CN108067311A - A kind of micro-fluidic chip and detection method for Microcystin detection - Google Patents
A kind of micro-fluidic chip and detection method for Microcystin detection Download PDFInfo
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- CN108067311A CN108067311A CN201611005361.0A CN201611005361A CN108067311A CN 108067311 A CN108067311 A CN 108067311A CN 201611005361 A CN201611005361 A CN 201611005361A CN 108067311 A CN108067311 A CN 108067311A
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- SRUWWOSWHXIIIA-UKPGNTDSSA-N Cyanoginosin Chemical compound N1C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](C)[C@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C)NC(=O)C(=C)N(C)C(=O)CC[C@H](C(O)=O)N(C)C(=O)[C@@H](C)[C@@H]1\C=C\C(\C)=C\[C@H](C)[C@@H](O)CC1=CC=CC=C1 SRUWWOSWHXIIIA-UKPGNTDSSA-N 0.000 title claims abstract description 49
- 108010067094 microcystin Proteins 0.000 title claims abstract description 49
- 238000001514 detection method Methods 0.000 title claims abstract description 46
- 239000000758 substrate Substances 0.000 claims abstract description 71
- 238000006243 chemical reaction Methods 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 38
- 239000007788 liquid Substances 0.000 claims abstract description 22
- 239000011521 glass Substances 0.000 claims abstract description 19
- 239000003153 chemical reaction reagent Substances 0.000 claims abstract description 17
- 239000003053 toxin Substances 0.000 claims abstract description 17
- 231100000765 toxin Toxicity 0.000 claims abstract description 16
- 108700012359 toxins Proteins 0.000 claims abstract description 16
- 239000000427 antigen Substances 0.000 claims abstract description 10
- 108091007433 antigens Proteins 0.000 claims abstract description 10
- 102000036639 antigens Human genes 0.000 claims abstract description 10
- 238000007385 chemical modification Methods 0.000 claims abstract description 5
- 239000002699 waste material Substances 0.000 claims abstract description 4
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- 229910021641 deionized water Inorganic materials 0.000 claims description 30
- 239000000243 solution Substances 0.000 claims description 28
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 20
- 239000000523 sample Substances 0.000 claims description 20
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid Substances OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 18
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 14
- 229920002120 photoresistant polymer Polymers 0.000 claims description 14
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 12
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- 108010049746 Microcystins Proteins 0.000 claims description 11
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 10
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- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
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- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- 241000192700 Cyanobacteria Species 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 108010073357 cyanoginosin LR Proteins 0.000 description 2
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- RZYKUPXRYIOEME-UHFFFAOYSA-N CCCCCCCCCCCC[S] Chemical compound CCCCCCCCCCCC[S] RZYKUPXRYIOEME-UHFFFAOYSA-N 0.000 description 1
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- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 1
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- JIGDOBKZMULDHS-UUHBQKJESA-N microcystin RR Chemical compound C([C@H](OC)[C@@H](C)\C=C(/C)\C=C\[C@H]1[C@@H](C(=O)N[C@H](CCC(=O)N(C)C(=C)C(=O)N[C@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@H]([C@H](C)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N1)C(O)=O)C(O)=O)C)C1=CC=CC=C1 JIGDOBKZMULDHS-UUHBQKJESA-N 0.000 description 1
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- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
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Classifications
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
- B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
- B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
- B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
- B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
- B01L2200/00—Solutions for specific problems relating to chemical or physical laboratory apparatus
- B01L2200/08—Ergonomic or safety aspects of handling devices
- B01L2200/087—Ergonomic aspects
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N2021/7756—Sensor type
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- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Analytical Chemistry (AREA)
- Clinical Laboratory Science (AREA)
- Hematology (AREA)
- Dispersion Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of micro-fluidic chips for Microcystin detection, including organic glass system into substrate and cover plate, the cover plate hot key is together on the substrate, microfluidic circuit is carved on the substrate, the microfluidic circuit includes 1 entrance of reagent, hybrid channel, 2 entrance of reagent, reaction channel, sense channel and waste liquid outlet successively, the hybrid channel and the reaction channel are snakelike meander-like, and the reaction channel inner surface is by chemical modification and is fixed with microcystic toxin antigens.The invention also discloses the microcystic toxins checking methods based on above-mentioned micro-fluidic chip.Micro-fluidic chip disclosed by the invention is at low cost, and chip repeats utilization, and detection method has the advantages that precise and high efficiency, quick, detection limit is low.
Description
Technical field
The present invention relates to enviromental monitoring equipment field more particularly to it is a kind of for Microcystin detection it is micro-fluidic
Chip and detection method.
Background technology
In recent years, with water pollution and the aggravation of eutrophic extent, the degree of algae and water pollution is also deepened year by year,
Red tide or wawter bloom frequently occur in the world be algae pollution degree deepen direct performance.The occurrence frequency of wawter bloom and rule
Mould also getting worse, not only Microcystin (Microcystins, MCs) content raises in surface water and source water, Er Qiewei
Capsule algae toxin can also be enriched in fishes and shrimps body, be then enriched in human body by food chain, damaged to human health, drawn
Play worldwide concern.The key character of cyanobacterial bloom pollution is exactly the malicious feature of production, and the wawter bloom of 50-70% can generate poison all over the world
Element, wherein the malicious cyanobacteria of main production is some strains in anabena, synnema algae and microcystis kutz.In above-mentioned toxin, Microcystin
It is widely distributed, toxicity is larger, harm is the most serious, thus be concerned.
Microcystin (Microcystins, MCs) is a kind of monocyclic heptapeptide substance generated by cyanobacteria, is had apparent
Hepatotoxicity, the hepatotoxin of strong carcinogenesis.MCs has water-soluble and heat resistance, is soluble in methanol or acetone, non-volatile,
The characteristics such as anti-pH variation and chemical property quite stable, be distributed in cyanobacterial bloom it is most wide, endanger most serious.It is presently found
Species more there are content and larger toxicity has 3 kinds of MC-LR, MC-RR and MC-YR in 90 various microcystins.In water body
MC drinks humans and animals
Safety and aquatic ecosystem are stabilized serious threat.The World Health Organization provides at present：Drinking water
In MC must not be higher than 1 μ g/L, what China 2006 promulgated《Standards for drinking water quality》(GB5749-2006) provide, drink
1 μ g/L are must not exceed with the concentration of the MC-LR in water.
Relevant patent of invention 200910247559.3 disclose it is a kind of quickly detected for Microcystin it is micro-fluidic
Chip and preparation method thereof.Method of molding is used to have example reaction microchannel with materials such as optically transparent dimethyl silicone polymers
Layer, valve key-course and substrate layer are formed.Contain example enrichment and immunoassay module in chip, the module is by one or several
The immune chromatograph position analysis room composition of a nano-volumes, each analysis room is fixed with antibody protein or antigen.The chip village then this
Complicated for operation, detection needs complicated signal acquisition module, microprocessor and database, also uses chromatogram analysis method, examines
Survey difficulty is big, speed is slow etc..
Bioanalysis, physical chemistry and immunoassay three are broadly divided into the technology that Microcystin is detected both at home and abroad
Class.For bioassay technique mainly using zoopery method and Cytotoxicity assays, the former is the conventional toxicity point used earliest
Analysis method, it is necessary to consume more toxin, sensitivity and specificity not high, and can not accurate quantification, can not distinguish isomers
Type etc..The latter is mainly detected using the toxic action of endotoxins on cells, although sensitivity is higher, but accurate quantification is still
Operate it is relatively cumbersome, and in the elementary step.For the preferable physical-chemical process of reappearance, the functions of the equipments that use are single,
Volume is larger and expensive, and portability and selectivity are poor.
Compared with preceding two classes detection technique, immunoassay is a kind of very potential method, and specificity is strong, sensitive
Degree is high.It is mainly carried out qualitative and quantitative using contratoxin the characteristics of antigen and the single-minded of antibody, specific binding.It is common to exempt from
Epidemic disease detection method has competition detection method, indirect method, " sandwich method " etc..The commercial kit that immunoassay uses is expensive, hand
Work operate and it is comparatively laborious, its degree of automation need to be improved.
Detection technique in relation to Microcystin is towards continuous detection, quick analysis, high-throughput and increasingly automated direction
Development.And the microsensor or analytical instrument based on micro-fluidic are following development trends, and conventional method of analysis ratio,
Microfluidic chip technology has analyze speed fast, and work efficiency is high, reagent consumption is few, system altitude is concentrated, high degree of automation
The advantages that.Micro-fluidic chip and immunoassay are combined, the reaction speed of antigen and antibody is not only increased, also reduces sample
With the consumption of reagent.
Therefore, those skilled in the art be directed to developing it is a kind of immunoassay and microflow control technique are combined, establish one
The method that kind efficiently, fast and automatically detects Microcystins in Water.
The content of the invention
In view of the drawbacks described above of the prior art, the present invention provides a kind of micro-fluidic cores for Microcystin detection
Piece, including organic glass system into substrate and cover plate, the cover plate hot key is carved with miniflow together on the substrate on the substrate
Road, the microfluidic circuit go out successively including 1 entrance of reagent, hybrid channel, 2 entrance of reagent, reaction channel, sense channel and waste liquid
Mouthful, the Microcystin solution inlet and PBS that 1 entrance of reagent includes sample water inlet to be measured, horseradish peroxidase marks
Solution inlet, 2 entrance of reagent include dodecyl sodium sulfate entrance, enzyme reaction substrate entrance, bovine serum albumin entrance, two
Anti- entrance and Microcystin antibody-solutions entrance, the hybrid channel and the reaction channel are snakelike meander-like, described anti-
Channel inner surface is answered by chemical modification and is fixed with microcystic toxin antigens.
Preferably, the substrate microfluidic circuit uses photolithography method, includes the following steps：
(1) surface treatment of glass substrate：Substrate is put into the concentrated sulfuric acid and is boiled 30 minutes, isopropyl is put it into after bath
Alcohol is 1 with acetone:It impregnates 10 minutes, is then ultrasonically treated 10 minutes in ethanol, after deionized water rinsing in 1 mixed solution
Nitrogen dries up, be put into baking oven 150 DEG C dry 1 it is small when；
(2) gluing：By the spin-coated machine spin coatings of negtive photoresist RFJ-220, gluing rotating speed is 1000rpm, and after 30 seconds, glue thickness is 5 μm；
Substrate is placed on front baking 6 minutes on hot plate, evaporates remaining solvent in photoresist；
(3) it is exposed and developed：Uv-exposure, exposure energy 18.9MJ/cm2 are carried out to substrate, the time for exposure is 9 seconds；
Glass substrate after exposure developing liquid developing 2 minutes, after developer solution naturally dry, 150 DEG C of post bakes 35 minutes；
(4) primer is swept：Glass substrate is put into and carries out within 2 minutes in plasma degumming machine sweeping primer, removal development rear surface
Remaining photoresist；
(5) etch：It carries out at room temperature, corrosive liquid etches for the different degrees of diluted buffer oxide of addition 10%HCl
Liquid often etches post bake 10 minutes after twenty minutes, continues to etch；After the completion of etching, photoresist is removed with fuming nitric aicd.
Preferably, the thermal bonding of the substrate and the cover plate includes the following steps：
(1) the affine processing in surface：Substrate surface is cleaned successively with deionized water, acetone and absolute ethyl alcohol, deionized water, then
With deionized water rinsing after dilute HF acid rinses, then with volume ratio 1：1 ammonium hydroxide：Hydrogen peroxide cleans, and finally uses deionized water rinsing；
(2) pre- bonding：Under no super-clean environment, by substrate and not stained new cover plate is directed at fitting in ultrapure water environment
Afterwards, substrate is made to be fitted closely with cover plate together with and slided without mutual, vacuum drying chamber, dry 1h are put in taking-up into；
(3) high temperature is bonded：Substrate after pre- bonding and cover plate are lain in high temperature furnace, it is each in substrate and cover plate upper and lower
One piece of polished graphite cake is put, presses one block of stainless steel on graphite cake above again；High temperature furnace heating rate is 10 DEG C/min, is selected
It selects in 620 DEG C of heat preservation a period of times, then room temperature is cooled to 10 DEG C/min.
Preferably, the chemical modification includes the following steps：
(1) clean：It is 1 that volume ratio is first used in reaction channel described in substrate:The mixed liquor of 1 methanol and hydrochloric acid is cleaned by ultrasonic
30min is dried up after deionized water cleaning；
(2) substrate activates：It is 3 by volume ratio:1 concentrated sulfuric acid and the mixed liquor of hydrogen peroxide are passed into chip channel, and 80
DEG C or so boil 40min；
(3) end deionized water rinsing is reacted, then chip is dried up with nitrogen；
(4) silanization：Under nitrogen protection, it is 1 by volume ratio:9 3- aminopropyl triethoxysilanes and acetone it is mixed
It closes liquid and is passed through reaction channel, for 24 hours, after natural cooling, 60 DEG C of dry 2h after being cleaned with deionized water will contain excessive for 50 DEG C of reactions
The phosphate buffer of glutaraldehyde is passed through in reaction channel, and 4 DEG C of reaction 1h, 20 DEG C are reacted 1h, are rushed after reaction with deionized water
Wash clean.
Preferably, microcystic toxin antigens fixation includes the following steps：
(1) BSA of 200 μ L is cleaned twice with 400 μ L Boratexes, dobell's solution concentration is 50mM, pH=8-
8.2；Then cleaned with PBS phosphate buffers；
(2) BSA after cleaning is passed through reaction channel and be full of, reaction 45 minutes are stood at room temperature, then with PBS phosphoric acid
Salt buffer cleans reaction channel, nitrogen drying；
(3) secondary antibody 10 times excessive being dissolved into dobell's solution, injected in reaction channel, room temperature submerges 45 minutes,
Then extra liquid is removed；Then cleaned with PBS phosphate buffers；
(4) 5 μ L, 50% glutaraldehydes are added in reaction channel, when reaction 1 is small at room temperature；Then delayed with PBS phosphate
Fliud flushing is cleaned；
(5) finally cleaned successively with 1M NaCl, 0.1M glycine, pure water, PBS phosphate buffers.
Preferably, the reaction channel length is with the hybrid channel length ratio：1.5-2：1.
The invention also discloses a kind of microcystic toxins checking method based on micro-fluidic chip, using claim 1-6
Any one of them micro-fluidic chip, detection method include the following steps：
(1) Microcystin antibody-solutions are added in the reaction channel of the micro-fluidic chip, are incubated 10-20 minutes,
PBS buffer solutions are added in afterwards to be cleaned；
(2) Microcystin that horseradish peroxidase marks with water sample is uniformly mixed, added in reaction channel, reaction
15 minutes, the rear PBS buffer solutions that add in were cleaned；
(3) enzyme reaction substrate is added in, reacts at room temperature and waits to develop the color, the rear sulfuric acid color development stopping reaction for adding in 1M；
(4) color product push-in sense channel is subjected to optical signal detecting again and obtains optical signal value F；
(5) after detecting, SDS is sequentially added, pure water cleans chip channel；
(6) blank control is done with deionized water, according to the detection of (1) under the step-(5) flow without Microcystin blank
Relative light intensity F ' is calculated finally by formula F '=(Fc-F)/Fc in the optical signal value Fc of sample, finally by different micro-
The concentration C x of capsule algae toxin calculates the Microcystins Concentration C in water sample on the linear equation of relative light intensity F '.
Preferably, the volume of the enzyme reaction substrate and the sulfuric acid is all higher than being equal to the Microcystin antibody-solutions
Volume.
Preferably, the Microcystin of the horseradish peroxidase mark and the volume of water sample ratio are 1:1.
Preferably, the pH=1.9 of the SDS, mass concentration 5%.
The invention has the advantages that：
In the present invention using it is cheap, be easy to get and easy to process and modified glass material makes chip, making
There is some superiority in cost.Secondly the detection of Microcystin is using competitiveness enzyme-linked immunological technique, by table in passage
Face is modified, immobilized antigen carrier, takes full advantage of the surface characteristic of chip.In addition immunoreaction process and detection process separate into
Row, reduces the interference of other reagents in reaction process.Finally, recycling can be achieved by cleaning in the chip of making, repeats
It is 50 times using number, avoids the drawbacks of conventional die is disposable.
Description of the drawings
Fig. 1 is the microfluidic chip structure schematic diagram of the preferred embodiment of the present invention；
Fig. 2 is the detection method canonical plotting of the preferred embodiment of the present invention.
Specific embodiment
The technique effect of the design of the present invention, concrete structure and generation is described further below with reference to attached drawing, with
It is fully understood from the purpose of the present invention, feature and effect.
Microcystin detection micro-fluidic chip includes the cover plate of the substrate for being carved with microfluidic circuit and sealing covering thereon.Base
The material that piece and cover plate use is organic glass, microfluidic circuit is carved with using photoetching process on substrate, using thermal bonding technology to base
Piece is bonded with cover plate.Microfluidic circuit on substrate as shown in Figure 1, from left to right include successively 1 entrance of reagent, hybrid channel 2,
2 entrance of reagent, reaction channel 7, sense channel 5 and waste liquid outlet 6.1 entrance of reagent includes sample water inlet 1 to be measured, horseradish peroxide
Change Microcystin solution inlet 11 and the PBS solution entrance 12 that hydrogen enzyme (HRP) marks, 2 entrance of reagent includes dodecyl sulphur
Sour sodium (SDS) entrance 3, enzyme reaction substrate entrance 4, bovine serum albumin BSA entrance 9, secondary antibody entrance 8, Microcystin antibody are molten
Liquid entrance 10.Hybrid channel 2 and reaction channel 7 are arranged to snakelike tortuous passageway, increase the time of immune response, make immune response
More completely, 7 length of reaction channel is more than hybrid channel length 2, and in this example, the two length ratio is：1.5-2：1.Mixing
Passage 2 is mainly used for the Microcystin solution mixing of water sample and HRP mark to be measured, and reaction channel 7, which is mainly used for antigen, to be fixed
And immune response.
Using the slide of pmma material as micro-fluidic chip base material in the present embodiment, and using the side of photoetching
Method carves microfluidic circuit on the glass sheet, and substrate is bonded with cover plate using low-temperature bonding.It is as follows：
(1) surface treatment of glass substrate：Substrate is put into the concentrated sulfuric acid and is boiled 30 minutes, isopropyl is put it into after bath
Alcohol is 1 with acetone:It impregnates 10 minutes, is then ultrasonically treated 10 minutes in ethanol, after deionized water rinsing in 1 mixed solution
Nitrogen dries up, be put into baking oven 150 DEG C dry 1 it is small when or so.
(2) gluing：By the spin-coated machine spin coatings of negtive photoresist RFJ-220, gluing rotating speed is 1000rpm, and after 30 seconds, glue thickness is 5 μm；
Substrate is placed on front baking 6 minutes on hot plate, evaporates remaining solvent in photoresist.
(3) it is exposed and developed：Uv-exposure, exposure energy 18.9MJ/cm are carried out to substrate2, the time for exposure is 9 seconds；
Glass substrate after exposure developing liquid developing 2 minutes, after developer solution naturally dry, 150 DEG C of post bakes 35 minutes make photoresist
It is full cross-linked.
(4) primer is swept：Glass substrate is put into and carries out within 2 minutes in plasma degumming machine sweeping primer, removal development rear surface
Remaining photoresist, while substrate surface is made to be conducive to contact of the corrosive liquid with substrate for hydrophily.
(5) etch：It using wet etching, carries out at room temperature, corrosive liquid is diluted BOE solution (buffering in various degree
Oxide etch liquid), and 10%HCl is added to improve the quality of etching surface.In order to extend tolerance of the photoresist in etching liquid
Time, the method for employing multiple post bake etching often etch post bake 10 minutes after twenty minutes, continue to etch.Etching reaches requirement
Depth after, with fuming nitric aicd remove photoresist.
Then cover plate is bonded on substrate, using thermal bonding technology, the step of substrate is with cover plate thermal bonding is as follows：
(1) the affine processing in surface：First substrate surface is cleaned, with deionized water, acetone and absolute ethyl alcohol, deionization
Water cleans successively.Again with deionized water rinsing after dilute HF acid rinses, then with volume ratio 1：1 ammonium hydroxide：Hydrogen peroxide cleans, and finally uses
Deionized water rinsing to increase the activity of glass surface, makes bonding surface adsorb more hydroxyl groups.
(2) pre- bonding：Under no super-clean environment, pre- bond sequence is selected to be made.By substrate and not stained new lid
After piece is directed at fitting in ultrapure water environment, substrate is made to be fitted closely with cover plate together with and slided without mutual, taking-up is put into true
Empty drying box, dry 1h.
(3) high temperature is bonded：Substrate and cover plate are lain in high temperature furnace after pre- bonding, respectively put in substrate and cover plate upper and lower
One piece of polished graphite cake presses one block of stainless steel on graphite cake above again, and the matter of stainless steel is selected according to the size of chip
It measures as 500g.High temperature furnace heating rate is 10 DEG C/min, and to eliminate the internal stress of glass, hydrogen bond density gradually increases during this
Greatly, bond strength increases.Again temperature is continued to raise, occur polymerisation in the process, the converting hydrogen bonds between interface are silicon
Oxygen key.When temperature reaches 400 DEG C -500 DEG C, this polymerisation is basically completed, and bonding reaches saturation, what polymerisation generated
Hydrone is spread around at high temperature, bonding area increase.Since the annealing point of glass is at 520 DEG C -530 DEG C or so, selection
Room temperature is cooled in 620 DEG C of held for some time, then with 10 DEG C/min, bonding is completed.
Micro-fluidic chip component surface has large specific surface area, and skin effect is notable；Material diversification and demand diversification
Feature, untreated chip surface single property do not adapt to various chip applications, surface is modified predominantly
Reduction surface is non-specific, increases surface specific and improves surface stability.Glass is inorganic salt blended silica,
It is noncrystal, good heat conductivity, it is cheap, it is easy to etching and sealing-in.Common method of modifying is Silanization reaction, principle
It is the silicone hydroxyl dehydration condensation using silicon oxygen bond hydrolysis generation silicone hydroxyl and chip surface, in chip surface coupling function
Siloxanes, functional group can be used for react in next step.Substrate surface modification step is as follows：
1) clean：It is 1 that volume ratio is first used in substrate reaction channel:The mixed liquor of 1 methanol and hydrochloric acid is in supersonic wave cleaning machine
Middle ultrasonic cleaning micro-fluidic chip 30min is dried up after deionized water cleaning
2) substrate activates：It is 3 by volume ratio:1 concentrated sulfuric acid and the mixed liquor of hydrogen peroxide are passed into chip channel, 80 DEG C
40min is boiled in left and right, makes passage surface hydroxylation；
3) end deionized water rinsing is reacted, then chip is dried up with nitrogen, silanization is carried out at once after dry.
4) silanization：Volume ratio is 1 by silanization under nitrogen protection:9 3- aminopropyl triethoxysilanes
(APTES) and the mixed liquor of acetone is passed through conversion zone (in snake bend passage), and 50 DEG C of reactions for 24 hours, make chip internal-surface ammonia
After base natural cooling, 60 DEG C of dry 2h, core is passed through by the phosphate buffer containing excessive glutaraldehyde after being cleaned with deionized water
In in piece passage, 4 DEG C of reaction 1h, 20 DEG C are reacted 1h, are rinsed well after reaction with deionized water.
The present invention is using competitive immunization absorption method, mainly by modified reaction channel inner surface to the spy of protein
Opposite sex effect, as solid phase carrier.Secondary antibody is combined with the PROTEIN B SA being fixed in passage, at the same secondary antibody again can with it is micro-
The antibody of capsule algae toxin carries out immune response.It is as follows to the fixation procedure of antigen：
1) a-protein (bovine serum albumin BSA) of 200 μ L is carried out with 400 μ L Boratexes (50mM, pH=8-8.2) clear
It washes twice.Then cleaned with PBS phosphate buffers.
2) BSA after cleaning is added in into micro-fluidic chip, ensures to stand reaction 45 at room temperature full of BSA in reaction channel
Minute, BSA is made to be sufficiently secured on conduit wall.Then cleaned with PBS phosphate buffers, nitrogen drying.
3) secondary antibody 10 times excessive is dissolved into dobell's solution, injected in chip channel, room temperature submergence 45 minutes, so
Extra liquid is removed afterwards.Then cleaned with PBS phosphate buffers.
4) 5 μ L, 50% glutaraldehydes are added in pipeline, when reaction 1 is small at room temperature.Then it is clear with PBS phosphate buffers
It washes.
5) 1M NaCl, 0.1M glycine is finally used, pure water is cleaned successively.Then it is clear with PBS phosphate buffers
It washes.
In the present embodiment, the pH=7.2-7.4 of above-mentioned PBS phosphate buffers, concentration 0.01M.In a kind of implementation
In mode, PBS phosphate-buffered liquid and preparation method thereofs are as follows：Weigh 8g NaCl, 0.2g KCl, 1.44g Na2HPO4And 0.24g
KH2PO4, it is dissolved in 800ml deionized waters, the pH value 7.2-7.4 of solution is adjusted with HCl, finally plus deionized water is settled to 1L i.e.
It can.Steam sterilization (at least 20 minutes) under high pressure are stored in room temperature or 4 DEG C of refrigerators.
Micro-fluidic chip immune response and testing process in this patent are adapted to automatically detect, and are firstly added microcystin
Plain antibody-solutions are incubated, then add in 1 simultaneously:Microcystin that 1 mark has and unlabelled
Water sample after mixing, two resistance to surface is adsorbed in into immune response passage.Then excessive enzyme reaction substrate is added in, after reaction
Colour developing.Final color product enters sense channel and is detected.It is micro- that the mark being adsorbed on antibody can be obtained according to optical signal
Capsule algae toxin, and then understand the content of unlabelled Microcystin, luminous intensity and the Microcystins Concentration in water sample are into negative
It is related.
Detailed immune response and detection process is as follows：
(1) 50 μ L Microcystin antibody-solutions are added in reaction channel, be incubated 15 minutes.It is molten to add in PBS bufferings
Liquid is cleaned.
(2) by the Microcystin of appropriate horseradish peroxidase (HRP) mark and water sample with 1:1(V:V) it is uniformly mixed,
It adds in reaction channel, reacts 15 minutes.PBS buffer solutions are added in be cleaned.
(3) 50 μ L enzyme reaction substrates are eventually adding, reacts at room temperature 10 minutes and develops the color, add in the reaction of 1M sulfuric acid color development stopping.
(4) color product push-in sense channel is subjected to optical signal detecting again and obtains optical signal value F.
(5) after detecting, pH=1.9 is sequentially added, 5%SDS, pure water clean chip channel, in favor of core
The recycling of piece.
(6) blank control is done with deionized water.According to light of the flow detection in (1)-(5) without Microcystin blank sample
Relative light intensity F ' is calculated finally by formula F '=(Fc-F)/Fc, finally by different Microcystins in signal value
Concentration C x calculates the Microcystins Concentration C in water sample on the linear equation of relative light intensity F '.
In above-mentioned detection process, to react fully, the volume of enzyme reaction substrate and sulfuric acid is all higher than being equal to microcystin
The volume of plain antibody-solutions.
The drafting of standard curve：In the case where many experiments obtain optimum reaction condition, chip is carried out to Microcystin and is determined
Amount analysis first by microcystin-LR standards solution analysis, makes standard curve.Prepare 0,0.50,1.00,2.00,
4.00th, 8.00 and 10.00 μ g/L Microcystin standard solution is carried out according to above-mentioned testing process, and various concentration is done puts down twice
Row experiment.Light intensity is detected, it is weaker with the increase optical signal of concentration, and the error of parallel laboratory test is in the range of limitation.
Finally obtain the concentration C of different Microcystins (microcystin-LR)xLinear equation on relative light intensity F '.The inspection
The detection of survey method is limited to 0.5 μ g/L, and detection time is 10min or so.Detection range is 0.50 μ g/L-20.00 μ g/L.Y=
(2.41572-0.20893X R=-0.9929).Canonical plotting is as shown in Figure 2.
The contrasting detection of water sample glyphosate content to be measured：Using this method and national standard method simultaneously to containing Microcystin
Identical water sample to be measured be detected, in water sample Microcystins Concentration be higher than detection range when, first carry out gradient dilution.By
After two kinds of detection method detections, this method detects the 32.25 μ g/L of concentration of Microcystin in water sample, National Standard Method method detection knot
Fruit is then 32.13 μ g/L.The data of two methods detection gained do not have notable difference, show the detection method accuracy of the present invention
It is very high.In addition, compared to National Standard Method, the method for the present invention is 0.50 μ g/L to the minimum detectability of Microcystin, and detection range is
0.50 μ g/L-25.00 μ g/L, and detection time is short, entire reaction and detection process are taken as 45min or so.
The preferred embodiment of the present invention described in detail above.It should be appreciated that those of ordinary skill in the art without
Creative work is needed according to the present invention can to conceive and makes many modifications and variations.Therefore, all technologies in the art
Personnel are available by logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Technical solution, all should be in the protection domain being defined in the patent claims.
Claims (10)
1. it is a kind of for Microcystin detection micro-fluidic chip, including organic glass system into substrate and cover plate, the lid
Piece hot key is together on the substrate, which is characterized in that microfluidic circuit is carved on the substrate, the microfluidic circuit includes reagent 1 successively
Entrance, hybrid channel, 2 entrance of reagent, reaction channel, sense channel and waste liquid outlet, 1 entrance of reagent include water sample to be measured
The Microcystin solution inlet and PBS solution entrance of entrance, horseradish peroxidase mark, 2 entrance of reagent include ten
Dialkyl sulfonates entrance, enzyme reaction substrate entrance, bovine serum albumin entrance, secondary antibody entrance and Microcystin antibody-solutions enter
Mouthful, the hybrid channel and the reaction channel are snakelike meander-like, and the reaction channel inner surface is by chemical modification and consolidates
Surely there are microcystic toxin antigens.
2. as described in claim 1 for the micro-fluidic chip of Microcystin detection, which is characterized in that the substrate miniflow
Road uses photolithography method, includes the following steps：
(1) surface treatment of glass substrate：Substrate is put into the concentrated sulfuric acid and is boiled 30 minutes, put it into after bath isopropanol with
Acetone is 1:It impregnates 10 minutes, is then ultrasonically treated 10 minutes in ethanol, nitrogen after deionized water rinsing in 1 mixed solution
Drying, be put into baking oven 150 DEG C dry 1 it is small when；
(2) gluing：By the spin-coated machine spin coatings of negtive photoresist RFJ-220, gluing rotating speed is 1000rpm, and after 30 seconds, glue thickness is 5 μm；By base
Piece is placed on front baking 6 minutes on hot plate, evaporates remaining solvent in photoresist；
(3) it is exposed and developed：Uv-exposure, exposure energy 18.9MJ/cm are carried out to substrate2, the time for exposure is 9 seconds；After exposure
Glass substrate developing liquid developing 2 minutes, after developer solution naturally dry, 150 DEG C of post bakes 35 minutes；
(4) primer is swept：Glass substrate is put into and carries out within 2 minutes in plasma degumming machine sweeping primer, removal development rear surface residual
Photoresist；
(5) etch：It carries out at room temperature, corrosive liquid is adds the different degrees of diluted buffer oxide etching liquid of 10%HCl, often
Post bake 10 minutes after twenty minutes are etched, continue to etch；After the completion of etching, photoresist is removed with fuming nitric aicd.
3. as described in claim 1 for the micro-fluidic chip of Microcystin detection, which is characterized in that the substrate and institute
The thermal bonding for stating cover plate includes the following steps：
(1) the affine processing in surface：Substrate surface deionized water, acetone and absolute ethyl alcohol mixed solution, deionized water are clear successively
It washes, then with deionized water rinsing after dilute HF acid rinses, then with volume ratio 1：1 ammonium hydroxide：Hydrogen peroxide cleans, and finally uses deionized water
It rinses；
(2) pre- bonding：Under no super-clean environment, after by substrate and not stained new cover plate is directed at fitting in ultrapure water environment,
It is slided together with substrate is made to be fitted closely with cover plate and without mutual, vacuum drying chamber, dry 1h are put in taking-up into；
(3) high temperature is bonded：Substrate after pre- bonding and cover plate are lain in high temperature furnace, one is respectively put in substrate and cover plate upper and lower
The polished graphite cake of block presses one block of stainless steel on graphite cake above again；High temperature furnace heating rate is 10 DEG C/min, is selected
620 DEG C of heat preservation a period of times, then room temperature is cooled to 10 DEG C/min.
4. as described in claim 1 for the micro-fluidic chip of Microcystin detection, which is characterized in that the chemical modification
Include the following steps：
(1) clean：It is 1 that volume ratio is first used in reaction channel described in substrate:The mixed liquor of 1 methanol and hydrochloric acid is cleaned by ultrasonic 30min,
It is dried up after deionized water cleaning；
(2) substrate activates：It is 3 by volume ratio:1 concentrated sulfuric acid and the mixed liquor of hydrogen peroxide are passed into chip channel, 80 DEG C of left sides
Boil 40min in the right side；
(3) end deionized water rinsing is reacted, then chip is dried up with nitrogen；
(4) silanization：Under nitrogen protection, it is 1 by volume ratio:9 3- aminopropyl triethoxysilanes and the mixed liquor of acetone
Reaction channel is passed through, for 24 hours, after natural cooling, 60 DEG C of dry 2h after being cleaned with deionized water will contain excessive penta 2 for 50 DEG C of reactions
The phosphate buffer of aldehyde is passed through in reaction channel, and 4 DEG C of reaction 1h, 20 DEG C are reacted 1h, are done after reaction with deionized water rinsing
Only.
5. as described in claim 1 for the micro-fluidic chip of Microcystin detection, which is characterized in that Microcystin resists
Original fixation includes the following steps：
(1) BSA of 200 μ L is cleaned twice with 400 μ L Boratexes, dobell's solution concentration is 50mM, pH=8-8.2；
Then cleaned with PBS phosphate buffers；
(2) BSA after cleaning is passed through reaction channel and be full of, stood reaction 45 minutes at room temperature, then delayed with PBS phosphate
Fliud flushing cleans reaction channel, nitrogen drying；
(3) secondary antibody 10 times excessive is dissolved into dobell's solution, injected in reaction channel, room temperature submergence 45 minutes, then
Remove extra liquid；Then cleaned with PBS phosphate buffers；
(4) 5 μ L, 50% glutaraldehydes are added in reaction channel, when reaction 1 is small at room temperature；Then PBS phosphate buffers are used
Cleaning；
(5) finally cleaned successively with 1M NaCl, 0.1M glycine, pure water, PBS phosphate buffers.
6. as described in claim 1 for the micro-fluidic chip of Microcystin detection, which is characterized in that the reaction channel
Length is with the hybrid channel length ratio：1.5-2：1.
7. a kind of microcystic toxins checking method based on micro-fluidic chip, which is characterized in that using any one of claim 1-6
The micro-fluidic chip, detection method include the following steps：
(1) Microcystin antibody-solutions are added in the reaction channel of the micro-fluidic chip, are incubated 10-20 minutes, rear to add
Enter PBS buffer solutions to be cleaned；
(2) Microcystin that horseradish peroxidase marks with water sample is uniformly mixed, added in reaction channel, react 15 points
Clock, the rear PBS buffer solutions that add in are cleaned；
(3) enzyme reaction substrate is added in, reacts at room temperature and waits to develop the color, the rear sulfuric acid color development stopping reaction for adding in 1M；
(4) color product push-in sense channel is subjected to optical signal detecting again and obtains optical signal value F；
(5) after detecting, SDS is sequentially added, pure water cleans chip channel；
(6) blank control is done with deionized water, according to the detection of (1) under the step-(5) flow without Microcystin blank sample
Optical signal value Fc, relative light intensity F ' is calculated finally by formula F '=(Fc-F)/Fc, finally by different Microcystis aeruginosas
The concentration C of toxinxLinear equation on relative light intensity F ' calculates the Microcystins Concentration C in water sample.
8. the microcystic toxins checking method based on micro-fluidic chip as claimed in claim 7, which is characterized in that the enzyme is anti-
The volume of substrate and the sulfuric acid is answered to be all higher than the volume equal to the Microcystin antibody-solutions.
9. the microcystic toxins checking method based on micro-fluidic chip as claimed in claim 7, which is characterized in that the horseradish
The Microcystin of catalase mark is 1 with the volume of water sample ratio:1.
10. the microcystic toxins checking method based on micro-fluidic chip as claimed in claim 7, which is characterized in that the SDS
PH=1.9, mass concentration 5%.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611005361.0A CN108067311A (en) | 2016-11-15 | 2016-11-15 | A kind of micro-fluidic chip and detection method for Microcystin detection |
Applications Claiming Priority (1)
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| CN114062465A (en) * | 2021-11-12 | 2022-02-18 | 福州大学 | Device for rapidly, highly sensitively and high-flux detecting organophosphorus and carbamate pesticide residues and using method thereof |
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| CN115779987A (en) * | 2022-12-06 | 2023-03-14 | 海南医学院 | PDMS micro-fluidic chip and application thereof |
| CN115779987B (en) * | 2022-12-06 | 2023-09-08 | 海南医学院 | PDMS micro-fluidic chip and application thereof |
| CN116874000A (en) * | 2023-08-09 | 2023-10-13 | 湖北微流控科技有限公司 | Recycling method of micro-fluidic disk chip for total nitrogen water quality detection |
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