CN111077325A - Microporous membrane interception and aggregation biochemical detection device and detection method thereof - Google Patents
Microporous membrane interception and aggregation biochemical detection device and detection method thereof Download PDFInfo
- Publication number
- CN111077325A CN111077325A CN201811224877.3A CN201811224877A CN111077325A CN 111077325 A CN111077325 A CN 111077325A CN 201811224877 A CN201811224877 A CN 201811224877A CN 111077325 A CN111077325 A CN 111077325A
- Authority
- CN
- China
- Prior art keywords
- solution
- membrane
- microporous membrane
- reaction
- microspheres
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000012982 microporous membrane Substances 0.000 title claims abstract description 218
- 238000001514 detection method Methods 0.000 title claims abstract description 109
- 230000002776 aggregation Effects 0.000 title claims abstract description 19
- 238000004220 aggregation Methods 0.000 title claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 215
- 239000012528 membrane Substances 0.000 claims abstract description 211
- 239000004005 microsphere Substances 0.000 claims abstract description 152
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 134
- 150000001875 compounds Chemical class 0.000 claims abstract description 72
- 238000000034 method Methods 0.000 claims abstract description 44
- 239000000126 substance Substances 0.000 claims abstract description 41
- 239000000523 sample Substances 0.000 claims abstract description 40
- 239000011148 porous material Substances 0.000 claims abstract description 35
- 238000002372 labelling Methods 0.000 claims abstract description 31
- 239000003446 ligand Substances 0.000 claims abstract description 24
- 239000012488 sample solution Substances 0.000 claims abstract description 17
- 238000009739 binding Methods 0.000 claims abstract description 14
- 239000000243 solution Substances 0.000 claims description 299
- 239000002245 particle Substances 0.000 claims description 48
- 239000007788 liquid Substances 0.000 claims description 38
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 27
- 230000001681 protective effect Effects 0.000 claims description 16
- 238000009825 accumulation Methods 0.000 claims description 14
- 238000005406 washing Methods 0.000 claims description 14
- 230000005660 hydrophilic surface Effects 0.000 claims description 13
- 239000002096 quantum dot Substances 0.000 claims description 12
- 230000003595 spectral effect Effects 0.000 claims description 12
- 239000000020 Nitrocellulose Substances 0.000 claims description 9
- 239000011358 absorbing material Substances 0.000 claims description 9
- 229920001220 nitrocellulos Polymers 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 5
- 239000006229 carbon black Substances 0.000 claims description 5
- 102000034287 fluorescent proteins Human genes 0.000 claims description 5
- 108091006047 fluorescent proteins Proteins 0.000 claims description 5
- 239000004816 latex Substances 0.000 claims description 5
- 229920000126 latex Polymers 0.000 claims description 5
- 239000002105 nanoparticle Substances 0.000 claims description 5
- 230000000903 blocking effect Effects 0.000 claims description 4
- 239000000835 fiber Substances 0.000 claims description 4
- 239000011888 foil Substances 0.000 claims description 3
- 239000003365 glass fiber Substances 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 230000000717 retained effect Effects 0.000 claims description 3
- 238000009991 scouring Methods 0.000 claims description 3
- 238000001228 spectrum Methods 0.000 claims description 2
- 239000002250 absorbent Substances 0.000 claims 1
- 230000002745 absorbent Effects 0.000 claims 1
- 230000000694 effects Effects 0.000 abstract description 17
- 230000008569 process Effects 0.000 abstract description 5
- 239000000427 antigen Substances 0.000 description 31
- 102000036639 antigens Human genes 0.000 description 29
- 108091007433 antigens Proteins 0.000 description 29
- 230000036046 immunoreaction Effects 0.000 description 23
- 238000005516 engineering process Methods 0.000 description 21
- 239000011248 coating agent Substances 0.000 description 20
- 238000000576 coating method Methods 0.000 description 20
- 230000035945 sensitivity Effects 0.000 description 19
- 102000004169 proteins and genes Human genes 0.000 description 15
- 108090000623 proteins and genes Proteins 0.000 description 15
- 238000010521 absorption reaction Methods 0.000 description 14
- 238000003018 immunoassay Methods 0.000 description 14
- 238000011534 incubation Methods 0.000 description 13
- 238000002965 ELISA Methods 0.000 description 12
- 101100327917 Caenorhabditis elegans chup-1 gene Proteins 0.000 description 11
- 238000004458 analytical method Methods 0.000 description 11
- 238000004587 chromatography analysis Methods 0.000 description 11
- 238000003317 immunochromatography Methods 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 230000003068 static effect Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 230000009471 action Effects 0.000 description 7
- 229960001399 clenbuterol hydrochloride Drugs 0.000 description 7
- OPXKTCUYRHXSBK-UHFFFAOYSA-N clenbuterol hydrochloride Chemical compound Cl.CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 OPXKTCUYRHXSBK-UHFFFAOYSA-N 0.000 description 7
- 230000027455 binding Effects 0.000 description 6
- 238000002156 mixing Methods 0.000 description 6
- 238000003556 assay Methods 0.000 description 5
- 238000010227 cup method (microbiological evaluation) Methods 0.000 description 5
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- 230000004931 aggregating effect Effects 0.000 description 4
- 230000002860 competitive effect Effects 0.000 description 4
- 238000011026 diafiltration Methods 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001917 fluorescence detection Methods 0.000 description 4
- 239000007850 fluorescent dye Substances 0.000 description 4
- 230000001900 immune effect Effects 0.000 description 4
- 230000014759 maintenance of location Effects 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 229930195730 Aflatoxin Natural products 0.000 description 3
- XWIYFDMXXLINPU-UHFFFAOYSA-N Aflatoxin G Chemical compound O=C1OCCC2=C1C(=O)OC1=C2C(OC)=CC2=C1C1C=COC1O2 XWIYFDMXXLINPU-UHFFFAOYSA-N 0.000 description 3
- 239000005409 aflatoxin Substances 0.000 description 3
- MJBWDEQAUQTVKK-IAGOWNOFSA-N aflatoxin M1 Chemical compound C=1([C@]2(O)C=CO[C@@H]2OC=1C=C(C1=2)OC)C=2OC(=O)C2=C1CCC2=O MJBWDEQAUQTVKK-IAGOWNOFSA-N 0.000 description 3
- 229960001117 clenbuterol Drugs 0.000 description 3
- STJMRWALKKWQGH-UHFFFAOYSA-N clenbuterol Chemical compound CC(C)(C)NCC(O)C1=CC(Cl)=C(N)C(Cl)=C1 STJMRWALKKWQGH-UHFFFAOYSA-N 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 3
- 238000001215 fluorescent labelling Methods 0.000 description 3
- 235000013305 food Nutrition 0.000 description 3
- 230000008105 immune reaction Effects 0.000 description 3
- 239000010410 layer Substances 0.000 description 3
- 239000003550 marker Substances 0.000 description 3
- 238000004445 quantitative analysis Methods 0.000 description 3
- 238000003127 radioimmunoassay Methods 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 239000002344 surface layer Substances 0.000 description 3
- 238000000108 ultra-filtration Methods 0.000 description 3
- 210000002700 urine Anatomy 0.000 description 3
- 239000003814 drug Substances 0.000 description 2
- 238000011984 electrochemiluminescence immunoassay Methods 0.000 description 2
- 230000002349 favourable effect Effects 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000003292 glue Substances 0.000 description 2
- 230000005484 gravity Effects 0.000 description 2
- 230000036039 immunity Effects 0.000 description 2
- 238000010166 immunofluorescence Methods 0.000 description 2
- 238000004452 microanalysis Methods 0.000 description 2
- 239000008267 milk Substances 0.000 description 2
- 235000013336 milk Nutrition 0.000 description 2
- 210000004080 milk Anatomy 0.000 description 2
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- 238000005325 percolation Methods 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 230000002829 reductive effect Effects 0.000 description 2
- 239000012465 retentate Substances 0.000 description 2
- 150000003384 small molecules Chemical class 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 239000007790 solid phase Substances 0.000 description 2
- 238000000967 suction filtration Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- 238000008157 ELISA kit Methods 0.000 description 1
- 230000005526 G1 to G0 transition Effects 0.000 description 1
- 241001529936 Murinae Species 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 239000004820 Pressure-sensitive adhesive Substances 0.000 description 1
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 244000052616 bacterial pathogen Species 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000012295 chemical reaction liquid Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 230000009137 competitive binding Effects 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000003745 diagnosis Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003640 drug residue Substances 0.000 description 1
- 238000010828 elution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002550 fecal effect Effects 0.000 description 1
- 208000002672 hepatitis B Diseases 0.000 description 1
- 238000000265 homogenisation Methods 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 230000000984 immunochemical effect Effects 0.000 description 1
- 230000010354 integration Effects 0.000 description 1
- 238000002032 lab-on-a-chip Methods 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 238000007422 luminescence assay Methods 0.000 description 1
- 239000000891 luminescent agent Substances 0.000 description 1
- 238000004020 luminiscence type Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- GBMDVOWEEQVZKZ-UHFFFAOYSA-N methanol;hydrate Chemical compound O.OC GBMDVOWEEQVZKZ-UHFFFAOYSA-N 0.000 description 1
- 239000011859 microparticle Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 230000035515 penetration Effects 0.000 description 1
- 239000012466 permeate Substances 0.000 description 1
- 238000003752 polymerase chain reaction Methods 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 238000003908 quality control method Methods 0.000 description 1
- 238000000163 radioactive labelling Methods 0.000 description 1
- 239000000941 radioactive substance Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012764 semi-quantitative analysis Methods 0.000 description 1
- 230000000405 serological effect Effects 0.000 description 1
- 229910052710 silicon Inorganic materials 0.000 description 1
- 239000010703 silicon Substances 0.000 description 1
- 239000012086 standard solution Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 235000013618 yogurt Nutrition 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
Abstract
The invention discloses a biochemical detection device for intercepting aggregated microspheres by microporous membranes, which comprises a solution reaction zone, wherein one end surface of the solution reaction zone is provided with a solution outlet, and the surface of the solution outlet is provided with microporous membranes with basically uniform pore diameters; the solution reaction zone is provided with coated microspheres, a capture reagent fixed on the surfaces of the coated microspheres and a marking reagent; the capture reagent and the labeling reagent have substances in direct or indirect ligand relationship with each other; after the sample solution containing the substance to be detected is added, the capture reagent, the labeling reagent and the sample to be detected are subjected to a binding reaction in the solution, a ligand reaction compound is formed on the surface of the coated microsphere, in the process that the solution flows out through the microporous membrane, the free labeling reagent flows out through the microporous membrane, and the coated microsphere carrying the ligand reaction compound is intercepted on the surface by the microporous membrane, so that an aggregation and concentration effect is formed.
Description
Technical Field
The invention relates to the technical field of biochemical immunoassay, in particular to a microporous membrane interception and accumulation biochemical detection device and a detection method thereof.
Background
The immunoassay technology utilizes the recognition and combination reaction generated by high specificity between antigen and antibody to realize the detection of biological molecules, has the advantages of high sensitivity, strong specificity, wide application range, simple required equipment, wider linear range and the like, becomes one of the most competitive and challenging analysis and test technologies at present, and is widely applied to the fields of life science, clinical medicine, environment, food, medicine and the like. The immune marker analysis technology mainly comprises the following steps: radioactive labeling, enzyme labeling, luminescent labeling, fluorescent labeling, and the like. Radioimmunoassay (RIA) developed by labeling an antigen or antibody with a radioactive substance is a microanalysis created by Yalow and Berson in 1959 in the united states science, which is a new method created by combining a radionuclide tracing technique with high sensitivity and a specific immunochemical technique. The technology utilizes the amplification effect of the nuclide marker, improves the lower detection limit of the object to be detected, and simultaneously takes the antibody or the antigen as a binding reagent, thereby greatly improving the specificity of the detection method.
Fluorescence Immunoassay (FIA) is a labeling immunological technique originated from Conn et al in the 40 th century, and the used labels are fluorescein and fluorescent dye, and is a detection method for detecting fluorescence intensity and fluorescence phenomenon by combining an antigen or an antibody labeled with a fluorescent substance and a corresponding antigen or antibody under a fluorescence microscope or ultraviolet irradiation. Fluorescence labeling immunoassays have high sensitivity, but fluorescein often generates biological toxicity, resulting in decreased sensitivity and selectivity of antibodies or antigens.
The enzyme labeling assay technology is a novel serological technology developed after immunofluorescence antibody technology and radioimmunoassay. In 1966, Nakane et al and Avrameas et al reported that an enzyme was used instead of a fluorescein-labeled antibody, and an enzyme-labeled antibody technique (enzyme-labeled antibody technique) was established for localization and identification of antigens in biological tissues. In 1971, Engval Van Weemen et al reported enzyme-linked immunosorbent assay, thereby establishing a quantitative detection technology of enzyme-labeled antibodies. In the 20 th century and the 80 th era, immuno-transfer technology based on enzyme-labeled antibodies for detection and identification of protein molecules was developed. At present, the immunoassay labeling technique has become one of the most widely used immunological methods in immunodiagnosis, detection and molecular biology research.
Luminescent labeling analysis was in the end of the 80 th 20 th century, and antigens or antibodies were labeled with chemiluminescent reagents from abroad, thereby establishing a luminescent immunoassay technique. The Luminescence Immunoassay (LIA) in the narrow sense is mainly referred to as chemiluminescence immunoassay (CLIA). In addition, there are enzyme-amplified chemiluminescence immunoassay and electrochemiluminescence immunoassay (ECLIA). CLIA was established by Sohrochler and Halman in the late 70's 20 th century, a method combining the high sensitivity of luminescence assays with the specificity of the immune response. The basic principle is the same as enzyme labeling analysis method, and the method is that the antigen or antibody is labeled by a chemical luminescent reaction reagent (such as a luminescent agent or a catalyst), the labeled antigen or antibody and a substance to be detected are subjected to a series of immunoreaction and physicochemical steps (such as centrifugal separation, washing and the like), and finally the detection is carried out in the form of measuring the luminous intensity.
The prior immunoassay technology mainly adopts a heterogeneous analysis mode which takes a microporous plate as an experimental platform, needs multi-step operations such as embedding, elution, separation and the like, has a complex analysis process and long analysis time, and cannot meet the requirements of rapid detection and diagnosis. The penetration of proteins and micro-particles with different molecular weights through a microporous membrane in biological reaction is a very common technical means, in particular to a centrifugal filter tube commonly used in protein purification. However, the centrifugal filter tube is generally added by hand with the solution, and the protein passing through is dissolved and recovered by adding the complex solution after centrifugation.
The immune lateral chromatography technology is an analysis method combining the immune technology and the chromatographic chromatography technology, which is developed at the end of the 20 th century, has the characteristics of specificity, simple operation, rapidness and the like, and is widely applied to important fields of clinical diagnosis, environmental monitoring, food safety and the like. The traditional immunochromatography technology takes colloidal gold as a marker, and qualitative detection or semi-quantitative analysis is carried out on a target object through strip color development. As a novel immunoassay technology, the fluorescence immunochromatography technology not only has the advantages of on-site rapid detection of the traditional colloidal gold test strip, but also develops the high sensitivity characteristic of the fluorescence technology.
Immunochromatography is based on antigen-antibody specific reactions. The technology uses a strip-shaped fiber chromatography material fixed with a detection line (coated antibody or coated antigen, also marked as T line) and a quality control line (anti-antibody, also marked as C line) as a stationary phase, a test solution as a mobile phase, and a labeled antibody or antigen fixed on a connecting pad, so that an analyte moves on the chromatography strip through capillary action. For macromolecular antigens (proteins, viruses, pathogenic bacteria and the like) with a plurality of antigenic determinants, a sandwich-type double-antibody sandwich immunochromatography method is generally adopted, namely, an object to be detected is firstly combined with a fluorescence labeling antibody under the action of a mobile phase, and then is dynamically captured and combined by a coating antibody to form a sandwich-type double-antibody sandwich reaction compound when reaching a detection line. Typically, the ELISA is incubated for 10 minutes and the reaction is complete. The length of the developed solid phase membrane of the immune lateral chromatography is generally more than 2cm, the width of a T line is not more than 1mm, the time of the liquid advancing for 1mm is only about 3 seconds, and the protein to be detected needs to be captured and combined by the dynamic capture protein within 3 seconds. Therefore, the immunoaffinity requirement of the coating protein is high, and many proteins are good in the enzyme-linked immunosorbent assay (ELISA) of static capture but cannot be adopted in the application of immune lateral chromatography. Therefore, in many cases, the detection sensitivity is improved by mixing the labeled antibody with the sample solution in advance, or by increasing the distance between the coated reagent and the labeled reagent for the purpose of more complete binding reaction, but additional steps are added. If the dynamic protein capture can be abandoned in the immunochromatography method and the immune binding capture is carried out by adopting a mixed incubation mode biased to static incubation, the application effect is better.
The dot immunogold diafiltration sandwich assay is performed by dropping purified antibody onto the center of the nitrocellulose membrane, where it is adsorbed by the membrane. When the specimen liquid dripped on the membrane permeates through the membrane, the antigen contained in the specimen is captured by the antibody on the membrane, and other irrelevant proteins and the like are not filtered out of the membrane. The colloidal gold label added thereafter also binds to the antigen already bound to the membrane during diafiltration. Since colloidal gold itself is red, a positive reaction shows a red spot in the center of the membrane. Compared with a nitrocellulose membrane with a pore size of 5-20um in the immune lateral chromatography, the pore size of the membrane in the diafiltration method is generally 0.45um, so the solution flowing speed passing through the membrane is slow, and the immune reaction with weaker affinity can obtain a proper result in the diafiltration experiment. However, in the dot immunogold filtration assay, multiple solution addition procedures are required, and it would be preferable if a protocol that is simple and convenient for one-step loading could be devised.
In recent years, the micro-fluidic chip technology is rapidly popularized, is a brand-new microanalysis technology, and can realize the miniaturization, automation, integration and portability from sample processing to detection, so that the micro-fluidic chip technology can show strong development vigor in the aspect of food safety detection, and provides a brand-new technical tool and platform. The micro-fluidic chip is characterized in that a multifunctional integrated system and a micro total analysis system with a plurality of composite systems can be formed on one chip. The microreactor is a structure commonly used in a lab-on-a-chip for biochemical reactions, such as a microreactor for capillary electrophoresis, a polymerase chain reaction, an enzyme reaction and a DNA hybridization reaction, an immunological detection reaction, and the like. In both the conventional ELISA method and the emerging chemiluminescence method, most of the immunoassay methods require the addition and mixing of multiple solutions, and thus require multiple steps. How to reduce the types of solutions in the microfluidic chip as much as possible and enable the microfluidic immune chip to operate more automatically and reduce manual operations of personnel is an important development direction at present. The design of a suitable microfluidic immune chip under the target guidance of few solutions and few manual intervention steps is also a quite challenging direction for better detection of relevant immunological signals.
The domestic patent document CN201310228708.8 discloses a quantitative detection device based on fibrous membrane trapping and separation and a detection method thereof: the method comprises the following steps of putting coating microspheres for marking protein into a deep-hole filter plate, carrying out static mixing incubation reaction with a marking reagent, then completely flowing out a sample solution and a washing solution from a filter membrane of the filter plate under the action of centrifugal force, enabling small molecules or small-particle-size substances such as the marking reagent to flow out of the deep-hole filter plate along with the solution, partially intercepting the coating microspheres when the coating microspheres pass through a bottom filter membrane, and then detecting the surface of the membrane through optics to obtain an experiment of related detection signals. The method has the significance that the immunoreaction reagent is statically incubated in a homogeneous environment, so that the requirement on the affinity of the immune antibody antigen is further reduced; meanwhile, after incubation reaction, the coated microspheres carrying the reaction compound in the solution are gathered in the filter membrane, so that a good reaction substance concentration effect is achieved. However, the relationship between the particle size of the coated microspheres and the pore size of the filter membrane in the patent document is not clearly defined, and the microspheres will penetrate into the filter membrane to a certain depth, which will cause part of the microspheres to pass through the filter membrane, thereby causing inaccurate detection results. At the same time, differences in the spectral signals reflected by the microspheres at different depths of the filter will also result.
Disclosure of Invention
The invention aims to provide a microporous membrane interception and aggregation biochemical detection device and a detection method thereof, and aims to solve the problem of poor detection effect of the conventional microporous membrane interception and aggregation biochemical detection device.
In order to achieve the purpose, the invention adopts the following technical scheme:
as a first aspect of the invention, a micro-porous membrane interception aggregation biochemical detection device comprises a solution reaction area, wherein a solution outlet with the area smaller than 12 square millimeters is arranged on one end surface of the solution reaction area, the end surface of the solution outlet is attached with a micro-porous membrane with basically uniform pore diameter, and the micro-porous membrane is attached to cover the solution outlet; at least one kind of microballoon with homogeneous diameter and pore size greater than that of the microporous membrane, capture reagent fixed onto the microballoon surface and marking reagent with particle size smaller than that of the microporous membrane are set in the solution reaction area, and the capture reagent and the marking reagent have coordination compounds in direct or indirect ligand relationship.
According to the invention, the reaction mode of the coated microspheres and the marking reagent in the solution reaction zone and the microscopic molecular size matching relationship thereof are as follows: when the solution enters the solution reaction zone, the capture reagent, the labeling reagent and the sample to be detected are subjected to a binding reaction in the solution, and a ligand reaction compound is formed on the surface of the microsphere; when the solution flows out from the solution outlet, the coating microspheres and the microspheres formed by the reaction compound of the coating microspheres and the labeled reagent form accumulation on the surface of the filter membrane, the free labeled reagent can pass through the pores between the accumulated microspheres again after being carried by the solution, and the blocking residue cannot be formed.
According to the invention, the labeling reagent is derived from a fluorescent substance or a color particle directly visible to the naked eye; the color particles are selected from one or more of nano colloidal gold, latex microspheres, carbon black particles and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and microspheres thereof, quantum dot microspheres, up-conversion luminescent microspheres, time-resolved fluorescent molecules and microspheres thereof, and fluorescent protein.
According to the invention, the biochemical detection device for intercepting and gathering the microporous membrane also comprises detection equipment for optically detecting at least one wavelength signal for the spectral signal of the labeled reagent in the reaction compound gathered and accumulated on the surface of the microporous membrane; the microporous membrane is irradiated by light with a specific wavelength, the fluorescent substance gathered on the surface is excited to emit a fluorescent signal with the specific wavelength, the intensity of the fluorescent signal has a specific corresponding relation with the number of the reaction compounds gathered on the surface of the microporous membrane, the fluorescent spectrometer receives the fluorescent signal and analyzes the fluorescence of the corresponding wave peak through the microprocessor to detect different microsphere reaction compounds, and the concentration of different substances to be detected is calculated.
Furthermore, the reaction compound trapped on the surface layer of the microporous membrane emits a fluorescent signal with a specific wavelength after being excited by the irradiation of light with a specific wavelength.
According to the present invention, the solution reaction zone comprises an immune sidestream solution flow path, the solution outlet port is provided at one end of the immune sidestream solution flow path, and the microporous membrane is bonded to the solution outlet port.
Further, the immune lateral flow solution flow channel comprises a hydrophilic porous membrane capable of directionally transmitting the aqueous solution, wherein the hydrophilic porous membrane is selected from one or more of a nitrocellulose membrane, a glass fiber membrane of a sample absorption pad, a chemical fiber membrane and filter paper; the immune lateral flow solution flow channel is a gap channel formed by clamping the hydrophilic porous membrane by the hydrophilic surfaces of two hydrophilic membranes, and the solution flows from one end of the gap channel to the other end.
Further, two hydrophilic membranes are arranged at the tail ends of the immune lateral flow channels, the hydrophilic surfaces of the two hydrophilic membranes clamp the upper surface and the lower surface of a hydrophilic porous membrane, the lower surface of the hydrophilic membrane at the bottom of the two hydrophilic membranes is attached with the microporous membrane, and a notch with the area not more than 12 square millimeters is arranged in the middle of the hydrophilic membrane attached to the upper surface of the microporous membrane and serves as a solution outlet;
or the hydrophilic surfaces of the two hydrophilic membranes can also clamp the left side and the right side of the hydrophilic porous membrane, and the middle part of the bottom surface of the gap channel formed by clamping the two hydrophilic membranes is provided with a gap as a solution outlet;
the solution outflow opening comprises at least one gap shape with a maximum gap of not more than 1.5mm or an included angle with an angle of not more than 95 degrees.
Furthermore, the lower surface of the microporous membrane is adhered with a water absorbing material, and the solution flowing into the hydrophilic membrane gap passes through the microporous membrane and is absorbed and transferred by the water absorbing material.
According to the invention, the solution reaction zone comprises an open micro-pore cup with the volume not more than 550 microliters, a through hole with the area not more than 12 square millimeters is arranged in the middle of the bottom of the open micro-pore cup, and the bottom plane is arranged to form a certain conical inclination angle by taking the center of the through hole as an axis; the microporous membrane with basically uniform aperture and the aperture range of 0.02-1.2um is attached to the surface of the through hole. When the solution flows out through the microporous membrane, the free labeled reagent flows out, the labeled reagent forming the ligand reaction compound and the coated microspheres are intercepted on the surface by the microporous membrane to form an aggregation concentration effect, so that the concentration effect from liquid to the membrane surface is realized, the area concentration effect is also realized on the section of a detection area, and the good effect of improving the sensitivity is achieved. Therefore, a rapid reaction method with two-step operation of two solutions can be realized, the step of immunoreaction is simplified into static incubation of antibody and antigen, and then signal detection can be carried out after washing the reaction area by a washing solution. Meanwhile, when immunoreaction microsphere compounds formed in the static incubation immunoreaction reach the interception area of the filter membrane, the marking reagent with the diameter smaller than the aperture of the filter membrane can freely pass through the filter membrane, but the coated microspheres with the particle size larger than the aperture of the filter membrane and the immunoreaction compounds thereof are intercepted and concentrated on the surface of the cultivation filter membrane, so the invention can greatly improve the efficiency and the sensitivity of immunoreaction.
Furthermore, a counter bore is arranged below the through hole at the bottom of the micropore cup, the shape of the counter bore is matched with that of the micropore membrane, and the lower surface of the micropore membrane is basically flush with the lower surface of the micropore cup after the counter bore accommodates the micropore membrane; a protective membrane with a heat conduction function is arranged below the microporous membrane, the protective membrane is made of metal foil or an organic heat conduction membrane, the heat conduction function of the protective membrane is favorable for controlling the temperature of the microporous cup, so that the temperature of immunoreaction of each pore is accurate and consistent, and the precision of the obtained result is better; one part of the protective membrane is attached to the bottom surface of the microporous cup, and the other part of the protective membrane is attached to the microporous membrane; the middle part of the protective membrane is provided with an opening which is positioned right below the through hole of the microporous cup.
According to the invention, the microporous membrane interception and aggregation biochemical detection device comprises a reaction container, wherein the reaction container is a centrifugal microfluidic chip with a reaction volume not more than 100 microliters, the centrifugal microfluidic chip comprises a solution reaction area, a solution outlet and a liquid inlet, the solution reaction area comprises a first solution reaction area, the solution outlet is arranged at the bottom of the center of the first solution reaction area, the area of the solution outlet is not more than 12 square millimeters, the liquid inlet is arranged at the edge area of the bottom of the first solution reaction area, a microporous membrane is attached to one end face of the first solution reaction area, and the microporous membrane covers the solution outlet and separates the solution outlet from the first solution reaction area.
Furthermore, the volume of the first solution reaction area is not more than 100 microliters, the first solution reaction area is also provided with an exhaust port, and the exhaust port is arranged at a position where the solution flowing into the first solution reaction area is filled with the detection area finally;
the first solution reaction zone is at least provided with two liquid inlets, and when solution flows into the first solution reaction zone from different liquid inlets, the uniform scouring result on the surface of the microporous membrane at the bottom or the top of the first solution reaction zone is achieved.
Furthermore, the centrifugal microfluidic chip further comprises a second solution reaction area, a second liquid outlet and a second liquid inlet, wherein the second solution reaction area is filled with the solution from the second liquid inlet, and the solution is discharged from the second liquid outlet and then flows to the first solution reaction area.
Furthermore, the centrifugal micro-fluidic chip is also provided with a constant volume point and an overflow area; the constant volume point is arranged at the position filled with the solution finally in the solution reaction area, and after the solution amount entering the second solution reaction area exceeds the solution amount, the redundant solution is reserved from the constant volume point to the overflow area.
Furthermore, the second liquid outlet is provided with a control area, and after the solution enters the second solution reaction area, the solution can flow out of the second liquid outlet after the control area is opened.
In a second aspect of the present invention, a detection method of the above-mentioned biochemical detection device for trapping and aggregating microporous membrane includes adding a sample solution into the microporous cup, allowing the sample solution to flow out of the through hole of the filter membrane, adding a washing solution to wash and suspend the microspheres accumulated on the microporous membrane, allowing the washing solution to flow out of the through hole of the filter membrane, and detecting a spectroscopic signal of the microspheres accumulated on the surface of the microporous membrane again.
The invention relates to a microporous membrane interception and aggregation biochemical detection device, which has the beneficial effects that:
1. the sensitivity of the reaction is improved, and the dependence of ligand affinity in the immune reaction is reduced:
(1) by adopting the mode of intercepting the immunoreaction compound based on the coated microspheres by the microporous membrane in the solution reaction zone, the spectral signals of the coated microspheres are gathered on the surface of the microporous membrane, thereby not only realizing the concentration effect of the spectral signals from the liquid to the membrane surface, but also realizing the area concentration effect on the section of the detection zone, and achieving the good effect of improving the sensitivity;
(2) the microsphere reaction compound of the immunochromatographic test paper is captured or microsphere-trapped and gathered through a microporous membrane arranged in a lateral flow channel, and the signal of the immunochromatographic test paper can be trapped and concentrated on the basis of one-step immunochromatography, so that a result of higher detection sensitivity is achieved; the dynamic capture mode of the immunoreaction compound is fundamentally changed, the coating reagent and the marking reagent are fully mixed and react freely in the solution flow along with the sample to be detected, the ligand of dynamic chromatography is not captured by the coating protein of the microporous membrane in the reaction, but the reaction compound is trapped by the microporous membrane, and the reaction sensitivity is greatly improved;
(3) meanwhile, reaction compounds intercepted by the microporous membrane are gathered at the same position, so that the opportunity of single-point multiple immunofluorescence detection is provided;
(4) the capture mode of the immune complex in the immunochromatography reaction can be changed, namely the ligand of dynamic chromatography is not captured by the coating ligand of the microporous membrane any more, but the reaction complex after the full mixed coordination reaction is realized in the chromatography process is physically trapped and captured by the microporous membrane, so that the reaction complex does not appear depending on the affinity of the ligand;
(5) the microsphere reaction compound of the immunochromatographic test paper is trapped and gathered by a microporous membrane arranged in a lateral flow channel, and a signal of the immunochromatographic compound can be trapped and concentrated on the basis of one-step immunochromatography, so that a result of higher detection sensitivity is achieved;
(6) the method is a better implementation mode of immunochromatography detection, a new functional structure, namely a percolation microporous membrane is added on the basis of the immunochromatography detection, the reaction compound which is fully mixed and incubated in the lateral flow process is captured or physically trapped by the microporous membrane, and if the reaction compound is physically trapped, the reaction compound does not depend on the affinity of a ligand any more;
2. the capture reagent is coated on microspheres which can be suspended in an aqueous solution, the microspheres are arranged on a solution flow channel, when a sample solution to be detected enters a lateral solution flow channel, the capture reagent and the marking reagent are respectively mixed and dissolved, a substance to be detected, the capture reagent and the marking reagent in the sample solution are subjected to mixed incubation and immune combination reaction in the lateral flow of the solution, an immune or ligand reaction compound is formed on the surfaces of the microspheres, and after immune lateral flow at a certain distance, all components in the solution sequentially reach a microporous membrane, and the components with the particle size smaller than the pore size of the microporous membrane pass through the microporous membrane and flow to a water absorbing material such as filter paper; the diameter of the coated microspheres is larger than the pore diameter of the microporous membrane, and the free coated microspheres and the coated microspheres forming the reaction compound are intercepted by the microporous membrane at the solution outlet.
3. The quick reaction method of two-step operation of two solutions is realized, the step of immunoreaction is simplified into static incubation of antibody and antigen, and then signal detection can be carried out after a washing solution washes a reaction area; meanwhile, when immunoreaction microsphere compounds formed in the static incubation immunoreaction reach the interception area of the filter membrane, the marking reagent with the diameter smaller than the aperture of the filter membrane can freely pass through the filter membrane, but the coated microspheres with the particle size larger than the aperture of the filter membrane and the immunoreaction compounds thereof are intercepted and concentrated on the surface of the cultivation filter membrane, so the invention can greatly improve the efficiency and the sensitivity of immunoreaction.
4. The micro-porous cup is used for immunodetection, the reaction of the immunodetection is finished in liquid phase incubation, the detected microsphere reaction compound passes through the micro-porous membrane along with the flowing of a solution and is trapped and gathered on the surface of the micro-porous membrane by the micro-porous membrane on the surface of a through hole at the bottom of the cup, the microsphere reaction compound can be suspended and fully washed, only microspheres with the particle size larger than the pore size of the micro-porous membrane are left, and the rest part of the microsphere reaction compound passes through the micro-porous membrane and flows away, and.
Drawings
FIG. 1 is a schematic diagram of the microporous membrane retention and accumulation detection device of example 1.
Fig. 2 is a schematic top view of the microporous membrane rejection and accumulation detection device of fig. 1.
Fig. 3 is a schematic diagram of the structure of the microporous membrane rejection concentration detection device of example 2.
Fig. 4 is another schematic diagram of the microporous membrane retention and accumulation detection device of example 2.
Fig. 5 is a schematic partial top view of the microporous membrane retention and accumulation detection device of example 2.
Fig. 6 is a schematic structural view of a solution outflow port of embodiment 2.
Fig. 7 is another schematic structural view of the solution outflow port of embodiment 2.
Fig. 8 is a schematic structural view of the amphiphilic water membrane of the microporous membrane interception aggregation detection apparatus of example 2.
Fig. 9 is a schematic top view of the microporous membrane rejection and accumulation detection device of example 3.
Fig. 10 is a schematic top view of the microporous membrane rejection and accumulation detection device of example 3.
Fig. 11 is a schematic top view of the microporous membrane rejection and accumulation detection device of example 3.
FIG. 12 is a schematic view showing the structure of the microporous membrane entrapping aggregation detecting device of example 3 in which the second solution reaction zone is connected to the first solution reaction zone.
FIG. 13 is a schematic view showing the binding of a sample to be measured, a capture reagent-immobilized microsphere, and a labeled reagent.
FIG. 14 is another schematic view showing the binding of a sample to be measured, a capture reagent-immobilized microsphere, and a labeling reagent.
FIG. 15 is a quantitative calibration graph of example 9.
FIG. 16 is a quantitative calibration graph of example 10.
Detailed Description
In order to make the aforementioned objects, features and advantages of the present invention comprehensible, specific embodiments accompanied with figures are described in detail below, and it is apparent that the described embodiments are a part of the embodiments of the present invention, not all of the embodiments. All other embodiments, which can be obtained by a person skilled in the art without making creative efforts based on the embodiments of the present invention, shall fall within the protection scope of the present invention.
In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention, but the present invention may be practiced in other ways than those specifically described and will be readily apparent to those of ordinary skill in the art without departing from the spirit of the present invention, and therefore the present invention is not limited to the specific embodiments disclosed below.
Meanwhile, in the description of the present invention, it should be noted that the terms "upper, lower, inner and outer" and the like indicate orientations or positional relationships based on the orientations or positional relationships shown in the drawings, and are only for convenience of describing the present invention and simplifying the description, but do not indicate or imply that the referred device or element must have a specific orientation, be constructed in a specific orientation and operate, and thus, cannot be construed as limiting the present invention.
Example 1
As shown in figures 1 and 2, the biochemical detection device for intercepting and aggregating microporous membrane of the invention comprises a solution reaction zone, wherein the solution reaction zone comprises an open microporous cup 1 with a volume not exceeding 550 microliter, a through hole 2 with an area not exceeding 12 square millimeters is arranged at the middle position of the bottom of the solution reaction zone, and the plane of the bottom is arranged to form a certain conical inclination angle by taking the center of the through hole 2 as an axis; a microporous membrane 3 with basically uniform aperture and aperture larger than 20nm is attached to the surface of the through hole 2, and the microporous membrane 3 is attached to cover the through hole; at least one kind of microballoon with homogeneous diameter and pore size greater than that of the microporous membrane, capture reagent fixed onto the microballoon surface and marking reagent with particle size smaller than that of the microporous membrane are set in the solution reaction area, and the capture reagent and the marking reagent have coordination compounds in direct or indirect ligand relationship. When the solution flows out through the microporous membrane, the free labeled reagent flows out, the labeled reagent forming the ligand reaction compound and the coated microspheres are intercepted on the surface by the microporous membrane to form an aggregation concentration effect, so that the concentration effect from liquid to the membrane surface is realized, the area concentration effect is also realized on the section of a detection area, and the good effect of improving the sensitivity is achieved. Therefore, a rapid reaction method with two-step operation of two solutions can be realized, the step of immunoreaction is simplified into static incubation of antibody and antigen, and then signal detection can be carried out after washing the reaction area by a washing solution. Meanwhile, when immunoreaction microsphere compounds formed in the static incubation immunoreaction reach the interception area of the filter membrane, the marking reagent with the diameter smaller than the aperture of the filter membrane can freely pass through the filter membrane, but the coated microspheres with the particle size larger than the aperture of the filter membrane and the immunoreaction compounds thereof are intercepted and concentrated on the surface of the cultivation filter membrane, so the invention can greatly improve the efficiency and the sensitivity of immunoreaction.
And a counter bore 6 is arranged below the through hole 2 at the bottom of the micropore cup, the shape of the counter bore 6 is matched with that of the micropore membrane 3, and after the counter bore 6 accommodates the micropore membrane 3, the lower surface of the micropore membrane 3 is basically flush with the lower surface of the micropore cup 1. A protective membrane 4 with a heat conduction function is arranged below the microporous membrane 3, the protective membrane 4 is made of metal foil or an organic heat conduction membrane, and the heat conduction function of the protective membrane 4 is favorable for controlling the temperature of the microporous cup 1, so that the temperature of immunoreactions of each pore is accurate and consistent, and the precision of the obtained result is better; one part of the protective membrane 4 is attached to the bottom surface of the microporous cup 1, and the other part is attached to the microporous membrane 3; the middle part of the protective membrane 4 is provided with an opening 5, and the opening 5 is positioned right below the through hole 2 of the microporous cup 1.
The cross section of the micropore cup 1 is circular, at least one pair of lower convex points 7 with the same height are symmetrically arranged on the lower portion of the outer wall of the micropore cup 1, at least one pair of upper convex points 8 with the same height are symmetrically arranged on the upper portion of the outer wall of the micropore cup 1, and the upper convex points 8 and the lower convex points 7 of the micropore cup are matched with a perforated plate for solid phase aggregation immunoassay. During the use, through upper and lower protruding point and perforated plate cooperation, can carry out solitary highly uniform's fixed to every micropore cup, can carry out warm bath contact and other contacts to the micropore cup bottom through a plane, if carry out solution extraction to micropore cup bottom through vacuum suction nozzle. When the microporous cup is inserted into a perforated plate matched with the shape of the microporous cup, the microporous cup 1 can be suspended by the upper convex points 8; after the micro-hole cup 1 is rotated by a certain angle, the lower convex point 7 of the micro-hole cup is contacted with the lower surface of the perforated plate, the upper convex point 8 is contacted with the upper surface of the perforated plate, and at the moment, the micro-hole cup cannot descend or be pulled out, so that the function of fixing the micro-hole cup is achieved.
The reaction mode of the coated microspheres and the marking reagent in the solution reaction zone and the microscopic molecular size matching relationship thereof are as follows: when the solution enters the solution reaction zone, the capture reagent, the labeling reagent and the sample to be detected are subjected to a binding reaction in the solution, and a ligand reaction compound is formed on the surface of the microsphere; when the solution flows out from the solution outlet, the coated microspheres and the microspheres formed by the reaction compound of the coated microspheres and the labeled reagent form accumulation on the surface of the filter membrane, and the free labeled reagent can pass through the pores between the accumulated microspheres and the micropores of the filter membrane again after being carried by the solution, so that no blocking residue is formed.
The labeling reagent is derived from fluorescent substances or color particles directly visible to the naked eye; the color particles are selected from one or more of nano colloidal gold, latex microspheres, carbon black particles and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and microspheres thereof, quantum dot microspheres, up-conversion luminescent microspheres, time-resolved fluorescent molecules and microspheres thereof, and fluorescent protein.
The biochemical detection device for intercepting and aggregating the microporous membrane also comprises detection equipment for carrying out optical detection on at least one wavelength signal on the spectral signal of the labeled reagent in the reaction compound aggregated and accumulated on the surface of the microporous membrane; the microporous membrane is irradiated by light with a specific wavelength, the fluorescent substance gathered on the surface is excited to emit a fluorescent signal with the specific wavelength, the intensity of the fluorescent signal has a specific corresponding relation with the number of the reaction compounds gathered on the surface of the microporous membrane, the fluorescent spectrometer receives the fluorescent signal and analyzes the fluorescence of the corresponding wave peak through the microprocessor to detect different microsphere reaction compounds, and the concentration of different substances to be detected is calculated.
The reaction compound trapped on the surface layer of the microporous membrane emits a fluorescent signal with a specific wavelength after being excited by the irradiation of light with the specific wavelength.
Example 2
As shown in fig. 3, the microporous membrane interception and aggregation detection device of the present invention comprises a solution reaction zone, wherein the solution reaction zone comprises an immune lateral flow channel and a microporous membrane 23 with a pore size smaller than 1.2um, a solution in the immune lateral flow channel directly flows into a solution outlet 29 arranged at the end of the immune lateral flow channel, and the microporous membrane 23 is attached to the solution outlet 29 for the solution to penetrate and percolate. At least one marking reagent is arranged in the immune lateral flow passage, and a capture reagent is coated on the microporous membrane; the capture reagent and the labeling reagent are provided with substances which are in direct or indirect ligand relationship with each other.
As shown in fig. 4, another microporous membrane interception and accumulation detection device of the present invention comprises a solution reaction zone, wherein the solution reaction zone comprises an immunity lateral flow solution flow channel, a solution outlet 29 is arranged at one end of the immunity lateral flow solution flow channel, and a microporous membrane 23 with a substantially uniform pore size and a pore size range of 0.02-1.2um is attached to the solution outlet 29; the immune lateral flow channel is internally provided with at least one coating microsphere with basically uniform diameter and larger than the pore diameter of the microporous membrane, a capture reagent fixed on the surface of the microsphere and a marking reagent with the diameter smaller than the pore diameter of the microporous membrane; the capture reagent and the labeling reagent are provided with substances which are in direct or indirect ligand relationship with each other.
Specifically, the lower surface of the hydrophilic membrane 24 attached to the microporous membrane 23 in the immune lateral flow channel is coated with glue or non-setting adhesive, so that microspheres in the solution cannot flow away from the attachment gap between the hydrophilic membrane 24 and the microporous membrane 23, and only flow out from the microporous membrane 23 attached to the notch of the hydrophilic membrane serving as the solution outflow port 29. As shown in fig. 5, the notch may be polygonal, circular or rectangular, and when the notch is circular, at least one of the maximum gap 291 (as shown in fig. 6 and 7) of the notch is not more than 1.5mm or the angle is not more than 95 degrees, so that the solution flowing to the edge of the notch flows into the notch along the side edge and contacts with the microporous membrane 23 with good hydrophilicity attached to the bottom of the side edge of the hydrophilic membrane; when the gap is rectangular, its width can be set to 0.2-2.5mm, and the solution on the surface of its two sides will be pulled forward from the sides across the gap due to surface tension, at this time, the solution across the sides will sink to contact the hydrophilic membrane at the bottom under the action of gravity, and then the liquid will enter the gap to cross the microporous membrane 23 under the action of hydrophilic pulling force of the hydrophilic porous membrane.
When the microporous membrane is used, the hydrophilic surface of the hydrophilic membrane 24 can transmit solution to the edge of the notch, but the side edge of the notch of the hydrophilic membrane 24 is difficult to treat to achieve the same surface hydrophilicity, so that the solution is difficult to flow into the notch along the side edge and contacts the microporous membrane which is attached to the bottom of the side edge of the hydrophilic membrane and has good hydrophilicity. When the critical condition caused by the gap or angle of the gap is reached, namely the shape of the gap with the maximum gap not exceeding 1.5mm or the included angle with the angle not exceeding 95 degrees is set, the solution on the surfaces of the two side edges close to the gap will pull the solution to cross the gap from the side edges forwards due to the surface tension, at the moment, the solution crossing the side edges will sink and contact the hydrophilic membrane at the bottom under the action of gravity, and then under the action of the hydrophilic pulling force of the microporous membrane, the liquid will enter the gap and cross the microporous membrane 23. When two hydrophilic membranes 24 are sandwiched to form a lateral flow channel, at least the hydrophilic membrane opposite to the hydrophilic membrane with the gap is transparent (i.e. the upper hydrophilic membrane of the two hydrophilic membranes is transparent), and fluorescence or other spectrum detection can be performed on the surface of the microporous membrane opposite to the gap of the hydrophilic membrane.
The immune lateral flow solution flow channel comprises a hydrophilic porous membrane 21 capable of directionally transmitting an aqueous solution, wherein the hydrophilic porous membrane 21 is selected from one or more of a nitrocellulose membrane, a sample absorption pad glass fiber membrane, a chemical fiber membrane and filter paper; the immune lateral flow solution flow channel is a gap channel constructed by clamping the hydrophilic porous membrane 21 by the hydrophilic surfaces of two hydrophilic membranes 24, and the solution flows from one end of the gap channel to the other end by using the surface tension of the solution on the surfaces of the hydrophilic membranes 24 as the power for liquid flow.
As shown in fig. 1 or 4, two hydrophilic membranes 24 are disposed at the end of the immune lateral flow channel, the hydrophilic surfaces of the two hydrophilic membranes 24 sandwich the upper and lower surfaces of the hydrophilic porous membrane 21, the microporous membrane 23 is attached to the lower surface of the hydrophilic membrane at the bottom of the two hydrophilic membranes 24, and a notch with an area not exceeding 12 square millimeters is disposed in the middle of the hydrophilic membrane 24 attached to the upper surface of the microporous membrane 23 as a solution outlet 29. As shown in fig. 8, the hydrophilic surfaces of the two hydrophilic membranes 24 may also sandwich the left and right sides of the hydrophilic porous membrane 21, and the middle of the bottom surface of the gap channel formed by sandwiching the two hydrophilic membranes 24 is provided with a notch as a solution outlet 29. In order to construct the gap channel, the two hydrophilic membranes can clamp the hydrophilic porous membrane 1 to form the gap channel, the tail end of the channel can be attached with a double-sided adhesive tape or provided with other plugging materials, and the gap between the two hydrophilic membranes is highly fixed and plugged, so that the solution in the gap channel does not flow outwards any more.
The solution outflow opening 29 comprises at least one gap shape 291 whose maximum gap is not more than 1.5mm or an included angle of not more than 95 degrees in its shape.
The lower surface of the microporous membrane 23 is adhered with the water absorbing material 22, and the solution flowing into the gap of the hydrophilic membrane passes through the microporous membrane 23 and is absorbed and transmitted by the water absorbing material 22.
The reaction mode of the coated microspheres and the marking reagent in the solution reaction zone and the microscopic molecular size matching relationship thereof are as follows: when the solution enters the solution reaction zone, the capture reagent, the labeling reagent and the sample to be detected are subjected to a binding reaction in the solution, and a ligand reaction compound is formed on the surface of the microsphere; when the solution flows out from the solution outlet, the coated microspheres and the microspheres formed by the reaction compound of the coated microspheres and the labeled reagent form accumulation on the surface of the filter membrane, and the free labeled reagent can pass through the pores between the accumulated microspheres and the micropores of the filter membrane again after being carried by the solution, so that no blocking residue is formed.
The lower surface of the microporous membrane 23 is attached with a water absorbing material 22 such as filter paper, and the solution flowing into the hydrophilic membrane 24 from the gap passes through the microporous membrane 23 and is absorbed and transferred by the water absorbing material 22 such as filter paper. The market also has the microporous membrane 23 directly formed on the surface of the filter paper, so that the commercialized product can be directly adopted, and the hydrophilic membrane 24 with glue on the surface is attached to the surface of the microporous membrane 23, so that the water absorption effect can be better ensured.
As shown in fig. 3 and 4, the head end of the hydrophilic porous membrane 21 is further provided with a pre-treatment microporous membrane 28 having a pore size smaller than that of the microporous membrane; the sample solution flows from the sample absorption pad 26 through the pretreatment microporous membrane 28, then flows to the reagent binding pad 27, and finally flows to the hydrophilic porous membrane 21; the hydrophilic porous membrane to which the labeling agent is fixed is provided at the rear end of the pretreatment microporous membrane 28, and flows through the hydrophilic porous membrane 21 after being dissolved by the solution of the pretreatment microporous membrane 28.
The labeling reagent is derived from fluorescent substances or color particles directly visible to the naked eye; the color particles are selected from one or more of nano colloidal gold, latex microspheres, carbon black particles and other nanoparticles with color signals; the fluorescent substance is selected from one or more of fluorescent molecules and microspheres thereof, quantum dot microspheres, up-conversion luminescent microspheres, time-resolved fluorescent molecules and microspheres thereof, and fluorescent protein.
Wherein the labeling reagent can be suspended in a solution and added into the lateral flow channel; the labeling reagent may also be coated on a porous membrane that is immobilized in the flow channel or on a surface of the lateral flow channel, and then dissolved by the solution followed by the solution flowing forward. The coated microspheres can be suspended in a solution and added into the lateral flow channel; the coated microspheres may also be coated on a porous membrane that is solidified in the flow channels or coated on a surface of the lateral flow channels, and then dissolved by the solution followed by the solution flowing forward.
The biochemical detection device for intercepting and aggregating the microporous membrane further comprises detection equipment for optically detecting at least one wavelength signal simultaneously for the labeled reagent spectral signal in the reaction compound aggregated and accumulated on the surface of the microporous membrane; the microporous membrane is irradiated by light with a specific wavelength, the fluorescent substance gathered on the surface is excited to emit a fluorescent signal with the specific wavelength, the intensity of the fluorescent signal has a specific corresponding relation with the number of the reaction compounds gathered on the surface of the microporous membrane, the fluorescent spectrometer receives the fluorescent signal and analyzes the fluorescence of the corresponding wave peak through the microprocessor to detect different microsphere reaction compounds, and the concentration of different substances to be detected is calculated.
The reaction compound trapped on the surface layer of the microporous membrane emits a fluorescent signal with a specific wavelength after being excited by the irradiation of light with the specific wavelength.
Example 3
As shown in fig. 9-12, a microporous membrane interception and aggregation detection apparatus according to the present invention includes a reaction vessel, the reaction vessel is a centrifugal microfluidic chip with a reaction volume of not more than 100 microliters, the centrifugal microfluidic chip includes a solution reaction region, the solution reaction region includes a first solution reaction region 200, the first solution reaction region 200 is provided with a solution outlet 202 and a liquid inlet 203, the solution outlet 202 is disposed at a central bottom of the first solution reaction region 200 and has an area of not more than 12 square millimeters, the liquid inlet 203 is disposed at a bottom edge region of the first solution reaction region 200, one end surface of the first solution reaction region 200 is attached with a microporous membrane 300, and the microporous membrane 300 covers the solution outlet 202 and separates the solution outlet 202 from the first solution reaction region 200.
The volume of the first solution reaction zone 200 is not more than 100 microliters, the first solution reaction zone 200 is provided with an exhaust port 201, the exhaust port 201 is arranged at a position where the solution flowing into the first solution reaction zone 200 fills the first solution reaction zone 200 at last, and the solution outlet 202 is arranged at the central bottom of the first solution reaction zone 200.
The first solution reaction zone 200 is provided with at least two liquid inlets 203, and when the solution flows into the first solution reaction zone 200 from different liquid inlets 203, the uniform scouring result is achieved on the surface of the microporous membrane 300 at the bottom or the top of the first solution reaction zone 200.
The centrifugal microfluidic chip further comprises a second solution reaction area 600, a second exhaust port 601, a second liquid outlet 602 and a second liquid inlet 603 are arranged on the second solution reaction area 600, and the second exhaust port 601 is arranged at a position which is filled with the solution in the second solution reaction area 600 after entering.
The centrifugal microfluidic chip is also provided with a constant volume point 604 and an overflow area 1000; the constant volume point 604 is arranged at a position where the solution flowing into the second solution reaction area 600 is filled up at last, and after the solution amount entering the second solution reaction area 600 exceeds the solution amount, the redundant solution is reserved from the constant volume point to the overflow area 1000.
The second liquid outlet 602 is provided with a control area 800, and after the solution enters the second solution reaction area 600, the control area 800 is opened, so that the solution can flow out from the second liquid outlet 602.
The flow channel of the micro-fluidic chip is also added with at least one coating microsphere (the grain diameter is 800nm) with the diameter basically uniform and larger than the aperture of the filter membrane, a ligand fixed on the surface of the coating microsphere, such as monoclonal antibody A of certain protein, and a monoclonal antibody B of hepatitis B surface antigen protein marked by quantum dot particles (the diameter is 20nm) with the diameter smaller than the aperture of the microporous membrane; when the sample solution of the protein is added into the microfluidic chip, the coated microspheres, the monoclonal antibody A and the monoclonal antibody B marked with quantum dot particles enter a first solution reaction area together for mixed incubation reaction. When the solution is driven by a driving force, the solution flows out from the outlet, the monoclonal antibody B marked by the quantum dot particles with the diameter smaller than the aperture of the filter membrane can freely pass through the filter membrane, but the coated microspheres with the particle size larger than the aperture of the filter membrane, the monoclonal antibody A and the antibody immunoreaction compound (the coated microspheres and the quantum dot particles) are intercepted and concentrated on the surface of the filter membrane.
The capture reagent, the labeling reagent of the present embodiment is immobilized in the second solution reaction zone 600 or at least one of the capture reagent and the labeling reagent is immobilized upstream of the second solution reaction zone 600, and at least one of the capture reagent and the labeling reagent flows in from the second liquid inlet of the second solution reaction zone 600.
In the detection method of the microfluidic chip with the micro-porous membrane intercepting aggregated microspheres, after a sample solution flows into the solution inlet 203 of the first solution reaction area 200 and fills the first solution reaction area 200, a capture reagent, a labeling reagent and a sample to be detected are subjected to a binding reaction in the sample solution, and a ligand reaction compound is formed on the surface of the coated microspheres; when the sample solution is driven to pass through the microporous membrane from the solution outlet, the other washing solution flows into the first solution reaction zone from the solution inlet, continuously passes through the microporous membrane and washes microspheres gathered on the surface of the microporous membrane, the free marking reagent flows out, the marking reagent forming the ligand reaction compound and the coated microspheres are intercepted by the microporous membrane on the surface, and the free marking reagent is prevented from being intercepted and remained on the surface of the microporous membrane.
The particle size difference relationship between the coated microspheres and the quantum dot particles of the present embodiment is as follows: when the free coating microspheres and the reaction compound microspheres form aggregation and accumulation on the surface of the filter membrane, free quantum dot particles can pass through the gaps of the accumulated microspheres by being carried by a solution from the pores between the accumulated microspheres and then flow out of the filter membrane again, no interception residue is formed, so that the quantum dot particles intercepted on the surface of the filter membrane belong to the reaction compound, and when fluorescence is excited, the fluorescence intensity and the content of the protein have a specific corresponding relation.
The coated microspheres have the characteristic of aqueous solution dissolution and suspension, and are selected from one or more of polystyrene microspheres, magnetic microspheres, silicon microspheres and microspheres with other nano materials as cores and surfaces modified by hydrophilicity.
The labeling reagent comprises fluorescent substances or color particles capable of presenting a spectral signal, and the color particles are selected from one or more of nano colloidal gold, latex microspheres, carbon black particles, other nano particles with color signals and the like; the fluorescent substance is selected from one or more of fluorescent molecules and microspheres thereof, quantum dot particles and microspheres thereof, up-conversion luminescent particles and microspheres thereof, time-resolved fluorescent molecules and microspheres thereof, fluorescent protein and the like.
The micro-fluidic chip for intercepting the aggregated microspheres by the microporous membrane further comprises detection equipment for optically detecting a labeled reagent spectral signal in a reaction compound aggregated and accumulated on the surface of the microporous membrane; the microporous membrane is irradiated by light with a specific wavelength, the fluorescent substance of the labeling reagent gathered on the surface of the microsphere emits a fluorescent signal with the specific wavelength after being excited, and the intensity of the fluorescent signal has a specific corresponding relation with the quantity of the reaction compound gathered on the surface of the microporous membrane.
The micro-fluidic chip for intercepting the aggregated microspheres by the microporous membrane also comprises a detection device for simultaneously carrying out multiple wavelength signal optical detection on the spectral signal of the marking reagent in the reaction compound intercepted on the surface of the microporous membrane.
The capture reagent, the labeling reagent, and the sample to be tested in examples 1 to 3 have three binding reactions in the solution:
(1) competitive binding of a sample to be detected and a marking reagent to microspheres fixed with a capture reagent in a solution reaction area to form a reaction compound on the surfaces of the microspheres, and after the solution fully reacts, when the reaction liquid flows through a microporous membrane, the microspheres fixed with the capture reagent and the reaction compound are intercepted on the surface of the microporous membrane;
(2) as shown in fig. 13, a sample 15 to be tested competes with the microspheres 11 immobilized with the capture reagent 12 for binding with the labeled reagent 14, a reaction complex 13 is formed on the surfaces of the microspheres 11, and after the solution is sufficiently reacted, when the reaction solution flows through the microporous membrane, the microspheres 11 immobilized with the capture reagent 12 and the reaction complex 13 are retained on the surface of the microporous membrane;
(3) as shown in fig. 14, a sample 15 to be measured, microspheres 11 immobilized with a capture reagent 12, and a labeled reagent 14 are combined to form a reaction complex 13, and after the solution is sufficiently reacted, when the reaction solution flows through a microporous membrane, the microspheres 11 immobilized with the capture reagent 12 and the reaction complex 13 are retained on the surface of the microporous membrane.
In the competitive immune reaction, the capture reagent can be a competitive antigen coated on the microporous membrane, such as a small molecular antigen coupled with carrier protein, and the marking reagent is an antibody of a substance to be detected marked with fluorescence or other spectral signals; when the free antigen in the solution reacts with all the labeled reagents in the solution, the capture reagent does not have immunoreaction complex any more; when the free antigen in the solution reacts with a portion of the labeled reagent in the solution, the capture reagent on the microporous membrane will produce a portion of the immunoreactive complex, and thus a portion of the immunoreactive complex will be captured on the microporous membrane.
In the reaction of the immune sandwich method, a capture reagent can be an antibody A coated on a microporous membrane, and a marking reagent is an antibody B marked with fluorescence or other spectral signals; when the antigen in the solution flows in the mixing way in the lateral flowing solution, the antigen reacts with the labeling reagent and the antibody B which are simultaneously dissolved in the solution, and when the solution flows on the microporous membrane in a percolation way, the solution is captured by the antibody A on the microporous membrane and is trapped on the microporous membrane.
Example 4 assembling method of a biochemical detecting apparatus with microporous membrane for intercepting aggregated microspheres
As shown in fig. 3 and 4, a biochemical detection device for retaining aggregated microspheres by microporous membrane, wherein a nitrocellulose membrane (i.e. a porous hydrophilic membrane 21) is adhered on a PVC base plate coated with a pressure-sensitive adhesive, and a reagent combination pad 27, a pretreatment microporous membrane 28 and a sample absorption pad 26 are adhered on the lower edge of the front end of the nitrocellulose membrane in sequence, wherein the sample absorption pad 16mm overlaps the pretreatment microporous membrane 28 by 2mm, and the pretreatment microporous membrane 28 with a length of 12mm overlaps the reagent combination pad 27 by 2mm, thereby completely shielding the overlapping part of the sample absorption pad 26 and the reagent combination pad 27; the overlapping part of the reagent combination pad 27 with the length of 8mm and the nitrocellulose membrane with the length of 20mm is 1mm, the nitrocellulose membrane is clamped by 1-10mm by the upper hydrophilic membrane 24 and the lower hydrophilic membrane 24, the length of a formed hydrophilic membrane gap channel is 5-30mm, the position of a notch at the bottom of the hydrophilic membrane 24 is shown in figure 4 (a notch 29 is arranged on the hydrophilic membrane below), the width of the notch is 0.2-1.5mm, the interception microporous filter membrane is tightly attached to the notch of the hydrophilic membrane, the width of the microporous membrane 23 is 2-8mm, when the two are assembled, as shown in figure 4, at the moment, the sample solution flows to the microporous membrane 23 from the immune side flow channel, and all the solution can only flow through.
Example 5A microporous membrane entraps gather biochemical detection device of microballon is used for detecting HCG protein in urine
As shown in fig. 3 and fig. 4, the biochemical detection device for retaining the aggregated microspheres by the microporous membrane comprises two parts, namely an immune lateral flow channel and the microporous membrane 23 with the pore diameter of 0.45um, wherein the immune lateral flow channel mainly comprises a sample absorption pad 26, a reagent combination pad 27, a hydrophilic porous membrane 21 and the microporous membrane 23, the sample absorption pad 26, the reagent combination pad 27, the hydrophilic porous membrane 21 and the microporous membrane 23 are sequentially arranged from front to back, the hydrophilic porous membrane 21 is formed by oppositely clamping the hydrophilic surfaces of two layers of hydrophilic membranes 24, and keeping a gap of 0.15mm to extend forwards for about 5mm, the solution in the hydrophilic porous membrane 21 is guided out by the hydrophilic membranes 24 which vertically clamp the hydrophilic porous membrane 21, the solution directly flows into a solution outlet 29 arranged at the tail end of the bottom hydrophilic membrane, and the microporous membrane 23 is attached to the solution outlet 29 and has the pore diameter of 0.45.
A reagent combination pad of the immune lateral flow channel is added with HCG monoclonal antibody A marked colloidal gold particles, and a microporous membrane 23 is coated with HCG monoclonal antibody B; when the solution containing HCG protein flows in the immune lateral flow channel, HCG protein is reacted and combined by HCG monoclonal antibody A, and then when the solution passes through the microporous membrane 23, the reaction complex is captured by HCG monoclonal antibody B again, and a reaction complex of two monoclonal antibody sandwich HCG protein is formed on the membrane surface of the microporous membrane. The reaction compound can judge and analyze the concentration condition of HCG in the solution through the naked eye of a user or a matched colloidal gold quantitative analysis instrument.
Example 6A Biochemical detection device with microporous membrane for trapping aggregated microspheres for detecting HCG protein in urine
As shown in fig. 3 and fig. 4, the biochemical detection device for retaining the aggregated microspheres by the microporous membrane comprises two parts, namely an immune lateral flow channel and the microporous membrane 23 with the pore diameter of 0.22um, wherein the immune lateral flow channel mainly comprises a sample absorption pad 26, a reagent combination pad 27, a hydrophilic porous membrane 21 and the microporous membrane 23, the sample absorption pad 26, the reagent combination pad 27, the hydrophilic porous membrane 21 and the microporous membrane 23 are sequentially arranged from front to back, the hydrophilic porous membrane 21 is formed by oppositely clamping the hydrophilic surfaces of two layers of hydrophilic membranes 24, and keeping a gap of 0.15mm to extend forwards for about 5mm, the solution in the hydrophilic porous membrane 21 is guided out by the hydrophilic membranes 24 which vertically clamp the hydrophilic porous membrane 21, the solution directly flows into a solution outlet 29 arranged at the tail end of the bottom hydrophilic membrane, and the microporous membrane 23 is attached to the solution outlet 29.
Example 7A microporous membrane entraps gather biochemical detection device of microballon is used for detecting FOB protein in faecal sample
A biochemical detection device for intercepting aggregated microspheres by a microporous membrane comprises an immune lateral flow channel and a microporous membrane with the pore diameter of 0.22um, wherein the immune lateral flow channel mainly comprises a sample absorption pad, a marking reagent combination pad, a microporous membrane with the pore diameter of 0.22um, a coating reagent combination pad and a hydrophilic porous membrane, the sample absorption pad, the marking reagent combination pad, the coating reagent combination pad, a hydrophilic porous membrane and the microporous membrane are sequentially arranged from front to back, the hydrophilic porous membrane is formed by oppositely clamping hydrophilic surfaces of two layers of hydrophilic membranes, a gap with the thickness of 0.15mm is kept to extend forwards by about 5mm, a solution of the hydrophilic porous membrane is guided out by the hydrophilic membranes which vertically clamp chromatographic membranes, the solution directly flows into a solution outlet arranged at the tail end of the hydrophilic membrane at the bottom, and the solution outlet is attached with the microporous membrane with the pore diameter of 0.22 um.
The FOB monoclonal antibody A marked colloidal gold particles with the particle size of about 40nm are solidified on the marking reagent bonding pad in the immune lateral flow channel, and the coating reagent bonding pad is solidified with a coating microsphere with the diameter of 1.5um and the surface of which is fixedly coated with the FOB monoclonal antibody A. When the fecal sample solution is dripped into the detection device, FOB protein flows in the immune lateral flow channel, the solution mixes, reacts and combines FOB monoclonal antibody A-labeled colloidal gold particles, then the solution passes through a 0.22um filtering microporous membrane, all the magazine particles with the particle size larger than the aperture are intercepted, the primarily reacted colloidal gold particle compound is further mixed, reacts and combines with FOB monoclonal antibody B corresponding coated microspheres to form two monoclonal antibody sandwich HCG protein reaction compounds, then when the solution passes through the 0.22um microporous membrane, free HCG monoclonal antibody A-labeled colloidal gold particles with the particle size of about 40nm flow away through the microporous membrane, the coated microspheres and the free coated microspheres of the reaction compounds are intercepted on the surface of the microporous membrane, the reaction compound can judge and analyze the concentration condition of HCG in the solution through the naked eye of a user or a matched colloidal gold quantitative analysis instrument.
Example 8
As shown in FIG. 1, a 500nm SIO of BSA-AFM1 antigen was coupled to the bottom of the microwell cup 1 using a filter 3 with a pore size of 0.45 μm2Microspheres and 400nm fluorescent microspheres coupled with AFM1 murine monoclonal antibody.
When the immunoassay is carried out, the method comprises the following steps:
i. the solution containing the substance to be detected was put into the cuvette 1 and 500nm SIO conjugated with BSA-AFM1 antigen2Dissolving the microsphere and 400nm fluorescent microsphere coupled with AFM1 mouse monoclonal antibody, and dissolving to obtain 500nm SIO of the substance to be detected and the coupled BSA-AFM1 antigen2The microspheres compete to bind with 400nm fluorescent microspheres coupled with AFM1 mouse monoclonal antibodies;
ii.after the solution has reacted sufficiently, flowing the reactant through the microporous membrane, the pore size of which is substantially uniform, to achieve a 500nm SIO coupled to the BSA-AFM1 antigen2The microspheres and the reaction compound are trapped on the surface of the microporous membrane;
and iii, detecting the marking signal of the retentate on the surface of the microporous membrane, and calculating to obtain the concentration of the to-be-detected substance.
The detection of the labeled signal of the retentate on the surface of the microporous membrane comprises the following steps: the microporous membrane is irradiated by light with a specific wavelength, and the fluorescent substance carried by the intercepted substance on the surface of the microporous membrane emits a fluorescent signal with the specific wavelength after being excited.
The concentration of the substance to be detected is calculated based on: the intensity of the fluorescence signal has a specific corresponding relation with the amount of the reaction compound accumulated on the surface of the microporous membrane.
Example 9 measurement of aflatoxin content in milk and the like
A. Accurately sucking 500 mu L of milk (sample 1) and yoghourt (sample 2) into a 15mL centrifuge tube, and adding 9.5mL 35% methanol water; oscillating and mixing evenly, centrifuging at room temperature of 5000rpm for 5 min; 50 μ L of the supernatant was taken for analysis.
B. And taking out the micropore cup, standing to room temperature, and respectively adding the standard substance and the sample to be detected.
C. The reaction vessel was incubated at 37 ℃ for 15 min.
D. Putting into a reaction vessel ultrafiltration system, and performing suction filtration for 20 s.
E. The reaction vessel was removed from the millipore cup ultrafiltration system and 300uL of wash solution was added.
F. Putting into a reaction vessel ultrafiltration system, and performing suction filtration for 45 s.
G. And putting the sample into a fluorescence detection system to read fluorescence values.
The results of the experiment are shown in table 1.
TABLE 1 fluorescence values
Then, the OD value of the standard was plotted against the average OD value to obtain the following equation, y-0.3999 x +2.9025, R2The results are shown in fig. 15, 0.9991.
The concentration of the sample was calculated as (ppb) according to the curve method.
The aflatoxin concentration of sample 1 was calculated to be 0.005074ppb, and the aflatoxin concentration of sample 2 was calculated to be 0.014189 ppb.
And (4) conclusion: the microporous membrane interception and aggregation detection device has good linearity in immunodetection tests, and shows that the detection device and the detection have good consistency and accuracy. The detection concentration of this example was 0.005ppb and was the lower limit of detection.
Example 10 detection of drug residue small molecule clenbuterol by Competition assay
1 materials and methods
1.1 materials and reagents
A coated microsphere coated with a clenbuterol competitive antigen, a fluorescent microsphere marked with a clenbuterol antibody and a 0.45um filter membrane.
1.2 apparatus and instruments
A pipettor; a pH acidimeter; a fluorescence detection system; filter membrane retaining type micropore cup (solidified with antigen coupled by coating microballoon and fluorescent microballoon coupled with antibody).
1.3 methods
1.3.1 treatment of samples
Accurately weighing 1 plus or minus 0.01g of homogeneous substances (blood water in a sample is filled before homogenization) of 5 samples in total into a 5ml centrifuge tube, adding 1.5ml of 0.05M HCl, adding 1.5ml of 2% NaCl, shaking and uniformly mixing, centrifuging at the room temperature of 5000rpm for 5min, taking 1ml of supernatant into a 2ml centrifuge tube, adjusting the pH value to 7-8, and detecting.
1.3.2 Experimental procedures
(1) And taking out the filter membrane interception type micropore cup, placing to room temperature, and adding 45ul of standard substance and 45ul of sample to be detected into the sample adding pool respectively.
(2) Placing the filter membrane interception type microporous cup into a centrifuge to centrifuge for 10s at 900rpm, incubating in a reaction tank for 30min, and centrifuging to a waste liquid tank at 1200 rpm.
(3) Taking out the filter membrane interception type micropore cup from the micropore cup working system, adding 60ul of washing liquid into the washing liquid pool, putting the micropore cup into the micropore cup working system, and centrifuging at 3500rpm for 30 s.
(4) And putting the sample into a fluorescence detection system to read fluorescence values.
1.3.3 drawing of Standard Curve
Respectively preparing standard solutions of clenbuterol hydrochloride 0, 0.002, 0.01, 0.05, 0.25 and 1.25ppb by using 4% NaCl, adding 45ul of filter membrane interception type micropore cups, operating as 1.3.2, and drawing a standard curve through fluorescence values read by a fluorescence detection system.
1.3.4 clenbuterol hydrochloride ELISA and colloidal gold detection
The ELISA detection kit and the colloidal gold test strip for clenbuterol hydrochloride of Shanghai Kuailing Biotechnology Limited are used for detecting 5 samples to be detected according to the operation instruction.
1.4 results and analysis
1.4.1 plotting Standard Curve, the plotting Standard Curve is shown in FIG. 16.
1.4.2 detection results of filter membrane interception type micropore cup method
The content of clenbuterol hydrochloride in 5 samples to be detected was determined by a filter membrane-retention type micropore cup method, and the results are shown in table 2.
TABLE 2 detection results of filter membrane retention type micropore cup method
1.4.3 clenbuterol hydrochloride ELISA method and colloidal gold method detection results
The ELISA method and the colloidal gold method are used for detecting the content of the clenbuterol hydrochloride in 5 samples to be detected, and the detection results are shown in Table 3.
TABLE 3 results of ELISA and colloidal gold assay
1.5 results
The test adopts a filter membrane interception type micropore cup method to detect the content of the clenbuterol hydrochloride in 5 samples, the detection results are respectively 0.01, 0.018, 0.032, 3.15 and 2.82ppb, the test results are simultaneously detected by purchasing an ELISA kit and colloidal gold of Shanghai Kuailing Biotech limited company, the test results of the ELISA method are that 3 samples to be detected exceed the detection range, 2 samples to be detected are respectively 1.78 and 1.98ppb, compared with the ELISA method, the detection sensitivity of the microfluidic chip method is improved by 25 times, the detection result of the colloidal gold method is that 3 samples to be detected have no signals, 2 samples to be detected have signals, compared with the colloidal gold method, the sensitivity of the filter membrane interception type micropore cup method is improved by 100 times and 1000 times.
Claims (16)
1. A micro-porous membrane interception and aggregation biochemical detection device is characterized by comprising a solution reaction area, wherein a signal detection area is arranged on one end face of the solution reaction area, a solution outlet with the area smaller than 12 square millimeters is arranged on the signal detection area, the end face of the solution outlet is attached to a micro-porous membrane with basically uniform pore diameter, and the micro-porous membrane is attached to cover the solution outlet; at least one kind of microballoon with homogeneous diameter and pore size greater than that of the microporous membrane, capture reagent fixed onto the microballoon surface and marking reagent with particle size smaller than that of the microporous membrane are set in the solution reaction area, and the capture reagent and the marking reagent have coordination compounds in direct or indirect ligand relationship.
2. The biochemical detector of claim 1, wherein the coated microspheres and the labeled reagent in the solution reaction zone are reacted in a manner and in a microscopic molecular size matching relationship: when the solution enters the solution reaction zone, the capture reagent, the labeling reagent and the sample to be detected are subjected to a binding reaction in the solution, and a ligand reaction compound is formed on the surface of the microsphere; when the solution flows out from the solution outlet, the coated microspheres and the microspheres formed by the reaction compound of the coated microspheres and the labeled reagent form accumulation on the surface of the filter membrane, and the free labeled reagent can pass through the pores between the accumulated microspheres and the micropores of the filter membrane again after being carried by the solution, so that no blocking residue is formed.
3. The microporous membrane trapped mass biochemical detection device of claim 1, wherein the labeled reagent is derived from a fluorescent substance or a colored particle directly visible to the naked eye; the color particles are selected from one or more of nano colloidal gold, latex microspheres, carbon black particles and other nanoparticles with color signals; the fluorescent substance package is selected from one or more of fluorescent molecules and microspheres thereof, quantum dot microspheres, up-conversion luminescent microspheres, time-resolved fluorescent molecules and microspheres thereof, and fluorescent protein.
4. The biochemical detector of claim 2, further comprising a detector for optically detecting at least one wavelength signal of the labeled reagent spectrum signal of the reaction complex accumulated and accumulated on the surface of the microporous membrane; the microporous membrane is irradiated by light with specific wavelength, the fluorescent substance gathered on the surface can be excited to emit fluorescent signals with specific wavelength, and the intensity of the fluorescent signals and the number of the reaction compounds gathered on the surface of the microporous membrane have a specific corresponding relationship.
5. The apparatus of claim 1, wherein the solution reaction zone comprises an immune lateral flow solution channel, wherein a distal end of the immune lateral flow solution channel is provided with the solution outlet, and the microporous membrane is attached to the solution outlet.
6. The microporous membrane-trapped biochemical detection device according to claim 5, wherein the immune lateral flow solution flow channel comprises a hydrophilic porous membrane capable of directionally transmitting an aqueous solution, the hydrophilic porous membrane being selected from one or more of a nitrocellulose membrane, a glass fiber membrane of a sample absorbent pad, a chemical fiber membrane, and filter paper; the immune lateral flow solution flow channel is a gap channel formed by clamping the hydrophilic porous membrane by the hydrophilic surfaces of two hydrophilic membranes, and the solution flows from one end of the gap channel to the other end.
7. The biochemical detector of claim 6, wherein two hydrophilic membranes are disposed at the end of the immune lateral flow channel, the hydrophilic surfaces of the two hydrophilic membranes hold the upper and lower surfaces of the hydrophilic porous membrane, the lower surface of the bottom hydrophilic membrane of the two hydrophilic membranes is attached with the microporous membrane, and the middle of the hydrophilic membrane attached to the upper surface of the microporous membrane is provided with a gap with an area not more than 12 square millimeters as a solution outlet;
or the hydrophilic surfaces of the two hydrophilic membranes can also clamp the left side and the right side of the hydrophilic porous membrane, and the middle part of the bottom surface of the gap channel formed by clamping the two hydrophilic membranes is provided with a gap as a solution outlet;
the solution outflow opening comprises at least one gap shape with a maximum gap of not more than 1.5mm or an included angle with an angle of not more than 95 degrees.
8. The biochemical detector of claim 7, wherein the lower surface of the microporous membrane is attached with a water-absorbing material, and the solution flowing into the hydrophilic membrane gap passes through the microporous membrane and is absorbed and transmitted by the water-absorbing material.
9. The biochemical detector of claim 1, wherein the solution reaction zone comprises an open micro-porous cup with a volume of no more than 550 μ l, a through hole with an area of no more than 12mm is arranged at the middle of the bottom of the cup, and the bottom plane is arranged with a certain cone-shaped inclination angle around the center of the through hole; the microporous membrane with basically uniform aperture and the aperture range of 0.02-1.2um is attached to the surface of the through hole.
10. The biochemical detection device for the interception and aggregation of the microporous membrane as claimed in claim 9, wherein a counter bore is arranged below the through hole at the bottom of the microporous cup, the shape of the counter bore is matched with the shape of the microporous membrane, and the lower surface of the microporous membrane is basically flush with the lower surface of the microporous cup after the counter bore receives the microporous membrane; a protective membrane with a heat conduction function is arranged below the microporous membrane, the protective membrane is made of metal foil or an organic heat conduction membrane, one part of the protective membrane is attached to the bottom surface of the microporous cup, and the other part of the protective membrane is attached to the microporous membrane; the middle part of the protective membrane is provided with an opening which is positioned right below the hole of the micropore cup through 2.
11. The apparatus of claim 1, wherein the reaction vessel is a centrifugal microfluidic chip having a reaction volume of not more than 100 μ l, the centrifugal microfluidic chip comprises the solution reaction region, the solution outlet and a liquid inlet, the solution reaction region comprises a first solution reaction region, the solution outlet is disposed at a central bottom of the first solution reaction region and has an area of not more than 12mm, the liquid inlet is disposed at a bottom edge region of the first solution reaction region, and a microporous membrane is attached to one end surface of the first solution reaction region, covers the solution outlet and separates the solution outlet from the first solution reaction region.
12. The biochemical detector of claim 11, wherein the first solution reaction zone has a volume of no more than 100 μ l, and further comprises an exhaust port disposed at a position where the detection zone is filled with the solution flowing into the first solution reaction zone;
the first solution reaction zone is at least provided with two liquid inlets, and when solution flows into the first solution reaction zone from different liquid inlets, the uniform scouring result on the surface of the microporous membrane at the bottom or the top of the first solution reaction zone is achieved.
13. The apparatus of claim 11, wherein the microfluidic chip further comprises a second solution reaction area, a second liquid outlet, and a second liquid inlet, wherein the second liquid inlet fills the second solution reaction area, and the solution is discharged from the second liquid outlet and flows to the first solution reaction area.
14. The apparatus according to claim 13, wherein the centrifugal microfluidic chip further comprises a constant volume point and an overflow area; the constant volume point is arranged at the position filled with the solution finally in the solution reaction area, and after the solution amount entering the second solution reaction area exceeds the solution amount, the redundant solution is reserved from the constant volume point to the overflow area.
15. The microfluidic chip with a concentrated microsphere retained by a microporous membrane according to claim 13, wherein the second outlet port is provided with a control zone, and the solution in the second solution reaction zone enters the control zone and then flows out of the second outlet port after the control zone is opened.
16. The method for detecting the biochemical detection device intercepted and concentrated by the microporous membrane as claimed in claim 9 or 10, wherein a sample solution is added into the microporous cup, after the sample solution is drained from the through hole of the attached filter membrane, a washing solution is additionally added to wash and suspend the microspheres concentrated on the microporous membrane, and then the microspheres concentrated on the surface of the microporous membrane again are subjected to the spectral signal detection after the washing solution is drained from the through hole of the filter membrane.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811224877.3A CN111077325A (en) | 2018-10-19 | 2018-10-19 | Microporous membrane interception and aggregation biochemical detection device and detection method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811224877.3A CN111077325A (en) | 2018-10-19 | 2018-10-19 | Microporous membrane interception and aggregation biochemical detection device and detection method thereof |
Publications (1)
Publication Number | Publication Date |
---|---|
CN111077325A true CN111077325A (en) | 2020-04-28 |
Family
ID=70308395
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811224877.3A Pending CN111077325A (en) | 2018-10-19 | 2018-10-19 | Microporous membrane interception and aggregation biochemical detection device and detection method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN111077325A (en) |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113008654A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting whole course C-reactive protein in sweat, detection kit comprising concentration device and application of concentration device |
CN113009150A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting urine microalbumin in sweat, detection kit comprising same and application thereof |
CN113008655A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting serum amyloid A in sweat, detection kit comprising concentration device and application of concentration device |
CN117554606A (en) * | 2023-10-13 | 2024-02-13 | 北京大学 | Magnetic bead method immunodetection method assisted by porous filter membrane |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5783087A (en) * | 1994-04-08 | 1998-07-21 | Millipore Investment Holdings Limited | Method and apparatus for isolation and purification of immune complexes |
US5958714A (en) * | 1996-10-02 | 1999-09-28 | Safety Associates, Inc. | Test kits for determining at least two specific analytes in foods and other complex matrices |
CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
CN103018224A (en) * | 2012-12-14 | 2013-04-03 | 中国科学院上海微***与信息技术研究所 | Separate detection system and method of rare cells based on centrifugal micro-fluidic technology |
KR20140036827A (en) * | 2012-09-18 | 2014-03-26 | 한국생산기술연구원 | Microfluidic channel detection kit and manufacturing method of the same |
US20150330879A1 (en) * | 2014-05-16 | 2015-11-19 | Junyu Mai | Method and apparatus for biomolecule analysis |
WO2018177445A1 (en) * | 2017-04-01 | 2018-10-04 | 北京康华源科技发展有限公司 | Centrifugation immunochromatography detection method and apparatus |
CN209590052U (en) * | 2018-10-19 | 2019-11-05 | 上海快灵生物科技有限公司 | Biochemical detection device is gathered in microporous barrier retention |
-
2018
- 2018-10-19 CN CN201811224877.3A patent/CN111077325A/en active Pending
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5783087A (en) * | 1994-04-08 | 1998-07-21 | Millipore Investment Holdings Limited | Method and apparatus for isolation and purification of immune complexes |
US5958714A (en) * | 1996-10-02 | 1999-09-28 | Safety Associates, Inc. | Test kits for determining at least two specific analytes in foods and other complex matrices |
CN102053153A (en) * | 2010-11-25 | 2011-05-11 | 西安微通生物技术有限公司 | Dot immuno gold directed infiltration detection kit and application thereof |
KR20140036827A (en) * | 2012-09-18 | 2014-03-26 | 한국생산기술연구원 | Microfluidic channel detection kit and manufacturing method of the same |
CN103018224A (en) * | 2012-12-14 | 2013-04-03 | 中国科学院上海微***与信息技术研究所 | Separate detection system and method of rare cells based on centrifugal micro-fluidic technology |
US20150330879A1 (en) * | 2014-05-16 | 2015-11-19 | Junyu Mai | Method and apparatus for biomolecule analysis |
WO2018177445A1 (en) * | 2017-04-01 | 2018-10-04 | 北京康华源科技发展有限公司 | Centrifugation immunochromatography detection method and apparatus |
CN209590052U (en) * | 2018-10-19 | 2019-11-05 | 上海快灵生物科技有限公司 | Biochemical detection device is gathered in microporous barrier retention |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN113008654A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting whole course C-reactive protein in sweat, detection kit comprising concentration device and application of concentration device |
CN113009150A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting urine microalbumin in sweat, detection kit comprising same and application thereof |
CN113008655A (en) * | 2021-02-20 | 2021-06-22 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting serum amyloid A in sweat, detection kit comprising concentration device and application of concentration device |
CN113008654B (en) * | 2021-02-20 | 2021-11-23 | 北京华科泰生物技术股份有限公司 | Concentration device for collecting whole course C-reactive protein in sweat, detection kit comprising concentration device and application of concentration device |
CN117554606A (en) * | 2023-10-13 | 2024-02-13 | 北京大学 | Magnetic bead method immunodetection method assisted by porous filter membrane |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN111077325A (en) | Microporous membrane interception and aggregation biochemical detection device and detection method thereof | |
US11123731B2 (en) | Microfluidic detection chip, preparation method thereof, fixing device and centrifugal detection device | |
EP1957984B1 (en) | Test device for rapid diagnostics | |
KR101519379B1 (en) | Centrifugal Micro-fluidic Device and Method for immunoassay | |
JP4672263B2 (en) | Simple detection method, detection device, detection kit and production method thereof | |
US20110008776A1 (en) | Integrated separation and detection cartridge using magnetic particles with bimodal size distribution | |
CA2664565A1 (en) | Rotatable test element | |
CN101166974A (en) | Flow control technique for assay devices | |
EP3833735A1 (en) | Lateral flow immunoassay device with separation membrane | |
CN103018231A (en) | Preparation method and application of composite nano material paper chip electrochemical luminescence immunosensor | |
JP3813150B2 (en) | Biosensor and blood component analysis method using the same | |
CN111751525A (en) | Lateral flow immune test strip based on ordered micro-nano structure | |
KR20200102420A (en) | Immunochromatography device | |
WO2010086942A1 (en) | Automatic analyzer | |
CN209590052U (en) | Biochemical detection device is gathered in microporous barrier retention | |
CN108212228B (en) | Whole blood separation micro-fluidic chip, detection device and whole blood detection method | |
CN112014369B (en) | System and method for ultrasensitive digital chromatography rapid detection of analytes | |
JP4980944B2 (en) | Immunological measurement method | |
Sun et al. | Design and fabrication of a microfluidic chip to detect tumor markers | |
CN109954522A (en) | More fluid passage lateral chromatography quantitative testing devices and preparation method | |
CN111068798A (en) | Micro-fluidic chip with micro-porous membrane for intercepting aggregated microspheres and detection method thereof | |
CN109158136B (en) | Micro-fluid chip intercepted by microporous membrane and solution flow path control method thereof | |
CN109939751B (en) | Microfluidic chip, detection device and detection method for whole blood detection | |
CN210159631U (en) | Micro-fluidic chip with micro-porous membrane for intercepting aggregated microspheres | |
KR102631862B1 (en) | Method of enhancing the signal intensity in immunochromatographic assay |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
CB02 | Change of applicant information |
Address after: 201314 3rd floor, building 2, no.48-52, GUCUI Road, Xinchang Town, Pudong New Area, Shanghai Applicant after: QUICKING BIOTECH Co.,Ltd. Applicant after: SHANGHAI KUAILING BIOLOGY ENGINEERING Co.,Ltd. Address before: 201314 room 276, No. 187, Sun Nan Road, Xinchang Town, Pudong New Area, Shanghai Applicant before: QUICKING BIOTECH Co.,Ltd. Applicant before: SHANGHAI KUAILING BIOLOGY ENGINEERING Co.,Ltd. |
|
CB02 | Change of applicant information | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination |