CN104597230B - A kind of functional polymer film, preparation method and applications - Google Patents
A kind of functional polymer film, preparation method and applications Download PDFInfo
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- CN104597230B CN104597230B CN201510045459.8A CN201510045459A CN104597230B CN 104597230 B CN104597230 B CN 104597230B CN 201510045459 A CN201510045459 A CN 201510045459A CN 104597230 B CN104597230 B CN 104597230B
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Landscapes
- Apparatus Associated With Microorganisms And Enzymes (AREA)
Abstract
The invention provides a kind of functional polymer film, preparation method and applications.Methods described comprises the steps：1) photocrosslinking agent is modified in substrate surface；2) polymer solution is spin-coated on into step 1) form surface；3) reacted with ultraviolet light, make polymer covalence graft thin polymer film be formed to substrate；4) carry out to the further functionalization of polymer or directly bio-sensing application.It is simple, quick, efficient that the method prepares three-dimensional macromolecule surface; can be with the high molecular density in precise control surface and thickness by regulating and controlling photocrosslinking agent density, polymer molecular weight, spin coating parameters etc.; and, methods described is workable, reproducible, is adapted to large-scale production.The polymeric film surface for obtaining is high to the fixed amount of biomolecule, there is good anti-non-specific adsorption ability simultaneously, the detection performance of biology sensor (such as surface plasmon resonance imaging, fluorescence, QCM and electrochemical sensor etc.) can be improved.
Description
Technical field
The present invention relates to a kind of simple, quick, general macromolecule membrane, preparation method and its answering in bio-sensing
With the particularly application in surface plasmon resonance imaging.
Background technology
Biochip mainly by the surface chemical modification to substrate, by biomolecule such as protein, polypeptide, nucleic acid and thin
The biological samples such as born of the same parents, tissue are fixed on chip surface, and then realize to the accurate, quick of targeted biological component, large information capacity inspection
Survey, with the features such as height collimation, diversity, miniaturization and automation.Rational surface chemical modification can make biology
It is highly efficient, stable that sample is fixed.In order to improve crystallized ability of the biochip to biological sample, non-specific suction is reduced
It is attached, improve detection signal strength signal intensity, and then improve instrument detection sensitivity, people to surface chemistry in terms of carried out substantial amounts of grinding
Study carefully.
At present, self assembled monolayer is relatively broad in bio-sensing and chip technology field application, and having an advantage in that can
To form stable, homogeneous, fine and close molecular layer on surface, not only can in detection process protection group bottom, reduce non-specific
Absorption, it is also possible to possess diversified terminal functional groups, do and further modify；In addition, self assembled monolayer modification operation
It is extremely simple, modify reproducible.But, self assembled monolayer, is examined because its biomolecule fixation amount is little as two-dimensional surface
When surveying bio-molecular interaction, signal is weak so as to which application is greatly limited.In order to further enhance biochip opposite
The fixed amount of thing molecule, so as to improve biology sensor detection sensitivity, many researchs employ the chip surface of three-dimensional structure
Method of modifying.One layer of macromolecule membrane rich in various active groups with three-dimensional structure is built in biochip substrate,
Surface prepared by this kind of modification mode is commonly referred to as three-dimensional surface, because its three dimensions surface has a large amount of biomolecule
Binding site, substantially increases the fixed amount of biomolecule, can reach the tens of or even hundreds times of common chip.
The existing research with regard to three-dimensional structure surface modification includes the three-dimensional surface of various polymer films, such as glucan,
Polyacrylic acid, celluloid, nylon, agarose, polyacrylamide, the polyethylene glycol hydrogel of polylysine modification, micro-/ nano
Level rough surface structure and some Supramolecular self assembly three-dimensional structures etc., wherein glucan three-dimensional surface are most widely used
(Johnsson B.,et al.,Comparison of methods for immobilization to carboxymethyl
dextran sensor surfaces by analysis of the specific activity of monoclonal
antibodies.Journal of Molecular Recognition,1995.8(1-2):p.125-131.).In addition, being based on
The research of polymer brush shaped polymer prepared by ATRP (ATRP) method also because its reaction condition it is gentle,
It is easy to operate, the advantages of anti-non-specific adsorption ability is strong, receive significant attention (Wang, J.-S.and K.Matyjaszewski,
Controlled/"living"radical polymerization.Atom transfer radical
polymerization in the presence of transition-metal complexes.Journal of the
American Chemical Society,1995.117(20):p.5614-5615.).And, the Biocompatibility is good,
End function group is enriched, and with good characteristics such as anti-non-specific adsorptions, is widely applied in biosensor surface.
In polymer growth, strand typically has two methods with the connection at surface or interface：One kind is " being grafted to "
(graft-to) method, the method (graft-from) that another is " from surface grafting ".In " from surface grafting " method,
Polymer directly grows on the surface of the substrate, and generally first the initiator of polymer is fixed on surface, then by surface
Initiator causes next step polymerization, the method control polymer thickness by adjusting monomer and reaction time, and by adjusting
The surface coverage of whole initiator is controlling the grafting density of polymer.However, the method experimental procedure is complex, in regulation and control
During surface polymer density, repeatability is relatively poor, and production cost is higher, generally has bio-toxicity using catalyst, and easily remains
On surface, it is unfavorable for that biomolecule activity keeps.And " being grafted to " method is simple, quick due to preparing, easily entered using material
The antenatal Quality Control of row, is favored by people.But polymer reaction functional group is bonded by chemistry, for different functional groups will be adopted
Differential responses system, it is more with respect to Consideration, and for nano-crystal, reaction rate is not generally high, the time compared with
Long, macromolecule is moved in the solution compared with being difficult to and self space steric hindrance is big, causes local reaction to be limited, and shortcoming is projected all the more.
The content of the invention
For the problem of prior art, an object of the present invention is to provide a kind of preparation side of functional polymer film
Polymer is randomly covalently fixed on substrate surface using the mode of ultraviolet light cross-linking for method, the method, obtains macromolecule membrane,
Then the capture and detection of biomolecule and drug molecule etc. are carried out to its further functionalization or directly.
To achieve these goals, present invention employs following technical scheme：
A kind of preparation method of functional polymer film, the method comprising the steps of：
(1) self assembled monolayer of end-functionalization is formed in biochip substrate surface；
(2) photocrosslinking agent is grafted to into self assembled monolayer end by chemical bonding；
(3) Polymer Solution is spun to into the surface that step (2) is formed under the conditions of lucifuge, is then dried；
(4) there is high molecular biochip that ultraviolet lighting is carried out under inert gas shielding surface spin coating, in ultraviolet light
Under the conditions of be chemically bonded, make macromolecular grafted to surface, form macromolecule membrane；
Optionally, carry out step (5)：
(5) macromolecule is carried out into end-functionalization, forms functional polymer film, which is to catch biomolecule and medicine
Molecule etc., and carry out high flux detection.
The present invention combines spin coating technique and photo-crosslinking technology, establishes a kind of simple, quick and general biochip table
Face chemical modification method, has prepared (function) macromolecule membrane.The main method using photo-crosslinking of the invention is in biological core
Piece surface is chemically modified, and to the three-dimensional surface for forming high density grafting, further functionalization profit is then carried out to which
Little point of biomolecule (such as albumen, polypeptide, polysaccharide, nucleic acid) or medicine etc. is realized on SPRi instruments with the functionalization group of end
The fixation and detection of son directly carry out the fixation and detection of biomolecule or drug molecule.
Preferably, biological chip base is pre-processed as follows before step (1)：Biochip substrate is cleaned up.
Preferably, the biochip substrate, including all can be used for preparing the material of biochip holder, for example
Glass, silicon chip and quartz；The macromolecule class such as dimethyl silicone polymer, polystyrene, Merlon and polymethyl methacrylate
Film；The metals such as golden film, silverskin and di-aluminium trioxide film and metal-oxide film etc..
Preferably, the cleaning way of the biochip substrate including but not limited to organic solvent (as ethanol, methyl alcohol and
DMF etc.) or deionized water be rinsed, shake and wash or be cleaned by ultrasonic, also include entering using plasma cleaning instrument
Row surface clean, or arbitrarily both or three kinds of modes therein are applied in combination.
Preferably, in step (1), the reagent of self assembled monolayer includes but is not limited to single mercaptan, dithiol and silanization
In reagent etc. any one or at least two mix reagent.
Preferably, in step (1), the group of end-functionalization includes but is not limited to alkoxyl, hydroxyl, carboxyl, amino, ring
Epoxide or cyano group etc..
Preferably, described in step (2), photocrosslinking agent refers to containing photosensitive sensitive group, can under specific wavelength (energy) illumination
To carry out the reagent of chemical reaction, such as any one in azirine, diazonium, acetophenone, benzophenone or Anthraquinones etc. or
At least two combination.
Preferably, it is described to make photocrosslinking agent by chemical bonding, be grafted to the method for self assembled monolayer end according to
Biochip substrate surface functionalization group and photocrosslinking agent functional end-group different and adopt different bonding methods, such as
Amidatioon, esterification, open loop or nucleophilic substitution etc..
Such as adoptable photocrosslinking agent structural formula of the invention is as shown in Fig. 2 for (a) structure, connect using esterification
Branch arrives self assembled monolayer end；For (b) structure, self assembled monolayer end is grafted to using amidation process.With
As a example by carboxyl terminal photocrosslinking agent to be grafted to esterification process hydroxy thiol surface, due to first containing F and N in above-mentioned photocrosslinking agent
Element, and the surface before graft esterification photocrosslinking agent has no two kinds of elements and exists, it is possible to according to two kinds of element peaks of F and N
Occur in and judge whether photocrosslinking agent is grafted successfully.As shown in figure 3, there is F and N in the surface XPS data displays after esterification simultaneously
Two obvious peaks, and two elements are had no in compareing, it was demonstrated that photocrosslinking agent has successfully been grafted to surface.
Preferably, the macromolecule refer mainly to biocompatibility and active end group functional polymer, including
But it is not limited to the macromolecule with good anti-non-specific binding performance, such as polyethylene glycol and its derivative, fluoropolymer, poly-
Acrylic acid, polystyrene and PLA etc.；Both sexes betaines polymer etc.；Macromolecule with good biocompatibility, such as
Nitrocellulose, shitosan, glucan and its derivative etc..
Preferably, step (4) inert gas includes but is not limited to the inert gas such as nitrogen or/and argon gas.
Preferably, a length of 365nm of step (4) ultraviolet light wave.
As shown in figure 4, by macromolecule after 365nm ultraviolet light cross-linkings to surface, with FT-IR (sweep angle annex) to chip
Surface is characterized.For the surface of poly- (methacrylic acid macrogol ester) is grafted, 3500cm-1There is O-H to stretch
Vibration peak, 3000-3200cm-1There is CH in place2Vibration it is bimodal, 1740cm-1There are obvious C=O stretching vibration peaks in left and right, and
Lower wave number region, such as 1100cm-1There is the peak of obvious C-O and C-C vibrations in left and right.Similarly, for the table of grafting glucan
For face, 3500cm-1There is-OH characteristic peaks, 3000-3200cm-1There is CH in place2Vibration it is bimodal, 1100cm-1Left and right occurs
The peak of obvious C-O-C stretching vibrations.Above evidence prove macromolecule by successful photo-crosslinking to biochip surface, also demonstrate that
The feasibility of the technical scheme.
Preferably, in step (5), the group of end-functionalization includes but is not limited to hydroxyl, carboxyl, aldehyde radical, amino, epoxy
Base, cyano group, alkynyl, azido or azirine etc..
Preferably, by regulating and controlling photocrosslinking agent density (1mM～100mM), macromolecule number-average molecular weight (100,000～200
Ten thousand), spin coating parameters (1000rpm～8000rpm) etc. can be with the density of precise control surface macromolecule membrane and thickness.The method
Prepare three-dimensional macromolecule surface simply, quickly and efficiently.
The second object of the present invention is to provide a kind of functional polymer film obtained by method as defined above.
The third object of the present invention is to provide a kind of purposes of functional polymer film as above, and which is used for biology
The fixation of molecule and drug molecule and high flux detection.
Preferably, heretofore described biomolecule include but is not limited to albumen, polypeptide, nucleic acid (DNA, RNA, cDNA and
Peptide nucleic acid etc.) and sugared equimolecular.Drug molecule is mainly the pharmaceutical active compounds and Separation of Natural Products of chemical synthesis
Active component that purification is obtained etc..
Heretofore described high-flux detection method, mainly includes surface plasmon resonance imaging technique, fluorescence mark
Note detection technique, fluorescence intensity, fluorescence polarization, FRET, fluorescent quenching, QCM technology and
The method such as various high-throughout electrochemical measuring techniques and hot detection technique.
Compared with the prior art, the present invention has the advantages that：
The invention provides a kind of new " being grafted to " method, has obtained universality using spin coating technique and photo-crosslinking technology
The high molecular method of fixation, it is possible to use macromolecule is non-selectively covalently fixed on one step of ultraviolet light surface, directly use
In three-dimensional surface biochip of the preparation with photo-crosslinking structure.
Additionally, the method for the invention produces the Cabbeen of high reaction activity under ultraviolet light using photocrosslinking agent, its
Reactivity is strong, and the reaction time is short, can carry out intercalation reaction with macromolecule at random, it is adaptable to all macromolecules, with universality.
In addition, spin coating technique of the method based on mature and reliable, required instrument and equipment has popularization and versatility.Profit
The fixation of various biological detection things and detection signal are significantly improved with biochip prepared by this method, practicality
By force, it is adapted to large-scale promotion and commercial applications, with boundless application prospect.
Additionally, present invention preferably employs relatively long ultraviolet light (365nm), side reaction is few, to other modifications point of surface
Sub (such as mercaptan etc.) is without injury.
Description of the drawings
Fig. 1 is the functional polymer three-dimensional surface modification flow chart based on photo-crosslinking method；
Fig. 2 is the photocrosslinking agent molecular structural formula in the present invention, wherein, (a) carboxyl terminal photocrosslinking agent；(b) amino end
End photocrosslinking agent；
Fig. 3 is x-ray photoelectron power spectrum (XPS) to the surface-element analysis result being grafted before and after photocrosslinking agent, wherein, figure
A () and (b) is respectively the XPS figures before and after the grafting of N1s elements, scheme (c) and figure (d) is respectively the XPS before and after the grafting of F1s elements
Figure；
Fig. 4 is that Fourier transform infrared spectroscopy (FT-IR) is characterized to the surface of photo-crosslinking grafting polymer, (a) is gathered
(methacrylic acid macrogol ester)；(b) glucan)；
Fig. 5 is the glucan three-dimensional surface bio-sensing chip prepared based on photo-crosslinking method, is fixed in the way of circulating
Streptavidin (SA), and detect 4nM biotins (Biotin)；
Fig. 6 is on poly- (methacrylic acid macrogol ester) surface to fix rapamycin, is detected using SPRi and 100nM
FKBP12 (PBS, pH7.4,0.5%T20) albumen interaction, wherein, rapamycin -1, rapamycin -2 and Lei Pa are mould
Element -3 represents the sample that three repetition experiments are adopted respectively；
Fig. 7 is agglutinin microarray surface plasma resonance imaging figure；
Fig. 8 is aggegation pixel array to serum screening result.
Specific embodiment
Technical scheme is further illustrated below in conjunction with the accompanying drawings and by specific embodiment.
(small molecule is detected 1. dextran surface of embodiment：The fixed SA of circulation, detects Biotin)
(1) prepare the gold of the layers of chrome and one layer of 47nm thickness of one layer of 3nm thickness on the glass substrate with the method for hot evaporation
Layer, as the substrate of biochip.
(2) the ethanol solution HS- (CH of C-terminal mercaptan are prepared2)11- EG6-OH, concentration are 1mM.
(3) biochip substrate ethanol or deionized water are cleaned up, biochip is put into into plasma then
Clean 3 minutes in cleaning device.
(4) biochip is immersed in ready thiol solution, is incubated 12 hours at 4 DEG C, reaches the scheduled time
Afterwards, biochip is taken out, ethanol and deionized water are alternately cleaned, and nitrogen is dried up.
(5) solution 20mL needed for esterification connection photocrosslinking agent, carboxyl terminal photocrosslinking agent 10mM, 1- (3- diformazan ammonia are prepared
Base propyl group) -3- ethyl carbodiimides (EDC) 10mM, DMAP (DMAP) 1mM, solvent is N, N- dimethyl formyls
Amine (DMF).
(6), in the esterification solution for preparing biochip immersion, room temperature lucifuge is reacted 4 hours, after reaching the scheduled time,
Biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen dries up standby.
(7) glucan that molecular weight is 2000KDa is configured to the aqueous solution of mass concentration 40%, is stirred, is removed
Bubble is to water white uniform solution.
(8) dextran solution for preparing is paved with biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute,
After spin coating, by biochip, lucifuge standing is dried for 1 hour at room temperature.
(9) biochip is put in ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2)。
(10) after reaching the scheduled time, biochip is shaken repeatedly with 50 DEG C of hot water and is washed 1 hour, remove surface not covalent
Fixed dextran molecule, period change water 3-5 time, and after biochip cleaning, nitrogen dries up standby.
(11) biochip is immersed the DMF solution of succinic anhydride (10mg/mL) and DMAP (15mg/mL)
In, (25 DEG C) of room temperature is reacted 16 hours, after reaching predetermined reaction time, biochip is taken out, successively with DMF, ethanol, go from
Sub- water cleaning, nitrogen are dried up.
(12) sodium acetate buffer (pH=4.5) for preparing 50ug/mL Streptavidins (SA) is standby.
(13) SPRi optical positions are adjusted, SA solution is passed to into biochip surface with the speed of 3uL/s, is persistently combined
100s。
(14) repeat step 12, until surface, fixed amount reaches saturation.
(15) change System Solution into PBS (containing 1%DMSO), adjust optical position, be passed through the biotin containing 4nM
PBS solution (1%DMSO), with reference to 300s, dissociate 300s, 1:100 phosphate aqueous solutions are lived again, and the 10mM NaOH aqueous solution is lived again.
The results show, the biochip prepared with the method have very high proteopexy amount, it is sufficient to detect and egg
The white small molecule for combining (such as Fig. 5).
Poly- (methacrylic acid macrogol ester) surface (array of small molecules) of embodiment 2.
(1) prepare the gold of the layers of chrome and one layer of 47nm thickness of one layer of 3nm thickness on the glass substrate with the method for hot evaporation
Layer, as the substrate of biochip.
(2) ethanol solution (HS- (CH of the mercaptan of C-terminal and carboxyl terminal are prepared2)11- EG6-OH and HS-
(CH2)11- EG6-COOH), concentration is 1mM, by both the above thiol solution according to 999:1 (v/v) mixes, standby.
(3) biochip ethanol or deionized water are cleaned up, biochip is put into into plasma clean then
Clean 3 minutes in instrument.
(4) biochip is immersed in the thiol solution for mixing, is incubated 12 hours at 4 DEG C, reaches the scheduled time
Afterwards, biochip is taken out, ethanol and deionized water are alternately cleaned, and nitrogen is dried up.
(5) biochip surface carboxyl is activated, then is immersed 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
(EDC), and in the aqueous solution of N-hydroxy-succinamide (NHS) (0.4M and 0.1M), it is incubated at room temperature 30 minutes, reaches pre- timing
Between, being cleaned with water, nitrogen dries up standby.
(6) biochip is immersed in DMF (DMF) solution of 10mM amino terminal photocrosslinking agents, room
Warm lucifuge is reacted 4 hours, after reaching the scheduled time, biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen
Air-blowing is done standby.
(7) synthesize poly- (methacrylic acid macrogol ester) macromolecule using atomic radicals transfer polymerization (ATRP) method
The methanol/water solution of polymer.By copper chloride (CuCl2) solution mixed with its part second bipyridine (Bpy), stirs 15min.It is first
First by monomer OEGMA and monomer HEMA with 1:1 (mol ratio) adds first alcohol and water (1：1, volume ratio) mixed solution, ultrasound
15min, is continually fed into nitrogen 30min.The aqueous ascorbic acid of 0.04M newly configurations is added, stirring 10min is mixed, then
The ethanol solution of the dithiol initiator of 1mM is added, wherein initiator and the mol ratio of monomer are 1:25000, it is continually fed into nitrogen
Gas, reacts 16 hours.
(8) ATRP macromolecules stoste revolving (35 DEG C) for obtaining step 7, removes methyl alcohol, remaining liq dichloromethane
Solvent extraction, centrifugation.Lower floor's dichloromethane solution part is taken out, is spin-dried for.
(9) with the macromolecule of purifying in ethanol dissolving step (8), obtain 2 μM of poly- (methacrylic acid macrogol ester)
The ethanol solution of high molecular polymer, it is standby.
(10) Polymer Solution for having configured is paved with biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute,
After spin coating, by biochip, lucifuge standing is dried for 1 hour at room temperature, and after reaching the scheduled time, second alcohol and water is alternately cleaned, nitrogen
Air-blowing is done.
(11) biochip is put in ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2)。
(12) after reaching the scheduled time, biochip is cleaned by ultrasonic with second alcohol and water, nitrogen dries up standby core.
(13) biochip is immersed the DMF solution of succinic anhydride (10mg/mL) and DMAP (15mg/mL)
In, room temperature reaction 12 hours, after reaching predetermined reaction time, biochip is taken out, successively with DMF, ethanol and deionized water
Cleaning, nitrogen are dried up.
(14) biochip surface being layered on the ethanol solution of the EG3 of 1mM, closing 30min, then second alcohol and water is alternately clear
Wash, nitrogen is dried up.So far, poly- (methacrylic acid macrogol ester) the surface chip that prepared by photo-crosslinking method has been completed, then
Secondary utilization photo-crosslinking method is fixed small molecule and its GAP-associated protein GAP is detected using SPRi, as follows：
(15) biochip surface is activated again, is immersed 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides
(EDC), and in the aqueous solution of N-hydroxy-succinamide (NHS) (0.4M and 0.1M), it is incubated at room temperature 30 minutes, reaches pre- timing
Between, being cleaned with water, nitrogen dries up standby.
(16) biochip is again dipped in DMF (DMF) solution of 10mM photocrosslinking agents, room temperature
Lucifuge is reacted 4 hours, after reaching the scheduled time, biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen
Dry up standby.
(17) 100nM rapamycins (Rapamycin) small molecule solution and 100ug/mLFKBP12 albumen are prepared
PBST buffer solutions (wherein tween 0.05%), it is standby.
(18) surface that small molecule solution is prepared in step (16) by point sample instrument point sample, vacuum drying.
(19) chip is placed again in ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2), carry out photo-crosslinking.
(20) SPRi optical positions are adjusted, FKBP12 protein solutions is passed to into chip surface with the speed of 3uL/s, continued
With reference to 300s, dissociate 300s, and glycine-HCI is lived again.
The results show, the biochip prepared with the method have very high small molecule fixed amount, it is sufficient to detect little
Molecule protein bound interaction (as shown in Figure 6) associated therewith.
3. dextran surface of embodiment (antigen-antibody fluoroscopic examination)
(1) substrate of glass ethanol or deionized water are cleaned up, and biochip is put in plasma clean instrument clearly
Wash 3 minutes, as the substrate of biochip.
(2) prepare 1:In the 3- aminopropyl triethoxysilanes (APES) of 50 acetone dilution, take out after 20-30s, then with pure
Acetone soln removes the APES not tied, and APES is combined with glass, forms one layer of monolayer in glass surface.
(3) soak 30 minutes in 25% glutaraldehyde, cleaned with acetone, it is hydroxyl that the one end for immersing 1mM is amino one end
In the aqueous solution of the PEG of base, under room temperature, 1h is incubated, after reaching the scheduled time, biochip is taken out, deionized water cleaning,
Nitrogen is dried up.
(4) solution 20mL needed for esterification connection photocrosslinking agent, carboxyl terminal photocrosslinking agent 10mM, 1- (3- diformazan ammonia are prepared
Base propyl group) -3- ethyl carbodiimides (EDC) 10mM, DMAP (DMAP) 1mM, solvent is N, N- dimethyl formyls
Amine (DMF).
(5), in the esterification solution for preparing biochip immersion, room temperature lucifuge is reacted 4 hours, after reaching the scheduled time,
Biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen dries up standby.
(6) glucan that molecular weight is 2000KDa is configured to the aqueous solution of mass concentration 40%, is stirred, is removed
Bubble is to water white uniform solution.
(7) dextran solution for preparing is paved with biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute,
After spin coating, by chip, lucifuge standing is dried for 1 hour at room temperature.
(8) biochip is put in ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2)。
(9) after reaching the scheduled time, biochip is shaken repeatedly with 50 DEG C of hot water and is washed 1 hour, remove surface not covalent
Fixed dextran molecule, period change water 3-5 time, and after biochip cleaning, nitrogen dries up standby.
(10) biochip is immersed the DMF solution of succinic anhydride (10mg/mL) and DMAP (15mg/mL)
In, (25 DEG C) of room temperature is reacted 16 hours, after reaching predetermined reaction time, biochip is taken out, successively with DMF, ethanol, go from
Sub- water cleaning, nitrogen is dried up, standby.
(11) human IgG (sodium acetate solution of the solvent for pH value 4.5) that the 1mg/ml of 3 μ l is taken with liquid-transfering gun drops in step
(10) on the table obtained in, and be aided with 1mg/ml BSA be negative control, be incubated at room temperature 1 hour, with containing 0.0005g/ml's
The phosphate buffer of Tween-20 cleans surface twice (every time 5 minutes), and after deionized water cleaning, nitrogen is dried up.
(12) (0.1M that 5g skimmed milk powers are dissolved in 100ml is weighed with the skimmed milk solution (Skim milk) of 0.05g/ml
Phosphate buffer be prepared from) surface is carried out room temperature close 1 hour after, with the phosphorus of the Tween-20 containing 0.0005g/ml
Twice (every time 5 minutes), deionized water cleaning, nitrogen are dried up on acid buffer cleaning surface.
(13) Goat anti human that fluorescein isothiocynate (Fluorescein isothiocyanate, FITC) is marked
IgG is with 1：50 extension rate is diluted in the skim milk aqueous solution of 0.005mg/ml, and by after step 12 is processed
Chip is immersed in the skim milk, and room temperature is placed 1 hour, carries out antigen-antibody reaction, with the Tween-20 of 0.0005g/ml
Phosphate buffer cleaning surface twice (every time 5 minutes), and deionized water cleaning after, nitrogen is dried up.
(14) by the chip after step 13 is processed, by using Leica M-6000 fluorescence microscopes in 480nm halogen
With the 300ms time for exposure under plain light lamp, light intensity be 5 under conditions of carry out fluorescence imaging with 20 times of object lens.
Poly- (methacrylic acid macrogol ester) surface of embodiment 4. (SPRi glycoprotein array detection serum)
(1) prepare the gold of the layers of chrome and one layer of 47nm thickness of one layer of 3nm thickness on the glass substrate with the method for hot evaporation
Layer, as the substrate of biochip.
(2) ethanol solution (HS- (CH of the mercaptan of C-terminal and carboxyl terminal are prepared2)11- EG6-OH and HS-
(CH2)11- EG6-COOH), concentration is 1mM, by both the above thiol solution according to 999:1 (v/v) mixes, standby.
(3) biochip ethanol or deionized water are cleaned up, biochip is put into into plasma clean then
Clean 3 minutes in instrument.
(4) biochip is immersed in the thiol solution for mixing, is incubated 12 hours at 4 DEG C, reaches the scheduled time
Afterwards, biochip is taken out, ethanol and deionized water are alternately cleaned, and nitrogen is dried up.
(5) biochip surface carboxyl is activated, chip is immersed into 1- (3- dimethylamino-propyls) -3- ethyls carbon two
In the aqueous solution of imines (EDC) and N-hydroxy-succinamide (NHS) (0.4M and 0.1M), it is incubated at room temperature 30 minutes, reaches pre-
Fix time, cleaned with water, nitrogen dries up standby.
(6) biochip is immersed in DMF (DMF) solution of 10mM photocrosslinking agents, room temperature lucifuge is anti-
Answer 4 hours, after reaching the scheduled time, biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen dries up standby
With.
(7) synthesize poly- (methacrylic acid macrogol ester) macromolecule using atomic radicals transfer polymerization (ATRP) method
The methanol/water solution of polymer.By copper chloride (CuCl2) solution mixed with its part second bipyridine, stirs 15min.First will
Monomer OEGMA and monomer HEMA is with 1:1 (mol ratio) adds first alcohol and water (1：1, volume ratio) mixed solution, ultrasonic 15min,
It is continually fed into nitrogen 30min.The aqueous ascorbic acid of 0.04M newly configurations is added, stirring 10min is mixed.It is subsequently adding 1mM
Dithiol initiator ethanol solution, the wherein mol ratio of initiator and monomer is 1:25000, nitrogen is continually fed into, is reacted
16 hours.
(8) ATRP macromolecules stoste revolving (35 degree) for obtaining step (7), removes methyl alcohol, remaining liq dichloromethane
Alkane solvents are extracted, centrifugation, are taken out lower floor's dichloromethane solution part, are spin-dried for.
(9) with the macromolecule of purifying in ethanol dissolving step (8), obtain poly- (the methacrylic acid macrogol ester) for purifying
The ethanol solution of high molecular polymer, it is standby.
(10) Polymer Solution for having configured is paved with biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute,
After spin coating, by biochip, lucifuge standing is dried for 1 hour at room temperature.
(11) biochip is put in ultraviolet light cross-linking instrument, in nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2)。
(12) after reaching the scheduled time, biochip is cleaned by ultrasonic with second alcohol and water, nitrogen dries up standby.
(13) biochip is immersed the DMF solution of succinic anhydride (10mg/mL) and DMAP (15mg/mL)
In, room temperature reaction 16 hours, after reaching predetermined reaction time, biochip is taken out, clear with DMF, ethanol, deionized water successively
Wash, nitrogen is dried up.So far, poly- (methacrylic acid macrogol ester) the surface chip that prepared by photo-crosslinking method has been completed, profit
Carried out with point sample instrument agglutinin (lectin) fixation and using SPRi to type 1 diabetes (T1), diabetes B (T2) and
The coherent detection of 1.5 patients with type Ⅰ DM (LADA) human serum, and with normal human serum as control (C).
(14) biochip surface is activated, is immersed 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC)
With (0.4M and 0.1M) in the aqueous solution of N-hydroxy-succinamide (NHS), it is incubated at room temperature 30 minutes, reaches the scheduled time, uses
Water is cleaned, and nitrogen dries up standby.
(15) using point sample instrument by 49 kinds of difference lectin point samples in the chip surface for preparing, vacuum drying is stand-by.
(16) chip in step (15) is closed with 2% skim milk (PBS solution), under the conditions of 4 degree overnight,
10*PBST, 1*PBST, 0.1*PBST and water cleaning 10min are used successively, and nitrogen is dried up, stand-by.
(17) with HEPES buffer solution by 4 kinds of different blood serum samples with 1:4000 are diluted, standby.
(18) SPRi optical positions are adjusted, HEPES buffer solution 10min is passed through with the flow velocity of 4ul/s first, then with 1:200 salt
Acid (add 0.05% tween) is lived again surface 3 times, and blood serum sample solution is passed to chip with the speed random sequence of 3uL/s
Surface, persistently with reference to 300s, dissociate 300s, and 1:200 hydrochloric acid (adding 0.05% tween) are lived again.
The results show, the chip prepared with the method have very high lectin fixed amounts, it is possible to detect well
To variety classes diabetes serum (such as Fig. 7 and Fig. 8).
5. polyacrylic acid surface (PAA) of embodiment prepares protein microarray
(1) prepare the gold of the layers of chrome and one layer of 47nm thickness of one layer of 3nm thickness on the glass substrate with the method for hot evaporation
Layer, as the substrate of biochip.
(2) ethanol solution (HS- (CH of the mercaptan of C-terminal and carboxyl terminal are prepared2)11- EG6-OH and HS-
(CH2)11- EG6-COOH), concentration is 1mM.By two kinds of thiol solution (EG6-OH：EG6-COOH) according to 999:1 mixing, it is standby
With.
(3) biochip ethanol or deionized water are cleaned up, biochip is put into into plasma clean then
Clean 3 minutes in instrument.
(4) biochip is immersed in the thiol solution for mixing, is incubated 12 hours at 4 DEG C, reaches the scheduled time
Afterwards, biochip is taken out, ethanol and deionized water are alternately cleaned, and nitrogen is dried up.
(5) biochip surface carboxyl is activated, biochip is immersed into 1- (3- dimethylamino-propyls) -3- ethyls
In the aqueous solution of carbodiimide (EDC) and N-hydroxy-succinamide (NHS) (0.4M and 0.1M), it is incubated at room temperature 30 minutes, reaches
To the scheduled time, cleaned with water, nitrogen dries up standby.
(6) biochip is immersed in DMF (DMF) solution of 10mM carboxyl terminal photocrosslinking agents, room
Warm lucifuge is reacted 4 hours, after reaching the scheduled time, biochip is cleaned with DMF, ethanol and deionized water successively, nitrogen
Air-blowing is done standby.
(7) polyacrylic acid macromolecule (PAA) that molecular weight is 140000 is configured to the aqueous solution of volumetric concentration 1%, is stirred
Mix uniform, bubble is removed to water white uniform solution.
(8) the PAA solution for having configured is paved with biochip surface, with the rotating speed of 8000rpm, spin coating 1 minute, spin coating
Afterwards, by biochip, lucifuge standing is dried for 1 hour at room temperature.
(9) biochip is put in ultraviolet light cross-linking instrument, under nitrogen environment, with 365nm ultraviolet lights 15 minutes
(2.4J/cm2)。
(10) after reaching the scheduled time, biochip is cleaned with water, nitrogen dries up standby.
(11) biochip surface is activated, is immersed 1- (3- dimethylamino-propyls) -3- ethyl carbodiimides (EDC)
With (0.4M and 0.1M) in the aqueous solution of N-hydroxy-succinamide (NHS), it is incubated at room temperature 30 minutes.The scheduled time is reached, is used
Water is cleaned, and nitrogen dries up standby.So far, the polyacrylic acid surface chip that prepared by photo-crosslinking method has been completed, and is entered using SPRi
The fixation of row antigen and the detection of antibody.
(12) PBS solution of the Goat-anti-H-IgG of the PBS solution and 100ug/ml of the H-IgG of 1mg/ml is configured,
It is standby.
(13) chip surface that the H-IgG solution point for preparing ocean is prepared in step (11), is dried, stand-by.
(14) 10min, PBS solution and water cleaning are closed under the BSA solution room temperatures by biochip with 1mg/ml, nitrogen blows
It is dry, it is standby.
(15) SPRi optical positions are adjusted, the PBS solution of Goat-anti-H-IgG is passed to into core with the speed of 3uL/s
Piece surface, persistently with reference to 300s, dissociate 300s, and NaOH solution is lived again.
The results show, the biochip prepared with the method have very high antigen fixed amount, can be very good detection
To the interaction of antigen and its antibody.
Applicant states that the present invention illustrates the method detailed of the present invention, but the present invention not office by above-described embodiment
It is limited to above-mentioned method detailed, that is, does not mean that the present invention has to rely on above-mentioned method detailed and could implement.Art
Technical staff it will be clearly understood that any improvement in the present invention, to the equivalence replacement and auxiliary element of each raw material of product of the present invention
Addition, selection of concrete mode etc., within the scope of all falling within protection scope of the present invention and disclosure.
Claims (14)
1. a kind of preparation method of functional polymer film, it is characterised in that the method comprising the steps of：
(1) self assembled monolayer of end-functionalization is formed in biochip substrate surface；
(2) photocrosslinking agent is grafted to into self assembled monolayer end by chemical bonding；
(3) Polymer Solution is spun to into the surface that step (2) is formed under the conditions of lucifuge, is then dried；
(4) there is high molecular biochip that ultraviolet lighting is carried out under inert gas shielding surface spin coating, in ultraviolet light conditions
Under be chemically bonded, make macromolecular grafted to surface, form macromolecule membrane；
Optionally, carry out step (5)：
(5) macromolecule is carried out into end-functionalization, forms functional polymer film；
Photocrosslinking agent density is 1mM～100mM, and macromolecule number-average molecular weight is 100,000～2,000,000, and spin coating rotating speed is 1000rpm
～8000rpm；In step (1), the reagent of self assembled monolayer is any one in single mercaptan, dithiol and silylating reagent
Kind or at least two mix reagent, in step (5) group of end-functionalization be hydroxyl, carboxyl, aldehyde radical, amino, epoxy
Base, cyano group, alkynyl, azido or azirine.
2. the method for claim 1, it is characterised in that biological chip base is pre-processed as follows before step (1)：
Biochip substrate is cleaned up.
3. the method for claim 1, it is characterised in that the biochip substrate be glass, silicon chip, quartz, poly- two
Methylsiloxane, polystyrene, Merlon, polymethyl methacrylate, golden film, silverskin or di-aluminium trioxide film.
4. the method for claim 1, it is characterised in that the cleaning way of the biochip substrate is to use organic solvent
Or deionized water is rinsed, shakes and wash or be cleaned by ultrasonic, or surface clean, or its combination are carried out using plasma cleaning instrument.
5. the method for claim 1, it is characterised in that the group of end-functionalization is alkoxyl, hydroxyl in step (1)
Base, carboxyl, amino, epoxy radicals or cyano group.
6. the method for claim 1, it is characterised in that photocrosslinking agent described in step (2) is azirine, diazonium, second
In acyl benzene, benzophenone or Anthraquinones any one or at least two combination.
7. the method for claim 1, it is characterised in that make photocrosslinking agent by chemical bonding, be grafted to self assembly list
The method of molecular layer end is amidatioon, esterification, open loop or nucleophilic substitution.
8. the method for claim 1, it is characterised in that the macromolecule is polyethylene glycol and its derivative, fluorine-containing poly-
Compound, polyacrylic acid, polystyrene, PLA, both sexes betaines polymer, nitrocellulose, shitosan or glucan and its
Derivative.
9. the method for claim 1, it is characterised in that step (4) inert gas is nitrogen or/and argon gas.
10. the method for claim 1, it is characterised in that a length of 365nm of step (4) ultraviolet light wave.
A kind of 11. functional polymer films obtained by one of claim 1-10 methods described.
A kind of 12. purposes of functional polymer film as claimed in claim 11, which is used for biomolecule and drug molecule
Fixed and high flux is detected.
13. purposes as claimed in claim 12, it is characterised in that the biomolecule is albumen, polypeptide, nucleic acid and sugar；
The drug molecule purifies the active component for obtaining for the pharmaceutical active compounds and Separation of Natural Products of chemical synthesis.
14. purposes as claimed in claim 12, it is characterised in that high flux detection include surface plasmon resonance into
As technology, fluorescence labeling detection technique, fluorescence intensity, fluorescence polarization, FRET, fluorescent quenching, quartz crystal
Micro- balance technology, high-throughout electrochemical measuring technique and high-throughout hot detection technique.
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| CN104897617A (en) * | 2015-05-19 | 2015-09-09 | 国家纳米科学中心 | Micro-array biochip, and preparation method and application of micro-array biochip |
| CN108885212B (en) | 2016-02-22 | 2022-05-31 | 勃林格殷格翰维特梅迪卡有限公司 | Method for immobilizing biomolecules |
| CN106383224A (en) * | 2016-08-31 | 2017-02-08 | 中国科学院长春应用化学研究所 | Biological detection element and preparation method thereof |
| CN106990079B (en) * | 2017-03-20 | 2019-06-25 | 中国科学院化学研究所 | A kind of surface multifunctional coating and the preparation method and application thereof |
| CN106896087B (en) * | 2017-03-31 | 2019-04-23 | 丁利 | The preparation method of surface plasma resonance instrument chip based on hyperbranched amphoteric ion polymethyl base cysteine modified |
| CN109900814B (en) * | 2017-12-08 | 2021-06-08 | 中国科学院大连化学物理研究所 | Analysis method and application of fragmentable chemical cross-linking agent based on glycosidic bond mass spectrum |
| CN110282997A (en) * | 2019-07-10 | 2019-09-27 | 浙江理工大学 | A kind of polystyrene laminated film and its preparation method and application |
| CN111497366B (en) * | 2020-04-07 | 2021-06-15 | 上海交通大学 | Interface-controllable non-layered multi-level graphene conformal folds and preparation method thereof |
| CN112982012B (en) * | 2021-02-09 | 2022-05-10 | 杭州梓晶生物有限公司 | Polyacrylamide derivative modified filter paper and application thereof in nucleic acid separation and enrichment |
| CN115677922B (en) * | 2022-09-28 | 2024-01-26 | 深圳市曙芯生物科技有限公司 | Dual-functional polymer and modification method |
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| CN101078730A (en) * | 2006-05-22 | 2007-11-28 | 杜宏武 | Enzyme labeled chip method capable of in situ detecting multiple ordinary small molecule drugs |
| JP5679253B2 (en) * | 2009-05-26 | 2015-03-04 | 国立大学法人東京工業大学 | Self-supporting polymer thin film |
| CN102242053B (en) * | 2011-04-01 | 2014-06-04 | 沈越 | Biochip with polymer three-dimensional nanostructure |
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