CN1254845A - Microfluid biochip detection-analysis board and its detection method - Google Patents
Microfluid biochip detection-analysis board and its detection method Download PDFInfo
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Abstract
A microfluid biochip detection-analysis board for using microfluid biochip in the chemical or biological analysis system is composed of chip board and cover plate. On said chip board, microslot channels connected to reaction sites are distributed and transparent cover plate is used to seal the chip board. Or, reaction sites are distributed on chip board and microslots are arranged on cover plate. Its detection method is that different kinds of probes or label moleculae are fixed at said reaction sites and different components in same specimen are analyzed on one chip at same time.
Description
Microfluid biochip detection-analysis board of the present invention and detection method thereof relate to is a kind of microfluid biochip to be applied to the check and analysis plate of chemistry or bioanalysis and reaction system, especially adopts microfluidic channel to connect the new design of many reflecting points.In very short time, can analyze the required result who obtains multiple composition simultaneously for same raw material or determinand.
Biochip mainly is meant by Micrometer-Nanometer Processing Technology and supermolecule self-assembling technique, in microanalysis unit and the system that the solid chip surface makes up.Biochip can integrate many difference in functionality devices, for example, the pre-service of biological sample, the extraction of inhereditary material, the amplification of specific gene fragment, biological probe array and Capillary Electrophoresis form whole microfluid system, with realize to compound, protein, nucleic acid, cell and other biological components accurately, fast, the screening or the detection of large information capacity.Genetic chip is a most important class biochip, and it the is integrated gene probe of dense arrangement in a large number can be analyzed a large amount of genes at short notice, makes people can promptly read and analyze the program of life.
Biochip has extremely important meaning on biological detection, medical test and medical diagnosis on disease, drug screening and gene sequencing.For example in biology, along with molecular biological continuous development, since the Human Genome Project particularly highly visible was implemented, the data of related nucleic acid, protein sequence and structure were exponential increase.And the most challenging work of next century is exactly after the Human Genome Project is finished, and promptly at the back era gene, how we use a large amount of biomolecule information services in human society, and makes medical diagnosis and treatment produce fundamental change.In medical science, " the subordinate phase medical science on system, organ, tissue, the cell level " transforms to " on the gene level; DNA → RNA → protein → protein and nucleic acid interaction, and the phase III medical science on they and the environmental interaction level ".This gene diagnosis of carrying out on molecular level and gene therapy will be familiar with the root that disease produces at all, and will be hopeful basic understanding and treat the major disease that comprises cancer.The fundamental change of these biology, medical science, a mensuration and the analysis that basic prerequisite is a gene order.Can carry out gene sequencing and analysis effectively apace, will have influence on the enforcement of the Human Genome Project, thereby influence further developing of biology, medical science.The methods that biomolecule detection adopted such as tradition gene comprise a series of numerous and diverse steps such as chemical reaction, gel electrophoresis, these method spended times are longer, and operate complicated, especially time-consuming and be not suitable for portability and check order fast aspect large scale sequencing.Traditional gene tester is being carried out in the improved process, is that the emerging technology of representative is arisen at the historic moment with the biochip.Biochip technology is with many discontinuous analytic process related in the life science, and as specimen preparation, chemical reaction and analyzing and testing etc. are by adopting microelectronics, technologies such as micromechanics are integrated in the chip, make it serialization, integrated, microminiaturization and robotization.The strong means that provide are obtained and analyzed to the maturation of this technology and use and will bring a revolution in medical diagnosis on disease and the life science association areas such as treatment, new drug development, judicial expertise, food and environment of next century for biological information.
96 orifice plates are existing a kind of conventional utensils that are used for the medical science biochemistry detection that generally use.96 reaction tanks on it are isolated from each other and do not pollute mutually, can select the number of reaction tank arbitrarily for use according to user's needs.But required amount of reagent is still bigger, also is not easy to continued operation simultaneously, more is not easy to simultaneously to multi-component continuous fast detecting in the sample.
The objective of the invention is weak point at conventional utensil existence in the present clinical medicine routine inspection, a kind of microfluid biochip detection-analysis board and detection method thereof are proposed, can carry out the continuous express-analysis of many index based on this check and analysis plate, and sample and reagent consumption are little, the operation simple and feasible.
Microfluid biochip detection-analysis board and detection method thereof take following scheme to realize:
Its structure of microfluid biochip detection-analysis board is made of chip board, cover plate, be furnished with little gutter channel on the chip board, the passage first line of a couplet site that responds, transparent cover plate covers the sealing chip board, or on chip board, be furnished with reaction site, and transparent cover plate is provided with little groove, and cover plate is to have little groove one cover on chip board, little groove on the cover plate joins the reaction site on the chip board, forms a sealing microfluid biochip detection-analysis board with inflow and outflow port.
The microfluid biochip detection-analysis board that the present invention proposes can carry out multi-component continuous express-analysis to micro sample, detect simple, the following realization of microfluid biochip detection-analysis board detection method:
The probe molecule or the marker molecules of multiple biological or chemical are placed respectively and are fixed on little reaction site of microfluid biochip detection-analysis board, each little reaction site is only fixed a kind of, every kind of probe molecule or marker molecules only with determinand in a certain component hybridize, crosslinked or specific chemical reaction takes place.The sample of detected analysis and various analytical reagent inject according to the program of setting and flow into port, the required little reaction site of flowing through, and produce corresponding effect.Above-mentioned specific reaction, hybridization or crosslinked will not be disturbed in the absorption of other materials.Even interference is arranged, also can be got rid of by methods such as washings.Little reaction site also can take following method to obtain: promptly can constitute by in advance probe substance or label being fixed on the minute solid phase carrier, putting on the site that is provided with on the analysis plates again.Since the probe molecule of each little reaction site or marker molecules only with determinand in specific component react, do not disturb mutually again each other, just can carry out qualitative or quantitative test to each component in the same sample simultaneously on chip piece, the gained result can detect with optics, electrical method.For example available microscope, CCD observation or surperficial excimer resonance (SPR), surface interfere reflection optics, electric field technology such as (RIFS) to observe and provide the result, also available other mark or cold method detection.Thereby increase work efficiency greatly, reduce reagent dosage, really accomplish quick, accurate, robotization and pollution-free.Probe molecule is meant and can produces the interactional material with single-minded recognition material function of specificity with specifying chemistry and biological substance, can adopt biomacromolecule material (as protein, nucleic acid, DNA, PNA, cell etc.), chemicals and polymer substance probe, label can adopt biological substance, chemical substance.
The microfluid biochip detection-analysis board that the present invention proposes, little reaction of being carried out on each little reflecting point can be quickened and regulates and control by physical methods such as electricity, light, sound, for example, can on each little reflecting point, microelectrode be set respectively, control little reaction on the reaction site by the regulation and control electric field.By the size and Orientation of Control current, the required reactant of characteristic reaction is assembled fast, unwanted material leaves fast, thereby adds fast response, improves detection sensitivity and shortens detection time.
The invention will be further described below with reference to accompanying drawing.
One of microfluid biochip detection-analysis board that Fig. 1 proposes for the present invention.
The cover plate corresponding that Fig. 2 proposes for the present invention with microfluid biochip detection-analysis board shown in Figure 1.
Two of the microfluid biochip detection-analysis board that Fig. 3 proposes for the present invention.
The cover plate corresponding that Fig. 4 proposes for the present invention with microfluid biochip detection-analysis board shown in Figure 3.
Three of the microfluid biochip detection-analysis board that Fig. 5 proposes for the present invention.
The cover plate corresponding that Fig. 6 proposes for the present invention with Fig. 5 microfluid biochip detection-analysis board.
Four of the microfluid biochip detection-analysis board that Fig. 7 proposes for the present invention.
The cover plate corresponding that Fig. 8 proposes for the present invention with Fig. 7 microfluid biochip detection-analysis board.
Five used diaphragms of the microfluid biochip detection-analysis board that Fig. 9 proposes for the present invention.
Five chip analysis plate of the microfluid biochip detection-analysis board that Figure 10 proposes for the present invention.
Figure 11 is the cover plate of the microfluid biochip detection-analysis board corresponding with Fig. 9, Figure 10 of the present invention's proposition.
Six of the microfluid biochip detection-analysis board that Figure 12 proposes for the present invention.
The cover plate corresponding that Figure 13 proposes for the present invention with microfluid biochip detection-analysis board shown in Figure 12.
With reference to accompanying drawing 1 is a kind of tandem type plastics (or metal and other material) microfluid biochip detection-analysis boards (abbreviation micro fluidic plate), on this fluid plate, be furnished with little gutter channel with inflow and outflow port, the passage first line of a couplet respond site a, b, c, d ..., these reaction site can be micro reaction pool, reactive tank or other forms.
With reference to accompanying drawing 2 is the chip transparent cover plate (glass or plastics) corresponding with Fig. 1, can cover sealing A plate, makes it to form the micro fluidic plate of sealing.
With reference to accompanying drawing 3,4 is another kind of chip design method, and characteristics are to be furnished with uniform little reaction site on chip board shown in Figure 3.A, b, c, d ..., this slightly reaction site can be regular shapes such as small sircle hole, strip groove, also can be other form.
Fig. 4 is the transparent cover plate corresponding with Fig. 3 chip board, and this cover plate is provided with little groove, when cover plate when having little groove one cover on the A plate, the little groove on the cover plate just in time joins the reflecting point on the chip board.Make the reaction site on the chip board connect to the analysis plates that an envelope increases with microfluid inflow and outflow port by little gutter channel.
With reference to accompanying drawing 5,6, be another kind of chip analysis plate design.On the chip board of Fig. 5, be furnished with a plurality of little grooves, each little groove several little reaction site of connecting.Fig. 6 is the cover plate with the corresponding chip board of Fig. 5.With reference to accompanying drawing 7,8, be another kind of chip board design.On chip analysis plate shown in Figure 7, be furnished with several rows reaction site independent of each other, several reaction site independent of each other arranged with delegation.Fig. 8 is and the corresponding cover plate of Fig. 7 chip analysis plate.Cover and be furnished with some independently little grooves.When cover plate covered on the chip board shown in Figure 7 with the one side that little groove is arranged, each little groove just in time got up delegation's reaction site series connection.
Fig. 9,10,11 is the design of another kind of microfluid biochip detection-analysis board.But Fig. 9 is the diaphragm of fixing biological molecules such as common nylon, fixes some kinds of different molecular probes and biological substance in the above with conventional method.Figure 10 is distributed with microwell array and is associated with the micropore transparent cover plate of pipeline, and Figure 11 is a base plate, diaphragm is clipped between transparent micropore cover plate and the base plate, and the biological fixation site on the diaphragm is overlapped with micropore on the transparent micropore cover plate.Make integral body become a micro fluidic plate.Can the specific reaction on the diaphragm be detected by the optics aspect equally.
Figure 12,13 is the design of another kind of microfluid biochip detection-analysis board.On the chip board of Figure 12, be furnished with the parallel little reflecting point of some row, whenever the little reflecting point that lists does not link to each other each other mutually, Figure 13 is the cover plate corresponding with Figure 12 chip board, on the cover board be furnished with the parallel minitype channel of several rows, 1,2 ... n, be drilled with some micropores between each passage and the cover plate face, microchannel and cover plate face are coupled together.This one side that is drilled with micropore at cover plate is furnished with the little groove of bent type some parallel to each other arranged side by side with above-mentioned microchannel capwise in addition, this slightly groove do not communicate with microchannel and micropore.When this that will have micropore and little groove earlier simultaneously covers on the chip board shown in Figure 12, each row micropore is just in time corresponding identical with little reflecting point of respectively being listed as on the same position of little reflecting point on the chip board, for example, the first row micropore is respectively with respectively to be listed as first little reflecting point of little reflecting point corresponding identical overlapping on the chip board on the cover plate, if the direction of cover plate along curved little groove moved a certain distance, micropore on the cover plate no longer with chip board on little reflecting point overlapping, then with the curved little groove on the cover plate each the little reflecting point on the chip board is coupled together, each curved little groove connects row.If continue to move a certain distance along same direction again, little groove on the cover plate and the little reflecting point on micropore and the chip board will be not overlapping.During use, the little groove on can cover plate links up the little reaction site that respectively is listed as on the chip board, by inlet 1,2 ... n feeds reactant, and these reactants can be identical, also can be different.The translation cover plate is with the micropore on the cover plate, on chip board then.Little reaction site is overlapping, again by the microchannel a on the cover plate
1, a
2, a
3A
n, feed second kind of reactant, these reactants can be identical, also can be different, Using such method, also can continue to introduce the third, the 4th kind ... reactant.If need, a kind of reactant of every adding can make little groove or the little reaction site on micropore and the chip board on the cover plate not overlapping by mobile again cover plate, and each little reaction site is isolated each other fully, reacts certain hour under the condition of setting.Above-mentioned this design can be avoided cross pollution mutually between each little reaction site.After reaction finishes, can cover plate on little groove each little reaction site on the chip board is linked up, feed washing reagent or distilled water washs by inlet.The end product of reaction can detect and provide the result with the method for optics or electricity.
To further specify the microfluid biochip detection-analysis board detection method that the present invention proposes with embodiment below.
Embodiment 2: series connection detects when using enzyme-linked immunosorbent assay for measuring to multiple infectious diseases.
The antibody of disease to be measured is coated on reaction site a, b, c, d on the microfluid biochip detection-analysis board A shown in Fig. 1 (or Fig. 3) that the present invention proposes respectively ... on, each little Chi Bao is by a kind of antibody, use the airtight covering of cover plate of Fig. 2 (or Fig. 4) then, heating is hatched again.Feeding confining liquid by inlet again seals and logical N
2After the air dry, feed antigen reagent, hatch certain hour for 37 ℃ by inlet, feed lavation buffer solution and deionized water wash again after, feed drying nitrogen by inlet and carry out drying.Feed elisa reagent by inlet at last, shake mixing, hatch certain hour for 37 ℃, feed lavation buffer solution by inlet again and wash, feed the chromogenic reagent solution colour developing then, and hatch certain hour in 37 ℃, feed stop buffer again.After washing drying, observe and provide the result with microscope or RIFS or SPR or other method.
Embodiment 3: the rapid biochip detection of human body blood group, with the anti-A of blood of human body, the anti-albumen of ABS respectively chemical coupling be fixed on micro reaction pool a, the b of chip board shown in the figure (or Fig. 7), and cover with cover plate shown in Fig. 6 (or Fig. 8) and to close envelope, insulation is at 37 ℃.Then sample blood to be measured is introduced from inlet.If blood is the A type, the blood clotting phenomenon will take place in reaction site a, if blood is Type B, the blood clotting phenomenon will appear in reaction site b.If the O type the blood clotting phenomenon can not occur in a, b reaction site; If the AB type then all the blood clotting phenomenon will occur in a, b reaction site.The appearance of blood clotting phenomenon will cause the change of penetrability, also will cause the change of material thickness in a, the b micro reaction pool.Detect and provide the result with spectroanalysis instrument or RIFS.Using this method can carry out the blood group analysis to a plurality of blood samples simultaneously, only needs blood sample 1,2,3 ... respectively from entering the mouth 1,2,3 ... inject and get final product.
Embodiment 4 detects when using diaphragm-type check and analysis plate to multiple infectious diseases.
The antibody that detects disease is coated on site a, b, c, d on the fiber diaphragms such as nylon as shown in Figure 9 that the present invention proposes or acetate fibre paper respectively ... on, each site bag is by a kind of antibody, then the biological fixation site on the above-mentioned diaphragm that is coated with antibody is overlapped with micropore on the transparent micropore cover plate, make integral body become a micro fluidic plate.Heating is hatched then.Feed confining liquid by inlet again and seal and feed N
2After the air dry, feed antigen reagent, hatch certain hour for 37 ℃, feed cleansing solution again behind liquid and deionized water wash, feed dry N by inlet by inlet
2Carry out drying, feed elisa reagent by inlet at last, shake mixing, hatch certain hour for 37 ℃, feed cleansing solution by inlet again and wash towards liquid.Then, feed the chromogenic reagent solution colour developing, and hatch certain hour in 37 ℃.Feed stop buffer again, after washing drying, observe and provide the result with microscope or RIFs or SPR or other method.
Claims (10)
1. microfluid biochip detection-analysis board, it is characterized in that: its structure of analysis plates is made of chip board, cover plate, be furnished with little gutter channel on the chip board, the passage first line of a couplet site that responds, transparent cover plate covers the sealing chip board, or is furnished with reaction site on chip board, and transparent cover plate is provided with little groove, cover plate is to have little groove one cover on chip board, and the little grooved surface on the cover plate joins the reaction site on the chip board.
2. microfluid biochip detection-analysis board according to claim 1 is characterized in that: (a) preparation has a series of little reaction site on same chip board; (b) this slightly reaction site have by what little gutter channel connected to a sealing that microfluid flows into and the analysis plates of outflow port.
3. microfluid biochip detection-analysis board according to claim 1 is characterized in that reaction site can be micro reaction pool, reactive tank or other forms.
4. microfluid biochip detection-analysis board according to claim 1, but it is characterized in that between chip board and cover plate clamping has the diaphragm of fixing biological molecules.
5. microfluid biochip detection-analysis board detection method, it is characterized in that multiple probe substance or marker molecules are separately fixed on the reaction site of microfluid biochip detection-analysis board, each little reaction site is only fixed a kind of, every kind of probe substance or label only with determinand in a certain component hybridize, crosslinked or specific chemical reaction takes place; The sample of detected analysis and various analytical reagent inject according to the program of setting and flow into port, the required little reaction site of flowing through, and produce corresponding effect; Simultaneously each component in the same sample is carried out qualitative or quantitative test on chip piece, the gained result can detect with optics, electrical method.
6. according to the described microfluid biochip detection-analysis board detection method of claim 5, it is characterized in that can be on analysis plates directly stationary probe material or label, also can be by in advance probe substance or label being fixed on the minute solid phase carrier, be placed on again to be provided with on the site on the analysis plates and constitute.
7. according to claim 5, described microfluid biochip detection-analysis board detection method is characterized in that biological substance fixing on its reflecting point can be protein, nucleic acid, PNA, cell.
8. according to the described microfluid biochip detection-analysis board detection method of claim 5, it is characterized in that multiple probe substance that is used to detect and biological substance can be in advance according to the array and the sizes that design, be fixed on nylon membrane, on the fiber diaphragms such as acetate fibre paper, be placed on then between chip board and the cover plate, probe on the fiber diaphragm or the reaction site on biological substance dot matrix and chip board or the cover plate are coincided, make it to constitute a miniflow system.
9. according to claim 5, described microfluid biochip detection-analysis board detection method is characterized in that each little reaction site can be furnished with microelectrode, controls little reaction on the reaction site by the regulation and control electric field.
10. according to claim 5, described microfluid biochip detection-analysis board detection method is characterized in that the characteristic reaction that takes place on little reaction site on the chip, and the result is observed and provided to available microscope, optics, electric field instrument analysis technology.
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| CN 99114512 CN1117284C (en) | 1999-10-27 | 1999-10-27 | Microfluid biochip detection-analysis board and its detection method |
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| CN 99114512 CN1117284C (en) | 1999-10-27 | 1999-10-27 | Microfluid biochip detection-analysis board and its detection method |
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| CN100394184C (en) * | 2002-09-27 | 2008-06-11 | 洹艺科技股份有限公司 | Micro-fluid transfer and analysis system |
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