CN103665117A - 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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- CN103665117A CN103665117A CN201310637210.7A CN201310637210A CN103665117A CN 103665117 A CN103665117 A CN 103665117A CN 201310637210 A CN201310637210 A CN 201310637210A CN 103665117 A CN103665117 A CN 103665117A
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- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 title claims abstract description 38
- 238000000034 method Methods 0.000 title claims abstract description 38
- 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 31
- 238000000703 high-speed centrifugation Methods 0.000 claims abstract description 6
- 239000002245 particle Substances 0.000 claims description 96
- 108010090804 Streptavidin Proteins 0.000 claims description 58
- 239000000243 solution Substances 0.000 claims description 35
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 claims description 28
- -1 polyoxyethylene Polymers 0.000 claims description 24
- 238000006243 chemical reaction Methods 0.000 claims description 20
- 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
- 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
- 230000002209 hydrophobic effect Effects 0.000 claims description 7
- CCMKPCBRNXKTKV-UHFFFAOYSA-N 1-hydroxy-5-sulfanylidenepyrrolidin-2-one Chemical compound ON1C(=O)CCC1=S CCMKPCBRNXKTKV-UHFFFAOYSA-N 0.000 claims description 6
- 230000004913 activation Effects 0.000 claims description 6
- RAABOESOVLLHRU-UHFFFAOYSA-N diazene Chemical compound N=N RAABOESOVLLHRU-UHFFFAOYSA-N 0.000 claims description 6
- 229910000071 diazene Inorganic materials 0.000 claims description 6
- 239000002953 phosphate buffered saline Substances 0.000 claims description 6
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 claims description 5
- 230000002378 acidificating effect Effects 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
- 235000011187 glycerol Nutrition 0.000 claims description 4
- 230000003993 interaction Effects 0.000 claims description 4
- 238000001556 precipitation Methods 0.000 claims description 4
- 238000007789 sealing Methods 0.000 claims description 4
- 230000004048 modification Effects 0.000 claims description 3
- 238000012986 modification Methods 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
- 239000002244 precipitate Substances 0.000 claims description 2
- 230000003287 optical effect Effects 0.000 abstract description 8
- 238000000926 separation method Methods 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 3
- 239000002202 Polyethylene glycol Substances 0.000 abstract description 2
- 229920001223 polyethylene glycol Polymers 0.000 abstract description 2
- 230000004071 biological effect Effects 0.000 abstract 1
- 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
- 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
- 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
- 238000010168 coupling process Methods 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 230000008901 benefit Effects 0.000 description 6
- 230000008878 coupling Effects 0.000 description 6
- 238000005859 coupling reaction Methods 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
- 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
- 239000002082 metal nanoparticle Substances 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
- 238000004519 manufacturing process Methods 0.000 description 3
- 229910052751 metal Inorganic materials 0.000 description 3
- 239000002184 metal Substances 0.000 description 3
- 239000002086 nanomaterial 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
- 230000008569 process Effects 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
- 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
- GOQYKNQRPGWPLP-UHFFFAOYSA-N n-heptadecyl alcohol Natural products CCCCCCCCCCCCCCCCCO GOQYKNQRPGWPLP-UHFFFAOYSA-N 0.000 description 2
- GLDOVTGHNKAZLK-UHFFFAOYSA-N n-octadecyl alcohol Natural products CCCCCCCCCCCCCCCCCCO GLDOVTGHNKAZLK-UHFFFAOYSA-N 0.000 description 2
- 229910000510 noble metal Inorganic materials 0.000 description 2
- 239000003921 oil Substances 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
- 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
- 238000013459 approach 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
- 239000000178 monomer 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
Images
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)
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- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
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 that have similar biological characteristics, can high degree of specificity ground in conjunction with a kind of water miscible VITAMIN: Bio, the avidity of itself and vitamin H is very strong, the Kd value between them approaches 10
-15m.The tetramer that complete Streptavidin is comprised of four identical peptide chains, total molecular weight is about 66.5kDa, and each Streptavidin molecule four biotin molecules of can combining closely, average every peptide chain (monomer) can be 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, make vitamin H-Streptavidin be widely used in immunology, the field such as molecular biology and histological chemistry, and obtained good effect.
The noble metal nano particles that the gold and silver nano particle of take is representative, has the character such as special optical, electrical, magnetic, has 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 have replaced traditional fluorescent marker, in fields such as cell imaging, DNA hybridization, protein interactions, are with a wide range of applications.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 100 times of left and right.The gold and silver nano particle of 20 nm of take is example, and the optical extinction coefficient of gold nano grain is 8.78 * 10
8m
-1cm
-1, and silver nano-grain has reached 2.87 * 10
10m
-1cm
-1.So strong scattered signal provides good signal to noise ratio for detecting, the sensitivity that effectively reduces detectability, improved Single Molecule Detection, for DNA/RNA, the trace such as medical inspection of live body Virus Sample and the detection of trace provide good label probe.
Metal nanoparticle and surface thereof show various and complicated optical characteristics.The rich shade that comprises noble metal nano particles is color, the surface plasmon resonance absorption of thin metal layer, and near the cancellation of the dyestuff that is stimulated metallic surface etc.Recent findings, the interaction of fluorescent marker and metal nanoparticle or precious metal surface is used to obtain near the experimental phenomenas such as directed radiation of fluorescent marker enhancing, the analysis based on the cancellation of gold nano colloid and the thin metal layer of fluorescent signal.Silver nano-grain is also showing very large advantage aspect metal-enhancing fluorescence.Except highlighting, metal-fluorescent mark objects system also shows the advantages such as minimizing of the shortening of fluorescence lifetime and the increase of stability and photobleaching.Distance between metal nano material and fluorescent marker has directly determined the efficiency that fluorescence strengthens, and it is generally acknowledged, 10 nm are distance optimum values that a fluorescence strengthens efficiency.But, some high molecular weight proteins as green fluorescent protein and immune system in, silver nano-grain also exists the characteristic of metal-enhancing fluorescence.In a word, as a kind of novel biomarker, nano-Ag particles is combined with after organic dye or fluorescence dye, and 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 carboxyl function is rolled into a ball surperficial water-soluble nano silver particle and is most widely used, and the amphiphilic polymers that adopts many carboxyls and hydrophobic chain is the water-soluble nano silver particle most popular method that oil-soluble nano silver particle is converted into carboxyl surface.
Because Streptavidin is similar to the affine effect of chemical coupling in conjunction with vitamin H, so the common carrier that 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, free Streptavidin in nano-Ag particles Streptavidin conjugate and solution is carried out to high efficiency separation, be the prerequisite that guarantees 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 silver particle nano particle diameter is little, specific surface area is large, a large amount of carboxyls is contained on nano material and Streptavidin conjugate surface, therefore adopt conventional centrifugal (lower than 30000 g centrifugal force), nano-Ag particles Streptavidin conjugate organic efficiency is (being less than 50%) on the low side generally, if nano-Ag particles Streptavidin conjugate organic efficiency will be 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 molecular weight difference (showing as the difference of optics and aquation particle diameter) that the method is utilized nano-Ag particles Streptavidin conjugate and Streptavidin, utilizes exclusion chromatography principle to carry out separation.The third conventional separation method is ultra-filtration and separation method.This method utilizes the ultra-filtration membrane of the super filter tube molecular weight difference that dams that Streptavidin is carried out to a kind of separated method with nano-Ag particles Streptavidin conjugate.Also has the method for purifying and separating based on sepharose and the gel electrophoresis of agarose-polyacrylamide heterozygosis etc. simultaneously.In a word, although utilize above purification process can obtain the nano-Ag particles Streptavidin conjugate of better quality, but still exist operational condition harsh, the defect such as flow process is complicated, and yield is low, is difficult to accomplish scale production.
Summary of the invention
Water-soluble nano silver particle 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, can be widely used in the numerous areas such as fluorescent microscopic imaging and immune quick diagnosis in cell marking, active somatic cell imaging, living animal body.But there is the defects such as operational condition is harsh, and flow process is complicated, and yield is low and be difficult to accomplish scale production in traditional nano-Ag particles Streptavidin conjugate purification method.
The object of this invention is to provide a kind of easy and simple to handle, separation efficiency is high, the novel method of purifying nano-Ag particles 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 activation that (1) modifies water-soluble carboxyl, add solution of streptavidin, adjust behind pH value of solution to 7.0～9.0 linked reaction; (2) after linked reaction finishes, in solution, add the carboxyl of nano-Ag particles remained on surface in single-ended amination polyoxyethylene glycol sealing coupled product, reacting solution pH value is adjusted to slightly acidic; (3) high speed centrifugation, abandons supernatant liquor, gets precipitation.
After step (3), will precipitate in addition with containing 25% glycerine, 0.05 mol/L pH 7.0～7.5 phosphate buffered saline buffer dissolving steps of 0.01% NaN3.
The nano-Ag particles that water-soluble nano silver particle used is nucleocapsid structure, shell is comprised of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyls surfaces, and internal layer is that long-chain hydrophobic grouping is wrapped in shell inside by the hydrophobic interaction with trioctylphosphine oxide by oil-soluble nano silver particle.
In described step (1), activation is for being dissolved in pH 5.0～6.0 by the nano-Ag particles of water-soluble carboxyl modification, in 0.05 mol/L borate buffer solution, add respectively 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide, 37 ℃ 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 finishes, in solution, adding single-ended amination polyoxyethylene glycol to single-ended amination polyoxyethylene glycol final concentration is 1～2%, fully mixes 15～30 minutes.
Step is adjusted to slightly acidic for pH value is adjusted to 4.5～5.0 by reacting solution pH value in (2), 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 buffers dissolve standby step.
Step (1) is adjusted pH value of solution to 7.5～8.5 with 0.1～1.0 M NaOH solution.
Principle is shown in Fig. 1.
The nano-Ag particles preparation method that described water-soluble carboxyl is modified is:
By concentration, be 98% vitriol oil (H
2sO
4) and concentration be 30% hydrogen peroxide (H
2o
2) with after the even mixing of volume ratio (1:3), put electric stove wire and be heated to boiling.Get a certain amount of quartz and silicon wafer and slowly join in above-mentioned boil-off liquid, after reaction 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), reaction 20 min.Above-mentioned reaction soln is joined to 10 mM Ti (SO
4)
2the aqueous solution (0.1 M H
2sO
4) in, reaction 5 min.Then response matrix is transferred in phosphoric acid buffer (pH 4.0), placed after several seconds and transfer to again in another phosphoric acid buffer (pH 4.0), place 5 min.Finally, with a large amount of distilled water rinsings and be placed in nitrogen (N
2) in dry, form silicon substrate.At 50 ℃, above-mentioned silicon substrate is immersed in to 10 mM AgNO
3solution, after reaction 24 h with distilled water rinsing 1 min and be placed in nitrogen (N
2) in dry, form the phospho-peptide substrate tablet of silver ions doping.Above-mentioned substrate tablet is immersed in to 10 mM NaBH of fresh preparation
45 min in solution, then with distilled water rinsing 1 min and be placed in nitrogen (N
2) in dry, obtain nano-Ag particles.Getting respectively 1 g polymaleic anhydride stearyl alcohol ester, 1.2 g 2-(2-amino ethoxy) ethanol and 1.26 g nano-Ag particles is dissolved in 5 mL 96% ethanolic solns, be placed in and at 70 ℃, react 1 h, heating volatilization ethanol, finally obtains water-soluble carboxyl nano-Ag particles.
The nano-Ag particles that the nano-Ag particles that water-soluble carboxyl is modified is nucleocapsid structure, shell is comprised of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyls surfaces, and internal layer is long-chain hydrophobic grouping, by the hydrophobic interaction with trioctylphosphine oxide, oil-soluble nano silver particle is wrapped in to shell inside.Nano-Ag particles is dissolved in to pH 6.0, in 0.05 mol/L borate buffer solution, adding mol ratio is the 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide of 100～200:1,37 ℃ are reacted 2 hours, and nano-Ag particles surface carboxyl is converted into active ester stable under acidic conditions.Add solution of streptavidin, wherein Streptavidin and nano-Ag particles mol ratio are 10:1, with 1 M NaOH solution, adjust pH value of solution to 8.0～9.0.Under pH 8.0～9.0 conditions, 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 coupled, thereby Streptavidin is coupled to nano-Ag particles surface.After nano-Ag particles and Streptavidin coupling, due to sterically hindered reason, still can the be residual a large amount of not coupled carboxyls in nano-Ag particles surface, therefore nano-Ag particles Streptavidin conjugate still keeps larger zeta-potential, therefore adopts common centrifugal method cannot nano-Ag particles Streptavidin conjugate and unmarked Streptavidin be carried out effectively separated.In order to reduce the zeta-potential on nano-Ag particles Streptavidin conjugate surface, the present invention is by amino and the free carboxy generation ion-exchange of nano-Ag particles surface of single-ended amination polyoxyethylene glycol, make the residual carboxyl of nano-Ag particles Streptavidin conjugate be converted to hydroxyl, improve nano-Ag particles Streptavidin conjugate iso-electric point, further by the single-ended amination polyoxyethylene glycol of high density (1%～2%), destroy the hydration layer on Streptavidin surface simultaneously, when pH is adjusted to 4.5, nano-Ag particles Streptavidin conjugate is under common high speed centrifugation (18, 000 g～20, 000 g) can be with in solution, the realization of coupling Streptavidin be effectively not separated, 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 can effectively reduce the zeta-potential of carboxylated nano-Ag particles, we take carboxylated water-soluble nano silver particle is raw material, the single-ended amination polyoxyethylene glycol of take is encapsulant, adopts EDC method sealing nano-Ag particles surface carboxyl.Then adopt 0.1M HCl or NaOH to adjust respectively to 2 of carboxylated nano-Ag particles (concentration is 0.1 μ M) pH, 3,4,5,6 and 7, add the 0.1M HCl of isodose or NaOH solution to the hydroxylation nano-Ag particles solution of same concentrations simultaneously.The nano-Ag particles of the different pH values of above two classes is analyzed through Ma Erwen surface potential particle instrument, the results are shown in Table 1.As known from Table 1, under identical pH value, the polyethyleneglycol modified hydroxylation nano-Ag particles surface potential of single-ended amination is significantly lower than hydroxylation nano-Ag particles, and when 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 |
Adopt technical solution of the present invention to there is following beneficial effect:
1, the carboxyl that the inventive method is not reacted by adding single-ended amination polyoxyethylene glycol sealing nano-Ag particles surface, make carboxyl 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, by acid adjustment, changes reacting solution pH value to 4.5, and water-soluble nano silver particle Streptavidin conjugate is easily separated out in solution.
3, the technology of the present invention is by adding single-ended amination polyoxyethylene glycol and acid adjustment, make water-soluble nano silver particle Streptavidin conjugate be more prone to separate out from reaction soln, adopt common centrifugal method 28,000 g～30,000 g just can carry out high efficiency separation (separation efficiency reaches more than 90%) by water-soluble nano silver particle Streptavidin conjugate and coupling Streptavidin not.Compare 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 etc.
Accompanying drawing explanation
Fig. 1 the inventive method principle schematic.
Embodiment
In order to make the present invention clearer, 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, is not intended to limit the present invention.
The collocation method of phosphate buffered saline buffer (pH 7.4 for PBS, 0.05 M): NaCl 40 g, Na
2hPO
413.5 g, KH
2pO
41.0 g, KCl 1.0 g are 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.
Embodiment 1 is synthetic take the water-soluble carboxyl nano-Ag particles that amphiphilic polymer is shell
By concentration, be 98% vitriol oil (H
2sO
4) and concentration be 30% hydrogen peroxide (H
2o
2) with after the even mixing of volume ratio (1:3), put electric stove wire and be heated to boiling.Get a certain amount of quartz and silicon wafer and slowly join in above-mentioned boil-off liquid, after reaction 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), reaction 20 min.Above-mentioned reaction soln is joined to 10 mM Ti (SO
4)
2the aqueous solution (0.1 M H
2sO
4) in, reaction 5 min.Then response matrix is transferred in phosphoric acid buffer (pH 4.0), placed after several seconds and transfer to again in another phosphoric acid buffer (pH 4.0), place 5 min.Finally, with a large amount of distilled water rinsings and be placed in nitrogen (N
2) in dry, form silicon substrate.At 50 ℃, above-mentioned silicon substrate is immersed in to 10 mM AgNO
3solution, after reaction 24 h with distilled water rinsing 1 min and be placed in nitrogen (N
2) in dry, form the phospho-peptide substrate tablet of silver ions doping.Above-mentioned substrate tablet is immersed in to 10 mM NaBH of fresh preparation
45 min in solution, then with distilled water rinsing 1 min and be placed in nitrogen (N
2) in dry, obtain nano-Ag particles.Getting respectively 1 g polymaleic anhydride stearyl alcohol ester, 1.2 g 2-(2-amino ethoxy) ethanol and 1.26 g nano-Ag particles is dissolved in 5 mL 96% ethanolic solns, be placed in and at 70 ℃, react 1 h, heating volatilization ethanol, finally obtains water-soluble carboxyl nano-Ag particles.
It is 10 ± 0.7 nm that water-soluble carboxyl nano-Ag particles (0.5 nmol/L) after synthetic is measured its particle diameter through transmission electron microscope (JEOL 2100F).
Embodiment 2 water-soluble nano silver particle Streptavidin conjugate and purifying process
Getting the carboxylated water-soluble nano silver particle of 5 mL (concentration is 50 nmol/L) mixes with the 0.05 mol/L borate buffer solution of equal-volume pH 5.5; The 1-ethyl that to add respectively with nano-Ag particles mol ratio be 150:1-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide, 37 ℃ of reactions 2 hours; The anti-solution of streptavidin that to add with nano-Ag particles mol ratio be 20:1, adjusts behind pH value of solution to 8.0～9.0 room temperature reaction 3 hours with 1 M NaOH solution; In the most backward solution, adding final concentration is 1.5% single-ended amination polyoxyethylene glycol, further with 1 M HCl solution, adjusts pH to 4.5.18,000 rpm(approximately 29,000 g) 4 ℃ 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 the water-soluble nano silver particle Streptavidin conjugate obtained containing free Streptavidin.Experimental result, the synthetic water-soluble nano silver particle Streptavidin conjugate of technical solution of the present invention is after 1.5% single-ended amination polyoxyethylene glycol is processed, and the rate of recovery of conjugate centrifugal purification is 93.7 ± 1.2%.
Claims (8)
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 activation that (1) modifies water-soluble carboxyl, add solution of streptavidin, adjust behind pH value of solution to 7.0～9.0 linked reaction; (2) after linked reaction finishes, in solution, add the carboxyl of nano-Ag particles remained on surface in single-ended amination polyoxyethylene glycol sealing coupled product, reacting solution pH value is adjusted to slightly acidic; (3) high speed centrifugation, abandons supernatant liquor, gets precipitation.
2. the method for claim 1, is characterized in that after step (3), will precipitate in addition with containing 25% glycerine, 0.01% NaN
30.05 mol/L pH 7.0～7.5 phosphate buffered saline buffer dissolving steps.
3. method according to claim 1, it is characterized in that water-soluble nano silver particle is the nano-Ag particles of nucleocapsid structure, shell is comprised of parents' polymkeric substance, outside is a large amount of hydrophilic carboxyls surfaces, and internal layer is that long-chain hydrophobic grouping is wrapped in shell inside by the hydrophobic interaction with trioctylphosphine oxide by oil-soluble nano silver particle.
4. method according to claim 1, it is characterized in that in described step (1) that activation is for being dissolved in pH 5.0～6.0 by the nano-Ag particles of water-soluble carboxyl modification, in 0.05 mol/L borate buffer solution, add respectively 1-ethyl-(3-dimethylaminopropyl) phosphinylidyne diimine and N-hydroxy thiosuccinimide, 37 ℃ 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, is preferably 150:1; The mol ratio of described Streptavidin and nano-Ag particles is 1～10:1.
6. the method for claim 1, after it is characterized in that linked reaction finishes, in solution, adding single-ended amination polyoxyethylene glycol to single-ended amination polyoxyethylene glycol final concentration is 1～2%, fully mixes 15～30 minutes.
7. 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 4.5～5.0, is preferably 4.5.
8. 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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| CN104313130A (en) * | 2014-09-23 | 2015-01-28 | 华南师范大学 | Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof |
| CN107942069A (en) * | 2017-11-15 | 2018-04-20 | 捷和泰（北京）生物科技有限公司 | A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof |
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|---|---|---|---|---|
| WO2001078786A2 (en) * | 2000-04-14 | 2001-10-25 | Alnis Biosciences, Inc. | High affinity peptide-containing nanoparticles |
| CN1431070A (en) * | 2003-01-21 | 2003-07-23 | 武汉大学 | Method for preparing water-soluble nano particles |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001078786A2 (en) * | 2000-04-14 | 2001-10-25 | Alnis Biosciences, Inc. | High affinity peptide-containing nanoparticles |
| CN1431070A (en) * | 2003-01-21 | 2003-07-23 | 武汉大学 | Method for preparing water-soluble nano particles |
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|---|---|---|---|---|
| CN104313130A (en) * | 2014-09-23 | 2015-01-28 | 华南师范大学 | Functionalized magnetic nanoparticles for efficiently enriching microorganisms, preparation and applications thereof |
| CN107942069A (en) * | 2017-11-15 | 2018-04-20 | 捷和泰（北京）生物科技有限公司 | A kind of NGAL latex immunoturbidimetries detection kit and preparation method thereof |
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