CN107064072A - The preparation method of surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified - Google Patents
The preparation method of surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified Download PDFInfo
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- 235000018417 cysteine Nutrition 0.000 title claims abstract description 35
- 241001044369 Amphion Species 0.000 title claims abstract description 32
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 title claims abstract description 29
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- -1 mercapto alkene Chemical class 0.000 claims abstract description 21
- 239000000178 monomer Substances 0.000 claims abstract description 16
- 229920001690 polydopamine Polymers 0.000 claims abstract description 8
- 239000003999 initiator Substances 0.000 claims abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 90
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 54
- 229910052757 nitrogen Inorganic materials 0.000 claims description 42
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 36
- 239000000243 solution Substances 0.000 claims description 31
- 239000008367 deionised water Substances 0.000 claims description 28
- 229910021641 deionized water Inorganic materials 0.000 claims description 28
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 24
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 20
- VYFYYTLLBUKUHU-UHFFFAOYSA-N dopamine Chemical compound NCCC1=CC=C(O)C(O)=C1 VYFYYTLLBUKUHU-UHFFFAOYSA-N 0.000 claims description 16
- 239000000758 substrate Substances 0.000 claims description 15
- 238000003756 stirring Methods 0.000 claims description 14
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims description 13
- 238000007872 degassing Methods 0.000 claims description 12
- QAOWNCQODCNURD-UHFFFAOYSA-N sulfuric acid group Chemical class S(O)(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 12
- 238000007654 immersion Methods 0.000 claims description 10
- 239000000843 powder Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 8
- 229960003638 dopamine Drugs 0.000 claims description 8
- 238000005516 engineering process Methods 0.000 claims description 8
- 239000011259 mixed solution Substances 0.000 claims description 8
- 239000011521 glass Substances 0.000 claims description 7
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical class CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 6
- 239000007983 Tris buffer Substances 0.000 claims description 6
- 150000001649 bromium compounds Chemical class 0.000 claims description 6
- QTMDXZNDVAMKGV-UHFFFAOYSA-L copper(ii) bromide Chemical class [Cu+2].[Br-].[Br-] QTMDXZNDVAMKGV-UHFFFAOYSA-L 0.000 claims description 6
- 150000001945 cysteines Chemical class 0.000 claims description 6
- 230000008021 deposition Effects 0.000 claims description 6
- 229910001873 dinitrogen Inorganic materials 0.000 claims description 6
- 238000005566 electron beam evaporation Methods 0.000 claims description 6
- 230000008014 freezing Effects 0.000 claims description 6
- 238000007710 freezing Methods 0.000 claims description 6
- 229910052737 gold Inorganic materials 0.000 claims description 6
- 239000010931 gold Substances 0.000 claims description 6
- 229910052698 phosphorus Inorganic materials 0.000 claims description 6
- 239000011574 phosphorus Substances 0.000 claims description 6
- QQONPFPTGQHPMA-UHFFFAOYSA-N propylene Natural products CC=C QQONPFPTGQHPMA-UHFFFAOYSA-N 0.000 claims description 6
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 claims description 6
- 239000003643 water by type Substances 0.000 claims description 6
- 241000252506 Characiformes Species 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 239000006210 lotion Substances 0.000 claims description 4
- 238000003786 synthesis reaction Methods 0.000 claims description 4
- 238000005406 washing Methods 0.000 claims description 4
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 2
- 239000013307 optical fiber Substances 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- DWNBOPVKNPVNQG-LURJTMIESA-N (2s)-4-hydroxy-2-(propylamino)butanoic acid Chemical compound CCCN[C@H](C(O)=O)CCO DWNBOPVKNPVNQG-LURJTMIESA-N 0.000 claims 1
- NIXOWILDQLNWCW-UHFFFAOYSA-N 2-Propenoic acid Natural products OC(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 claims 1
- 150000001875 compounds Chemical class 0.000 claims 1
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims 1
- 238000000034 method Methods 0.000 claims 1
- 239000012460 protein solution Substances 0.000 abstract description 8
- 230000003373 anti-fouling effect Effects 0.000 abstract description 6
- 238000001514 detection method Methods 0.000 abstract description 4
- 238000012650 click reaction Methods 0.000 abstract 1
- 238000010526 radical polymerization reaction Methods 0.000 abstract 1
- 230000001960 triggered effect Effects 0.000 abstract 1
- 241001494479 Pecora Species 0.000 description 18
- 108060003951 Immunoglobulin Proteins 0.000 description 16
- 102000018358 immunoglobulin Human genes 0.000 description 16
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 11
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 10
- 238000001035 drying Methods 0.000 description 9
- 235000013336 milk Nutrition 0.000 description 9
- 239000008267 milk Substances 0.000 description 9
- 210000004080 milk Anatomy 0.000 description 9
- 238000001179 sorption measurement Methods 0.000 description 9
- 238000004140 cleaning Methods 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 6
- 229940098773 bovine serum albumin Drugs 0.000 description 6
- 235000010469 Glycine max Nutrition 0.000 description 5
- 244000068988 Glycine max Species 0.000 description 5
- 102000016943 Muramidase Human genes 0.000 description 5
- 108010014251 Muramidase Proteins 0.000 description 5
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 description 5
- 239000002253 acid Substances 0.000 description 5
- 229910021529 ammonia Inorganic materials 0.000 description 5
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 5
- 239000004325 lysozyme Substances 0.000 description 5
- 229960000274 lysozyme Drugs 0.000 description 5
- 235000010335 lysozyme Nutrition 0.000 description 5
- 230000004044 response Effects 0.000 description 5
- 210000002966 serum Anatomy 0.000 description 5
- 102000008946 Fibrinogen Human genes 0.000 description 4
- 108010049003 Fibrinogen Proteins 0.000 description 4
- 230000001143 conditioned effect Effects 0.000 description 4
- 229940012952 fibrinogen Drugs 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical class ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 description 2
- HZAXFHJVJLSVMW-UHFFFAOYSA-N 2-Aminoethan-1-ol Chemical compound NCCO HZAXFHJVJLSVMW-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 102000006395 Globulins Human genes 0.000 description 2
- 108010044091 Globulins Proteins 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 238000002242 deionisation method Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000010355 oscillation Effects 0.000 description 2
- 235000018102 proteins Nutrition 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 235000013322 soy milk Nutrition 0.000 description 2
- 101001065501 Escherichia phage MS2 Lysis protein Proteins 0.000 description 1
- AMPHKYRLSOPVBX-YFKPBYRVSA-N allylcysteine Chemical compound OC(=O)[C@H](CS)NCC=C AMPHKYRLSOPVBX-YFKPBYRVSA-N 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 238000003759 clinical diagnosis Methods 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
- 238000010168 coupling process Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 235000020247 cow milk Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 235000021393 food security Nutrition 0.000 description 1
- 230000009878 intermolecular interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000007800 oxidant agent Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/55—Specular reflectivity
- G01N21/552—Attenuated total reflection
- G01N21/553—Attenuated total reflection and using surface plasmons
Landscapes
- Physics & Mathematics (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a kind of preparation method of the surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified, methylpropenyl cysteine monomer is obtained by " mercapto alkene " click-reaction, initiator is modified to surface by the poly-dopamine on golden film surface, and then the Transfer Radical Polymerization triggered by surface triggers monomer in golden film surface aggregate, so as to obtain a kind of surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified, chip in single protein solution and natural complex sample system to being respectively provided with very strong antifouling property, and there is high supported quantity and good compatibility to large biological molecule such as antibody, target molecule is highly sensitive in achievable complexity albumen system, unmarked quick detection；With very strong antifouling property, the advantages of high stability and high supported quantity, there are good feasibility and practicality.
Description
Technical field
It is more particularly to a kind of to be based on both sexes the present invention relates to surface plasma resonance spectrometer antipollution chip preparation field
The preparation method of the surface plasma resonance instrument chip of ion polymethyl base cysteine modified.
Background technology
Surface plasma resonance (Surface Plasmon Resonance, abbreviation SPR) instrument is that occur 1980s
A kind of biosensor technology, when incident light incides low refractive index dielectric from high refractive index medium, it may occur that total reflection,
If now plating layer of metal film (gold or silver) at dielectric interface, the evanescent wave that incident light is produced can cause metal surface certainly
Occur collective oscillation by electronics, and then form plasma, when evanescent wave and the frequency and wave number phase of surface plasmon oscillations
Deng when, that is, produce covibration (SPR), cause energy from evanescent wave is transferred to surface plasma-wave, make the intensity of reflected light
Weaken significantly, decay total reflection phenomenon, the incidence angle referred to as resonance angle when the intensity of reflected light is zero is presented.Resonance angle and gold
Belong to film interface medium refraction index relevant, and refractive index changes with combination in the molecular mass of metal film surfaces, therefore can
The information of intermolecular interaction is obtained by analyzing resonance angle, because it has detection quick and the features such as need not mark,
It has been widely used in the fields such as proteomics, medicament research and development, clinical diagnosis, food security and environmental monitoring, and has shown
Go out wide application prospect.
SPR chips be surface plasma resonance instrument core the most component there is provided produce spr signal necessary physics bar
Part, mainly including coupled apparatus, metal film and surface matrix.However, when carrying out analysis test using surface plasma resonance instrument,
The surface contamination of sensing chip is a common problem, the non-specificity of biomolecule or microorganism in sample
The accuracy quantitatively detected will be reduced by adsorbing, and produce false positive results.Therefore, exploitation is effective against the surface of non-specific adsorption
It is the matter of utmost importance for improving surface plasma resonance sensor sensitivity and accuracy.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of based on the Guang ammonia of amphion polymethyl base half
The preparation method of the surface plasma resonance instrument chip of acid modification.
The present invention is achieved by the following technical solutions：
A kind of preparation method of the surface plasma resonance instrument chip based on amphion polymethyl base cysteine,
Comprise the following steps：
A) prepared by naked golden chip
The thick layers of chrome of one layer of 2-3nm is coated in substrate using electron beam evaporation deposition technology, one is coated in the layers of chrome
Golden film thick layer 45-50nm, obtains naked golden chip；
B) naked golden chip pretreatment
In the naked golden chip immersion Piranha washing lotion that step a) is obtained, 1-3h, taking-up deionized water are soaked under normal temperature
Dried up after cleaning 3 times with nitrogen；
C) synthesis of amphion methylpropenyl cysteine monomer
By 24.98mmol cysteines with after 20mL deionized water dissolvings, add thereto 27.47mmol 2- acrylic acid-
2- hydroxyls -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 2-4h at 20-30 DEG C, reacted solution
It is extracted with ethyl acetate once, dichloromethane is extracted twice, the amphion methylpropenyl of white powder is obtained after freezing
Cysteine monomer；
D) naked golden chip surface dopamine polymerization
1-3mg/mL dopamine solutions are prepared with pH=8.5 TRIS buffer, b) step is obtained
Naked golden chip be immersed, in being incubated 1-2h at 40-60 DEG C, taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；
E) poly-dopamine chip surface bromo isobutyl acylbromide initiator is modified
The substrate immersion 20ml for the chip that d) step is obtained contains in the dichloromethane of 1.0ml triethylamines, in ice bath and
The dichloromethane that 10ml contains 0.9ml bromo isobutyl acylbromides is added dropwise under stirring condition, stirring reaction 12-24h, takes at room temperature
Go out to be cleaned successively with ethanol and deionized water after 3 times and dried up with nitrogen；
F) naked golden chip surface polymethyl base cysteine synthesis
The chip that e) step is obtained is put into the Shi Lunke bottles of advance three circulating degasifications and inflated with nitrogen；C) step is obtained
To 5mmol monomers be dissolved in 4ml deionized waters, deaerated 10-30min with nitrogen, add thereto 0.5mmol bipyridyls,
0.167mmol cuprous bromides and 0.083mmol copper bromides, with ultrasonically treated 5-10min is used after nitrogen degassing 10-30min, are incited somebody to action
The solution arrived moves into Shi Lunke bottles so that solution submerges the chip in bottle, and chip is taken out after 2-4h is reacted under condition of nitrogen gas,
Cleaned after 3 times and dried up with nitrogen successively with ethanol and deionized water.
Moreover, above-mentioned substrate is glass chip bottom or optical fiber substrate.
Moreover, above-mentioned golden film, which can also be silverskin or silver/gold, meets film.
Moreover, above-mentioned Piranha washing lotion is the mixed solution of the 98wt.% concentrated sulfuric acids and 30wt.% hydrogen peroxide, the concentrated sulfuric acid
Volume ratio with hydrogen peroxide is 7:3.
Advantages of the present invention and beneficial effect are：
1) surface plasma resonance instrument based on amphion polymethyl base cysteine modified prepared by the present invention
Chip has good antifouling property；
2) surface plasma resonance instrument based on amphion polymethyl base cysteine modified prepared by the present invention
Chip has biological functional effect, such as by immobilized sheep immune globulin antibody and then detects its antigen；
3) surface plasma resonance instrument based on amphion polymethyl base cysteine modified prepared by the present invention
Chip has high stability, stores 1 month in the dry state or after storage during pH is 7.4 phosphate buffer 7 days,
Antifouling property is unchanged；
4) the of the invention surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified
Preparation method has certain universality, is not limited by base material property.
Brief description of the drawings
Fig. 1 is the surface plasma resonance instrument chip of the invention based on amphion polymethyl base cysteine modified
Structural representation；
Fig. 2 a are the surface plasma resonance instrument core of the invention based on amphion polymethyl base cysteine modified
Piece is to single albumen system (1mg/mL lysozyme (Lysozyme), bovine serum albumin (BSA), fibrinogen
(fibrinogen) the non-specific adsorption amount of albumen in)；
Fig. 2 b are the surface plasma resonance instrument core of the invention based on amphion polymethyl base cysteine modified
Piece is to egg in complex system (100% human serum (Blood serum), soya-bean milk (Soybean milk), milk (Cow milk))
White non-specific adsorption amount；
Wherein：
1st, glass chip bottom；2nd, layers of chrome；3rd, golden membranous layer；4th, poly-dopamine coupling triggers oxidant layer；5th, the poly- methyl of amphion
Allyl cysteine layer.
Embodiment
The present invention is described in further detail by following examples.It should be noted that：Following embodiments are illustrative, are not
Limited, it is impossible to limit protection scope of the present invention with following embodiments.
Embodiment 1
The thick layers of chrome of one layer of 2nm is coated in BK7 substrate of glass using electron beam evaporation deposition technology, is applied in the layers of chrome
The thick golden films of one layer of 48nm are covered, naked golden chip is obtained；By the mixed of above-mentioned naked golden chip 98% concentrated sulfuric acid of immersion and 30% hydrogen peroxide
Close in solution, the volume ratio of 98% concentrated sulfuric acid and 30% hydrogen peroxide is 7:3, in soaking 2h under normal temperature.Then take out and use deionization
Water is dried up standby with nitrogen after cleaning 3 times.
By 24.98mmol cysteines with after 20mL deionized water dissolvings, add thereto 27.47mmol 2- acrylic acid-
2- hydroxyls -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 4h at 20 DEG C, reacted solution acetic acid
Ethyl ester is extracted once, and dichloromethane is extracted twice, and the Guang ammonia of amphion methylpropenyl half of white powder is obtained after freezing
Acid monomers；
2mg/mL dopamine solutions are prepared with pH=8.5 TRIS buffer, above-mentioned drying is standby
Naked golden chip be immersed, in being incubated 2h at 40 DEG C, taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；It will be modified with
The substrate immersion of poly-dopamine includes in the 20ml dichloromethane of 1.0ml triethylamines, adds dropwise under ice bath and stirring condition
Enter the dichloromethane that 10ml includes 0.9ml bromo isobutyl acylbromides, conditioned response 24h, taking-up second is then stirred at room temperature
Alcohol and deionized water are cleaned after 3 times and dried up with nitrogen successively, and obtained chip is put into advance three circulating degasification inflated with nitrogen
In Shi Lunke bottles, take the white powder amphion methylpropenyl cysteine monomer 5mmol obtained after above-mentioned freeze molten
Solution is deaerated 15min in 4ml deionized waters with nitrogen, add thereto 0.5mmol bipyridyls, 0.167mmol cuprous bromides and
0.083mmol copper bromides, then with nitrogen degassing 10min, ultrasonically treated 5min is used afterwards, it will obtain solution moving into Shi Lunke bottles
In and chip is submerged, take out chip after 2h is reacted under condition of nitrogen gas, successively with respectively cleaning 3 times of ethanol and deionized water, use
Nitrogen drying is standby.
By the surface plasma resonance chip pitch of the amphion methylpropenyl cysteine modified of above-mentioned acquisition
On the prism for fitting to surface plasma resonance system, using pH be 7.4 phosphate buffer as mobile phase, selection flow velocity be 10 μ
L/min.After baseline is steady, 100 μ L 1mg/mL protein solution is injected separately into quantitative loop：Bovine serum albumin(BSA), bacteriolyze
Enzyme, fibrinogen, 100% serum, soymilk supernatant and milk supernatant, promote above-mentioned each protein solution to reach through mobile phase
Chip surface, by surface plasma resonance system measurement resonance angle real-time change curve, surface plasma resonance is read after 20min
Angle changing value, and calculate non-specific adsorption amount, respectively 1.40ng/cm2、0.81ng/cm2、1.89ng/cm2、12.81ng/
cm2、6.08ng/cm2、7.15ng/cm2, non-specific adsorption amount of its protein adsorption quantity far below each albumen of naked gold surface.
Embodiment 2
The thick layers of chrome of one layer of 3nm is coated in BK7 substrate of glass using electron beam evaporation deposition technology, is applied in the layers of chrome
The thick golden films of one layer of 47nm are covered, naked golden chip is obtained；By the mixed of above-mentioned naked golden chip 98% concentrated sulfuric acid of immersion and 30% hydrogen peroxide
Close in solution, the volume ratio of 98% concentrated sulfuric acid and 30% hydrogen peroxide is 7:3, in soaking 1h under normal temperature.Then take out and use deionization
Water is dried up standby with nitrogen after cleaning 3 times.
By 24.98mmol cysteines with after 20mL deionized water dissolvings, add thereto 27.47mmol 2- acrylic acid-
2- hydroxyls -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 2h at 30 DEG C, reacted solution acetic acid
Ethyl ester is extracted once, and dichloromethane is extracted twice, and the Guang ammonia of amphion methylpropenyl half of white powder is obtained after freezing
Acid monomers；
3mg/mL dopamine solutions are prepared with pH=8.5 TRIS buffer, above-mentioned drying is standby
Naked golden chip be immersed, in being incubated 1h at 60 DEG C, taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；It will be modified with
The substrate immersion of poly-dopamine includes in the 20ml dichloromethane of 1.0ml triethylamines, adds dropwise under ice bath and stirring condition
Enter the dichloromethane that 10ml includes 0.9ml bromo isobutyl acylbromides, conditioned response 18h, taking-up second is then stirred at room temperature
Alcohol and deionized water are cleaned after 3 times and dried up with nitrogen successively, and obtained chip is put into advance three circulating degasification inflated with nitrogen
In Shi Lunke bottles, take the white powder amphion methylpropenyl cysteine monomer 5mmol obtained after above-mentioned freeze molten
Solution is deaerated 20min in 4ml deionized waters with nitrogen, add thereto 0.5mmol bipyridyls, 0.167mmol cuprous bromides and
0.083mmol copper bromides, then with nitrogen degassing 20min, ultrasonically treated 10min is used afterwards, obtained solution is moved into Shi Lunke bottles
In and chip is submerged, take out chip after 3h is reacted under condition of nitrogen gas, successively with respectively cleaning 3 times of ethanol and deionized water, use
Nitrogen drying is standby.
By the surface plasma resonance chip pitch of the amphion methylpropenyl cysteine modified of above-mentioned acquisition
On the prism for fitting to surface plasma resonance system, using pH be 7.4 phosphate buffer as mobile phase, selection flow velocity be 10 μ
L/min.After baseline is steady, 100 μ L 1mg/mL protein solution is injected separately into quantitative loop：Bovine serum albumin(BSA), bacteriolyze
Enzyme, fibrinogen, 100% serum, soymilk supernatant and milk supernatant, promote above-mentioned each protein solution to reach through mobile phase
Chip surface, by surface plasma resonance system measurement resonance angle real-time change curve, surface plasma resonance is read after 20min
Angle changing value, and calculate non-specific adsorption amount, respectively 1.95ng/cm2、1.02ng/cm2、2.20ng/cm2、14.25ng/
cm2、6.95ng/cm2、9.05ng/cm2, non-specific adsorption amount of its protein adsorption quantity far below each albumen of naked gold surface.
Embodiment 3
The thick layers of chrome of one layer of 2nm is coated in BK7 substrate of glass using electron beam evaporation deposition technology, is applied in the layers of chrome
The thick golden films of one layer of 48nm are covered, naked golden chip is obtained；Above-mentioned naked golden chip is immersed into 98% concentrated sulfuric acid and 30% hydrogen peroxide (volume
Than 7:3) in mixed solution, the volume ratio of 98% concentrated sulfuric acid and 30% hydrogen peroxide is 7:3, in soaking 2h under normal temperature.Then take
Go out and clean standby with nitrogen drying after 3 times with deionized water.
By 24.98mmol cysteines with after 20mL deionized water dissolvings, add thereto 27.47mmol 2- acrylic acid-
2- hydroxyls -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 4h at 20 DEG C, reacted solution acetic acid
Ethyl ester is extracted once, and dichloromethane is extracted twice, and the Guang ammonia of amphion methylpropenyl half of white powder is obtained after freezing
Acid monomers；
2mg/mL dopamine solutions are prepared with pH=8.5 TRIS buffer, above-mentioned drying is standby
Naked golden chip be immersed, in being incubated 2h at 40 DEG C, taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；It will be modified with
The substrate immersion of poly-dopamine includes in the 20ml dichloromethane of 1.0ml triethylamines, adds dropwise under ice bath and stirring condition
Enter the dichloromethane that 10ml includes 0.9ml bromo isobutyl acylbromides, conditioned response 24h, taking-up second is then stirred at room temperature
Alcohol and deionized water are cleaned after 3 times and dried up with nitrogen successively, and obtained chip is put into advance three circulating degasification inflated with nitrogen
In Shi Lunke bottles, take the white powder amphion methylpropenyl cysteine monomer 5mmol obtained after above-mentioned freeze molten
Solution is deaerated 15min in 4ml deionized waters with nitrogen, add thereto 0.5mmol bipyridyls, 0.167mmol cuprous bromides and
0.083mmol copper bromides, then with nitrogen degassing 10min, ultrasonically treated 5min is used afterwards, it will obtain solution moving into Shi Lunke bottles
In and chip is submerged, take out chip after 2h is reacted under condition of nitrogen gas, successively with respectively cleaning 3 times of ethanol and deionized water, use
Nitrogen drying is standby.
By the surface plasma resonance chip pitch of the amphion methylpropenyl cysteine modified of above-mentioned acquisition
On the prism for fitting to surface plasma resonance system, using pH be 7.4 phosphate buffer as mobile phase, selection flow velocity be 10 μ
L/min.After baseline is steady, into quantitative loop, 100 μ L 1- ethyls -3- (3- dimethylaminopropyls) phosphinylidyne of interior injection two is sub-
The mixed solution of amine (EDC, concentration is 0.4M) and N- hydroxy thiosuccinimides (NHS, concentration is 0.1M), is pushed away through mobile phase
Dynamic above-mentioned mixed solution reaches chip surface.100 μ L sheep immunoglobulin (anti-IgG, 1mg/mL) solution are injected after 15min,
Promote sheep immunoglobulin solution to reach chip surface through mobile phase, then closed through monoethanolamine, immobilized sheep is obtained after 30min and is immunized
The surface plasma resonance sensing chip of globulin.
The surface plasma resonance sensing chip of the immobilized sheep immunoglobulin of above-mentioned acquisition is fitted into surface with pitch
On the prism of plasmon resonance system.Using pH be 7.4 phosphate buffer as mobile phase, selection flow velocity be 10 μ L/min.Treat base
After line is steady, 100 μ L protein solutions are injected separately into quantitative loop：Lysozyme (1mg/mL), soya-bean milk (10mg/mL), milk
(30mg/mL) and 100% serum, promotes above-mentioned each protein solution to reach chip surface, by surface plasma resonance through mobile phase
Surface plasma resonance angle changing value is read after system measurement resonance angle real-time change curve, 20min, and calculates non-specific suction
Attached amount, respectively 1.21ng/cm2、6.88ng/cm2、8.35ng/cm2、14.51ng/cm2。
Embodiment 4
The thick layers of chrome of one layer of 2nm is coated in BK7 substrate of glass using electron beam evaporation deposition technology, is applied in the layers of chrome
The thick golden films of one layer of 48nm are covered, naked golden chip is obtained；Above-mentioned naked golden chip is immersed into 98% concentrated sulfuric acid and 30% hydrogen peroxide (volume
Than 7:3) in mixed solution, the volume ratio of 98% concentrated sulfuric acid and 30% hydrogen peroxide is 7:3, in soaking 2h under normal temperature.Then take
Go out and clean standby with nitrogen drying after 3 times with deionized water.
By 24.98mmol cysteines with after 20mL deionized water dissolvings, add thereto 27.47mmol 2- acrylic acid-
2- hydroxyls -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 4h at 20 DEG C, reacted solution acetic acid
Ethyl ester is extracted once, and dichloromethane is extracted twice, and the Guang ammonia of amphion methylpropenyl half of white powder is obtained after freezing
Acid monomers；
2mg/mL dopamine solutions are prepared with pH=8.5 TRIS buffer, above-mentioned drying is standby
Naked golden chip be immersed, in being incubated 2h at 40 DEG C, taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；It will be modified with
The substrate immersion of poly-dopamine includes in the 20ml dichloromethane of 1.0ml triethylamines, adds dropwise under ice bath and stirring condition
Enter the dichloromethane that 10ml includes 0.9ml bromo isobutyl acylbromides, conditioned response 24h, taking-up second is then stirred at room temperature
Alcohol and deionized water are cleaned after 3 times and dried up with nitrogen successively, and obtained chip is put into advance three circulating degasification inflated with nitrogen
In Shi Lunke bottles, take the white powder amphion methylpropenyl cysteine monomer 5mmol obtained after above-mentioned freeze molten
Solution is deaerated 15min in 4ml deionized waters with nitrogen, add thereto 0.5mmol bipyridyls, 0.167mmol cuprous bromides and
0.083mmol copper bromides, then with nitrogen degassing 10min, ultrasonically treated 5min is used afterwards, obtained solution is moved into Shi Lunke bottles
And submerge chip, chip is taken out after 2h is reacted under condition of nitrogen gas, successively with respectively cleaning 3 times of ethanol and deionized water, nitrogen is used
Air-blowing is done standby.
By the surface plasma resonance chip pitch of the amphion methylpropenyl cysteine modified of above-mentioned acquisition
On the prism for fitting to surface plasma resonance system.Using pH be 7.4 phosphate buffer as mobile phase, selection flow velocity be 10 μ
L/min.After baseline is steady, into quantitative loop, 100 μ L 1- ethyls -3- (3- dimethylaminopropyls) phosphinylidyne of interior injection two is sub-
The mixed solution of amine (EDC, concentration is 0.4M) and N- hydroxy thiosuccinimides (NHS, concentration is 0.1M), is pushed away through mobile phase
Dynamic above-mentioned mixed solution reaches chip surface.100 μ L sheep immunoglobulin (anti-IgG, 1mg/mL) solution are injected after 15min,
Promote sheep immunoglobulin solution to reach chip surface through mobile phase, then closed through monoethanolamine, obtaining immobilized sheep after 30min exempts from
The surface plasma resonance sensing chip of epidemic disease globulin, is injected separately into the protein solution of 100 μ L various concentrations：Lysozyme (1mg/
ML), soya-bean milk (10mg/mL), sheep immunoglobulin (15nM), sheep immunoglobulin (75nM), sheep immunoglobulin (150nM),
Sheep immunoglobulin (300nM), sheep immunoglobulin (450nM), sheep immunoglobulin (700nM), sheep immunoglobulin
The mixed liquor of (15nM) and lysozyme (1mg/mL) and the mixed liquor of sheep immunoglobulin (15nM) and soya-bean milk (10mg/mL),
By SPR response signals with obtain sheep immunoglobulin (IgG) detection standard curve.
Surface plasma resonance instrument core based on amphion polymethyl base cysteine modified prepared by the present invention
Piece has good antifouling property；With biological functional effect, immobilized sheep immune globulin antibody and then detection can be passed through
Its antigen；With high stability, store 1 month or stored 7 days in pH is 7.4 phosphate buffer in the dry state
Afterwards, antifouling property is unchanged；And preparation method has certain universality, do not limited by base material property.
Exemplary description is done to the present invention above, it should explanation, in the situation for the core for not departing from the present invention
Under, any simple deformation, modification or other skilled in the art can not spend the equivalent substitution of creative work equal
Fall into protection scope of the present invention.
Claims (4)
1. a kind of preparation side of the surface plasma resonance instrument chip based on amphion polymethyl base cysteine modified
Method, it is characterised in that comprise the following steps：
A) prepared by naked golden chip
The thick layers of chrome of one layer of 2-3nm is coated in substrate using electron beam evaporation deposition technology, one layer of 45- is coated in the layers of chrome
Golden film thick 50nm, obtains naked golden chip；
B) naked golden chip pretreatment
In the naked golden chip immersion Piranha washing lotion that step a) is obtained, 1-3h is soaked under normal temperature, taking-up cleans 3 with deionized water
Dried up after secondary with nitrogen；
C) synthesis of amphion methylpropenyl cysteine monomer
By 24.98mmol cysteines with after 20mL deionized water dissolvings, 27.47mmol 2- acrylic acid -2- hydroxyls are added thereto
Base -1,3- propylene diester and 29.4 μm of ol xylyl phosphorus, in stirring reaction 2-4h at 20-30 DEG C, reacted solution second
Acetoacetic ester is extracted once, and dichloromethane is extracted twice, and the Guang of amphion methylpropenyl half of white powder is obtained after freezing
Propylhomoserin monomer；
D) naked golden chip surface dopamine polymerization
Prepare 1-3mg/mL dopamine solutions with pH=8.5 TRIS buffer, by b) step obtain it is naked
Golden chip is immersed, in being incubated 1-2h at 40-60 DEG C, and taking-up is cleaned after 3 times with deionized water and dried up with nitrogen；
E) poly-dopamine chip surface bromo isobutyl acylbromide initiator is modified
The substrate immersion 20ml for the chip that d) step is obtained contains in the dichloromethane of 1.0ml triethylamines, in ice bath and stirring
Under the conditions of the dichloromethane that 10ml contains 0.9ml bromo isobutyl acylbromides is added dropwise, stirring reaction 12-24h at room temperature takes out and used
Ethanol and deionized water are cleaned after 3 times and dried up with nitrogen successively；
F) naked golden chip surface polymethyl base cysteine synthesis
The chip that e) step is obtained is put into the Shi Lunke bottles of advance three circulating degasifications and inflated with nitrogen；C) step is obtained
5mmol monomers are dissolved in 4ml deionized waters, are deaerated 10-30min with nitrogen, add thereto 0.5mmol bipyridyls,
0.167mmol cuprous bromides and 0.083mmol copper bromides, with ultrasonically treated 5-10min is used after nitrogen degassing 10-30min, are incited somebody to action
The solution arrived moves into Shi Lunke bottles so that solution submerges the chip in bottle, and chip is taken out after 2-4h is reacted under condition of nitrogen gas,
Cleaned after 3 times and dried up with nitrogen successively with ethanol and deionized water.
2. preparation method according to claim 1, it is characterised in that described substrate is glass chip bottom or optical fiber base
Bottom.
3. preparation method according to claim 1, it is characterised in that described golden film can also be that silverskin or silver/gold are compound
Film.
4. preparation method according to claim 1, it is characterised in that the Piranha washing lotion be the 98wt.% concentrated sulfuric acids with
The volume ratio of the mixed solution of 30wt.% hydrogen peroxide, the concentrated sulfuric acid and hydrogen peroxide is 7:3.
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