CN109975563B - Integrated reagent device for microfluid immunoassay - Google Patents
Integrated reagent device for microfluid immunoassay Download PDFInfo
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- CN109975563B CN109975563B CN201910217608.2A CN201910217608A CN109975563B CN 109975563 B CN109975563 B CN 109975563B CN 201910217608 A CN201910217608 A CN 201910217608A CN 109975563 B CN109975563 B CN 109975563B
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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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
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- G01N33/544—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being organic
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- G01N2035/00237—Handling microquantities of analyte, e.g. microvalves, capillary networks
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Abstract
The invention relates to a microfluid immunoassay integrated reagent device, which comprises a base, an upper cover and a characteristic marker detection reaction tube, wherein a washing liquid storage tank, a marker film sample loading liquid guide structure, a reaction tube mounting seat and a waste liquid storage tank are sequentially arranged on the base along the flow direction of a reagent, the upper cover is combined with the base in a jogged manner, the characteristic marker detection reaction tube is formed by injection molding of PS (polystyrene) plastics, is fixedly embedded in the reaction tube mounting seat, has an observation surface facing upwards and is provided with a slit-shaped inner cavity, two ends of the inner cavity are respectively a liquid inlet and a liquid outlet, the marker film sample guide structure fixedly installs the marker film in the liquid inlet and is provided with a liquid guide cavity, a washing liquid inlet and a sample inlet which are communicated with the liquid guide cavity, the washing liquid storage tank is communicated with the liquid inlet through a washing liquid inlet, the liquid guide cavity is communicated with the liquid inlet through a fluid, the waste liquid storage tank is communicated with the liquid outlet through a fluid, and a water absorption material is filled in the waste liquid storage tank. The microfluidic immunoassay integrated reagent device has a compact structure and is convenient to use.
Description
Technical Field
The invention relates to a structure of a reagent tube for instant detection, in particular to a microfluid immunoassay integrated reagent device for realizing high-sensitivity instant detection in medical diagnosis, health monitoring, food safety and other emergency places.
Background
In medical diagnosis, health monitoring, food safety and other emergency places, high-sensitivity instant detection always has a real and vigorous demand.
Currently, the labeling immune instant detection based on lateral chromatography is widely applied, and labels relate to colloidal gold, fluorescence, lanthanide rare earth element Eu2+, quantum dots and the like. The method has the greatest advantages that the detection process is simple and convenient to operate, the structure of the used instrument is relatively short, and the method can meet the requirement of instant detection in many places.
The labeled immunoreagent material based on lateral chromatography is poor in quality controllability, large in uncertainty of production process control, unstable in property of reagent assembly materials, uncontrollable in detection process, low in sensitivity and large in variation of detection results, and prone to occurrence of conditions such as missed diagnosis and misdiagnosis in clinical application, so that the labeled immunoreagent material is only used for screening detection of high-risk people.
The detection test application of enzyme linked immunosorbent assay based on PS (polystyrene) microporous plate has spanned half century, and is still widely applied so far. Countless facts prove that the reagent structure which takes the PS as the solid phase and is detected by the enzyme-linked immunosorbent assay is an immunoassay device with simple structure, easy processing and low cost and high signal-to-noise ratio.
The reaction area of the solid-liquid interface of the reaction liquid is increased by increasing the surface area of the micropore inner cavity of the micropore plate, the detection sensitivity is improved, and the detection time is shortened. The invention is found in Chinese invention patents CN101533009A and CN104977405A. The application of related products verifies the innovative value of the invention, more effectively improves the detection capability of low values and high values, and the reaction behavior is more complete and close to the liquid-liquid homogeneous reaction.
However, the object of the present invention is to provide a detection device which is designed and processed from a PS material in a simpler manner, satisfies an instantaneous detection mode, and has high sensitivity.
Disclosure of Invention
The invention aims to provide a microfluid immunoassay integrated reagent device based on labeled immunoassay, which utilizes capillary action and centrifugal force to drive a reaction process, can realize rapid manual or automatic sample loading and automatic control of the reaction process, can automatically detect an analysis result through an instrument, and particularly can finish automatic washing of a detection tube to separate reacted components from unreacted components, thereby greatly improving the detection sensitivity.
In order to achieve the above object, the present invention provides an integrated reagent device for microfluidic immunoassay, comprising: the reaction tube comprises a washing liquid storage tank, a marker film sample-loading liquid guide structure, a reaction tube mounting seat and a waste liquid storage tank, wherein the washing liquid storage tank, the marker film sample-loading liquid guide structure, the reaction tube mounting seat and the waste liquid storage tank are sequentially arranged on the base along the reagent flowing direction, the upper cover is combined with the base in an embedded mode, the characteristic marker detection reaction tube is formed by injection molding of PS (polystyrene) plastics, is fixedly embedded in the reaction tube mounting seat, has an observation surface facing upwards and is provided with a slit-shaped inner cavity, so that a capillary action is generated to propel a reaction process, two ends of the inner cavity are respectively provided with a liquid inlet and a liquid outlet, the marker film sample-loading liquid guide structure fixedly installs the marker film in the liquid inlet and is provided with a liquid guide cavity, a washing liquid inlet and a sample inlet which are communicated with the liquid guide cavity, the washing liquid storage tank is communicated with the liquid guide cavity through the washing liquid, the liquid guide cavity is communicated with the liquid inlet, the waste liquid inlet is communicated with the liquid inlet, the waste liquid outlet is communicated with the liquid, and water absorption materials are filled in the waste liquid storage tank.
Furthermore, the washing liquid storage tank is an oval cavity structure, a sealed washing liquid bag is placed in the washing liquid storage tank, an opening is formed in one side, facing the sample loading and liquid guiding structure of the marker membrane, of the washing liquid storage tank, the opening is in fluid communication with the washing liquid introducing port and is internally provided with a puncture needle, and the puncture needle is used for puncturing the washing liquid bag.
Further, the volume of the wash solution in the wash solution bag is 1 ml.
Further, the marker membrane sample loading and drainage structure comprises a marker membrane fixing clamp, and the marker membrane fixing clamp is used for pushing and fixing the marker membrane at the marker membrane position of the liquid inlet.
Furthermore, the sample introducing port is of a cross-shaped capping opening and closing structure, the sample is introduced after the sampling needle is ejected, and the opening is closed after the sampling needle is withdrawn.
Further, the waste reservoir is in fluid communication with the liquid outlet via a isthmus.
Furthermore, a luminescent substrate liquid leading-in hole is formed in the characteristic marker detection reaction tube close to the liquid outlet, and the luminescent substrate liquid leading-in hole is used for adding luminescent substrate liquid.
Furthermore, a clamping edge is arranged on the reaction tube mounting seat and used for fixing the characteristic marker detection reaction tube.
Further, the cross section of the inner cavity of the characteristic marker detection reaction tube is circular, semicircular, elliptical, rectangular or trapezoidal.
Further, the wall surface of the inner cavity can be provided with an arc-shaped or corrugated structure so as to increase the wall surface area of the inner cavity.
Further, the base is integrally formed by injection molding of a PP material.
By adopting the technical scheme, the invention has the beneficial effects that: the microfluid immunoassay integrated reagent device has compact structure and convenient use, utilizes PS plastic with large specific surface area and high signal-to-noise ratio as a solid phase carrier, utilizes capillary action to drive reaction, utilizes centrifugal force to wash a reaction area, and realizes high-efficiency and controllable reaction and separation of the whole reaction at an interface with high adsorption capacity, thereby achieving high-sensitivity qualitative and quantitative instant detection.
Drawings
To further illustrate the various embodiments, the invention provides the accompanying drawings. The accompanying drawings, which are incorporated in and constitute a part of this disclosure, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the embodiments. Those skilled in the art will appreciate still other possible embodiments and advantages of the present invention with reference to these figures. Elements in the figures are not drawn to scale and like reference numerals are generally used to indicate like elements.
FIG. 1 is a schematic structural diagram of a microfluidic immunoassay integrated reagent device according to the present invention;
FIG. 2 is a schematic view of the structure of the characteristic marker detection reaction tube in FIG. 1;
FIGS. 3a, 3b, 3c and 3d show different cross-sections of the characteristic marker detection reaction tube described in FIG. 2, respectively.
Detailed Description
The invention will now be further described with reference to the accompanying drawings and detailed description.
As shown in fig. 1 and 2, a microfluidic immunoassay integrated reagent device may include a base 1, an upper cover (not shown), and a characteristic marker detection reaction tube 2. The base 1 is formed by pp plastics through integral injection molding, and can comprise a washing liquid storage tank 11, a marker membrane sample loading and liquid guiding structure 12, a reaction tube mounting seat 13 and a waste liquid storage tank 14 which are sequentially arranged along the flowing direction of a reagent. The upper cover is consistent with the shape of the base 1 and is combined with the base 1 in a mosaic mode to form a closed washing liquid storage tank 11, a marker membrane sample loading liquid guide structure 12 and a waste liquid storage tank 14. For the sake of clarity of orientation, the base 1 is narrower at one end of the lotion reservoir 11. Further, for the convenience of grasping, the middle portion of the base 1 is provided with a concave portion 15. The washing reagent reservoir 11, the label membrane loading and drainage structure 12, the reaction tube holder 13, the waste liquid reservoir 14, and the characteristic marker detection reaction tube 2 will be described in detail below.
As shown in FIG. 1, the washing liquid storage tank 11 is an oval cavity structure and is located at one end of the base 1. An opening 111 is provided in the side of the washing solution reservoir facing the label membrane loading guide structure 12, and a sharp-edged protrusion is provided in the opening, and a needle 112 is provided therein. A sealed wash solution bag 113 is placed in the wash solution tank 11. During centrifugation, the washing solution bag 113 is pushed to the puncture needle 112 by centrifugal force, the puncture needle 112 punctures the washing solution bag 113, and the washing solution flows out of the washing solution bag 113, passes through the opening 111 and the marker membrane sample-loading liquid guide structure 12, and enters the feature marker detection reaction tube 2, thereby completing the washing function. The washing solution bag 113 contains a washing solution sufficient to wash the characteristic marker detection reaction tube 2 sufficiently. In one embodiment, the volume of wash solution in the wash solution bag 113 is about 1 mL.
The labeled membrane loading and liquid guiding structure 12 is located between the washing solution reservoir 11 and the reaction tube holder 13, and may include a labeled membrane fixing clip 121, a liquid guiding chamber 122, a sample introducing port 123, and a washing solution introducing port 124. The label membrane fixing clip 121 is used to push the label membrane 125 against the label membrane position in the liquid inlet 211 of the inner cavity 21 of the characteristic marker detection reaction tube 2, and further fixed to the label membrane position by physical structure acting force. The drainage lumen 122 is used to allow a washing solution and a sample to be introduced and flow through the marker membrane to the characteristic marker detection reaction tube 2. That is, the drainage lumen 122 is in fluid communication with the sample introduction port 123 and the washing liquid introduction port 124. The sample introducing port 123 is a cross-shaped capping opening and closing structure, a sample is introduced after the sampling needle is ejected, and the opening is closed after the sampling needle is ejected, so that the washing liquid is prevented from being led out during centrifugation. The washing liquid introduction port 124 is in fluid communication with a washing liquid reservoir (specifically, the opening 111), and the washing liquid is introduced into the liquid guide chamber 122 by high-speed centrifugal force.
As shown in fig. 1 to 3d, the characteristic marker detection reaction tube 2 is injection-molded from PS plastic and is light-permeable. The characteristic marker detection reaction tube 2 is embedded in the reaction tube mounting seat 13. Specifically, the reaction tube mounting base 13 is provided with a clamping edge 131, and the characteristic marker detection reaction tube 2 is fixed by the clamping edge 131. Therefore, the characteristic marker detection reaction tube 2 does not move during centrifugation. The characteristic marker detection reaction tube 2 has a slit-shaped inner cavity 21, and this structure is advantageous in promoting the progress of the reaction by generating capillary action. Namely, the capillary action can be formed in the cavity, which is beneficial to the solid phase process and the driving detection reaction. The cross-section of the lumen 21 may be circular, semi-circular (as shown in fig. 3 a), rectangular (as shown in fig. 3 b), oval (as shown in fig. 3 c), or trapezoidal (as shown in fig. 3 d), etc. The wall surface 213 of the inner chamber 21 is a reaction surface, and the upper surface 22 of the characteristic marker detection reaction tube 2 is an observation surface. Preferably, the wall surface of the inner cavity 21 is provided with an arc or a corrugated structure to increase the surface area, i.e. the reaction surface area, and further increase the reaction speed. The characteristic marker detection reaction tube 2 has two flush ends, i.e., the two ends of the inner cavity 21 are a liquid inlet 211 and a liquid outlet 212, respectively. The inlet port 211 is in fluid communication with the drainage lumen 122 of the label film loading drainage structure 12, and the outlet port 212 is in fluid communication with the waste reservoir 14. The inlet port 211 is flared to better fill the marker membrane 125.
In the illustrated embodiment, the characteristic marker-detecting reaction tube 2 is provided, near the liquid outlet, with a luminescent substrate liquid introduction hole 23, and the luminescent substrate liquid introduction hole 23 is used for introducing a luminescent substrate liquid. The luminescent substrate liquid introduction hole 23 is provided with a sealing means (e.g., a sealing film) which is opened when the luminescent substrate liquid is required to be introduced.
The characteristic marker detection reaction tube 2 may further comprise a protective edge 24, the protective edge 24 being adapted to protect the characteristic marker detection reaction tube 2 on the one hand and to cooperate with a clamping edge of the base on the other hand to better fix the characteristic marker detection reaction tube 2.
As shown in fig. 1, the waste liquid tank 14 is a hollow structure having a flat horseshoe shape and one closed end. Of course, the waste reservoir 14 may take other configurations. The waste liquid tank 14 is located at the rear end of the characteristic marker detection reaction tube 2, and is filled with a water absorbing material 141. The water absorbing material 141 serves to absorb reaction waste and washing liquid to prevent backflow. The water absorbing material 141 may be a sponge, a resin, or the like. Preferably, the waste reservoir 14 is connected to the characteristic marker detection reaction tube 2 (specifically, the liquid outlet 212) through the isthmus 142 to prevent backflow of unabsorbed reaction waste and washing liquid.
The working principle of the present invention is explained in detail below. When in use, the microfluidic fluorescence immunoassay integrated reagent device is firstly placed in a reaction detection disc by a bayonet and is symmetrically placed in sequence. When the odd microfluid fluorescence immunoassay integrated reagent device is used, a blank control device is arranged to balance the centrifugal force; secondly, the sample is led into the cross seal 123 from the sample through the automatic sample introduction device and is added to the liquid guide cavity 122, then the marker on the marker membrane 125 is dissolved, the compound formed by the target object and the marker in the sample is transferred into the inner cavity 21 of the reaction tube 2 by means of capillary force and is combined and reacted with the solid phase object on the reaction surface 213 to form a reaction detection chain, the reaction lasts for a certain time by means of capillary force or centrifugal force until the reaction tube 2 is completely filled with the reaction liquid and lasts for a specified time, and under the action of the capillary force or centrifugal force, the reactant flows to the waste liquid storage tank 14 through the gorges 142 and is absorbed and retained by the water absorption material 141; then washing, starting centrifugation, pushing the washing liquid bag 113 in the washing liquid storage tank 11 to the puncture needle 112 by means of centrifugal force under the short-term rotation of a certain rotation speed (a rotation speed working point needs to be established), and puncturing, wherein the washing liquid flows to the marker membrane 125 through the flow guide cavity 122, washes the marker membrane 125, flows to the inner cavity 21 of the reaction tube 2, the effective reactant continuously reacts and combines with the uncombined point on the reaction surface 213, and rotates for a certain time at a low speed to fully carry out the reaction; then, the reaction detection disc rotates intermittently at a high speed, the reaction surface 213 is fully washed, and the reaction detection disc stops rotating and is static after the reaction surface 213 is fully washed; and finally, performing fluorescence detection, namely scanning the reaction surface 213 of the inner cavity 21 from the observation surface 22 in a segmented manner by the fluorescence detection device from near to far, measuring the fluorescence value of each segment, and calculating the fluorescence value of each segment and the ratio of the fluorescence values of each segment.
It should be noted that, in the luminescence detection, since the luminescent substrate needs to be added, the sealing film on the luminescent substrate liquid introduction hole 23 needs to be torn off and added, and the luminescence detection is performed after the diffusion thereof is completed.
According to the established and stored standard curve, calculating the concentration value of the whole blood sample corresponding to each representative index of the microfluidic fluorescence/luminescence immunoassay integrated reagent, converting the plasma concentration value according to the hematocrit ratio, and respectively reporting the plasma or whole blood concentration value.
While the invention has been particularly shown and described with reference to a preferred embodiment, it will be understood by those skilled in the art that various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (8)
1. An integrated reagent device for microfluidic immunoassay, comprising: the reagent sample injection device comprises a base, an upper cover and a characteristic marker detection reaction tube, wherein a washing liquid storage tank, a marker membrane sample injection and guide structure, a reaction tube mounting seat and a waste liquid storage tank are sequentially arranged on the base along the reagent flowing direction, the upper cover is combined with the base in an embedded manner, the characteristic marker detection reaction tube is formed by injection molding of PS (polystyrene) plastics, is fixedly embedded in the reaction tube mounting seat, has an observation surface facing upwards and is provided with a slit-shaped inner cavity, and has a liquid inlet and a liquid outlet at two ends for generating capillary action to propel a reaction process, the marker membrane sample injection and guide structure fixedly installs a marker membrane in the liquid inlet and is provided with a liquid guide cavity, a washing liquid inlet and a sample inlet which are communicated with the liquid guide cavity, the washing liquid storage tank is communicated with the liquid guide cavity through the washing liquid, the liquid guide cavity is communicated with the liquid inlet through the fluid of the liquid inlet, the waste liquid storage tank is communicated with the liquid outlet through the fluid, a water absorption material is filled in the waste liquid guide cavity, the washing liquid storage tank is of an elliptic cavity structure, a sealed washing liquid bag is arranged in the washing liquid guide cavity, an opening and a needle is arranged at one side facing the marker membrane sample injection needle, the washing liquid guide structure is communicated with the liquid inlet, and is used for opening and closing the sample injection needle, and closing the needle for closing the needle, and closing the needle is used for closing the sample injection needle, and withdrawing the needle for withdrawing the sample injection needle, and the needle for withdrawing the sample injection needle for injecting the sample injection needle; the base is narrower at one end of the washing liquid storage tank, and a concave portion is provided at the middle portion of the base.
2. The microfluidic immunoassay integrated reagent device of claim 1, wherein: the marker membrane sample loading and drainage structure comprises a marker membrane fixing clamp, and the marker membrane fixing clamp is used for pushing and fixing the marker membrane on the marker membrane position of the liquid inlet.
3. The microfluidic immunoassay integrated reagent device of claim 1, wherein: the waste reservoir is in fluid communication with the liquid outlet via a isthmus.
4. The microfluidic immunoassay integrated reagent device of claim 1, wherein: and a luminescent substrate liquid leading-in hole is formed in the characteristic marker reaction tube close to the liquid outlet, and is used for adding luminescent substrate liquid.
5. The microfluidic immunoassay integrated reagent device of claim 1, wherein: and a clamping edge is arranged on the reaction tube mounting seat and used for fixing the characteristic marker reaction tube.
6. The microfluidic immunoassay integrated reagent device of claim 1, wherein: the cross section of the inner cavity of the characteristic marker reaction tube is circular, semicircular, elliptical, rectangular or trapezoidal.
7. The microfluidic immunoassay integrated reagent device of claim 1, wherein: the wall surface of the inner cavity is provided with an arc-shaped or corrugated structure.
8. The microfluidic immunoassay integrated reagent device of any one of claims 1 to 7, wherein: the base is integrally formed by injection molding of a PP material.
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| CN110508338B (en) * | 2019-08-30 | 2024-07-02 | 烟台芥子生物技术有限公司 | Liquid bag for microfluidic chip |
| CN113419073B (en) * | 2020-03-30 | 2024-05-31 | 厦门先明生物技术有限公司 | High-flux full-automatic microfluidic fluorescence immunoassay device |
| CN111659477B (en) * | 2020-06-11 | 2022-02-18 | 厦门先明生物技术有限公司 | Independent sample introduction micro-fluidic immunoassay joint inspection device |
| CN111665354B (en) * | 2020-06-11 | 2023-06-09 | 厦门先明生物技术有限公司 | Shared sample injection microfluidic immunoassay joint inspection device |
| CN113281499A (en) * | 2021-05-18 | 2021-08-20 | 厦门先明生物技术有限公司 | Microfluidic immunoassay joint detection device and using method thereof |
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| CN102680441A (en) * | 2011-03-16 | 2012-09-19 | 罗姆股份有限公司 | Analysis chip, analysis system, and analysis method |
| US10493448B2 (en) * | 2015-03-13 | 2019-12-03 | Bio-Rad Laboratories, Inc. | Assay cartridge |
| CN105435867B (en) * | 2015-10-26 | 2018-05-22 | 深圳华迈兴微医疗科技有限公司 | Detect the magnetic microparticle chemiluminescence micro-fluidic chip of creatine kinase isozyme in whole blood |
| CN106807461B (en) * | 2017-01-10 | 2018-02-13 | 北京华科泰生物技术有限公司 | A kind of micro-fluidic chip for fluorescence immunoassay detection and preparation method thereof |
| CN109061207A (en) * | 2018-06-21 | 2018-12-21 | 微粒云科技（北京）有限公司 | A kind of biochip for heart infarction marker detection, detection method |
| CN109126916A (en) * | 2018-09-30 | 2019-01-04 | 厦门为正生物科技股份有限公司 | A kind of reagent bottle |
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