2. The microfluidic chip according to claim 1, wherein,
when the cover plate covers the substrate, the protrusions on the substrate are embedded into the grooves on the cover plate, so that the substrate and the cover plate are positioned.
3. The microfluidic chip according to claim 1, wherein the bypass flowThe number N of the channels, the radius R1 of the branch channel and the radius R2 of the trunk channel satisfy the following relation: r2=r1×n 1/2 The method comprises the steps of carrying out a first treatment on the surface of the The radius of the trunk flow passage is 200 micrometers-500 micrometers.
4. The microfluidic chip according to claim 1, wherein,
one end of the sample injection region for flowing out the sample is connected with a second end of the trunk flow channel for flowing in the sample.
5. The microfluidic chip according to claim 4, wherein the sample filtration zone is disposed between the sample injection zone and the dry channel flow channel; the sample filtration zone is provided with a plurality of posts extending from the substrate surface.
6. The microfluidic chip according to claim 5, wherein the radius of the convex columns is 15-20 microns, and the distance between two adjacent convex columns is 5-10 microns.
7. The microfluidic chip according to claim 4, wherein,
and a through hole is formed in the cover plate at a position corresponding to the sample injection region, and the through hole is communicated with the sample injection region, so that a sample enters the sample injection region through the through hole.
8. The microfluidic chip according to claim 1, wherein,
the bottom of the capture zone is provided with a recess of microarray arrangement for capturing micro particulate matter in a sample entering the capture zone, the recess having a space size that can only accommodate one or two micro particulate matter.
9. The microfluidic chip according to claim 8, wherein the recess has a circular, triangular, rectangular, or diamond-shaped cross section; the diameter of the circle is greater than or equal to the diameter of the micro particulate matter and less than or equal to twice the diameter of the micro particulate matter; the diameter of the round shape is 10 micrometers-30 micrometers; the microparticulate material is magnetic beads, microbeads, or cells.
10. An immunofluorescence analyzer, characterized in that it comprises a microfluidic chip according to any one of claims 1-9.
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| CN110161260A (en) * | 2019-06-19 | 2019-08-23 | 深圳市一正科技有限公司 | A kind of flow-path module, microfluidic analysis device and measuring method |
| EP4140584A4 (en) * | 2021-02-05 | 2023-08-02 | BOE Technology Group Co., Ltd. | Microfluidic chip |
| CN114367321A (en) * | 2022-01-06 | 2022-04-19 | 上海交通大学 | Reusable multi-index microfluidic detection system based on coding microspheres and use method |
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