CN117471105B - Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application - Google Patents
Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application Download PDFInfo
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- 239000000758 substrate Substances 0.000 claims abstract description 22
- 238000003908 quality control method Methods 0.000 claims abstract description 18
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 7
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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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- 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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- 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
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract
The invention provides a microfluidic chip for detecting a bidirectional double-drive sequential platelet HLA antibody and application thereof, wherein the microfluidic chip comprises a substrate and a cover plate pressed on the substrate, the substrate and the cover plate are enclosed to form a microchannel, the left end of the microchannel is communicated with a second sample adding hole formed in the cover plate, the right end of the microchannel is communicated with a second waste liquid area, a marking area, a detection area and a quality control area are sequentially arranged on the microchannel from left to right, a first sample adding hole is further formed in the cover plate, a first waste liquid area is arranged between the marking area and the detection area, the first waste liquid area is arranged at two ends of the microchannel in the width direction and is communicated with the microchannel through a unidirectional liquid flow channel arranged on the microchannel, the marking area is provided with a marked anti-human IgG antibody, the detection area is coated with a platelet capturing antibody, and the microfluidic chip is used for detecting the platelet antibody. Can simplify the platelet antibody detection process and shorten the detection time.
Description
Technical Field
The invention belongs to the technical field of in-vitro diagnosis and immunodetection, and particularly relates to a microfluidic chip for detecting a bidirectional double-drive sequential platelet HLA antibody and application thereof.
Background
At present, component transfusion is increasingly widely applied to clinic, and the transfusions of platelets are used as important clinical transfusion treatment means, and have important value for treating bleeding tendency and platelet dysfunction caused by platelet deficiency. Platelet antibody detection is often used in medical diagnosis, and the platelet antibody detection is performed before blood transfusion particularly important for improving blood transfusion effect because part of patients generate platelet antibodies in vivo to cause ineffective blood transfusion, so that blood transfusion efficiency is low, blood resources are wasted, and serious complications, such as post-transfusion purpura, immune thrombocytopenic purpura, severe hemorrhage and the like, are caused even. Furthermore, in routine examination of gestational women, detection of platelet antibodies helps to avoid the occurrence of neonatal alloimmune thrombocytopenia.
Platelets express HLA-class I antigens, encoded by HLA complexes. Since HLA complexes are extremely abundant in polymorphism, there are almost no identical individuals among individuals who are unrelated. Thus, different individual HLA's in the population have different antigen specificities. For this reason, any individual in the presence of cells exposed to a heterologous HLA antigen will result in the production of HLA antibodies, such as in pregnant women, in allogeneic transfusion history, and the like. Platelets express only HLA-class I antigens, women with history of pregnancy, or individuals with history of blood transfusion, and are likely to produce antibodies against HLA-class I antigens, hereinafter abbreviated as platelet HLA antibodies. When a patient infuses platelets, it is desirable to detect the presence of antibodies in the patient against the donor's platelet HLA, or to select donor platelets that match the patient's serum, and is therefore commonly referred to as a platelet matching test.
At present, more than 80% of platelet infusion is ineffective because of platelet HLA antibodies, and most of the detection methods used are solid-phase agglutination methods, the method needs a large amount of serum, the actual operation needs more than one hour, the method comprises repeated washing steps, and the result interpretation mode is subjective, is easy to influence and has low accuracy. In addition, the method needs professional operators, can not immediately complete blood matching detection in a hospital, needs to be sent to a blood station for uniform blood matching, and can not realize rapid blood transfusion for patients. The platelet antibody detection gold standard is a monoclonal antibody specific platelet antigen immobilization test (Monoclonal Immobilization of Platelet Antigen, MAIPA), and the test has better accuracy, but has complicated operation steps, extremely long time and high requirements on operators, and is only limited to being developed in a laboratory and cannot be popularized to the clinic.
Disclosure of Invention
Aiming at the defect of conventional detection of the prior platelet HLA antibody, the invention provides a microfluidic chip for detecting the bidirectional double-drive sequential platelet HLA antibody and application thereof, and the chip can be used for perfectly integrating the complex flow of conventional platelet HLA antibody detection, thereby greatly reducing the time required by platelet antibody detection and enabling the platelet antibody detection work in the early-stage recipients of clinical blood transfusion to be faster and more convenient.
In order to achieve the above object, the technical scheme of the invention is that the microfluidic chip for detecting the platelet HLA antibody comprises a substrate and a cover plate pressed on the substrate, wherein the substrate and the cover plate are enclosed to form a microchannel, the left end of the microchannel is communicated with a second sample adding hole formed in the cover plate, the right end of the microchannel is communicated with a second waste liquid area, the second waste liquid area is provided with a movable filter paper sheet, the filter paper sheet contacts or is far away from the microchannel when moving, the microchannel is sequentially provided with a marking area, a detection area and a quality control area from left to right, the cover plate is also provided with a first sample adding hole, the first sample adding hole is arranged above the microchannel between the quality control area and the second waste liquid area and is communicated with the microchannel, a first waste liquid area is arranged between the marking area and the detection area, the first waste liquid area is arranged at two ends of the width direction of the microchannel and is communicated with the microchannel through a unidirectional liquid channel arranged on the microchannel, the marking area is provided with a mark anti-human platelet antibody capture area.
In one embodiment of the invention, the lower surface of the cover plate is provided with a groove along the length direction, and the cover plate and the upper surface of the substrate are enclosed to form a micro-channel through the groove.
In one embodiment of the invention, the unidirectional liquid flow channel is arranged on the lower surface of the cover plate, two ends of the unidirectional liquid flow channel are communicated with the micro-channel, the width of the part, where the left end of the unidirectional liquid flow channel is communicated with the micro-channel, is smaller than the width of other positions of the micro-channel, the unidirectional liquid flow channel gradually narrows from left to right, and the first waste liquid area is communicated with the front end and the rear end of the widest part of the unidirectional liquid flow channel.
In one embodiment of the invention, the first waste liquid zone is provided on the lower surface of the cover sheet, the first waste liquid zone being provided with a water absorbent material.
In one embodiment of the invention, the marking area, the detection area, the quality control area and the second waste liquid area are all arranged on the upper surface of the substrate.
In one embodiment of the invention, the upper surface of the substrate is provided with buffer solution dry powder at the positions corresponding to the first sample adding hole and the second sample adding hole.
On the other hand, the invention also provides an application of the microfluidic chip in any one of the technical schemes to detection of platelet HLA antibodies, which comprises at least the following steps:
1) Dripping platelets to be detected into the first sample adding hole, wherein the flowing direction of the liquid is from right to left, the platelets are combined with platelet capture antibodies of the detection area in a side flow mode preferentially, and the rest samples or other substances in the samples are collected in a first waste liquid area;
2) Dropwise adding serum to be detected into the first sample adding hole, wherein the flowing direction of the liquid is from right to left, the serum is combined with platelets combined with the detection area in a lateral flow mode preferentially, and the rest samples or other substances in the samples are collected in a first waste liquid area;
3) Filling distilled water into the second sample adding hole, and simultaneously moving the filter paper sheet positioned in the second waste liquid area leftwards to contact with liquid in the micro-channel, wherein the liquid moves from left to right, the labeled anti-human IgG antibody in the labeled area is dissolved, the labeled antibody continues to move to the detection area and the quality control area to be combined with the detection area and the quality control area, and the excessive labeled antibody is collected in the second waste liquid area along with the liquid flow;
4. and reading signal values of the detection area and the quality control area according to the conventional micro-flow control.
The micro-fluidic chip for detecting the two-way double-drive sequential platelet HLA antibody, which is obtained through the technical scheme, and the application thereof have the beneficial effects that:
1. simplifying platelet antibody detection process and shortening detection time
The micro-size and the precise manufacturing of the micro-fluidic chip can realize accurate fluid manipulation and reaction control, can reach micrometer-level precision, is more accurate than the traditional experimental method, and is more suitable for POCT analysis modes. The microfluidic chip is used for integrating complex experimental steps, only a small amount of serum samples are needed for detection and no professional operation is needed, compared with the existing method, the experimental time can be greatly shortened, the working efficiency is improved, portability is provided for clinical diagnosis, the result is presented in a signal value mode, and compared with a solid-phase agglutination rule, the method is more accurate and is not influenced by subjective judgment of experimental operators.
2. Avoidance of non-specific IgG interference
In a conventional microfluidic detection mode, serum to be detected is preferentially mixed with phycoerythrin-labeled mouse anti-human IgG to form a complex and then flows through a detection area, a large amount of non-specific IgG antibodies exist in the serum to be detected, and the labeled secondary antibodies are combined with the non-specific antibodies to interfere with the detection result of the antibody to be detected. The invention adopts a bidirectional liquid flow mode, so that platelets and serum to be detected sequentially flow through a detection area from right to left, the platelets and the serum to be detected are captured gradually, and after the nonspecific IgG antibody is absorbed by a waste liquid pool, the labeled secondary antibody flows through the detection area from left to right, thereby avoiding the interference of nonspecific IgG and improving the detection sensitivity.
Drawings
FIG. 1 is a schematic structural diagram of a microfluidic chip according to the present invention;
FIG. 2 is a schematic view of the structure of the upper surface of the substrate according to the present invention;
FIG. 3 is a schematic view of the structure of the lower surface of the cover sheet according to the present invention;
FIG. 4 is a schematic diagram of the flow direction of the filter paper sheet driving liquid in the second liquid waste region according to the present invention;
fig. 5 is a schematic structural diagram of a platelet HLA antibody positive serum detection microfluidic chip according to one of the embodiments of the present invention;
FIG. 6 is a schematic diagram of the present invention for dropping platelets to be tested on the chip of FIG. 5;
FIG. 7 is a schematic diagram of the present invention after dropping serum to be tested on the chip of FIG. 5;
FIG. 8 is a schematic diagram of the present invention after distilled water is added dropwise to the chip of FIG. 5.
Detailed Description
It should be noted that, without conflict, the embodiments of the present invention and features of the embodiments may be combined with each other.
Unless defined otherwise, technical terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified.
The invention relates to the technical field of immunodetection, in particular to platelet HLA antibody detection, and the core of the invention is based on a microfluidic technology, and designs a bidirectional and double-drive immunomicrofluidic chip which is used for perfectly integrating the complex flow of conventional platelet HLA antibody detection, so that the time required for platelet antibody detection is greatly reduced, and the platelet antibody detection work in a clinical transfusion early-stage recipient is faster and more convenient.
The invention is further illustrated below with reference to examples and figures, it being understood that the invention is not limited to the specific embodiments described.
As shown in fig. 1, the invention provides a microfluidic chip for detecting a bidirectional double-drive sequential platelet HLA antibody, which comprises a substrate and a cover plate pressed on the substrate, wherein the substrate and the cover plate are surrounded to form a microchannel, the left end of the microchannel is communicated with a second sample adding hole B formed in the cover plate, the right end of the microchannel is communicated with a second waste liquid area W2, the second waste liquid area W2 is provided with a movable filter paper sheet, the filter paper sheet contacts or is far away from the microchannel when moving, the microchannel is used for controlling the flow of liquid in the microchannel to the second waste liquid area W2 by moving the filter paper sheet, a labeling area L, a detection area T and a quality control area C are sequentially arranged in the microchannel from left to right, a first sample adding hole S is further formed in the cover plate, the first sample adding hole S is arranged above the microchannel between the quality control area C and the second waste liquid area W2 and is communicated with the microchannel, a first waste liquid area W1 is arranged between the labeling area L and the detection area W2, the first area W1 is arranged in the width direction of the microchannel and is contacted with or far away from the microchannel, the microchannel is arranged on the side of the microchannel, when the microchannel is not contacted with the microchannel, the microchannel is contacted with the microchannel, the liquid flow of the liquid from the detection area D, and the microchannel is one-way channel is arranged on the side of the microchannel, and the channel is one-way, and the liquid flow channel is one-way or the liquid flow can flow through the channel, and the channel is one-way, and the channel can flow through the channel and the channel has the one-way direction channel and the flow direction channel.
The quality control region C is conventionally provided, for example, coated with an anti-antibody that specifically binds to a labeled anti-human IgG antibody, for determining whether the detection process is effective.
As shown in fig. 3, the lower surface of the cover plate is provided with a groove along the length direction, and the cover plate and the upper surface of the substrate are enclosed to form a micro-channel through the groove.
The one-way liquid flow channel D is arranged on the lower surface of the cover plate, two ends of the one-way liquid flow channel D are communicated with the micro-channel, the width of the communication part between the left end of the one-way liquid flow channel D and the micro-channel is smaller than the width of other positions of the micro-channel, the one-way liquid flow channel D is gradually narrowed from left to right, and the front end and the rear end of the widest part of the first waste liquid area W1 are communicated with the widest part of the one-way liquid flow channel D.
The first waste liquid area W1 is arranged on the lower surface of the cover plate, and the first waste liquid area W1 is provided with a water absorbing material.
When liquid flows to the right side from the micro-channel at the left side of the unidirectional liquid flow channel D, the opening of the micro-channel is smaller, so that the liquid is accelerated to flow into the unidirectional liquid flow channel D under the siphon action and continuously enters the micro-channel at the right side of the unidirectional liquid flow channel D, and the liquid cannot enter the first waste liquid area; when the liquid flows from the micro channel on the right side of the unidirectional flow channel D to the left side, the flow rate of the liquid entering the unidirectional flow channel D is reduced due to the gradual widening of the unidirectional flow channel D, and the liquid spreads in the width direction of the unidirectional flow channel D, and at this time, the liquid is adsorbed by the water-absorbent material of the first waste liquid area W1 connected to both ends of the unidirectional flow channel D and flows into the first waste liquid area W1.
As shown in fig. 2, the marking area L, the detecting area T, the quality control area C, and the second waste liquid area W2 are all disposed on the upper surface of the substrate.
And buffer solution dry powder is arranged on the upper surface of the substrate at positions corresponding to the first sample adding hole B and the second sample adding hole S.
The labeled anti-human IgG antibody is an phycoerythrin labeled antibody.
Examples
Taking platelet HLA antibody positive serum detection as an example, the detection principle of the bidirectional double-drive microfluidic chip is described:
the structure of the microfluidic chip is shown in figure 5,
marking area: coating phycoerythrin marked mouse anti-human IgG antibody.
Detection area: the detection zone is coated with GPIIb/IIIa antibodies (platelet capture antibodies).
And a quality control area: rabbit anti-murine antibodies (which can bind to phycoerythrin-labeled murine anti-human IgG antibody molecules).
As shown in fig. 4 and 6, during detection, the filter paper sheet of the second waste liquid area is firstly placed on the right side and is not connected with the channel, platelets to be detected are added into the first sample adding hole, flow from right to left under the action of the driving force of the micro-channel, are combined with the GPIIb/IIIa antibody of the detection area, and unbound substances flow into the first waste liquid area and are collected under the driving of the filter paper sheet.
As shown in FIG. 7, the serum to be tested containing positive antibodies was added to the first well and likewise flowed to the left, and the platelet antibodies were attached to the platelets bound to the detection zone, and unbound material flowed into the first waste zone and collected.
As shown in fig. 4 and 8, the filter paper sheet in the second waste liquid area is pushed to the left and combined with the microfluidic channel, distilled water is added to the second sample Kong Di at this time to rapidly dissolve the buffer solution dry powder, the liquid moves to the right under the driving force of the microchannel, the murine anti-human IgG antibody coated with phycoerythrin is dissolved, the solution continues to flow to the right through the detection area and the quality control area, and the remaining labeled antibody flows to the second waste liquid area.
At this time, a detecting instrument is used to detect the signal intensity of the detection area and the quality control area and output the detection result (the detecting instrument is a conventional technology, for example, a fluorescence detector, and is consistent with the signal carried by the labeled antibody).
The above technical solution only represents the preferred technical solution of the present invention, and some changes that may be made by those skilled in the art to some parts of the technical solution represent the principles of the present invention, and the technical solution falls within the scope of the present invention.
Claims (7)
1. The utility model provides a two-way two-drive sequential formula platelet HLA antibody detection's micro-fluidic chip, includes substrate and pressfitting cover plate on the substrate, and substrate and cover plate enclose and close the microchannel that forms, its characterized in that, the second application of sample hole intercommunication of seting up on microchannel left end and the cover plate, the right-hand member and second waste liquid district intercommunication, the second waste liquid district is provided with mobilizable filter paper piece, contact or keep away from when the filter paper piece removes the microchannel, the microchannel has from left to right set gradually mark district, detection zone, matter control zone, still set up first application of sample hole on the cover plate, first application of sample hole sets up matter control zone and second waste liquid district's microchannel top to be linked together with the microchannel, be provided with first waste liquid district between mark district and the detection zone, first waste liquid district sets up the both ends of microchannel width direction, and through the one-way flow channel that sets up on the microchannel with the microchannel is linked together, one-way flow channel both ends intercommunication the microchannel, one-way flow channel left end and width from left to right and the other places of microchannel are less than the microchannel, the one-way is caught by the sign the antibody of the miniascaper, the one-way is located from left to right and is the width of the microchannel is gradually from left to the front to the side to the microchannel, the sign the anti-human body is located to the sign.
2. The microfluidic chip for detecting the two-way double-drive sequential platelet HLA antibody according to claim 1, wherein the lower surface of the cover plate is provided with a groove along the length direction thereof, and the cover plate and the upper surface of the substrate are enclosed to form a micro channel through the groove.
3. The microfluidic chip for bidirectional dual drive sequential platelet HLA antibody detection according to claim 2, wherein the unidirectional flow channel is disposed on the lower surface of the cover sheet.
4. The microfluidic chip for bidirectional dual drive sequential platelet HLA antibody detection according to claim 2, wherein the first waste liquid region is disposed on the lower surface of the cover sheet, and the first waste liquid region is provided with a water absorbing material.
5. The microfluidic chip for detecting the two-way double-drive sequential platelet HLA antibody according to claim 2, wherein the labeling area, the detection area, the quality control area and the second waste liquid area are all arranged on the upper surface of the substrate.
6. The microfluidic chip for detecting the two-way double-drive sequential platelet HLA antibody according to claim 2, wherein buffer dry powders are respectively arranged on the upper surface of the substrate at positions corresponding to the first sample adding hole and the second sample adding hole.
7. Use of a bi-directional double-drive sequential platelet HLA antibody detection, characterized in that the detection is performed using a microfluidic chip according to any one of claims 1-6, comprising at least the following steps:
1) Dripping platelets to be detected into the first sample adding hole, wherein the flowing direction of the liquid is from right to left, the platelets are combined with platelet capture antibodies of the detection area in a side flow mode preferentially, and the rest samples or other substances in the samples are collected in a first waste liquid area;
2) Dropwise adding serum to be detected into the first sample adding hole, wherein the flowing direction of the liquid is from right to left, the serum is combined with platelets combined with the detection area in a lateral flow mode preferentially, and the rest samples or other substances in the samples are collected in a first waste liquid area;
3) Filling distilled water into the second sample adding hole, and simultaneously moving the filter paper sheet positioned in the second waste liquid area leftwards to contact with liquid in the micro-channel, wherein the liquid moves from left to right, the labeled anti-human IgG antibody in the labeled area is dissolved, the labeled antibody continues to move to the detection area and the quality control area to be combined with the detection area and the quality control area, and the excessive labeled antibody is collected in the second waste liquid area along with the liquid flow;
4) And reading signal values of the detection area and the quality control area according to the conventional micro-flow control.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311810362.2A CN117471105B (en) | 2023-12-27 | 2023-12-27 | Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311810362.2A CN117471105B (en) | 2023-12-27 | 2023-12-27 | Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application |
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| Publication Number | Publication Date |
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| CN117471105A CN117471105A (en) | 2024-01-30 |
| CN117471105B true CN117471105B (en) | 2024-03-26 |
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| CN202311810362.2A Active CN117471105B (en) | 2023-12-27 | 2023-12-27 | Microfluidic chip for bidirectional double-drive sequential platelet HLA antibody detection and application |
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