CN105728071A - Microfluidic chip and application thereof - Google Patents
Microfluidic chip and application thereof Download PDFInfo
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- CN105728071A CN105728071A CN201610080054.2A CN201610080054A CN105728071A CN 105728071 A CN105728071 A CN 105728071A CN 201610080054 A CN201610080054 A CN 201610080054A CN 105728071 A CN105728071 A CN 105728071A
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- 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
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- 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
- G01N21/76—Chemiluminescence; Bioluminescence
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- 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
- B01L2200/10—Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
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Abstract
The invention relates to a microfluidic chip.The microfluidic chip is formed by a polydimethylsiloxane chip and a glass substrate in a bonding mode.The polydimethylsiloxane chip is provided with a spiral microchannel, the inlet end of the spiral microchannel is provided with three inlet channels, and the first inlet channel and the second inlet channel are joined and then joined with the third inlet channel.The invention further relates to a method for detecting the content of nitrite with the microfluidic chip.The method includes the steps that a carbon dot solution and a nitrite solution are injected through the first inlet channel and the second inlet channel respectively, and an acidified hydrogen dioxide solution is injected into the microfluidic chip through the third inlet channel; a mixed solution of the carbon dot solution and the nitrite solution and the acidified hydrogen dioxide solution are subjected to a chemiluminescence reaction in the microfluidic chip, and the content of nitrite is determined by measuring the chemiluminescence intensity of a solution generated in the reaction.
Description
Technical field
The present invention relates to the detection field of nitrite, be specifically related to a kind of micro-fluidic chip and application thereof.
Background technology
Nitrite is widely present in nature, and is present in whole nitrogen cycle.Nitrite is often used as the preservative in agricultural fertilizer or food.These formulation fertilizer containing nitrogens are an important polluter in environment.The nitrite absorbed from food then plays important physiological action in human body.They can react with two grades in human body or tertiary amine and generate the nitrosamine with carcinogenesis, bring significant damage to human body.Under the effect of nitrite, hemoglobin can be changed into metahemoglobin and cause that the ability of blood-transmitted oxygen declines.Therefore, the detection of nitrite is particularly significant for environmental and human health impacts.
At present, the detection method of existing multiple nitrite is seen in report.Document 1 have detected the nitrite in sea water and nitrate with isotope dilution mass spectrometry simultaneously.Document 2 is by preparing into a kind of electrochemical sensor on modified hemoglobin to glass-carbon electrode, for the detection of nitrite.Document 3, using graphite and argentum powder as " ink ", prepares into the sensor of a kind of " writing type ", can on-line checking nitrite.But, the detecting instrument of these methods or complex operation or needs costliness.Chemiluminescence as a kind of analysis method of maturation, shows that cost is low, highly sensitive, easily operated and feature from ambient interferences.Using Flow Injection Analysis is usually used in detecting inorganic ions with chemoluminescence method, but, the reagent consumption of the method is relatively big, and it is relatively slow to analyze speed, and automaticity is not high enough.
Document 1:High-precisionquadrupleisotopedilutionmethodforsimulta neousdeterminationofnitriteandnitrateinseawaterbyGCMSaft erderivatizationwithtriethyloxoniumtetrafluoroborate (PaglianoE.；MeijaJ.；MesterZ.；Anal.Chim.Acta,2014,824,36-41).
Document 2:Enhancingsensitivityofhemoglobin-basedelectrochemicalb iosensorbyusingproteinconformationalintermediate (WuH.；WangX.；QiaoM.；ZhangH.；JinX.；FanS.；Sens.ActuatorB-Chem.,2015,221,694-699).
Document 3:WritingSensorsonSolidAgriculturalProductsforInSituDete ction (TangW.；WuJ.；YingY.；LiuY.；Anal.Chem.,2015,87,10703-10707).
Document 4:Anaptamer-functionalizedchemomechanicallymodulatedbiom oleculecatch-and-releasesystem (ShastriA.；McGregorL.M.；LiuY.；HarrisV.；NanH.；MujicaM.；VasquezY.；BhattacharyaA.；MaY.；AizenbergM.；KuksenokO.；BalazsA.C.；AizenbergJ.；HeX.；Nat.Chem.,2015,7,447-454).
Summary of the invention
In view of above-mentioned technical problem, it is an object of the invention to provide a kind of micro-fluidic chip, by the chemiluminescence reaction that the specific microchannel arranged on micro-fluidic chip realizes, it is possible to the content of nitrite in water body and various environment is detected and analyzed.
An embodiment of the invention is in that to provide a kind of micro-fluidic chip, including the polymer chip being bonded with substrate of glass, described polymer chip is provided with spiral type microchannel, arrival end in described spiral type microchannel is provided with three access roades, wherein, the first access road and the second access road cross with the 3rd access road after crossing again.
According to the present invention, design by spiral type microchannel, the area making described micro-fluidic chip is used more effectively, the time that reaction carries out is extended considerably, further, three access roades of spiral type microchannel can make multiple reaction reagent respectively enter, and crosses with the 3rd access road after crossing especially by making the first access road and the second access road again, specific components can be made preferentially to mix, be conducive to controlling reaction process is more accurately effective and regulating.Additionally, by the present invention micro-fluidic chip, it is possible to less reagent consumption complete reaction, it is easy to control and operation.
One of the present invention preferred embodiment in, the outlet of described spiral type microchannel is located at the center of described spiral type microchannel.
The present invention another preferred embodiment in, the width of described access road is 0.5-2mm, and the width of described spiral type microchannel is 0.5-1mm, the spiral number of turns be 4-10 circle.
The present invention another preferred embodiment in, the degree of depth of described access road and spiral type microchannel is 20-100 μm, it is preferred to 20-50 μm.
According to the present invention, by being limited in above-mentioned scope by the width of the width of spiral type microchannel, the spiral number of turns and access road, be conducive to making chemical reagent mix more fully within the scope of micro-fluidic chip, reacting.
The present invention another preferred embodiment in, described polymer chip is polydimethylsiloxane (PDMS) chip.
Another embodiment of the invention is in that to provide the method utilizing above-mentioned micro-fluidic chip detection content of nitrite, including:
Carbon point (CDs) solution and nitrite solution are injected respectively through the first and second access roades, the hydrogen peroxide (H of acidifying2O2) solution by the 3rd access road inject micro-fluidic chip；
There is chemiluminescence reaction in described carbon point solution and the mixed solution of nitrite solution and the hydrogenperoxide steam generator of described acidifying, generated the chemiluminescence intensity of solution by assaying reaction, it is determined that the content of nitrite in micro-fluidic chip.
Inventor it have been investigated that, as it is shown in figure 1, be no matter that CDs solution is added H2O2And NaNO2In mixed liquor, or by NaNO2Solution adds CDs and NaNO2In mixed liquor, the chemiluminescence obtained all is not so good as H2O2Solution adds CDs and NaNO2In mixed liquor, the chemiluminescence intensity of gained is strong.Inventor speculate, this be due to peroxynitrite (ONOOH) through HOONO* (i.e. peroxynitrite excited state) be changed into nitric acid be generation chemiluminescent single step reaction, peroxynitrite is then the H of acidifying2O2With NaNO2The product of reaction.By CDs solution and nitrite solution being pre-mixed, add H2O2Solution, the peroxynitrite of generation can react the chemiluminescence producing to strengthen immediately with CDs.Determine the content of nitrite based on this chemiluminescence intensity, there is higher sensitivity and accuracy.
One of the present invention preferred embodiment in, in the hydrogenperoxide steam generator of described acidifying, the concentration of hydrogen peroxide is 0.01-5mol/L, it is preferred to 1-5mol/L.
In a preferred embodiment of the present invention, the hydrogenperoxide steam generator of described acidifying is the mixed liquor of hydrogen peroxide stock solution and sulfuric acid solution, and the concentration of described sulfuric acid solution is 0.0025mol/L-0.1mol/L, it is preferred to 0.005mol/L-0.02mol/L.The sulfuric acid solution of the hydrogen peroxide in above-mentioned concentration range by being mixed to get with the sulfuric acid solution of above-mentioned concentration and hydrogenperoxide steam generator, is conducive to improving the chemiluminescence intensity of the solution that reaction produces.
The present invention another preferred embodiment in, the volumetric concentration of described carbon point solution is 10%-20%.Namely described carbon point and water are mixed to get carbon point solution according to the volume ratio of 1-2:10.By the volumetric concentration of carbon point solution being limited in above-mentioned scope, be conducive to improving the chemiluminescence intensity of the solution that reaction produces.
The present invention another preferred embodiment in, the flow velocity injected by the hydrogenperoxide steam generator of described carbon point solution, nitrite solution, acidifying is 100-300 μ L/min, it is preferred to 100-200 μ L/min.By by the injection flow speed control of each reagent solution in above-mentioned scope, be conducive to improving the chemiluminescence intensity of solution that reaction produces.
Yet further embodiment of the invention is in that to provide said apparatus and said method application in environment and food inspection.
According to micro-fluidic chip provided by the present invention, by the design of spiral type microchannel, making the area of chip be used more effectively, the time that reaction carries out is extended considerably.When being applied to the assay of nitrite, it is pre-mixed the mode mixed again with the hydrogenperoxide steam generator of acidifying by carbon point solution and nitrite solution, the inverting solution with higher chemiluminescence intensity can be produced in the short period of time, determine the content of nitrite based on this chemiluminescence intensity, there is higher sensitivity and accuracy.Additionally, adopt the detection that micro-fluidic chip carries out, it is possible to complete reaction with less reagent consumption, it is easy to control and operation.
Accompanying drawing explanation
That Fig. 1 represents is CDs-NaNO2-H2O2(in Fig. 1, three peak values are from left to right followed successively by and CDs are injected into NaNO the chemiluminescence kinetic curve of system2And H2O2Mixed liquor in produce chemiluminescence intensity, by NaNO2It is injected into H2O2With in the mixed liquor of CDs produce chemiluminescence intensity and by H2O2It is injected into CDs and NaNO2The chemiluminescence intensity of the generation in mixed liquor).
What Fig. 2 represented is the schematic diagram of the spiral type microchannel pattern in embodiments of the invention 2 on mask.
What Fig. 3 represented is the structural representation of the micro-fluidic chip in an embodiment of the invention, in figure 3, and 1-the first access road；2-the second access road, 3-the 3rd access road, 4-conduit, 5-spiral type microchannel, 6-exports, 7-PDMS chip, 8-substrate of glass.
For detecting the standard curve of Nitrite content in the embodiment of the present invention 8 that Fig. 4 shows.
Detailed description of the invention
The present invention is described in detail by the following examples, but protection scope of the present invention is not limited to the description below.If without specified otherwise, it it is experimentally normal experiment method；The reagent used and material etc. all can pass through to be either commercially available.
The manufacture method of embodiment 1 micro-fluidic chip and structure thereof
(making of formpiston)
After being cleaned processing with the circular silicon chip that diameter is 75mm by water tiger acid solution, dry, the negative optical cement of SU-82050 being 30 μm with the rotating speed of 1000rpm in surface spin coating a layer thickness with sol evenning machine, toasts 20min at 65 DEG C.
Drawing spiral type microchannel pattern as shown in Figure 2 on mask, wherein the spiral number of turns of spiral microchannel is 4 circles, and width is 0.5mm, and the width of three access roades is 0.5mm.This mask is covered on the silicon chip scribbling negative optical cement, carry out uv-exposure 3 times, each 30s；Then take mask off and develop with developer solution；After drying up with nitrogen again, namely obtain the formpiston for forming spiral type microchannel.
(making of micro-fluidic chip)
Prepared formpiston is carried out silanization treatment and makes its surface hydrophobicity.PDMS prepolymer and initiator being mixed with mass ratio 10:1 pours in formpiston, places 2h at 75 DEG C after bubble removing.The PDMS chip being complementary to that polymerization is formed is taken off, cuts into cuboid-type (2cm × 4cm × 5mm).With the tack syringe needle beginning place and the center punching of spiral microchannel at three access roades of this PDMS chip, formed for three entrance and exits being connected with conduit, namely obtain the PDMS chip with spiral type microchannel.Being processed by oxygen plasma, making this have, the PDMS chip of spiral type microchannel is irreversible with microscope slide is bonded, and namely completes the making of micro-fluidic chip.
Embodiment 2-7 and comparative example 1-4
The chemiluminescence intensity that sodium nitrite solution produces in micro-fluidic chip is measured respectively by embodiment 2-7 and comparative example 1-4.
Configuration concentration be the sodium nitrite solution of 1mol/L, 1mol/L (i.e. 1M) hydrogenperoxide steam generator (wherein, except the solvent of the hydrogenperoxide steam generator of comparative example 4 is except distilled water, the hydrogenperoxide steam generator of remaining embodiment and comparative example is the sulfuric acid solution of hydrogen peroxide) and according to concentration configuration carbon point solution as shown in table 1, and sodium nitrite solution, hydrogenperoxide steam generator and carbon point solution are distinguished in inhalation syringe, and syringe is connected with syringe pump.
As shown in Figure 3, the entrance of the first access road is connected with the syringe filling sodium nitrite solution by conduit, the entrance of the second access road is connected with the syringe filling carbon point solution by conduit, the entrance of the 3rd access road is connected with the syringe filling hydrogenperoxide steam generator by conduit, and the outlet of spiral type microchannel is connected with waste liquid cylinder by conduit.After completing connection, micro-fluidic chip is positioned over the surface of the photomultiplier tube of Chemiluminescence Apparatus (the ultraweak chemiluminescent analyzer of BPCL, Chinese Academy of Sciences's biophysics Research Institute).
Open three syringe pumps, three kinds of solution are injected in micro-fluidic chip with identical flow velocity (as shown in table 1), the biochemical luminescence-producing reaction of hybrid concurrency in micro-fluidic chip of three kinds of solution, read the chemiluminescence intensity of the luminescent substance that reaction generates by Chemiluminescence Apparatus, record in Table 1 after numerical stability.The above-mentioned process injecting, react and obtaining chemiluminescence intensity in micro-fluidic chip can be passed through computer and control to be automatically performed.Detection only need to consume the sodium nitrite solution of 1 μ about L every time, and the detection time is less than 1min.
The reaction condition of table 1 embodiment and comparative example and chemiluminescence intensity
As shown in table 1, when testing with the sodium nitrite solution of same concentrations and hydrogenperoxide steam generator, luminous intensity obtained for embodiments of the invention 2-7 is apparently higher than comparative example 1-4, thus being conducive to improving sensitivity and the accuracy of the content based on this luminous intensity detection nitrite.
Embodiment 8
Preparation series concentration (0,5,10,25,50,75,100 μm of ol/L) sodium nitrite solution, as standard substance, its chemiluminescence intensity is tested according to the reaction condition of embodiment 2, and with the concentration of sodium nitrite solution for abscissa, chemiluminescence intensity is that vertical coordinate prepares standard curve, as shown in Figure 4.Can be seen that along with NaNO2The increase of concentration, chemiluminescence intensity strengthens gradually and shows good linear (y=9.044x+46.75, R2=0.9973).Thus can at NaNO2Concentration be 5-100 μm of ol/L scope in realize the detection of high accuracy.
Utilize this standard curve, measure the content of four kinds of water sample (pond water, boiling water, tap water and spring water) nitrites.Specifically, gather four kinds of water samples (boiling water is cooled to room temperature) respectively, with the membrane filtration of 0.22 μm, then add in every 1mL sample solution 100 μ L, 1.0 × 10-5The EDTA of mol/L shields transition metal ions therein.
Testing its chemiluminescence intensity according to the reaction condition implementing 2, test result is as shown in table 2.
2 four kinds of water samples of table content of nitrite
Remarks: a data obtain (n=6) by three independent experiments.
As shown in table 2, the content of four kinds of water sample nitrites is from high to low: pond water > boiling water > tap water > spring water.This conclusion is at Peroxynitrous-Acid-InducedChemiluminescenceofFluorescent CarbonDotsforNitriteSensing (Z.Lin, W.Xue, H.Chen, J.Lin, Anal.Chem., 83 (2011) 8245-8251.) document in obtain checking.Additionally, experiment has good repeatability, all relative standard deviation values (RSDs) are less than 10%.
It should be noted that, embodiment described above is only used for explaining the present invention, it is not intended that any limitation of the invention.By referring to exemplary embodiments, invention has been described, it should be appreciated that word wherein used is descriptive and explanatory vocabulary, rather than limited vocabulary.Within the scope of the claims the present invention can be modified by regulation, and in without departing substantially from scope and spirit of the present invention, the present invention be revised.Although the present invention described in it relates to specific method, material and embodiment, it is not intended that the present invention is limited to wherein disclosed particular case, on the contrary, the present invention can be extended to other all methods and applications with identical function.
Claims (10)
1. a micro-fluidic chip, including the polymer chip being bonded with substrate of glass, described polymer chip is provided with spiral type microchannel, arrival end in described spiral type microchannel is provided with three access roades, wherein, the first access road and the second access road cross with the 3rd access road after crossing again.
2. micro-fluidic chip according to claim 1, it is characterised in that the outlet of described spiral type microchannel is located at the center of described spiral type microchannel.
3. micro-fluidic chip according to claim 1 and 2, it is characterised in that the width of described access road is 0.5-2mm, the width of described spiral type microchannel is 0.5-1mm, and the spiral number of turns is 4-10 circle.
4. the micro-fluidic chip according to any one of claim 1-3, it is characterised in that the degree of depth of described access road and spiral type microchannel is 20-100 μm, it is preferred to 20-50 μm.
5. the micro-fluidic chip according to any one of claim 1-3, it is characterised in that described polymer chip is polydimethylsiloxanechip chip.
6. utilize the method that the micro-fluidic chip according to any one of claim 1-5 detects content of nitrite, including:
Carbon point solution and nitrite solution being injected respectively through the first and second access roades, the hydrogenperoxide steam generator of acidifying injects micro-fluidic chip by the 3rd access road；
There is chemiluminescence reaction in described carbon point solution and the mixed solution of nitrite solution and the hydrogenperoxide steam generator of described acidifying, generated the chemiluminescence intensity of solution by assaying reaction, it is determined that the content of nitrite in micro-fluidic chip.
7. method according to claim 6, it is characterised in that in the hydrogenperoxide steam generator of described acidifying, the concentration of hydrogen peroxide is 0.01-5mol/L, it is preferred to 1-5mol/L.
8. method according to claim 7, it is characterised in that the hydrogenperoxide steam generator of described acidifying is the mixed liquor of hydrogen peroxide stock solution and sulfuric acid solution, the concentration of described sulfuric acid solution is 0.0025mol/L-0.1mol/L, it is preferred to 0.005mol/L-0.02mol/L.
9. the method according to any one of claim 6-8, it is characterised in that the volumetric concentration of described carbon point solution is 1%-20%, it is preferred to 10%-20%.
10. the method according to any one of claim 6-9, it is characterised in that the flow velocity injected by the hydrogenperoxide steam generator of described carbon point solution, nitrite solution, acidifying is 100-300 μ L/min, it is preferred to 100-200 μ L/min.
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| CN106622414A (en) * | 2017-01-23 | 2017-05-10 | 广东顺德工业设计研究院(广东顺德创新设计研究院) | Microfluidic chip |
| CN108593618A (en) * | 2018-05-04 | 2018-09-28 | 南京工业大学 | Method for detecting nitrite ions based on polymer carbon dot fluorescence colorimetry |
| CN109967149A (en) * | 2019-04-24 | 2019-07-05 | 中国农业大学 | Micro-fluidic chip |
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| CN108593618B (en) * | 2018-05-04 | 2020-08-04 | 南京工业大学 | Method for detecting nitrite ions based on polymer carbon dot fluorescence colorimetry |
| CN109967149A (en) * | 2019-04-24 | 2019-07-05 | 中国农业大学 | Micro-fluidic chip |
| CN109967149B (en) * | 2019-04-24 | 2020-07-28 | 中国农业大学 | Micro-fluidic chip |
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Application publication date: 20160706 |