CN212845420U - Double-layer micro-fluidic chip for antibody detection - Google Patents
Double-layer micro-fluidic chip for antibody detection Download PDFInfo
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Abstract
The utility model discloses a double-layer micro-fluidic chip for antibody detection, which comprises a reaction layer substrate, a microcavity sample adding layer and a capping layer from bottom to top in sequence, wherein the reaction layer substrate comprises a glass slide substrate and a glutaraldehyde substrate assembled on the glass slide substrate, and a strip-shaped detection zone and a quality control zone are printed on the glutaraldehyde substrate; a plurality of through holes which are communicated up and down are arranged on the microcavity sample adding layer, and the through holes correspond to the strip-shaped detection zones and the quality control zones one by one to form a plurality of reaction tanks; the bottom of the sealing cover layer is provided with a plurality of micro-channel grooves, the micro-channel grooves correspond to the reaction tanks one by one, and inflow holes and outflow holes which penetrate through the sealing cover layer from top to bottom are formed in two ends of each micro-channel groove respectively. The utility model discloses a chip can realize quick, sensitive, accurate high flux detection to the platelet antibody.
Description
Technical Field
The utility model relates to a micro-fluidic chip technical field, in particular to double-deck micro-fluidic chip for antibody detection.
Background
With the remarkable increase of patients with tumor and blood system diseases and the rapid development of clinical transfusion medicine, the transfusion dosage is rising year by year. Blood transfusion is used as an effective measure for improving platelet count, preventing bleeding and guaranteeing life safety of patients, and safe, scientific and effective infusion of the blood transfusion is an inevitable requirement for development of clinical blood transfusion technology. However, most of the blood transfusions are still random transfusions at present, and the incompatible blood is rapidly destroyed and removed by the immune system of the body after being transfused into the body of a patient, so that the platelet transfusion ineffectiveness frequently occurs, not only is precious blood resources wasted, but also the optimal treatment time of the patient is delayed, and the pain and the economic burden of the patient are increased.
In order to improve the current situation and improve the blood transfusion effect of patients, antibody detection items are newly added and developed in part of clinical hospitals. The existing blood type detection technologies at present are as follows: immunofluorescence assay, enzyme-linked immunosorbent assay; the mixed passive blood coagulation technology, the mixed cell adhesion test (solid phase technology), the specific monoclonal antibody to antigen fixation test and the like, but the operation procedures of the tests are complex, the required time is long, and some required equipment and instruments are expensive, so that blood type matching is not a routine detection project of clinical blood transfusion at present, and part of projects cannot meet the safe blood transfusion requirement due to accuracy and sensitivity in the clinical development process, are complicated to operate and are gradually eliminated clinically. At present, the solid-phase red cell adsorption test (SPRCA) and the monoclonal-specific antigen-immobilization test (MAIPA) are commonly used in clinic, the SPRCA technology is that an anti-blood monoclonal antibody is coated in a reaction plate, and a blood monolayer can be formed on the bottom of a reaction well after the blood suspension is centrifugally washed. Serum or plasma is added and after incubation in the wells, if the serum or plasma contains antibodies, the antibodies bind to the blood monolayer in the reaction wells and unbound components are removed by washing. Anti-human IgG and human IgG sensitized erythrocytes (indicator erythrocytes) are added, and after centrifugation, the indicator erythrocytes are bound to the antibody on the blood monolayer through the bridging of the anti-human IgG, so that a positive reaction is that the indicator erythrocytes are tiled on the bottom surface of the reaction well. The negative reaction indicates that the red blood cells are gathered at the center of the bottom of the reaction hole under the action of centrifugal force; MAIPA technology principle is that platelets first bind to human alloantibodies and then are incubated with different mouse anti-human monoclonal antibodies against blood membrane glycoproteins (anti-GPIb/CD 42b, GPIIb/CD41a, GPIIIa, GPIX, HLA, etc.). After washing, the blood is cracked, the product is transferred into a coated goat anti-mouse IgG microporous plate, goat anti-human IgG is labeled by horseradish peroxidase, and the specific homogeneous antibody of the membrane glycoprotein can be detected through enzyme substrate color development.
When the method is used for qualitatively detecting the antibody, the sensitivity and the specificity are only 30-40 percent, the result interpretation has human errors, the detection can be finished within about several hours, and the rapid requirements of clinicians cannot be met, and meanwhile, the technology does not have a corresponding quality control technology in order to overcome the practical problems of low sensitivity, poor specificity, complex clinical operation, overlong detection time, difficult result interpretation, lack of corresponding reagent quality control and the like of other clinical antibody detection methods; the existing micro-column gel method for detecting the antibody indicates that red blood cells are generally subjected to hydroformylation treatment by adopting a chemical reagent (such as glutaraldehyde) and then coated with anti-human IgG to indicate a reaction result.
Therefore, there is a need to establish an antibody detection system that is sensitive, rapid, low cost and easy to operate.
SUMMERY OF THE UTILITY MODEL
In order to solve the technical problem, the utility model provides a double-deck micro-fluidic chip for antibody detection to reach the purpose of quick, sensitive, accurate high flux detection of platelet antibody.
In order to achieve the above purpose, the technical scheme of the utility model is as follows:
a double-layer micro-fluidic chip for antibody detection sequentially comprises a reaction layer substrate, a micro-cavity sampling layer and a capping layer from bottom to top, wherein the reaction layer substrate comprises a glass slide substrate and a glutaraldehyde substrate assembled on the glass slide substrate, and a strip-shaped detection band and a quality control band are printed on the glutaraldehyde substrate; a plurality of through holes which are communicated up and down are arranged on the microcavity sample adding layer, and the through holes correspond to the strip-shaped detection zones and the quality control zones one by one to form a plurality of reaction tanks; the bottom of the sealing cover layer is provided with a plurality of micro-channel grooves, the micro-channel grooves correspond to the reaction tanks one by one, and inflow holes and outflow holes which penetrate through the sealing cover layer from top to bottom are formed in two ends of each micro-channel groove respectively.
In the above-mentioned scheme, a plurality of the bar is detected the area and is taken with quality control and evenly arrange periodically, and is a plurality of the bar is detected the area and is taken with quality control parallel interval and set up, and is a plurality of the through-hole align to grid, it is a plurality of the microchannel groove align to grid corresponds a plurality of respectively the through-hole.
In the scheme, the glass slide substrate is made of calcium silicate, sodium silicate or silicon dioxide.
In the scheme, the glutaraldehyde substrate is prepared by immersing the glass slide substrate into an APTES absolute ethanol solution to prepare a silanized substrate; and then immersing the silanized substrate into a glutaraldehyde aqueous solution, reacting, washing and airing to obtain the silanized substrate.
In the scheme, the plurality of strip-shaped detection bands and the plurality of quality control bands are periodic strip-shaped detection bands and periodic quality control bands formed by printing a zigzag microfluidic chip on the glutaraldehyde substrate, and the periodic strip-shaped detection bands and the periodic quality control bands form stable chemical bonds through covalent crosslinking of amino groups in biomolecules and aldehyde groups of glutaraldehyde and are fixed on the glass slide substrate.
In the above scheme, the microcavity sample-adding layer and the capping layer are made of polydimethylsiloxane material by casting or injection molding.
A preparation method of a double-layer micro-fluidic chip for antibody detection comprises the following steps:
(1) respectively preparing a reaction layer substrate, a microcavity sampling layer and a capping layer:
the reaction layer substrate is prepared into a silanized substrate by immersing a glass slide substrate into an APTES absolute ethyl alcohol solution; then immersing the silanized substrate into a glutaraldehyde aqueous solution, reacting, washing and airing to obtain a glutaraldehyde substrate attached to the glass slide substrate, and then printing a purified antigen strip-shaped detection band and a quality control band on the glutaraldehyde substrate;
the microcavity sample-adding layer is made of polydimethylsiloxane material by casting or injection molding, and a plurality of through holes which are communicated up and down are formed in the microcavity sample-adding layer;
the cover layer is made of polydimethylsiloxane material by adopting a casting or injection molding mode, a plurality of micro-channel grooves are processed at the bottom of the cover layer, and meanwhile, inflow holes and outflow holes which penetrate through the cover layer up and down are respectively processed at two ends of the micro-channel grooves;
(2) assembling the double-layer micro-fluidic chip:
overlapping the microcavity sample adding layer and the reaction layer substrate and positioning the microcavity sample adding layer and the reaction layer substrate on the reaction layer substrate, so that a plurality of through holes of the microcavity sample adding layer are in one-to-one correspondence with the plurality of strip-shaped detection bands and the plurality of quality control bands respectively to form a plurality of reaction cells; and overlapping the bottom of the sealing cover layer with the microcavity sampling layer and positioning the sealing cover layer on the microcavity sampling layer to enable the plurality of micro-channel grooves to correspond to the plurality of reaction cells one by one, thereby obtaining the double-layer micro-fluidic chip for antibody detection.
Through the technical scheme, the utility model provides a pair of a double-deck micro-fluidic chip for antibody detection has following beneficial effect:
the self-assembly glutaraldehyde substrate of the utility model is integrated with a micro-fluidic chip, the micro-fluidic chip comprises a plurality of reaction tanks, and a plurality of biochemical or chemical indexes can be detected for a sample at the same time; when the chip is used, only the outflow hole and the inflow hole are communicated with the outside, most of reaction processes are finished in a closed micro-channel system, and the interference of the external environment on the reaction and detection processes is effectively reduced; and the chip has simple structure, and is easy to integrate and realize automatic detection by combining with matched automatic equipment. The chip can control the flow through the micro-channel, has strong controllability, and is matched with different fluorescence labeling technologies, thereby achieving the rapid, sensitive and accurate high-flux detection of the antibody.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
Fig. 1 is a schematic view of an overall structure of a double-layer microfluidic chip disclosed in an embodiment of the present invention;
FIG. 2 is a schematic structural diagram of a reaction layer substrate disclosed in an embodiment of the present invention;
fig. 3 is a schematic view of a microcavity sample-adding layer structure disclosed in an embodiment of the present invention;
fig. 4 is a schematic view of a back structure of a capping layer according to an embodiment of the present invention.
In the figure, 1, a reaction layer substrate; 2. a microcavity sample-adding layer; 3. a capping layer; 4. detecting a belt; 5. a quality control band; 6. a through hole; 7. a micro flow channel groove; 8. an inflow hole; 9. and flows out of the hole.
Detailed Description
The technical solution in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.
The utility model provides a double-deck micro-fluidic chip for antibody detection, like the structure shown in figure 1, this chip from the bottom up includes reaction layer substrate 1, microcavity application of sample layer 2 and capping layer 3 in proper order.
As shown in fig. 2, the reaction layer substrate 1 includes a glass slide substrate and a glutaraldehyde substrate assembled on the glass slide substrate, wherein the glutaraldehyde substrate is prepared by immersing the glass slide substrate in an APTES absolute ethanol solution to prepare a silanized substrate; and then immersing the silanized substrate into a glutaraldehyde aqueous solution, reacting, washing and airing to obtain the silanized substrate.
Strip-shaped detection belts 4 and quality control belts 5 are printed on the glutaraldehyde substrate, the strip-shaped detection belts 4 and the quality control belts 5 are uniformly and periodically arranged, and the strip-shaped detection belts 4 and the quality control belts 5 are arranged in parallel at intervals.
The strip-shaped detection belt 4 and the quality control belt 5 are periodic strip-shaped detection belts 4 and periodic quality control belts 5 formed by printing a zigzag microfluidic chip on a glutaraldehyde substrate, and the periodic strip-shaped detection belts 4 and the periodic quality control belts 5 form stable chemical bonds by covalent crosslinking of amino groups in biomolecules and aldehyde groups of glutaraldehyde and are fixed on a glass slide substrate.
The glass slide substrate is made of calcium silicate, sodium silicate or silicon dioxide.
As shown in fig. 3, a plurality of square through holes 6 which are through up and down are arranged on the microcavity sample-adding layer 2, the square through holes 6 are uniformly arranged, and correspond to the strip-shaped detection zones 4 and the quality control zones 5 one by one to form a plurality of reaction cells; the array of X X Y can be designed on the same chip according to the number of samples to be detected, the value range of X is 1-5, the value range of Y is 1-12, and the array is mainly used for detecting serum samples.
As shown in fig. 4, the bottom of the capping layer 3 is provided with a plurality of micro-channel grooves 7 which are uniformly arranged, the plurality of micro-channel grooves 7 correspond to the plurality of reaction cells one by one, and both ends of the micro-channel grooves 7 are respectively provided with an inflow hole 8 and an outflow hole 9 which vertically penetrate through the capping layer 3. In this embodiment, the outlet 9 is located above the reaction cell.
The microcavity sample-adding layer 2 and the capping layer 3 are Polydimethylsiloxane (PDMS) material and a curing agent matched with the PDMS material, and the ratio of the PDMS material to the curing agent is as follows 10: 1, and processing by adopting a casting or injection molding mode.
A preparation method of a double-layer micro-fluidic chip for antibody detection comprises the following steps:
(1) respectively preparing a reaction layer substrate 1, a microcavity sampling layer 2 and a capping layer 3:
the reaction layer substrate 1 is prepared into a silanized substrate by immersing a glass slide substrate into an APTES absolute ethyl alcohol solution; then immersing the silanized substrate into a glutaraldehyde aqueous solution, reacting, washing and airing to obtain a glutaraldehyde substrate attached to the glass slide substrate, and then printing a purified antigen strip-shaped detection band 4 and a quality control band 5 on the glutaraldehyde substrate;
the microcavity sample-adding layer 2 is made of polydimethylsiloxane material by casting or injection molding, and a plurality of through holes 6 which are communicated up and down are formed in the microcavity sample-adding layer;
the sealing cover layer 3 is made of polydimethylsiloxane material by adopting a casting or injection molding mode, a plurality of micro-channel grooves 7 are processed at the bottom of the sealing cover layer, and meanwhile, inflow holes 8 and outflow holes 9 which penetrate through the sealing cover layer 3 up and down are respectively processed at two ends of each micro-channel groove 7;
(2) assembling the double-layer micro-fluidic chip:
overlapping the microcavity sample adding layer 2 and the reaction layer substrate 1 and positioning the microcavity sample adding layer on the reaction layer substrate 1, so that a plurality of through holes 6 of the microcavity sample adding layer 2 correspond to a plurality of strip-shaped detection bands 4 and quality control bands 5 one by one to form a plurality of reaction cells; and overlapping the bottom of the cover layer 3 with the microcavity sampling layer 2 and positioning the cover layer on the microcavity sampling layer 2, so that the plurality of micro-channel grooves 7 correspond to the plurality of reaction cells one by one, thereby obtaining the double-layer micro-fluidic chip for antibody detection.
The utility model also provides an application of detecting micro-fluidic chip in the detection of platelet antibody as above.
Firstly, respectively injecting 2uL of different samples to be detected and monoclonal detection antibodies from the inflow hole 8, filling the samples to be detected and the monoclonal detection antibodies into the reaction tank through the micro-channel groove 7, incubating for 20min to ensure that the antibodies and the monoclonal detection antibodies in the samples to be detected are fully combined with the specific antigens on the reaction layer substrate 1, after the incubation is finished, pumping the redundant liquid from the outflow hole 9, flushing the liquid with BSA with the mass concentration of 1%, and flushing the antibodies to be detected which are not adsorbed on the glutaraldehyde substrate. At this time, the antibody to be detected is combined with the specific antigen and fixed on the reaction layer substrate 1;
then injecting 2uL of the same goat anti-mouse fluorescent secondary antibody from the inflow hole 8 respectively, filling the goat anti-mouse fluorescent secondary antibody into the reaction tank through the micro-channel groove 7, incubating for 20min to ensure that the goat anti-mouse fluorescent secondary antibody is fully combined with the antibody specificity in the sample to be detected on the reaction layer substrate 1, after the incubation is finished, draining the redundant liquid from the outflow hole 9, washing the BSA with the mass concentration of 1%, and washing the goat anti-mouse fluorescent secondary antibody which is not adsorbed on the glutaraldehyde substrate. At this time, the goat anti-mouse fluorescent secondary antibody is specifically bound and fixed on the substrate with the antibody in the sample to be detected. Thereby realizing the fluorescent detection of the platelet antibody.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (5)
1. A double-layer micro-fluidic chip for antibody detection is characterized by sequentially comprising a reaction layer substrate, a micro-cavity sampling layer and a capping layer from bottom to top, wherein the reaction layer substrate comprises a glass slide substrate and a glutaraldehyde substrate assembled on the glass slide substrate, and a strip-shaped detection band and a quality control band are printed on the glutaraldehyde substrate; a plurality of through holes which are communicated up and down are arranged on the microcavity sample adding layer, and the through holes correspond to the strip-shaped detection zones and the quality control zones one by one to form a plurality of reaction tanks; the bottom of the sealing cover layer is provided with a plurality of micro-channel grooves, the micro-channel grooves correspond to the reaction tanks one by one, and inflow holes and outflow holes which penetrate through the sealing cover layer from top to bottom are formed in two ends of each micro-channel groove respectively.
2. The dual-layered microfluidic chip for antibody detection according to claim 1, wherein a plurality of the strip-shaped detection bands and the quality control bands are uniformly and periodically arranged, a plurality of the strip-shaped detection bands and the quality control bands are arranged in parallel at intervals, a plurality of the through holes are uniformly arranged, and a plurality of the micro-channel grooves are uniformly arranged to correspond to the plurality of the through holes respectively.
3. The double-layer microfluidic chip for antibody detection according to claim 1, wherein the material of the glass slide substrate is calcium silicate, sodium silicate or silicon dioxide.
4. The double-layered microfluidic chip for antibody detection according to claim 1, wherein the plurality of strip-shaped detection bands and quality control bands are periodic strip-shaped detection bands and quality control bands formed by printing a zigzag microfluidic chip on the glutaraldehyde substrate, and the periodic strip-shaped detection bands and the periodic quality control bands are fixed on the glass slide substrate by forming stable chemical bonds through covalent crosslinking of amino groups in biomolecules and aldehyde groups of glutaraldehyde.
5. The double-layer microfluidic chip for antibody detection according to claim 1, wherein the microcavity sample-adding layer and the capping layer are made of polydimethylsiloxane material by casting or injection molding.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113083386A (en) * | 2021-04-02 | 2021-07-09 | 重庆大学 | Simple and rapid liquid sample discretization chip and using method thereof |
| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
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2020
- 2020-06-17 CN CN202021122339.6U patent/CN212845420U/en active Active
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113083386A (en) * | 2021-04-02 | 2021-07-09 | 重庆大学 | Simple and rapid liquid sample discretization chip and using method thereof |
| CN113311160A (en) * | 2021-06-17 | 2021-08-27 | 山东科讯生物芯片技术有限公司 | Micro-fluidic biochip for rapidly detecting SARS-CoV-2 antigen and IgG/IgM antibody |
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