CN107860934A - A kind of micro-fluidic chip and method of modifying and detection food bacterial number on apply - Google Patents
A kind of micro-fluidic chip and method of modifying and detection food bacterial number on apply Download PDFInfo
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- CN107860934A CN107860934A CN201711035097.XA CN201711035097A CN107860934A CN 107860934 A CN107860934 A CN 107860934A CN 201711035097 A CN201711035097 A CN 201711035097A CN 107860934 A CN107860934 A CN 107860934A
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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
- 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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
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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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- 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
- B01L2200/12—Specific details about manufacturing devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
- G01N2035/00099—Characterised by type of test elements
- G01N2035/00158—Elements containing microarrays, i.e. "biochip"
Abstract
Applied the invention discloses a kind of micro-fluidic chip and its method of modifying and on detection food bacterial number, the method for modifying comprises the following steps：(1) by-CH of the microchannel inner surface on micro-fluidic chip3Group modified is OH groups；(2) after step (1) reaction is completed, aminosilane reagents is passed through into the microchannel, carry out amino silane reaction；(3) after drying, the dendrimer that surface was modified via COOH is passed through into the microchannel, carries out coupling reaction；(4) after the step (3) reaction is completed, be passed through into the microchannel there is specific recognition performance to bacterium to be measured and 5 ' ends by amino modified mistake adaptation liquid solution so that aptamers are grafted to the dendrimer surface.The present invention micro-fluidic chip is modified, can quick detection food bacterial number, improve detection efficiency, and entirely detect environment individual secure, prevent cross pollution.
Description
Technical field
The invention belongs to technical field of food detection, and in particular to a kind of micro-fluidic chip and its method of modifying and detect
Applied on food bacterial number.
Background technology
In the last few years, disease increasingly attracted people's attention as caused by food.Therefore led for inspection for food hygiene
Domain, quick and sensitive detection device is highly important.Similarly for food processing enterprises and Food Monitoring department, if energy
Target bacteria quantity in enough quickly detection food, it is possible to the monitoring of food quality is improved, so as to effectively implement to public
The guarantee of food sanitation and safety.
Bacteria Detection means traditionally are the methods using bacterium colony culture, and this kind of method can determine food very accurately
In the target bacteria that is harmful to it is whether exceeded, but result overlong time is to wait for the shortcomings that this method, it usually needs several days
Time just can determine that testing result；Meanwhile this method needs the substantial length of operation of professional and technical personnel, cause cost higher.
In the last few years, also there are the device and method of other quick detection bacteriums in the market, as enzyme connects reaction reagent
Box, PCR kit, electrophoretic analysis chip, flow cytometer etc., these detecting instruments be able to can generally be completed within several hours
As a result reading, there is disposal ability quickly in the reading of final result, but these methods still need the sample of complexity
Product processing early stage and enrichment work, so overall detection time is still long (usual 2 days or so).Meanwhile some equipment
Sensitivity is inadequate, and detection limit is too high (such as electrophoretic analysis chip), and detection error is larger.
Because micro-fluidic chip can be integrated in the chemical process of complexity in one chip, also occur in the last few years not
The equipment of few quick detection based on micro-fluid chip.Such as electrochemical microfluidic body chip, impedance micro-fluid chip etc..These
The Cleaning Principle of chip is the change by measuring electric current or resistance of the microfluidic device before and after target bacteria is detected mostly
Change to be finally reached the purpose of detection bacterium quantity.The advantage of this equipment is can to analyze the bacterium of extremely low quantity, but this
Kind equipment is not easy to operate, and the price of equipment entirety is relatively expensive.
Another equipment in detection based on microfluid is made with optics (fluorescent staining, or the change of color)
For Cleaning Principle.The advantages of this detection mechanism is that detection signal intuitively can be observed and detected very, so as to realize pair
Target bacteria quantifies.Although such a method equipment is relatively cheap, in order to reduce the non-targeted particle of other in food samples
Caused noise signal and the interference to detection signal, this method are modified to the surface of microfluid material and require higher.
The content of the invention
In order to solve the above problems, the invention provides a kind of method of modifying of micro-fluidic chip, to existing micro-fluidic core
Piece is modified, be prepared it is a kind of can quick detection food bacterial number micro-fluidic chip.
The technical scheme is that：A kind of method of modifying of micro-fluidic chip, comprises the following steps：
(1) by-CH of the microchannel inner surface on micro-fluidic chip3Group modified is-OH groups；
(2) after step (1) reaction is completed, aminosilane reagents is passed through into the microchannel, carry out amino
Silane reaction；
(3) after drying, the dendrimer that surface was modified via-COOH is passed through into the microchannel, is carried out
Coupling reaction；
(4) after step (3) reaction is completed, be passed through into the microchannel has specific recognition to bacterium to be measured
Performance and 5 ' end by amino modified mistake adaptation liquid solution so that aptamers are grafted to the dendrimer surface.
The present invention is first by the-CH of microchannel inner surface3Group modified is-OH groups, then by the step (2)
Amino silane reaction introduce amino group, then the dendrimer being modified via-COOH is passed through in microchannel so that
- COOH on dendrimer reacts with amino group, so that access dendrimer, most backward miniflow in microchannel
Adaptation liquid solution is passed through in pipeline, aptamers are grafted to dendritic macromole surface.Because aptamers have spy to bacterium to be measured
Different in nature identification function, therefore the inspection of bacterial number can be carried out to bacterium to be measured by the micro-fluidic chip that the present invention is prepared
Survey.
By-CH in wherein described step (1)3The group modified method for-OH groups have it is a variety of, such as can use chemistry
Method is modified, and is reacted preferably, micro-fluidic chip is placed in plasma generator in the step (1),
Wherein plasma generator pressure is 100mTorr, and power 118W, the reaction time is 10~20 seconds.
Amino silane reaction can use existing conventional method in the present invention, and the species of aminosilane reagents have it is more
Kind, preferably, in the step (2), the aminosilane reagents are 3- aminopropyl trimethoxysilanes, N- (2- ammonia second
Base) -3- aminopropyltriethoxy dimethoxysilanes,N- (2- aminoethyls) -3- aminopropyl triethoxysilanes, N- (2- aminoethyls)-
At least one of 3- aminopropyl trimethoxysilanes.
As further preferred, in the step (2), the aminosilane reagents are 3- aminopropyl trimethoxysilanes,
The mixed solution of 3- aminopropyl trimethoxysilanes and absolute ethyl alcohol is passed through in microchannel, carries out amino silane reaction, its
Described in 3- aminopropyl trimethoxysilanes content be 1~5%.
As still more preferably, the content of the 3- aminopropyl trimethoxysilanes is 5%.
Preferably, dendrimer is the dendrimer that monomer is PAMAM in the step (3).
As further preferred, dendrimer is tree-shaped big point of the 7th generation that monomer is PAMAM in the step (3)
Son.
Preferably, the preparation method of identification solution is in the step (4)：By 8 μM of aptamers, 4.0 μM of surfaces
Quantum dot nano particle, 2.0mM NHS with-COOH and 1.0mM EDC are dissolved in 0.1M and pH is in 6.0 MES buffer solutions,
1h is reacted at 30 DEG C, then, 10000 turns/min of centrifuge is centrifuged 10 minutes, removes supernatant, and it is 6.0MES to add 1mL pH
Buffer solution, obtain the identification solution.
Preferably, by 4.0 μM of dendrimers, 1.74mM NHS in the step (3)) it is dissolved in 1.04mM EDC
0.1M and pH are in 6.0 MES buffer solutions, and then MES buffer solutions are passed through in microchannel, react 2h at 30 DEG C.
Preferably, in the step (4), 5.2mM NHS and 0.26M EDC are added into the MES that 0.1M and pH are 6.0 and delayed
In fliud flushing, MES buffer solutions are then passed through microchannel, and react 1h at room temperature, the identification for being subsequently passed 2 μM of 0.5ml is molten
Liquid, react at room temperature 30 minutes.So that aptamers solution graft copolymerization is to the dendrimer surface.
Preferably, the bacterium to be measured is Escherichia coli, salmonella, staphylococcus, lactobacillus acidophilus or golden yellow Portugal
Grape coccus, the corresponding aptamers relation of the bacterium to be measured are：
Preferably, the pH of the adaptation liquid solution of the Escherichia coli is 7.5.
The present invention also provides the micro-fluidic chip that a kind of above-mentioned method of modifying is modified to obtain.
Present invention also offers a kind of application of above-mentioned micro-fluidic chip on detection food bacterial number.
Preferably, application of the micro-fluidic chip on detection food bacterial number, including：
(1) it is passed through testing sample into the microchannel of micro-fluidic chip；
(2) after step (1) reaction is completed, buffer solution for cleaning microchannel is passed through, is then passed through again corresponding
The quantum dot nano particle solution with aptamers.
(3) micro-fluidic chip is finally placed in fluorescence viewed under light microscopy and records fluorescence points, you can obtained to be measured
Contained bacterium number amount to be measured in sample solution.
During present invention detection, testing sample is passed through into the microchannel of micro-fluidic chip so that treated in testing sample
Survey bacterium and microchannel surface have it is specific be adapted to precursor reactant, after reaction completion, be passed through buffer solution for cleaning microchannel.
Then be passed through the quantum dot nano particle solution combined with the aptamers again, the conjugate of aptamers and quantum dot with it is to be measured
Bacterium carries out specific reaction, to be dyed to bacterium to be measured.Present invention use first identifies to be dyed again, can be effectively reduced and examined
The infection risk that bacterium brings to testing staff during survey.
Preferably, the reaction condition in the step (2) is：It is passed through 2 μM of the 0.5ml quantum dot with aptamers
Nano-particle solution, react at room temperature 30 minutes.Under the conditions of being somebody's turn to do aptamers solution graft copolymerization can be made fully to be reacted with the bacterium that is captured.
Preferably, the preparation method of the quantum dot nano particle solution with aptamers is in the step (2)：By 8 μ
Aptamers described in M, 4.0 μM of surfaces quantum dot nano particle, 2.0mM NHS and 1.0mM EDC with-COOH be dissolved in 0.1M and
PH is in 6.0 MES buffer solutions, and 1h is reacted at 30 DEG C, and then, 10000 turns/min of centrifuge is centrifuged 10 minutes, removes supernatant
Liquid, it is 6.0MES buffer solutions to be eventually adding 1mL pH.
Compared with prior art, beneficial effects of the present invention are embodied in：
(1) compared with other optics microfluid detection techniques, detection time of the invention is fast, such as the inspection to Escherichia coli
Rising limit is 102Cells/ml, detection time are that the detection of most of optics microfluidic devices is limited to 10 within 4 hours3To 104
Cells/ml, the present invention is far beyond existing detection device.
(2) design concept of the invention is to catch tested target cell using multiple aptamers, such as according to the 7th generation
During dendrimer, each PAMAM G7 (the 7th generation PAMAM dendrimer) has 512 branches, if each branch can
An aptamers are enough combined, one can be grafted on each PAMAM G7 in theory and share 512 aptamers.Although it is contemplated that space
Steric hindrance and other factors, but the present invention still can increase substantially the quantity of aptamers grafting, so as to highly effective seizure quilt
Examine target cell.
(3) compared with the detection performance of the similar microfluidic channel using antibody graft modification, the detection of this detection method
Performance is higher, such as when dendrimer is the 7th generation PAMAM dendrimer, antibody is modified detection and is limited to 104, its reason
It is due to that antibody volume is larger, each PAMAM G7 macromoleculars can about be grafted an antibody (about molecular dimension：PAMAM G7
=8nm；Aptamers=2nm；Antibody is=10~15nm).
(4) Detection results in the present invention using the 7th generation PAMAM dendrimer access aptamers are especially prominent, such as
Its fluorescence intensity is higher by nearly twice of the fluorescence intensity of PAMAM G4 (forth generation PAMAM dendrimers) access aptamers, and this says
Bright PAMAM G7 are higher than the aptamers number being grafted on PAMAM G4, and Detection results are especially prominent.
(5) dendrimer is combined with aptamers and can produce multiple binding sites in the present invention, raising aptamers number
The efficiency of target cell is caught so as to improve it, the non-specificity that can reduce or be completely eliminated impurity in microfluidic surface is inhaled
It is attached, so as to reduce detection noise, realize quick and sensitive measurement.
(6) present invention is caught to bacterium by microfluid system, dyes, detects, with traditional first to Bacterial stain
For the system detected again, the present invention can effectively reduce the infection that bacterium brings to testing staff in detection process
Risk.Microfluid system in the present invention is a relatively independent and closed system, therefore can be reduced in whole detection process
Pollution of the testing staff to test sample.Simultaneously as this present invention is simple to operate, detection process need not have higher correlation
Technical staff.
Brief description of the drawings
Fig. 1 is the reaction principle figure of Example 1 and Example 2 of the present invention.
Fig. 2 is the fluorescence intensity introduced in the present invention after amino.
Fig. 3 is the Surface Characterization that the 7th generation PAMAM dendrimer being modified via-COOH group is introduced in the present invention
Situation.
Fig. 4 is by the variation diagram of chemical element before and after X-ray photoelectron spectroscopic analysis material modification in the present invention.
Fig. 5 is the contrast for the fluorescence intensity that the embodiment of the present invention 1, embodiment 2 and embodiment 3 are accessed before and after aptamers
Figure.
Embodiment
Embodiment 1
A kind of method of modifying of micro-fluidic chip, comprises the following steps：
(1) micro-fluidic chip of dimethyl silicone polymer (PDMS) is put into plasma generator and reacted, reacted
Condition is：Pressure is 100mTorr, power 118W, 10 seconds reaction time, the step for purpose be table in microchannel
- the CH in face3Group transformations are into-OH groups；
(2) after the step (1) reaction is completed, by the mixed of APTMS (aminopropyl trimethoxysilane) and absolute ethyl alcohol
Close solution and be passed through progress Silanization reaction in the microchannel, wherein APTMS mass ratio is 5%, after reacting 30 seconds, is led to
Enter the mixed solution of residual in air blow drying microchannel, and spontaneously dry at room temperature, this step can be on microchannel surface
Introduce amino group.
(3) after drying, it is tree-shaped that the 7th generation PAMAM that surface was modified via-COOH is passed through into the microchannel
Macromolecular, carry out coupling reaction；By 4.0 μM of dendrimers, 1.74mM NHS (n-hydroxysuccinimide) and 1.04mM
It is in 6.0 MES buffer solutions that EDC, which is dissolved in 0.1M and pH, and then MES buffer solutions are passed through in microchannel, reacted at 30 DEG C
The amino group on original microchannel surface can be converted into the 7th generation PAMAM dendrimer by 2h, this reactions steps.
(4) after step (3) reaction is completed, it is 6.0 that 5.2mM NHS and 0.26M EDC are added into 0.1M and pH
In MES buffer solutions, MES buffer solutions are then passed through microchannel, and react 1h at room temperature, it is suitable to be subsequently passed 10 μM of 100 μ L
Ligand solution, so that aptamers solution graft copolymerization is to the dendrimer surface, wherein adaptation liquid solution is the IDTE that pH is 7.5
Solution, it has the adaptation liquid solution of specific recognition function to bacterium to be measured, and the sequence of wherein aptamers is：SEQ ID No.1.
After above-mentioned modified completion, the micro-fluidic chip that above-mentioned modification is obtained is used to detect the large intestine bar in testing sample
Bacterium, specific steps include：
(1) it is passed through testing sample into the microchannel of micro-fluidic chip；
(2) after step (1) reaction is completed, buffer solution for cleaning microchannel is passed through, is then passed through again corresponding
The quantum dot nano particle solution with aptamers.
(3) micro-fluidic chip is finally placed in fluorescence viewed under light microscopy and records fluorescence points, you can obtained to be measured
Contained bacterium number amount to be measured in sample solution.
Reaction condition wherein in above-mentioned steps (2) is：It is passed through 2 μM of the 0.5ml quantum dot nano grain with aptamers
Sub- solution, react at room temperature 30 minutes.Under the conditions of being somebody's turn to do aptamers solution graft copolymerization can be made fully to be reacted with the bacterium that is captured.
The preparation method of the quantum dot nano particle solution with aptamers is in above-mentioned steps (2)：By 8 μM of adaptations
It is 6.0 that the quantum dot nano particle of body, 4.0 μM of surfaces with-COOH, 2.0mM NHS and 1.0mM EDC, which are dissolved in 0.1M and pH,
In MES buffer solutions, 1h is reacted at 30 DEG C, then, 10000 turns/min of centrifuge is centrifuged 10 minutes, removes supernatant, is finally added
It is 6.0MES buffer solutions to enter 1mL pH.
Above-mentioned testing result is shown in Table 1.
The reaction principle figure of above-mentioned steps, may refer to Fig. 1.Wherein Oxygen plasma:Oxygen gas plasma generator
Processing；APTMS treatment：APTMS processing；PAMAM engraftment：PAMAM is grafted；Generation 4/
Generation 7：The generation of forth generation/the 7th；PAMAM G4engraftment：PAMAM forth generations are grafted；PAMAM
G7engraftment：The pickup branches of PAMAM the 7th；Aptamer engraftment:Aptamers are grafted；
E.coli.detection:Escherichia coli monitor；64sites：64 connection sites；512sites:512 connection sites；
PDMS surface：PDMS surfaces；PAMAM G4：PAMAM forth generations；PAMAM G7：The generations of PAMAM the 7th；Aptamer：Adaptation
Body；E.coli.：Escherichia coli；Other particles：Other impurities particle；Aptamer-QD：Aptamers --- quantum dot
Nano-particle conjugate；Quantum dot；Quantum dot nano-particle.
Tested for the microchannel Surface Characterization in above-mentioned steps (1) and step (2)：
Characterize principle：Rhodamine with-NHS groups can react with amino group, therefore reaction surface
By this chemical reaction handling, then cleaned with deionized water, the fluorescence intensity of reaction surface is then characterized, if surface has ammonia
Base group, then have higher fluorescence intensity.
Figure it is seen that the fluorescence intensity ratio on microchannel surface treated APTMS does not make what APTMS was treated
The fluorescence intensity on microchannel surface is high, it may be said that surface success grafted amino group group treated bright APTMS.
And " blank PDMS surfaces (micro-fluidic chip of mistake not modified by this invention) soaks in NHS- rhodamine liquors
30 minutes " and " the treated PDMS surfaces of plasma generator are (through the micro-fluidic core that step (1) was modified in embodiment 1
Piece) soaked 30 minutes in NHS- rhodamine liquors " compare, wherein " plasma generator treated PDMS surfaces " it is glimmering
The fluorescence intensity of light strength ratio " blank PDMS surfaces " is high, and this phenomenon may be considered as absorption of the surface to fluorescent balls.
PAMAM G7 (tree-shaped big point of the 7th generation PAMAM) table is introduced for the microchannel surface in above-mentioned steps (3)
Sign test：
Characterization test method is as follows：
1st, by AFM surface sweeping material surface, compare via PAMAM G7 before modified after change
From figure 3, it can be seen that the microchannel surface roughness being modified via PAMAM G7 has obvious increase.Phase
It increased compared with the microchannel surface roughness that APTMS was modified, therefore the microchannel surface that APTMS was modified is not
There is the microchannel surface being modified via PAMAM G7 increased obvious.
2nd, by X-ray photoelectron spectroscopic analysis microchannel via the PAMAM G7 changes of chemical element afterwards before modified.
From Fig. 4 (Intensity：Intensity；Binding energy：Bond energy) in as can be seen that be shown via PAMAM
G7 before modified after N 1s and C 1s XPS peaks spectrum.I and III compares the change of nitrogen, it can be found that nitrogen has substantially
Change.There is no the spectrogram of nitrogen in I, and in III, by grafting PAMAM G7, produced at 401.2eV and 286.4eV
A peak has all been given birth to, has corresponded to-CONH- and C-N structures respectively.The immanent structure that two chemical bonds are respectively present in APTMS
With in PAMAM G7 immanent structure.Compare the change of II and IV carbons again, before modification in II, it can be seen that surface
Chemical constitution is C-H and C-Si structures, and it is 285.0eV and 284.5eV to correspond to energy respectively., can be with the modified IV in surface
See that C-Si structure disappears substantially, this is due to that PAMAM G7 have been grafted on surface, and PAMAM G7 cover former PDMS tables
Face structure.Simultaneously, it can be seen that it is more C-H (C) (285.0eV) on surface texture, C-N (286.4eV), C=O (288eV) with
(C=O) chemical constitution such as-O (289.1eV).These structures are all G7 internal structures.
Embodiment 2
Using method same as Example 1, unique difference is, the dendrimer being grafted is PAMAM G4
(forth generation PAMAM dendrimers).Reaction principle figure, may refer to Fig. 1.
After above-mentioned modified completion, Escherichia coli in testing sample are entered using detection method same as Example 1
Row detection, testing result are shown in Table 1.
Embodiment 3
Using method same as Example 1, unique difference is, the dendrimer being grafted is PAMAM G9
(the 9th generation PAMAM dendrimer).
After above-mentioned modified completion, Escherichia coli in testing sample are entered using detection method same as Example 1
Row detection, testing result are shown in Table 1.
From fig. 5, it can be seen that detections of the grafting PAMAM G7 to Escherichia coli is limited to 102Cells/ml, detection time are
Within 4 hours, the detection of existing most of optics microfluidic devices is limited to 104Cells/ml, therefore via the embodiment of the present invention
Can be far beyond existing detection device after 1 modification.
The detection performance of the wherein embodiment of the present invention 1 is higher, and (antibody is modified detection and is limited to 104), its reason is due to antibody
Volume is larger, and each G7 macromoleculars can about be grafted an antibody (about molecular dimension：G7=8nm；Aptamers=2nm；Antibody
For=10~15nm).
The present invention has found that the fluorescence intensity after being modified in embodiment 1 is higher by reality by the fluorescence intensity of embodiment 1~3
Nearly twice of the fluorescence intensity of example 2 is applied, this explanation PAMAM G7 are higher than the aptamers number being grafted on PAMAM G4.
Table 1 is testing result
Absorption situation of the Escherichia coli in the microchannel after different modifications, each microchannel are divided into two portions
Point：Pipeline situation and Escherichia coli quantity.Wherein the situation of Escherichia coli absorption is shown in " situation "；" quantity " is shown
The quantity that Escherichia coli are specifically adsorbed；These quantity are manually to count to afterbody from the head of pipeline.
As it can be seen from table 1 wherein microchannel surface can significantly reduce the non-of impurity with PAMAM G7 are modified
Specific adsorption, noise is reduced, so as to improve signal to noise ratio, and then effectively raises the sensitivity of detection.Can be with from upper table 1
Microchannel that PAMAM G7 were modified is found out all without the absorption of Escherichia coli, without modified microchannel in large intestine bar
Bacterium number reaches 105Non-specific adsorption is just had during the above.Therefore, it may indicate that the microfluidic channel that PAMAMG7 was modified can
To effectively reduce detection noise.
Sensitivity of the detection method except effectively raising detection, reduce the time of detection, also for its
His chip functions integrated (such as sample separation and concentration of upstream) and detection automation provide possibility.
Embodiment 4
Using method same as Example 1, unique difference is, the aptamers sequence of aptamers is in the present embodiment
SEQ ID No.2, the present embodiment are used to detect salmonella.
After above-mentioned modified completion, using detection method same as Example 1 to 102In cells/ml testing sample
Salmonella detected, you can obtain in testing sample solution the quantity of contained salmonella, testing result 21+/-
4 cells are.
Embodiment 5
Using method same as Example 1, unique difference is, the aptamers sequence of aptamers is in the present embodiment
SEQ ID No.3, the present embodiment are used to detect staphylococcus
After above-mentioned modified completion, using detection method same as Example 1 to 102In cells/ml testing sample
Staphylococcus detected, you can obtain in testing sample solution contained staphylococcic quantity, testing result is shown in 25
+/- 3 cells.
Embodiment 6
Using method same as Example 1, unique difference is, using method same as Example 1, uniquely not
It is that the aptamers sequence of aptamers is SEQ ID No.4 in the present embodiment with point, the present embodiment is used to detect acidophilus breast bar
Bacterium.
After above-mentioned modified completion, using detection method same as Example 1 to 102In cells/ml testing sample
Lactobacillus acidophilus detected, you can obtain in testing sample solution the quantity of contained lactobacillus acidophilus, testing result
See 18+/- 4 cell.
Sequence table
<110>Hainan Institute of Science ＆ Technology
Lv Chengnongke detection techniques Co., Ltd
<120>A kind of micro-fluidic chip and its method of modifying and detection food bacterial number on apply
<160> 5
<170> SIPOSequenceListing 1.0
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<211> 42
<212> DNA
<213>Artificial synthesized sequence (unkonw)
<400> 1
ccggacgctt atgccttgcc atctacagag caggtgtgac gg 42
<210> 2
<211> 89
<212> RNA
<213>Artificial synthesized sequence (unkonw)
<400> 2
gggagagcgg aagcgugcug gguaguguga gagccgugag ugaaaggccg cgacaaagau 60
cggacauaac ccagaggucg auggucccc 89
<210> 3
<211> 92
<212> RNA
<213>Artificial synthesized sequence (unkonw)
<400> 3
gggagagcgg aagcgugcug ggccgggaag uuuugauacg gcuucaugca aguaauguuu 60
uuaucauaac ccagaggucg auggaucccc cc 92
<210> 4
<211> 73
<212> DNA
<213>Artificial synthesized sequence (unkonw)
<400> 4
atccgtcaca cctgctctac ggcgctccca acaggcctct ccttacggca tattatggtg 60
ttggctcccg tat 73
<210> 5
<211> 88
<212> DNA
<213>Artificial synthesized sequence (unkonw)
<400> 5
gcaatggtac ggtacttcct cggcacgttc tcagtagcgc tcgctggtca tcccacagct 60
acgtcaaaag tgcacgctac tttgctaa 88
Claims (10)
1. a kind of method of modifying of micro-fluidic chip, it is characterised in that comprise the following steps：
(1) by-CH of the microchannel inner surface on micro-fluidic chip3Group modified is-OH groups；
(2) after step (1) reaction is completed, aminosilane reagents is passed through into the microchannel, carry out amino silane
Reaction；
(3) after drying, the dendrimer that surface was modified via-COOH is passed through into the microchannel, is coupled
Reaction；
(4) after step (3) reaction is completed, be passed through into the microchannel has specific recognition performance to bacterium to be measured
And 5 ' end by amino modified mistake adaptation liquid solution so that aptamers are grafted to the dendrimer surface.
2. the method for modifying of micro-fluidic chip as claimed in claim 1, it is characterised in that will be micro-fluidic in the step (1)
Chip, which is placed in plasma generator, to be reacted, and wherein plasma generator pressure is 100mTorr, power 118W,
Reaction time is 10~20 seconds.
3. the method for modifying of micro-fluidic chip as claimed in claim 1, it is characterised in that in the step (2), the amino
Silane reagent is 3- aminopropyl trimethoxysilanes, N- (2- aminoethyls) -3- aminopropyltriethoxy dimethoxysilanes, N- (2- ammonia
Ethyl) at least one of -3- aminopropyl triethoxysilanes, N- (2- aminoethyls) -3- aminopropyl trimethoxysilanes.
4. the method for modifying of micro-fluidic chip as claimed in claim 3, it is characterised in that in the step (2), the amino
Silane reagent is 3- aminopropyl trimethoxysilanes, and the mixed solution of 3- aminopropyl trimethoxysilanes and absolute ethyl alcohol is passed through
In microchannel, amino silane reaction is carried out, wherein the content of the 3- aminopropyl trimethoxysilanes is 1~5%.
5. the method for modifying of the micro-fluidic chip as described in Claims 1 to 4 is any, it is characterised in that set in the step (3)
Shape macromolecular is the 7th generation dendrimer that monomer is PAMAM.
6. the method for modifying of micro-fluidic chip as claimed in claim 5, it is characterised in that by 4.0 μM of trees in the step (3)
It is in 6.0 MES buffer solutions, then by MES buffer solutions that shape macromolecular, 1.74mM NHS and 1.04mM EDC, which are dissolved in 0.1M and pH,
It is passed through in microchannel, reacts 2h at 30 DEG C.
7. the method for modifying of micro-fluidic chip as claimed in claim 6, it is characterised in that in the step (4), by 5.2mM
NHS and 0.26M EDC are added in the MES buffer solutions that 0.1M and pH are 6.0, and MES buffer solutions then are passed through into microchannel, and
1h is reacted in room temperature, is subsequently passed 10 μM of 100 μ L adaptation liquid solutions, so that aptamers solution graft copolymerization is to the dendrimer
Surface.
8. the method for modifying of micro-fluidic chip as claimed in claim 1, it is characterised in that the bacterium to be measured be Escherichia coli,
Salmonella, staphylococcus, lactobacillus acidophilus or staphylococcus aureus, the corresponding aptamers relation of the bacterium to be measured
For：
9. a kind of method of modifying as described in claim 1~8 is any is modified obtained micro-fluidic chip.
A kind of 10. application of the micro-fluidic chip on detection food bacterial number described in claim 9.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753929A (en) * | 2018-04-24 | 2018-11-06 | 绿城农科检测技术有限公司 | A kind of micro-fluid chip and its method of modifying and the application on detection food bacterial number |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN113125716A (en) * | 2021-03-23 | 2021-07-16 | 中国农业科学院农产品加工研究所 | Method for simultaneously killing and detecting microorganisms |
| WO2023039862A1 (en) * | 2021-09-17 | 2023-03-23 | 绿城农科检测技术有限公司 | Dendritic macromolecule-based method for modifying solid-phase carrier |
-
2017
- 2017-10-30 CN CN201711035097.XA patent/CN107860934A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108753929A (en) * | 2018-04-24 | 2018-11-06 | 绿城农科检测技术有限公司 | A kind of micro-fluid chip and its method of modifying and the application on detection food bacterial number |
| CN110632302A (en) * | 2019-10-30 | 2019-12-31 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN110632302B (en) * | 2019-10-30 | 2023-07-18 | 中国农业科学院农产品加工研究所 | Method for simultaneously detecting contents of escherichia coli and salmonella in sample to be detected |
| CN113125716A (en) * | 2021-03-23 | 2021-07-16 | 中国农业科学院农产品加工研究所 | Method for simultaneously killing and detecting microorganisms |
| WO2023039862A1 (en) * | 2021-09-17 | 2023-03-23 | 绿城农科检测技术有限公司 | Dendritic macromolecule-based method for modifying solid-phase carrier |
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