CN102011193B - Protein modified GaN nanowire array as well as preparation method and application thereof - Google Patents

Abstract

Translated fromChinese

一种蛋白质修饰的GaN纳米线阵列，它是以直立的GaN纳米线阵列作为基片，纳米线表面经化学氧化或等离子氧化产生Ga-OH官能团；或者经化学气相沉积法在纳米线表面沉积SiO₂、TiO₂或Al₂O₃，经水解在在纳米线表面产生-OH官能团，GaN纳米线表面的-OH官能团与SiCl₄反应形成-O-Si-Cl键，最后通过聚乙二醇分子与蛋白质分子相连接形成蛋白质修饰的GaN纳米线阵列。它可以应用于蛋白质分离提纯、识别或检测。本发明公开了其制法。A protein-modified GaN nanowire array, which uses an upright GaN nanowire array as a substrate, and the surface of the nanowire is chemically oxidized or plasma oxidized to generate Ga-OH functional groups; or SiO is deposited on the surface of the nanowire by chemical vapor deposition_2. TiO₂ or Al₂ O₃ generates -OH functional groups on the surface of nanowires through hydrolysis, and the -OH functional groups on the surface of GaN nanowires react with SiCl₄ to form -O-Si-Cl bonds, and finally through polyethylene glycol molecules Linked with protein molecules to form a protein-modified GaN nanowire array. It can be applied to protein separation and purification, identification or detection. The invention discloses its preparation method.

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 method for making and the purposes of protein modification.

Background technology

The same zinc oxide of gan (GaN) nano wire (ZnO), silicon nanowires (Si), the carbon nanotube (CNT), it 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 preparediameter 5～500nm, the nano wire of grade 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 process to produce activity-OH functional group, but this technology produce-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 chemical vapor 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 with organic molecule on active function groups the Ou Lian reaction occurs, thereby 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 greatly improves the selectivity of biomolecules.Simultaneously, with respect to basic biosensors such as the silicon of porous, 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 the 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 GaN nano wire of ALD deposition also can be used Infrared Characterization.

Some trace in the organism exists albumen that physiological function is played an important role, and purifies, identifies, 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, just can mutually identification occur with correspondingly biomolecules (such as antibody-antigen, acceptor-give body, enzyme-substrate etc.), then by some detection meanss, the information acquisition of these biomolecules out.Because the identification of biomolecules belongs to reversible, controlled, this array can repeatedly use.By the identification of biomolecules, 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 the high cost of biochemical separation to drop into.

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 occur in its interference fringe, gathers its interference data, calculates its optical thickness, can quantitatively obtain grafting in the grafting density of 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 such 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 GaN nano wire in the application of LED aspect.Chinese patent 200510048111 relates to the preparation of GaN nano wire.Chinese patent 200780016946 relates to 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 with it separation, identification or detection for protein molecule.

Technical scheme of the present invention is as follows:

A kind of GaN nano-wire array of protein modification, it be with upright GaN nano-wire array as substrate, nanowire surface produces Ga-OH functional group through chemical oxidation or plasma oxidation; Perhaps deposit SiO through atomic layer deposition method in nanowire surface₂, TiO₂Or Al₂O₃Film, its surface has-OH functional group through hydrolysis, the GaN nanowire surface-OH functional group and SiCl₄React formation-O-Si-Cl key, be connected to form at last the GaN nano-wire array of protein modification 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 upright GaN nano-wire array;

Step 2. has the surface thatstep 1 obtains-the GaN nano-wire array of OH functional group in soaking at room temperature at pure SiCl₄In the liquid 0.5-2 hour, make nanowire surface-the OH base is converted into-O-SiCl₃

Step 3. has the surface thatstep 2 obtains-O-SiCl₃The GaN nano-wire array place the end of 0.1mol/L with 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 the GaN nano wire of NHS activation;

The GaN nano wire that the NHS that step 4. obtainsstep 3 activates places water or the PBS (phosphate buffer solution of protein, 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 ofstep 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), 80 ℃ oflower heating 4～10 hours, take out and clean, dry up.

The method of the GaN nano-wire array of above-mentioned preparation protein modification, describedstep 1 can substitute with the following method:

Step 1 ' upright GaN nano-wire array is deposited SiO with atomic layer deposition method in nanowire surface₂, TiO₂Or Al₂O₃Film, its surface is through hydrolysis generation-OH functional group.

The method of the GaN nano-wire array of above-mentioned preparation protein modification, describedstep 3,4 can substitute with following two steps:

Step 3 ' surface thatstep 2 is obtained has-O-SiCl₃The GaN nano-wire array place the end of 0.1mol/L with 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 (such as Si-Cl) of GaN surface-OH reaction, an other end with contain-NH₂Crosslinked functional group's (such as NHS ester) occurs in functional group.Here first GaN array room temperature is placed pure SiCl₄Solution 0.5～2 hour, the GaN nanowire surface-OH is just and SiCl₄Reaction, product is-O-Si-Cl₃, unnecessary Si-Cl can with PEG one end-OH reaction, thereby with the PEG grafting on the GaN nano wire, (CONH-) be connected with vitamin H or avidin molecule, thereby they be fixed on the surface of GaN by amido linkage other a section of PEG.Here, adopt respectively two kinds to modify route according to the functional group of vitamin H and avidin is different.Then a kind of first fixed biologically element identifies avidin; Another kind is fixing avidin first, then identifies vitamin H.

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-NH₂On-NH₂Reaction, thereby synthetic PEG-Biotin:

The Si-Cl of GaN nanowire surface and the OH of the other end of PEG-Biotin are reacted, thus vitamin H is surperficial at GaN by the amido linkage grafting.At aqueous phase, the avidin molecule is easy to identify 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-NH₂On-NH₂With double amber imide (SC, bis (N-succinimidyl) carbonate) reaction, generate active PEG-NHS ester:

This NHS ester can with the avidin molecule on another-NH₂Crosslinking reaction occurs.Under the water, the GaN of grafting avidin can identify 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 (such 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 identified by corresponding antigen molecule (such 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 then first immobilized antigen is identified with their antibody, 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 protein 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 the affine separation of protein.The GaN nano-wire array that vitamin H is fixing places 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, slightly heat (50 ℃) half an hour, 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, the sample of PEG-NHS ester activation also can be first fixing avidin, thereby the purification vitamin H.In like manner, place respectively the PBS buffered soln of antibody (such as antibody, the mouse monoclonal antibody of brain natriuretic peptide (BNP)) to hatch one hour the GaN chip of NHS ester activation, cushion and washed with de-ionized water with PBS after taking out, drying, on these two kinds of antibody-NH₂With the NHS effect, thereby they are fixed on the GaN chip.Chip is placed the PBS buffered soln that contains corresponding antigen (such 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, the antigen BNP that wash-out is fixed and IgG molecule.Freezing, drying can obtain highly purified antigen BNP and IgG.Otherwise, first immobilized antigen, rear 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 sensor.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 λ₁λ₂)/2 (λ₁-λ₂), n is the mean refractive index of GaN, d is the thickness of film, λ₁And λ₂Be 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 (about 10nm of protein) of biomolecules self, interfering layer thickness changes, and its variable quantity comes from fixing biomolecules.Gather respectively the interference spectrum of grafting front and back, with data substitution formula, calculated respectively the variable quantity of optical thickness, weigh according to this grafting density of institute's grafting protein molecule, also namely obtain the concentration of target biological molecules.

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 fixing ratio, like this by fluorescence measurement, can qualitative, quantitatively obtain the biomolecules quantity that the GaN nano wire is fixed.

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 the GaN chip of NHS ester activation, at first fixedly avidin, brain natriuretic peptide (BNP) or mouse monoclonal antibody, then affine concentrated fixed biologically element, brain natriuretic peptide (BNP), goat mouse-anti immune protein (IGg).Place MALDI-ToF-MS to detect in the sample of drying, 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 non-target protein molecule in the non-specific adsorption of nanowire surface, can screen highly concentrated target biological molecules by antibody-AI, thereby obtain better detectability.

The GaN chip of NHS ester activation of the present invention can also detect unknown protein molecule.The GaN chip of NHS ester activation 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 the sequence of protein.Equally, also contain-NH owing to the unknown protein molecule surface₂, 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 orthographic plan; B is sectional view.

Fig. 4, PEG-Biotin synthesize infrared figure

Fig. 5, sulfuric acid/hydrogen peroxide are processed, and SiO after the vapour deposition₂, Al₂O₃, TiO₂The IR spectrum of the GaN nano wire that covers, a are that sulfuric acid/hydrogen peroxide is processed; The TiO of b for covering₂C is Al₂O₃Cover; D is SiO₂Cover.

The OH content balance figure of four samples among Fig. 6, Fig. 5.

Fig. 7, at SiO₂Cover 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 that separate on Figure 12, nano-wire array surface.

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 plane and the section SEM picture of GaN nano-wire array (USA, NIST).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 the position upright with the supporting pieces base.

The GaN nano-wire array placed 80 ℃ sulfuric acid: the solution (5mL) of hydrogen peroxide (3: 1), processed 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 CCl₂H₂In the solution, with pipettor with Solution Dispersion on the KBr of drying 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-wirearray 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 CCl₂H₂In the solution, with pipettor with Solution Dispersion on the KBr of drying 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 difference₂, Al₂O₃, SiO₂Deposition ALD film.Under the high vacuum, pass into first precursor Al (OR)₃With high purity water steam, form one deck Al in the GaN nanowire surface₂O₃Film, its surface has OH, then passes into respectively TiCl₄, SiCl₄With high purity water steam, form respectively TiO₂, SiO₂Film, its surface has OH.Like this, zone of oxidation just is grown in the surface of GaN nano wire, and the thickness of zone of oxidation is relevant with the cycle index that passes into 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 CCl₂H₂In the solution, with pipettor with Solution Dispersion on the KBr of drying 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, then drop on the TEM copper mesh, make high resolution TEM picture, see Fig. 8, a is that sulfuric acid/hydrogen peroxide is processed, and b, c, d are respectively SiO₂, Al₂O₃, TiO₂Cover 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 DMF (dimethyl formamide) solution that the 0.5g vitamin H is dissolved in the 50mL drying, put into 0.51g DCC (N, the N-dicyclohexylcarbodiimide) and 0.26g NHS, 50 ℃ oflower stirrings 16 hours, 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, 3228,3108,3063,2939,2915,2875,2846,1818,1787,1745,1729,1696cm occurred^-1Absorption peak.

Embodiment 3. synthetic PEG-Biotin

Get in the chloroformic solution of 0.225g biotin-NHS adding 50mL drying, add 1.0g PEG-NH₂(M=3000) (Sigma-Adrich buys) and a small amount 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, 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm occurred^-1Absorption peak.Show and synthesized PEG-Biotin.

Get in the chloroformic solution of 0.225g biotin-NHS adding 50mL drying, add 0.2g PEG-NH₂(M=600) (Sigma-Adrich buys) and a small amount of triethylamine stirred 1 hour, filtered insolubles, added excessive dry ether, made it to produce white precipitate.Medicine is done infrared spectra, 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm occurred^-1Absorption peak.Show and synthesized PEG-Biotin.

Get in the chloroformic solution of 0.225g biotin-NHS adding 50mL drying, add 0.33g PEG-NH₂(M=1000) (Sigma-Adrich buys) and a small amount of triethylamine stirred 2 hours, filtered insolubles, added excessive dry ether, made it to produce white precipitate.Medicine is done infrared spectra, 3340,2978,2945,2883,2857,2806,2739,1702,1698,1667cm occurred^-1Absorption peak.Show and synthesized PEG-Biotin.

The preparation of the GaN nano-wire array ofembodiment 4. biotin modifications

With SiO₂The GaN nano-wire array of functionalization (sample corresponding diagram 5d) places pure SiCl₄Placed 1 hour in the solution, sample washing is dry to be placed on the DMF solution of the PEG (M=3000) of 0.1Mol/L-Biotin (vitamin H), 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.PEG (2887,2860cm has appearred^-1) and Biotin (1665,1652cm^-1) charateristic avsorption band.Show that PEG-Biotin has been grafted the surface at the GaN nano wire.The GaN nano-wire array of the biotin modification that equally preparation PEGM=1000, and PEG M=600 is corresponding carries out Infrared spectroscopy, the charateristic avsorption band of PEG-Biotin all occurred.

The GaN nano-wire array ofembodiment 5. usefulness biotin modifications separates avidin

The sample of gained in above-describedembodiment 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 infrared middle acid amides absorption peak that avidin occurred.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.Use PEG M=1000 equally, and the GaN nano-wire array of biotin modification corresponding to PEG M=600, carrying out identical experiment, the acid amides absorption peak of avidin has all appearred.

The GaN nano-wire array ofembodiment 6. usefulness biotin modifications separates avidin

GaN nano-wire array and covering Al with sulfuric acid-hydrogen peroxide processing₂O₃And TiO₂The GaN nano-wire array carry out respectively the experiment ofembodiment 4 and 5, sample is done infrared analysis, PEG all occurred, PEG (2887,2860cm has appearred respectively in the charateristic avsorption band of vitamin H and avidin in their infrared spectra^-1), Biotin (1665,1652cm^-1), avidin (1543,1652cm^-1) charateristic avsorption band.

Embodiment 7. synthetic PEG-NHS

Get 3g PEG-NH₂(M=1500) place dry 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, 2939,2915,2875,2846,1818,1775,1735,1729cm occurred^-1Absorption peak.Show the NHS ester that generates PEG.

Get 2g PEG-NH₂(M=2000) place dry 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, at 1775,1735cm^-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 processing with sulfuric acid-hydrogen peroxide among theembodiment 1 is placed pure SiCl₄Placed 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 the dry PEG-NHS of washing 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 CCl₂H₂In the solution, with pipettor with Solution Dispersion on the KBr of drying crystal wafer, sample was placed 80 ℃ of baking boxs dry 4 hours, sample is done Infrared spectroscopy.2939,2915,2875,2846,1818,1775,1735,1729,534cm appears in the infrared spectra^-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 ofembodiment 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) processes 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 have been grafted the surface at nano wire.

The GaN nano-wire array ofembodiment 10. usefulness protein modifications separates corresponding protein

Under the room temperature, the monoclonal antibody of brain natriuretic peptide in above-described embodiment 9, the GaN nano-wire array of mouse monoclonal antibody modification are placed respectively the solution of brain natriuretic peptide (1nMol/mL), goat mouse-anti immune protein (5nMol/mL), 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 have been grafted the surface at nano wire.

Embodiment 11.

The GaN nano-wire array that is fixed with the biotin modification of avidin among above-describedembodiment 5 and theembodiment 6 is dyeed with uranyl acetate, take out, cleaning, dry, behind the peeling GaN nano wire, do tem analysis, the pattern (seeing Fig. 9) of avidin molecule has appearred in sample.Fig. 5 a-d (process, SiO by sulfuric acid/hydrogen peroxide₂, Al₂O₃, TiO₂Covering 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 above-describedembodiment 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 (process, SiO by sulfuric acid/hydrogen peroxide among above-mentioned Fig. 5 a-d₂, Al₂O₃, TiO₂Covering 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 above-describedembodiment 5 and 6 gained is made respectively laser interference spectrum, 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, namely after the grafting of avidin molecule, optical thickness increased value ((nd₂-nd₁)/nd₁* be 14.5% 100%).

Embodiment 14

The avidin that above-described embodiment 9 is obtained, peptide and bovine serum modify GaN nano-wire array sample do laser interference spectrum.Utilize formula, optical thickness increases respectively 22.5%, 11.3% and 14.2% after calculating avidin, peptide and the bovine serum grafting.

Embodiment 15

The bovine serum albumin that above-described embodiment 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 fixing 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 above-described embodiment 9 is obtained is modified²) infiltrate with 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 above-describedembodiment 12 is obtained places respectively the deionized water of 50 ℃ of 5mL, after hatching 1 hour, get respectively two kinds of solution of 10 μ L and drop on the silicon chip of 2 cleanings, dry gas dries up, do 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 that Figure 11 l is corresponding 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, first fixing avidin molecule, rear identification biotin molecule just can obtain highly purified vitamin H.In like manner, highly purified antibody (such as brain natriuretic peptide monoclonal antibody, mouse monoclonal antibody) and antigen (such as brain natriuretic peptide, goat mouse-anti immune protein) also can be used Methods For Purification of the same race.

Claims (10)

Translated fromChinese

1.一种蛋白质修饰的GaN纳米线阵列，其特征是：它是以直立的GaN纳米线阵列作为基片，纳米线表面经化学氧化或等离子氧化产生Ga-OH官能团；或者经化学气相沉积法在纳米线表面沉积SiO₂、TiO₂或Al₂O₃，经水解在表面产生-OH官能团，GaN纳米线表面的-OH官能团与SiCl₄反应形成-O-Si-Cl键，最后通过聚乙二醇分子与蛋白质分子相连接形成蛋白质修饰的GaN纳米线阵列。1. A protein-modified GaN nanowire array, characterized in that: it is based on an upright GaN nanowire array as a substrate, and the surface of the nanowire produces Ga-OH functional groups through chemical oxidation or plasma oxidation; or through chemical vapor deposition Deposit SiO₂ , TiO₂ or Al₂ O₃ on the surface of nanowires, and generate -OH functional groups on the surface through hydrolysis, and the -OH functional groups on the surface of GaN nanowires react with SiCl₄ to form -O-Si-Cl bonds, and finally through polyethylene Diol molecules are linked with protein molecules to form protein-modified GaN nanowire arrays.2.根据权利要求1所述的蛋白质修饰的GaN纳米线阵列，其特征是：所述的蛋白质分子是任何蛋白质分子。2. The protein-modified GaN nanowire array according to claim 1, characterized in that: said protein molecule is any protein molecule.3.根据权利要求1所述的蛋白质修饰的GaN纳米线阵列，其特征是：所述的蛋白质分子是亲和素、小鼠单克隆抗体或脑利尿钠肽的单克隆抗体。3. The protein-modified GaN nanowire array according to claim 1, characterized in that: the protein molecule is avidin, a mouse monoclonal antibody or a brain natriuretic peptide monoclonal antibody.4.根据权利要求1所述的蛋白质修饰的GaN纳米线阵列，其特征是：所述的聚乙二醇是数均分子量为600-3000的聚乙二醇。4. The protein-modified GaN nanowire array according to claim 1, characterized in that: said polyethylene glycol is polyethylene glycol with a number average molecular weight of 600-3000.5.一种制备权利要求1所述的蛋白质修饰的GaN纳米线阵列的方法，其特征是它包括下列步骤：5. A method for preparing the protein-modified GaN nanowire array of claim 1, characterized in that it comprises the following steps:步骤1.将直立的GaN纳米线阵列用化学氧化法氧化或等离子辐射氧化，使纳米线表面产生-OH官能团；Step 1. The upright GaN nanowire array is oxidized by chemical oxidation or plasma radiation to generate -OH functional groups on the surface of the nanowires;步骤2.将步骤1得到的表面具有-OH官能团的GaN纳米线阵列在室温下浸泡在纯SiCl₄液体中0.5-2小时，使纳米线表面的-OH基转化为-O-SiCl₃；Step 2. Soak the GaN nanowire array with -OH functional groups on the surface obtained in step 1 in pure SiCl₄ liquid for 0.5-2 hours at room temperature, so that the -OH groups on the surface of the nanowires are converted into -O-SiCl₃ ;步骤3.将步骤2得到的表面具有-O-SiCl₃的GaN纳米线阵列置于0.1mol/L的一端带有NHS(N-OH琥珀酰亚胺)的聚乙二醇的氯仿溶液中，在黑暗中放置0.5小时，取出GaN纳米线阵列，清洗，干燥，得到NHS活化的GaN纳米线；Step 3. the surface that step 2 obtains has -O-SiCl_The GaN nanowire array that one end of 0.1mol/L has NHS (N-OH succinimide) in the chloroform solution of the polyethylene glycol of NHS (N-OH succinimide), Place in the dark for 0.5 hours, take out the GaN nanowire array, wash and dry to obtain NHS-activated GaN nanowires;步骤4.将步骤3得到的NHS活化的GaN纳米线置于蛋白质的水或浓度为10-50nmol/L、pH＝7～10的磷酸缓冲溶液中放置0.5～1小时，取出GaN纳米线阵列，清洗，干燥，得到蛋白质修饰的GaN纳米线。Step 4. Place the NHS-activated GaN nanowires obtained in step 3 in water of protein or in a phosphate buffer solution with a concentration of 10-50 nmol/L and pH=7-10 for 0.5-1 hour, and take out the GaN nanowire array, Wash and dry to obtain protein-modified GaN nanowires.6.根据权利要求5所述的制备蛋白质修饰的GaN纳米线阵列的方法，其特征是：步骤1所述的化学氧化法氧化是将GaN纳米线阵列置于硫酸和双氧水混合溶液或硫酸与硝酸混合溶液中，在80℃下加热4～10小时，取出清洗，吹干。6. The method for preparing a protein-modified GaN nanowire array according to claim 5, characterized in that: the chemical oxidation method oxidation described in step 1 is to place the GaN nanowire array in a mixed solution of sulfuric acid and hydrogen peroxide or sulfuric acid and nitric acid In the mixed solution, heat at 80°C for 4 to 10 hours, take it out, wash it, and dry it.7.根据权利要求5所述的制备蛋白质修饰的GaN纳米线阵列的方法，其特征是所述的步骤1用如下方法替代：7. The method for preparing protein-modified GaN nanowire arrays according to claim 5, wherein the step 1 is replaced by the following method:步骤1’将直立的GaN纳米线阵列用化学气相沉积法在纳米线表面沉积SiO₂、TiO₂或Al₂O₃，经水解在外表面产生-OH官能团。Step 1' Deposit SiO₂ , TiO₂ or Al₂ O₃ on the surface of the upright GaN nanowire array by chemical vapor deposition, and generate -OH functional groups on the outer surface through hydrolysis.8.根据权利要求5所述的制备蛋白质修饰的GaN纳米线阵列的方法，其特征是所述的步骤3和4可以用如下两个步骤替代：8. The method for preparing protein-modified GaN nanowire arrays according to claim 5, characterized in that described steps 3 and 4 can be replaced by the following two steps:步骤3’将步骤2得到的表面具有-O-SiCl₃的GaN纳米线阵列置于0.1mol/L的一端带有生物素的聚乙二醇DMF溶液中，加入少许三乙胺，放置0.5小时，清洗，干燥，得到生物素修饰的GaN纳米线；Step 3' Place the GaN nanowire array with -O-_SiCl3 on the surface obtained in step 2 in a 0.1 mol/L polyethylene glycol DMF solution with biotin at one end, add a little triethylamine, and leave it for 0.5 hours , washed and dried to obtain biotin-modified GaN nanowires;步骤4’将步骤3’得到的生物素修饰的GaN纳米线置于浓度为10-50nmol/mL的亲和素水或PBS溶液中，放置0.5小时，清洗，干燥，得到亲和素修饰的GaN纳米线。Step 4' put the biotin-modified GaN nanowires obtained in step 3' in avidin water or PBS solution with a concentration of 10-50nmol/mL, leave it for 0.5 hours, wash, and dry to obtain avidin-modified GaN Nanowires.9.根据权利要求5-8所述的任一制备蛋白质修饰的GaN纳米线阵列的方法，所述的蛋白质是任何蛋白质分子。9. The method for preparing a protein-modified GaN nanowire array according to any one of claims 5-8, wherein the protein is any protein molecule.10.权利要求1所述的蛋白质修饰的GaN纳米线阵列在蛋白质分离提纯、识别或检测中的应用。10. The application of the protein-modified GaN nanowire array of claim 1 in protein separation and purification, identification or detection.

Images