CN1265199C - Micro fluidic biological chip based on micro balls - Google Patents
Micro fluidic biological chip based on micro balls Download PDFInfo
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- CN1265199C CN1265199C CN 200410041366 CN200410041366A CN1265199C CN 1265199 C CN1265199 C CN 1265199C CN 200410041366 CN200410041366 CN 200410041366 CN 200410041366 A CN200410041366 A CN 200410041366A CN 1265199 C CN1265199 C CN 1265199C
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Abstract
The present invention relates to a microfluidic biological chip on the basis of microspheres, which can be used to detect proteins, nucleic acids and other biological macromolecules, and can be widely used in the fields of clinical detection, inspection and quarantine, environmental monitoring, drug screening, microbiological assay, the function analyses of nucleic acids and albumins, etc. The biochip is composed of a cover sheet (1) arranged on the upper part and a base sheet (2) arranged on the lower part, wherein the inner part of the cover sheet (1) is provided with a stepped microchannel, the microchannel is provided with the microspheres with different sizes used as the carriers of probe molecules, and both ends of the microchannel are provided with an inlet (16) and an outlet (17); the stepped microchannel has at least two steps and at least two microspheres with different sizes, each step corresponds to a microsphere, and the sizes of the microspheres can be used as codes to distinguish different probe molecules fixed to the microspheres.
Description
Technical field
What the present invention relates to is a kind of microflow controlled biochip based on microballoon and preparation method thereof.It can be used for detecting biomacromolecules such as protein, nucleic acid, can be widely used in fields such as clinical detection, inspection and quarantine, environmental monitoring, drug screening, microbial identification and nucleic acid and protein function analysis.
Background technology
Biochip is meant the miniature analytic system that makes up on the solid-phase media surface of square centimeter size by microelectronics, micro-processing technology, to realize quick, efficient, sensitive processing and the analysis to DNA, protein and other biological component in tissue and the cell.It is a kind of Measurement for Biotechnique that grows up early 1990s, is the product of multidisciplinary cross-synthesis development.Its principle is at about 1～2cm
2Glass sheet, silicon chip, nylon membrane, gel or the metal etc. of size are above the carrier material, form different probe biomolecule (protein, nucleic acid etc.) diverse location is fixing with large scale array, these probe molecules can interact (as hybridization, immune response etc.) with purpose target molecule in the solution, react finish after by the unreacted molecule of the flush away of developing a film.The purpose target molecule is mark in advance, also can dye after reacting with probe molecule.Have or not power machine data analysis as calculated, the biological information that can obtain being correlated with by what signal collection equipment (as fluorescent microscope, laser confocal microscope, chip scanner etc.) obtained reaction signal.Because the characteristics of biochip high-throughput, parallelization, simplify and shortened the experimental study process greatly, obtained significant progress in recent years, particularly the Human Genome Project fulfil ahead of schedule start with post genome project after, Application of Biochips and demand have all had significant increase.Difference according to the fixing molecule of chip surface generally can be divided into it genetic chip and protein-chip.According to the difference of solid phase surface molecule fixing means, can be divided into point sample synthetic method and in-situ synthesis again.Though the sheet base of most biochips adopts low-cost glass sheet, because the chip preparation process needs special equipment and technology, so cause the cost of chip higher.In addition, the chip use is that the surface point at substrate adds the solution that contains the purpose target molecule, ten hundreds of different probes and purpose target molecule solid-liquid interface are interacted, the purpose target molecule often will just can be diffused into the position at correspondent probe molecule place through a lot of reflecting points, the reaction detection time is longer, and sensitivity remains further to be improved.Therefore how to reduce the cost of chip and the speed and the sensitivity of raising chip detection is the focus and the direction of biochip research always.
Micro-fluidic chip is current most active fields and a frontier development in the micro-total analysis system (Micro Total Analysis System, μ-TAS also claims Lab-On-A-chip), and its aim is that the function of assay laboratory is transferred on the chip.It is a kind of analytical technology that grows up in the analytical chemistry field mid-term early 1990s, based on analytical chemistry and analytical biochemistry, use microelectronic processing technique, on microchip, process microstructure networks such as micron-sized container, pump, valve, pipeline, this process of preparation, reaction and the detection of sample is carried out integrated micro-total analysis system.The size of chip is about several square centimeters, select for use silicon chip, glass, silicon rubber, plastic or other material as substrate and cover plate, by methods such as etching, photoetching or die processing microchannel, adopt modes such as electricity, pressure, gravity to drive the interior fluid of passage, adopt chemiluminescence, galvanochemistry, fluorescence detector etc. to detect at last.Micro-fluidic chip has been sent out application widely and development fast at biochemical analysis and environmental analysis aspects now.Because the process that micro-fluidic chip is analyzed whole sample, comprise sampling and processing, preconcentration, dilution and the mixing of sample, separate, chemical reaction and input be whole integrated on a little chip, it compares reagent and the sample consumption that has realized the sub-micro liter even received upgrading with traditional analytical equipment, the homogeneity that makes reaction of dwindling greatly of reaction compartment improves, reaction velocity is accelerated, and has reduced production cost simultaneously and is easy to carry.Therefore enjoy gazing at of domestic and international AC, correlative study also to become an important directions of present analysis chemical developer in recent years.But micro-fluidic chip seldom has the example that is applied to analyses such as nucleic acid hybridization at present, and its reason is to lack a kind of carrier of the different probe molecule of encoding.
Summary of the invention
Technical matters: the purpose of this invention is to provide that a kind of detection speed is fast, highly sensitive, the sample requirement is little, detect the low a kind of microflow controlled biochip of cost based on microballoon.
Technical scheme: the present invention fixes different probe molecules on the different microballoon of particle size, then that particle size is different microballoons is fixed on the biochip of making in the stepped microchannel based on microballoon.
This biochip is made up of cover plate that is positioned at the first half and the substrate that is positioned at Lower Half; In cover plate, be provided with stair-stepping microchannel, in the microchannel, be provided with the microballoon that varies in size, be provided with an import and an outlet at two of microchannel as the probe molecule carrier.Wherein, the ladder in the stepped microchannel has two-stage at least, and the microballoon that varies in size has two kinds at least, and each grade ladder is corresponding a kind of microballoon respectively, distinguishes the different probe molecule that is fixed on the microballoon with the size of microballoon as coding.By glass or polystyrene or PMMA (polymethylmethacrylate) or PDMS (dimethyl silicone polymer) preparation, the size of microballoon is between 30～100 μ m as the microballoon of probe molecule carrier.The length of each step in the stepped microchannel is at 500 μ m～1000 μ m, and the width of stepped microchannel is 105～205 μ m, each step from the distance of substrate at 25～105 μ m.
Its principle of work is: stair-stepping microchannel can be used as highly different dams, can fix the microballoon that varies in size, and makes it be unlikely to flow out the microchannel with fluid.When micro-creep pump driving hybridization solution cycles through the microchannel, have fluorescently-labeled purpose target molecule in the hybridization solution and just carry out hybridization reaction with the probe on the microballoon.The required temperature conditions of hybridization reaction can be by water-bath, and the box of constent temperature heater or controllable temperature provides.After hybridization reaction is intact, drives elution buffer with the micro-creep pump wash-out is carried out in microchannel and microballoon.Drain the eluent fluorescence intensity of detection microballoon under fluorescent microscope or laser confocal microscope afterwards, to determine the hybridization reaction result.
Beneficial effect: according to the present invention, utilize the different microballoon of stair-stepping microchannel fixed size, utilize the size of the microballoon different probe molecule of encoding, determine that by the fluorescence intensity that detects microballoon in the microchannel reaction result has the following advantages: (1) detection speed is fast: because hybridization reaction only carries out in the microchannel, hybridization solution circulates, can reduce the volatilization of solution and purpose target molecule time, improve the speed of reaction, shorten detection time to tat probe.(2) can realize high throughput testing: can there be the different dam of some height each stepped microchannel, can fix some glass microspheres that vary in size, and can contact or be in parallel in several stepped microchannels simultaneously, therefore can realize high-throughout detection.(3) highly sensitive: as because the surface of stationary probe molecule is spherical, increases than the circular surface of generic array chip is long-pending, thereby improved the sensitivity that detects, can detect the purpose target molecule of low concentration.(4) the sample requirement is little: because microchannel and microballoon all are micron-sized, therefore can realize receiving the sample introduction of upgrading.(5) extensibility height: owing to adopted the form of micro-fluidic chip, can be integrated with micro-fluidic chips such as sample pre-service easily, promoted the microminiaturization and the robotization of analytic system.
The objective of the invention is to make full use of the advantage of biochip and micro-fluidic chip, adopt the fixing different nucleic acid or the protein molecules of microballoon of different-grain diameter size, in microfluidic channel, carry out hybridization reaction, detect cost with fast reaction speed and reduction with the purpose target molecule.
Description of drawings
Fig. 1 is the cross-sectional view of chip of the present invention.
Fig. 2 for chip of the present invention vertical view.
When Fig. 3 uses for chip of the present invention, the structural representation of whole device.
Have among the above figure: cover plate 1, first ladder 11, second ladder 12, the 3rd ladder 13, four-step 14, the 5th ladder 15, import 16, outlet 17; Substrate 2; First microballoon 31, second microballoon 32, the 3rd microballoon 33, the 4th microballoon 34; Micro-creep pump 4, pipeline 5.
Embodiment
The present invention is a kind of microflow controlled biochip based on microballoon, and this biochip is made up of cover plate 1 that is positioned at the first half and the substrate 2 that is positioned at Lower Half; In cover plate 1, be provided with stair-stepping microchannel, in the microchannel, be provided with the microballoon that varies in size, be provided with an import 16 and an outlet 17 at two of microchannel as the probe molecule carrier.Ladder in the stepped microchannel has two-stage at least, and the microballoon that varies in size has two kinds at least, and each grade ladder is corresponding a kind of microballoon respectively, distinguishes the different probe molecule that is fixed on the microballoon with the size of microballoon as coding.Its material of microballoon as the probe molecule carrier can be glass, polystyrene or rubber etc., and the size of microballoon is between 30～100 μ m.The length of each step in the stepped microchannel is about 500 μ m, and the width of stepped microchannel is 105～205 μ m, each step from the distance of substrate at 25～105 μ m.
As chip material, to carry out biological detection be that example illustrates that its embodiment is as follows to the immobilized oligonucleotide probe on glass microsphere with PMMA (polymethylmethacrylate) for cover plate 1, substrate 2:
The preparation of a, cover plate: adopt laser micromachining methods.By AutoCAD design microchannel figure; But convert the CAD figure to the laser micro-machining system recognition instruction; Adopt laser micro-machining system to process stair-stepping microchannel on substrate or cover plate, punching two ends is respectively as injection port and outlet.
The preparation of b, substrate: select with the onesize PMMA slice, thin piece of cover plate as substrate.
The encapsulation of c, cover plate and substrate: carry out the encapsulation of substrate and cover plate by glassy state hot key and method.
Stationary probe on d, the microballoon: clean microballoon is modified through silanization and bifunctional reagent, then different probe molecules is fixed on the glass microsphere that varies in size.
E, microballoon fixing in the microchannel: draw an amount of microballoon and pre-reaction liquid with liquid-transfering gun or syringe etc., inject successively in the microchannel according to order from small to large, the ladder in the microchannel can be fixed on different positions to the microballoon that varies in size successively.
The connection of f, micro-fluidic chip: connect micro-creep pump 4 and chip successively by pipeline 5.
Hybridization reaction in g, the microchannel: open pipeline interface, the fluorescently-labeled sample that has that will anticipate with the micro-creep pump sucks the microchannel, is circulated in the stream of closure by micro-creep pump drive fluid then
H, the mobile reaction, reaction is opened pipeline interface after finishing, and pumps reactant liquor, sucks the eluent wash-out simultaneously.If the temperature that reaction needed is special can be carried out in the box of water-bath or controllable temperature.
H, detection: after hybridization reaction finishes, observe microballoon in the microchannel down, judge having or not and what of target molecule according to having or not of microsphere surface fluorescence signal and intensity with fluorescent microscope or laser co-focusing fluorescent microscope.
Claims (4)
1, a kind of microflow controlled biochip based on microballoon is characterized in that: this biochip is made up of cover plate that is positioned at the first half (1) and the substrate (2) that is positioned at Lower Half; Be provided with stair-stepping microchannel in cover plate (1), be provided with the microballoon as the probe molecule carrier that varies in size in the microchannel, be provided with an import (16) of microchannel, the other end of microchannel is provided with an outlet (17).
2, a kind of microflow controlled biochip according to claim 1 based on microballoon, it is characterized in that the ladder in the stepped microchannel has two-stage at least, the microballoon that varies in size has two kinds at least, each grade ladder is corresponding a kind of microballoon respectively, distinguishes the different probe molecule that is fixed on the microballoon with the size of microballoon as coding.
3, a kind of microflow controlled biochip based on microballoon according to claim 1 and 2 is characterized in that its material of microballoon as the probe molecule carrier is glass or rubber, and the size of microballoon is between 30～100 μ m.
4, a kind of microflow controlled biochip according to claim 1 based on microballoon, the length that it is characterized in that each step in the stepped microchannel is at 500 μ m～1000 μ m, the width of stepped microchannel is 105～205 μ m, each step from the distance of substrate at 25～105 μ m.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200410041366 CN1265199C (en) | 2004-07-13 | 2004-07-13 | Micro fluidic biological chip based on micro balls |
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| Application Number | Priority Date | Filing Date | Title |
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| CN 200410041366 CN1265199C (en) | 2004-07-13 | 2004-07-13 | Micro fluidic biological chip based on micro balls |
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| CN1595149A CN1595149A (en) | 2005-03-16 |
| CN1265199C true CN1265199C (en) | 2006-07-19 |
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| CN 200410041366 Expired - Fee Related CN1265199C (en) | 2004-07-13 | 2004-07-13 | Micro fluidic biological chip based on micro balls |
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Families Citing this family (19)
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| CN100360940C (en) * | 2005-05-31 | 2008-01-09 | 武汉大学 | Method and apparatus for screening G protein coupled receptor medicine |
| CN1948966B (en) * | 2005-10-13 | 2010-09-01 | 财团法人工业技术研究院 | Micro passageway biological chip |
| CN100406887C (en) * | 2006-01-10 | 2008-07-30 | 华东理工大学 | Tubular biochip |
| CN101221168B (en) * | 2008-01-08 | 2011-08-10 | 东南大学 | Microfluidic chip based on microsphere biological detection |
| EP2587248A1 (en) * | 2011-10-25 | 2013-05-01 | Koninklijke Philips Electronics N.V. | Filtering particles from blood or other media |
| CN102925337B (en) * | 2012-11-08 | 2014-06-18 | 武汉友芝友生物制药有限公司 | Microfluid cell capturing chip and manufacture method thereof |
| CN104138844B (en) * | 2014-06-27 | 2015-11-18 | 东南大学 | A kind of micro-fluidic medicaments sifting chip |
| CN104407036B (en) * | 2014-11-06 | 2017-12-08 | 上海慧观贸易有限公司 | Preparation and its application for the electrochemical microfluidic control device of nucleic acid isothermal amplification |
| CN104549589B (en) * | 2015-01-20 | 2016-05-25 | 重庆科技学院 | A kind of three substrate microballoon screening chip and usings method |
| KR101799192B1 (en) * | 2016-03-24 | 2017-11-17 | 고려대학교 산학협력단 | Micro-fluidic apparatus for detecting target gene |
| MX2019012509A (en) | 2017-04-21 | 2019-12-02 | Abay Sa | Systems, devices and methods for microfluidic analysis. |
| CN107271421A (en) * | 2017-07-27 | 2017-10-20 | 深圳中科芯海智能科技有限公司 | A kind of microparticle fluorescence detection means in fluid sample |
| CN108344866B (en) * | 2018-01-12 | 2020-07-28 | 天津大学 | Micro-fluidic chip detection system and method for detecting sample based on same |
| CN108459162A (en) * | 2018-02-07 | 2018-08-28 | 深圳赛斯鹏芯生物技术有限公司 | Detect the method and its kit of inflammation biomarker |
| CN109682962B (en) * | 2019-01-15 | 2024-02-23 | 中南大学 | Immunofluorescence detection system and detection method based on microfluidic chip |
| PL3721980T3 (en) * | 2019-04-12 | 2023-04-17 | Paris Sciences Et Lettres | Emulsion production microfluidic device |
| CN112114133A (en) * | 2020-09-03 | 2020-12-22 | 武汉纺织大学 | Particle arrangement method for multiple biochemical detection |
| CN112415058B (en) * | 2020-11-09 | 2021-12-14 | 华中农业大学 | Biosensing detection method based on microchannel resistance change caused by concentration change of insulating microspheres |
| CN113945551A (en) * | 2021-10-19 | 2022-01-18 | 重庆医科大学附属永川医院 | Microfluidic analysis and detection model for platelet function |
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