CN103665117B - Method for high-efficiency purification of water soluble nano silver particle-streptavidin conjugate - Google Patents
Method for high-efficiency purification of water soluble nano silver particle-streptavidin conjugate Download PDFInfo
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- CN103665117B CN103665117B CN201310637210.7A CN201310637210A CN103665117B CN 103665117 B CN103665117 B CN 103665117B CN 201310637210 A CN201310637210 A CN 201310637210A CN 103665117 B CN103665117 B CN 103665117B
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- 238000000034 method Methods 0.000 title claims abstract description 40
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 39
- 238000000746 purification Methods 0.000 title abstract description 13
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 title abstract description 12
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims abstract description 28
- 238000000703 high-speed centrifugation Methods 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims description 87
- 108010090804 Streptavidin Proteins 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 38
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 28
- -1 polyoxyethylene Polymers 0.000 claims description 23
- 238000005576 amination reaction Methods 0.000 claims description 15
- 229920003171 Poly (ethylene oxide) Polymers 0.000 claims description 14
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 12
- 239000000872 buffer Substances 0.000 claims description 9
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 8
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 7
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims description 7
- 229910000071 diazene Inorganic materials 0.000 claims description 7
- 230000002378 acidificating effect Effects 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- 238000001556 precipitation Methods 0.000 claims description 6
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 5
- 230000004913 activation Effects 0.000 claims description 4
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 230000002209 hydrophobic effect Effects 0.000 claims description 4
- 230000003993 interaction Effects 0.000 claims description 4
- 238000005538 encapsulation Methods 0.000 claims description 3
- 125000001165 hydrophobic group Chemical group 0.000 claims description 3
- 239000000047 product Substances 0.000 claims description 3
- 239000000126 substance Substances 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- ZMBHCYHQLYEYDV-UHFFFAOYSA-N trioctylphosphine oxide Chemical compound CCCCCCCCP(=O)(CCCCCCCC)CCCCCCCC ZMBHCYHQLYEYDV-UHFFFAOYSA-N 0.000 claims description 3
- 230000003287 optical effect Effects 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 7
- 238000011084 recovery Methods 0.000 abstract description 3
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 2
- 230000004071 biological effect Effects 0.000 abstract description 2
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 2
- 238000011031 large-scale manufacturing process Methods 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 12
- 229910052709 silver Inorganic materials 0.000 description 9
- 239000004332 silver Substances 0.000 description 9
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 8
- XUIMIQQOPSSXEZ-UHFFFAOYSA-N Silicon Chemical compound [Si] XUIMIQQOPSSXEZ-UHFFFAOYSA-N 0.000 description 8
- 238000010168 coupling process Methods 0.000 description 8
- 239000012153 distilled water Substances 0.000 description 8
- 229910052710 silicon Inorganic materials 0.000 description 8
- 239000010703 silicon Substances 0.000 description 8
- 239000000758 substrate Substances 0.000 description 8
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 7
- 230000008878 coupling Effects 0.000 description 7
- 238000005859 coupling reaction Methods 0.000 description 7
- 230000008901 benefit Effects 0.000 description 6
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 6
- 229910052737 gold Inorganic materials 0.000 description 6
- 239000010931 gold Substances 0.000 description 6
- 230000033444 hydroxylation Effects 0.000 description 6
- 238000005805 hydroxylation reaction Methods 0.000 description 6
- 229910052757 nitrogen Inorganic materials 0.000 description 6
- 238000003384 imaging method Methods 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000002105 nanoparticle Substances 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 4
- 239000007864 aqueous solution Substances 0.000 description 4
- 230000008033 biological extinction Effects 0.000 description 4
- 229910052751 metal Inorganic materials 0.000 description 4
- 239000002184 metal Substances 0.000 description 4
- 239000002082 metal nanoparticle Substances 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000010453 quartz Substances 0.000 description 4
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N silicon dioxide Inorganic materials O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 4
- 229930003756 Vitamin B7 Natural products 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000001965 increasing effect Effects 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000002086 nanomaterial Substances 0.000 description 3
- 239000003921 oil Substances 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 3
- 239000011782 vitamin Substances 0.000 description 3
- 229930003231 vitamin Natural products 0.000 description 3
- 235000013343 vitamin Nutrition 0.000 description 3
- 229940088594 vitamin Drugs 0.000 description 3
- 239000011735 vitamin B7 Substances 0.000 description 3
- 235000011912 vitamin B7 Nutrition 0.000 description 3
- 150000003722 vitamin derivatives Chemical class 0.000 description 3
- PXFBZOLANLWPMH-UHFFFAOYSA-N 16-Epiaffinine Natural products C1C(C2=CC=CC=C2N2)=C2C(=O)CC2C(=CC)CN(C)C1C2CO PXFBZOLANLWPMH-UHFFFAOYSA-N 0.000 description 2
- GIAFURWZWWWBQT-UHFFFAOYSA-N 2-(2-aminoethoxy)ethanol Chemical compound NCCOCCO GIAFURWZWWWBQT-UHFFFAOYSA-N 0.000 description 2
- GFLJTEHFZZNCTR-UHFFFAOYSA-N 3-prop-2-enoyloxypropyl prop-2-enoate Chemical compound C=CC(=O)OCCCOC(=O)C=C GFLJTEHFZZNCTR-UHFFFAOYSA-N 0.000 description 2
- 101710134784 Agnoprotein Proteins 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 108010001441 Phosphopeptides Proteins 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- NJSSICCENMLTKO-HRCBOCMUSA-N [(1r,2s,4r,5r)-3-hydroxy-4-(4-methylphenyl)sulfonyloxy-6,8-dioxabicyclo[3.2.1]octan-2-yl] 4-methylbenzenesulfonate Chemical compound C1=CC(C)=CC=C1S(=O)(=O)O[C@H]1C(O)[C@@H](OS(=O)(=O)C=2C=CC(C)=CC=2)[C@@H]2OC[C@H]1O2 NJSSICCENMLTKO-HRCBOCMUSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 229960002685 biotin Drugs 0.000 description 2
- 235000020958 biotin Nutrition 0.000 description 2
- 239000011616 biotin Substances 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000000084 colloidal system Substances 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 230000036571 hydration Effects 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910000510 noble metal Inorganic materials 0.000 description 2
- 229920000371 poly(diallyldimethylammonium chloride) polymer Polymers 0.000 description 2
- 229920000141 poly(maleic anhydride) Polymers 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000004044 response Effects 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 210000001082 somatic cell Anatomy 0.000 description 2
- 125000004079 stearyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229910021642 ultra pure water Inorganic materials 0.000 description 2
- 238000000108 ultra-filtration Methods 0.000 description 2
- 239000012498 ultrapure water Substances 0.000 description 2
- 101710153593 Albumin A Proteins 0.000 description 1
- 108010043121 Green Fluorescent Proteins Proteins 0.000 description 1
- 102000004144 Green Fluorescent Proteins Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 101710120037 Toxin CcdB Proteins 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012412 chemical coupling Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000008393 encapsulating agent Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000032050 esterification Effects 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- 238000002270 exclusion chromatography Methods 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 238000005227 gel permeation chromatography Methods 0.000 description 1
- 239000005090 green fluorescent protein Substances 0.000 description 1
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 210000000987 immune system Anatomy 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 238000002558 medical inspection Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 239000002071 nanotube Substances 0.000 description 1
- 239000000101 novel biomarker Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 229920002401 polyacrylamide Polymers 0.000 description 1
- 239000010970 precious metal Substances 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 108020001775 protein parts Proteins 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 238000004557 single molecule detection Methods 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 1
- 238000002198 surface plasmon resonance spectroscopy Methods 0.000 description 1
- 238000005199 ultracentrifugation Methods 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
Abstract
The invention discloses a new method suitable for large-scale high-efficiency purification of a water soluble nano silver particle-streptavidin conjugate, and belongs to the field of biotechnology. In allusion to the disadvantages that a conventional nano silver particle-streptavidin conjugate has complex purification process and low recovery rate and difficultly realizes large-scale production, single-terminal aminated polyethylene glycol is adopted to seal residual carboxyl of the nano silver particle-streptavidin conjugate, a surface zeta potential of the conjugate is reduced; by adjusting the pH value of a solution to 4.5, the net charge content of the conjugate in the solution is further reduced, and the large-scale high-efficiency purification of the nano silver particle-streptavidin conjugate is achieved by an ordinary high-speed centrifugation method. The method simplifies experimental operation procedures of the nano silver particle-streptavidin conjugate, reduces requirements on separation equipment, is suitable for large-batch purification of the nano silver particle-streptavidin conjugate, allows the yield to be more than 90%, and allows optical characteristics and biological activity of the conjugate to have no obvious changes.
Description
Technical field
The invention belongs to biological technical field, relate to the novel method of the modification of nano material biology and conjugate purification.
Background technology
Streptavidin is a kind of and affine protein have similar biological properties, can high degree of specificity in conjunction with a kind of water miscible VITAMIN: Bio, the avidity of itself and vitamin H is very strong, and the Kd value between them is close to 10
-15m.The tetramer that complete Streptavidin is made up of four identical peptide chains, total molecular weight is about 66.5kDa, four biotin molecules and each Streptavidin molecule can be combined closely, average every bar peptide chain (monomer) can in conjunction with a biotin molecule.Complete tetramer Streptavidin also has Heat stability is good, to strong acid, sodium laurylsulfonate, urea, the feature such as the tolerance of Guanidinium hydrochloride is strong, these features make vitamin H-Streptavidin system have high-affinity, highly sensitive, high specificity, good stability, numerous advantages such as signal amplification, in addition vitamin H has and is highly susceptible to miscellaneous bioactive molecules (as albumen, nucleic acid and carbohydrate etc.) there is the advantage of coupling, vitamin H-Streptavidin is made to be widely used in immunology, the field such as molecular biology and histological chemistry, and obtained good effect.
With the noble metal nano particles that gold and silver nano particle is representative, there is the character such as special optical, electrical, magnetic, developed into a kind of very effective bioprobe.At present, gold nano grain is with its good biocompatibility and optical stability, very high optical extinction coefficient, the advantages such as scattered light intensity instead of traditional fluorescent marker, are with a wide range of applications in fields such as cell imaging, DNA hybridization, protein interactions.Silver nano-grain (Sliver nanoparticle, SNP) be a kind of metallic colloid, it is the same with gold nano grain has great optical extinction coefficient, scattered signal is strong, but by comparison, two kinds of nano particles of equal particle diameter, the optical extinction coefficient of silver nano-grain is higher, is generally its about 100 times.For the gold and silver nano particle of 20 nm, the optical extinction coefficient of gold nano grain is 8.78 × 10
8m
-1cm
-1, and silver nano-grain reaches 2.87 × 10
10m
-1cm
-1.So strong scattered signal is detect to provide good signal to noise ratio, effectively reduce detectability, improve the sensitivity of Single Molecule Detection, for the trace such as medical inspection of DNA/RNA, life virus sample and the detection of trace provide good label probe.
Metal nanoparticle and surface exhibits thereof go out the various and optical characteristics of complexity.The rich shade comprising noble metal nano particles is color, the surface plasmon resonance absorption of thin metal layer, and the cancellation etc. of the dyestuff that is stimulated near metal surface.Nearest discovery, the interaction of fluorescent marker and metal nanoparticle or precious metal surface is used to obtain the enhancing of fluorescent signal, the experimental phenomena such as directed radiation based on fluorescent marker near the analysis of gold nano colloid cancellation and thin metal layer.Silver nano-grain also shows very large advantage in metal-enhancing fluorescence.Except highlighting, metal-fluorescent mark objects system also shows the advantages such as the minimizing of the shortening of fluorescence lifetime and the increase of stability and photobleaching.Distance between metal nano material and fluorescent marker directly determines the efficiency of Fluorescence Increasing, it is generally acknowledged, 10 nm are distance optimum values of a Fluorescence Increasing efficiency.But at some high molecular weight proteins as in green fluorescent protein and immune system, silver nano-grain also also exists the characteristic of metal-enhancing fluorescence.In a word, as a kind of novel biomarker, after nano-Ag particles is combined with organic dye or fluorescence dye, in cell marking, active somatic cell imaging, living animal body, the field such as fluorescent microscopic imaging and fluorescently-labeled immunology quick diagnosis technology has more significant advantage.Wherein the water-soluble nano Argent grain on carboxyl function group surface is most widely used, and adopts the amphiphilic polymers of many carboxyls and hydrophobic chain to be the water-soluble nano Argent grain most popular method that oil solubility nanometer Argent grain is converted into carboxyl surface.
Because Streptavidin to be similar to the affine effect of chemical coupling in conjunction with vitamin H, the common carrier that therefore nano-Ag particles Streptavidin coupled product is considered to prepare different kind organism fluorescent probe is widely used.Carboxylated nano-Ag particles Streptavidin coupling common method is the active ester method of EDC/NHSS mediation.In nano-Ag particles and Streptavidin coupling process, Streptavidin free in nano-Ag particles Streptavidin conjugate and solution being carried out high efficiency separation, is the prerequisite ensureing nano-Ag particles Streptavidin conjugate service efficiency.At present, nano-Ag particles Streptavidin conjugate purification method mainly comprises following several.The first, Ultracentrifugation Method.Because water-soluble nano Argent grain nano particle diameter is little, specific surface area is large, nano material and Streptavidin conjugate surface are containing a large amount of carboxyls, therefore conventional centrifugal (lower than 30000 g centrifugal force) is adopted, nano-Ag particles Streptavidin conjugate organic efficiency generally (being less than 50%) on the low side, to nano-Ag particles Streptavidin conjugate organic efficiency is increased to more than 90%, centrifugal speed generally need be greater than 50,000 g.The common purification process of the second is gel chromatography.The method utilizes the molecular weight difference of nano-Ag particles Streptavidin conjugate and Streptavidin (showing as the difference of optics and aquation particle diameter), utilizes exclusion chromatography principle to be separated.The third conventional separation method is ultra-filtration and separation method.This method utilize the ultra-filtration membrane of super filter tube to dam a kind of method that Streptavidin and nano-Ag particles Streptavidin conjugate carry out being separated by molecular weight difference.Also has the method for purifying and separating based on sepharose and agarose-polyacrylamide hybrid gel electrophoresis etc. simultaneously.In a word, although utilize above purification process can obtain the nano-Ag particles Streptavidin conjugate of better quality, but still there is operational condition harshness, flow process is complicated, and the defects such as yield is low, are difficult to accomplish scale production.
Summary of the invention
Water-soluble nano Argent grain is a kind of good nano fluorescent marker material, this material by with antibody, Streptavidin, albumin A, Protein G and part or acceptor molecule coupling, the numerous areas such as fluorescent microscopic imaging and immune quick diagnosis in cell marking, active somatic cell imaging, living animal body can be widely used in.But traditional nano-Ag particles Streptavidin conjugate purification method exists operational condition harshness, flow process is complicated, and yield is low and be difficult to defects such as accomplishing scale production.
The object of this invention is to provide a kind of easy and simple to handle, separation efficiency is high, the novel method of purified nanotubes Argent grain Streptavidin conjugate in enormous quantities, specifically comprise the following steps:
A kind of novel method of efficiently purifying water-soluble nano silver particle Streptavidin conjugate, it is characterized in that comprising the following steps: the nano-Ag particles of water-soluble carboxyl modified activates by (1), add solution of streptavidin, behind adjustment pH value of solution to 7.0 ~ 9.0, linked reaction; (2), after linked reaction terminates, add the carboxyl that nano-Ag particles remained on surface in coupled product closed by single-ended amination polyoxyethylene glycol in solution, reacting solution pH value is adjusted to slightly acidic; (3) high speed centrifugation, abandons supernatant liquor, gets precipitation.
After step (3), also have precipitation with containing 25% glycerine, 0.05 mol/L pH 7.0 ~ 7.5 phosphate buffered saline buffer dissolving step of 0.01% NaN3.
Water-soluble nano Argent grain used is the nano-Ag particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, and internal layer is that long chain hydrophobic group is by inner at shell by oil-soluble nano silver particle encapsulation with the hydrophobic interaction of trioctylphosphine oxide.
In described step (1), activation is for be dissolved in pH 5.0 ~ 6.0 by the nano-Ag particles of water-soluble carboxyl modified, in 0.05 mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide respectively, 37 DEG C are reacted 2 hours, activation nano-Ag particles carboxyl.
The mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and nano-Ag particles is 100 ~ 200:1, is preferably 150:1; The mol ratio of described Streptavidin and nano-Ag particles is 1 ~ 10:1.
After linked reaction terminates, adding single-ended amination polyoxyethylene glycol to single-ended amination polyoxyethylene glycol final concentration in solution is 1 ~ 2%, fully mixes 15 ~ 30 minutes.
Reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 4.5 ~ 5.0 in (2) by step, is preferably 4.5.
The described high speed centrifugation centrifugal force of step (3) is 28,000 ~ 30,000g.
Also comprise precipitation with containing 25% glycerine, 0.01% NaN
30.05 mol/L pH 7.0 ~ 7.5 phosphate buffered saline buffer dissolve step for subsequent use.
Step (1) 0.1 ~ 1.0 M NaOH solution adjustment pH value of solution to 7.5 ~ 8.5.
Principle is shown in Fig. 1.
The nano-Ag particles preparation method of described water-soluble carboxyl modified is:
Be 98% vitriol oil (H by concentration
2sO
4) and concentration be 30% hydrogen peroxide (H
2o
2) with after volume ratio (1:3) Homogeneous phase mixing, put electric stove wire and be heated to boiling.Get a certain amount of quartz and silicon wafer slowly joins in above-mentioned boil-off liquid, after reacting 20 min, with a large amount of distilled water rinsings.Quartz after rinsing and silicon wafer are immersed in the diallyl dimethyl ammoniumchloride aqueous solution (PDDA, 1.0 mg/mL), react 20 min.Above-mentioned reaction soln is joined 10 mM Ti (SO
4)
2the aqueous solution (0.1 M H
2sO
4) in, react 5 min.Then response matrix is transferred in phosphoric acid buffer (pH 4.0), place after several seconds and transfer to again in another phosphoric acid buffer (pH 4.0), place 5 min.Finally, nitrogen (N is placed in a large amount of distilled water rinsings
2) middle dry, form silicon substrate.At 50 DEG C, above-mentioned silicon substrate is immersed in 10 mM AgNO
3solution, is placed in nitrogen (N with distilled water rinsing 1 min after reacting 24 h
2) middle dry, form the phospho-peptide substrate tablet of silver ions doping.Above-mentioned substrate tablet is immersed in 10 mM NaBH of fresh preparation
45 min in solution, are then placed in nitrogen (N with distilled water rinsing 1 min
2) middle dry, namely obtain nano-Ag particles.Getting 1 g polymaleic anhydride octadecyl, 1.2 g 2-(2-amino ethoxy) ethanol and 1.26 g nano-Ag particles is respectively dissolved in 5 mL 96% ethanolic solns, 1 h is reacted at being placed in 70 DEG C, heating volatilization ethanol, finally obtains water-soluble carboxylated nano-Ag particles.
The nano-Ag particles of water-soluble carboxyl modified is the nano-Ag particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, and internal layer is long chain hydrophobic group, by oil-soluble nano silver particle encapsulation is inner at shell with the hydrophobic interaction of trioctylphosphine oxide.Nano-Ag particles is dissolved in pH 6.0, in 0.05 mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide that mol ratio is 100 ~ 200:1,37 DEG C are reacted 2 hours, nano-Ag particles surface carboxyl groups are converted into the active ester of stable under acidic conditions.Add solution of streptavidin, wherein Streptavidin and nano-Ag particles mol ratio are 10:1, with 1 M NaOH solution adjustment pH value of solution to 8.0 ~ 9.0.Under pH 8.0 ~ 9.0 condition, amino in Streptavidin amino-acid residue is easy to protonated, and the carboxyl of the active esterification in nano-Ag particles surface issues unboiled water solution at weak basic condition, form stable amido linkage with protonated amino coupling, thus Streptavidin is coupled to nano-Ag particles surface.After nano-Ag particles and Streptavidin coupling, due to sterically hindered reason, nano-Ag particles surface still can remain a large amount of not coupled carboxyl, therefore nano-Ag particles Streptavidin conjugate still keeps larger zeta-potential, therefore adopts common centrifugal method nano-Ag particles Streptavidin conjugate effectively cannot be separated with unmarked Streptavidin.In order to reduce the zeta-potential on nano-Ag particles Streptavidin conjugate surface, the present invention is by the amino of single-ended amination polyoxyethylene glycol and the free carboxy generation ion-exchange of nano-Ag particles surface, make nano-Ag particles Streptavidin conjugate remain carboxyl and be converted to hydroxyl, improve nano-Ag particles Streptavidin conjugate iso-electric point, destroy the hydration layer on Streptavidin surface further by the single-ended amination polyoxyethylene glycol (1% ~ 2%) of high density simultaneously, when solution ph is adjusted to 4.5, nano-Ag particles Streptavidin conjugate is under common high speed centrifugation (18, 000 g ~ 20, 000 g) can realize effectively being separated with coupling Streptavidin non-in solution, wherein the rate of recovery of nano-Ag particles and Streptavidin conjugate is greater than 90%.
In order to verify whether single-ended amination polyoxyethylene glycol effectively can reduce the zeta-potential of carboxylated nano-Ag particles, we with carboxylated water-soluble nano Argent grain for raw material, with single-ended amination polyoxyethylene glycol for encapsulant, EDC method is adopted to close nano-Ag particles surface carboxyl groups.Then adopt 0.1M HCl or NaOH to adjust carboxylated nano-Ag particles (concentration is 0.1 μM) pH respectively to 2,3,4,5,6 and 7, add the 0.1M HCl of isodose or NaOH solution in the hydroxylation nano-Ag particles solution of same concentrations simultaneously.The nano-Ag particles of the different pH value of above two classes is analyzed through Malvern surface potential particle instrument, the results are shown in Table 1.As known from Table 1, under same pH, the polyethyleneglycol modified hydroxylation nano-Ag particles surface potential of single-ended amination is significantly lower than hydroxylation nano-Ag particles, and when solution ph is down between 4 ~ 5, hydroxylation nano-Ag particles zeta-potential declines obviously.
Under table 1 condition of different pH, the surface potential of carboxylated and hydroxylation nano-Ag particles
| pH 2 | pH 3 | pH 4 | pH 5 | pH 6 | pH 7 | |
| Carboxylated nano-Ag particles | -6.23 | -15.21 | -14.19 | -34.5 | -49.11 | -61.19 |
| Hydroxylation nano-Ag particles | 11.201 | -7.15 | -4.63 | -31.66 | -34.34 | -53.28 |
Technical solution of the present invention is adopted to have following beneficial effect:
1, the inventive method closes nano-Ag particles surface not by the carboxyl reacted by adding single-ended amination polyoxyethylene glycol, carboxyl is made to change carboxyl into, reduce the zeta-potential on water-soluble nano silver particle Streptavidin conjugate surface, destroy the hydration layer on Streptavidin surface simultaneously, be conducive to water-soluble nano silver particle Streptavidin conjugate and separate out from reaction soln.
2, the inventive method is by acid adjustment, changes reacting solution pH value to 4.5, water-soluble nano silver particle Streptavidin conjugate is easily separated out in the solution.
3, the technology of the present invention is by adding single-ended amination polyoxyethylene glycol and acid adjustment, water-soluble nano silver particle Streptavidin conjugate is made to be more prone to separate out from reaction soln, adopt common centrifugal method 28,000 g ~ 30, water-soluble nano silver particle Streptavidin conjugate and non-coupling Streptavidin just can be carried out high efficiency separation (separation efficiency reaches more than 90%) by 000 g.Compared with traditional water-soluble nano silver particle Streptavidin conjugate purification method, have simple to operate, equipment requirements low (common laboratory all can reach), purification efficiency high (more than 90%) and can accomplishing scale production.
Accompanying drawing explanation
Fig. 1 the inventive method principle schematic.
Embodiment
In order to make the present invention clearly understand, below in conjunction with embodiment, the present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to explain the present invention, be not intended to limit the present invention.
The collocation method of phosphate buffered saline buffer (PBS, 0.05 M, pH 7.4): NaCl 40 g, Na
2hPO
413.5 g, KH
2pO
41.0 g, KCl 1.0 g be dissolved in 1 L ultrapure water.With 0.1 M NaOH adjust pH to 8.0 ~ 9.0.
The compound method of borate buffer solution (0.05 M, pH 6.0): get 1.0 g boric acid and be dissolved in 1 L ultrapure water.Adjust pH to 6.0.
It is the water-soluble carboxyl nano-Ag particles of shell that embodiment 1 is synthesized with amphiphilic polymer
Be 98% vitriol oil (H by concentration
2sO
4) and concentration be 30% hydrogen peroxide (H
2o
2) with after volume ratio (1:3) Homogeneous phase mixing, put electric stove wire and be heated to boiling.Get a certain amount of quartz and silicon wafer slowly joins in above-mentioned boil-off liquid, after reacting 20 min, with a large amount of distilled water rinsings.Quartz after rinsing and silicon wafer are immersed in the diallyl dimethyl ammoniumchloride aqueous solution (PDDA, 1.0 mg/mL), react 20 min.Above-mentioned reaction soln is joined 10 mM Ti (SO
4)
2the aqueous solution (0.1 M H
2sO
4) in, react 5 min.Then response matrix is transferred in phosphoric acid buffer (pH 4.0), place after several seconds and transfer to again in another phosphoric acid buffer (pH 4.0), place 5 min.Finally, nitrogen (N is placed in a large amount of distilled water rinsings
2) middle dry, form silicon substrate.At 50 DEG C, above-mentioned silicon substrate is immersed in 10 mM AgNO
3solution, is placed in nitrogen (N with distilled water rinsing 1 min after reacting 24 h
2) middle dry, form the phospho-peptide substrate tablet of silver ions doping.Above-mentioned substrate tablet is immersed in 10 mM NaBH of fresh preparation
45 min in solution, are then placed in nitrogen (N with distilled water rinsing 1 min
2) middle dry, namely obtain nano-Ag particles.Getting 1 g polymaleic anhydride octadecyl, 1.2 g 2-(2-amino ethoxy) ethanol and 1.26 g nano-Ag particles is respectively dissolved in 5 mL 96% ethanolic solns, 1 h is reacted at being placed in 70 DEG C, heating volatilization ethanol, finally obtains water-soluble carboxylated nano-Ag particles.
It is 10 ± 0.7 nm that water-soluble carboxylated nano-Ag particles (0.5 nmol/L) after synthesis measures its particle diameter through transmission electron microscope (JEOL 2100F).
Embodiment 2 water-soluble nano silver particle Streptavidin conjugate and purifying process
Get the carboxylated water-soluble nano Argent grain of 5 mL (concentration is 50 nmol/L) to mix with the 0.05 mol/L borate buffer solution of equal-volume pH 5.5; Adding respectively with nano-Ag particles mol ratio is 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and the N-hydroxy thiosuccinimide of 150:1,37 DEG C of reactions 2 hours; Adding with nano-Ag particles mol ratio is the anti-solution of streptavidin of 20:1, behind 1 M NaOH solution adjustment pH value of solution to 8.0 ~ 9.0, and room temperature reaction 3 hours; Adding final concentration in the most backward solution is 1.5% single-ended amination polyoxyethylene glycol, adjusts pH to 4.5 further with 1 M HCl solution.18,000 rpm(about 29,000 g) 4 DEG C of centrifugal 30 min, abandon supernatant, and precipitation is with containing 25% glycerine, 0.01% NaN
30.05 mol/L phosphate buffered saline buffer (pH 7.0 ~ 7.5) dissolve and namely obtain not containing the water-soluble nano silver particle Streptavidin conjugate of free Streptavidin.Experimental result, the water-soluble nano silver particle Streptavidin conjugate of technical solution of the present invention synthesis is after 1.5% single-ended amination polyoxyethylene glycol process, and the rate of recovery of conjugate centrifugal purification is 93.7 ± 1.2%.
Claims (9)
1. the method for an efficiently purifying water-soluble nano silver particle Streptavidin conjugate, it is characterized in that comprising the following steps: the nano-Ag particles of water-soluble carboxyl modified activates by (1), add solution of streptavidin, behind adjustment pH value of solution to 7.0 ~ 9.0, linked reaction; (2), after linked reaction terminates, add the carboxyl that nano-Ag particles remained on surface in coupled product closed by single-ended amination polyoxyethylene glycol in solution, reacting solution pH value is adjusted to slightly acidic; (3) high speed centrifugation, abandons supernatant liquor, gets precipitation;
Reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 4.5 ~ 5.0 in (2) by step.
2. the method for claim 1, after it is characterized in that step (3), also has precipitation with containing 25% glycerine, 0.01% NaN
30.05 mol/L pH 7.0 ~ 7.5 phosphate buffered saline buffer dissolving step.
3. method according to claim 1, it is characterized in that water-soluble nano Argent grain is the nano-Ag particles of nucleocapsid structure, shell is made up of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyl surfaces, and internal layer is that long chain hydrophobic group is by inner at shell by oil-soluble nano silver particle encapsulation with the hydrophobic interaction of trioctylphosphine oxide.
4. method according to claim 1, it is characterized in that in described step (1), activation is for be dissolved in pH 5.0 ~ 6.0 by the nano-Ag particles of water-soluble carboxyl modified, in 0.05 mol/L borate buffer solution, add 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide respectively, 37 DEG C are reacted 2 hours, activation nano-Ag particles carboxyl.
5. method according to claim 4, is characterized in that the mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and nano-Ag particles is 100 ~ 200:1; The mol ratio of described Streptavidin and nano-Ag particles is 1 ~ 10:1.
6. method according to claim 5, is characterized in that the mol ratio of described 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide and nano-Ag particles is 150:1.
7. the method for claim 1, after it is characterized in that linked reaction terminates, adding single-ended amination polyoxyethylene glycol to single-ended amination polyoxyethylene glycol final concentration in solution is 1 ~ 2%, fully mixes 15 ~ 30 minutes.
8. method according to claim 1, is characterized in that, in step (2), reacting solution pH value is adjusted to slightly acidic for pH value is adjusted to 4.5.
9. method according to claim 1, is characterized in that the described high speed centrifugation centrifugal force of step (3) is 28,000 ~ 30,000g.
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