CN103191792B - Microfluidic chip for microspheric multi-element biological detection - Google Patents
Microfluidic chip for microspheric multi-element biological detection Download PDFInfo
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- CN103191792B CN103191792B CN201310135485.0A CN201310135485A CN103191792B CN 103191792 B CN103191792 B CN 103191792B CN 201310135485 A CN201310135485 A CN 201310135485A CN 103191792 B CN103191792 B CN 103191792B
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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 for microspheric multi-element biological detection and preparation method thereof.This chip, for detecting the biomolecule such as protein, DNA, can be widely used in clinical detection, inspection and quarantine, environmental monitoring, drug screening, microbial identification and the field such as nucleic acid and protein-function assays.
Background technology
Microfluidic analysis chip refers to and on chip, builds by Micrometer-Nanometer Processing Technology the microflow path system be made up of liquid storage tank, micro-reative cell, microchannel etc. micro-function element, after loading biological sample and reactant liquor, microfluidic circuit is formed under compression pump or electric field action, a kind of or multiple continuously reaction is carried out, to realize the high flux rapid analysis to DNA, protein and other biological component in tissue and cell on chip.It is a kind of analytical technology grown up in analytical chemistry field mid-term early 1990s, based on analytical chemistry and analytical biochemistry, apply micro electronmechanical process technology, microchip processes the micro-structural networks such as micron-sized container, pump, valve, pipeline, the processes such as sampling, dilution, reagent adding, reaction, separation, detection are carried out integrated micro-total analysis system.The size of chip is at about several square centimeters, select the materials such as silicon chip, glass, quartz, high polymer as chip underlying carrier, by method processing microchannels such as etching, photoetching or dies, the modes such as employing electricity, pressure, gravity, centrifugal force drive the fluid in passage, finally adopt chemiluminescence, electrochemistry, absorbance, fluorescence detector etc. to detect.The principal character of this chip on device is the resulting structure (comprising its passage, reative cell and other functional part) of its containing fluid is at least micron order yardstick in a dimension.Compared with the experimental provision of macro-scale, the micron scale construction of micro-fluidic chip significantly increases the specific area of fluid environment.This change causes the peculiar effect of a series of decision its property relevant with body surface in microfluidic system, as 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 and the volume of analytical equipment can be made to reduce, and equip more integrated automation, can significantly improve analysis efficiency and make sample and reagent consumption significantly descend degradation.Laboratory main equipment is integrated on operating platform little as far as possible, in order to complete different experimentations, and the technology can analyzed product.It not only makes the consumption of reagent reduce, and speed of experiment is improved, and expense reduces, and has fully demonstrated the development trend of microminiaturized, the integrated and portability of current laboratory equipment.Present micro-fluidic chip has been sent out application widely at biochemical analysis and environmental analysis aspects and has been developed fast.Because micro-fluidic chip is by the process of whole sample analysis, comprise sampling with the process of sample, preconcentration, dilution with mixing, be separated, chemical reaction and signal detection all integrated on the chip of a piece little, it achieves sub-micro and rises the reagent and sample consumption even receiving and upgrade compared with traditional analytical equipment, greatly reducing of reaction compartment makes the homogeneity of reaction improve, reaction speed is accelerated, and reduces production cost simultaneously and is easy to carry.
Summary of the invention
technical problem:the object of this invention is to provide a kind of take microballoon as the micro-fluidic chip that solid phase carrier carries out biomolecule detection.The coding microball being fixed with bioprobe molecule is carried out sample introduction by chip, reacted with testing sample, wash and detect, a kind of chip platform detecting analysis is provided.
technical scheme:object of the present invention can be achieved through the following technical solutions:
A kind of micro-fluidic chip detected for microspheric multi-element biological, it is characterized in that: this micro-fluidic chip comprises cover plate, substrate and square hole array, the reaction tank described cover plate being provided with multiple sample intake passage arranged side by side and being communicated with described sample intake passage, the outlet of this reaction tank is positioned on the inner surface of described cover plate; The sample-out pool described substrate being provided with multiple sample output passage arranged side by side and being communicated with described sample output passage, the entrance of this sample-out pool is positioned on the inner surface of described substrate; Described square hole array is between the reaction tank and the sample-out pool of substrate of described cover plate; A drop delivery line is also connected with at the outlet of described substrate sample output passage; In described reaction tank, be provided with the microballoon of coding, be fixed with probe biomolecule on the surface of this microballoon; The aperture of described square hole array is less than the diameter of described microballoon.
The size of microballoon is between 45 μm ~ 300 μm; The diameter of reaction tank and sample-out pool is all between 1.5mm ~ 2.5mm, and the height of reaction tank and sample-out pool is all between 300 μm ~ 1200 μm, and the injection port diameter of sample intake passage is between 200 μm ~ 1000 μm, and length is between 5mm ~ 20mm; The aperture of square hole array is between 40 μm ~ 290 μm.
Described probe biomolecule is nucleic acid, protein or polypeptide.
The material of described microballoon is silica or polystyrene and polyethyleneglycol diacrylate 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 encoding, 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 the injection port of its vehicle buffer by sample intake passage, inject sample-out pool.Size due to square hole array is less than the diameter of coding microball, and therefore microballoon is stayed in the reaction tank above square hole array, and waste liquid is derived by drop delivery line by the outlet of sample output passage;
B) after, drop delivery line is connected with vavuum pump, drains the residual liquid in chip;
C) now, inject testing sample solution with liquid-transfering gun by injection port, make sample be full of sample-out pool above square hole array, testing sample solution and microballoon are fully reacted;
D) react complete, continue through injection port and inject lavation buffer solution;
E) after washing completely, drop delivery line is connected with vavuum pump, bleed 5s ~ 10s, make microballoon dry and snap in square hole array under airflow function, make it in monolayer distribution on square hole array by the quantity controlling microballoon, finally the reaction result of microsphere surface detects in side under the die.
beneficial effect:according to the present invention, utilize chip to carry out injection microballoon, application of sample, reaction, washing and the continuous operation detected are that the solid phase carrier of biomolecule detection has the following advantages with 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, avoids the generation of sample dead volume in external pipeline, decreases reagent consumption;
(2) diversification is detected: the microballoon as solid phase carrier has Multi-encoding, fixes different probe molecules respectively, can detect the multiple indexs in same sample simultaneously.Ball type carrier specific area is large simultaneously, and detection sensitivity is high, and reaction speed is fast;
(3) flux is detected high: chip can integrated multiple construction unit, carries out the detection analysis of multiple sample simultaneously.
(3) degree of accuracy is high: the limitation effect of microballoon due to mesh above square hole array is separated mutually, ordered arrangement, and the fluorescence between microballoon does not have an impact mutually, and be convenient to image procossing and decoding, detection accuracy is high;
(4) fluorescence background is low: reaction reagent is trapped in above square hole array, does not contact square hole array lower surface, produces fluorescence background hardly when being detected by beneath chips.
(3) extensibility is high: owing to have employed the form of micro-fluidic chip, can be integrated with micro-fluidic chips such as sample pretreatments easily, facilitates microminiaturization and the automation of analytical system;
(3) the present invention combines biological detection advantage that is microsphere supported and micro-fluidic chip simultaneously, simple to operate to undersized microballoon, has distinct advantage.
accompanying drawing explanation
Fig. 1 is the profile of chip of the present invention.
Fig. 2 is the top view of the cover plate of chip of the present invention.
Fig. 3 is the top view of the substrate of chip of the present invention.
When Fig. 4 is chip detection of the present invention, the detection index path of single microballoon.
When Fig. 5 is chip detection of the present invention, microballoon and square hole array top view.
Have in above figure: 1, cover plate, 11, sample intake passage, 12, reaction tank, 2, substrate, 21, sample output passage, 22, sample-out pool, 3, square hole array, 4, drop delivery line.
detailed description of the invention
Below in conjunction with accompanying drawing, elaborate to the present invention, biochip of the present invention is made up of three parts, as Fig. 1, comprises the substrate 2 of the cover plate 1 of the superiors, the square hole array 3 in intermediate layer and lower floor; Cover plate 1 and substrate 2 structure are as shown in Figure 2 and Figure 3, multiple sample intake passage 11 arranged side by side and reaction tank 12 is provided with in cover plate 1, be provided with multiple sample output passage 21 arranged side by side and sample-out pool 22 in 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 form a construction unit, and this biochip is made up of multiple construction unit.Have the microballoon of different coding, its particle size is at 45 μm ~ 300 μm, and the material of preparing of microballoon is silica, polystyrene and polyethyleneglycol diacrylate (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 between 40 μm ~ 290 μm; The diameter of reaction tank is between 1.5mm ~ 2.5mm, and the height of reaction tank is between 300 μm ~ 1200 μm, and injection port diameter is between 200 μm ~ 1000 μm, 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: using PMMA(polymethyl methacrylate) as chip material, on photonic crystal coding glass microsphere, stationary probe carries out biological detection, and microsphere diameter is 50 μm, and square hole array is the polycarbonate membrane of Laser Micro-Machining, and aperture is 40 μm.
The preparation of a, cover plate: adopt laser micromachining methods.By AutoCAD designed channel and reaction tank figure, CAD Graphic Exchanging is become the instruction of laser micro-machining system identifiable design; Adopt the structure that laser micro-machining system needs at the upper machining of substrate (or cover plate), wherein, sample intake passage diameter is 0.8mm, long 5mm, and reaction tank diameter is 2mm, is highly 1.2mm;
The preparation of b, substrate: identical with 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: the PDMS (reserving reaction tank part) being coated with layer on substrate, put it into 75 DEG C of baking oven precuring 5min afterwards, the PDMS of semi-solid preparation places square hole array, and by two panels chip substrate alignment bonding, fix two panels substrate with clip and put into 75 DEG C of curing oven 1h;
Stationary probe on d, microballoon: different probe molecules, through hydrophilic treated and silanization, is then fixed on the photon crystal micro-ball of different coding by clean microballoon;
The connection of e, micro-fluidic chip: drop delivery line 4 connects vavuum pump;
F, sample introduction: the injection port by liquid-transfering gun, coding microball being injected sample intake passage 11 together with its vehicle buffer, then enter sample-out pool 12, finally drain vehicle buffer, coding microball is stayed in reaction tank 12;
G, reaction: the injection port by liquid-transfering gun, solution to be detected being injected sample intake passage 11, is finally entered in reaction tank 12 and react with the probe molecule on coding microball surface;
H, washing: after completion of the reaction, inject lavation buffer solution by liquid-transfering gun from the injection port of sample intake passage 11 and fully wash to reaction tank 12 pairs of microballoons;
I, detection: drain the lavation buffer solution in reaction tank 12, bleed 5s ~ 10s, and make microballoon dry and snap in square hole array under airflow function, finally the reaction result of microsphere surface detects in side under the die.
Embodiment two: using PDMS and glass as chip material, stationary probe carries out biological detection on quantum-dot coding polystyrene microsphere, and microsphere diameter is 210 μm, and square hole array is copper mesh, and aperture is 200 μm.
The preparation of a, substrate: select the matrix of PDMS and the ratio of curing agent to be 10:1(mass ratio), after mixing, through vacuum suction with after removing bubble, be cast in the container of arrangement draw point, carry out being heating and curing 60min, wherein, draw point length is 2cm, and diameter is 0.8mm.After being cured, PDMS is taken off from container, namely make substrate at draw point end punching (aperture 2.5mm);
The preparation of b, cover plate: identical with substrate preparation method, has prepared rear alcohol dampening cover plate, has pulled out draw point;
The involution of c, cover plate and substrate: the glass that the substrate be cured and cover plate and two panels are cleaned is after oxygen plasma treatment, substrate is placed square hole array, substrate and cover plate are clipped between two sheet glass and fit, then the direct encapsulation being carried out substrate and cover plate by thermal bonding method;
Stationary probe on d, microballoon: the Surfaces of Polystyrene Microparticles different probe molecules being fixed to different coding;
The connection of e, micro-fluidic chip: drop delivery line 4 connects vavuum pump;
F, sample introduction: the injection port by liquid-transfering gun, coding microball being injected sample intake passage 11 together with its vehicle buffer, then enter reaction tank 12, finally drain vehicle buffer, coding microball is stayed in reaction tank 12;
G, reaction: the injection port by liquid-transfering gun, solution to be detected being injected sample intake passage 11, is finally entered in reaction tank 12 and react with the probe molecule on coding microball surface;
H, washing: after completion of the reaction, inject lavation buffer solution by liquid-transfering gun from the injection port of sample intake passage 11 and fully wash to reaction tank 12 pairs of microballoons;
I, detection: drain the lavation buffer solution in reaction tank 12, bleed 5s ~ 10s, and make microballoon dry and snap in square hole array under airflow function, finally the reaction result of microsphere surface detects in side under the die.
Claims (5)
1. the micro-fluidic chip detected for microspheric multi-element biological, it is characterized in that: this micro-fluidic chip comprises cover plate (1), substrate (2) and square hole array (3), the reaction tank (12) described cover plate (1) being provided with multiple sample intake passage arranged side by side (11) and being communicated with described sample intake passage (11), the outlet of this reaction tank (12) is positioned on the inner surface of described cover plate (1); The sample-out pool (22) described substrate (2) being provided with multiple sample output passage arranged side by side (21) and being communicated with described sample output passage (21), 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 reaction tank (12) of described cover plate (1) and the sample-out pool (22) of substrate (2); A drop delivery line (4) is also connected with at the outlet of described substrate (2) sample output passage (22); In described reaction tank (12), be provided with the microballoon of coding, be fixed with probe biomolecule on the surface of this microballoon; The aperture of described square hole array is less than the diameter of described microballoon.
2. a kind of micro-fluidic chip detected for microspheric multi-element biological according to claim 1, is characterized in that: the size of microballoon is between 45 μm ~ 300 μm; 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 μm ~ 1200 μm, the injection port diameter of sample intake passage (11) is between 200 μm ~ 1000 μm, and length is between 5mm ~ 20mm; The aperture of square hole array (3) is between 40 μm ~ 290 μm.
3. a kind of micro-fluidic chip detected for microspheric multi-element biological according to claim 1 and 2, is characterized in that: described probe biomolecule is nucleic acid, protein or polypeptide.
4. a kind of micro-fluidic chip detected for microspheric multi-element biological according to claim 1 and 2, is characterized in that: the material of described microballoon is silica or polystyrene or polyethyleneglycol diacrylate 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 detected for microspheric multi-element biological 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 encoding, size coding or position encoded.
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| CN105021654B (en) * | 2015-04-17 | 2017-10-03 | 东南大学 | The preparation method and application method of quantitative detection system, mercury ion detecting chip |
| CN105203523A (en) * | 2015-09-25 | 2015-12-30 | 中国计量学院 | Microflow device based on SERS (surface enhanced Raman scattering) technology to detect specific antigens in serum |
| CN106918693A (en) * | 2015-12-24 | 2017-07-04 | 上海微柯力高分子材料有限公司 | A kind of microballoon micro-fluidic detection technology based on specific dimensions microballoon and plastic basis material |
| CN105689028B (en) * | 2016-01-20 | 2018-06-19 | 中国科学院上海微系统与信息技术研究所 | Micro-fluid chip, method and its application for the single distribution of immune microsphere |
| CN109126913A (en) * | 2018-08-06 | 2019-01-04 | 陈思 | A kind of porous micro-fluid chip |
| CN111077303A (en) * | 2019-12-25 | 2020-04-28 | 兰州大学 | Vertical channel array microchip and imaging device and method for immunodetection |
| CN112114133A (en) * | 2020-09-03 | 2020-12-22 | 武汉纺织大学 | Particle arrangement method for multiple biochemical detection |
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