CN100414296C - Embedded high-pass three-dimensional biological detecting technique and agent box - Google Patents
Embedded high-pass three-dimensional biological detecting technique and agent box Download PDFInfo
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- CN100414296C CN100414296C CNB021213259A CN02121325A CN100414296C CN 100414296 C CN100414296 C CN 100414296C CN B021213259 A CNB021213259 A CN B021213259A CN 02121325 A CN02121325 A CN 02121325A CN 100414296 C CN100414296 C CN 100414296C
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Abstract
The present invention discloses an embedded high-flux 3D cube biological detecting technique and a method for producing a test kit. The technique has the advantages of high sensitivity, high speed, large information quantity, convenient and simple production and low cost; the technique can simultaneously detect and analyze various different biological and chemical components, cells or microbes, etc., in a sample; the present invention has the technical scheme that micro balls coated with different chemical or biological molecules are respectively embedded in different micropores in a detection plate, and the micropores are connected by micropipes; the micropipes, a liquid inlet and a liquid outlet are formed into a microflow path which is communicated with the outside; the sample is reacted with the coated molecules on the surface of the micro balls through the microflow path; the result is recorded and is analyzed by eyes or a device. The present invention can be widely used for research and development in the field of environmental detection, medical research, disease diagnosis, protrinology, etc.
Description
Technical field
The present invention relates to a kind of novel embedded high-pass three-dimensional biological detecting technique and the preparation method of kit.This technology is sensitive fast, contain much information, the preparation method is easy, cost is low, compositions such as multiple different chemical substance, biomolecule, microorganism or cell in the test sample can be widely used in fields such as biology, medicine and pharmacology, preventive medicine, zoology and botany, farming and animal husbandry, food and health, the energy and chemical industry, environmental monitoring and medical diagnosis and detection simultaneously.
Background technology
Solid phase adsorption test often is used to detect or analyzes test sample or sample solution, biological fluid (as blood, serum, blood plasma, cerebrospinal fluid, urine etc., juices such as tear, sweat, digestive juice and seminal fluid, tissue fluid, transudate, vomitus, ight soil, tissue/cell homogenate etc.) in chemical substance, biomolecule and microorganism or biosome such as cell.
Microwell plate, microparticle or microballon in the solid phase adsorption test often is used to by absorption or in conjunction with catching analyte, so that object is separated from complicated sample potpourri or suspension as solid phase.Experimental difference, solid microparticulate can be made as glass, plastics, latex, glucosan, agarose, magnetic material, pottery, metal etc. with multiple different material with microwell plate.In traditional solid phase adsorption test, microwell plate, microparticle mainly are by its absorption or binding ability as solid phase, corresponding molecule in the aided capture liquid phase, by gravity, pressure, centrifugal, filtration, magnetic force etc., very easily unconjugated molecule (liquid phase or claim free phase) is separated with binding molecule (solid phase) through a step or multistep operation.
In the check and analysis test of sample, need be in same container under most of situation, carry out two or multinomial experiment simultaneously, so that the having or not of multiple heterogeneity in the sample, content and source etc. are made check and analysis such as parallel qualitative, quantitative, location; Or the multiple different qualities (having a plurality of different epi-positions or antigenic determinant as single protein molecular) of single kind of molecule carried out parallel detection and analysis.In the case, traditional solid phase adsorption is tested existing problem and is: to surpassing the different detected materials more than 3 kinds, owing to be difficult to distinguish, be difficult to carry out simultaneously.That is to say that with traditional solid phase adsorption test method, the quantity that will detect or analyze detected material in a test is subjected to the restriction or the restriction of solid phase system.
In modern immune detection and proteome analysis and research, need carry out quick, sensitive high throughput testing to compositions such as a large amount of in the same sample, multiple different chemical material, antigen, antibody or protein, with analytical chemistry goods and materials, antigen, antibody or kinds of proteins, quantity, composition, variation and polymorphism, posttranslational modification, individual Infection Status or the functional status of individual immunity system etc.The tradition solution is at first by technology and technologies such as separation and purification, the a large amount of various bioprobe of difference purifying, as drug molecule, protease molecule, growth factor and acceptor, antigen and various not homospecific antibody etc., again respectively with the surface of bioprobe immobilization at solid-phase matrix such as different microwell plates or microballons, respectively with sample in corresponding chemical substance, antibody or/and antigen react respectively, carry out check and analysis more respectively, therefore just need carry out tens different experiments as detecting tens kinds of compositions, unusual very complicated.Prior biological chip technology (technical scheme that provides as Affymatrix company etc.) though check and analysis are become one, is simplified the high throughput testing of sample, and the preparation of its bioprobe and immobilization technology and technology are still very complicated.The detection technique that Illumina company sets up by a kind of microballon that has electromagnetic wave launcher, though further simplified the preparation and the immobilization technology of bioprobe, but because the existence of electromagnetic wave launcher, not only manufacturing cost is higher, also make simultaneously the capacity of bioprobe in the size of microballon and the solid phase and detection sensitivity etc. be restricted, also need solve problems such as electromagnetic screen simultaneously.Purpose of the present invention will provide a kind of technology and technology more simple exactly, can overcome above-mentioned all puzzlements, promptly easy sensitivity, fast in time, the technology of multiple different material composition in the test sample simultaneously again.
Summary of the invention
Major technique feature of the present invention is, the surface is coated with different biological molecules respectively or has the microballon of different chemical material, be embedded in respectively in the different micropores of check-out console, there is microtubule to link to each other between micropore, and with advance/liquid outlet forms microfluidic circuit and communicates with the external world, the bag of sample by microfluidic circuit and bead surface is by molecular reaction, the reaction result in each hole can be analyzed with range estimation or with instrument record, arrangement position and the order in check-out console according to microballon or micropore is to the chemical substance in the sample, bio-target molecule, having or not of multiple heterogeneity such as cell and microorganism, character, content, the source, identification and analysis simultaneously carries out for tissue and cytology location etc.
This invention also provides a kind of method for preparing kit based on embedded high-pass three-dimensional biological detecting technique simultaneously, its major technique is characterised in that: kit is by being inlaid with microballon, and the porous check-out console and the various relevant matched reagent that have microfluidic circuit are formed respectively.According to having or not of each micropore reaction of check-out console with strong and weak, and arrangement position and the order of each micropore in check-out console, can compare simultaneously to multiple heterogeneity in the different samples, the sample composition that reagent that utilizes in the kit to be provided and method also can be caught each hole in the check-out console is done molecular weight, isoelectric point, glycosylation, phosphorylation and amino acid sequence etc. and is further analyzed.This kit has not only been simplified the operation steps that same sample carries out multifactor check and analysis greatly, shortened the running time, reduced working strength, accuracy, sensitivity, reappearance and the comparability of testing result have more been increased, but also can do further different the analysis to multiple different material composition in the test sample simultaneously, use more convenient, operate fasterly, and can be widely used in technology, research fields such as environment measuring, medical diagnosis on disease, new drug development, vaccine research and proteomics.
The technical solution adopted in the present invention in order to achieve the above object, its essential characteristic is that the preparation of embedded high-pass three-dimensional biological detecting technique and kit comprises the steps and composition at least:
1. check-out console is made up of microwell plate, microfluidic circuit and microballon etc.; Microwell plate is made up of plate body (1) He Bangai (2); Microfluidic circuit is made up of micropore (3), microflute (4) and/or microtubule (5), inlet opening (6), fluid hole (7), O-ring seal structures such as (8), can be respectively or be positioned at jointly on plate body (1) and/or the plate lid (2); Microballon (9) can have bags such as the arm molecule of different activities group or coating (10) by different chemical molecular (11) or biomolecule (12) by the surface, be embedded in respectively in sequence in the different micropore of check-out console and (see Fig. 1-5).Detected object has binding specificity (as antigen/haptens and antibody, hormone/cell factor and acceptor, aglucon and part etc.) or biospecificity (as enzyme-to-substrate, medicine and target molecule) etc. respectively in the chemistry of bead surface bag quilt or biomolecule and the sample;
2. test sample, as various liquid such as water source, biological fluids, direct or process is handled, as the cracking of tissue/cell through lysate, by inlet opening (6) enter check-out console, along microfluidic circuit, mix through the flow through chemical substance (11) of each micropore (3) and microballon (9) pan coating or biomolecule (12) etc. of microflute (4) and/or microtubule (5), and interaction or specificity combination; Unconjugated educt flows out through fluid hole (7) along microfluidic circuit in the sample;
3. use labeled molecule (13), as fluorescein (FITC, Cy3, Cy5, Texas Red etc.), isotope (
125I,
32P,
35S etc.), biotin and haptens, enzyme (HRP, AP, galactosidase, glucose-6-phosphate dehydrogenase (G6PD), urea plum and luciferase etc.), collaurum, rare earth ion (as Eu, Sm, Tb and Dy etc.) and chelate thereof are (as Eu
3+-DTPA) wait the direct mark test sample of material; Or with the different marked product-labels (14) of labeled molecule, as fluorescence antibody, enzyme labelled antibody, biotinylated antibody, biotinylation enzyme, Eu
3+Labels such as-DTPA-antibody, mark test sample (indirect labelling);
4. washing lotion stream flush away removes residual and label non-specific binding (14), sample or sample label etc. in the check-out console;
5. visual observations or with labeled molecule (13) in the detector analyzing and testing plate micropore as fluorescence, colour developing product or luminous product, radioactively have or not, power or the depth, and analyze the having or not of checking matter in the test sample, content, composition, source, tissue/cell in view of the above and learn the location or put in order etc.
When 6. label is enzyme (15), as HRP (horseradish peroxidase), AP (alkaline phosphatase), luciferase (Luciferase) etc., need before the observations to add substrate reagents (16) such as developer or luminescence reagent earlier, as OPD (o-phenylenediamine), DAB (diaminobenzidine) or luminescence reagent (as luminol, methyl umbelliferone phosphate, p-hydroxyphenylaceticacid) etc.
Feature of the present invention also is: microwell plate is made up of plate body (1) He Bangai (2), and plate body and plate cover can have micropore (3) respectively, microflute (4) and/or microtubule (5), inlet opening (6), fluid hole (7), O-ring seal (8), drive hole (19), pilot hole (20) and form part or all of structures such as (21); Micropore (3) has microflute (4) or microtubule (5) directly or indirectly to link to each other in twos therebetween by certain format permutation; Plate body (1) He Bangai (2) but be any machine-shaping, printing opacity, lighttight or reflective hard or soft material, as glass, plastics, high molecular synthetic material, pottery, metal, multiple different materials such as nonmetal or compound substance etc., the entity structure (seeing Fig. 1-4) of, rectangle square, collar plate shape or other face shaping through having of forming of finishing.1. plate body (1) He Bangai (2) difference or common through different chemical modifications or surface treatment produces reflector layer, reflected light signal; 2. obtain the inertia immersion coating, handle to reduce the non-specific adsorption to plate body and plate lid such as sample and/or label (14) as octylame, Tai Fulong (Teflon) coating, silication etc.; 3. obtain to have the molecular surface or the coating of differential responses reactive group; (see Fig. 6 as amino, carboxyl, aldehyde radical, hydroxyl, sulfydryl, acetyl bromide, hydrazide group, cyclisation imines carbonate group etc.; Fig. 7); so that different chemical molecular (11); as hapten molecules such as medicine, hormones, or the bag of different biological molecules (12) such as proteinase, growth factor, antibody is cooperated with microballon by (see figure 8); increase specific surface area, to improve detection sensitivity.
Feature of the present invention also is: microfluidic circuit is by micropore (3), microflute (4) and/or microtubule (5), form jointly with extraneous inlet opening (6), fluid hole (7), the O-ring seal structures such as (8) that communicates; Said structure can lay respectively at plate body or plate covers, and also can all be positioned at plate body or plate covers, and it is in diverse location and combination that plate body or plate cover, can form to have different geometric properties, satisfies the microfluidic circuit of different experiments demand, sees Fig. 3-5.
Feature of the present invention also is: microballon (9) but be material by any machine-shaping, as glass, plastics, glucosan, ficoll, agarose, high molecular synthetic material, cellulose, latex, silica gel, chromatography substrate, pottery, metal, nonmetal, magnetic material or compound substance etc., form big or small homogeneous, porous or solid with different face shapings through finishing; Can directly adsorb chemistry and biomolecule; Or after radiation exposure, chemical modification or surface treatment, acquisition has the arm molecule surface or the coating (10) of different chemical reactive group, can be further and different biomolecule (12) by the activity chemistry group microballon on the arm molecule, as antigen, antibody, hormone, cell factor, acceptor, aglucon and part etc., or chemical molecular connections such as (11) is coated on bead surface with it, generation has the medicine microballon (17) and the biological microballon (18) of different binding characteristics, as (seeing Fig. 6-8) such as immune microballon, albumen microballon, nucleic acid microballons.
Feature of the present invention also is: microballon (9) and micropore (3) all can or respectively by its arm molecule coating or the chemical active radical that had of surface, form amido link, peptide bond, ehter bond, disulfide bond respectively as the reactive group on acetyl bromide, hydrazide group, cyclisation imines carbonate group, amino, aldehyde radical, sulfydryl etc. and chemistry/drug molecule, the protein molecular such as carboxyl, amino, aldehyde radical, sulfydryl etc.; Or the hydrophobic grouping by hydrophobic surface and protein molecule interacts, connect respectively or bag by different chemical moleculars or biomolecule, (see Fig. 7, Fig. 8) with the microballon or the micropore that obtain different chemical or biomolecule are had different binding abilities respectively; When micropore (3) also wraps when processed, the chemistry (11) or the biomolecule (12) of its surface quilt that wraps are consistent with the chemistry (11) or the biomolecule (12) of microballon (9) quilt that wraps that embeds this hole, be in the same hole, the bag of micropore and bead surface is identical by molecule.
Feature of the present invention is that also biological microballon (18) can also prepare with following method, and its basic step comprises at least:
The antibody microballon (23) that 1. will be coated with antigen-specific antibodies (22) is educated altogether with the compound that contains excessive specific antigen (24), by antigen-antibody reaction antigen (24) is adsorbed on bead surface, after treating fully reaction, with the abundant rinsing of damping fluid to remove impurity and unconjugated antigen;
2. add bi-functional cross-linking agents such as glutaraldehyde or charcoal diimine, make antigen and antibody linked, to avoid or to reduce desorption or come off (the seeing Fig. 9 A) of the specific antigen molecule of antibodies;
3. the abundant rinsing of damping fluid after reaction is finished is removed crosslinking chemical and is sealed residual crosslink sites, obtains to have the biological microballon-antigen immune microballon (25) of specific antigen.
Feature of the present invention is that also biological microballon (18) also can prepare with following method, and its basic step comprises at least:
1. will be coated with anti-mouse immuning ball protein Fc section antibody (26) or biological microballon (27) educate altogether with the Hybridoma Cell Culture liquid or the ascites that contain mouse monoclonal antibody (28), by antigen-antibody reaction monoclonal antibody is adsorbed on bead surface, after treating fully reaction, use the damping fluid rinsing;
2. add bi-functional cross-linking agents (seeing Fig. 9 B) such as glutaraldehyde or charcoal diimine;
3. the abundant rinsing of damping fluid promptly obtains to be coated with the biological microballon-antibody mediated immunity microballon (29) of mouse anti specific antigen monoclonal antibody to remove crosslinking chemical and to seal residual crosslink sites.
Feature of the present invention also is: can adopt following method to carry out quantitative test: 1. bag can be had multiple hole more than 1 or 1 with the micropore of a kind of chemistry or biomolecule and the quantity of inlaying microballon on same microwell plate, when sample pursues orifice flow through each micropore and multiple hole thereof along microfluidic circuit, corresponding detected material is by constantly combination, and constantly from specimen fluids, remove, therefore, the binding capacity in each micropore and multiple hole thereof, the difference that puts in order because of front and back reduces gradually, according to having or not and strong and weak variation that react in each micropore and multiple hole, can carry out quantitative test; 2. when carrying out quantitative test, according to said method established standards tester or object of reference can be drawn checking matter concentration-reacting hole quantity and strong and weak change curve thereof by Computer Analysis, in conjunction with the sample volume, obtain quantitative analysis results.
Feature of the present invention also is: kit of the present invention is equipped with a check-out console at least, at least inlay a microballon in each micropore, bead surface is wrapped respectively by certain chemical molecular or biomolecule, and can form different kits respectively or jointly with following compositions:
1. have one tubing/cell or microbiological specimens treating fluid at least, mainly contain an amount of scaling agent in the treating fluid, as Triton X-100, NP40, Chaps etc. and protease inhibitors, as PMSF, EDTA, TPCK, TLCK, aprotinin.leupeptin, antipain etc., to guarantee abundant cracking of sample and dissolving, guarantee that simultaneously protein molecular is not degraded;
2. have a pipe at least for protein concentration check and analysis reagent, as the Folin-phenol reagent, Bradford reagent etc.;
3. have a kind of reagent that contains labeled molecule or label at least, as FITC, Cy3, Cy5, Texas Red,
125I,
32P,
35S, digoxin, biotin-avidin system, fluorescence antibody, enzyme labelled antibody etc. can be used for the direct or indirect mark of sample;
4. have pipe cleansing solution or its concentrate at least, as contain the PBST of an amount of BSA or calf serum, skim milk, Tween 20 etc., or the TBST damping fluid, the rinsing or the stream that can be used for check-out console are washed, to remove various non-specific adsorption things;
5. when label is enzyme, have a kind of chromogenic substrate or luminous substrate at least, as OPD or DAB and hydrogen peroxide/urea, luminol, methyl umbelliferone phosphate, ATP etc.;
6. have at least a pipe that the surface is housed and had certain chemical active radical or wrapped quilt the microballon of certain biomolecule, as wrapping by anti-CEA, anti-AFP, anti-P53, the monoclonal antibody of anti-Erb-2 etc. or the glass microballoon of polyclonal antibody, SepheroseCL-4B or polystyrene microbeads etc.;
7. have a tubulin matter analysis of Phosphorylation reagent at least, as bacterial alkaline phosphatase, potato phosphatase etc.;
8. have a pipe at least for proteolytic enzyme, as trypsase, chymotrypsin, carboxypeptidases etc. are used for microballon to catch clearing up of albumen, so that carry out the mass spectrophotometry or the amino acid sequence analysis of protein;
9. have a pipe at least for the electrophoresis sample buffer, as contain the SDS-PAGE electrophoresis sample buffer of different proteins denaturants such as an amount of SDS or urea, guanidine hydrochloride or contain urea or the IEF electrophoresis sample buffer of Chaps etc.;
10. has a pipe at least for the protein glycosylation analytical reagent, as PNGase F etc.;
Feature of the present invention also is: after finishing basic check and analysis, the reagent of going back in the available reagent box is done the further preceding processing of analysis such as mass spectrophotometry, determined amino acid sequence, protein phosphorylation and glycosylation by following diverse ways respectively to the sample composition of the variant hole of check-out console institute combination:
1. directly in interested each micropore, add proteolytic enzyme or phosphatase or reagent such as electrophoresis sample buffer or protein glycosylation analysis, the sample preparation before the bond of micropore and microballon is further analyzed in position;
2. the biological microballon in the micropore is taken out, in the micro-reaction container, add the sample preparation before proteolytic enzyme or phosphatase or reagent such as electrophoresis sample buffer or protein glycosylation analysis are further analyzed respectively;
3. fresh sample is educated altogether with the duplicate microballon of micropore interested respectively, repeat the operation steps of microwell plate with same condition, interested material composition in the sample is separated with sample liquid, add proteolytic enzyme or phosphatase or reagent such as electrophoresis sample buffer or protein glycosylation analysis, the sample preparation before bond is further analyzed.
The present invention has the following advantages: 1 contains much information.Sample can obtain the parallel analysis result and the data information of thousands of kinds of heterogeneities simultaneously through the one-time detection analysis, has solved the little problem of traditional experiment technical information amount.2. be easy to preparation, cost is low.It is starting material that microballon of the present invention can select for use various merchants to sell product, as the host material Bio-Glas used as the polypeptide solid phase synthesis, polystyrene bead etc., various chromatography substrates such as the Sepharose-4B of cyanogen bromide-activated, silica-gel bead etc., preparation with biological microballon such as the separation and purification of biomolecule and solid phase synthesis is combined together, omitted numerous and diverse bio-separation purge process; 3. independent assortment is variable flexibly.Can be fully according to the difference of application target, independent assortment, or change any hole in the microwell plate neatly and inlay the kind of microballon and binding characteristic to change the content that detects all puts in order and form and need not change; 4. highly sensitive.The used solid-phase matrix of present technique method has adopted 3-D solid structure, and promptly microballon and micropore all can or be made solid phase respectively.Each microballon has been equivalent to solidify the affinity chromatography matrices of antigen/antibody or aglucon/part, compares with classic method, bigger specific surface area is arranged and compare binding capacity.Owing to connect medicine or bioprotein molecule, eliminated sterically hindered influence simultaneously, further improved and compared binding capacity the molecule combination by the chemical active radical on the arm molecule.For improving reaction sensitivity, also can adopt porous beads and micropore to make solid phase, make specific surface area considerably beyond prior art and method.When sample is crossed the micropore that beading sample arranges by orifice flow, can with the bioprobe specific bond (have filtration, affinity chromatography, separate and concentrated effectiveness) in the solid phase; No longer is mixed simultaneously, reduced detected material (as antigen/antibody etc.), so sensitivity more because of diffusion repeatedly, the diluted influence of balance with sample stoste by the sample liquid after the solid phase adsorption.In addition, owing to omitted complicated separation and purification and harsh desorption process, so the biologically active of the immobilised coated thing that is obtained will be higher, purity also will further improve simultaneously, and this factor also will further improve the sensitivity of check and analysis; 5. easy and simple to handle, save sample and reagent.Adopt high density microwell plate of the present invention to carry out check and analysis, sample, sample, label, washing lotion and substrate are along microfluidic circuit each hole of flowing through one by one, changed tradition and prior art reacts respectively, cleans or the mode of operation of reacting and embathing in bigger container, therefore can solve puzzlement because of sample is difficult for obtaining and label costs an arm and a leg and brought, can save reagent in a large number, and be easy to realize full-automatic operation; 6. quantitative test is more accurate.This method concentrates on the check and analysis of tens of kinds even thousands of kinds of chemical substances and biomolecule on the check-out console carries out simultaneously, according to fluorescence, collaurum deposition, luminous having or not in each micropore in the check-out console, or the generation of quality product whether, contrast its arrangement position, not only can determine the composition of sample and source, the content of each component, tissue/cell location is convenient to the parallel comparison between different sample rooms and same sample heterogeneity simultaneously again.The bag of microballon is by can disposable a large amount of preparations in same test tube, thereby can guarantee the parallel unanimity of the testing result between each multiple hole and each check-out console.When needs carry out quantitative test to a certain composition,, cooperate microfluidic circuit to solve the problem of quantitative test by increasing the multiple hole number of the parallel bag quilt of identical encrusting substance; 7. tradition and existing detection method good reproducibility have been kept, excellent characteristics such as accuracy height, good reliability.8. purposes is more extensive.The present invention not only can be directly used in the research and development of modern life sciences such as the exploitation of diagnosis, new drug of disease and proteomics; But also can also can directly in check-out console, carry out glycosylation, phosphorylation and molecular function and the tissue/cell and the further analysis such as sub-micro or ultrastructural Location of amino acid sequence and variation, protein to interested micropore.
Description of drawings
Fig. 1 is a check-out console basic structure synoptic diagram.
Be plate body 1, plate lid 2, micropore 3, microflute 4 shown in the figure, be embedded in microballon 9 and structures such as the inlet opening 6 that communicates with micropore 3 through microflute 4, fluid hole 7 and sealing gasket 8 in the micropore 3; Plate lid 2 can form microtubule 5 with microflute 4, and micropore 3 forms microfluidic circuit with microflute 4, inlet opening 6, fluid hole 7 and sealing gasket 8 among the figure.
Fig. 2 is the surface structure synoptic diagram of 3 kinds of typical check-out consoles
2A among the figure, 2B, 2C are respectively the surface structure synoptic diagram of 3 kinds of typical check-out consoles, are inlet opening 6, fluid hole 7 shown in the figure, drive hole 19, structures such as pilot hole 20 and form 21.
Fig. 3 is the enlarged drawing of frame of broken lines A part among Fig. 2 A.
Fig. 3 A is a floor map, is plate body 1, micropore 3, discontinuous microflute 4 shown in the figure, is embedded in the microballon 9 in the micropore 3, and the fluid hole 7 that communicates with microflute 4, sealing gasket 8;
Fig. 3 B is the A-A sectional view of Fig. 3 A, is plate body 1, plate lid 2, micropore 3, microflute 4 shown in the figure, and microtubule 5 is embedded in the microballon 9 in the micropore 3, fluid hole 7, sealing gasket 8 etc.; The microflute 4 of micropore 3 upper ends forms microtubule 5 with plate lid 2 among the figure, and with the microtubule 5 of lower end micropore 3 is linked to each other in twos, forms complete microfluidic circuit with inlet opening 6, fluid hole 7 etc.
Fig. 4 is the enlarged drawing of frame of broken lines B part among Fig. 2 A.
Fig. 4 A is a floor map, is micropore 3, microflute 4 shown in the figure, is embedded in the microballon 9 in the micropore 3;
Fig. 4 B is the A-A sectional view of Fig. 4 A, is plate body 1, plate lid 2, micropore 3 shown in the figure, is embedded in the microballon 9 in the micropore 3; The microflute 4 at micropore 3 two ends links to each other micropore 3 in twos with plate lid formation microtubule 5 among the figure, and with inlet opening 6, fluid hole 7 grades form complete microfluidic circuit.
Fig. 5 is the partial enlarged drawing with check-out console of different microfluidic circuit.
Be micropore 3, microflute 4 shown in the figure, be embedded in the microballon 9 in the micropore 3; Microtubule 5 is arranged on the sidewall of two adjacent micropores among the figure, and the opening of its upper end communicates with microflute 4, and microflute 4 forms microtubules 5 with plate lid 2, microtubule 5 about openings at two ends micropore 3 is continuous in twos, and with advance/the complete microfluidic circuit of formation such as fluid hole.
Fig. 6 is the structural representation of microballon 9.
Fig. 6 A demonstration is polyhedral microballon 9, and the surface is connected with the arm molecule 10 of band reactive group R;
Fig. 6 B is the sectional view of polyhedron microballon 9, shows porous structure, and the surface of microballon 9 connects chemistry/drug molecule 11 by the arm molecule 10 of band reactive group, generates medicine microballon 17;
Fig. 6 C is the surface structure synoptic diagram of spheroid microballon 9, and the surface connects biomolecule 12 by the arm molecule 10 of band reactive group, obtains biological microballon 18.
Fig. 6 D is the sheet microballon made from tunica fibrosa 9, and surface adsorption biomolecule 12 produces biological microballon 18.
Fig. 7 is the structural representation of part arm molecule.
Be example with the microballon among the figure, show the surface molecular of microballon 9 or coating 10 with the molecular structure of various different chemical groups.10 for having the arm molecule of chemical active radical among the figure; Among the figure each microballon arm molecule 10 with reactive group be respectively: Fig. 7 .1 is an aldehyde radical; Fig. 7 .2 is a carboxyl; Fig. 7 .3 is an epoxy ethyl; Fig. 7 .4 is an ethylene glycol; Fig. 7 .5 is amino; Fig. 7 .6 is a sulfydryl; Fig. 7 .7 is the azlactone group; Fig. 7 .8 is a cyanate group; Fig. 7 .9 is a cyclisation imines carbonate group; Fig. 7 .10 is the acylimidazole group; Fig. 7 .11 is an imidazoles carbonate group; Fig. 7 .12 is the iodacetyl group; Fig. 7 .13 is the acetyl bromide group; Fig. 7 .14 two thiopyridines groups; Fig. 7 .15 is a dithio-nitrobenzene formic acid group; Fig. 7 .16 is the iminodiacetic acid (salt) acid groups; Fig. 7 .17 is the diazo salt group; Fig. 7 .18 is the octylame group; Fig. 7 .19 is the NHS group; Fig. 7 .20 is a trifluoroacetyl sulfonic acid chloride group; Fig. 7 .21 is a p-methylphenyl sulfonic acid chloride group; Fig. 7 .22 is the diazanyl group; Fig. 7 .23 is the hydrazide group group; Fig. 7 .24 is the cyanuric chloride group;
Fig. 8. be albumen or antibody sandwich reaction synoptic diagram
Show the chemical active radical-acetyl bromide group and the reaction of the sulfydryl on the protein molecular 12 of the arm molecule 10 on microballon 9 surfaces among Fig. 8 A, protein molecular is connected bead surface, generate biological microballon (18);
Show the chemical active radical-hydrazides group and the reaction of the aldehyde radical on the haptens drug molecule (11) of microballon 9 surperficial arm molecules 10 among Fig. 8 B, drug molecule is connected bead surface, generate medicine microballon (17);
Show the chemical active radical-azlactone group and the reaction of the amino on the antibody molecule (22) of microballon 9 surperficial arm molecules 10 among Fig. 8 C, the antibody protein molecule is connected bead surface;
Show the chemical active radical-cyclisation imines carbonate group and the reaction of the amino on the antibody molecule (22) of microballon 9 surperficial arm molecules 10 among Fig. 8 D, protein molecular is connected bead surface; The schematic arrangement of Fig. 9 antigen immune microballon and antibody mediated immunity microballon
Fig. 9 A is the schematic arrangement of antigen immune microballon 25, and microballon 9 is coated on its surface by arm molecule 10 with antigen-specific antibodies 22 among the figure; Antigen 24 is by antigen-antibody reaction, is adsorbed on the surface of microballon 9 by antibody 22, through the protein coupling reaction antigen 24 delivered a child into antigen immune microballon 25 with antibody 22 by chemical bond-linking again;
Fig. 9 B is the schematic arrangement of antibody mediated immunity microballon 29, microballon 9 will resist mouse immuning ball protein Fc section antibody 26 (antiantibodys) to be coated on its surface by arm molecule 10 among the figure, mouse monoclonal antibody 28 (is antigen at this) is adsorbed on the surface of microballon 9 by antiantibody 26 by antigen-antibody reaction, through the protein coupling reaction, mouse monoclonal antibody 28 is delivered a child into antibody mediated immunity microballon 29 with antiantibody 26 by chemical bond-linking again.
Figure 10. be several representative experimental technique basic principle schematic
Figure 10 .1 is a direct method.Figure 10 .2 is an indirect method.Figure 10 .3 is a sandwich method.Figure 10 .4 and Figure 10 .5 are competition law, and the latter is a double label method.Figure 10 .6 is bridging method (a directly mark).Show microballon 9 among the figure, the arm molecule 10 of band reactive group, haptens drug molecule 11, different labeled molecule 13 and 13 ', different specific marker things 14 and 14 ', HRP enzyme 15, substrate 16, known specific antibody 22, known specific antigen 24, unknown tested antigen 30, unknown tested antibody 31, the target protein 32 of haptens drug molecule, affinity element or streptavidin 33, biotin 34, product or luminous product 35 add lustre to.
Embodiment
The present invention's basic operational steps and method in force is as follows:
One. the preparation of check-out console: the preparation method of check-out console comprises following basic skills and step:
1. surface treatment: the surface treatment of microwell plate and microballon is different slightly different because of selected materials, every kind of its concrete disposal route of material and condition all have multiple, the present invention at this with " biomolecule mobilization technology and application " (Jiang Zhonghua etc.,, chemical publishing house in 1998) and China Patent No. be 01207812.3 and application number be that 00120798.9 patent is the main reference document.Use known various biomacromolecule immobilization technology, handle the microballon and the microwell plate of various materials, make its surface have specific reactive group (as hydroxyl, carboxyl, carbonyl, aldehyde radical, amino, sulfydryl, hydrophilic/hydrophobic group, anionic/cationic and metal group etc., see Fig. 7), can react with the reactive group on chemical molecular, drug molecule and the biomolecule, and have specific adsorptive power.Embathe with dimethylbenzene after strictness is cleaned as porous glass beads, again through 70% ethanol rinsing, oven dry; Then at 2% the third amino-triethyl silicane anhydrous propanone solution (APES, 3-Aminopropyltriethoxysilane, Sigma company, catalog number (Cat.No.) A3648) soaked 30 seconds in, with anhydrous propanone and the rinsing of distilled water order, dry (Weetall, H.H., Biochem.Biophys.Acta, 1970; 212:1).Or the surface treated various chromatography substrates of selling with the merchant (as the Sepharose-4B of cyanogen bromide-activated), the matrix that polypeptide and oligonucleotides solid phase synthesis are used is with the solid-phase matrix material as microballon such as beaded glass (Sigma company) of various reactive groups.
Microwell plate also can be done different surface treatments according to requirement of experiment.As carry out silicidation or the processing of Tai Fulong (Teflon) coating, as just reaction vessel or microfluidic circuit, to reduce the non-specific adsorption in the experiment.
2. bag quilt: the method for coating of microballon and microwell plate mainly is a 1. biomolecule, be dissolved in respectively as protein moleculars such as antigen, antibody, cell factor and enzymes in the different damping fluids such as phosphate, carbonate, directly educate altogether with the Sepharose-4B of surface treated polystyrene microbeads, beaded glass, cyanogen bromide-activated etc., make and wait to wrap the biomolecule of quilt and the reactive group of bead surface fully reacts, and by the surface of immobilization at microballon and microwell plate, water or damping fluid fully stream are washed, to remove not immobilised free biomolecule; 2. to biological micromolecule, can directly be connected on the microballon by reactive group as chemical moleculars such as haptens, drug molecule, cortin, steroid hormones; 3. be connected earlier on the protein molecular carrier, again carrier molecule be connected on the microballon; 4. original position is synthetic.Utilization solid state chemistry synthetic technology, little peptide, drug molecule, lead compound etc. are directly synthetic in bead surface.
3. inlay microballon: artificial or use the full-automatic mechanical hand system, difference is wrapped processed microballon (having different binding characteristics), embed respectively in certain sequence in each micropore of microwell plate.
4. capping: make micropore, microflute and advance/fluid hole forms microfluidic circuit, and can only by advance/fluid hole communicates with the external world.
5. sealing: through advance/fluid hole removes microballon, microwell plate or cover plate (or epiphragma) with the sealing damping fluid with sealing and waits reactive group or the nonspecific binding sites that the surface may be residual, to reduce the non-specific binding that may occur in the detection.Promptly obtain thus can with chemical molecular, biomolecule, antibody and/or antigen, specificity combinations such as enzyme/substrate or antagonist, aglucon/part, nucleic acid probe, nucleic acid/albumen, and can be used for 3 D stereo braided type high flux biological detection plate that bond is detected.Confining liquid adjuvant commonly used has: bovine serum albumin(BSA) (0.5-5%), skim milk (1-6%), casein (0.5-2%), gelatin (0.1-0.5%), Normal animal serum (0.5-5%) and scaling agent etc. are (as Tween 20, NP40, TritonX-100) etc.But cellular check-out console does not have microtubule, microflute or does not have into/fluid hole yet, needs sealing before the capping, and the epiphragma of taking seal during use off can use, and rinse method adopts and embathes.
Two. the check and analysis of sample
The check and analysis of sample generally comprise following basic step and method:
1. the preparation of sample: according to the difference of testing goal, biological sample will carry out different processing respectively before detection.Common sample treatment comprises: 1. add appropriate amount of buffer solution to regulate sample concentration or protein concentration, ionic strength and potential of hydrogen etc.; 2. particulate antigen such as cell, microorganism cooperates processing such as homogenate or Ultrasonic Pulverization to make it to become true property solution with lysis buffer; 3. centrifugal or filter and remove suspended substances or particle are as haemocyte etc.
2. the mark of sample: 1. direct mark.Use known biomolecular labeling technology, use labeled molecule such as material or derivatives thereof such as biotin, enzyme, fluorescein, collaurum, isotope or rare earth ion and sequestrant thereof respectively as direct mark sample of indicator or sample to be checked.2. adopt indirect labelling.With the label mark sample of above-mentioned labeled molecule, but labeled reactant is to carry out after sample to be checked and stream such as wash at step finishing to add.
3. add sample to be checked: with sample or handle sample and add in the check-out console through inlet opening, make it with each hole in oneself immobilised different chemical molecule of bead surface, biomolecule etc. react.The biomolecule corresponding with solid phase is hunted down and specific bond or absorption in the test sample, and remaining and unconjugated part flows out check-out console through fluid hole.
4. stream is washed or is embathed: by the microfluidic circuit of check-out console, wash with washing lotion stream, cellular check-out console can by advance/the fluid orifice flow washes or embathes through form (21), to remove test sample residual in the plate (not bond or claim educt) or label etc.
5. interpretation of result: according to the difference of labeled molecule, range estimation or with observation of use instrument such as scanner and write down having or not and strong and weak (power as the depth that generates the product color, collaurum deposition, fluorescence intensity, activity etc. changes) of each hole reaction.The arrangement position of contrast microballon promptly can be determined in this sample the composition of detected material and each components contents by Computer Analysis.But when label is proteinase, must increases operation stepss such as adding substrate in the operation and could observe testing result, its testing result is color reaction or luminescence-producing reaction.
The present invention in force, the aforesaid operations step is different according to its encrusting substance, detected material, label and display result etc., and multiple array mode can be arranged.Detection with antigen-antibody is an example, describes in detail below in conjunction with accompanying drawing:
1. direct method: will wrap by the biological microballon-antibody microballon of different specific antibodies (22,22 ', 22 ") (23,23 ', 23 ") and be embedded in respectively in the different micropore (3), capping adds the sealing fluid-tight and closes nonspecific binding site.Behind sample usefulness labeled molecule (fluorescein 13) mark, directly add in the check-out console through inlet opening (6).Sample flow when micropore, unknown antigen (30,30 ' wherein, 30 ") with each micropore in the corresponding antibody molecule (22 of antibody microballon (23,23 ', 23 ") pan coating, 22 ', 22 ") combination respectively, unconjugated part flows out through fluid hole (7) with sample liquid.Wash with washing lotion (as PBST, 50mM PB, pH7.0-7.6,100Mm NaCl, 0.05% Tween 20) stream, remove the residual sample and the sample of non-specific bond, fluorescent microscope or scanner are observed and the record testing result.Fluorescence is arranged in the micropore, and expression has the unknown antigen corresponding with antibody (22,22 ', 22 ") (30,30 ', 30 ") in the test sample, otherwise then negative.Analyze the power of positive hole reaction, promptly know the variation of test sample component content in the sample.See Figure 10 .1.
2. indirect method: with hepatitis B 24
A, third liver 24
D, acquired immune deficiency syndrome (AIDS) 24
G, syphilis 24
K, plasmodium 24
NSynthesize the antigen coated biological microballon (18 of acquisition etc. different specific antigen polypeptide (24) by solid-phase polypeptide synthesizer original position
A, 18
D, 18
G, 18
K, 18
NDeng), being embedded in respectively in the different micropore (3), capping adds the sealing fluid-tight and closes nonspecific binding site.With the sample of handling well (add the patients serum, contain unknown antibody 31), mark does not directly react in inlet opening (6) adds check-out console, sample flow when each micropore, unknown antibody (31 wherein
A, 31
D, 31
G, 31
K, 31
N) combine with the antigen molecule (24) of antigen microballon (18) pan coating in each micropore (3), unconjugated part flows out through fluid hole (7) with sample liquid.Stream is washed to remove residual and unreacted sample, adds label (14) reaction of the anti-human immunoglobulin(HIg) antibody (antiantibody) of labeled molecule (fluorescein 13) mark, and the stream flush away removes unconjugated free label, observes also record testing result.As only inlaying bag by hepatitis B antigen 24
AThe hole reacting positive at antigen microballon place, just representing has the unknown antibody 31 corresponding with hepatitis B antigen in examinee's serum
A, deducibility thus, the patient has or had the infection of hepatitis B, does not have the infection of other corresponding pathogen.See Figure 10 .2.
3. sandwich method: will wrap by different specific antibodies (22,22 ', 22 ");, be embedded in respectively in the different micropore (3) as the cellulose diaphragm of the monoclonal antibody of rat anti-mouse inhomogeneity, subclass, type and hypotype antibody (antibody microballon 23; 23 ', 23 "), capping adds the sealing fluid-tight and closes nonspecific binding site.Through inlet opening (6) sample to be checked (the Hybridoma Cell Culture liquid that contains mouse monoclonal antibody) is added reaction in the check-out console (available cellular microwell plate), sample flow is when micropore, unknown antibody (30 wherein, 30 ', 30 ") in (at this as antigen) and each micropore (3) on the cellulose diaphragm antibody molecule (22) of bag quilt combine respectively, the part of last combination flows out through fluid hole (7) with sample liquid.Stream is washed or is embathed to remove unreacted free antigen-mouse monoclonal antibody (30), the antibody (species specificity antiantibody 14 ') that adds the anti-mouse immuning ball protein of rabbit (or sheep etc.) of collaurum (13 ') mark again reacts, the stream flush away removes unconjugated free label, estimate the reaction in each hole, positive hole had both represented that the monoclonal antibody in the tested nutrient solution was the antibody of this type and this hypotype.See Figure 10 .3.
4. competition law: will wrap by the antibody microballon (23) of different specific antibodies (22) be embedded in respectively in the different micropore (3), capping adds the sealing fluid-tight and closes nonspecific binding site.The known sample (antigenic label 24) of fluorescein Cy3 (labeled molecule 13) mark is mixed with unlabelled sample to be checked (antigen 30), add check-out console then, sample to be checked (antigen 30) combines the immobilization antibody (22) of bag quilt in the check-out console with known antigens (31) competition of mark, stream is washed or is embathed and remove free known sample (antigenic label 31) and sample to be checked (antigen 30), write down having or not and strong and weak variation of each hole fluorescence reaction with observation of use instrument such as fluorescent microscope or scanners, see Figure 10 .4.In the expression that fluorescence the is strong sample to be checked this antigen do not have or content lower, otherwise show that then content is higher.The reference standard curve can be made comparatively accurate quantitative test.Also can as carrying out, see Figure 10 .5 in the enforcement respectively with the fluorescein (13 ') of different colours with the experiment of both double-taggings, or multiple labeling of Cy5 mark sample to be checked (antigen 30).
5. bridging method: will wrap by the medicine microballon (17) of different chemical drug molecule (haptens 11) be embedded in respectively in the different micropore (3), capping adds the sealing fluid-tight and closes nonspecific binding site.The sample to be checked (protein target molecule 32 of medicine 11) that adds affinity element or streptavidin (33) mark, damping fluid stream are washed or are embathed and remove unconjugated free drug protein target molecule in the sample, add the HRP (enzyme 15) of biotin (34) mark again; Damping fluid stream is washed or is embathed and remove unconjugated resolvase label, adds substrate (16), and positive hole generates coloured product or luminous product (35), observes the change color or the luminous power in each hole, the discriminatory analysis result.In aforesaid method of operating 1-4, all can adopt the bridging method to carry out, improve the sensitivity that detects to use its amplification.
In the enforcement, in same detection system, often need carry out corresponding antigen and detection of antibodies simultaneously, to judge patient's immunity, Infection Status and infectiousness.The simplest and the most direct solution is direct method and direct bridging method, but direct method is low because of sensitivity, and less employing; Next is double-tagging or multiple labeling method, secondly is again to adopt subregion plate method (application number is 00120798.9).The present invention also can select for use in force China Patent No. be 01207812.3 and application number be 00120798.9 described microwell plate and dedicated detector board and technical scheme.With the enzyme be label and need be further analyzed interested reacting hole the time, preferred check-out console of the present invention, the check-out console that particularly has microfluidic circuit feature shown in Figure 5.
In the check-out console inlay microballon when carrying out immunology detection with preferred porous and solid polystyrene bead and beaded glass.Polystyrene microbeads will adopt processing such as isotope irradiation, the concentrated sulphuric acid/potassium dichromate or nitric acid dousing; And handle according to the different poly-D-lysine or the glutaraldehydes of adopting respectively of needs such as sensitivity and subsequent analysis processing, also can select for use the merchant to sell the solid-phase matrix that polystyrene bead, beaded glass and polypeptide solid phase synthesis are used.
The present invention is described further below in conjunction with embodiment.
Example I: the application of 3 D stereo braided type high flux Measurement for Biotechnique in prevention congenital malformation and intrauterine infection.
Rubella virus, cytomegalovirus, herpes simplex virus, Coxsackie virus, toxoplasm are the main pathogens that causes intrauterine infection and cause fetal anomaly; Hepatitis B, syphilis and acquired immune deficiency syndrome (AIDS) etc. are the pathogen that can cause the communicable disease of infection of newborn by vertical transmission.The detection diagnosis of reproduction age and gravid woman being carried out above-mentioned pathogenic infection is the important means of prevention congenital abnormality and infection of newborn, and concrete operations step in force is as follows:
1. wrap by the preparation of microballon:
1. the preparation of microballon can be different because of the difference of selected solid-phase matrix material, only is example with the polystyrene microbeads at this.Through polystyrene plastics microballon washing lotion (concentrated sulphuric acid and the potassium bichromate solution) soaked overnight of isotope irradiation, distilled water rinsing, oven dry.With the glutaraldehyde solution immersion treatment of 0.1-5%, rinsing, be sub-packed in the different test tubes; Or with the poly-D-lysine immersion treatment of 0.05-0.5mg/ml, add respectively contain above-mentioned pathogenic microorganism not the carbonic acid buffer of synantigen (0.5-100ug/ml) (pH8.6-10.0), normal temperature is hatched and was spent the night in 6 hours or 4 ℃ for CB, 50mM.After the abundant rinsing of CB, add confining liquid (NaCl 100mM contains 1-5% normal serum and other confining liquid adjuvant for PBS, 50mM pH6.8-7.8) sealing nonspecific binding site.
2. be antigen with the little peptide of specificity haptens of amino acid synthesizer by the above-mentioned pathogenic microorganism of solid phase synthesis, serve as that bag is by microballon (handling without cracking or chain rupture) directly with its solid phase carrier microballon and synthetic product, be sub-packed in and add confining liquid (PBS in the micro-reaction pipe, 50mM pH6.8-7.8, NaCl 100mM contains 1-5% normal serum and other confining liquid adjuvant) the sealing nonspecific binding site.
2. the preparation of check-out console:
From inlet opening (6) end, the microballon that above-mentioned sealing is handled embeds in the micropore of microwell plate in regular turn, adds a cover shrouding.Add confining liquid through inlet opening, normal temperature is hatched and was spent the night in 6 hours or 4 ℃.
3. test sample:
Person's serum 20ul to be checked, PBS dilutes in right amount, gets 100ul and adds in the check-out console incubated at room 20 minutes through inlet opening; PBST (PBS contains 0.01-0.2%Tween 20) stream was washed 5-10 minute; Add and contain the goat anti-human igg of Cy3 mark and the goat-anti people IgM mixed liquor 100ul of Cy5 mark, incubated at room 20 minutes; PBSA (PBST contains 2%BSA) stream was washed 5-10 minute.
4. observations:
Check-out console is put fluorescent microscope or scanner analysis result.Cy3 is positive, and expression has the infection history, and the value of being above standard prompting has recent infection; The Cy5 positive shows recent infection, may be in carrier state, is infectious, unsuitable gestation.
Example II: the application of 3 D stereo braided type high flux Measurement for Biotechnique in the research of cell protein group express spectra.
For individual, although the cell genes carried of each histoorgan of whole body is all identical, expressed proteins kind and quantity then vary.Research cell protein group express spectra not only helps to understand the normal function and the regulatory mechanism of cell, but also can help human functional status and the regulatory mechanism of understanding under pathology or the abnormality, for diagnosis, the treatment of disease provides marker molecule or target molecule.
1. the preparation of microballon:
1. the preparation of microballon is solid-phase matrix at this to sell Bio-Glas through merchant of further particle screening, by manufacturer's product description requirement the monoclonal antibody of the anti-different proteins antigen of purifying is educated with it altogether, after reaction is finished, abundant rinsing is also sealed residual activated group, and the bag that sealing is handled is sub-packed in the different test tubes by microballon.
The antiantibody microballon (27) that 2. will be coated with anti-mouse immuning ball protein Fc section antibody (26) is educated altogether with the Hybridoma Cell Culture liquid or the ascites that contain mouse monoclonal antibody (28), by antigen-antibody reaction monoclonal antibody is adsorbed on bead surface, after treating fully reaction, use the damping fluid rinsing; Add glutaraldehyde and handled 30-60 minute, the abundant rinsing of Tris damping fluid, acquisition is coated with the biological microballon-antibody mediated immunity microballon (29) of mouse anti specific antigen monoclonal antibody.
2. the preparation of check-out console:
From the feed liquor nose end, the microballon that is coated with different monoclonal antibody specifics is embedded in the micropore of microwell plate in regular turn, add a cover shrouding, add confining liquid through inlet opening, normal temperature is hatched and was spent the night in 6 hours or 4 ℃.
3. test sample:
Tissue or cell liquid that cracking is handled are got an amount of mensuration protein concentration, behind fluorescence labeling, get an amount of adjustment volume to 300-500ul, slowly add in the check-out console incubated at room 30-60 minute through inlet opening; Stream was washed 5-10 minute.
4. observations:
Fluorescent microscope or scanner analysis result.Relatively separate sources or through the histiocytic proteomic expression profile of different disposal is analyzed the relation of difference bands of a spectrum and function or disease, determines the protein of interest bands of a spectrum.
5. mass spectrophotometry:
To protein of interest bands of a spectrum in analyzing, the arrangement of microballon in the control test plate is taken out corresponding microballon and is put in the micro-reaction test tube, adds the desorb attached liquid, gets then and carries out mass spectrophotometry in right amount to measure the molecular weight of protein; Or in former reacting hole, adding trypsase (proteolytic enzyme), room temperature or 37 ℃ of reactions 2-6 hour or digestion are spent the night, and get an amount of point sample and carry out mass spectrophotometry, obtain protein fingerprint spectrum, by the amino acid sequence of computer search with analysing protein.
6. protein phosphorylation analysis:
Get an amount of enzyme hydrolyzate, add the bacterial alkaline phosphatase (be dissolved in HEPES 20mmol/L, pH7.0 contains NaCl 150mmol/L, 0.1%Triton X-100,10% glycerine) of an amount of volume, 30 ℃ were reacted 60 minutes, and got and carry out mass spectrophotometry in right amount; The microballon taking-up that maybe will answer in the hole is put in the micro-reaction test tube, add phosphatase, question response thoroughly back adds the desorb attached liquid, gets then and carries out mass spectrophotometry in right amount, contrast has or not the possible position of phosphorylation and phosphorylation without the microballon analysis result of phosphatase processing with mensuration and analysing protein.
7. protein glycosylation analysis:
The microballon taking-up is put in the micro-reaction test tube or in foramen primum, adding PNGase reagent (is dissolved in the Tris-HCl damping fluid, 20mmol/L, pH7.2, contain 50mmol/L NaCl, 1mmol/L EDTA) an amount of, 37 ℃ were reacted 60-120 minute, get and carry out mass spectrum or stratographic analysis in right amount, have or not glycosylation and glycosylated degree with mensuration and analysing protein.
8. isoelectric points of proteins and molecular weight determination
Get an amount of cell pyrolysis liquid, according to the analysis result of step 4, with corresponding microballon reaction, fully after the rinsing, be sub-packed in the different microtest tubes, add special-purpose isoelectric focusing and SDS-PAGE electrophoresis sample buffer respectively, take out an amount of electrophoresis respectively, to measure isoelectric point and molecular weight.
EXAMPLE III: the application of 3 D stereo braided type high flux Measurement for Biotechnique in new drug development research.
In the research and development of new drug, normally brilliant in structure or known drug molecular structure according to the three-dimensional of protein target molecule, the synthetic new compound-lead compound that has different fine structure differences in a large number.Need carry out adhesion to new synthetic lead compound, binding specificity, the high throughput analysis of metabolic pathway and accretion rate etc., with the chemical constitution of analyzing new synthetic lead compound and drug effect and the property of medicine and with the relation of toxicity.
1. the preparation of microballon:
The activation that the preparation of microballon is sold with the merchant or the glass microballoon (Sigma company) of modification, resins such as polystyrene are carrier matrix, use different medicine route of synthesis and technology path to carry out solid phase synthesis, can obtain to have the various lead compounds of different chemical group and molecular structure.The new lead compound that obtains, is directly used by microballon as bag without the processing of unwinding together with solid-phase synthesized carrier.
2. the preparation of check-out console:
From the feed liquor nose end, the resin particle or the microballon that will have lead compound embed respectively in the different micropores of microwell plate in regular turn, add epiphragma or cover plate shrouding, add confining liquid through inlet opening, the sealing nonspecific binding site.
3. test sample:
Fluorescently-labeled protein target molecule is dissolved in PBS or the TBS suitable damping fluids such as (Tris-HCl), gets an amount of volume (50-500ul) and adds in the check-out console through inlet opening, incubated at room 30-60 minute, washes 5-10 minute with PBS or TBS stream.
4. observations:
With laser scanning confocal microscopy, scanner or fluorescent microscope record result.
5. stream is washed: use the Laemmli buffer system Laemmli (as acetate buffer solution, PBS or TBS etc.) and the damping fluid stream of different ionic strength of different pH values to wash respectively 5-10 minute.
6. each stream is washed the back and is write down the result with laser scanning confocal microscopy, scanner or fluorescent microscope.Each hole of analyzing and testing plate under various flows is washed condition, the variation of fluorescence intensity, obtaining the affinity of variant lead compound and protein target molecule, combination stability, fine structure changes the influence to lead compound and target molecule combination stability.
The present invention separates technology and the analysis technology phases such as biochip to solid phase synthesis technique with adsorption chromatography, Capillary Electrophoresis etc. In conjunction with, be a kind of biological analysis detection technique of novel concept, integrate the advantage of few techniques, have contain much information, The outstanding advantages such as quantitatively accurate, highly sensitive, low cost of manufacture can be widely used in biology, medical science, materia medica, environment The fields such as science.
Claims (10)
1. the embedded high-pass three-dimensional biological detecting method of chemical molecular in the test sample or biomolecule, cell, bacterium and virus or other microbial body, this method comprises following compositions or step at least;
1. check-out console is made up of microwell plate, microfluidic circuit and microballon; Microwell plate is made up of plate body (1) He Bangai (2); Microfluidic circuit is made up of micropore (3), microflute (4) and/or microtubule (5), inlet opening (6), fluid hole (7), O-ring seal (8), is positioned at respectively or jointly on plate body (1) or the plate lid (2); Microballon (9) wraps by different chemical molecular (11) or biomolecule (12) by arm molecule or coating (10) that the surface has the different activities group, and is embedded in respectively in sequence in the different micropore (3);
2. test sample directly or after treatment by inlet opening (6) enter microwell plate, along microfluidic circuit, flowing through through microflute (4) and/or microtubule (5), micropore (3) interacts with chemical molecular (11), the biomolecule (12) of microballon (9) pan coating or specificity combines; Unconjugated sample composition flows out through fluid hole (7) along microfluidic circuit;
3. use labeled molecule (13) or its different labels (14) mark test sample;
4. non-specific binding and residual sample, label (14) or sample label in the check-out console are removed in the washing lotion rinsing;
5. detect having or not and content of labeled molecule in each micropore (13) with range estimation, scanner or other known method, analyze the having or not of checking matter in the test sample, content, composition, source, tissue/cell and learn the location or put in order;
6. or add substrate (16) colour developing of enzyme (15), observations.
2. detection method according to claim 1, it is characterized in that microwell plate is made up of plate body (1) He Bangai (2), plate body and plate cover have micropore (3) respectively, microflute (4) or microtubule (5), inlet opening (6), fluid hole (7), O-ring seal (8), drive hole (19), pilot hole (20) and form (21) structure or part-structure; Micropore has microflute (4) or microtubule (5) directly or indirectly to link to each other in twos therebetween by certain format permutation; Plate body (1) He Bangai (2) is square, rectangle, garden dish-type or hard other face shaping printing opacity, lighttight or reflective or soft material; Plate body (1) He Bangai (2) obtains to have the molecular surface or the coating (10) of inertia or reactive group through different chemical modifications or surface treatment respectively or jointly, the coating of reactive group or molecular surface are through further handling, and bag is by different chemical molecular (11) or biomolecule (12).
3. detection method according to claim 1 is characterized in that microfluidic circuit is by micropore (3), microflute (4) or microtubule (5), form with extraneous inlet opening (6), fluid hole (7) and O-ring seal (8) structure that communicates; Said structure lays respectively at plate body or plate covers, or all is positioned on plate body (1) or the plate lid (2).
4. detection method according to claim 1 is characterized in that microballon (9) processes from strand, big or small homogeneous, the porous with different face shapings or solid; After radiation exposure, chemical modification or surface treatment, the surface obtains to have the arm molecule or the coating (10) of different chemical reactive group; Activity chemistry group on the arm molecule further connects with different chemical molecular (11) or biomolecule (12), and it is coated on bead surface, generates medicine microballon (17) and biological microballon (18) with different binding characteristics.
5. according to claim 1, claim 2 or the described detection method of claim 4, it is characterized in that bag is meant on the coating or arm molecule (10) of microballon (9) and micropore (3), connect different chemical molecular (11) or biomolecule (12) respectively, obtain to have the microballon or the micropore of different binding characteristics.
6. detection method according to claim 1 is characterized in that microballon prepares with following method, and its step comprises at least:
The antibody microballon (23) that 1. will be coated with the specific antibody (22) of antigen is educated altogether with the damping fluid that contains excessive specific antigen (24), by antigen-antibody reaction specific antigen (24) is adsorbed on bead surface, after treating fully reaction, with the abundant rinsing of damping fluid;
2. add glutaraldehyde, charcoal diimine or other bi-functional cross-linking agent;
3. the abundant rinsing of damping fluid, acquisition has the biological microballon-antigen immune microballon (25) of specific antigen.
7. detection method according to claim 4 is characterized in that microballon prepares with following method, and its step comprises at least:
The antiantibody microballon (27) that 1. will be coated with anti-mouse immuning ball protein Fc section antibody (26) is educated altogether with the Hybridoma Cell Culture liquid or the ascites that contain mouse monoclonal antibody (28), by antigen-antibody reaction monoclonal antibody is adsorbed on bead surface, after treating fully reaction, use the damping fluid rinsing;
2. add glutaraldehyde or charcoal diimine or other bi-functional cross-linking agent;
3. fully rinsing, acquisition is coated with the biological microballon-antibody mediated immunity microballon (29) of mouse anti specific antigen monoclonal antibody.
8. detection method according to claim 1, it is characterized in that adopting following method to carry out quantitative test: bag is had more than 1 or 1 with the micropore (2) of a kind of chemistry or biomolecule and the quantity of inlaying microballon (4) on same microwell plate, when sample pursues orifice flow through each micropore and multiple hole thereof along microfluidic circuit, corresponding detected material is by constantly combination, and constantly from specimen fluids, remove, therefore, the binding capacity in each micropore and multiple hole thereof, the difference that puts in order because of front and back reduces gradually, according to having or not and strong and weak variation that react in each micropore and multiple hole, carry out quantitative test; When carrying out quantitative test, according to said method established standards tester or object of reference are drawn checking matter concentration-reacting hole quantity and strong and weak change curve thereof by Computer Analysis, in conjunction with the sample volume, obtain quantitative analysis results.
9. the embedded high-pass three-dimensional biological detection reagent kit of chemical molecular in the test sample or biomolecule, cell, bacterium and virus and other microbial body, it is characterized in that kit is equipped with the described check-out console of claim 1 more than 1 or 1, at least inlay a microballon in its each micropore, bead surface is wrapped respectively by certain chemistry or biomolecule, and forms different kits respectively or jointly with following compositions:
1. have a tubing or cell or microbiological specimens treating fluid at least;
2. have at least a pipe to be protein concentration check and analysis reagent;
3. has a kind of sample labelled reagent that contains labeled molecule or label at least;
4. have pipe cleansing solution or its concentrate at least;
5. have a kind of the be chromogenic substrate of enzyme or luminous substrate at least;
6. have at least a pipe that the surface is housed and had certain chemical active radical or wrapped quilt the microballon of certain biomolecule;
7. has a tubulin matter analysis of Phosphorylation reagent at least;
8. have at least a pipe to be proteolytic enzyme;
9. have at least a pipe to be the electrophoresis sample buffer;
10. have at least a pipe to be the protein glycosylation analytical reagent.
10. detection kit according to claim 9, it is characterized in that, after finishing check and analysis, pass through following diverse ways, with the reagent that kit provided, the sample composition of institute's combination in the variant hole of microwell plate is done mass spectrophotometry, determined amino acid sequence, protein phosphorylation and glycosylation or other further preceding processing of analysis respectively:
(1) directly in interested micropore, adds proteolytic enzyme or phosphatase or electrophoresis sample buffer or protein glycosylation analytical reagent, the sample preparation before the bond of micropore and microballon is further analyzed in position;
(2) the biological microballon in the micropore is taken out, in the micro-reaction container, add the sample preparation before proteolytic enzyme or phosphatase or electrophoresis sample buffer or protein glycosylation analytical reagent are further analyzed respectively;
(3) fresh sample is educated altogether with the duplicate microballon of micropore interested respectively, repeat the operation steps of microwell plate with same condition, interested material composition in the sample is adsorbed on the microballon, fully unconjugated sample mixture is removed in rinsing, add proteolytic enzyme or phosphatase or electrophoresis sample buffer or protein glycosylation analytical reagent, the sample preparation before bond is further analyzed.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021213259A CN100414296C (en) | 2002-06-14 | 2002-06-14 | Embedded high-pass three-dimensional biological detecting technique and agent box |
Applications Claiming Priority (1)
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| CN101180530B (en) * | 2005-05-20 | 2011-11-09 | 日立化成工业株式会社 | Method of analyzing biochemical substance, analyzing reagent kit and analyzer |
| CN102721817B (en) * | 2012-06-27 | 2014-08-13 | 中国医学科学院输血研究所 | Method for detecting biological activity of Fc segment of human immunoglobulin by high throughput method |
| CN107389549A (en) * | 2017-08-17 | 2017-11-24 | 武汉璟泓万方堂医药科技股份有限公司 | Rotating disc type collaurum/fluorescent test paper chip |
| CN111197002A (en) * | 2018-11-19 | 2020-05-26 | 武汉菲思特生物科技有限公司 | High-throughput hybridization recognizing and reading integrated machine |
| CN109870582B (en) * | 2019-02-27 | 2020-07-10 | 华中科技大学 | Multi-target magnetic immunochemistry luminescence microfluidic chip detection platform and method |
| WO2021233303A1 (en) * | 2020-05-18 | 2021-11-25 | Shanghai Polaris Biology Co., Ltd. | Microbeads and uses thereof |
| CN115015547B (en) * | 2021-12-30 | 2024-04-12 | 上海云泽生物科技有限公司 | Kit for quantitatively determining tacrolimus in whole blood by low-steric-hindrance latex-enhanced immune competition method |
Citations (5)
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|---|---|---|---|---|
| WO1999067641A2 (en) * | 1998-06-24 | 1999-12-29 | Illumina, Inc. | Decoding of array sensors with microspheres |
| CN1320820A (en) * | 2000-04-27 | 2001-11-07 | 赵翀 | Immunodetection method with rich information and its special detection board |
| US6327410B1 (en) * | 1997-03-14 | 2001-12-04 | The Trustees Of Tufts College | Target analyte sensors utilizing Microspheres |
| CN2478109Y (en) * | 2001-03-09 | 2002-02-20 | 赵翀 | Immunity pick-up plate |
| WO2002021128A2 (en) * | 2000-09-05 | 2002-03-14 | Illumina, Inc. | Cellular arrays comprising encoded cells |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6327410B1 (en) * | 1997-03-14 | 2001-12-04 | The Trustees Of Tufts College | Target analyte sensors utilizing Microspheres |
| WO1999067641A2 (en) * | 1998-06-24 | 1999-12-29 | Illumina, Inc. | Decoding of array sensors with microspheres |
| CN1320820A (en) * | 2000-04-27 | 2001-11-07 | 赵翀 | Immunodetection method with rich information and its special detection board |
| WO2002021128A2 (en) * | 2000-09-05 | 2002-03-14 | Illumina, Inc. | Cellular arrays comprising encoded cells |
| CN2478109Y (en) * | 2001-03-09 | 2002-02-20 | 赵翀 | Immunity pick-up plate |
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