CN103772728B - Dimethyl silicone polymer surface method of modifying based on hydrophobin/methylcellulose - Google Patents
Dimethyl silicone polymer surface method of modifying based on hydrophobin/methylcellulose Download PDFInfo
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- CN103772728B CN103772728B CN201410023787.3A CN201410023787A CN103772728B CN 103772728 B CN103772728 B CN 103772728B CN 201410023787 A CN201410023787 A CN 201410023787A CN 103772728 B CN103772728 B CN 103772728B
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- silicone polymer
- dimethyl silicone
- methylcellulose
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Abstract
The invention discloses a kind of dimethyl silicone polymer surface method of modifying based on hydrophobin/methylcellulose, taking hydrophobin SC3 and methylcellulose as surface modifier, form amphipathic monomolecular film through physical absorption in the self assembly of dimethyl silicone polymer microchannel surface, then heating chip passage, the configuration of methylcellulose Induced drainage Protein S C3 is transformed, form stable amphipathic monomolecular film in dimethyl silicone polymer microchannel surface. Adopt the inventive method can strengthen the coating stability of dimethyl silicone polymer, strengthen the surface hydrophilicity of its passage, CKIs matter is in the non-specific adsorption of microchannel surface effectively, improve the contamination resistance of dimethyl silicone polymer surface, the efficient reproduction of Realization analysis thing separates simultaneously.
Description
Technical field
The invention belongs to micro-fluidic chip separation technology field, be specifically related to a kind of dimethyl silicone polymer micro flow control coreThe surface modifying method of sheet.
Background technology
Micro-fluidic chip claims again micro-total analysis system, be the one that grows up the nineties in 20th century by sample preparation,Reaction, separation and detection etc. are integrated in the new technology on single microchip substantially, have been widely used at present life sectionMany research fields such as, biological medicine, environmental science and chemistry. Dimethyl silicone polymer (PDMS) is because of itThere is the advantages such as cheap, stable chemical nature, bio-compatibility be good, thereby be commonly used for the base of micro-fluidic chipBody material, but because it has stronger hydrophobicity, EOF is unstable and the multiple analytes matter such as protein are hadStronger adsorptivity, causes the reduction of PDMS micro-fluidic chip performance, and then affects its range of application.
Physical absorption coating is a kind of microchannel surface method of modifying that current PDMS micro-fluidic chip is conventional. The partyMethod be surface active molecules in the spontaneous adsorption phenomena of solid/liquid interfaces, form physics absorbent coating at the surface of solids, fromAnd overcome the non-specific adsorption of sample molecule on chip channel surface, and the method is easy and simple to handle, and coating is easily regenerated.It is as better in 0.5% methylcellulose (MC) modification effect with high-concentration water-soluble polymer in conventional coating material,But high-concentration water-soluble polymer makes solution viscosity become large, for experimental implementation is made troubles, and to CKIs matterNon-specific adsorption effect is undesirable. In addition, the unstability of the coating forming due to physical absorption, soModify after chip not treatedly when directly analyte is separated, can make the separative efficiency of analyte be subject to certain journeyThe impact of degree.
Summary of the invention
Technical problem to be solved by this invention is to overcome the thing that existing micro-fluidic chip surface modifying method existsReason absorbent coating unsettled shortcoming, provides that a kind of chip surface hydrophily is strong, contamination resistance is high, coating is stableDimethyl silicone polymer micro flow control chip surface modifying method.
Solving the problems of the technologies described above adopted technical scheme is: hydrophobin SC3 and methylcellulose are added to fortuneIn row buffering liquid, in every liter of running buffer, add 0.5～2.5mg hydrophobin SC3,0.5g methylcellulose,Be mixed with surface modifier, in the microchannel of the dimethyl silicone polymer micro flow control chip after NaOH activation processingInjection surface modification agent, normal temperature is hatched 10～20 minutes, and 60～70 DEG C are heated 10～30 minutes, obtain surfaceThe dimethyl silicone polymer of modification.
The present invention preferably adds hydrophobin SC3 and methylcellulose in running buffer, every liter of running bufferIn add 1.0mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modifier.
Above-mentioned running buffer is that 20mmol/LpH value is 7.4～9.4 PBS buffer solution, 10mmol/LpHValue is the Tris-HCl buffer solution that 8.0～11.0 borate buffer solution or 50mmol/LpH value are 7.0～9.0; InstituteThe hydrophobin SC3 stating is provided by sigma company, its article No. 68795.
The present invention is taking hydrophobin SC3 and methylcellulose as surface modifier, through physical absorption at poly dimethyl siliconThe self assembly of oxygen alkane microchannel surface forms amphipathic monomolecular film, and then heating chip passage, makes methylcelluloseThe configuration of Induced drainage Protein S C3 transforms, and forms stable amphipathic in dimethyl silicone polymer microchannel surfaceMonomolecular film, the method can strengthen the coating stability of dimethyl silicone polymer and the surface hydrophilicity of passage,CKIs matter, in the non-specific adsorption of microchannel surface, improves the anti-soil of dimethyl silicone polymer surface effectivelyDye ability. Experimental result shows, dimethyl silicone polymer micro flow control chip separation egg after the modification of employing the inventive methodIn vain, polypeptide, the chromatographic peak type of analyte is significantly improved, post effect and separating degree are significantly improved, realThe efficient reproduction that has showed analyte separates.
Brief description of the drawings
Fig. 1 is the dimethyl silicone polymer of the surface modification that obtains of dimethyl silicone polymer (a), comparative example 1And the infrared spectrogram of the dimethyl silicone polymer (c) of the surface modification that obtains of embodiment 1 (b).
Fig. 2 is that the bovine serum albumin(BSA) of marked by fluorescein isothiocyanate is in polydimethylsiloxanechip chip microchannelFluorescence indicator adsorption photo.
Fig. 3 is poly-two of the surface modification that obtains at embodiment 1 of the bovine serum albumin(BSA) of marked by fluorescein isothiocyanateFluorescence indicator adsorption photo in methylsiloxane chip microchannel.
Fig. 4 is that the lysozyme of the marked by fluorescein isothiocyanate fluorescence in polydimethylsiloxanechip chip microchannel is inhaledAttached photo.
Fig. 5 is the poly dimethyl silicon of the surface modification that obtains at embodiment 1 of the lysozyme of marked by fluorescein isothiocyanateIn oxygen alkane chip microchannel fluorescence indicator adsorption photo.
Fig. 6 be marked by fluorescein isothiocyanate bovine serum albumin(BSA), lysozyme respectively dimethyl silicone polymer,Relative fluorescence adsorption strength in the polydimethylsiloxanechip chip microchannel of the surface modification that embodiment 1 obtains.
Fig. 7 be in blood red blood cell at the scanning electron microscope (SEM) photograph of polydimethylsiloxanechip chip adsorption situation.
Fig. 8 is that inhale on the polydimethylsiloxanechip chip surface of the surface modification that in blood, red blood cell obtains at embodiment 1The scanning electron microscope (SEM) photograph of attached situation.
Fig. 9 is the dimethyl silicone polymer of the surface modification that obtains at embodiment 2 of neurotensin and methionine enkephalin,MEKChip obtains electrophoretogram while separation.
Figure 10 is the poly dimethyl silica of the surface modification that obtains at embodiment 2 of myosin and bovine serum albumin(BSA)The electrophoretogram that alkane chip obtains while separation.
Detailed description of the invention
Below in conjunction with drawings and Examples, the present invention is described in more detail, but the present invention is not limited only to these in factExecute example.
Embodiment 1
Polydimethylsiloxanechip chip microchannel is first used to the NaOH aqueous solution soaking 5 minutes of 1mol/L, carried outActivation processing, then extremely neutral with deionized water rinsing, then the PBS that is 7.4 by 20mmol/LpH value bufferingLiquid rinses, and obtains the dimethyl silicone polymer micro flow control chip after NaOH activation processing. By hydrophobin SC3Adding 20mmol/LpH value with methylcellulose is in 7.4 PBS buffer solution, in every liter of PBS buffer solution, addsEnter 1.0mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modifier, use syringe to NaOHInjection surface modification agent in the microchannel of the dimethyl silicone polymer micro flow control chip after activation processing, normal temperature hatches 15Minute, 65 DEG C are heated 30 minutes, obtain the dimethyl silicone polymer of surface modification.
Comparative example 1
It is in 7.4 PBS buffer solution that hydrophobin SC3 and methylcellulose are added to 20mmol/LpH value,In every liter of PBS buffer solution, add 1.0mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modificationAgent is injected table with syringe in the microchannel of the dimethyl silicone polymer micro flow control chip after NaOH activation processingFace modifier, normal temperature is hatched 15 minutes, obtains the dimethyl silicone polymer of surface modification.
Inventor adopts respectively the method for Fourier transformation attenuate total reflection and contact angle measurement to poly dimethyl silicaThe table that the polydimethylsiloxanechip chip of the surface modification that alkane chip, embodiment 1 obtain and comparative example 1 obtainThe polydimethylsiloxanechip chip of face modification characterizes, and the results are shown in Figure 1. As seen from the figure, comparative example 1 obtainsTo the polydimethylsiloxanechip chip of surface modification be 1653cm in wave number-1And 1679cm-1The functional group at place dividesDo not represent that hydrophobin exists alpha-helix state and free state, and the poly dimethyl silicon of the surface modification that embodiment 1 obtainsOxygen alkane chip is only 1653cm in wave number-1The functional group at place, this surface modifying method that shows embodiment 1 makesMethylcellulose has induced the configuration of hydrophobin SC3 to transform, thereby the coating of enhancing dimethyl silicone polymer is stableProperty. In addition, the contact angle that obtains polydimethylsiloxanechip chip by contact angle experiments of measuring is 119.4 ± 2.6 °,The contact angle of the polydimethylsiloxanechip chip of the surface modification that comparative example 1 obtains is 43.2 ± 0.8 °, implementsThe contact angle of the polydimethylsiloxanechip chip of the surface modification that example 1 obtains is 37.6 ± 0.9 °, illustrates of the present inventionSurface modifying method can strengthen the surface hydrophilicity of polydimethylsiloxanechip chip microchannel.
Embodiment 2
The method of NaOH activation processing dimethyl silicone polymer micro flow control chip and embodiment 1 phase in the present embodimentWith. It is in 9.5 borate buffer solution that hydrophobin SC3 and methylcellulose are added to 10mmol/LpH value,In every liter of borate buffer solution, add 1.0mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modificationAgent is injected table with syringe in the microchannel of the dimethyl silicone polymer micro flow control chip after NaOH activation processingFace modifier, normal temperature is hatched 15 minutes, and 65 DEG C are heated 30 minutes, obtain the poly dimethyl silica of surface modificationAlkane.
Embodiment 3
The method of NaOH activation processing dimethyl silicone polymer micro flow control chip and embodiment 1 phase in the present embodimentWith. It is 8.0 Tris-HCl buffer solution that hydrophobin SC3 and methylcellulose are added to 50mmol/LpH valueIn, in every liter of borate buffer solution, add 0.5mg hydrophobin SC3,0.5g methylcellulose, be mixed with surfaceModifier is noted with syringe in the microchannel of the dimethyl silicone polymer micro flow control chip after NaOH activation processingEnter surface modifier, normal temperature is hatched 20 minutes, and 60 DEG C are heated 30 minutes, obtain the poly dimethyl of surface modificationSiloxanes.
Embodiment 4
The method of NaOH activation processing dimethyl silicone polymer micro flow control chip and embodiment 1 phase in the present embodimentWith. It is in 8.7 PBS buffer solution that hydrophobin SC3 and methylcellulose are added to 20mmol/LpH value,In every liter of PBS buffer solution, add 2.5mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modificationAgent is injected table with syringe in the microchannel of the dimethyl silicone polymer micro flow control chip after NaOH activation processingFace modifier, normal temperature is hatched 10 minutes, and 70 DEG C are heated 10 minutes, obtain the poly dimethyl silica of surface modificationAlkane.
In order to prove beneficial effect of the present invention, inventor adopts poly-two of surface modification that embodiment 1 and 2 obtainsMethylsiloxane has carried out a large amount of laboratory research tests, and various test situation are as follows:
1, surperficial anti-adsorptivity test
The bovine serum albumin(BSA) of 1mg/mL marked by fluorescein isothiocyanate and lysozyme are injected respectively to poly dimethylIn the polydimethylsiloxanechip chip microchannel of the surface modification that siloxanes chip microchannel and embodiment 1 obtain,Hatch 1 hour for 37 DEG C, the PBS buffer solution that is then 7.4 by 20mmol/LpH value rinses 10 minutes, usesInverted fluorescence microscope records the absorption situation of bovine serum albumin(BSA) in microchannel, the results are shown in Figure 2～6.
As seen from the figure, in polydimethylsiloxanechip chip microchannel, the ox blood of marked by fluorescein isothiocyanate is pureThe relative intensity of fluorescence of albumen and lysozyme is stronger, and the dimethyl silicone polymer of the surface modification that embodiment 1 obtainsIn chip microchannel, the bovine serum albumin(BSA) of marked by fluorescein isothiocyanate and the relative intensity of fluorescence of lysozyme are very weak,The polydimethylsiloxanechip chip that the surface modification that embodiment 1 obtains is described plays the non-specific adsorption of proteinGood inhibitory action.
2, surface anti-pollution test
The polydimethylsiloxanechip chip of the surface modification that polydimethylsiloxanechip chip and embodiment 1 are obtained respectivelyImmerse in the platelet rich plasma obtaining after centrifugal blood, 37 DEG C adsorb 3 hours, then use 20mmol/LpHValue is 7.4 PBS buffer solution rinsing 3 times, then soaks 30 in the glutaraldehyde water solution that is 2.5% at mass fractionMinute, the PBS buffer solution rinsing that is 7.4 by 20mmol/LpH value 3 times, drying at room temperature, uses ESEMCharacterize, the results are shown in Figure 7 and Fig. 8.
As seen from the figure, on polydimethylsiloxanechip chip, adsorb a large amount of red blood cells, and embodiment 1 obtainsOn the polydimethylsiloxanechip chip of surface modification, almost without red blood cell absorption, illustrate that the surface that embodiment 1 obtains changesThe polydimethylsiloxanechip chip of property has good contamination resistance.
3, protein isolate and polypeptide
The polydimethylsiloxanechip chip of the surface modification that employing embodiment 2 obtains is to neurotensin and first sulphur brain coffeePeptide, myosin and bovine serum albumin(BSA) carry out electrophoretic analysis. Result is shown in respectively Fig. 9 and Figure 10.
As seen from the figure, neurotensin and methionine enkephalin,MEK, myosin and bovine serum albumin(BSA) obtain respectively fineGround separates, and illustrates that the polydimethylsiloxanechip chip of the surface modification that embodiment 1 obtains can be to polypeptide, proteinRealize the efficient separation that reappears Deng large biological molecule.
Claims (2)
1. the dimethyl silicone polymer surface method of modifying based on hydrophobin/methylcellulose, its feature existsIn: hydrophobin SC3 and methylcellulose are added in running buffer, in every liter of running buffer, add 0.5～2.5mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modifier, to NaOH activation processingAfter the microchannel of dimethyl silicone polymer micro flow control chip in injection surface modification agent, normal temperature is hatched 10～20 pointsClock, 60～70 DEG C are heated 10～30 minutes, obtain the dimethyl silicone polymer of surface modification;
Above-mentioned running buffer is that 20mmol/LpH value is 7.4～9.4 PBS buffer solution, 10mmol/LpHValue is the Tris-HCl buffer solution that 8.0～11.0 borate buffer solution or 50mmol/LpH value are 7.0～9.0.
2. the dimethyl silicone polymer surface based on hydrophobin/methylcellulose according to claim 1 changesProperty method, is characterized in that: hydrophobin SC3 and methylcellulose are added in running buffer to every elevator rowIn buffer solution, add 1.0mg hydrophobin SC3,0.5g methylcellulose, be mixed with surface modifier.
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| CN103331097A (en) * | 2013-05-27 | 2013-10-02 | 陕西师范大学 | Application of polydimethylsiloxane micro fluidic chip in separating oligosaccharide and polysaccharide |
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| CN103331097A (en) * | 2013-05-27 | 2013-10-02 | 陕西师范大学 | Application of polydimethylsiloxane micro fluidic chip in separating oligosaccharide and polysaccharide |
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| "Hydrophobin can prevent secondary protein adsorption on hydrophobic substrates without exchange";Rui Xu等;《Anal Bioanal Chem》;20110405;第2031-2040页 * |
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