CN1460855A - Microfluid biological sensor chip device and its application - Google Patents
Microfluid biological sensor chip device and its application Download PDFInfo
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- CN1460855A CN1460855A CN 03142327 CN03142327A CN1460855A CN 1460855 A CN1460855 A CN 1460855A CN 03142327 CN03142327 CN 03142327 CN 03142327 A CN03142327 A CN 03142327A CN 1460855 A CN1460855 A CN 1460855A
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Abstract
The present invention uses the replaseable biosensor chip technology as core, and uses the related MEMS system formed from microreactor and microflow cell to make determination of environment pollution. After the harmfule molecule (for example influenza virus or SARS virus) is combined on the functional chip, said polluted molecule can store the information, and can release it in specific environment, and its released information is directly propertional to the polluted extent, so that it can implement the determination of environmental pollution.
Description
Technical field:
The invention belongs to the biosensor technology field.Concrete will be referred to the micro-system that biochip that a kind of utilization has difference in functionality detects the different pollution source of environment.Utilize detection system of the present invention to can be used for to the chemical molecular in the environment (poison) gas, biomacromolecule, as: viruses molecule, pathogen etc. effectively detects.
Technical background:
The city dweller has time of 70% to spend indoor, and the condition of indoor environment has substantial connection to people's health.Though and the domestic and international at present determination techniques for environmental pollution has advanced large-scale instruments such as HPLC, mass spectrum, but mensuration in the face of a large amount of environmental pollutions, use not only cost height of large-scale instrument, and the secondary pollution that produces a large amount of organic wastewaters simultaneously in the process of measuring.Therefore, in the face of the sample concentration of environmental pollution is lower, enormous amount, and require the technical requirement height measured: promptly have fast, one of sensitive, the low-cost difficult point problem that has become hi-tech in the world.So far, yet there are no the technology report that solves this difficult problem both at home and abroad.
Summary of the invention:
Based on to above-mentioned in-problem consideration, the object of the present invention is to provide a kind of effectively, fast, the determination techniques of sensitive, environmental pollution cheaply, promptly created a kind of little detection system device that yet there are no the microfluid biological function chip of report so far both at home and abroad, utilize this little detection system to chemical drugs gas, liquid and biomacromolecule in the environment, as: virus comprises and can effectively detect pollutions such as popular SARS virus, cause of disease mattresses.
Little detection system device of microfluid biological function chip of the present invention is made up of biological function chip, sensor and microfluid.Its ultimate principle is as follows: after harmful molecule (or virus) is attached to biochip in the environment, this sensor can get off information storage, and in particular environment, the information that stores is discharged with technology such as physics, the degree that the information of its release is polluted with it is proportional, and can it be read and write out the high sensitivity mode easily, thereby finish the mensuration of environmental pollution.
Little detection system device of microfluid biological function chip of the present invention can be used to solve environmental pollution and measure key issue, can make present environment measuring reach a new height.
Technical characterictic of the present invention:
(1) function that possesses of biochip of the present invention has: (1) plays fixed support function; (2) functional molecular fixedly secures function; (3) on fixed support, has the fixing auxiliary agent of functional molecular; (4) keep the active function of biomolecule; (5) sample application wide ranges, wide adaptability does not need pre-service, and is easy to use, realizes robotization easily; (6) it can accept the information of environmental pollution, comprising: chemical toxic gas, liquid, biomacromolecule, antigenic virus, gasoloid, pathogen and chemical and biological weapons etc. also store the function of transmitting and reading easily with the information of polluting.
(2) basic function of sensor of the present invention: the filterableness absorbent material is as fixed support, and fixedly secures auxiliary agent at support with the help function molecule on this.In order to keep the active function of biomolecule, the filterableness material to be packed in the interlayer, the face that will contain functional molecular is the front of chip, towards sample inlet direction to be measured, the back side and polytetrafluoroethylene filter disc are combined.Be full of solution between functional biological chip and polytetrafluoroethylene sheet bilayer, purpose provides solution in order to keep bio-sensing chip, makes this biologic sensor chip (gas, liquid, liquid/solid interface) and keep it to have physiological function in a continuous medium system.Topped in the chip front side of functional molecular by the fine little plain filter disc of nitric acid.Biologic sensor chip can be replaced by the object difference of sample survey, as Fig. 1 and shown in Figure 5.When the sample gas that pollutes or liquid or antigenic virus molecule and pathogen during by this chip, the functional molecular of this chip can optionally be combined in testing molecule by requirement in advance looks like antigen, acceptor, part etc., in particular environment, it is discharged then and read, thereby finish the mensuration of environmental pollution.The sample determination overall schematic is Fig. 3, Fig. 5.
A kind of convenience that the present invention provides microfluid and two kinds of technical combinations of functional biological chip together, fast, sensitive biologic sensor chip is little detection system device of core technology, it is characterized in that it being that replaceable biological function chip and three major parts compositions of sensor are installed in the microfluid of its device:
(1) biological function chip: solid support by filter aspiration water material as macromolecular material, cellulose etc., the firmly fixed of auxiliary agent that functional molecular is fixing and functional molecular form.On the front of this solid support, scribble uniform positively charged molecule as poly-D-lysine; Or the fine little plain molecule of nitric acid; Protein A etc. helps the help agent of functional molecular strong bonded on solid support, and with the fixedly securing of functional molecular.The solid support front is a biologically functional molecule, and this functional molecular can be a protein molecule as antibody; The lipase body; Acceptors etc., the back side and another polytetrafluoroethylene filter disc are combined.And be full of solution between their bilayers, purpose provides solution environmental in order to keep biologically functional molecule, makes this biologic sensor chip at a continuous medium gas, keeps it to have physiological function in the liquid/solid interface system.At the fine little plain filter disc of a positive topped nitric acid of functional molecular.Assembling kit such as Fig. 1 of functional chip; Fig. 3 and shown in Figure 5.
(2) preparation of sensor: the functional protein molecule chip for preparing on above-mentioned filter aspiration water material is called biologic sensor chip, be assembled into this chip as shown in Figure 5 replaceable: the said chip of packing in the pocket with double-deck sandwich material sealing, double-deck sandwich material is by polytetrafluoroethylene film, the fine mems thin film of acetic acid and fine mems thin film (NC) of nitric acid and the common composition of functional molecular be called sensor.Obviously, can prepare different sensors according to sample to be checked, can prepare becomes the replaceable sensor of series.According to the replaceable sensor with the different biological functions of biomacromolecule of above-mentioned technology preparation of the present invention, can realize for the those of ordinary skill of technical field.
(3) being connected of the preparation of microfluid and sensor: microfluid with macromolecular material (organic glass) as material preparation as shown in Figure 5, runner one side in organic glass is connected with sample inlet, opposite side is the outlet duct of sample, embeds sensor in the inside in outlet duct.Above-mentioned replaceable sensor and microfluidic channel plate are packaged together, the rubber ring of sealing of the binding between sensor embedding and the microfluid, connection one end of sample export connects the microkinetic device.Connect with emulsion tube respectively being connected of microkinetic device and surge chamber as: shown in Figure 5.
(3) mensuration of sample sensor:
Above-mentioned replaceable sample sensor can be noted the information of polluting, and carries out Fast Reading and record for different samples with different modes: as the available immunological technique of polluting for antibody antigen of mensuration; Mensuration available light physical technique such as synoptic diagram for poisonous chemical gas are shown in Figure 4.
Aforesaid replaceable bio-sensing chip, biology sensor and described microfluid, to the gas in the contaminated environment, chemical moleculars such as liquid, gasoloid, biomacromolecule, as: the application during virus, pathogen detect.The mensuration that this technology popularization can be polluted to other chemical drugs simultaneously, as gas Trucks waste gas, also can Application and Development in the monitoring of the poisonous gas of military affairs, or food preservation monitoring and Feng Wei Monitoring fixed on and the screening of clinical medical.
Description of drawings:
The assembly drawing of Fig. 1 bio-sensing chip and microreactor:
The fine little plain film of 1 chip surface acetic acid; 2 base chips (the polytetrafluoroethylene filter disc is directly through 23mm); 3 bio-sensing chip NC films (fixed support has the fixing auxiliary agent of functional molecular on functional molecular and the fixed support); 4,5 microreactor sequences; With the shadow figure after the microreactor assembling.
Fig. 2 formaldehyde determination experimental result
C: be control group; A: at formaldehyde air pollution set of circumstances 10min.; B: be formaldehyde air pollution set of circumstances 20min.;
Each sample is 10 mean values; After the line focus of illumination condition 150W Halogen lamp LED, apart from the 50cm place of sample, according to 1min.
Fig. 3 sample determination process flow diagram
A: sample (can be liquid or gas); B: microfluid and microreactor; C: be power source
The connection of A and B or B and C is common medical sebific duct
Fig. 4 low light level is measured synoptic diagram
A: visible light source; B: biologic sensor chip; C: low light level analyzer
Fig. 5 microfluid and microreactor assembling:
Unit: mm material: organic glass ratio 1:1
(A) top (B) bottom
1 gas feed; 2 microfluid tops; 3 encapsulants; 4 microreactors and combination biologic sensor chip; 5 fixed supports; 6 microfluids bottom; 7 is gas vent
The connection place of A and B is with the rubber ring of sealing, and seals with screw thread rotation.
Fig. 6 influenza antigen is measured (hydrometry A figure; Eudiometry B figure).
The ultrasonic gasification synoptic diagram of Fig. 7 fluid sample: 1 air intake opening; 2 ultrasonic covers; 3 connect biologic sensor chip and air extractor; The atomizing of 4 samples; 5 ultrasonic generator heads; 6 Ultrasound Instrument.
Embodiment:
Biological function sensing chip apparatus for a better understanding of the present invention and concrete the detection further explain by following examples, but listed examples are not to be limitation of the invention.
Embodiment: the mensuration of 1 formaldehyde contaminated environment gas:
Ultimate principle: at filterableness absorbent material (fixed support, with cellulose (No. 1) filter paper, diameter is 6mm), and be adsorbed with poly-D-lysine (every of 5ul/ in this filter disc front, poly-D-lysine concentration 0.01mg/ml) after to be dried, fixedly secure on filter paper with photosynthesis composite body (Chromatophores) (protein complex) after three hours in baking under 90 ℃.In order to keep the active function of photosynthesis compound, the filter paper sheet material to be packed in the interlayer, the face that will contain functional molecular is the front of chip, towards sample inlet direction to be measured, the back side and polytetrafluoroethylene filter disc are combined.Be full of solution between functional biological chip and polytetrafluoroethylene sheet bilayer, purpose provides solution in order to keep bio-sensing chip, makes this biologic sensor chip (gas, liquid, liquid/solid interface) and keep it to have physiological function in a continuous medium system.Topped in the chip front side of functional molecular by the fine little plain filter disc of nitric acid.The functional chip assembling becomes replaceable, as Fig. 1.When the formaldehyde gas molecule passes through above-mentioned functional chip, formaldehyde molecule combines with amino acid in the photosynthetic albumen (LH1-RC), information behind this protein combination formaldehyde molecule can store easily, transmit, and this photosynthesis produces phosphorylation (producing ATP) under the condition of illumination, available chemiluminescence method is measured, and the ATP ability of its generation is formed significant difference with contrast, thereby finishes the mensuration of PARA FORMALDEHYDE PRILLS(91,95) environmental pollution.
(1) photophosphorylation sensitive material support C hromatophores preparation:
(1) photosynthetic bacteria is cultivated, photosynthetic bacteria (R.rubrum, ATCC NO.11170) cultivation and results: the cultivation temperature of bacterium is 28 ± 2 ℃, and high light (4,000Lx) cultivated 5-7 days down, 5,000g centrifugal collecting precipitation, the extraction sample of carrier (Chromatophores) TS damping fluid (50mMTricine-NaOH, pH8.0,0.25M sucrose, 5mM MgCl
2) wash once, and then after adding the TS buffer suspension of 15ml, add the lysozyme of 1mg/ml, in ice, hatched ultrasonic 10 minutes 30 minutes, 4 ℃, 25, centrifugal 30 minutes of 000g keeps supernatant again with supernatant 180,000g 4 ℃ centrifugal 90 minutes, precipitation is support C hromatophores (being called the photophosphorylation functional material).
(2) above-mentioned photophosphorylation functional material is prepared into the biological function chip sensor: the about 10ul of photophosphorylation material (Chromatophores) (protein concentration 5mg/ml) is evenly distributed on cellulose filter disc (6mm diameter, thick 0.1mm) and be securely fixed in the middle of the cellulose filter disc, (after this cellulose filter disc soaks 2 hours with 0.01% poly-D-lysine in advance, 90 ℃ keep 3 hours after), add then between the interlayer of polytetrafluoroethylene filter disc combination, and be embedded in the organic glass microtubule and can cooperate assembling with microfluid.
(3) assembling of biological function chip sensor and microfluid is seen shown in Figure 5 as previously mentioned.
(2) microfluid that is suitable for room air/liquid contamination designs.
(1) design of microfluid and manufacturing:
A. the flow scheme design planimetric map of microfluid: (Fig. 3)
B. the selection of base material: have advantages such as good, the easy processing of light transmission, cheapness with macromolecular material such as PMMA (organic glass).
C. the preparation of Biofunctional materials: cellulose tablet is equipped with functional material and is clipped between the polytetrafluoroethylene film and composite intermediate layer is packed into one prepare in advance in the Xiao Chi, the diameter of this Xiao Chi is 30mm.(Fig. 5) a netted organic glass sheet being housed at the back side of Xiao Chi uses as the fixed function material.
D. select the microminiature power source to connect the last interface of microfluid, the functional material composite intermediate layer put into and microfluid between binding with the sealing rubber ring, connection between air pump and the microfluid is with common medical sebific duct, indoor gas (or gas to be measured, liquid) is entered by flowing out from sample export behind the chip from the inflow point of sample.
(2) interconnection technique of microfluid encapsulation technology and research photophosphorylation sensitive material and substrate: choose the microfluid of 22 mm dias, top was fixed with connecting with screw thread of lower bottom part, middle rubber ring with sealing.Itself and microfluidic channel are packaged together.Form (Fig. 1) with seven 6 millimeters functional chips of diameter.Be convenient to putting into of sensitive material composite intermediate layer (Kit) and connecting of air pump.(Fig. 3; Fig. 5)
(3) low light level determination techniques
The low light level is measured: functional chip is fetched from microfluid put into a capsule, and (ATP synthesizes damping fluid: 50mM Tricine-NaOH (pH8.0), 5mM MgCl to add Buffer
2, 4mM NaH
2PO
415mM glucose, 5mM DTT. suspends centrifugal Chromatophores with above-mentioned synthetic damping fluid) add 2mM ADP after, interrupt reactant (4%TCA (trichloroacetic acid) cessation reaction that adds 1/10th volumes then) with visible light according to adding immediately behind the 1min., then sample is added luciferase (Luciferase) and fluorescein (luciferin) 1umol/L, survey the synthetic amount of ATP, carry out with the luminous detection instrument.The results are shown in Figure 3, the ATP amount that photophosphorylation produces after sample is exposed in formaldehyde or the air 10min. respectively is respectively: ten mean values of 3500nm and 7000nm. sample.And it is measured concentration and is exposed to time direct ratio in formaldehyde or the air, when be exposed to the formaldehyde time be 20min. after the ATP amount that produces of photophosphorylation be respectively 1700nm obviously shows this method PARA FORMALDEHYDE PRILLS(91,95) gas-sensitive from Fig. 3 measurement result.
Embodiment 2: the mensuration of Antibody of Influenza antigen
1. material: isolate among influenza virus A (shang hai/7/99) the influenza patient, through 9～10d instar chicken embryo allantoic cavity inoculation propagation, the fresh allantoic fluid of being gathered in the crops for experiment with virus.Above-mentioned strain is cultivated the polyclonal antibody that is prepared into this susceptible poison through inoculation SPF chicken immune at virus-culturing fluid.
2. the pre-service of chip is as follows: after the NC film soaked with 20% methyl alcohol 20mM phosphate solution 50mM pH7.4 in two hours, with 20mM phosphate solution 50mM pH7.4 washing three times, with ProteinA1mg/ml 100ul carbonate solution 50mM pH9.6,4 ℃ are spent the night, add after 4 ℃ of the every hole 10ug of polyclonal antibody spend the night, spend the night with the 10%BSA sealing.
3. fluorescent technique: chip taken out on fluorescence spectrophotometer, measure condition determination: excite 491nm, emission 518nm, slit: Ex:3nm; Em:10nm: the result is as follows: can see that from hydrometry Fig. 6 A or eudiometry 6B Antibody of Influenza antigen liquid or eudiometry dilution with antigen increases fluorescence signal and reduces.Provable thus this method can promote the use of the antibody antigen of virus and measure.
The mensuration process is as follows: add viral antigen (viral HA) respectively and be diluted to (HAU) on chip after the above-mentioned pre-service: 4,2,1,0.5 and 10
4, 10
3, 10
2, 10, the virion subnumber.(correspond among Fig. 5: 1,2,3,4,5,6,7,8).The room temperature microfluid is (flow velocity 1ml/ per minute) after 5 minutes, wash with phosphate solution 50mM pH7.4,10 times, behind the non-specific material of flush away, add antibody with the FITC mark, the microfluid room temperature is (flow velocity 1ml/ per minute) after 5 minutes, after phosphate solution 50mM pH7.4 washing, the chip taking-up is measured on fluorescence spectrophotometer.For the mensuration of influenza virus gas with the virus in the gas of this ultrasonic atomizatio of ultrasonic atomizing device (as Fig. 7) at once by in the chip after the pre-service in the microfluid of suction filtration in Fig. 3, and specific adsorption, wash with phosphate solution 50mM pH7.4 with molecule for non-specific adsorption, after 10 times, after the adding, add antibody with the FITC mark, the microfluid room temperature is (flow velocity 1ml/ per minute) after 5 minutes, wash the antibody of unconjugated FITC mark with phosphate solution 50mM pH7.4, the chip taking-up is measured on fluorescence spectrophotometer, condition determination: excite 491nm, emission 518nm, slit: Ex:3nm; Em:10nm: Fig. 6 A (or B) as a result, Antibody of Influenza antigen liquid (or gas) measure the dilution that can see with antigen to be increased fluorescence signal and reduces.Provable thus the present invention can promote the use of the antibody antigen of virus and measure.
Claims (7)
1, a kind of convenience, fast, the sensitive little detection system device of microfluid biological function chip, but it is characterized in that it being the information that can accept environmental pollution is installed and the information of polluting is stored easily, transmit, reads and the replaceable bio-sensing chip of measurement function in the microfluid of this device, and biology sensor, above-mentioned replaceable sensor and microfluidic channel plate are packaged together, binding between sensor embedding and the microfluid rubber ring of sealing, connection one end of sample export connects the microkinetic device.The microkinetic device connected as shown in Figure 5 with emulsion tube with being connected respectively of surge chamber, above-mentioned replaceable sample sensor can be noted the information of polluting, and carries out Fast Reading and record for different samples with different modes: as the available immunological technique of polluting for antibody antigen of mensuration; For the mensuration of toxic gas available photophosphorylation technology and the low light level determination techniques shown in Figure 4 as synoptic diagram.
2, the composition of replaceable bio-sensing chip according to claim 1 and biology sensor, it is characterized in that it being the macromolecular material material of filtering aspiration water by a kind of, as: cellulose, as solid support, scribble uniform positively charged molecule in the front of this solid support as poly-D-lysine; Or the fine little plain molecule of nitric acid; Protein A helps the help agent of functional molecular strong bonded on solid support, and its biologically functional molecule can be fixedly secured thereon, and this biologically functional molecule can be a biomacromolecule as antibody; The lipase body; Acceptors etc., the fixed support back side and another polytetrafluoroethylene filter disc are combined.And be full of buffer solution between their bilayers, purpose provides solution environmental in order to keep biologically functional molecule, and makes this biochip keep it to have physiological function in continuous gas or solid medium interface system.At the fine little plain filter disc of a positive topped nitric acid of biologically functional molecule.The assembling of functional chip is used polytetrafluoroethylene film as shown in Figure 1, and the said chip of packing in the pocket of the common sandwich material sealing of forming of fine mems thin film of acetic acid and the fine mems thin film of nitric acid is replaceable biology sensor.
3, the microfluid of this device according to claim 1 is a kind ofly to manufacture as material with the macromolecular material organic glass, runner one side that designs in organic glass is connected with sample inlet, opposite side is the outlet duct of sample, embeds with biology sensor in the inside in outlet duct to connect.Above-mentioned replaceable biology sensor and channel plate are packaged together, the rubber ring of sealing of the binding between sensor embedding and the passage, connection one end of sample export connects the microkinetic device.Connect with emulsion tube respectively being connected of microkinetic device and surge chamber as: device shown in Figure 5.
4, according to claim 1,2,3 described replaceable biology sensors, be meant difference, in microfluid, connect different biology sensors according to sample to be checked.
But 5, according to claim 1ly the information of polluting is stored easily, transmits, reads and the replaceable bio-sensing chip of measurement function, be meant that replaceable sample sensor can note the information of polluting, for different samples with different modes carry out Fast Reading and record as: for the available immunological technique as shown in Figure 6 of mensuration of antibody antigen pollution; Shown in Figure 4 for available other technology of the mensuration of toxic gas such as synoptic diagram.
6, the little detection system device of microfluid biological function chip according to claim 1 is to the gas in the contaminated environment, gas Trucks waste gas, liquid, aerosol chemistry molecule, biomacromolecule, food preservation monitoring and Feng Wei Monitoring is fixed, application in the screening of detection in the chemical drugs, virus, pathogen and clinical medical.
7, replaceable bio-sensing chip according to claim 2 and biology sensor be or/and microfluid according to claim 3, to the gas in the contaminated environment, gas Trucks waste gas, liquid, aerosol chemistry molecule, biomacromolecule, food preservation monitoring and Feng Wei Monitoring is fixed, application in the screening of detection in the chemical drugs, virus, pathogen and clinical medical.
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| CN1947011B (en) * | 2004-02-26 | 2011-06-01 | 丹麦台达电子光学声学公司 | Method, chip, device and integrated system for detection of biological particles |
| CN102174382A (en) * | 2011-01-18 | 2011-09-07 | 北京大学 | System and method for monitoring bioaerosol in real time |
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