CN1464071A - Braided type high flux gene chip detecting technique and reagent box - Google Patents

Abstract

The present invention discloses one kind of embedded high-flux gene chip detection technology and kit preparing process. The technological process is sensitive, fast, great in information amount, simple in manufacture and low in cost and may be used in simultaneous detection and analysis of several kinds of nucleic acids, proteins and chemical molecules in sample. The main feature is that micro beads connecting the nucleic acids of different sequences are embedded onto different pores in the detection board and communicated via microtubules and are connected with liquid inlets and outlets to form micro flow channels to the outside. Via the micro flow channels, the sample may be crossbred or combined with nucleic acid molecules on the surface of the micro beads. The detection result may be recorded and analyzed with some instrument. The present invention may be used widely in the research and development of environment science, medicine, etc.

Description

Braided type high flux gene chip detecting technique and test kit

The present invention relates to a kind of novel braided type high flux gene chip detecting technique and the preparation method of test kit.This technology is sensitive fast, contain much information, the preparation method is easy, cost is low, compositions such as multiple different nucleic acid molecule, biochemical molecule, microorganism or cell in the test sample can be widely used in fields such as biology, medicine and pharmacology, preventive medicine, zoology and botany, husbandry, food and health, the energy and chemical industry, environmental monitoring and medical diagnosis and detection simultaneously.

Solid phase adsorption, separation and synthetic technology at sample or sample solution, biological fluid (as blood, serum, blood plasma, cerebrospinal fluid, urine etc., juices such as tear, sweat, Digestive system and seminal fluid, tissue juice, transudate, vomitus, ight soil, tissue/cell homogenate etc.), the aspect such as synthetic of detection, analysis, separation, purifying and protein polypeptide, oligonucleotide, lead compound and the medicine of biochemical molecule such as the albumen in microorganism or the cell, nucleic acid widely used.

Biological solid phase technique mainly is to utilize insoluble solid-phase matrix material, by surface adsorption or in conjunction with to catch the liquid phase assay and to be purified target molecule or fixing new synthetic target molecule in the thing, by gravity, pressure, centrifugal, filtration, magnetic force, fluid etc., very easily unconjugated molecule (liquid phase, mutually free or moving phase) is separated with binding molecule (solid phase) through a step or multistep operation, so that obtain pure molecules of interest, or target molecule separated from complicated sample mixture or suspension, be used for further analysis and research.Experimental difference, solid-phase matrix can be with multiple different materials, as glass, plastics, latex, dextran, agarose, magneticsubstance, pottery, metal and flat board, porous plate, microparticle and the microporous particles etc. made such as nonmetal.

In the check and analysis of sample, separation and purification, experiment such as synthetic, 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 (as the mononucleotide diversity) of single kind of molecule carried out parallel detection and analysis; Or multiple material carried out separation and purification simultaneously, or carry out the synthetic of multiple differing molecular simultaneously.In the case, traditional solid phase adsorption is tested existing problem and is: to surpassing the different detected materials more than 3 kinds, purifying thing or synthetics are difficult to carry out simultaneously owing to be difficult to distinguish.That is to say that with traditional solid phase technique or method, the quantity of detected material, synthetics or isolate is subjected to the restriction or the restriction of solid phase system in a container or test, operations such as more can't simultaneously or synthesizing continuously, separate and detect.

In modern genomics, in the analysis and research and technological development of proteomics and functional group, need simultaneously to a large amount of in the same sample, multiple different biochemical carries out fast, the sensitive high-throughput is synthetic, separate, purifying and detection, kind with the analytical biochemistry goods and materials, quantity, form, variation and polymorphism, posttranslational modification, structure, putting in order of amino acid/Nucleotide/glycosyl etc., interaction between different biological molecules, or individual health, Infection Status and medication characteristic, the functional status of individual immunity system, hereditary difference etc.The tradition terms of settlement is at first by technology and technologies such as synthetic and separation and purification, synthesize respectively or a large amount of various bioprobe of purifying, as drug molecule, nucleic acid molecule, protein molecular, somatomedin 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, because it is synthetic, operation such as separation and purification and check and analysis disconnects mutually, therefore just need carry out tens different synthesizing as detecting tens kinds of compositions, separation and purification and check and analysis experiment, 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, technology are still very complicated, and efficient is low.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 shielding simultaneously.Purpose of the present invention will provide the more easy sensitivity of a kind of technology and technology, timely fast exactly, biosynthesizing, separation and purification, the detection of multiple different substances composition, particularly nucleic acid and protein molecular and the three can be organically combined perforation new high-throughput biotechnology together in the test sample simultaneously again.

Major technique feature of the present invention is, the surface is connected with the microballon of different IPs acid molecule, 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 nucleic acid molecule reaction that sample is connected with bead surface by microfluidic circuit, the reaction result in each hole can be analyzed with range estimation or with instrument record, according to arrangement position and the order of microballon in check-out console, to sample, chemical substance in the biological sample such as cell and microorganism, bio-target molecule, nucleic acid, oligonucleotide, having or not of multiple heterogeneity such as medicine, 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 test kit based on braided type high flux gene chip detecting technique simultaneously, its major technique is characterised in that: test 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 test kit to be provided and method also can be caught each hole in the check-out console is done molecular cloning, molecular structure, avidity, molecular weight, iso-electric point, glycosylation, phosphorylation, Nucleotide or determined amino acid sequence etc. and is further analyzed.This test kit has not only been simplified the operation steps that same sample carries out multifactor check and analysis greatly, shortened the operating time, reduced working strength, more increased the accuracy of detected result, sensitivity, circulation ratio and comparability, 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 environment measuring, medical diagnosis on disease, new drug development, vaccine research, bio-molecular interaction, genomics, technology such as proteomics and functional group, research field.

Its essential characteristic of the technical solution adopted in the present invention is in order to achieve the above object, and the preparation of braided type high flux gene chip detecting technique and test kit comprises following compositions or step at least:

1. a kind of check-out console of forming by microwell plate (1), microballon (2) etc.; Microwell plate is made up of structures such as plate body (3), plate lid (4), micropore (9) and microfluidic circuit; Microballon connects nucleic acid (13) by the active group on the arm molecule (12) or oligonucleotide (14) molecular probe forms the surface molecular coating, and is embedded in sequence in the different micropores;

2. test sample is handled as processes such as solid-liquid body sample, biological fluid, tissue, cell, microorganisms, as dilution, the cracking of removing particulate matter, tissue/cell, separation and purification, reverse transcription, pcr amplification, mark etc.; Or do not handle directly by inlet opening (5) and enter microwell plate, interact along microflute and/or the microtubule nucleic acid molecule that micropore is connected with bead surface of flowing through, make the albumen in the sample or sequence complementary nucleic acid molecule is hybridized under given conditions with the solid-phase nucleic acid molecular probe or specificity combines, unconjugated sample composition flows out through fluid hole (6);

3. use tagged molecule (15), as fluorescein, isotropic substance, vitamin H and haptens, material or derivatives thereofs such as Radioactive colloidal gold, enzyme, rare earth ion and inner complex thereof (as Cy5-dCTP, ³²P-dCTP etc.) direct mark test sample; Or with the different marked products of tagged molecule, as markers such as fluorescence antibody (16) mark test sample;

4. rinsing, remove in the microwell plate sample non-specific binding or residual or sample marker and marker (16), etc.;

5. estimate or having or not and content with tagged molecule (15) in each micropore of detections such as scanner, as fluorescence, luminous product, radioactive having or not, having or not of power or colour developing product, the depths of color etc. are analyzed the having or not of checking matter in the test sample, content, composition, source, molecular structure, avidity, tissue/cell and are learned location and Nucleotide or aminoacid sequence etc.;

When 6. marker is enzyme, as HRP (horseradish peroxidase), AP (alkaline phosphatase), luciferase (Luciferin) etc., need before the observations to add substrate reagents such as developer or luminescence reagent earlier, as OPD (O-Phenylene Diamine), DAB (diaminobenzidine) or luminescence reagent (as luminol,3-aminophthalic acid cyclic hydrazide, methyl umbelliferone phosphoric acid ester, p-hydroxyphenylaceticacid) etc.

Feature of the present invention also is: microwell plate is made up of plate body, Ban Gai, micropore and microfluidic circuit; But plate body and plate lid are any machine-shaping, printing opacity, lighttight or reflective hard or soft material, as glass, plastics, high molecular synthetic material, pottery, metal, multiple differing materials such as nonmetal or matrix material etc., can be processed into the entity structure of square, rectangle, garden dish-type or other face shaping; Cover at plate body and plate can have inlet opening respectively, part or all of structure such as fluid hole, sealing-ring (7), peristaltic tube (8), micropore, microflute (10), microtubule (11), drive hole, pilot hole and form; Micropore has microflute therebetween or/and microtubule directly or indirectly links to each other in twos by certain format permutation, communicates with the external world or forms closed circuit by microfluidic circuit; 1. plate body and plate lid difference or common through different chemically modifieds or surface treatment produce reflector layer, reflected light signal; 2. obtain the inertia chemical coating, handle to reduce the non-specific adsorption to plate body and plate lid such as sample and/or marker as octylame, Tai Fulong (Teflon) coating, silication etc.

Feature of the present invention also is: but microballon is the material by any machine-shaping, as glass, plastics, dextran, ficoll, agarose, high molecular synthetic material, Mierocrystalline cellulose, latex, silica gel, chromatography substrate, pottery, magneticsubstance, metal or multiple differing materials or matrix material such as nonmetal, process from strand big or small homogeneous, porous or solid with different face shapings; Microballon can obtain to have the arm molecule of different activities group through surface treatment; Active group on the arm molecule can be further and connections such as different nucleic acid molecule or oligonucleotide molecules probe, obtains to have the surface molecular coating of different binding characteristics.

Feature of the present invention also is: microfluidic circuit is made up of structures such as inlet opening, fluid hole, microflute, microtubule, sealing-ring, peristaltic tubes, plate body can be laid respectively at or plate covers, also can all be positioned at plate body or plate covers, different positions and combination that it covers at plate body or plate, can form and have different geometric properties, satisfy the microfluidic circuit of different experiments demand; Microfluidic circuit is passed through microflute or/and microtubule links to each other with micropore, and communicates with the external world by inlet opening and fluid hole; Peristaltic tube advancing/fluid hole between, microfluidic circuit is connected into closed circuit, so that liquid phase can fully be reacted with solid phase; Peristaltic tube can be positioned at also can be external on the check-out console, and cellular microwell plate can not have microflute and microtubule, also or do not have/structures such as fluid hole, can add sample and rinsing by form during use; Cellular microwell plate can not have microflute and microtubule, or does not have into/microfluidic circuit structures such as fluid hole yet; Microfluidic circuit can obtain different inactive surfaces coatings through surface treatment.

Feature of the present invention also is: the surface molecular coating is meant by the active group on the arm molecule, nucleic acid molecule with different sequences and length of Lian Jieing or oligonucleotide molecules probe respectively, the nucleic acid molecule surface that forms on the solid-phase matrix surface of microballon and micropore with certain binding characteristic; The kind and the preparation method of molecular coatings comprise at least:

1. directly synthesize by the nucleic acid synthesizer, obtain the surface molecular coating of micromolecular oligonucleotide molecules probe on the solid-phase matrix surface of microballon;

2. by the active group on the arm molecule, connect or adsorb various not homotactic Nucleotide or oligonucleotide probe, form the surface molecular coating;

3. the oligonucleotide molecules probe with bead surface is an Auele Specific Primer, and known array nucleic acid is template, prolongs oligonucleotide chain and amplification by the PCR reaction in test tube or microwell plate, obtains the top coat of long-chain nucleic acid molecular probe.

Feature of the present invention also is: test sample and long-chain nucleic acid molecular probe, and available Oligonucleolide primers, the PCR reaction by at least one loop cycle is increased or is prolonged, amplification or prolong reaction and can also can carry out in test tube in microwell plate.What this loop cycle comprised a) sample oligonucleotide adds the Hot sex change; B) annealing of the target nucleotide in primer and template or sample hybridization; C) prolong the target nucleotide chain-ordering with archaeal dna polymerase.

Feature of the present invention also is: with fluorescence dye (as FITC, Cy3, Cy5, fluoresceins such as Texas Red), isotropic substance ( ¹²⁵I, ³²P, ³⁵S, ³H etc.), vitamin H and haptens, Radioactive colloidal gold, enzyme (HRP, AP, tilactase, glucose-6-phosphate dehydrogenase (G6PD), urea plum and luciferase etc.), rare earth ion (as Eu, Sm, Tb and Dy etc.) and inner complex thereof are (as Eu ³⁺-DTPA) wait or derivatives thereof (as Cy5-dCTP, ³²P-dCTP etc.) direct mark test sample; Or with the different marked products of tagged molecule, as fluorescence antibody, enzyme labelled antibody, biotinylated antibody, biotinylation enzyme, Eu ³⁺-DTPA-antibody etc. can with checking matter specificity bonded marker (16) the mark test sample (indirect labelling) in the sample; Labeled reactant can carry out in microwell plate, also can carry out in test tube; But only adopt when in microwell plate, carrying out mark and mix mark, and do not use marking methods such as coupling or modification.

8. feature of the present invention also is: can adopt following method to carry out quantitative analysis: the micropore quantity that 1. connects the micropore of identical sequence nucleic acid molecule or inlay microballon on same microwell plate can have the multiple hole more than 1 or 1, when sample pursues orifice flow through each micropore and multiple hole thereof along microfluidic circuit, corresponding checking matter is by constantly combination, and constantly from sample liquid, 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, even carry out pcr amplification, also, cause the amplification amount that obvious difference is arranged because of the difference of each micropore in conjunction with template number; Therefore can change according to each micropore and having or not of multiple hole reaction, carry out quantitative analysis with strong and weak; 2. when carrying out quantitative analysis, established standards object of reference according to said method can be drawn the concentration-reacting hole quantity or the copy number change curve strong and weak with it of marker, reference standard curve by Computer Analysis, in conjunction with the sample volume, obtain accurate quantitative analysis results.

9. feature of the present invention also is: test kit is equipped with a check-out console at least, inlays a microballon in the micropore at least, and bead surface connects the nucleic acid molecule of certain particular sequence respectively, and forms different test kits respectively or jointly with following compositions:

1. have one tubing/cell or microbiological specimens treatment solution at least, be used for DNA, RNA or proteinic extraction; Treatment solution can contain an amount of washing agent respectively (as Triton X-100, NP40, Chaps etc.), nucleic acid inhibitor (as DNA enzyme and RNA enzyme inhibitors), proteinase inhibitor, as PMSF, EDTA, TPCK, TLCK, aprotinin, leupeptin, antipain etc. to guarantee abundant cracking of sample and dissolving, guarantee that simultaneously DNA, RNA or protein molecular etc. are not degraded;

2. have at least a pipe to be dNTP (deoxy-ribonucleoside triphosphate), primer is used for the amplification of sample or the gene clone of the sample composition that microballon is caught with the thermostable DNA polymerases that is used for pcr amplification;

3. have at least a pipe to be dNTP and ThermoScript II, be used for the synthetic of the complementary cDNA of sample mRNA;

4. have at least a pipe contain tagged molecule and derivative thereof (as FITC, Cy3, Cy5, Texas Red, digoxin and vitamin H, Cy5-dCTP, ³²P-dCTP etc.) or marker (as fluorescence antibody, enzyme labelled antibody etc.) be used for direct or indirect mark to sample;

5. when marker is enzyme, have a kind of chromogenic substrate or luminous substrate at least, as OPD or DAB and hydrogen peroxide/urea, luminol,3-aminophthalic acid cyclic hydrazide, methyl umbelliferone phosphoric acid ester, ATP etc.;

6. have pipe washings or its concentrated solution at least, as contain an amount of BSA or calf serum, salmon sperm DNA, the damping fluid of skim-milk, washing agent etc., the rinsing or the stream that can be used for check-out console are washed, to remove the various non-specific binding things in the reaction;

7. have a pipe at least for protein concn check and analysis reagent, as the Folin-phenol reagent, Bradford reagent etc.;

8. have at least a pipe for endonuclease (as EcoRI etc.), proteolytic ferment (as trypsinase, Quimotrase, carboxypeptidase etc.), be used for microballon capture nucleic acid or proteic clearing up, so that carry out proteinic mass spectroscopy, Nucleotide or amino acid sequence analysis;

9. have a pipe at least for the electrophoresis sample buffer, as contain the SDS-PAGE electrophoresis sample buffer of different proteins denaturing agents such as an amount of SDS or urea or contain urea or the IEF electrophoresis sample buffer of Chaps etc. and nucleic acid electrophoresis sample buffer etc. are used for electrophoretic analysis;

10. have a pipe at least for Protein Glycosylation Overview or protein phosphorylation analytical reagent, as PNGase F, Phosphoric acid esterase (as bacterial alkaline phosphatase, potato Phosphoric acid esterase 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 spectroscopy, Nucleotide/determined amino acid sequence, protein phosphorylation and glycosylation by following diverse ways respectively to the variant hole of check-out console institute bonded sample composition:

1. directly in interested each micropore, add endonuclease, proteolytic ferment, Phosphoric acid esterase or reagent such as electrophoresis sample buffer or Protein Glycosylation Overview analysis, the binding substances of micropore and microballon is done gene clone in position or further analyzed preceding sample preparation;

2. the biological microballon in the micropore is taken out, in microscale reactor, add corresponding reagent respectively and do gene clone or further analyze preceding sample preparation;

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 corresponding reagent, binding substances is done gene clone or further analyzed preceding sample preparation.

The present invention has the following advantages: 1 contains much information.The big I of microballon is from the millimeter to the nanometer, generally between tens microns to hundreds of microns, as pressing 50 microns calculating, then every square millimeter can be held 100 pieces to the solid-phase matrix particle that present various merchant sells at least, every square centimeter can be held about 100,000 pieces, is equivalent to 2-3 times of human full gene.Therefore, the full gene probe if can be connected bead surface, be used for the research of genetic expression,, just can obtain the result and the data information of the parallel check and analysis of tens thousand of kinds of different expressing genes simultaneously through the one-time detection analysis.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, the substrate material Bio-Glas of using as the oligonucleotide solid phase synthesis, polystyrene bead, various chromatography substrate, silica-gel bead etc., the present invention combines the preparation of solid phase synthesis, separation and purification and biological microballons such as the mark of synthetic, the sample of the amplification of nucleic acid probe, PCR primer and amplification together, has omitted numerous and diverse bioseparation 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 aglucon/part, compares with traditional method, bigger specific surface area is arranged and compare binding capacity.Simultaneously owing to connect nucleic acid molecule by the chemical active radical on the arm molecule, eliminated sterically hindered influence during to molecular hybridization, further improved and compared binding capacity.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 nucleic acid probe hybridization or the specific combination (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; 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, marker, 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 operating method of reacting and embathing in bigger container, therefore can solve puzzlement because of sample is difficult for obtaining and marker 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 analysis is more accurate.Present method concentrates on the check and analysis of tens of kinds even thousands of kinds of chemical substances and biomolecules on the check-out console carries out simultaneously, according to fluorescence, Radioactive colloidal gold 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 connection of the surface molecular of microballon, amplification and mark can disposable a large amount of preparations in same test tube, thereby can guarantee the parallel unanimity of the detected result between each multiple hole and each check-out console.When needs carry out quantitative analysis to a certain composition,, cooperate microfluidic circuit to solve the problem of quantitative analysis 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. the result is easy to analyze.Because the shape and size of microballon are identical, closedly inlay the noise signal that three-dimensional arrangement can avoid dust to cause, thereby can obtain all image background intensity of even minimum, the arrangement of each micropore and microballon can be consistent with the arrangement of photosignal conversion element simultaneously, therefore can obtain best gene chip image.9. 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; And also can directly in check-out console, carry out gene clone, Nucleotide/amino acid variation and sequential analysis, the protein-bonded glycosylation of DNA, phosphorylation and molecular structure, function, avidity and tissue/cell and sub-micro or ultrastructural Location etc. to interested micropore and further analyze.

Description of drawings Fig. 1 is a check-out console basic structure synoptic diagram.

Shown in the figure microwell plate 1, microballon 2, plate body 3, plate lid 4, inlet opening 5, fluid hole 6, sealing-ring 7, peristaltic tube 8, micropore 9, microflute 10, wherein microballon 2 is embedded in the micropore 9; Microflute 10 between each micropore is micropore 9 and inlet opening 5, fluid hole 6, link to each other and and structure composition microfluidic circuit such as sealing-ring 7; Micropore 9 forms microfluidic circuit with microflute 10, inlet opening 5, fluid hole 6 and sealing-ring 7 among the figure, constitutes closed circuit with peristaltic tube 8 again.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 microwell plate 1, inlet opening 5, fluid hole 6 shown in the figure, drive hole 20, structures such as pilot hole 21 and form 22.Check-out console shown in Fig. 2 C also can be cellular, can not have microflute or microtubule between micropore, but keeps inlet opening 5, fluid hole 6; Perhaps do not have inlet opening 5, structures such as fluid hole 6 are directly added sample and rinsing through form 22 during use.Fig. 3 is the enlarged view of frame of broken lines A part among Fig. 2 A.

Fig. 3 A is an orthographic plan, is microwell plate 1, micropore 9, discontinuous microflute 10 shown in the figure, is embedded in the microballon 2 in the micropore 9, and the inlet opening 5 that communicates with microflute 10, sealing-ring 7;

Fig. 3 B is the A-A sectional view of Fig. 3 A, is plate body 3, plate lid 4, micropore 9, microflute 10, microtubule 11 shown in the figure, is embedded in the microballon 2 in the micropore 9, inlet opening 5, sealing-ring 7 etc.; The microflute 10 of micropore 9 upper ends forms microtubule 11 with plate lid 4 among the figure, and with the microtubule 11 of lower end micropore 9 is linked to each other in twos, forms complete microfluidic circuit with inlet opening 5, fluid hole 6 etc.Fig. 4 is the enlarged view of frame of broken lines B part among Fig. 2 A.

Fig. 4 A is an orthographic plan, is microwell plate 1, micropore 9, microflute 10 shown in the figure, is embedded in the microballon 2 in the micropore 9;

Fig. 4 B is the A-A sectional view of Fig. 4 A, is plate body 3, plate lid 4, micropore 9 shown in the figure, is embedded in the microballon 2 in the micropore 9; The microflute at micropore 9 two ends and plate lid forms microtubule 11 among the figure, and micropore 9 is linked to each other in twos, and with inlet opening 5, fluid hole 6 grades form complete microfluidic circuit.Fig. 5 is the partial enlarged drawing with check-out console of different microfluidic circuit.

Be plate body 3, micropore 9, microflute 10 shown in the figure, be embedded in the microballon 2 in the micropore 9; Microtubule 11 is arranged on the sidewall of two adjacent micropores among the figure, and the opening of its upper end communicates with microflute 10, and microflute 10 forms microtubules 11 with plate lid 4, microtubule 11 about openings at two ends micropore 9 is continuous in twos, and can with inlet opening, the microfluidic circuit that formation such as fluid hole is complete.Fig. 6 is the structural representation of microballon 2.

Fig. 6 A shows polyhedron microballon 2, connects oligonucleotide molecules probe 14 by the active group on the arm molecule 12;

Fig. 6 B is the sheet microballon made from cellulose membrane or microporous membrane 2, surface adsorption nucleic acid 13 molecular probes.

Fig. 6 C is the sectional view of polyhedron microballon 2, shows vesicular structure, and the surface of microballon 2 connects nucleic acid molecular probe 13 by the arm molecule 12 of band active group;

Fig. 6 D is the surface structure synoptic diagram of spheroid microballon 2, and the surface is the oligonucleotide 14 molecular probe primers on the arm molecule 12, obtains product 25 and chimeric molecule nucleic acid molecular probe 23 through PCR prolongation, amplified reaction.Fig. 7 solid phase PCR prepares long-chain nucleic acid molecular probe principle schematic.

The surface of microballon 2 is oligonucleotide molecules probes 14 that arm molecule 12 solid phase synthesis or that pass through the band active group connects among the figure.Template 27 hybridization of oligonucleotide 14 molecular probes and known array prolong under the catalysis of archaeal dna polymerase, obtain and template 27 base sequence complementary long-chain nucleic acid molecule 23 (chimeric molecule of amplified production 25 and oligonucleotide molecules 14); Or be 26 pairings of primer and Oligonucleolide primers with oligonucleotide molecules probe 14, by PCR reaction amplification, finally obtain the long-chain sub-thread nucleic acid molecular probe 23 or the bifilar nucleic acid molecular probe 28 of capacity in bead surface.The inserted gene chip nucleic acid molecular probe of Fig. 8 principle of work synoptic diagram.

Behind tagged molecule 15 marks, with nucleic acid molecular probe 13 hybridization on the microballon 2 surperficial arm molecules 12, Za Jiao sample nucleic acid molecule is not removed through rinsing in test tube for Fig. 8 A, sample nucleic acid molecule 24, and the certification mark thing obtains analytical results.

Fig. 8 B, nucleic acid molecule 24 in the sample, 24 ' 24 " etc. in test tube behind tagged molecule 15 marks; respectively be embedded in different micropores in microballon 2; 2 ' 2 " etc. the nucleic acid molecular probe 13 on the surperficial arm molecule 12, hybridization such as 13 ' 13 ", Za Jiao nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains the high throughput analysis result.

Fig. 8 C, nucleic acid molecule 24 usefulness 5 ' end in test tube in the sample has the primer of tagged molecule 15, behind at least one round-robin pcr amplification, amplified production 25 is hybridized with the nucleic acid molecular probe 13 on the microballon 2 surperficial arm molecules 12, Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains analytical results.

Fig. 8 D, nucleic acid molecule 24 in the sample, 24 ' 24 " primer that etc. in test tube, has tagged molecule 15; behind at least one round-robin pcr amplification, amplified production 25,25 ' 25 with 5 ' end " etc. be embedded in different micropores in microballon 2, nucleic acid molecular probe 13 on the surperficial arm molecule 12 such as 2 ' 2 "; 13 ' 13 " etc. hybridization, Za Jiao sample nucleic acid molecule is not removed through rinsing, the certification mark thing obtains the high throughput analysis result.Fig. 9 is inserted gene chip oligonucleotide molecules probe principle of work synoptic diagram.

Behind tagged molecule 15 marks, with oligonucleotide molecules probe 14 hybridization on the microballon 2 surperficial arm molecules 12, Za Jiao sample nucleic acid molecule 24 is not removed through rinsing in test tube for Fig. 9 A, the nucleic acid molecule 24 in the sample, and the certification mark thing obtains analytical results.

Fig. 9 B, nucleic acid molecule 24 in the sample, 24 ' 24 " etc. in test tube behind tagged molecule 15 marks; be embedded in different micropores in microballon 2; 2 ' 2 " etc. the different IPs acid molecule 14 on the surperficial arm molecule 12, hybridization such as 14 ' 14 ", Za Jiao sample nucleic acid molecule can not removed through rinsing, and the certification mark thing obtains the high throughput analysis result.

Fig. 9 C, in test tube, nucleic acid molecule 24 usefulness 5 ' end in the sample has the primer of tagged molecule 15, behind at least one round-robin pcr amplification, amplified production 25 is hybridized with the oligonucleotide molecules probe 14 on the microballon 2 surperficial arm molecules 12, Za Jiao sample nucleic acid molecule can not removed through rinsing, and the certification mark thing obtains analytical results.

Fig. 9 D, in test tube, nucleic acid molecule 24 in the sample, 24 ' 24 " etc. usefulness 5 ' end has the primer of tagged molecule 15, behind at least one round-robin pcr amplification; amplified production 25; 25 ' 25 " wait and 2,2 ' 2 " etc. the oligonucleotide molecules probe 14 on the bead surface arm molecule 12,14 ' 14 " etc. hybridization, Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains the high throughput analysis result.

Fig. 9 E, nucleic acid molecule 24 in the sample and 14 hybridization of the oligonucleotide molecules probe on microballon 2 surfaces; With the nucleic acid molecule in the sample is template, the oligonucleotide 26 that the oligonucleotide molecules probe 14 on microballon 2 surfaces and 5 ' end have tagged molecule 15 is primer, behind at least one round-robin pcr amplification, oligonucleotide molecules probe 14 on amplified production 25 and the microballon 2 surperficial arm molecules 12 or Oligonucleolide primers 26 form chimeric molecules, Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains analytical results.

Nucleic acid molecule 24 in Fig. 9 F sample, 24 ' 24 " etc. with check-out console in microballon 2; 2 ' 2 " wait surperficial oligonucleotide molecules probe 14, hybridization such as 14 ' 14 "; with the nucleic acid molecule in the sample is template; with the oligonucleotide molecules probe 14 on microballon 2 surfaces; 14 ' 14 " and 5 ' end have the oligonucleotide 26 of tagged molecule 15, or/and 26 ' 26 " be primer etc.; behind at least one round-robin pcr amplification; amplified production 25; 25 ' 25 " etc. with the few nucleic acid molecular probe 14 on microballon 2 surfaces, 14 ' 14 " etc., oligonucleotide 26 is or/and 26 ' 26 " wait the formation chimeric molecule, Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains the high throughput analysis result.Figure 10 solid phase pcr amplification long-chain nucleic acid molecular probe principle of work synoptic diagram

Figure 10 A and Figure 10 B are at test tube or in check-out console, nucleic acid molecule 27 with known array is a template, the oligonucleotide molecules probe 14 on microballon 2 surfaces and oligonucleotide 26 are primer, after at least one round-robin PCR prolongs, increases, the oligonucleotide molecules probe 14 or the oligonucleotide 26 on amplified production and microballon 2 surfaces are chimeric, strand chimeric molecule 23 that forms or double-stranded chimeric molecule 28 are fixed on the surface of microballon 2; Nucleic acid molecule 24 in the sample is through mark, or with 5 ' end have the oligonucleotide molecules primer of tagged molecule 15 through at least one round-robin pcr amplification, product 25 is hybridized with the chimeric molecule 23 or 28 on microballon 2 surfaces, Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains analytical results.

Figure 10 C and Figure 10 D are at test tube or in check-out console, with N known not homotactic nucleic acid molecule 27,27 ' 27 " be template etc.; the different oligonucleotide molecules probes 14 of bead surface; 14 ' 14 " and oligonucleotide 26 or 26 ' 26 " etc. be primer; through at least one round-robin PCR prolong, amplification; the oligonucleotide molecules probe 14 on the amplified production of acquisition and microballon 2 surfaces; 14 ' 14 " or oligonucleotide 26 or 26 ' 26 " chimeric, the chimeric molecule 28 etc. that forms different strand chimeric molecules 23 or two strands is fixed on the surface of microballon; Different nucleic acid molecule 24 in the sample, 24 ' 24 " etc. through mark; or have the product 25 of the oligonucleotide molecules primer of tagged molecule 15; 25 ' 25 through at least one round-robin pcr amplification with 5 ' end " with microballon 2,2 ' 2 " Biao Mian different chimeric molecules 23 or 28 hybridization; Za Jiao sample nucleic acid molecule is not removed through rinsing, and the certification mark thing obtains the high throughput analysis result.Figure 11 enzyme labelling law technology principle schematic

Figure 11 A, nucleic acid molecule 24 in the sample is behind biotin molecule (30) mark, with nucleic acid molecular probe 13 hybridization on the microballon 2 surperficial arm molecules 12, Za Jiao sample nucleic acid molecule can not removed through rinsing, the affinity element or the streptavidin (31) that add enzyme (18) mark combine with vitamin H (30), and unconjugated enzyme labelling thing is removed through rinsing; Under the catalysis of enzyme (18), the substrate of adding (19) generates coloured product or luminous product (32), detects the depth of color that has that it's too late of coloured product in each hole or luminous product or luminous power, obtains analytical results.

Figure 11 B, nucleic acid molecule 24 in the sample is behind biotin molecule (30) mark, nucleic acid molecular probe 13 hybridization with microballon 2 surfaces, Za Jiao sample nucleic acid molecule can not removed through rinsing, the anti-biotin antibodies (33) that adds enzyme (18) mark combines with vitamin H (30), and unconjugated enzymic-labelled antibody is removed through rinsing; Under the catalysis of enzyme (18), substrate (19) generates coloured product or luminous product (32), detects the depth of color that has that it's too late of coloured product in each hole or luminous product or luminous power, obtains analytical results.Figure 12 nucleic acid-protein interaction check and analysis principle

Figure 12 A, the protein molecular 34 in the sample combine with the nucleic acid molecular probe 13 or the oligonucleotide 14 on microballon 2 surfaces behind fluorescein molecule (15) mark, and unconjugated sample protein molecule is removed through rinsing, detect fluorescence to have that it's too late strong and weak, obtain analytical results.

Figure 12 B, protein molecular 34 in the sample is behind biotin molecule (30) mark, combine with the nucleic acid molecular probe 13 or 14 on microballon 2 surfaces, unconjugated sample protein molecule is removed through rinsing, the anti-biotin antibodies (33) that adds enzyme (18) mark combines with vitamin H (30), and unconjugated enzymic-labelled antibody is removed through rinsing; Under the catalysis of enzyme (18), substrate (19) generates coloured product or luminous product (32), detects the depth of color that has that it's too late of coloured product in each hole or luminous product or luminous power, obtains analytical results.

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 place of material Reason method and condition have multiple, the present invention this with " biomolecule mobilization application " (Jiang Zhonghua etc., 1998 years, Chemistry publishing house) and China Patent No. be 01207812.3 and application number be that 00120798.9 patent is the main reference document. Use known various large biological molecule immobilization technology, process microballon and the microwell plate of various materials, make its surface with the spy Fixed active group with the active group reaction on the nucleic acid molecules, and has specific adsorption capacity. As with 2% the third amino-Triethyl silicane anhydrous propanone solution (APES, 3-Aminopropyltriethoxysilane, Sigma company, catalog number (Cat.No.) Porous glass beads (Weetall, H.H., Biochem.Biophys.Acta, 1970 of A3648) processing; 212:1). Or discuss the surface treated various chromatography substrates (such as the Sepharose-4B of cyanogen bromide-activated) of selling, the oligonucleotides solid phase is synthetic With polystyrene bead or control hole bead (Applied biosystems, Sigma company) etc.

Microwell plate also can be done different surface treatments according to requirement of experiment. As carry out silicidation or Tai Fulong (Teflon) is coated with Layer is processed, as just reaction vessel or microfluidic circuit, to reduce the non-specific adsorption in the experiment. 2. solid phase coating: different nucleic acid molecules is connected with the active group on microballon or microwell plate surface respectively, or at microballon or little It is synthetic to obtain the face coat of different IPs acid molecule that orifice surface carries out the original position solid phase. The preparation method of face coat mainly is 1. modify and connect. Utilize the active group on the solid phase surface arm molecule that nucleic acid molecules or oligonucleotide molecules are passed through chemical bond Be connected the solid-phase matrix surface; 2. solid phase is synthetic. Use the solid state chemistry synthetic technology, directly carry out few nucleosides in bead surface The solid phase of acid molecule synthetic (Edge R.Wang etc., 1998, synthetic, the mark of nucleic acid probe and application, Science Press); Synthetic reaction can be in test tube or is filled in the special-purpose cylinder of synthesizer and carry out, and also can carry out in microwell plate; 3. PCR Solid-phase amplification. Take the nucleic acid molecule of known array as template, a primer in its paired primer is connected on the microballon, Or carrying out pcr amplification take the synthetic oligonucleotide molecules of solid phase as primer, amplified production connects by immobilised primer and is solid Change mutually on the surface of microballon or micropore, can in different test tubes, carry out simultaneously respectively different or identical template during amplification and expand Increase, also can in microwell plate, carry out simultaneously different or identical template amplification; 3. inlay microballon: artificial or use the full-automatic mechanical hand system, embed respectively in certain sequence the microballon of solid phase coating processing little In each micropore of orifice plate, the microballon in each micropore has different binding characteristics; In cellular microwell plate, microballon also can Has identical binding characteristic, in order to different sample specimen is carried out identical detection analysis simultaneously. 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 the work that the surfaces such as microballon, microwell plate or microfluidic circuit may be residual with the sealing buffer solution with sealing The property group or nonspecific binding site, to reduce the non-specific binding that may occur in detecting. Namely obtain thus can with change Credit, biomolecule, antibody and/or antigen, the specific bindings such as enzyme, nucleic acid or oligonucleotide molecules, and can be used for right The 3 D stereo braided type high flux gene chip that bond detects. Confining liquid additive commonly used has: salmon essence or yeast DNA ficoll, heparin, polyvinylpyrrolidone, bovine serum albumin(BSA) (0.5-5%), skimmed milk power (0.1-6%), junket Albumen (0.5-2%), gelatin (0.1-0.5%), Normal animal serum (0.5-5%) and detergent etc. (such as Tween 20, NP40, Triton X-100, SDS) etc. But cellular check-out console does not have microtubule, microflute or does not have into/fluid hole yet, need capping before Sealing is taken the epiphragma of seal off or is removed plate and covers and can use during use, rinse method adopts and embathes. Two. the detection analysis of the detection analytic sample of sample generally comprises following basic step and method: 1. the preparation of sample: according to the difference of testing goal, biological sample will carry out respectively different processing before detection. Common sample The product processing method comprises: 1. nucleic acid extractive: process various biological samples, biological fluid or mark with DNA or RNA extract This is with the DNA in the extraction sample or RNA etc.; 2. the biotic component extract such as albumen: cooperate homogenate with histiocyte lysate capable Or the processing such as Ultrasonic Pulverization makes the histocyte cracking obtain true property protein solution or biological sample, biological fluid or biological sample etc. Separating extractive; 3. artificial synthetic product: use prodrug that various artificial synthetic technologys obtain, lead compound with Reach the synthetic product of Peptide synthesizer, nucleic acid synthesizer etc. etc.; 4. cDNA is synthesized in reverse transcription; 5. PCR or RT-PCR amplification. 2. the mark of sample: 1. direct mark. Use known biomolecular labeling technology, use respectively biotin, enzyme, fluorescein, The labelled molecule such as material or derivatives thereof such as collaurum, isotope or rare earth ion and chelating agent thereof are as the direct mark of indicator Sample or biological sample; 2. adopt indirect labelling. Use the interaction of biomolecule, the marked product of usefulness labelled molecule (as Fluorescence antibody, the labels such as biotin-HRP) the mark sample; 3. mix mark, use various synthetic technologys, close such as solid phase One-tenth technology, pcr amplification etc. mix labelled molecule or label and are labeled in the molecule. 3. add sample to be checked: 1. with sample or process sample and add in the check-out console through inlet opening, make it with each hole in bead surface Hybridization or the combinations such as immobilised different IPs acid molecule or oligonucleotide molecules. In the test sample with complementary or corresponding biology branch Son is by solid-phase capture or specific bond, and remaining and unconjugated part flows out check-out console through fluid hole. 2. with antisense oligonucleotide primer Thing, dNTP, the nucleic acid samples that can mix label and hot resistant DNA polymerase etc. and trace adds in the microwell plate, carries out at least The PCR of a circulation or RT-PCR amplified reaction. 4. rinsing: by the microfluidic circuit of check-out console, wash with washing lotion stream, cellular check-out console can through advance/the fluid orifice flow washes or passes through form (22) embathe, to remove test sample residual in the check-out console (not bond or title educt) or label etc. 5. interpretation of result: according to the difference of labelled molecule, range estimation or with the observation of use instrument such as scanner and record having of each hole reaction and be difficult And strong and weak (power such as the depth that generates the product color, collaurum deposition, fluorescence intensity, activity etc. changes). Contrast The arrangement position of microballon, computer analysis namely can determine in this sample, the containing of the composition of detected material and each component Amount, nucleotide sequence, the nucleotide sequence of protein combination or identification. But when label is protease, must increase in the operation The operating procedures such as interpolation substrate could be observed testing result, and its testing result is color reaction or luminescence-producing reaction.

The present invention in force, the aforesaid operations step is not according to solid phase probe, detected material, label and demonstration result etc. no With, multiple different compound mode can be arranged. Various biochemical reactions can carry out in microwell plate, also can be partly or entirely at test tube In carry out. When carrying out in microwell plate, the bead surface molecular coatings of inlaying in each micropore can identical (cellular microwell plate), For detection of the identical component in the different samples; Also can be different, be used for the heterogeneity of same sample is carried out high flux The detection analysis.

The present invention is described further below in conjunction with embodiment.Example I: the application of braided type high flux gene chip in prevention congenital malformation and intrauterine infection.

Rubella virus, cytomegalovirus, hsv, Coxsackie virus, toxoplasma are the main pathogens that causes intrauterine infection and cause fetal anomaly; Hepatitis B, syphilis and acquired immune deficiency syndrome (AIDS) etc. are the pathogenic agent 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. the preparation of microballon:

1. the preparation of oligonucleotide probe microballon: the pcr amplification primer of pathogenic micro-organism specific nucleic acid molecular probe is connected to by solid phase synthesis on the arm molecule of microballon, obtains to have the oligonucleotide molecules top coat of different complementary sequences.

2. the preparation of nucleic acid probe microballon: the specific nucleic acid molecular probe of above-mentioned pathogenic micro-organism is connected on the arm molecule of microballon, obtains to have the molecular surface coating of different complementary sequences.

3. pcr amplification: the oligonucleotide molecules with solid phase synthesis on the microballon arm molecule is a primer, and pathogenic micro-organism specific nucleic acid molecular probe is a template, by pcr amplification reaction, obtains to have the nucleic acid molecular probe top coat of different complementary sequences.2. the preparation of check-out console:

From the feed liquor nose end, the microballon of above-mentioned sealing treatment is embedded in the micropore of microwell plate in regular turn, add a cover shrouding.Add prehybridization solution sealing nonspecific binding site through inlet opening.3. test sample:

Person's serum to be checked adds the nucleic acid extract, extracts pathogen nucleic acid, is primer with many oligonucleotide molecules that end is had a pathogenic micro-organism particular sequence of tagged molecule; 1. nucleic acid extractive adds in the check-out console through inlet opening, with the immobilised oligonucleotide molecules hybridization of bead surface; Nucleic acid extractive carries out pcr amplification and mixes mark as template in check-out console; 2. nucleic acid extractive carries out pcr amplification and mixes mark as template in test tube, removes residual primer, dNTP etc., adds in the check-out console, with the immobilised making nucleic acid molecular hybridization of bead surface through inlet opening; The stream flush away removes the impurity of unconjugated and non-specific binding etc.4. observations:

Check-out console is put fluorescent microscope or scanner analytical results.Positive hole represents that infection is arranged, and is in carrier state, is infectious, unsuitable gestation.Example II: the application of braided type high flux gene chip in the research of cell differential expression.1. the preparation of microballon:

1. prepare during the oligonucleotide probe microballon: use the nucleic acid solid phase synthetic instrument, add 3-12 of its back mode of base N at random with the mRNA initiation codon, at the synthetic cover pcr amplification primer of bead surface.By this synthesis mode,, just total mRNA of amplification can be divided into 4 according to the added base length in initiation codon back ⁿIndividual group can have 4 if any the oligonucleotide of 8 bases ⁵Individual not homotactic oligonucleotide molecules, and can obtain 4 ⁵Plant the microballon of oligonucleotide molecules top coat with different complementary sequences.2. the preparation of check-out console:

From the feed liquor nose end, with above-mentioned 4 ⁿPlant synthetic microballon microballon and embed in regular turn in the micropore of microwell plate, add a cover shrouding.Add prehybridization solution sealing nonspecific binding site through inlet opening, just can obtain the oligonucleotide gene chip check-out console of known initiation codon end parts base sequence.3. test sample:

Sample to be checked adds the nucleic acid extract, extracts mRNA.Adding the individual base N at random of 1-8 with oligo dT back is primer, the N of base at random on each first behind oligo dT can not have the T, all can be respectively A, T, G, the coordination of four kinds of different bases of C is arranged, as add with 12 thymidine back 2 at random the oligonucleotide molecules of base be second primer, oligo dT back the 1st and 2 s' NN base is arranged as:

dT-AA，dT-AC，dT-AG，dT-AT

dT-CA，dT-CC，dT-CG，dT-CT

dT-GA，dT-GC，dT-GG，dT-GT

Can obtain 12 kinds of different wildcard primers of arranging thus.With the terminal fluorescently-labeled oligodT primer that has that obtains in this way the mRNA that extracts is carried out reverse transcription, and then be template with resulting cDNA, oligonucleotide molecules with same oligo dT and bead surface is a primer, add in the check-out console and carry out pcr amplification, for guaranteeing that the stream flush away removes residual primer, dNTP, the impurity of unconjugated and non-specific binding etc.4. observation analysis result:

Check-out console is put having or not of fluorescent microscope or each hole fluorescence of scanner check and analysis and power, and makes comparisons with the sample of different sources, the gene that just can find differences and express.5. clone and order-checking:

The position of contrast difference expressing gene in check-out console can detect the nucleic acid base sequence of initiation codon end, obtains the gene clone of differential expression by pcr amplification, and can further check order.EXAMPLE III: the application of braided type high flux gene chip in nucleic acid-protein interaction research.

Screen bioactive moleculess such as nucleic acid binding protein, cell growth factor, the little peptide of hormone is used widely at aspects such as preclinical medicine, clinical diagnosis and drug screenings with oligonucleotide.Use biochip technology of the present invention, to control solid phase synthesis dedicated substrates such as hole glass or polystyrene for inlaying microballon, it is enough big that application solid phase oligonucleotide synthetic technology is easy to make up storage capacity, the single stranded oligonucleotide library that sequence is determined, and the absolute quantity of each bead surface institute this kind of bonded oligonucleotide molecules is much larger than light-operated mark location parallel synthesis technique.1. the preparation of microballon:

The single stranded oligonucleotide molecule that on the solid phase synthesis dedicated substrate, synthesizes particular sequence on demand, the preparation of setting up combinatorial chemistry oligonucleotide microballon storehouse 2. check-out consoles:

From the feed liquor nose end, the oligonucleotide microballon is embedded in regular turn in the micropore of high-density micropore check-out console, add a cover shrouding, add confining liquid through inlet opening, obtain single stranded oligonucleotide library gene chip check-out console.3. test sample:

Tissue, cell, bacterium, virus or phage etc. or the albumen extract of handling through cracking, after carrying out mark with direct labeling technique or indirect labelling technology, get in right amount and slowly add in the check-out console through inlet opening, hatch, stream is washed to remove impurity residual or non-specific binding.4. observations:

The scanner analytical results, 1. relatively various flows is washed the influence to each positive hole bonding strength and binding capacity of condition and competition binding substances or antagonist, and provides the base sequence of each hole oligonucleotide; 2. different sources or relatively through the protein bound spectrums of tissue, cell or the bacterium etc. of different treatment, analyze difference bands of a spectrum and structure, function, gene regulating or with the relation of disease etc.5. mass spectroscopy:

To interested reacting hole in analyzing, take out corresponding microballon and put in the micro-reaction test tube, add the desorb attached liquid, get then and carry out mass spectroscopy in right amount to measure proteinic molecular weight; Or in former reacting hole, add the trypsin 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 spectroscopy, 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 spectroscopy in right amount; The microballon taking-up that maybe will answer in the hole is put in the micro-reaction test tube, add Phosphoric acid esterase, question response thoroughly back adds the desorb attached liquid, gets then and carries out mass spectroscopy in right amount, contrast has or not the possible position of phosphorylation and phosphorylation without the microballon analytical results of Phosphoric acid esterase processing with mensuration and analysing protein.7. Protein Glycosylation Overview 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 analytical results of step 4, with corresponding microballon reaction, fully after the rinsing, be sub-packed in the different microtest tubes, add special-purpose isoelectrofocusing and SDS-PAGE electrophoresis sample buffer respectively, take out an amount of electrophoresis respectively, to measure iso-electric point and molecular weight.9. gene clone

With this oligonucleotide molecules is Auele Specific Primer, uses round pcr and carries out the amplification of upstream and downstream nucleic acid fragment, with clone's total length or long-chain nucleic acid molecule fragment.

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, Quantitatively accurate, highly sensitive, low cost of manufacture, purposes extensively wait outstanding advantages, can be widely used in biology, medical science, medicine Learn the fields such as environmental science.

Claims (10)

1. But 1. the braided type high flux gene chip detecting technique of nucleic acid, albumen and the chemical molecular in the test sample and the preparation method of test kit, this technology and test kit comprise following compositions or step at least:

   1. a kind of check-out console is made up of microwell plate and microballon; Microwell plate is made up of structures such as plate body, Ban Gai, micropore and microfluidic circuit; Microballon connects similar and different nucleic acid by the active group on the arm molecule or the oligonucleotide molecules probe forms the surface molecular coating, and is embedded in sequence in the different micropores;

   2. sample is through handling or not treatedly directly enter microwell plate by inlet opening, the various nucleic acid molecule interactions that are connected with bead surface when flowing through micropore, and tested molecule combines with solid phase molecular hybridization or specificity; Unconjugated sample composition flows out through fluid hole;

   3. mark test sample;

   4. rinsing;

   5. range estimation or the having or not of tagged molecule in each micropore, power or content are detected and analyzes with scanner etc.;

   When 6. marker is enzyme, need to add substrate before the observations.
2. 2. microwell plate according to claim 1 is made up of structures such as plate body, Ban Gai, micropore and microfluidic circuit; But plate body and plate lid is any machine-shaping, printing opacity, lighttight or reflective hard or soft material, can be processed into square, rectangle, garden dish-type or other face shaping; Cover at plate body and plate and can have partly or entirely structure such as micropore, microfluidic circuit, drive hole, pilot hole and form respectively; Micropore is by certain format permutation, communicates with the external world or forms closed circuit by microfluidic circuit.
3. 3. but microballon according to claim 1 is by the material of any machine-shaping, processes from strand, big or small homogeneous, the porous with different face shapings or solid; Microballon can obtain to have the arm molecule of different activities group through surface treatment; Active group on the arm molecule can be further and connections such as different nucleic acid or oligonucleotide molecules probe, obtains to have the surface molecular coating of different binding characteristics.
4. 4. microfluidic circuit according to claim 1, can be laid respectively at plate body or plate to cover or/and structures such as microtubule, sealing-ring, peristaltic tube are formed by inlet opening, fluid hole, microflute, also can all be positioned at plate body or plate covers; Microfluidic circuit is passed through microflute or/and microtubule links to each other with micropore, and communicates with the external world by inlet opening and fluid hole; Peristaltic tube connects into closed circuit with microfluidic circuit between inlet opening and fluid hole; Peristaltic tube can be positioned at also can be external on the check-out console; Cellular microwell plate can not have microflute and microtubule, or does not have into/microfluidic circuit structures such as fluid hole yet; Microfluidic circuit can obtain different inactive surfaces coatings through surface treatment.
5. 5. according to claim 1 and the described surface molecular coating of claim 3, be meant by the active group on the arm molecule, nucleic acid molecule with different sequences and length of Lian Jieing or oligonucleotide molecules probe respectively, the surface molecular that forms on the solid-phase matrix surface of microballon and micropore with certain binding characteristic; The kind and the preparation method of molecular coatings comprise at least:

   1. directly synthetic on the solid-phase matrix surface of microballon by dna synthesizer, acquisition connects the surface molecular coating of oligonucleotide molecules probe;

   2. by the active group on the arm molecule, connect various not homotactic Nucleotide or oligonucleotide molecules probe;

   3. the oligonucleotide molecules probe with bead surface is an Auele Specific Primer, known array nucleic acid is template, in test tube or microwell plate, prolong oligonucleotide chain and further amplification, obtain long-chain nucleic acid molecular probe surface molecular coating by polymerase chain reaction (PCR).
6. 6. according to claim 1 and described sample of claim 5 and long-chain nucleic acid molecular probe, available Oligonucleolide primers, the PCR reaction by at least one loop cycle prolongs or increases; Prolong or amplified reaction can also can carry out in test tube in microwell plate, primer all dissolves in the liquid phase, or is connected solid-phase matrix surface such as microballon by arm molecule; The PCP reaction cycle cycle comprises a) thermally denature; B) annealing, the target nucleotide hybridization in primer and template or the sample; C) prolong the target nucleotide chain-ordering with archaeal dna polymerase.
7. 7. mark according to claim 1 is meant, with direct mark samples of tagged molecule or derivatives thereof such as fluorescence dye, enzyme, vitamin H, haptens, Radioactive colloidal gold, isotropic substance, rare earth ion and sequestrants thereof; Or with can with checking matter specificity bonded marker mark sample in the sample; Labeled reactant can carry out in test tube, also can carry out in microwell plate.
8. 8. detection method according to claim 1, can adopt following method to carry out quantitative analysis: the micropore quantity that connects the micropore of identical sequence nucleic acid molecule or inlay microballon on same microwell plate can have the multiple hole more than 1 or 1; When sample by orifice flow during through each micropore and multiple hole thereof, corresponding detected material is by constantly combination, and constantly removes from specimen fluids, 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 analysis; When carrying out quantitative analysis, according to said method the established standards object of reference is drawn standard reference substrate concentration-reacting hole quantity and strong and weak change curve thereof by Computer Analysis, and the reference standard curve obtains quantitative analysis results in conjunction with the sample volume.
9. 9. test kit according to claim 1 is equipped with a check-out console at least, inlays a microballon in the micropore at least, and bead surface connects the nucleic acid molecule of certain particular sequence respectively, and forms different test kits respectively or jointly with following compositions:

   1. have at least a pipe to be sample preparation liquid;

   2. having a pipe at least is dNTP, primer and the thermostable DNA polymerases that is used for pcr amplification;

   3. have at least a pipe to be dNTP and ThermoScript II;

   4. has a pipe at least for containing the labelled reagent of tagged molecule or marker;

   5. when marker is enzyme, have a kind of chromogenic substrate or luminous substrate at least;

   6. have at least a pipe to be washings or its concentrated solution;

   7. have at least a pipe to be protein concn check and analysis reagent;

   8. have at least a pipe to be endonuclease or proteolytic ferment;

   9. have at least a pipe to be the electrophoresis sample buffer;

   10. have at least a pipe to be Protein Glycosylation Overview or protein phosphorylation analytical reagent;
10. 10. according to claim 1 and described detection method of claim 9 and test kit, can do gene clone, mass spectroscopy, sequencing, protein phosphorylation, glycosylation or electrophoresis etc. by following diverse ways respectively to the variant hole of microwell plate institute bonded sample composition with the reagent in the test kit and further analyze preceding processing:

    (1) directly in interested micropore, adds PCR reagent or endonuclease, proteolytic ferment, Phosphoric acid esterase or electrophoresis sample buffer, Protein Glycosylation Overview etc. and analyze reagent, the sample preparation before binding substances is further analyzed in position;

    (2) microballon in the micropore is taken out, in microscale reactor, add the sample preparation before PCR reagent or endonuclease, proteolytic ferment, Phosphoric acid esterase or reagent such as electrophoresis sample buffer or Protein Glycosylation Overview analysis are further analyzed;

    (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 PCR reagent, endonuclease or proteolytic ferment, Phosphoric acid esterase or reagent such as electrophoresis sample buffer or Protein Glycosylation Overview analysis, the sample preparation before binding substances is further analyzed.

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