CN109187957A - A method of with lignin in-situ modification gold chip - Google Patents
A method of with lignin in-situ modification gold chip Download PDFInfo
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- CN109187957A CN109187957A CN201811053639.0A CN201811053639A CN109187957A CN 109187957 A CN109187957 A CN 109187957A CN 201811053639 A CN201811053639 A CN 201811053639A CN 109187957 A CN109187957 A CN 109187957A
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- 229920005610 lignin Polymers 0.000 title claims abstract description 44
- 230000004048 modification Effects 0.000 title claims abstract description 23
- 238000012986 modification Methods 0.000 title claims abstract description 20
- 238000011065 in-situ storage Methods 0.000 title claims abstract description 19
- 238000000034 method Methods 0.000 title claims abstract description 17
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 239000010931 gold Substances 0.000 title claims abstract description 16
- 229910052737 gold Inorganic materials 0.000 title claims abstract description 16
- 108091003079 Bovine Serum Albumin Proteins 0.000 claims abstract description 16
- 108010059892 Cellulase Proteins 0.000 claims abstract description 16
- 229940098773 bovine serum albumin Drugs 0.000 claims abstract description 16
- 229940106157 cellulase Drugs 0.000 claims abstract description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 34
- 239000000243 solution Substances 0.000 claims description 26
- DYAOREPNYXXCOA-UHFFFAOYSA-N 2-sulfanylundecanoic acid Chemical compound CCCCCCCCCC(S)C(O)=O DYAOREPNYXXCOA-UHFFFAOYSA-N 0.000 claims description 22
- 239000007788 liquid Substances 0.000 claims description 15
- 235000019441 ethanol Nutrition 0.000 claims description 14
- 238000004140 cleaning Methods 0.000 claims description 11
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
- DKIDEFUBRARXTE-UHFFFAOYSA-N 3-mercaptopropanoic acid Chemical compound OC(=O)CCS DKIDEFUBRARXTE-UHFFFAOYSA-N 0.000 claims description 7
- 229920001732 Lignosulfonate Polymers 0.000 claims description 7
- HWJJKWDAKXZNEA-UHFFFAOYSA-N [Na].ON1C(CCC1=O)=S Chemical compound [Na].ON1C(CCC1=O)=S HWJJKWDAKXZNEA-UHFFFAOYSA-N 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000002156 mixing Methods 0.000 claims description 6
- GWOLZNVIRIHJHB-UHFFFAOYSA-N 11-mercaptoundecanoic acid Chemical compound OC(=O)CCCCCCCCCCS GWOLZNVIRIHJHB-UHFFFAOYSA-N 0.000 claims description 5
- 238000011010 flushing procedure Methods 0.000 claims description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims description 5
- 238000002474 experimental method Methods 0.000 claims description 3
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 claims description 3
- 239000002904 solvent Substances 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 2
- 239000012888 bovine serum Substances 0.000 claims description 2
- 239000000835 fiber Substances 0.000 claims description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 2
- 239000010452 phosphate Substances 0.000 claims description 2
- 108090000790 Enzymes Proteins 0.000 claims 1
- 102000004190 Enzymes Human genes 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 claims 1
- 238000003380 quartz crystal microbalance Methods 0.000 abstract description 23
- 230000008859 change Effects 0.000 abstract description 9
- 238000001179 sorption measurement Methods 0.000 abstract description 9
- 230000021715 photosynthesis, light harvesting Effects 0.000 abstract description 6
- 238000005516 engineering process Methods 0.000 abstract description 3
- 229920002521 macromolecule Polymers 0.000 abstract description 3
- 239000013078 crystal Substances 0.000 abstract description 2
- 239000010453 quartz Substances 0.000 abstract description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 abstract description 2
- 239000007787 solid Substances 0.000 abstract description 2
- 238000004528 spin coating Methods 0.000 abstract description 2
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 abstract 2
- 150000002343 gold Chemical class 0.000 abstract 1
- -1 paper-making pulping Substances 0.000 description 7
- 230000004913 activation Effects 0.000 description 6
- 238000012544 monitoring process Methods 0.000 description 6
- 239000011259 mixed solution Substances 0.000 description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 4
- 238000010521 absorption reaction Methods 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 3
- 238000004537 pulping Methods 0.000 description 3
- 239000003513 alkali Substances 0.000 description 2
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 2
- 239000002585 base Substances 0.000 description 2
- QGBSISYHAICWAH-UHFFFAOYSA-N dicyandiamide Chemical compound NC(N)=NC#N QGBSISYHAICWAH-UHFFFAOYSA-N 0.000 description 2
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 239000002023 wood Substances 0.000 description 2
- 239000002028 Biomass Substances 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 229920000049 Carbon (fiber) Polymers 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 1
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 1
- 241001104043 Syringa Species 0.000 description 1
- 235000004338 Syringa vulgaris Nutrition 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000001476 alcoholic effect Effects 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006227 byproduct Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000004917 carbon fiber Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 238000002144 chemical decomposition reaction Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 229910001385 heavy metal Inorganic materials 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Chemical compound C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000004001 molecular interaction Effects 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004227 thermal cracking Methods 0.000 description 1
- QERYCTSHXKAMIS-UHFFFAOYSA-N thiophene-2-carboxylic acid Chemical compound OC(=O)C1=CC=CS1 QERYCTSHXKAMIS-UHFFFAOYSA-N 0.000 description 1
- 238000009941 weaving Methods 0.000 description 1
Classifications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—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
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N29/00—Investigating or analysing materials by the use of ultrasonic, sonic or infrasonic waves; Visualisation of the interior of objects by transmitting ultrasonic or sonic waves through the object
- G01N29/02—Analysing fluids
- G01N29/022—Fluid sensors based on microsensors, e.g. quartz crystal-microbalance [QCM], surface acoustic wave [SAW] devices, tuning forks, cantilevers, flexural plate wave [FPW] devices
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Chemical & Material Sciences (AREA)
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- Biotechnology (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
本发明涉及木质素原位修饰金芯片的方法。石英晶体微天平(QCM)和表面等离子体共振仪(SPR)技术是实时、原位研究生物大分子在固体界面的吸附是重要工具，前者同时检测石英晶体频率的变化(对应感应器上的重量)和吸附层的能量耗散值(对应感应器上薄膜的结构)的变化，后者只研究“干物质”的变化。传统的木质素修饰金芯片的方法是通过溶解木质素然后旋涂的方法得到，其缺点是粗糙度较高。本发明采用先在金芯片上预先修饰牛血清蛋白或者纤维素酶的方法，然后再在其表面原位吸附一层木质素以制备木质素传感器，制备所得芯片更加平滑。The present invention relates to a method for in-situ modification of gold chips with lignin. Quartz crystal microbalance (QCM) and surface plasmon resonance (SPR) technology are important tools to study the adsorption of biological macromolecules on solid interfaces in real time and in situ. The former simultaneously detects the change of quartz crystal frequency (corresponding to the weight on the sensor). ) and the energy dissipation value of the adsorption layer (corresponding to the structure of the film on the sensor), the latter only studies the change of "dry matter". The traditional method of lignin-modified gold chips is obtained by dissolving lignin and then spin coating, which has the disadvantage of high roughness. The invention adopts the method of pre-modifying bovine serum albumin or cellulase on the gold chip, and then in situ adsorbs a layer of lignin on the surface of the gold chip to prepare the lignin sensor, and the chip obtained is smoother.
Description
Technical field
The present invention relates to the methods of lignin in-situ modification gold chip, belong to instrument analysis field.
Background technique
Lignin is the second largest renewable resource and the most abundant natural aromatic high score that yield is only second to cellulose
Sub- compound.In the related industry using plant fiber as core material such as paper-making pulping, wood-based composites, weaving, wood
Quality is discharged mainly as by-product, wherein maximum with the alkali lignin quantity that paper-making pulping process generates, annual output is about
Ten thousand tons of 4000-5000.In recent years, as people are to the pay attention to day by day of renewable resource, application and basis in relation to lignin are ground
Study carefully all to obtain and flourish.
Lignin has three by what is formed to three kinds of hydroxyl, guaiacyl and lilac base phenylpropyl alcohol alkane basic structural units
Tie up the high-molecular compound of reticular structure.During soda pulping process, along with the fracture of lignin structure, what is ultimately generated
A certain amount of hydroxyl and carboxyl isoreactivity group are introduced in alkali lignin.
Based on its structure feature, lignin can prepare biomass chemicals by chemical degradation or thermal cracking and biology fires
Material prepares high molecular material by blending, chemically modified to prepare heavy metal absorbent, carbon fiber, nano material and all kinds of
Dispersion of industry agent.Studies have found that soluble lignin is added to inside enzymolysis liquid or even can be improved enzymolysis efficiency.Lignin
Extensive use also urgently need people to carry out more deep basic research for it.By constantly improve related lignin
Theoretical research system, can gradually deepen people to the understanding of lignin micro-structure and physicochemical properties, thus further
Push the development and application of lignin.
Quartz crystal microbalance (QCM) and surface plasma body resonant vibration instrument (SPR) technology are research large biological molecules solid
The absorption of body interface is very favorable tool, the former detects the variation (weight on corresponding inductor of quartz crystal frequency simultaneously
Amount) and adsorption layer energy dissipation value (structure of film on correspondence inductor) variation, the latter only studies the change of " dry matter "
Change.The method of traditional research lignin and other high molecular interactions is that lignin liquor is consolidated by the method for spin coating
It is scheduled on QCM or SPR chip, other macromolecules is then studied lignin and other high molecular phase interactions as mobile phase
With.
The present invention relates to a kind of methods of lignin-modified golden chip, first fix bovine serum albumin on chip, then lead to
The specific adsorption for crossing lignin and bovine serum albumin soluble lignin is in situ, online can be fixed on QCM or SPR chip
On, so that lignin sensor be prepared, it is then applied in the repercussion study of other macromolecules and lignin.
Summary of the invention
1.A kind of method of lignin in-situ modification gold chip, it is characterised in that include the following steps:
Step (1): the chip used QCM or SPR chip for gold surface will be blown after the cleaning of golden chip with nitrogen before experiment
It is dry, it is then placed in UV-ozone cleaning machine irradiation 10-30min；
Step (2): being the 11- Mercaptoundecanoic acid (MUA) and 3- mercaptopropionic acid that solvent prepares 40mM with 75% ethyl alcohol
(MPA):
Step (3): 1- ethyl-(the 3- dimethylamino of 2mM is prepared with the phosphate buffer solution of the pH 0.05mM for being 7.4
Base propyl) carbodiimide hydrochloride (EDC) and 5mM N- hydroxy thiosuccinimide sodium salt (NHSS) solution and 50 μ g/
Bovine serum albumin liquid, cellulase liquid and the lignosulfonates liquid of ml；
Step (4): the chip after step (1) is cleaned and irradiated is placed in QCM or SPR flow cell；
Step (5): be passed through first with the flow velocity of 0.1ml/min 75% ethanol solution baseline is walked it is flat；
Step (6): after steady, MPA the and MUA solution prepared in step (2) by volume 10: 1 ratio mix
Afterwards, QCM or SPR flow cell is passed through with the flow velocity of 0.1ml/min, it is to be detected it is signal-balanced after be passed through the flushing of 75% ethyl alcohol；
Step (7): next EDC the and NHSS solution prepared in step (3) with the mixing of volume ratio 1: 1, then with
The flow velocity of 0.1ml/min is passed through system, stops logical liquid after 15min；
Step (8): restarting pump after one hour, the phosphoric acid for the 0.05mM that pH is 7.4 is passed through with the flow velocity of 0.1ml/min
Salt buffer solution；
Step (9): the flow velocity after signal is steady with 0.1ml/min is passed through the bovine serum albumen solution of step (3) preparation
Or cellulase solution, be passed through after balance to be adsorbed phosphate buffer solution wash the bovine serum albumin not being firmly combined or
Cellulase；
Step (10): it is passed through the lignosulfonates liquid of step (3) preparation with the flow velocity of 0.1ml/min, finally obtains
The golden chip of lignin in-situ modification.
Specific embodiment
Experimental temperature is set as 25 DEG C, and flow velocity is disposed as 0.1ml/min.
Then the chip used QCM or SPR chip for gold surface, with being dried with nitrogen, will be put before experiment after the cleaning of golden chip
Enter UV-ozone cleaning machine irradiation 10-30min.
Solution is prepared: being the 11- Mercaptoundecanoic acid (MUA) and 3- mercaptopropionic acid that solvent prepares 40mM with 75% ethyl alcohol
(MPA)；1- ethyl-(3- dimethylaminopropyl) carbon two for preparing 2mM with the phosphate buffer solution of the 0.05mM of pH7.4 is sub-
The solution of the N- hydroxy thiosuccinimide sodium salt (NHSS) of amine hydrochlorate (EDC) and 5mM and the cow's serum egg of 50 μ g/ml
White solution and lignin liquor.
Modification program: the chip after cleaning and irradiation is placed in QCM or SPR flow cell；Pass first into 75% second
Alcoholic solution walks baseline flat；After steady, be passed through MPA/MUA mixed solution and chip just modified, it is to be detected it is signal-balanced after
It is passed through the flushing of 75% ethyl alcohol；Next it is passed through EDC and NHSS solution to activate MPA/MUA, it is slow that phosphate is passed through after activation
Solution is rushed to be rinsed；Then bovine serum albumin or cellulase solution are passed through, bovine serum albumin or cellulase and activation
MPA/MUA interaction, be adsorbed on chip；Phosphate buffer solution is passed through after balance to be adsorbed washes be not firmly combined
Bovine serum albumin or cellulase, the lignosulfonates liquid of preparation is finally passed through, due to lignin and cellulase
There is specific adsorption in adsorption zone or bovine serum albumin, obtains the golden chip of lignin in-situ modification.
Below with reference to embodiment, the present invention is described in further detail, but embodiments of the present invention are not limited to
This.
Embodiment 1
The chip used QCM chip for gold surface.
It is the preparation of golden chip first:, with being dried with nitrogen, the irradiation of UV-ozone cleaning machine will be put into after the cleaning of golden chip
Then chip is placed in QCM flow cell by 10-30min.
Then start in-situ modification program:
75% ethanol solution is passed through with the rate of 0.1ml/min first, until baseline is walked surely.Then with 0.1ml/min
Rate be passed through in 10: 1 (V: V) ratio mixing 40mM 11- Mercaptoundecanoic acid (MPA) and 40mM 3- mercaptopropionic acid
(MUA) mixed solution.QCM frequency falls to approximately -6Hz,.After balance, it is passed through the flushing of 75% ethyl alcohol, frequency retrieval to -4Hz
Left and right.Whole process energy dissipation value (D) is almost inconvenient.According to Sauerbrey equation calculation, MPA/MUA is on golden chip
Adsorbance about 1.1mg/m2, the thickness of film is about 0.6nm.
Next it is passed through 1- ethyl-(3- dimethylaminopropyl) the carbodiimide salt for being 1: 1 mixed 2mM with volume ratio
The mixed solution 15min of the N- hydroxy thiosuccinimide sodium salt (NHSS) of hydrochlorate (EDC) and 5mM.Then stop logical liquid,
Static 1h keeps activation abundant；QCM monitoring frequency rises to about 150Hz, and corresponding energy dissipation value be reduced to -55 ×
10-6.Frequency and the energy dissipation variation of this QCM has mainly been transformed into phosphate buffer from ethanol system due to dicyandiamide solution
Caused by system.Activation is passed through phosphate buffer solution after one hour and is rinsed.
Followed by the modification of cellulase: it is passed through the cellulase that concentration is 50 μ g/ml after ready to balance, is passed through again after balance
Phosphate buffer solution washes the cellulase not being firmly combined, finally obtains the golden chip of cellulase modification in situ.By
Drop to 100Hz or so from about 150Hz accordingly in the frequency of the absorption of lignin, QCM monitoring, according to Sauerbrey equation
It calculates, adsorbance of the cellulase on golden chip about 8mg/m2, the thickness of film is about 5nm.
It is finally the modification of lignin: is passed through the lignosulfonates that concentration is 50 μ g/ml after ready to balance, finally obtains original
The lignin-modified golden chip in position.Since adsorption zone of the lignin to cellulase has specific adsorption, the frequency of QCM monitoring becomes
Change less, only slightly reduces, but energy dissipation value is sharply from -55 × 10-6Rise to about -40 × 10-6.It can be with from signal
It learns that the lignin membrane of absorption is very soft, is not able to satisfy the calculating requirement of Sauerbrey equation completely.
The QCM monitoring signals such as attached drawing 1 of entire lignin in-situ modification QCM gold chip.
Embodiment 2
The chip used SPR chip for gold surface.
It is the preparation of golden chip first:, with being dried with nitrogen, the irradiation of UV-ozone cleaning machine will be put into after the cleaning of golden chip
Then chip is placed in SPR flow cell by 10-30min.
Then start in-situ modification program:
75% ethanol solution is passed through with the rate of 0.1ml/min first, until baseline is walked surely, SPR angle about exists
72.68°.Then the 11- Mercaptoundecanoic acid of the 40mM of the ratio mixing in 10: 1 (V: V) is passed through with the rate of 0.1ml/min
(MUA) with the mixed solution of the 3- mercaptopropionic acid (MPA) of 40mM.SPR angle rises to about 72.89 °.After balance, it is passed through
75% ethyl alcohol rinses, and angle is almost inconvenient.According to equation estimation, adsorption thickness of the MPA/MUA on golden chip is about
3.8nm。
Next it is passed through 1- ethyl-(3- dimethylaminopropyl) the carbodiimide salt for being 1: 1 mixed 2mM with volume ratio
The mixed solution of the N- hydroxy thiosuccinimide sodium salt (EDC) of hydrochlorate (NHSS) and 5mM stops logical liquid after 15min, quiet
Only 1h keeps activation abundant；SPR monitoring angle drops to 69.2 ° or so.This variation is mainly that dicyandiamide solution turns from ethanol system
Caused by changing the change to its refractive index of phosphoric acid buffer liquid system.Activation is passed through phosphate buffer solution flushing after one hour.
Followed by the modification of bovine serum albumin: the bovine serum albumin that concentration is 50 μ g/ml, SPR angle are passed through after ready to balance
Rapidly rise to about 70.8 °.Phosphate buffer solution is passed through after balance again and washes the bovine serum albumin not being firmly combined.SPR
Angle is slowly returned to 70.2 °.That is, SPR angle change caused by the amount that entire bovine serum albumin securely adsorbs is about
1 °, according to equation estimation, the thickness of this layer of bovine serum albumin is about 18nm.
It is finally the modification of lignin: is passed through the lignosulfonates that concentration is 50 μ g/ml after ready to balance, SPR angle is slow
Slowly about 70.5 ° are risen to.Due to the absorption of lignin, about 0.3 ° of SPR angle change.That is, according to equation estimation, the layer
The thickness of lignin is about 5.5nm.
SPR the monitoring signals variation such as attached drawing 2 of entire lignin in-situ modification SPR chip.
Detailed description of the invention
Attached drawing 1: quartz crystal microbalance (QCM) monitors the case where in-situ modification gold chip of lignin
Attached drawing 2: surface plasma resonance instrument (SPR) monitors the case where in-situ modification gold chip of lignin
Technical effect
The thickness of adsorbance or film is in accordance with dependent equation and estimates to obtain in embodiment, since lignin membrane is viscoelastic
Property film, is not rigid film, therefore compared to SPR method, seriously underestimates film with Sauerbrey equation in QCM method
Thickness.
In addition it declares, is only presently preferred embodiments of the present invention in specific embodiment, not the present invention is made any
Formal limitation, although the present invention has been disclosed as a preferred embodiment, however, it is not intended to limit the invention, any to be familiar with
Professional and technical personnel does in the range of not departing from technical solution of the present invention when using the technology contents of the disclosure above
A little equivalent embodiment changed or be modified to equivalent variations out, but without departing from the technical solutions of the present invention, according to this
The technical spirit of invention still falls within the present invention to any simple modification, equivalent change and modification made by above embodiment
In the range of technical solution.
Claims (1)
1. a kind of method of lignin in-situ modification gold chip, it is characterised in that include the following steps:
Step (1): the chip used QCM or SPR chip for gold surface, with being dried with nitrogen after cleaning golden chip before experiment, so
After be put into UV-ozone cleaning machine irradiation 10-30min；
Step (2): being the 11- Mercaptoundecanoic acid (MUA) and 3- mercaptopropionic acid (MPA) that solvent prepares 40mM with 75% ethyl alcohol；
Step (3): 1- ethyl-(the 3- dimethylamino third of 2mM is prepared with the phosphate buffer solution of the pH 0.05mM for being 7.4
Base) carbodiimide hydrochloride (EDC) and 5mM N- hydroxy thiosuccinimide sodium salt (NHSS) molten armpit and 50 μ g/ml
Bovine serum albumin liquid, cellulase liquid and lignosulfonates liquid；
Step (4): the chip after step (1) is cleaned and irradiated is placed in QCM or SPR flow cell；
Step (5): be passed through first with the flow velocity of 0.1ml/min 75% ethanol solution baseline is walked it is flat；
Step (6): after steady, after the MPA benefit MUA solution prepared in step (2) by volume 10: 1 ratio mixing, with
The flow velocity of 0.1ml/min is passed through QCM or SPR flow cell, it is to be detected it is signal-balanced after be passed through the flushing of 75% ethyl alcohol；
Step (7): next EDC the and NHSS solution prepared in step (3) with the mixing of volume ratio 1: 1, then with 0.1ml/
The flow velocity of min is passed through system, stops logical liquid after 15min；
Step (8): restarting pump after one hour, slow with the phosphate that the flow velocity of 0.1ml/min is passed through the 0.05mM that pH is 7.4
Rush solution；
Step (9): the flow velocity after signal is steady with 0.1ml/min be passed through step (3) preparation bovine serum albumen solution or
Cellulase solution is passed through phosphate buffer solution after balance to be adsorbed and washes the bovine serum albumin or fiber not being firmly combined
Plain enzyme；
Step (10): it is passed through the lignosulfonates liquid of step (3) preparation with the flow velocity of 0.1ml/min, finally obtains wooden
The golden chip of plain in-situ modification.
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