CN103191792A - Microfluidic chip for microspheric multi-element biological detection - Google Patents
Microfluidic chip for microspheric multi-element biological detection Download PDFInfo
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- CN103191792A CN103191792A CN2013101354850A CN201310135485A CN103191792A CN 103191792 A CN103191792 A CN 103191792A CN 2013101354850 A CN2013101354850 A CN 2013101354850A CN 201310135485 A CN201310135485 A CN 201310135485A CN 103191792 A CN103191792 A CN 103191792A
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Abstract
The invention discloses a microfluidic chip for microspheric multi-element biological detection. The microfluidic chip comprises a cover piece, a substrate and a square hole array, wherein a plurality of parallel sample feeding channels and a reaction tank communicated with the sample feeding channels are arranged on the cover piece, and the outlet of the reaction tank is located on the inner surface of the cover piece; a plurality of parallel sample discharging channels and a sample discharging pool communicated with the sample discharging channels are arranged on the substrate, and the inlet of the sample discharging pool is located on the inner surface of the substrate; the square hole array is arranged between the reaction tank of the cover piece and the sample discharging pool of the substrate; the sample outlets of the sample discharging channels of the substrate are further connected with a liquid drop eduction tube; encoded microspheres are arranged in the reaction tank, and biological molecular probes are fixed on the surfaces of the microspheres; and the aperture of the square hole array is smaller than the diameter of the microsphere. During the application process of the chip, the feeding, reacting, washing and detecting operations of the microspheres and the sample are performed on the chip, so that the microfluidic chip has the advantages of high integration degree, convenience in use, small sample consumption and high detection sensitivity, and the like.
Description
Technical field
What the present invention relates to is a kind of micro-fluidic chip that detects for the microballoon multi-element biologic and preparation method thereof.This chip 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 for detection of biomolecule such as protein, DNA.
Background technology
Microfluidic analysis chip refers to make up the microflow path system that is made of little function element such as liquid storage tank, little reative cell, microchannels at chip by Micrometer-Nanometer Processing Technology, after loading biological sample and reactant liquor, under compression pump or electric field action, form microfluidic circuit, on chip, carry out a kind of or multiple reaction continuously, to realize the high flux rapid 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 micro electronmechanical process technology, process micro-structural networks such as micron-sized container, pump, valve, pipeline at microchip, processes such as reagent, reaction, separation, detection are carried out integrated micro-total analysis system with sampling, dilute, adding.The size of chip is about several square centimeters, select for use materials such as silicon chip, glass, quartz, high polymer as the chip underlying carrier, by methods such as etching, photoetching or die processing microchannel, adopt modes such as electricity, pressure, gravity, centrifugal force to drive the interior fluid of passage, adopt chemiluminescence, electrochemistry, absorbance, 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, reative cell and other functional part) is the micron order yardstick a dimension at least.Compare with the experimental provision of macro-scale, the micron order structure of micro-fluidic chip has enlarged markedly the specific area 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 diffusion 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 automation, 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 signal detection 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 speed is accelerated, and has reduced production cost simultaneously and is easy to carry.
Summary of the invention
Technical problem:The purpose of this invention is to provide a kind of is the micro-fluidic chip that solid phase carrier carries out biomolecule detection with the microballoon.With fixedly there being the coding microball of bioprobe molecule to carry out sample introduction, react, wash and detect with testing sample by chip, provide a kind of chip platform that detects analysis.
Technical scheme:Purpose of the present invention can be achieved through the following technical solutions:
A kind of micro-fluidic chip for the detection of microballoon multi-element biologic, it is characterized in that: this micro-fluidic chip comprises cover plate, substrate and square hole array, described cover plate is provided with a plurality of sample intake passages arranged side by side and the reaction tank that is communicated with described sample intake passage, and the outlet of this reaction tank is positioned on the inner surface of described cover plate; Described substrate is provided with a plurality of sample output passages arranged side by side and the sample-out pool that is communicated with described sample output passage, and the entrance of this sample-out pool is positioned on the inner surface of described substrate; Described square hole array is between the sample-out pool of the reaction tank of described cover plate and substrate; Outlet at described substrate sample output passage also is connected with a drop delivery line; In described reaction tank, be provided with the microballoon of coding, on the surface of this microballoon probe biomolecule arranged fixedly; The aperture of described square hole array is less than described diameter of micro ball.
The size of microballoon is between 45 μ m～300 μ m; All between 1.5mm ~ 2.5mm, all between 300 μ m ~ 1200 μ m, the injection port diameter of sample intake passage is between 200 μ m～1000 μ m for the height of reaction tank and sample-out pool for the diameter of reaction tank and sample-out pool, and length is between 5mm～20mm; The aperture of square hole array is between 40 μ m ~ 290 μ m.
Described probe biomolecule is nucleic acid, protein or polypeptide.
The material of described microballoon is silica or polystyrene and polyethylene glycol double methacrylate hydrogel; The material of described cover plate is glass, polymethyl methacrylate, dimethyl silicone polymer or Merlon; The material of described substrate is glass, polymethyl methacrylate, dimethyl silicone polymer or Merlon; The material of square hole array is polymer film, silicon chip or copper mesh.
The coding of microballoon is barcode encoding, fluorescence-encoded, quantum-dot coding, photonic crystal coding, Raman tag coding, infrared spectrum coding, shape coding, radio frequency coding, size coding or position encoded.
Its operation principle is:
A) utilize liquid-transfering gun to draw a certain amount of coding microball, microballoon is connected its delivery buffer solution by the injection port of sample intake passage, inject sample-out pool.Because the size of square hole array is less than the diameter of coding microball, so microballoon stays in the reaction tank of square hole array top, and waste liquid is derived by the drop delivery line by the outlet of sample output passage;
B) afterwards, the drop delivery line is connected with vavuum pump, drains the residual liquid in the chip;
C) at this moment, inject testing sample solution with liquid-transfering gun by injection port, make sample be full of the sample-out pool of square hole array top, testing sample solution and microballoon are fully reacted;
D) reaction finishes, and continues to inject lavation buffer solution by injection port;
E) after the washing fully, the drop delivery line is connected with vavuum pump, and the 5s ~ 10s that bleeds makes microballoon dry and snap in the square hole array under the air-flow effect, quantity by the control microballoon makes it be the individual layer distribution at the square hole array, the last reaction result that detects microsphere surface below chip.
Beneficial effect:According to the present invention, utilize chip to inject microballoon, application of 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) reagent consumption is few: because hybridization reaction only carries out in reaction tank, reagent is full of reaction tank only needs 2 μ L ~ 10 μ L, has avoided the generation of sample dead volume in the external pipeline, has reduced reagent consumption;
(2) detect diversification: the microballoon as solid phase carrier has multiple coding, fixes different probe molecules respectively, can detect a plurality of indexs in the same sample simultaneously.The ball type carrier specific area is big simultaneously, the detection sensitivity height, and reaction speed is fast;
(3) detect the flux height: chip can integrated a plurality of construction units, carry out the detection analysis of a plurality of samples simultaneously.
(3) degree of accuracy height: microballoon because the limitation effect of mesh is separated mutually, arranged in order, and the fluorescence between the microballoon does not exert an influence mutually, is convenient to image processing and decoding, the detection accuracy height above the square hole array;
(4) fluorescence background is low: reaction reagent is trapped in square hole array top, does not contact square hole array lower surface, produces fluorescence background when detecting by the chip below hardly.
(3) extensibility height: owing to adopted the form of micro-fluidic chip, can be integrated with micro-fluidic chips such as sample preliminary treatment easily, promoted microminiaturization and the automation of analytical 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 is the profile of chip of the present invention.
Fig. 2 is the vertical view of the cover plate of chip of the present invention.
Fig. 3 is the vertical view of the substrate of chip of the present invention.
Fig. 4 is during for chip detection of the present invention, the detection index path of single microballoon.
Fig. 5 is during for chip detection of the present invention, microballoon and square hole array vertical view.
Have among the above figure: 1, cover plate, 11, sample intake passage, 12, reaction tank, 2, substrate, 21, sample output passage, 22, sample-out pool, 3, the square hole array, 4, the drop delivery line.
The specific embodiment
Below in conjunction with accompanying drawing, the present invention is elaborated, biochip of the present invention is made of three parts, as Fig. 1, comprises the cover plate 1 of the superiors, the square hole array 3 in intermediate layer and the substrate 2 of lower floor; Cover plate 1 and substrate 2 structures such as Fig. 2, shown in Figure 3, be provided with a plurality of sample intake passages arranged side by side 11 and reaction tank 12 in the cover plate 1, be provided with a plurality of sample output passages arranged side by side 21 and sample-out pool 22 in the substrate 2, substrate is connected with drop delivery line 4 at the outlet of sample passage 21; Sample intake passage 11, reaction tank 12, square hole array, sample-out pool 22, sample output passage 21 and a drop delivery line 4 constitute a construction unit, and this biochip is made up of a plurality of construction units.Microballoon with different coding, its particle size are at 45 μ m～300 μ m, and the preparation material of microballoon is silica, polystyrene and polyethylene glycol double methacrylate (PEGDA) hydrogel etc.The material of square hole array can be polymer film (as PE, PP, PVC, PET, PC), silicon chip, copper mesh etc.; The aperture of square hole array can be between 40 μ m ~ 290 μ m; The diameter of reaction tank is between 1.5mm ~ 2.5mm, and the height of reaction tank is between 300 μ m ~ 1200 μ m, and the injection port diameter is between 200 μ m～1000 μ m, and length is between 5mm～20mm.The host material of chip can be glass, polymethyl methacrylate (PMMA), dimethyl silicone polymer (PDMS) and Merlon (PC) etc.
Embodiment one: with the PMMA(polymethyl methacrylate) as chip material, stationary probe carries out biological detection on photonic crystal coding glass microsphere, and microsphere diameter is 50 μ m, and the square hole array is the polycarbonate membrane of Laser Micro-Machining, and the aperture is 40 μ m.
The preparation of a, cover plate: adopt laser micromachining methods.By AutoCAD designed channel and reaction tank figure, but convert the CAD figure to the laser micro-machining system recognition instruction; Adopt laser micro-machining system in the required structure of substrate (or cover plate) processing, wherein, the sample intake passage diameter is 0.8mm, and long 5mm, reaction tank diameter are 2mm, highly are 1.2mm;
The preparation of b, substrate: identical with the cover plate preparation process, inserting external diameter at the outlet of sample output passage is 0.8mm, and length is the PE pipe of 7mm;
The involution of c, cover plate and substrate: be coated with the thinner PDMS of one deck (reserving the reaction tank part) at substrate, put it into 75 ℃ of baking oven precuring 5min afterwards, PDMS at semi-solid preparation places the square hole array, and two chip substrate alignment are bonding, fix two substrates and put into 75 ℃ of curing oven 1h with clip;
Stationary probe on d, the microballoon: clean microballoon is fixed to different probe molecules on the photon crystal micro-ball of different coding then through hydrophilic treated and silanization;
The connection of e, micro-fluidic chip: drop delivery line 4 connects vavuum pump;
F, sample introduction: with the injection port of coding microball together with its delivery buffer solution injection sample intake passage 11, enter sample-out pool 12 by liquid-transfering gun then, drain the delivery buffer solution at last, coding microball is stayed in the reaction tank 12;
G, reaction: by liquid-transfering gun solution to be detected is injected the injection port of sample intake passage 11, the probe molecule that enters at last in the reaction tank 12 with the coding microball surface reacts;
H, washing: after reaction finishes, fully wash to 12 pairs of microballoons of reaction tank by the injection port injection lavation buffer solution of liquid-transfering gun from sample intake passage 11;
I, detection: drain the lavation buffer solution in the reaction tank 12, the 5s ~ 10s that bleeds makes microballoon dry and snap in the square hole array the last reaction result that detects microsphere surface below chip under the air-flow effect.
Embodiment two: as chip material, stationary probe carries out biological detection on the quantum-dot coding polystyrene microsphere with PDMS and glass, and microsphere diameter is 210 μ m, and the square hole array is copper mesh, and the aperture is 200 μ m.
The preparation of a, substrate: selecting the matrix of PDMS and the ratio of curing agent is the 10:1(mass ratio), after mixing, through vacuum suction with after removing bubble, be cast in the container of the draw point of arranging, the 60min that is heating and curing, wherein, draw point length is 2cm, and diameter is 0.8mm.After being cured PDMS is taken off from container, namely make substrate in the terminal punching of draw point (aperture 2.5mm);
The preparation of b, cover plate: identical with substrate preparation method, after finishing, preparation uses the alcohol dampening cover plate, pull out draw point;
The involution of c, cover plate and substrate: the substrate that is cured and cover plate and two washed glass are after oxygen plasma treatment, place the square hole array at substrate, substrate and cover plate be clipped between two sheet glass fit, directly carry out the encapsulation of substrate and cover plate by the thermal bonding method then;
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: drop delivery line 4 connects vavuum pump;
F, sample introduction: with the injection port of coding microball together with its delivery buffer solution injection sample intake passage 11, enter reaction tank 12 by liquid-transfering gun then, drain the delivery buffer solution at last, coding microball is stayed in the reaction tank 12;
G, reaction: by liquid-transfering gun solution to be detected is injected the injection port of sample intake passage 11, the probe molecule that enters at last in the reaction tank 12 with the coding microball surface reacts;
H, washing: after reaction finishes, fully wash to 12 pairs of microballoons of reaction tank by the injection port injection lavation buffer solution of liquid-transfering gun from sample intake passage 11;
I, detection: drain the lavation buffer solution in the reaction tank 12, the 5s ~ 10s that bleeds makes microballoon dry and snap in the square hole array the last reaction result that detects microsphere surface below chip under the air-flow effect.
Claims (5)
1. one kind is used for the micro-fluidic chip that the microballoon multi-element biologic detects, it is characterized in that: this micro-fluidic chip comprises cover plate (1), substrate (2) and square hole array (3), described cover plate (1) is provided with a plurality of sample intake passages (11) arranged side by side and the reaction tank (12) that is communicated with described sample intake passage (11), and the outlet of this reaction tank (12) is positioned on the inner surface of described cover plate (1); Described substrate (2) is provided with a plurality of sample output passages (21) arranged side by side and the sample-out pool (22) that is communicated with described sample output passage (21), and the entrance of this sample-out pool (22) is positioned on the inner surface of described substrate (2); Described square hole array (3) is positioned between the sample-out pool (22) of the reaction tank (12) of described cover plate (1) and substrate (2); Outlet at described substrate (2) sample output passage (22) also is connected with a drop delivery line (4); In described reaction tank (12), be provided with the microballoon of coding, on the surface of this microballoon probe biomolecule arranged fixedly; The aperture of described square hole array is less than described diameter of micro ball.
2. a kind of micro-fluidic chip that detects for the microballoon multi-element biologic according to claim 1, it is characterized in that: the size of microballoon is between 45 μ m～300 μ m; The diameter of reaction tank (12) and sample-out pool (22) is all between 1.5mm ~ 2.5mm, the height of reaction tank (12) and sample-out pool (22) is all between 300 μ m ~ 1200 μ m, the injection port diameter of sample intake passage (11) is between 200 μ m～1000 μ m, and length is between 5mm～20mm; The aperture of square hole array (3) is between 40 μ m ~ 290 μ m.
3. a kind of micro-fluidic chip that detects for the microballoon multi-element biologic according to claim 1 and 2, it is characterized in that: described probe biomolecule is nucleic acid, protein or polypeptide.
4. a kind of micro-fluidic chip that detects for the microballoon multi-element biologic according to claim 1 and 2, it is characterized in that: the material of described microballoon is silica or polystyrene and polyethylene glycol double methacrylate hydrogel; The material of described cover plate is glass, polymethyl methacrylate, dimethyl silicone polymer or Merlon; The material of described substrate is glass, polymethyl methacrylate, dimethyl silicone polymer or Merlon; The material of square hole array is polymer film, silicon chip or copper mesh.
5. a kind of micro-fluidic chip that detects for the microballoon multi-element biologic according to claim 1 and 2 is characterized in that: the coding of microballoon is barcode encoding, fluorescence-encoded, quantum-dot coding, photonic crystal coding, Raman tag coding, infrared spectrum coding, shape coding, radio frequency coding, size coding or position encoded.
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