CN101221168A - Microfluidic chip based on microsphere biological detection - Google Patents
Microfluidic chip based on microsphere biological detection Download PDFInfo
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Abstract
The invention relates to a microfluidic chip which is based on the microsphere biological detection and can be used for the detection of protein, nucleic acid and other biological macromolecules, so the invention has broad application prospect in clinical testing, inspection and quarantine, environmental monitoring, drug screening, microbial identification, nucleic acid and protein functional analysis and other fields. The biological chip is composed of a cover plate which is positioned at the upper half part and a substrate which is positioned at the lower half part; the interior of the substrate is provided with a micro-channel network, a solid phase carrier which is provided with the coded microspheres as probe molecule for biological detection is arranged in the micro-channel, the both ends of the micro-channel are provided with an inlet (1), an outlet (2), a long channel (3), a reaction pool (4), a group of auxiliary channels (5) and a screening pipeline (6). When in using process of the chip, the sample injection, the reaction and the detection of the reaction result of the sample are carried out on the chip, and the invention has the advantages of high integration, convenient usage, less sample consumption, high detection sensitivity and so on.
Description
Technical field
What the present invention relates to is a kind of micro-fluidic chip based on the microballoon biological detection 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
Microfluidic analysis chip 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 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.The principal character of this chip on device is that its resulting structure of holding fluid (comprising its passage, reaction chamber and other functional part) is the micron order yardstick at least on a dimension.Compare with the experimental provision of macro-size, the micron order structure of microfluidic analysis chip can enlarge markedly the ratio of the area/volume of fluid environment.This variation causes the peculiar effect of a series of decision its properties relevant with body surface in microfluidic system, as the laminar flow effect, and surface tension and capillary effect, Rapid Thermal conduction effect and diffusional effect etc.These effects can make the analytical performance of microfluid analysis chip be significantly improved, and comprise the volume of analytical equipment is reduced, and equip more integrated robotization, can significantly improve analysis efficiency and make sample and degradation is significantly descended in reagent consumption.The laboratory main equipment is integrated on the as far as possible little operating platform, in order to finishing different experimentations, and the technology that can analyze product.It not only makes the consumption of reagent reduce, and speed of experiment is improved, and expense reduces, and has demonstrated fully the development trend of current laboratory equipment microminiaturization, integrated and portability.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.
Summary of the invention
Technical matters: the purpose of this invention is to provide a kind of micro-fluidic chip based on the microballoon biological detection, being used for the microballoon is that solid phase carrier carries out biomolecule detection.The coding microball that is fixed with the bioprobe molecule is carried out sample introduction, reacts, washs and detect with testing sample by chip, a kind of chip platform of check and analysis is provided.
Technical scheme: purpose of the present invention can be achieved through the following technical solutions:
Micro-fluidic chip based on the microballoon biological detection of the present invention is made up of cover plate that is positioned at the first half and the substrate that is positioned at Lower Half; In substrate or cover plate, be provided with functional microchannel network, in the network of microchannel, be provided with microballoon with coding carrier as biomolecule detection, in the network of microchannel, the periphery of reaction tank is the screening pipeline, the periphery of screening pipeline is a subsidiary conduit, subsidiary conduit and outlet are joined, and reaction tank taps into mouth by long-channel.
As carrier, microballoon has coding to biomolecule detection with microballoon, is fixed with probe biomolecule on the surface of microballoon, and used probe biomolecule can be a kind of in nucleic acid, protein, the polypeptide.
In the chip reaction tank, carry out the reaction of microballoon and testing sample; Solution to be measured and lavation buffer solution enter reaction tank by import, and with microballoon reaction or washing microballoon, reaction finishes microballoon and is transfused to detection and output in the passage in reaction tank.
Microballoon is as the probe molecule carrier, and its material can be glass or polystyrene and rubber, and the size of microballoon is between 50 μ m～300 μ m; The host material of chip can be a kind of in glass, silicon, polymetylmethacrylate, dimethyl silicone polymer PDMS and the polycarbonate.
Coding microball be barcode encoding, fluorescence-encoded, quantum-dot coding, photonic crystal coding, Raman tag coding, infrared spectrum coding, shape coding, radio frequency coding, size coding and position encoded in a kind of.
The diameter of reaction tank is between 200 μ m～1200 μ m, and the long-channel width is between 60 μ m～550 μ m, and length is between 10mm～50mm.
Its principle of work is:
A) connect a reciprocation type syringe pump or peristaltic pump in the chip exit, the outer coding microball of chip is connected its delivery damping fluid by long-channel, sucting reaction pond.Because the size of screening passage is less than the diameter of coding microball, therefore by the continuation suction of pump, microballoon is stayed in the reaction tank, and the damping fluid of delivery microballoon is then blotted;
B) at this moment, sucking testing sample solution to reaction tank, testing sample solution and microballoon are fully reacted by import;
C) reaction finishes, and continues to suck lavation buffer solution by import;
D) after the washing fully, inject microballoon delivery damping fluid, make microballoon enter long-channel, during by long-channel, detect the reaction result of microsphere surface at microballoon with the delivery damping fluid by outlet.
Beneficial effect: according to the present invention, utilize chip to detect carrier, sample and annotate sample, reaction, washing and the continuous operation that detects are that the solid phase carrier of biomolecule detection has the following advantages with the coding microball:
(1) detection speed is fast: because hybridization reaction only carries out in the microchannel, hybridization solution circulates, and can reduce the volatilization of solution and purpose target molecule to the time of tat probe, improves the speed of reaction, shortening detection time;
(2) can realize that multivariate analysis detects: the microballoon of each sample introduction has coding, and the multi-element biologic Molecular Detection of therefore can encoding detects a plurality of indexs in the same sample simultaneously.Simultaneously to have specific surface area big for ball type carrier, characteristics such as can roll, so detection reaction is highly sensitive, the sample requirement is few, and reaction velocity is fast;
(3) accuracy height: microballoon is when entering long-channel, because channel size is slightly larger than microsphere diameter, much smaller than two diameter of micro ball, so microballoon can only singlely pass through, and is detected successively, the accuracy height;
(3) 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;
(3) the present invention combines the biological detection advantage of microsphere supported and micro-fluidic chip simultaneously, and is simple to operate to undersized microballoon, has distinct advantage.
Description of drawings
Fig. 1 for chip of the present invention vertical view.
When Fig. 2 uses for chip of the present invention, the structural representation of whole device.
Have among the above figure: import 1, outlet 2, long-channel 3, reaction tank 4, subsidiary conduit 5, screening pipeline 6.
Embodiment
The present invention is a kind of microfluidic analysis chip based on microballoon, and 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 substrate or cover plate, be provided with functional microchannel network, in the network of microchannel, be provided with microballoon with coding carrier as biomolecule detection, in the network of microchannel, the periphery of reaction tank 4 is screening pipelines 6, the periphery of screening pipeline 6 is subsidiary conduits 5, subsidiary conduit 5 joins with outlet 2, and reaction tank 4 taps into mouth 1 by long-channel 3.
As carrier, microballoon has coding to biomolecule detection with microballoon, is fixed with probe biomolecule on the surface of microballoon, and used probe biomolecule can be a kind of in nucleic acid, protein, the polypeptide.In chip reaction tank 4, carry out the reaction of microballoon and testing sample; Solution to be measured and lavation buffer solution enter reaction tank 4 by import 1, and with microballoon reaction or washing microballoon, reaction finishes microballoon and is transfused to detection and output in the passage 3 in reaction tank 4.Microballoon is as the probe molecule carrier, and its material can be glass or polystyrene and rubber, and the size of microballoon is between 50 μ m～300 μ m; The host material of chip can be a kind of in glass, silicon, polymetylmethacrylate, dimethyl silicone polymer PDMS and the polycarbonate.Coding microball be barcode encoding, fluorescence-encoded, quantum-dot coding, photonic crystal coding, Raman tag coding, infrared spectrum coding, shape coding, radio frequency coding, size coding and position encoded in a kind of.The diameter of reaction tank 4 is between 200 μ m～1200 μ m, and long-channel 3 width are between 60 μ m～550 μ m, and length is between 10mm～50mm.
Its material of microballoon as the probe molecule carrier can be glass, polystyrene or rubber etc.
Embodiment one: as chip material, stationary probe carries out biological detection on photonic crystal coding glass microsphere with PMMA (polymethylmethacrylate), and microsphere diameter is 50 μ m.
The preparation of a, substrate: 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 be processed into required network microchannel on substrate (or cover plate), wherein, the long-channel width is 60 μ m, and long 20mm, reaction tank diameter are 250 μ m, and sieving pipeline wide is 20 μ m, and the degree of depth of passage is 60 μ m.Punching two ends is respectively as injection port and outlet;
The preparation of b, cover plate: select with the onesize PMMA slice, thin piece of substrate as cover plate; The encapsulation of cover plate and substrate: directly carry out the encapsulation of substrate and cover plate by glassy state hot key and method;
Stationary probe on c, the microballoon: clean microballoon is modified through silanization and bifunctional reagent, then different probe molecules is fixed on the glass microsphere of photonic crystal coding;
The connection of d, micro-fluidic chip; Import 1 connects sample cell by pipeline, and outlet 2 is access to compound syringe pump;
E, sample introduction: by syringe pump coding microball is sucked long-channel 3 together with its delivery damping fluid, enter reaction tank 4 then, drain the delivery damping fluid at last, coding microball is stayed in the reaction tank 4;
F, reaction: by syringe pump solution to be detected is sucked long-channel 3, the probe molecule that enters at last in the reaction tank 4 with the coding microball surface reacts;
G, washing: after reaction finishes, fully wash to 4 pairs of microballoons of reaction tank from the 1 suction lavation buffer solution that enters the mouth by syringe pump;
H, detection: drain the lavation buffer solution in the reaction tank 4, utilize syringe pump from exporting the delivery damping fluid of 2 injection microballoons, the delivery damping fluid is brought microballoon into long-channel (3), and microballoon passes through long-channel successively
3), and detected successively in long-channel 3.
Embodiment two: as chip material, stationary probe carries out biological detection on the quantum-dot coding polystyrene microsphere with glass, and microsphere diameter is 100 μ m.
The preparation of a, substrate: adopt standard photolithography techniques to carry out wet etching.Plating layer of metal chromium on clean level and smooth glass substrate surface, on the chromium layer, be coated with one deck photoresist uniformly with photoresist spinner, with chip structure figure by photo etched mask with ultraviolet photoetching to photoresist, development is to remove the photoresist of exposure, spend chrome liquor again and corrode the layer that dechromises, show the planar figure of microchannel network; Can go out required microchannel by wet etching at last.Wherein, the long-channel width is 130 μ m, and long 30mm, reaction tank diameter are 500 μ m, and sieving pipeline wide is 40 μ m, and the degree of depth of passage is 130 μ m;
The preparation of b, cover plate: select with the onesize glass of substrate as cover plate;
The encapsulation of c, cover plate and substrate: after the cover glass of the glass substrate peace after import 1 and the punching of outlet 2 places cleans, can carry out the high-temperature pressurizing bonding;
Stationary probe on d, the microballoon: the polystyrene microsphere surface that different probe molecules is fixed to different coding;
The connection of e, micro-fluidic chip: import 1 connects sample cell by pipeline, and outlet 2 is access to compound micro-creep pump;
F, sample introduction: by peristaltic pump coding microball is sucked long-channel 3 together with its delivery damping fluid, enter reaction tank 4 then, drain the delivery damping fluid at last, coding microball is stayed in the reaction tank 4;
G, reaction: by peristaltic pump solution to be detected is sucked long-channel 3, the probe molecule that enters at last in the reaction tank 4 with the coding microball surface reacts;
H, washing: after reaction finishes, fully wash to 4 pairs of microballoons of reaction tank from the 1 suction lavation buffer solution that enters the mouth by peristaltic pump;
I, detection: drain the lavation buffer solution in the reaction tank 4, utilize peristaltic pump from exporting the delivery damping fluid of 2 injection microballoons, the delivery damping fluid is brought microballoon into long-channel 3, and microballoon passes through long-channel 3 successively, and detected successively in long-channel 3.
Embodiment three: as chip material, stationary probe carries out biological detection on fluorescence-encoded glass microsphere with PDMS (dimethyl silicone polymer), and microsphere diameter is 200 μ m.
The preparation of a, substrate: selecting the matrix of PDMS and the ratio of hardening agent is 10: 1 (mass ratio), after mixing,, be cast on the formpiston that processes in advance with after removing bubble through vacuum suction, 25min is heating and curing, wherein, the long-channel width is 250 μ m, and long 50mm, reaction tank diameter are 1200 μ m, sieving pipeline wide is 50 μ m, and the degree of depth of passage is 250 μ m.After being cured PDMS is taken off from masterplate, promptly make substrate;
The preparation of b, cover plate: selecting the matrix of PDMS and the ratio of hardening agent is 5: 1 (mass ratio), and after mixing, with after removing bubble, directly the horizontal positioned 20min that is heating and curing promptly makes cover plate through vacuum suction;
The encapsulation of c, cover plate and substrate: substrate that is cured and cover plate directly carry out the encapsulation of substrate and cover plate by the thermal bonding method then again with fitting after the oxygen plasma treatment;
Stationary probe on d, the microballoon: the glass microsphere surface that different probe molecules is fixed to different coding;
The connection of e, micro-fluidic chip: import 1 connects sample cell by pipeline, and outlet 2 is access to compound micro syringe pump;
F, sample introduction: by syringe pump coding microball is sucked long-channel 3 together with its delivery damping fluid, enter reaction tank 4 then, drain the delivery damping fluid at last, coding microball is stayed in the reaction tank 4;
G, reaction: by syringe pump solution to be detected is sucked long-channel 3, the probe molecule that enters at last in the reaction tank 4 with the coding microball surface reacts;
H, washing: after reaction finishes, fully wash to 4 pairs of microballoons of reaction tank from the 1 suction lavation buffer solution that enters the mouth by syringe pump;
I, detection: drain the lavation buffer solution in the reaction tank 4, utilize syringe pump from exporting the delivery damping fluid of 2 injection microballoons, the delivery damping fluid is brought microballoon into long-channel 3, and microballoon passes through long-channel 3 successively, and detected successively in long-channel 3.
Claims (6)
1. micro-fluidic chip based on the microballoon biological detection is characterized in that: 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 substrate or cover plate, be provided with functional microchannel network, in the network of microchannel, be provided with microballoon with coding carrier as biomolecule detection, in the network of microchannel, the periphery of reaction tank (4) is a screening pipeline (6), the periphery of screening pipeline (6) is subsidiary conduit (5), subsidiary conduit (5) joins with outlet (2), and reaction tank (4) taps into mouthful (1) by long-channel (3).
2. a kind of micro-fluidic chip according to claim 1 based on the microballoon biological detection, it is characterized in that biomolecule detection with microballoon as carrier, microballoon has coding, surface at microballoon is fixed with probe biomolecule, and used probe biomolecule can be a kind of in nucleic acid, protein, the polypeptide.
3. according to claim 1 and 2 described a kind of micro-fluidic chips, it is characterized in that in chip reaction tank (4), carrying out the reaction of microballoon and testing sample based on the microballoon biological detection; Solution to be measured and lavation buffer solution enter reaction tank (4) by import (1), react or the washing microballoon with microballoon in reaction tank (4), and reaction finishes microballoon and is transfused to detection and output in the passage (3).
4. a kind of micro-fluidic chip based on the microballoon biological detection according to claim 2 is characterized in that microballoon as the probe molecule carrier, and its material can be glass or polystyrene and rubber, and the size of microballoon is between 50 μ m～300 μ m; The host material of chip can be a kind of in glass, silicon, polymetylmethacrylate, dimethyl silicone polymer PDMS and the polycarbonate.
5. a kind of micro-fluidic chip according to claim 1 and 2 based on the microballoon biological detection, it is characterized in that coding microball be barcode encoding, fluorescence-encoded, quantum-dot coding, photonic crystal coding, Raman tag coding, infrared spectrum coding, shape coding, radio frequency coding, size coding and position encoded in a kind of.
6. a kind of micro-fluidic chip according to claim 1 based on the microballoon biological detection, the diameter that it is characterized in that reaction tank (4) is between 200 μ m～1200 μ m, and long-channel (3) width is between 60 μ m～550 μ m, and length is between 10mm～50mm.
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