CN1472339A - High-flux cell biological chip testing technology and reagent case - Google Patents

Abstract

A high-throughput biochip detection technique and its reagent kit feature that the microcarrier with different cells or microbes are respectively inserted in different microholes which are communicated to each other and with inlet/outlet to form microchannel, hydrization between specimen and said cells or microbes, and analyzing result.

Description

High-flux cell biological chip testing technology and test kit

The present invention relates to a kind of novel braided type high flux cell chip detection technique and the preparation method of test kit.This technology rapid sensitive, contain much information, the preparation method is easy, cost is low, multiple different albumen, nucleic acid molecule, biology, chemical molecular, medicine and microorganism or cell etc. in the test sample can be widely used in biological and fields such as 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 in the analyzing and testing of biology, chemical molecular and medicines etc. such as the albumen of water quality, water source, biomaterial, biological fluid (as blood, serum, blood plasma, cerebrospinal fluid, urine, tear, sweat, Digestive system, seminal fluid, juice, tissue juice, transudate, vomitus, ight soil), tissue/cell and microbial lytic liquid, different sources, nucleic acid, separate and the aspect such as synthetic of purifying and oligonucleotide, polypeptide, lead compound and medicine is widely used.

Biological immobilization technology 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.

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 parallel qualitative, quantitative, positioning analysis are made in the having or not of multiple heterogeneity in the sample, content and source etc.; 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, more can't simultaneously or synthesize continuously, separate and detect.

Microbies such as cell, bacterium, fungi, mycoplasma, Rickettsiae and virus carry a large amount of, relevant with its biological characteristics bioinformations by biomolecules such as nucleic acid, protein-polysaccharide, enzymes; The fungi of genetic engineering bacterium, biotechnology cell, transform bacteria, transfection and infection and animal and plant cells and recombinant plasmid or virus etc. are except that having the gene and biomolecules relevant with self biological characteristics; also can have and insert the special biomolecules that gene, recombination, infection or transfection virus etc. are given, therefore be widely used in the fields such as structure of biomedical detection and analysis and molecular library.

Analysis and research and fields such as biomedical diagnostic and new drug research in modern genomics, proteomics, functional group, need simultaneously to a large amount of in the same sample, multiple different biochemicals carry out fast, the separation of sensitive high-throughput, purifying and detection, with the interaction between kind, quantity, composition, variation and the polymorphism of analyzing biology, chemical goods and materials, posttranslational modification, structure, sequence, different biological molecules, or individual medication characteristic and Infection Status, the functional status of individual immunity system, hereditary difference etc.The tradition terms of settlement is at first by technical matters such as separation, extraction, purifying or synthetic, prepare a large amount of various bioprobe (as the biomolecules of various different qualities such as nucleic acid or oligonucleotide molecules, albumen and polypeptide, somatomedin and acceptor, antigen and antibody) respectively, by technology such as microarraies bioprobe is distinguished the surface of immobilization at solid-phase matrix (as slide glass, microwell plate or microcarrier etc.) again, again with sample in corresponding chemical substance, nucleic acid, antigen/antibody reaction, analyzing and testing.Since synthetic unrelated with each links such as preparation, separation and purification and check and analysis, therefore just need carry out tens different separation and purification, the operational anomaly very complicated as detecting tens kinds of compositions.Particularly in the mass preparation of monoclonal antibody, genetic engineering antibody and vaccine etc., need carry out high-throughput, extensive screening to a large amount of various display libraries such as cDNA library, antibody library and phage, bacterium and yeast; And existing technological method can only be cultivated one by one by methods such as immunocytochemistries, fixes, check and analysis, and operational anomaly complexity, loaded down with trivial details can't realize industrialization.Purpose of the present invention will be used a preface coding techniques and suspension culture and solid phase isolation technique exactly, provide a cover low cost of manufacture in the hope of isolation and purification, detection and screening etc. for multiple different substances composition, particularly nucleic acid, protein molecular and medicine and precursor thereof etc., be convenient to industrialization, and the more easy sensitivity of technology and technology, quick and precisely; The two can be organically combined again simultaneously, connect together new high flux screening and check and analysis technology.

Major technique feature of the present invention is, with bacterium, microbe such as virus or cell obtains different biological microspheres attached to the surface of microcarrier, after processing such as fixing, 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, sample is by microfluidic circuit and biological microsphere surface virus, biomolecular reaction in the microbe such as bacterium or cell, the reaction result in each hole can be analyzed with range estimation or with instrument record, according to arrangement position and the order (position preface coding) of biological microsphere in check-out console, to the chemical substance in sample or the biological sample, bio-target molecule, nucleic acid, oligonucleotide, having or not of multiple heterogeneity such as medicine, character, content, the source, tissue and cytology distribute, location etc. is carried out check and analysis simultaneously.

This invention also provides a kind of preparation method of the test kit based on the braided type high flux cell biological chip testing technology simultaneously, its major technique is characterised in that: test kit is by being inlaid with biological microsphere, 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, multiple heterogeneity in the different samples is compared simultaneously, the sample composition that reagent that utilizes in the test kit to be provided and method also can be caught each hole in sample and the check-out console is done analyses such as molecular cloning, molecular structure, avidity, molecular weight, iso-electric point, sequencing.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, accuracy, sensitivity, circulation ratio and the comparability of detected 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, can be widely used in technology, research fields such as environment measuring, medical diagnosis on disease, new drug development, vaccine research, bio-molecular interaction, genomics, proteomics and functional group.

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

1. a kind of check-out console: form by microwell plate (1), biological microsphere (2) etc.; Microwell plate is made up of structures such as plate body (3), plate lid (4), micropore (9) and microfluidic circuit; Biological microsphere is made up of the similar and different cell or the microorganism of microcarrier and its surface attachment, and is embedded in sequence in the different micropores;

2. sealing: handle biological microsphere with the damping fluid that contains compositions such as washing agent, albumin, skim-milk, with sealing with remove that the microcarrier surface may exist or residual nonspecific binding site or endogenous enzyme activity etc.;

3. sample: test sample, as solid-liquid body sample, biological fluid, tissue, cell, microorganism etc. through handling, as the homogenate of dilution, tissue/cell, cracking, removal particulate matter, separation and purification, reverse transcription, pcr amplification, mark etc.; Or do not handle directly and enter microwell plate by inlet opening along microfluidic circuit, when flowing through micropore and the various bio-molecular interactions of microbies such as biological microsphere superficial cell or bacterium; Tested molecule combines and is attracted to the biological microsphere surface with the biomolecules hybridization or the specificity of solid phase; Unconjugated sample composition flows out through fluid hole;

4. mark: use tagged molecule, 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 marker mark test samples such as fluorescence antibodies;

5. rinsing: by microfluidic circuit, use washing lotion, as contain the PBS of the Tween20 of 0.03-0.1% or Triton X-100 etc., Tris-HCl, damping fluids such as HEPES, stream wash to remove residual test sample, mark sample and marker etc. in the check-out console;

6. detect and analyze: range estimation or having or not and content with tagged molecule in each micropore of detections such as scanner, as having or not according to fluorescence, radioactivity, luminous product, 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 7. 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 structures such as plate body, Ban Gai, micropore and microfluidic circuit; But plate body and plate lid are hard any machine-shaping, printing opacity, lighttight, reflective or opaque 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 and can have partly or entirely structure such as inlet opening, fluid hole, sealing-ring, peristaltic tube, micropore, microtubule, 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.The micropore treated surface can adhere to animal and plant cells or microorganisms such as bacterium, virus; 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 of microfluidic circuit sample and/or marker etc. as octylame, Tai Fulong (Teflon) coating, silication etc.

Feature of the present invention also is: but microcarrier 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; The microcarrier treated surface can adhere to animal and plant cells or microorganism is made biological microsphere; The processing of various microcarriers comprises following different methods at least:

1. use the poly-lysine immersion treatment;

2. silanization is handled, as third amino-triethyl silicane (APES) etc.;

3. with the protein peptide isoreactivity fragment of solid phase synthesis technique directly synthetic promotion cell attachment on microcarrier, as the RGD fragment of extracellular matrix etc.;

4. with collagen protein or extracellular matrix coating microcarrier, as mouse tail collagen, fibronectin etc.;

5. form the surface molecular layer that one deck is made up of active group by chemically modified on the microcarrier surface, as the DEAE group;

6. add material in the processing starting material of microcarrier with chemical active radical (as DEAE group).

Feature of the present invention also is: microfluidic circuit is made up of structures such as inlet opening, fluid hole, 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 links to each other with micropore by microtubule, and communicates with the external world by inlet opening and fluid hole; Peristaltic tube advancing/fluid hole between, microfluidic circuit can be connected into closed circuit when needing, make liquid phase circulation so that the solid-liquid phase composition fully reacts; Sealing-ring and peristaltic tube can be positioned at also can be external on the check-out console; Cellular microwell plate can not have microtubule and microflute, also or does not have into/structures such as fluid hole yet, can add sample and rinsing by form during use; Microfluidic circuit can obtain different inactive surfaces coatings through surface treatment, the non-specific adsorption when reacting to reduce.

Feature of the present invention also is: biological microsphere is that microbies such as phalangeal cell or microorganism and microcarrier are cultivated altogether and be attached on the microcarrier, acquisition have a different chemical activity, can with the biological microsphere of checking matter specific combination in the sample; After microbe is attached to the microcarrier surface, can adds various reagent, medicine, biotic factor etc. and handle appropriate time, processing can be respectively in cultivating vessel or in microwell plate, carry out simultaneously; Microbe is also available organize the cell fixation liquid stationary liquid of ethanol, formaldehyde, glutaraldehyde etc. (as contain) to fix respectively to handle or in microwell plate fixing simultaneously the processing, make microbe anchor at the surface of microcarrier; The kind and the preparation method of biological microsphere comprise at least:

1. animal and plant cells is cultivated altogether with microcarrier in culture dish or fermentor tank, grows up to individual layer on the microcarrier surface, and merges in flakes, and thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Rinsing can be carried out respectively also can carrying out simultaneously in microwell plate in cultivating vessel with fixing;

2. animal and plant cells is cultivated altogether with microcarrier in culture dish or fermentor tank, grows up to individual layer on the microcarrier surface, and after reaching suitable density, gives the suitable processing of different chemical factors such as medicine, reagent, biotic factor, and thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Processing, rinsing and fixingly can in cultivating vessel, carry out respectively also can in microwell plate, carrying out simultaneously;

3. bacterium etc. is cultivated altogether with microcarrier, treats that bacterium grows up to individual layer on the microcarrier surface, and merges in flakes, and thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Rinsing can be carried out respectively also can carrying out simultaneously in microwell plate in cultivating vessel with fixing;

4. in culture dish or fermentor tank, cultivate altogether with microcarrier through virus, phage or plasmid infection, transfection or animal and plant cells that has transformed or bacterium etc., treat that the cell microorganism grows up to individual layer on the microcarrier surface, and merge in flakes, thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Rinsing can be carried out respectively also can carrying out simultaneously in microwell plate in cultivating vessel with fixing;

5. infection, transfection or the animal and plant cells that has transformed or bacterium and microcarrier are cultivated in culture dish or the fermentor tank altogether, treat that cell or microorganism etc. grow up to individual layer on the microcarrier surface, and after reaching suitable density, give the suitable processing of different chemical factors such as medicine, reagent, biotic factor, thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Processing, rinsing and fixingly can in cultivating vessel, carry out respectively also can in microwell plate, carrying out simultaneously;

6. various microbies are made into certain density suspension, educate altogether with microcarrier, after treating adhere firmly or sprawling, thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Rinsing can be carried out respectively also can carrying out simultaneously in microwell plate in cultivating vessel with fixing;

7. various microbies are made into certain density suspension, educate altogether with microcarrier, after treating adhere firmly or sprawling, give the suitable processing of different chemical factors such as medicine, reagent, biotic factor, thoroughly residual nutrient solution is removed in rinsing; Or the stationary liquid processing, residual stationary liquid is removed in rinsing; Processing, rinsing and fixingly can in cultivating vessel, carry out respectively also can in microwell plate, carrying out simultaneously;

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) etc. the tagged molecule 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 be with checking matter specificity bonded in the sample, with the marker mark sample (indirect labelling) of indicative tagged molecule mark; Mark can carry out in test tube, also can carry out in microwell plate.

Feature of the present invention also is: can adopt following method to carry out quantitative analysis: 1. the micropore of cell attachment or the hole count of inlaying biological microsphere can have multiple hole more than 1 or 1 on same microwell plate; 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; 2. when carrying out quantitative analysis, can be by 1. described method established standards object of reference, draw the concentration-reacting hole quantity and the strong and weak change curve thereof of marker, reference standard curve by Computer Analysis, in conjunction with the sample volume, obtain accurate sample quantitative analysis results.

Feature of the present invention also is: the 1. sample DNA of Ti Quing, and available Oligonucleolide primers, archaeal dna polymerase, dNTP and tagged molecule etc., the PCR reaction by at least one loop cycle prolongs or increases; The PCR reaction cycle cycle comprises a) heat denatured; B) annealing, the target nucleotide hybridization in primer and template or the sample; C) archaeal dna polymerase prolongs the target nucleotide chain-ordering; 2. the sample RNA or the mRNA of Ti Quing obtains complementary DNA (cDNA) through reverse transcription, and the PCR reaction by at least one loop cycle prolongs or increases again; The PCR reaction cycle cycle comprises a) heat denatured; B) annealing, the target nucleotide hybridization in primer and template or the sample; C) archaeal dna polymerase prolongs the target nucleotide chain-ordering; 3. sample extracts compositions such as nucleoprotein, plasmosin or embrane-associated protein according to a conventional method.

Feature of the present invention also is: test kit is equipped with a check-out console at least, at least inlay a microcarrier in each micropore, microbies such as different animal and plant cellss or bacterium are adhered on the microcarrier surface respectively, and form different test kits respectively or jointly with following compositions:

1. have one tubing/cell or microbiological specimens treatment solution at least, be used for cracking and the DNA or the RNA of microbe, the extraction of protein etc.; 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. at least respectively have a pipe to be respectively dNTP (deoxy-ribonucleoside triphosphate), primer and thermostable DNA polymerases etc. is used for the amplification of sample or the gene clone of the sample nucleic acid component of catching;

3. at least respectively there is a pipe to be respectively dNTP and ThermoScript II, is used for the synthetic of sample mRNA complementary DNA;

4. have at least a pipe contain the tagged molecule or derivatives 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 tagged molecule or marker are 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 Folin one phenol reagent, Bradford reagent etc.;

8. have a pipe at least for endonuclease (as EcoRI etc.), proteolytic ferment (as trypsinase, Quimotrase, carboxypeptidase etc.), be used for 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 staining fluid,, be used for the dyeing of bacterium or cell or redye as Gram stain, H-E dye liquor, Ji's nurse Sa dye liquor etc.;

10. 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.

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 PCR, RT-PCR amplification, mass spectroscopy, Nucleotide/determined amino acid sequence, protein phosphorylation and glycosylation by following diverse ways respectively to sample or 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 microballoon is done gene clone in position or further analyzed preceding sample preparation;

2. the biological microsphere 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 by the microcarrier of identical combination characteristic molecule monomer altogether with the duplicate microballoon or the bag of micropore interested respectively, repeat the operation steps of microwell plate with same condition, interested material composition in the sample is adsorbed on microsphere surface and separates with sample liquid, add corresponding reagent, binding substances is done gene clone or further analyzed preceding sample preparation.

(4) PCR of sample, processing such as RT-PCR amplified reaction and mark can be carried out in test tube, also can carry out in microwell plate.

The present invention has the following advantages: 1 contains much information.The big I of microballoon is from the millimeter to the micron, the special-purpose microcarrier of solid-phase matrix particle that present various merchant sells and cell cultures is generally between tens microns to hundreds of microns, as pressing 200 microns calculating, the cell chip of 1 standard slide glass size just can reach about 10,000 pieces.Therefore, as be used for transfection of attached cell surface display system or microbies such as cell transformed, bacterium, carry out the research of genetic expression and posttranslational modification etc.,, just can obtain the result and the data information of the parallel check and analysis of the different expressing genes of thousands of kinds simultaneously through the one-time detection analysis; Can obtain data informations such as qualitative, quantitative, cytology location and tissue distribution in addition simultaneously.2. be easy to preparation, cost is low.It is starting material that microcarrier of the present invention can select for use various merchants to sell product, as Bio-Glas, polystyrene bead, various chromatography substrate, silica-gel bead and cell cultures microcarrier Cytodex I, Cytodex II, Cytodex III etc., the present invention combines the separation and purification of suspension culture and biological products and the preparation of biological microsphere 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 the kind of the biological microsphere that inlay in any hole in the microwell plate and binding characteristic neatly 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 microcarrier and micropore all can or be made solid phase respectively.Each microcarrier 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.For improving reaction sensitivity, also can adopt macroporous microsphere or/and micropore is made 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 albumen, polysaccharide or nucleic acid molecule specific combination or the hybridization (have filtration, affinity chromatography, separate and concentrated effectiveness) of cell on the biological microsphere or bacterium etc.; 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 disposable a large amount of preparations in same cultivation vessel simultaneously of cell that biological microsphere adheres to or bacterium, thus the parallel unanimity of the detected result between each multiple hole and each check-out console can be guaranteed.When needs carry out quantitative analysis to a certain composition, inlay the multiple hole count of the identical biological microsphere that adheres to microbies such as same cell by increase, cooperate microfluidic circuit to solve accuracy, repeatability and the different experiments chamber of quantitative analysis and different batches check and analysis result's the property compared problem; 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 micropore and biological microsphere are identical, closedly inlay the noise signal that three-dimensional arrangement can avoid dust etc. to cause, thereby can obtain all image background intensity of even minimum, the arrangement of each micropore and microballoon can be consistent with the arrangement of photosignal conversion element simultaneously, therefore can obtain best cell 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 molecular weight, iso-electric point, structure, function, avidity, tissue/cell and the sub-micro of gene clone, nucleotide sequence analysis, binding substances or ultrastructural Location etc. to interested micropore and further analyze; The different elution requirements of binding substances be can be used for the separation and purification of follow-up target molecule.

Be further described below in conjunction with accompanying drawing

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

Shown in the figure microwell plate 1, biological microsphere 2, plate body 3, plate lid 4, inlet opening 5, fluid hole 6, sealing-ring 7, peristaltic tube 8, micropore 9, microtubule 10, wherein microballoon 2 is embedded in the micropore 9; Microtubule 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 microtubule 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 11, structures such as pilot hole 12 and form 13.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 13 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 micropore 9, discontinuous microtubule 10 shown in the figure, is embedded in biological microsphere 2, sealing-ring 7 in the micropore 9 and the inlet opening 5 that communicates with microtubule 10;

Fig. 3 B is the A-A sectional view of Fig. 3 A, is plate body 3, plate lid 4, micropore 9, microtubule 10 shown in the figure, is embedded in the biological microsphere 2 in the micropore 9, inlet opening 5, sealing-ring 7 etc.; The microflute 14 of micropore 9 upper ends forms microtubule 10 with plate lid 4 among the figure, and with the microtubule 10 of lower end micropore 9 is linked to each other in twos, forms complete microfluidic circuit with inlet opening 5, fluid hole 6 etc.In the enforcement, plate body 3 can be divided into distinct portions to be processed respectively, integrates one at last.Fig. 4 is the partial enlarged drawing of the microwell plate with different microfluidic circuit structures of frame of broken lines B part among Fig. 2 A.

Fig. 4 A is an orthographic plan, is microwell plate 1, micropore 9, microtubule 10 shown in the figure, is embedded in the microballoon 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 biological microsphere 2 in the micropore 9; The microflute 14 at micropore 9 two ends links to each other micropore 9 in twos with plate lid formation microtubule 10 among the figure, 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, microtubule 10 shown in the figure, be embedded in the biological microsphere 2 in the micropore 9; Microtubule 10 is arranged on the sidewall of two adjacent micropores among the figure, and the opening of its upper end communicates with microflute 14, and microflute 14 forms the microtubule 10 parallel with top and bottom with plate lid 4; Microtubule about in the of 10 openings at two ends micropore 9 is linked to each other in twos, and can with inlet opening, fluid holes etc. form complete microfluidic circuit.Can in microtubule 10, insert transmitter in the enforcement, miniature sampling apparatus, or at the microwell plate external installation external little proofing unit corresponding with microtubule up and down, with the physiology and biological chemistry variation of microbies such as observation of cell to various processing.Fig. 6 is microcarrier face shaping and structural representation.

Fig. 6 A shows polyhedron microcarrier 15, has the surface molecular layer 16 that active group can promote cell attachment;

Fig. 6 B is the sheet microcarrier made from cellulose membrane or microporous membrane 17;

Fig. 6 C is the sectional view of polyhedron microcarrier 15, shows vesicular structure, and the surface of microcarrier 15 can promote the active group or the formed surface molecular layer 16 of molecule of cell attachment;

Fig. 6 D is the surface structure synoptic diagram of spherical microcarrier 15, the 16th, can promote the active group of cell attachment or the surface molecular layer that molecule forms, and this molecular layer can be active group, protein molecular, polypeptide, poly-lysine or polysaccharide etc.Fig. 7 biological microsphere structural representation.

It among Fig. 7 A spherical microcarrier 15; Can promote the active group of cell attachment or the surface molecular layer 16 that molecule forms; By the cell 18 of surface molecular layer 16 attached to microcarrier 15 surfaces.

Fig. 7 B is the partial enlarged drawing of Fig. 7 A, solid phase microcarrier 15 among the figure; Promote the active group of cell attachment or the surface molecular layer 16 that molecule forms; Cell 18 attached to microcarrier 15 surfaces.The inserted cell biological chip nucleic acid detection assay of Fig. 8 principle of work synoptic diagram.

Fig. 8 A, sample nucleic acid molecule 20 in test tube behind fluorescein molecule 21 marks, nucleic acid molecule 19 hybridization with microcarrier 15 superficial cells 18, Za Jiao sample nucleic acid molecule is not removed through rinsing, certification mark molecule 21, according to the position preface coding in each positive hole obtain that sample nucleic acid molecule is handled with expression level or different pharmaceutical in the distribution of different cells, different action time is to the analysis results such as influence of similar and different cellular expression levels.

Fig. 8 B, sample nucleic acid molecule carry out pcr amplification with terminal primer through fluorescein molecule 21 marks in test tube; Amplified production 22 is hybridized with the nucleic acid molecule 19 of microcarrier 15 superficial cells 18, and Za Jiao sample nucleic acid molecule is not removed through rinsing, and certification mark molecule 21 obtains the check and analysis results.

Fig. 8 C, the DNA or the annealing of RNA molecule of oligonucleotide molecules primer 23 and 24 (terminal with fluorescein molecule 21 marks) and microcarrier 15 superficial cells 18, and be that template increases through PCR or RT-PCR; Amplified production 25 and 26 is bonded to the solid phase particle surface, and free primer is removed in rinsing, and certification mark molecule 21 obtains the check and analysis result.

Fig. 8 D and Fig. 8 E, sample nucleic acid molecule or its amplified production 20 in test tube behind biotin molecule 27 marks, nucleic acid molecule 19 hybridization with microcarrier 15 superficial cells 18, Za Jiao sample nucleic acid molecule is not removed through rinsing, the anti-biotin antibodies 28 or the affinity element 32 that add enzyme 29 marks, reaction is finished post rinsing and is removed unconjugated educt, adds substrate 30, observe add lustre to or luminous product 31 have or not or strong and weak; Position preface coding according to each positive hole obtains the check and analysis result.

Fig. 8 F, sample nucleic acid molecule or its amplified production 20 in test tube behind hapten molecule 33 marks, nucleic acid molecule 19 hybridization with microcarrier 15 superficial cells 18, Za Jiao sample nucleic acid molecule is not removed through rinsing, antihapten antibody 34 reactions that add enzyme 29 marks, unconjugated, free enzyme labelling thing is removed in rinsing, adds substrate 30, observe add lustre to or luminous product 31 have or not or strong and weak; Position preface coding according to each positive hole obtains the check and analysis result.

Fig. 8 G, sample nucleic acid molecule 20 in test tube behind hapten molecule 33 marks, nucleic acid molecule 19 hybridization with microcarrier 15 superficial cells 18, Za Jiao sample nucleic acid molecule is not removed through rinsing, antihapten antibody 34 reactions that add fluorescein 21 marks, unconjugated free label is removed in rinsing, and certification mark molecule 21 is according to the position preface coding acquisition check and analysis result in each positive hole.The inserted cell biological chip immunodetection of Fig. 9 analytical work principle schematic.

Fig. 9 A, behind sample antibody molecule 36 usefulness fluorescein molecules 21 marks, combine with antigen molecule 35 specificitys of microcarrier 15 superficial cells 18, unconjugated sample antibody molecule is removed through rinsing, certification mark molecule 21, according to the position preface coding in each positive hole obtain that the specific antigens molecule that sample antibody discerns is handled with expression level or different pharmaceutical in the distribution of different cells, different action time is to the analysis results such as influence of similar and different cell-specific antigen molecule expression level.

Fig. 9 B, after sample antibody molecule 36 and 38 is used fluorescein molecule 21 and 39 marks respectively, combine with the antigen molecule 35 and 37 specificitys of microcarrier 15 superficial cells 18, unconjugated sample antibody molecule is removed through rinsing, certification mark molecule 21 and 39 obtains specific antigens molecule 35 and 37 the distributions and expression level or different pharmaceutical processing at different cells that sample antibody is discerned according to the position preface coding in each positive hole, analyze coexpression or regulation relationship result and antigen molecule 35 and antigen molecule 37 between to the influence of similar and different cell-specific antigen molecule expression level etc. different action times.

Fig. 9 C, sample antibody molecule 36 combines with antigen molecule 35 specificitys of microcarrier 15 superficial cells 18, unconjugated sample antibody molecule 36 is removed through rinsing, anti-antibody 40 reactions that add enzyme 29 marks, unconjugated educt is removed in rinsing, add substrate 30, observe add lustre to or luminous product 31 have or not or strong and weak; Position preface coding according to each positive hole obtains the check and analysis result.

Fig. 9 D; behind sample protein molecule 42 usefulness fluorescein molecules 21 marks; target molecule 44 specificitys normal with microcarrier 15 surfaces or transfectional cell 43 expression or displaying combine; unconjugated protein molecular 42 is removed through rinsing; certification mark molecule 21, according to the position preface coding in each positive hole obtain that the specific target molecule 44 that sample protein molecule 42 discerned is handled with expression level or different pharmaceutical in the distribution of different cells, different action time is to the analysis results such as influence of its expression level.

Fig. 9 E, drug molecule 47 combine with the protein molecular 46 of microcarrier 15 superficial cells 45 behind fluorescein molecule 48 marks, and unconjugated sample protein molecule is removed through rinsing, certification mark molecule 48 to have that it's too late strong and weak, obtain analytical results.

Fig. 9 F is a matrix with the solid phase particle 49 that wraps up fluorescent substance, obtains to be connected the drug molecule 47 on fluorescent particle surface through solid phase synthesis; The medicine fluorescent particle is mixed with microcarrier 15, drug molecule 47 and the protein molecular 46 of microcarrier 15 superficial cells 45 combine and fluorescent particle 49 are adsorbed on the surface of microcarrier 15, unconjugated fluorescent particle 49 is removed through rinsing, detect fluorescent particle 49 to have that it's too late strong and weak, the result is analyzed in the histocyte distribution etc. that obtains drug targets molecule (protein molecular 46).

Fig. 9 G has protein molecular 46 reactions of drug molecule 47 with microcarrier 15 superficial cells 45 of molecular label 50, and unconjugated drug molecule 47 is removed through rinsing; The anti-molecular label antibody 51 that adds fluorescein molecule 21 marks, reaction is finished post rinsing and is removed unconjugated educt, and certification mark molecule 21 obtains the high throughput analysis result of drug molecule 47 according to the position preface coding in each positive hole.

Fig. 9 H, behind antibody molecule 54 usefulness fluorescein molecules 21 marks, with acceptor molecule 53 reactions on microcarrier 15 superficial cells 52, unconjugated antibody molecule 54 is removed through rinsing; Certification mark molecule 21 obtains the high throughput analysis result that acceptor molecule 53 histocytes distribute according to the position preface coding in each positive hole.

The inserted cell biological chip albumen one nucleic acid interaction check and analysis principle of work synoptic diagram of Figure 10.

Inlay the nuclear of microcarrier 15 superficial cells 55 among the figure in the sample protein molecule 57 of fluorescein molecule 21 marks and the micropore Acid molecule 56 specific bindings, unconjugated protein molecular 57 is removed through rinsing, and certification mark molecule 21 is according to each positive hole Position order coding obtain the high throughput analysis result that nucleic acid molecules 56 histocytes distribute.

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: microcarrier can directly select surface treated various merchant to sell chromatography substrate such as DEAE-Sephadex or cell Cultivate microcarrier Cytodex etc., also can select polystyrene bead, bead or control hole bead (u.s.a. applied biosystem Company, Sigma company) etc. by oneself. The surface treatment of microwell plate and microcarrier is different slightly different because of selected materials, Every kind of its concrete processing method of material and condition have multiple, the present invention at this with " biomolecule mobilization application " (Jiang China etc.,, chemical publishing house in 1998) and China Patent No. be 01207812.3 and application number be 00120798.9 patent Draw and be the main reference document. Use known various large biological molecule immobilization technology, process the microcarrier of various materials and little Orifice plate makes its surface with specific active group, so that the adhering to of the microbial bodies such as cell.

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. Polystyrene bead or control hole The concrete processing method of bead etc. is:

1. poly-D-lysine is processed: bead, control hole bead and polystyrene bead through thoroughly rinsing processing rely ammonia with poly Acid (10-200ug/ml) processing was spent the night drying for standby after distilled water or the buffer solution rinsing in 15-120 minute or 4 ℃.

2. silanization is processed; The cellular glass microcarrier of cleaning treatment (Weetall, H.H., Biochem.Biophys.Acta, 1970; 212:1) with 2% the third amino-triethyl silicane (APES, 3-Aminopropyltriethoxysilane, Sigma public affairs Department, catalog number (Cat.No.) A3648) anhydrous propanone solution-treated 20-120 second, drying for standby after the acetone rinsing; 2. the preparation of biological microsphere: different cells is educated altogether with microcarrier respectively, make cell be attached to the microcarrier surface, division increases Grow and reach certain cell density. The cultivation of attached cell, propagation, fixing and counting etc. are because of its kind and tissue-derived Deng the difference of biological characteristics and larger difference is arranged, quote the Cytodex of Amersham Pharmacia Biotech company at this Client's service manual of product is the main reference data. 3. inlay microballoon: artificial or use the full-automatic mechanical hand system, biological microsphere is embedded respectively each of microwell plate in certain sequence In the micropore, except microwell plate of the present invention, also can select in the enforcement China Patent No. be 01207812.3 and application number be 00120798.9 the microwell plate of the described various structures of patent. Biological microsphere in each micropore can be the same or different, so that Different sample specimen is carried out identical detection analysis simultaneously, or the same sample sample is carried out different detection analyses 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 advancing/fluid hole adding sealing buffer solution, remove the surface possibilities such as biological microsphere, microwell plate or microfluidic circuit with sealing Residual active group or nonspecific binding site are to reduce the non-specific binding that may occur in the detection. Namely obtain thus Can with the specific bindings such as chemical molecular, biomolecule, antibody and/or antigen, enzyme, nucleic acid or oligonucleotide molecules, and Can be used for braided type high flux cell biological chip that bond is detected. Additive commonly used in the confining liquid has: the salmon essence Or cerevisiae dna, ficoll, heparin, polyvinylpyrrolidone, bovine serum albumin(BSA) (0.5-5%), skimmed milk power (0.1-6 %), casein (0.5-2%), gelatin (0.1-0.5%), Normal animal serum (0.5-5%) and detergent etc. (as Tween 20, NP40, Triton X-100, SDS). When showing as a result with enzyme in force, the additive kind also needs to add, The reagent such as Sodium azide, hydrogen peroxide, biotin or antibiotin with sealing endogenous peroxydase, alkaline phosphatase and The activity of biotin etc., the subduction unspecific staining is to the impact of testing result. Cellular check-out console do not have microtubule/groove or do not have yet into / fluid hole needs to seal before the capping, takes the epiphragma of seal during use off or removes the use of plate lid, and 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 testing goal difference, sample will carry out respectively different processing before detection. Common sample treatment Method comprises: 1. nucleic acid extractive: with DNA or RNA extract process various biological samples, biological fluid or culture with DNA in the extraction sample or RNA etc.; 2. the extract such as protein: the branch of biological sample, biological fluid or biological sample etc. From extract or histiocyte lysate capable, Ultrasonic Pulverization or homogenate; 3. artificial synthetic product: uses the various skills of manually synthesizing The synthetic products such as prodrug, lead compound and the polypeptide that art obtains, oligonucleotides; 4. the synthetic cDNA of reverse transcription; 5. PCR or RT-PCR amplified production etc. 2. the mark of sample: 1. direct mark. Use various known biomolecular labeling technology, use respectively biotin, enzyme, glimmering The labelled molecule such as material or derivatives thereof such as light element, collaurum, isotope or rare earth ion and chelating agent thereof are straight as indicator Connect mark sample or biological sample; 2. adopt indirect labelling. Use the specificity interaction principle of biomolecule, use mark The marked product of molecule (such as fluorescence antibody, labels such as biotin-HRP) mark sample; 3. mix mark, use various Synthetic technology is mixed labelled molecule or label such as solid phase synthesis technique, pcr amplification etc. and to be 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 the microcarrier surface The hybridization such as the different IPs acid molecule of attached cell or oligonucleotide molecules. Biomolecule complementary or corresponding in the test sample is by solid Catch mutually or specific bond, remaining and unconjugated part flows out check-out console through fluid hole. 2. with Oligonucleolide primers, 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 one circulation PCR or RT-PCR amplified reaction. 3. the antigen-antibody in the sample, aglucon part, cell factor or drug molecule etc. with little year The different biological molecules combination of surface attached cell. 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 micropore or biological microsphere position order coding, computer analysis namely can determine in this sample, detected material The content of composition and each component, structure sequence etc.; Perhaps obtaining target molecule transfers in the distribution of different tissues cell, with expressing Control. But when label is protease, must increases operating procedures 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 according to the microbial bodies such as cell or solid phase probe, detected material, label With show the different of result etc., multiple different compound mode can be arranged. Various biochemical reactions can carry out in microwell plate, also can Partly or entirely in test tube, carry out. When carrying out in microwell plate, it is thin that the biological microsphere adhering on surface of inlaying in each micropore Born of the same parents can identical (cellular microwell plate), for detection of the identical component in the different samples; Also can be different, be used for same Heterogeneity in the sample is carried out high-throughout detection analysis.

The present invention is described further below in conjunction with embodiment.Example I: the application of braided type high flux cell biological chip in monoclonal antibody research.

In the research of mouse monoclonal antibody, usually need the histocyte distribution of the antigen target molecule of antagonist identification to identify, as the various acceptor molecules of cell surface or CD molecule etc., concrete operations step in force is as follows: 1. microcarrier is handled: the peace counterweight West Asia cell cultures microcarrier Cytodex of company no calcium magnesium PBS or Hanks liquid swelling, ethanol or autoclave sterilization, with aseptic no calcium magnesium PBS or Hanks liquid, nutrient solution rinsing in regular turn, 1-5mg/ml adds nutrient solution and puts in the culture vessel.2. the preparation of biological microsphere: different cells is pressed 10 ⁵/ ml adds respectively in the culture vessel and the abundant mixing of microcarrier, 37 ℃ leave standstill appropriate time (10-90 minute), make cell attachment on the microcarrier surface, supply nutrient solution by 10-80rpm/ minute stir culture (2-5 days) to required cell density, stop cultivating, but the replacing of collecting cell counting, and visual particular case therebetween nutrient solution.Leave standstill treat microcarrier deposition after, abandon supernatant liquor and add no calcium magnesium PBS or Hanks liquid rinsing several.After adding the stationary liquid PBS of the sucrose of 3.7% Paraformaldehyde 96 and 4% (as contain) suitably fixing (10-30 minute), rinsing promptly obtains the histiocytic biological microsphere through the fixing different sources of handling for several times.Add the no calcium magnesium Hanks liquid that contains sanitas (as sodium azide etc.) of proper volume, 4 ℃ of preservations are standby.3. inlay microballoon: artificial or use the full-automatic mechanical hand system, biological microsphere is embedded respectively in certain sequence in each micropore of microwell plate, except that selecting microwell plate of the present invention for use, also can select for use China Patent No. be 01207812.3 and application number be the microwell plate of the described various structures of 00120798.9 patent.4. capping: make microflute and advance/fluid hole forms microfluidic circuit, micropore can only by advance/fluid hole communicates with the external world.5. sealing: through advance/fluid hole is with sealing damping fluid (no calcium magnesium Hanks liquid contains the 1-3% bovine serum albumin), remove active group or nonspecific binding site that surfaces such as microballoon, microwell plate or microfluidic circuit may be residual with sealing, the non-specific binding that may occur in detecting with minimizing.Promptly obtain thus and can combine, and can be used for braided type high flux cell biological chip that binding substances is detected with specificity such as antibody.During in force as usefulness enzyme display result, also need add the activity of reagent such as sodium azide or hydrogen peroxide with the sealing endogenous peroxydase in the additive, the subduction unspecific staining is to the influence of detected result.6. adding sample: get the nutrient solution (containing antibody) of the hybridoma to be identified of an amount of volume (50-150ul), add in the microwell plate, hatch appropriate time (5-90 minute), the antigen molecule of antibody and cell surface is fully acted on through inlet opening.Be to improve detection sensitivity, reduce the nonspecific reaction that deposition and absorption cause, can by advancing/fluid hole between, the loop line by internal or external formula peristaltic tube forms circulates sample liquid in microfluidic circuit.7. rinsing:, wash to remove residual test sample (unconjugated antibody) in the check-out console with washing lotion (PBS contains Tween20 or the Triton X-100 of 0.03-0.1%) stream by microfluidic circuit.8. mark: add the anti-mouse Ig antibody of the FITC mark of 100-200ul through inlet opening, hatch appropriate time (15 minutes).9. rinsing:, wash to remove residual marker (the anti-mouse Ig of FITC-) in the check-out console with washing lotion stream by microfluidic circuit.10. interpretation of result: observation of use instrument such as fluorescent microscope or biochip scanner also writes down arrangement position or the biological microsphere position preface coding that having or not of each hole fluorescent reaction contrasts positive hole, the histocyte that promptly can determine the antigen molecule that this monoclonal antibody is discerned by Computer Analysis distributes, or the specificity of its histiocytic reaction.According to the strong and weak difference of fluorescent reaction, can infer the antigen presentation density of different cells.Example II: the application of braided type high flux cell biological chip aspect prenatal and postnatal care.

The expression in prokaryotic host cell of eukaryotic gene and viral protein is not only effective, and with low cost.Yet, expressed proteins incorrect or efficient low because of folding mode, usually activity is very low, even does not have activity.Particularly proteic translation post-treatment and modification, prokaryotic cell prokaryocyte can not be carried out at all.Rubella virus, cytomegalovirus, hsv, Coxsackie virus etc. 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.Therefore, the detection diagnosis that reproduction age and gravid woman are carried out above-mentioned pathogenic infection becomes the important means of prevention congenital abnormality and infection of newborn.Concrete operations step in force is as follows: 1. microcarrier is handled: cell cultures microcarrier Cytodex no calcium magnesium PBS or Hanks liquid swelling, ethanol or autoclave sterilization, with aseptic no calcium magnesium PBS or Hanks liquid, nutrient solution is rinsing in regular turn, and 1-5mg/ml adds nutrient solution and puts in the culture vessel.2. the preparation of biological microsphere: virus infected cell or gene transfecting cell add respectively in the culture vessel and the abundant mixing of microcarrier, 37 ℃ leave standstill appropriate time (10-90 minute), make cell attachment on the microcarrier surface, supplying nutrient solution stops cultivating by 10-80rpm/ minute stir culture to required cell density, but collecting cell is counted therebetween, detect the antigen presentation amount of infection or transfection virogene, and look particular case and change nutrient solution.Leave standstill treat microcarrier deposition after, abandon supernatant liquor and add no calcium magnesium PBS or Hanks liquid rinsing several.Add stationary liquid and fix, promptly obtain after the rinsing to handle, express and infect or the cell biological microballoon of transfection virogene through fixing.Add the no calcium magnesium Hanks liquid that contains sanitas of proper volume, 4 ℃ of preservations are standby.3. inlay microballoon: artificial or use the full-automatic mechanical hand system, biological microsphere is embedded in regular turn in each micropore of low density 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 residual active group or the nonspecific binding sites of surperficial possibility such as microballoon and microfluidic circuit with sealing damping fluid (no calcium magnesium Hanks liquid contains the 1-3% bovine serum albumin) sealing, to reduce the non-specific binding or the absorption that may occur in the detection, promptly obtain the braided type high flux cell biological chip of expression specificity virus antigen thus.6. adding sample: get person's serum to be checked (containing antibody) of an amount of volume (50-150ul), add in the microwell plate, hatch appropriate time (5-90 minute), the antigen molecule of antibody and cell surface is fully acted on through inlet opening.7. rinsing:, wash to remove residual test sample (unconjugated antibody) in the check-out console with washing lotion stream by microfluidic circuit.8. mark: add the Cy3 of 120ul and the anti-human IgG and the IgM antibody of Cy5 mark, suitably hatch (15 minutes).9. rinsing: washing lotion stream is washed to remove residual marker (the anti-mouse Ig of FITC-) in the check-out console.10. interpretation of result: observation of use instrument such as fluorescent microscope or biochip scanner also writes down having or not of each hole fluorescent reaction, contrast the arrangement position in positive hole or the position preface coding of biological microsphere, can determine promptly that by Computer Analysis this patient or examinee have or not corresponding virus infection; According to the variation of the difference (being content and the ratio of antigen-specific IgG and IgM) of Cy3 and Cy5 fluorescence power and multiple hole fluorescence power, can infer whether the examinee is in the early stage of infection, and the immunizing power etc. that has or not infectivity and body.

Inserted cell biological chip molecular library make up, the transfection of molecular surface display technique, recombination combines with the fast parallel analytical technology of the high-throughput of biochip by microcarrier suspension culture with expression etc., the advantage that integrates few techniques is a kind of high-throughput biological analysis detection technique of novel concept.Have contain much information, quantitatively accurate, highly sensitive, low cost of manufacture, purposes extensively waits outstanding advantage, can be widely used in biology, medicine and pharmacology, fields such as environmental science, the energy and Materials science.

Claims (10)

1. But 1. the high-flux cell biological chip testing technology of nucleic acid, albumen and the chemical molecular etc. 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: be made up of microwell plate and biological microsphere, microwell plate is made up of structures such as plate body, Ban Gai, micropore and microfluidic circuit; Biological microsphere is made up of microbies such as the similar and different bacterium of microcarrier and its surface attachment or cells, and is embedded in sequence in the different micropores; Usually only adhere to a kind of cell at a carrier surface;

   2. sealing: handle biological microsphere with confining liquid, sealing or removal nonspecific binding site;

   3. sample: sample is treated or not treated directly to enter microwell plate by inlet opening, when flowing through micropore and the various bio-molecular interactions of biological microsphere superficial cell or microorganism; Tested molecule combines and is attracted to the biological microsphere surface with the biomolecules hybridization or the specificity of solid phase; Xi Fu sample composition does not flow out through fluid hole;

   4. mark: test sample carries out mark with indicative tagged molecule and derivative or marker etc.;

   5. rinsing: combine with bioactive molecules on the cell or the various molecules of non-specific combination with washing lotion or elutriant wash-out;

   6. detect and analyze: the having or not of indicative tagged molecule in each micropore, power or content are detected and analyzes;

   When 7. 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 hard any machine-shaping, printing opacity, lighttight, reflective or opaque 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.The micropore treated surface also can adhere to microbies such as animal and plant cells or bacterium;
3. 3. but microcarrier 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; The microcarrier treated surface can adhere to animal and plant cells or microorganism obtains biological microsphere; The processing of various microcarriers comprises following different methods at least:

   1. use the poly-lysine immersion treatment;

   2. silanization is handled;

   3. with solid phase synthesis technique directly synthetic bioactive peptide fragment that promotes cell attachment on microcarrier;

   4. with collagen protein or extracellular matrix coating microcarrier;

   5. be formed with the surperficial unimolecular layer that certain active group is formed on the microcarrier surface by chemically modified;

   6. in the processing starting material of microcarrier, add material with chemical active radical.
4. 4. form by structures such as inlet opening, fluid hole, microtubule, sealing-ring, peristaltic tubes according to claim 1 and the described microfluidic circuit of claim 2, can lay respectively at plate body or plate to cover, also can all be positioned at plate body or plate covers; Microfluidic circuit links to each other with micropore by microtubule, 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 microtubule or microflute, 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. be meant that according to claim 1 and the described biological microsphere of claim 3 microbies such as animal and plant cells or bacterium, fungi, Rickettsiae, chlamydozoan, mycoplasma, virus and microcarrier cultivate altogether and be attached on the microcarrier, acquisition has the different chemical activity, can with the biological microsphere of checking matter specific combination in the sample; Attached to the microbe on microcarrier surface, processing such as available various reagent, medicine, biotic factor or physical factor, processing can be carried out respectively or carry out simultaneously in microwell plate in cultivate vessel; The also available cell fixation liquid of organizing of microbe is fixed processing respectively or fixing simultaneously processing in microwell plate, makes the surface of microbe immobilization at microcarrier; The kind and the preparation method of biological microsphere comprise at least:

   1. animal and plant cells is cultivated altogether with microcarrier in culture dish or fermentor tank, grows up to individual layer on the microcarrier surface, and merges in flakes; Thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate; Or with stationary liquid handle fixing after, with preserving the thorough rinsing of liquid, remove residual stationary liquid;

   2. animal and plant cells is cultivated altogether with microcarrier in culture dish or fermentor tank, grows up to individual layer on the microcarrier surface, and after reaching suitable density, adds different medicines, reagent, biotic factor etc. and give suitable processing respectively or simultaneously; Thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate; Add stationary liquid and fix, preserve the thorough rinsing of liquid, remove residual stationary liquid;

   3. bacterium etc. is cultivated altogether with microcarrier, treats that bacterium grows up to individual layer on the microcarrier surface, and merges in flakes, thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate, or the mutually beneficial fixed cell of fixed liquid phase, with preserving the thorough rinsing of liquid, remove residual stationary liquid;

   4. cultivate altogether with microcarrier through the animal and plant cells of infection, transfection or conversions such as virus, phage or plasmid or bacterium etc., treat that cell or microorganism grow up to individual layer on the microcarrier surface, and merge in flakes, thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate, or adding stationary liquid fixed cell, with preserving the thorough rinsing of liquid, remove residual stationary liquid;

   5. infection, transfection or animal and plant cells that has transformed or bacterium etc. are cultivated in culture dish or fermentor tank altogether with microcarrier, treat that cell or microorganism grow up to individual layer on the microcarrier surface, and after reaching suitable density, add after different medicine, reagent or biotic factor etc. suitably handle respectively or simultaneously, thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate; Or the stationary liquid processing, with preserving the thorough rinsing of liquid, remove residual stationary liquid;

   6. microbies such as various bacteriums, cell are made into certain density suspension, educate altogether with microcarrier, treat behind the adhere firmly or after sprawling, thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate; Or the stationary liquid processing, with preserving the thorough rinsing of liquid, remove residual stationary liquid;

   7. microbies such as various bacteriums, cell are made into certain density suspension, educate altogether, after treating adhere firmly or sprawling, add different medicine, reagent or biotic factor etc. and give suitable processing respectively or simultaneously with microcarrier; Thoroughly residual nutrient solution is removed in rinsing in cultivating vessel or microwell plate, or adds the stationary liquid fixed cell, with preserving the thorough rinsing of liquid, removes residual stationary liquid;
6. 6. mark according to claim 1 is meant, with direct mark samples of indicative 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 be with checking matter specificity bonded in the sample, with the marker mark sample of indicative tagged molecule mark; Mark can carry out in test tube, also can carry out in microwell plate.
7. 7. detection method according to claim 1 and test kit, can adopt following method to carry out quantitative analysis: the micropore of cell attachment or the hole count of inlaying biological microsphere can have the multiple hole more than 1 or 1 on same microwell plate; 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.
8. 8. according to claim 1, claim 6 and the described sample of claim 7, one of available following method is handled:

   1. in the sample DNA that extracts through conventional method, add PCR reagent, the PCR reaction by at least one loop cycle prolongs or increases; The PCR reaction cycle cycle comprises a) heat denatured; B) annealing, the target nucleotide hybridization in primer and template or the sample; C) archaeal dna polymerase prolongs the target nucleotide chain-ordering;

   2. sample RNA or the mRNA that extracts according to a conventional method obtains complementary DNA through reverse transcription, and the PCR reaction by at least one loop cycle prolongs or increases; The PCR reaction cycle cycle comprises a) heat denatured; B) annealing, the target nucleotide hybridization in primer and template or the sample; C) archaeal dna polymerase prolongs the target nucleotide chain-ordering;

   3. sample extracts nucleoprotein, plasmosin or embrane-associated protein according to a conventional method;
9. 9. according to claim 1 and the described test kit of claim 7, a check-out console is housed at least, at least inlay a microcarrier in each micropore, microbies such as identical or different animal and plant cells or bacterium are adhered on each microcarrier surface, hole respectively, and form different test kits respectively or jointly with following compositions:

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

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

   3. at least respectively there is a pipe to be primer, 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 staining fluid;

   10. have at least a pipe to be the electrophoresis sample buffer;
10. 10. according to claim 1, claim 6 and described detection method of claim 9 and test kit, can do the further preceding processing of analysis such as PCR, RT-PCR, gene clone, mass spectroscopy, sequencing, electrophoresis by following diverse ways respectively to respectively inlaying biological microsphere institute bonded sample composition in sample or the different holes of microwell plate with the reagent in the test kit:

    (1) directly in interested micropore, adds PCR reagent or reagent such as electrophoresis sample buffer, staining fluid, the sample preparation before binding substances is further analyzed in position;

    (2) microcarrier in the micropore is taken out, in microscale reactor, add the sample preparation before PCR reagent or reagent such as electrophoresis sample buffer or staining fluid are further analyzed binding substances;

    (3) fresh sample is educated by the microballoon of identical combination characteristic molecule monomer altogether with the duplicate biological microsphere or the bag of micropore interested respectively, repeat the operation steps of microwell plate with same condition, interested composition in the sample is adsorbed on the biological microsphere, fully unconjugated sample mixture is removed in rinsing, add PCR reagent, endonuclease or reagent such as proteolytic ferment, Phosphoric acid esterase, electrophoresis sample buffer, Protein Glycosylation Overview analysis or staining fluid, sample preparation and analysis before binding substances is further analyzed;

    (4) PCR of sample, processing such as RT-PCR amplified reaction and mark can be carried out in test tube, also can carry out in microwell plate.

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