CN102011193A - Protein modified GaN nanowire array as well as preparation method and application thereof - Google Patents

Protein modified GaN nanowire array as well as preparation method and application thereof Download PDF

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CN102011193A
CN102011193A CN 201010290192 CN201010290192A CN102011193A CN 102011193 A CN102011193 A CN 102011193A CN 201010290192 CN201010290192 CN 201010290192 CN 201010290192 A CN201010290192 A CN 201010290192A CN 102011193 A CN102011193 A CN 102011193A
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gan nano
wire array
gan
wire
protein
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CN102011193B (en
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郭东杰
陈亚清
张昊
谭华
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Nanjing University of Aeronautics and Astronautics
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Nanjing University of Aeronautics and Astronautics
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Abstract

The invention relates to a protein modified GaN nanowire array which is formed by the following steps of: using an upright GaN nanowire array as a substrate and performing chemical oxidation or plasma oxidation on the surface of the nanowire to generate Ga-OH functional groups; or depositing SiO2, TiO2 or Al2O3 on the surface of the nanowire by means of chemical vaporous deposition, hydrolyzing to generate -OH functional groups on the surface of the nanowire, reacting with SiC14 to form -O-Si-Cl bond and finally connecting the nanowire to protein molecules through polyethylene glycol molecules to form the protein modified GaN nanowire array. The invention can be applied to separation and purification, identification or detection of protein and discloses a preparation method of the protein modified GaN nanowire array.

Description

The GaN nano-wire array of protein modification and method for making and purposes
Technical field:
The present invention relates to the GaN nano-wire array, specifically, is GaN nano-wire array and the method for making and the purposes of protein modification.
Background technology
The same zinc oxide of gan (GaN) nano wire (ZnO), silicon nanowires (Si), (CNT) is the same for carbon nanotube, is a kind of important one dimension semiconductor material.In gallium nitride, form strong covalent bond between gallium element and the nitrogen element, make its show can with carbon nanotube compare favourably mechanical property.The GaN nano wire has metastable physicochemical property, and it can bear the erosion of acid, alkali, organic solvent, and the thermostability of height is arranged, and can bear 900 ℃ high temperature.Existing technology can skillfully prepare diameter 5~500nm, the nano wire of millimeter level length.
The GaN nano wire has the bio-compatible performance of height, can use in vivo.As a kind of monodimension nanometer material, it has high area/volume ratio.Undressed GaN nanowire surface does not have functional group, can not be used for the grafting organic monomolecular film.The GaN nanowire surface can by chemical oxidation for example sulfuric acid/hydrogen peroxide handle and produce activity-OH functional group, but this technology produced-OH quantity is relatively limited, it is very meaningful therefore to seek the preparation method who generates a large amount of-OH.Technique for atomic layer deposition (ALD) belongs to vacuum chemistry gas phase deposition technology (CVD), this technology is when producing compact oxidation layer, can produce a large amount of-OH functional group on its surface, this part-OH just can be used for modifying organic monomolecular film and then grafting biomolecules.
Biomolecule surface contains numerous functional groups, can the Ou Lian reaction take place with active function groups on the organic molecule, thus with the biomolecules grafting on the GaN nanowire surface.Diameter is between 100~500nm, spacing is in 200nm~and upright GaN nano-wire array between the 20 μ m is very favourable for the turnover of biomolecules, thus improve grafting or separation efficiency.Polyoxyethylene glycol (PEG) has the non-specific repulsion biomolecules ability of height, at the terminal fixing biological molecules of PEG, can avoid the physical property absorption of biomolecules, thereby improves the selectivity of biomolecules greatly.Simultaneously, with respect to basic biosensors such as porous silicon, aluminium, because the GaN nano-wire array is upright, solution is higher in the flowability on its surface, is convenient to grafting, separation of biomolecules.
The GaN nano wire can allow infrared spectra to pass, and functional group that it is surperficial and organic decoration thing comprise that biomolecules can absorb infrared light, so utilize infrared spectra can monitor the chemical reaction of GaN nanowire surface.The thickness of ALD settled layer is usually in 2~10 nanometers, and the infrared light of micron order wavelength can pass the ALD settled layer, so the sedimentary GaN nano wire of ALD also can be used Infrared Characterization.
Biological intravital some trace exists albumen that physiological function is played an important role, and purifies, discerns, clones them and have great importance.Because the GaN nano wire has high-specific surface area, can be used as the carrier of the highdensity biomolecules of grafting.After the biomolecules grafting, can identification mutually take place with correspondingly biomolecules (for example antibody-antigen, acceptor-give body, enzyme-substrate etc.) just, by some detection meanss, the information acquisition of these biomolecules is come out then.Because it is reversible, controlled that Recognition of Biomolecular belongs to, this array can repeat repeatedly to use.By Recognition of Biomolecular, a small amount of biomolecules that exists in the solution can be caught out, realize the purpose of concentrating and separating target biological molecules, avoided biochemical isolating high cost input.
Usually the GaN nano-wire array is a kind of nano wire of vertical stand-up, and laser is easy to interfere on its surface.Before and after biomolecules identification, considerable change will take place in its interference fringe, gathers its interference data, calculates its optical thickness, can quantitatively obtain the grafting density of grafting at its surface biological molecule.In addition, if adopt fluorescently-labeled biomolecules grafting on the surface of nano wire, its fluorescence intensity just can obtain by fluorescent scanning.Utilize fluorometry, can quantitatively obtain the quantity of fluorescence molecule, can derive the grafting density of biomolecules.
At present, the patent about the GaN nano wire mainly concentrates on photodiode and laser apparatus aspect.The multinomial patent of the U.S. is as 6818061,7335262 preparations that relate to the GaN nano wire.United States Patent (USP) 7421274,7420147,6949773, European patent EP 1145282A2, TaiWan, China patent TW569474 relates to the application of GaN nano wire at LED aspect.Chinese patent 200510048111 relates to the preparation of GaN nano wire.Chinese patent 200780016946 relates to the preparation and the application aspect photodiode and laser apparatus of GaN nano wire.
Summary of the invention:
The GaN nano-wire array and the method for making thereof that the purpose of this invention is to provide a kind of protein modification, and it is used for separation, identification or the detection of protein molecule.
Technical scheme of the present invention is as follows:
A kind of GaN nano-wire array of protein modification, it be with axial GaN nano-wire array as substrate, nanowire surface produces Ga-OH functional group through chemical oxidation or plasma oxidation; Perhaps deposit SiO in nanowire surface through atomic layer deposition method 2, TiO 2Or Al 2O 3Film, its surface has-OH functional group through hydrolysis, the GaN nanowire surface-OH functional group and SiCl 4React formation-O-Si-Cl key, be connected to form the GaN nano-wire array of protein modification at last by peg molecule and protein molecule.
The GaN nano-wire array of above-mentioned protein modification, described protein molecule can be any protein molecules, for example: the monoclonal antibody of avidin, mouse monoclonal antibody or brain natriuretic peptide.
The GaN nano-wire array of above-mentioned protein modification, described polyoxyethylene glycol are that number-average molecular weight is the polyoxyethylene glycol of 600-3000.
A kind of method for preparing the GaN nano-wire array of above-mentioned protein modification, it comprises the following steps:
Step 1. with chemical oxidization method oxidation or plasma radiation oxidation, makes nanowire surface generation-OH functional group with axial GaN nano-wire array;
Step 2. has the surface that step 1 obtains-the GaN nano-wire array of OH functional group in soaking at room temperature at pure SiCl 4In the liquid 0.5-2 hour, make nanowire surface-the OH base is converted into-O-SiCl 3
Step 3. has the surface that step 2 obtains-O-SiCl 3The GaN nano-wire array place the end of 0.1mol/L to have the chloroformic solution of the polyoxyethylene glycol of NHS (N-hydroxy-succinamide), placed in the dark 0.5 hour, take out the GaN nano-wire array, clean, drying obtains NHS activatory GaN nano wire;
Step 4. places proteinic water or PBS (phosphate buffer solution with the NHS activatory GaN nano wire that step 3 obtains, pH=7~10) placed 0.5~1 hour in the solution (10-50nmol/L), take out the GaN nano-wire array, clean, drying obtains the GaN nano wire of protein modification.
The method of the GaN nano-wire array of above-mentioned preparation protein modification, the described chemical oxidization method of step 1 is that the GaN nano-wire array is placed sulfuric acid and hydrogen peroxide mixed solution (sulfuric acid: hydrogen peroxide=3: 1v/v) or sulfuric acid and nitric acid mixing solutions (sulfuric acid: nitric acid=1: 1v/v), heated 4~10 hours down at 80 ℃, take out and clean, dry up.
The method of the GaN nano-wire array of above-mentioned preparation protein modification, described step 1 can substitute with the following method:
Step 1 ' axial GaN nano-wire array is deposited SiO with atomic layer deposition method in nanowire surface 2, TiO 2Or Al 2O 3Film, its surface is through hydrolysis generation-OH functional group.
The method of the GaN nano-wire array of above-mentioned preparation protein modification, described step 3,4 can substitute with following two steps:
Step 3 ' surface that step 2 is obtained has-O-SiCl 3The GaN nano-wire array place the end of 0.1mol/L to have polyoxyethylene glycol DMF (dimethyl formamide) solution of vitamin H, add a little triethylamine, placed 0.5~1 hour, clean, drying obtains the GaN nano wire of biotin modification.
It is avidin water or the PBS solution of 10-50nmol/mL that the GaN nano wire of the biotin modification that step 4 ' with step 3 ' obtains places concentration, places 0.5 hour, cleans, and drying obtains the GaN nano wire that avidin is modified.
The method of the GaN nano-wire array of above-mentioned preparation protein modification, described protein are any protein molecules, and they can be the monoclonal antibodies of avidin, mouse monoclonal antibody or brain natriuretic peptide.
Being used for the organic molecule of grafting organic molecule film among the present invention contains bifunctional, an end can with the functional group (as Si-Cl) of GaN surface-OH reaction, an other end with contain-NH 2Crosslinked functional group's (as NHS ester) takes place in functional group.Here earlier GaN array room temperature is placed pure SiCl 4Solution 0.5~2 hour, the GaN nanowire surface-OH is just and SiCl 4Reaction, product is-O-Si-Cl 3, unnecessary Si-Cl can with PEG one end-OH reaction, thereby with the PEG grafting on the GaN nano wire, (CONH-) be connected, thereby they be fixed on the surface of GaN by amido linkage other a section of PEG with vitamin H or avidin molecule.Here, adopt two kinds to modify route respectively according to the functional group of vitamin H and avidin is different.A kind of first fixed biologically element is discerned avidin then; Another kind of elder generation is avidin fixedly, discerns vitamin H then.
The present invention can at first synthesize PEG-Biotin, utilizes the activated carboxylic of biotin molecule one end to become the NHS ester, then with PEG-NH 2On-NH 2Reaction, thereby synthetic PEG-Biotin:
Figure BSA00000281260700041
The Si-Cl of GaN nanowire surface and the OH of the other end of PEG-Biotin are reacted, thereby vitamin H is passed through the amido linkage grafting on the GaN surface.At aqueous phase, the avidin molecule is easy to discern with biotin molecule, thereby avidin is fixed on the GaN nanowire surface.The intermolecular reactive force of vitamin H and avidin is hydrogen bond, Van der Waals force, electrostatic force.Its reaction process as shown in Figure 1.
The present invention also can at first synthesize the GaN-PEG-NHS ester, with PEG-NH 2On-NH 2With double amber imide (SC, bis (N-succinimidyl) carbonate) reaction, generate active PEG-NHS ester:
Figure BSA00000281260700051
This NHS ester can with the avidin molecule on another-NH 2Crosslinking reaction takes place.Under the water, the GaN of grafting avidin can discern with vitamin H, thereby vitamin H is shifted in the GaN nanowire surface.Its reaction process as shown in Figure 2.
In view of all containing a large amount of activity-NH in the surface of protein (as monoclonal antibody of mouse monoclonal antibody, brain natriuretic peptide etc.) 2So, all can use these protein molecules of aforesaid method grafting.After these antibody molecule graftings, nano wire just can be discerned by corresponding antigen molecule (as goat mouse-anti immune protein (IgG), brain natriuretic peptide (BNP)), thereby antigen molecule is transferred to the GaN nanowire surface from liquid phase.Otherwise first immobilized antigen is discerned with their antibody then, thereby antibody molecule is transferred to the GaN nanowire surface.Obviously, the amido linkage by covalency between the protein molecule that this method also can grafting the unknown, the biomolecules of institute's grafting and organic molecule links to each other.
The GaN nano-wire array of protein modification of the present invention can be applied to the biomolecule detection of 4 aspects.Comprise proteinic molecular recognition, and identification before and after the ground substance assistant laser of fluoroscopic examination, protein molecule of laser interference spectrum, the protein molecule flight time mass spectrum that dissociates detect.
The GaN nano-wire array of protein modification of the present invention can be used for proteinic affine separation.Vitamin H fixed GaN nano-wire array is placed the mixing solutions that contains avidin, rely on the specific identification of vitamin H and avidin, avidin in the solution and vitamin H are had an effect and are fixed on the GaN nano wire, this chip is taken out, place deionized water, heat (50 ℃) half an hour slightly, break away from the constraint of avidin vitamin H from chip, enter in the solution.With the solution cryodrying, just can obtain highly purified active avidin.Otherwise, PEG-NHS ester activatory sample also can be earlier fixing avidin, thereby the purification vitamin H.In like manner, place the PBS buffered soln of antibody (as antibody, the mouse monoclonal antibody of brain natriuretic peptide (BNP)) to hatch respectively one hour NHS ester activatory GaN chip, take out the back and cushion and washed with de-ionized water with PBS, drying, on these two kinds of antibody-NH 2With the NHS effect, thereby they are fixed on the GaN chip.Chip is placed the PBS buffered soln that contains corresponding antigen (as brain natriuretic peptide (BNP), goat mouse-anti immune protein (IgG)), rely on antibody-AI, BNP and IgG antigen molecule just are fixed on the GaN chip, place the PBS solution of certain pH value, fixed antigen BNP of wash-out institute and IgG molecule.Freezing, drying can obtain highly purified antigen BNP and IgG.Otherwise, first immobilized antigen, back identification antibody molecule just can the separation antibody molecule.
The GaN nano-wire array of protein modification of the present invention can be used for the laser interference transmitter.The upright hexagonal prism type GaN nano wire (the nano wire pattern is seen Fig. 3) of the equally distributed rule that the present invention uses, incident laser reflects on the surface of nano wire, the multi beam reflector laser interferes, and utilizes detector that interference fringe is gathered, and utilizes formula nd=(Δ k λ 1λ 2)/2 (λ 12), n is the mean refractive index of GaN, d is the thickness of film, λ 1And λ 2Be respectively the wavelength value of adjacent two crests or trough, and for adjacent two crests or trough, Δ k=1.0 in the formula can get the relative thickness of interfering layer.After biomolecules grafting or identification, because the size (proteinic about 10nm) of biomolecules self, interfering layer thickness changes, and its variable quantity comes from the fixed biomolecules.Gather the interference spectrum of grafting front and back respectively,, calculated the variable quantity of optical thickness respectively, weigh the grafting density of institute's grafting protein molecule according to this, also promptly obtain the concentration of target biological molecules data substitution formula.
The GaN nano-wire array of protein modification of the present invention can be used for fluoroscopic examination.Biomolecules is carried out fluorescent mark, utilize fluorescent scanning can obtain the fluorescence picture.According to existing fluorescence analysis, fluorescence intensity and fluorescence molecule number are linear corresponding relation under the finite concentration, and fluorescence molecule number and biomolecules number have the fixed ratio, like this by fluorescence measurement, can qualitative, quantitatively obtain GaN nano wire institute fixed biomolecules quantity.
The GaN nano-wire array of protein modification of the present invention can be used for ground substance assistant laser (MALDI-ToF-MS) mass spectrometric detection of dissociating the flight time.On NHS ester activatory GaN chip, at first fixedly avidin, brain natriuretic peptide (BNP) or mouse monoclonal antibody, affine then concentrated fixed biologically element, brain natriuretic peptide (BNP), goat mouse-anti immune protein (IGg).Place MALDI-ToF-MS to detect in the exsiccant sample, just can obtain the mass spectrum picture of vitamin H, brain natriuretic peptide, goat mouse-anti immune protein.At the bottom of common stainless steel lining, the GaN nano wire display of protein modification has much higher specific surface area, the PEG polymer chain can repel the non-specific adsorption of non-target protein molecule in nanowire surface, can screen highly spissated target biological molecules by antibody-AI, thereby obtain better detectability.
NHS ester activatory GaN chip of the present invention can also detect unknown protein molecule.NHS ester activatory GaN chip is placed the solution of bovine serum albumin or peptide (Fmoc-EAALKLAR), bovine serum albumin or peptide (Fmoc-EAALKLAR) just are fixed on the surface of GaN, test MALDI-ToF-MS, the mass spectrum of bovine serum albumin or peptide (Fmoc-EAALKLAR) will be gathered.Utilize the protein sequence database set up in the world relatively, utilize software, can analyze proteinic sequence.Equally, also contain-NH owing to the unknown protein molecule surface 2, they also can be fixed on the GaN nano wire, detect by MALDI-ToF-MS and obtain its sequence, thereby disclose agnoprotein.
Description of drawings:
Fig. 1, biotin/avidin molecule route.
Fig. 2, avidin/biotin molecule route.
The plane of Fig. 3, vertical-growth GaN nano wire and section SEM picture, a is an orthographic plan; B is a sectional view.
Fig. 4, PEG-Biotin synthesize infrared figure
SiO after that Fig. 5, sulfuric acid/hydrogen peroxide is handled and the vapour deposition 2, Al 2O 3, TiO 2The IR spectrum of the GaN nano wire that covers, a are that sulfuric acid/hydrogen peroxide is handled; The TiO of b for covering 2C is Al 2O 3Cover; D is SiO 2Cover.
The OH content comparison diagram of four samples among Fig. 6, Fig. 5.
Fig. 7, at SiO 2Cover the infrared spectra of GaN nanowire surface grafting PEG-Biotin and Streptavidin.
The TEM picture of four samples among Fig. 8, Fig. 5.
TEM picture among Fig. 9, Fig. 5 behind the four sample surfaces grafting avidins.
Laser interference curve before and after Figure 10, Fig. 9 sample grafting avidin.A is the laser interference curve of the GaN nano-wire array before avidin is modified; B is the laser interference curve of the GaN nano-wire array after avidin is modified.
The fluorescent scanning figure of four sample surfaces grafting fluorescent mark avidins among Figure 11, Fig. 5.
The fluoroscopic image of the fluorescent mark avidin of Figure 12, nano-wire array surface isolation.
The MALDI-Tof-MS test of Figure 13, bovine serum albumin BSA.
The MALDI-Tof-MS test of Figure 14, Fmoc-EAALKLAR.
Figure 15, brain natriuretic peptide the MALDI-Tof-MS test comparison.
Embodiment:
Embodiment 1. produces Ga-OH functional group in the GaN nanowire surface
Fig. 3 shows GaN nano-wire array (USA, plane NIST) and section SEM picture.As seen from the figure: the diameter of single GaN nano wire is between 50~500nm, highly is between 9~10 μ m, the spacing of nano-wire array is between 200nm~20 μ m, all GaN nano wires have all shown even curface, are in and the axial position of supporting pieces base.
The GaN nano-wire array placed 80 ℃ sulfuric acid: the solution (5mL) of hydrogen peroxide (3: 1), handled 4 hours, utilize and ultrasonic the GaN nano wire is peeled off from growth sheet base, the GaN nano wire of peeling off is dispersed in CCl 2H 2In the solution, with pipettor solution is dispersed on the exsiccant KBr crystal wafer, sample was placed 80 ℃ of baking boxs dry 4 hours, sample is done Infrared spectroscopy.Infrared spectra such as Fig. 5 a are 3400,1600cm -1The zone occurred-infrared absorption of OH.3168cm wherein -1The corresponding OH of absorption peak, this OH can be used for the grafting organic molecule film.
With GaN nano-wire array plasma treatment 2 minutes (Harrick plasma clean machine (PDC-002), 30W power, 10sccm air), utilize and ultrasonic the GaN nano wire is peeled off from growth sheet base, the GaN nano wire of peeling off is dispersed in CCl 2H 2In the solution, with pipettor solution is dispersed on the exsiccant KBr crystal wafer, sample was placed 80 ℃ of baking boxs dry 4 hours, sample is done Infrared spectroscopy.3400,1600cm -1The zone occurred-infrared absorption of OH.3168cm wherein -1The corresponding OH of absorption peak, this OH can be used for the grafting organic molecule film.
With GaN nano-wire array surface each depositing Ti O of difference 2, Al 2O 3, SiO 2Deposition ALD film.Under the high vacuum, feed precursor Al (OR) earlier 3With high purity water steam, form one deck Al in the GaN nanowire surface 2O 3Film, its surface has OH, feeds TiCl then respectively 4, SiCl 4With high purity water steam, form TiO respectively 2, SiO 2Film, its surface has OH.Like this, zone of oxidation just is grown in the surface of GaN nano wire, and thickness of oxide layer is relevant with the cycle index that feeds gas.Utilize and ultrasonic the GaN nano wire is peeled off from growth sheet base, the GaN nano wire of peeling off is dispersed in CCl 2H 2In the solution, with pipettor solution is dispersed on the exsiccant KBr crystal wafer, sample was placed 80 ℃ of baking boxs dry 4 hours, sample is done Infrared spectroscopy.The infrared spectra of three kinds of ALD deposition GaN nano wires is corresponding diagram 5b-5d respectively, 3400,1600cm -1The zone occurred-infrared absorption of OH.3168cm wherein -1The corresponding OH of absorption peak, this OH can be used for the grafting organic molecule film.
The infrared spectra curve of Fig. 5 a-d is carried out match, get OH relative content (O-H area/Ga-N area) and be respectively 6.70%, 47.32%, 69.86% and 90.32%.It the results are shown in Fig. 6, compares, and the ALD settled layer makes-and OH content increased by 6.06,9.43,12.48 times.
The GaN nano wire of above-mentioned 4 kinds of generation-OH is peeled off from the sheet base, be dispersed in the methyl alcohol, drop in then on the TEM copper mesh, make high resolution TEM picture, see Fig. 8, a is that sulfuric acid/hydrogen peroxide is handled, and b, c, d are respectively SiO 2, Al 2O 3, TiO 2Cover the GaN nano wire.Can get on scheming, three kinds of ALD coating cladding thicknesses are respectively 4~5nm, 5~6nm, 10~12nm.
Embodiment 2. synthetic Biotin-NHS esters
Get the 0.5g vitamin H and be dissolved in 50mL exsiccant DMF (dimethyl formamide) solution, put into 0.51g DCC (N, the N-dicyclohexylcarbodiimide) and 0.26g NHS, 50 ℃ were stirred 16 hours down, to not dissolve sedimentation and filtration removes, add excessive dry ether, make it to produce white precipitate, suction filtration, white Biotin-NHS product.Medicine is done infrared spectra, sees Fig. 4 b, has occurred 3228,3108,3063,2939,2915,2875,2846,1818,1787,1745,1729,1696cm -1Absorption peak.
Embodiment 3. synthetic PEG-Biotin
Get 0.225g biotin-NHS and add in the 50mL exsiccant chloroformic solution, add 1.0g PEG-NH 2(M=3000) (Sigma-Adrich buys) and a spot of triethylamine stirred 6 hours, filtered insolubles, added excessive dry ether, made it to produce white precipitate.Medicine is done infrared spectra, sees Fig. 4 d, has occurred 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm -1Absorption peak.Show and synthesized PEG-Biotin.
Get 0.225g biotin-NHS and add in the 50mL exsiccant chloroformic solution, add 0.2g PEG-NH 2(M=600) (Sigma-Adrich buys) and a spot of triethylamine stirred 1 hour, filtered insolubles, added excessive dry ether, made it to produce white precipitate.Medicine is done infrared spectra, has occurred 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm -1Absorption peak.Show and synthesized PEG-Biotin.
Get 0.225g biotin-NHS and add in the 50mL exsiccant chloroformic solution, add 0.33g PEG-NH 2(M=1000) (Sigma-Adrich buys) and a spot of triethylamine stirred 2 hours, filtered insolubles, added excessive dry ether, made it to produce white precipitate.Medicine is done infrared spectra, has occurred 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm -1Absorption peak.Show and synthesized PEG-Biotin.
The preparation of the GaN nano-wire array of embodiment 4. biotin modifications
With SiO 2The GaN nano-wire array of functionalization (sample corresponding diagram 5d) places pure SiCl 4Placed 1 hour in the solution, the dry DMF solution that is placed on PEG (M=3000)-Biotin (vitamin H) of 0.1Mol/L of sample washing, placed 0.5 hour in the dark place, take out the GaN nano-wire array, clean, drying is peeled off from the array chip base, do Infrared spectroscopy, infrared spectrogram is seen Fig. 7 h.Occurred PEG (2887,2860cm -1) and Biotin (1665,1652cm -1) charateristic avsorption band.Show that PEG-Biotin is by the surface of grafting at the GaN nano wire.The GaN nano-wire array of the biotin modification of same preparation PEGM=1000 and PEG M=600 correspondence carries out Infrared spectroscopy, the charateristic avsorption band of PEG-Biotin all occurred.
The GaN nano-wire array of embodiment 5. usefulness biotin modifications separates avidin
The sample of gained in the foregoing description 4 is placed Streptavidin (avidin) aqueous solution (10nMol/mL), hatch half an hour, take out sample, clean, dry up, stripping nano line from the sheet base is done infrared analysis (seeing Fig. 7 i).The acid amides absorption peak that has occurred avidin in infrared.1652cm -1Corresponding C=O peak from avidin, 1543cm -1Corresponding N-H peak from avidin.Show that avidin has been fixed on the surface of nano wire.Reactive force between avidin and the vitamin H derives from hydrogen bond, Van der Waals force, electrostatic force.The same GaN nano-wire array of using the biotin modification of PEG M=1000 and PEG M=600 correspondence carries out identical experiment, the acid amides absorption peak of avidin all occurred.
The GaN nano-wire array of embodiment 6. usefulness biotin modifications separates avidin
GaN nano-wire array and covering Al with sulfuric acid-hydrogen peroxide processing 2O 3And TiO 2The GaN nano-wire array carry out the experiment of embodiment 4 and 5 respectively, sample is done infrared analysis, PEG all occurred, the charateristic avsorption band of vitamin H and avidin, occurred respectively in their infrared spectra PEG (2887,2860cm -1), Biotin (1665,1652cm -1), avidin (1543,1652cm -1) charateristic avsorption band.
Embodiment 7. synthetic PEG-NHS
Get 3g PEG-NH 2(M=1500) place the exsiccant acetonitrile, add 0.52g SC (double amber imide) and the triethylamine of a little, stirred 0.5 hour evaporated under reduced pressure, the PEG-NHS of acetonitrile recrystallization gained under the room temperature.Sample is done infrared spectra, has occurred 2939,2915,2875,2846,1818,1775,1735,1729cm -1Absorption peak.Show the NHS ester that generates PEG.
Get 2g PEG-NH 2(M=2000) place the exsiccant acetonitrile, add 0.44g SC and the triethylamine of a little, stirred 2 hours evaporated under reduced pressure, the PEG-NHS of acetonitrile recrystallization gained under the room temperature.Sample is done infrared spectra, 1775, and 1735cm -1The charateristic avsorption band of NHS has appearred in the place.Show the NHS ester that generates PEG.
The preparation of the GaN nano-wire array that embodiment 8.PEG-NHS modifies
The GaN nano-wire array of generation-OH of handling with sulfuric acid-hydrogen peroxide among the embodiment 1 is placed pure SiCl 4Placed 1 hour in the solution, take out sample, clean, dry up, get Si-Cl functionalization sample.This sample is put into the chloroformic solution of the PEG-NHS (M=2000) of 0.1Mol/L, add a little triethylamine, reaction half an hour, take out sample, the GaN nano wire that washing exsiccant PEG-NHS modifies.Utilize and ultrasonic the GaN nano wire is peeled off from growth sheet base, the GaN nano wire of peeling off is dispersed in CCl 2H 2In the solution, with pipettor solution is dispersed on the exsiccant KBr crystal wafer, sample was placed 80 ℃ of baking boxs dry 4 hours, sample is done Infrared spectroscopy.Occur 2939,2915,2875,2846,1818,1775,1735,1729 in the infrared spectra, 534cm -1Absorption peak.Show that PEG-NHS is fixed on the GaN nano wire.
The preparation of the GaN nano-wire array of embodiment 9. protein modifications
Get GaN nano-wire array that the PEG-NHS of embodiment 8 preparation modifies place respectively avidin (10nMol/mL), bovine serum albumin (20nMol/mL), peptide (Fmoc-EAALKLAR) (50nMol/mL), the solution of brain natriuretic peptide monoclonal antibody (10nMol/mL), mouse monoclonal antibody (50nMol/mL) handles half an hour, take out, cleaning, drying, the peeling GaN nano wire is done red analysis.Acid amides I (~1560cm has all appearred in their infrared spectra -1) and II (~1650cm -1) infrared absorption peak.Show that these biomolecules are by the surface of grafting at nano wire.
The GaN nano-wire array of embodiment 10. usefulness protein modifications separates corresponding protein
Under the room temperature, the monoclonal antibody of brain natriuretic peptide in the foregoing description 9, the GaN nano-wire array of mouse monoclonal antibody modification are placed the solution of brain natriuretic peptide (1nMol/mL), goat mouse-anti immune protein (5nMol/mL) respectively, took out in 10 minutes, cleaning, drying, the peeling GaN nano wire is done red analysis.Acid amides the I (~1560cm that strengthens has appearred in their infrared spectra -1) and II (~1650cm -1) infrared absorption peak.Show that these two kinds of antigens are by the surface of grafting at nano wire.
Embodiment 11.
The GaN nano-wire array that is fixed with the biotin modification of avidin among the foregoing description 5 and the embodiment 6 is dyeed with uranyl acetate, take out, cleaning, drying behind the peeling GaN nano wire, is done tem analysis, and the pattern (see figure 9) of avidin molecule has appearred in sample.Fig. 5 a-d (handle, SiO by sulfuric acid/hydrogen peroxide 2, Al 2O 3, TiO 2Covering GaN nano wire) sample is Fig. 9 i-l corresponding to the TEM figure of the GaN nano-wire array of the biotin modification that is fixed with avidin.Because the covering of protein molecule, the N constituent content obviously increases.
Embodiment 12.
Replace not having fluorescently-labeled avidin with fluorescently-labeled avidin (1nMol/mL), repeat the experiment in the foregoing description 5 and 6.To be fixed the GaN nano-wire array cleaning of fluorescence avidin, drying separates the GaN nano wire, does fluorescent scanning, obtains the fluorescence picture and lists in Figure 11, and 4 samples (handle, SiO by sulfuric acid/hydrogen peroxide among above-mentioned Fig. 5 a-d 2, Al 2O 3, TiO 2Covering GaN nano wire) fluoroscopic image is corresponding diagram 11i-l respectively.Its average fluorescent strength is respectively 17.02,133.52, and 108.75,167.02.
Embodiment 13.
The sample of the foregoing description 5 and 6 gained is made laser interference spectrum respectively, obtain Figure 10.Utilize formula noted earlier to calculate optical thickness before and after the grafting of avidin molecule and be respectively 4800 and 5500nm, promptly after the grafting of avidin molecule, optical thickness increased value ((nd 2-nd 1)/nd 1* be 14.5% 100%).
Embodiment 14
The avidin that the foregoing description 9 is obtained, peptide and bovine serum modify GaN nano-wire array sample do laser interference spectrum.Utilize formula, optical thickness increases by 22.5%, 11.3% and 14.2% respectively after calculating avidin, peptide and the bovine serum grafting.
Embodiment 15
Bovine serum albumin that the foregoing description 9 is obtained and peptide (Fmoc-EAALKLAR) modify the GaN nano-wire array do the MALDI-ToF-MS test.Spectrogram such as Figure 13-14.Occurred bovine serum albumin and peptide molecule mass spectra peak in the spectrogram, shown that GaN nanowire surface fixed bovine serum albumin, peptide molecule can detect by MALDI-ToF-MS.Wherein, Figure 14 b resolves through international protein library, and the partial amino-acid series of this peptide is through resolving to EAALKLAR.In like manner, agnoprotein also can be fixed on the GaN nano wire, and its aminoacid sequence also can resolve and get by MALDI-ToF-MS.
Embodiment 16.
Sample (1 * 1cm that the brain natriuretic peptide monoclonal antibody that the foregoing description 9 is obtained is modified 2) soak into deionized water, to get 10 μ L brain natriuretic peptide antigenic solutions (1nMol/mL) and drip thereon, 10min is hatched in sealing, takes out GaN nano-wire array sample, cleans, and drying is done the MALDI-ToF-MS test.Spectrogram is seen Figure 15 a, has occurred strong brain natriuretic peptide mass spectra peak in the spectrogram.Get 10 μ L brain natriuretic peptide antigenic solutions (1nMol/mL) and drop on the stainless substrate, nitrogen dries up, and does the MALDI-ToF-MS test.Spectrogram is listed in Figure 15 b, contrasts two curves, and Figure 15 b has shown the noise jamming of height, obviously has the signal to noise ratio of height at the resulting spectrogram of GaN nano-wire array.
Embodiment 17.
The nano wire sample (Figure 11 i and 11l) that the foregoing description 12 is obtained places the deionized water of 50 ℃ of 5mL respectively, hatch 1 hour after, get two kinds of drips of solution of 10 μ L respectively on the silicon chip of 2 cleanings, dry gas dries up, and does fluorescent scanning, picture is seen Figure 12.Obviously, two surfaces have all presented the fluoroscopic image of different fluorescence intensities, show that avidin separates from the GaN nano-wire array.Wherein, the sample of Figure 11 l correspondence contains more avidin molecule, this with Figure 12 l sample in also have more fluorescently-labeled avidin consistent.With these two solution lyophilizes, can obtain highly purified avidin.Otherwise elder generation is the avidin molecule fixedly, and identification biotin molecule in back just can obtain highly purified vitamin H.In like manner, highly purified antibody (as brain natriuretic peptide monoclonal antibody, mouse monoclonal antibody) and antigen (as brain natriuretic peptide, goat mouse-anti immune protein) also can be purified with method of the same race.

Claims (10)

1. the GaN nano-wire array of a protein modification is characterized in that: it be with axial GaN nano-wire array as substrate, nanowire surface produces Ga-OH functional group through chemical oxidation or plasma oxidation; Perhaps deposit SiO in nanowire surface through chemical Vapor deposition process 2, TiO 2Or Al 2O 3, through hydrolysis in surface generation-OH functional group, the GaN nanowire surface-OH functional group and SiCl 4Reaction formation-O-Si-Cl key, the GaN nano-wire array that is connected with protein molecule and forms protein modification by peg molecule at last.
2. the GaN nano-wire array of protein modification according to claim 1, it is characterized in that: described protein molecule is any protein molecule.
3. the GaN nano-wire array of protein modification according to claim 1, it is characterized in that: described protein molecule is the monoclonal antibody of avidin, mouse monoclonal antibody or brain natriuretic peptide.
4. the GaN nano-wire array of protein modification according to claim 1, it is characterized in that: described polyoxyethylene glycol is that number-average molecular weight is the polyoxyethylene glycol of 600-3000.
5. a method for preparing the GaN nano-wire array of the described protein modification of claim 1 is characterized in that it comprises the following steps:
Step 1. with chemical oxidization method oxidation or plasma radiation oxidation, makes nanowire surface generation-OH functional group with axial GaN nano-wire array;
Step 2. has the surface that step 1 obtains-and the GaN nano-wire array of OH functional group at room temperature is immersed in pure SiCl 4In the liquid 0.5-2 hour, make nanowire surface-the OH base is converted into-O-SiCl 3
Step 3. has the surface that step 2 obtains-O-SiCl 3The GaN nano-wire array place the end of 0.1mol/L to have the chloroformic solution of the polyoxyethylene glycol of NHS (N-OH succinimide), placed in the dark 0.5 hour, take out the GaN nano-wire array, clean, drying obtains NHS activatory GaN nano wire;
Step 4. places proteinic water or PBS (phosphate buffer solution with the NHS activatory GaN nano wire that step 3 obtains, pH=7~10) placed 0.5~1 hour in the solution (10-50nmol/L), take out the GaN nano-wire array, clean, drying obtains the GaN nano wire of protein modification.
6. the method for the GaN nano-wire array of preparation protein modification according to claim 5, it is characterized in that: the described chemical oxidization method oxidation of step 1 is that the GaN nano-wire array is placed sulfuric acid and hydrogen peroxide mixed solution or sulfuric acid and nitric acid mixing solutions, heated 4~10 hours down at 80 ℃, take out and clean, dry up.
7. the method for the GaN nano-wire array of preparation protein modification according to claim 5 is characterized in that described step 1 substitutes with the following method:
Step 1 ' axial GaN nano-wire array is deposited SiO with chemical Vapor deposition process in nanowire surface 2, TiO 2Or Al 2O 3, through hydrolysis in outside surface generation-OH functional group.
8. the method for the GaN nano-wire array of preparation protein modification according to claim 5 is characterized in that described step 3 and 4 can substitute with following two steps:
Step 3 ' surface that step 2 is obtained has-O-SiCl 3The GaN nano-wire array place the end of 0.1mol/L to have the polyoxyethylene glycol DMF solution of vitamin H, add a little triethylamine, placed 0.5 hour, clean, drying obtains the GaN nano wire of biotin modification.
It is avidin water or the PBS solution of 10-50nmol/mL that the GaN nano wire of the biotin modification that step 4 ' with step 3 ' obtains places concentration, places 0.5 hour, cleans, and drying obtains the GaN nano wire that avidin is modified.
9. according to the method for the GaN nano-wire array of the described arbitrary preparation protein modification of claim 5-8, described protein is any protein molecule.
10. the application of the GaN nano-wire array of the described protein modification of claim 1 in protein separating purifying, identification or detection.
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