WO1990011128A1 - Polymer-coated noble metal particles and a method for their preparation - Google Patents
Polymer-coated noble metal particles and a method for their preparation Download PDFInfo
- Publication number
- WO1990011128A1 WO1990011128A1 PCT/DK1990/000080 DK9000080W WO9011128A1 WO 1990011128 A1 WO1990011128 A1 WO 1990011128A1 DK 9000080 W DK9000080 W DK 9000080W WO 9011128 A1 WO9011128 A1 WO 9011128A1
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- WIPO (PCT)
- Prior art keywords
- noble metal
- layer
- metal particles
- dispersion
- organic polymer
- Prior art date
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- 229910000510 noble metal Inorganic materials 0.000 title claims abstract description 91
- 239000002923 metal particle Substances 0.000 title claims abstract description 82
- 238000000034 method Methods 0.000 title claims abstract description 49
- 238000002360 preparation method Methods 0.000 title claims abstract description 29
- 229920000642 polymer Polymers 0.000 title abstract description 7
- 239000002245 particle Substances 0.000 claims abstract description 104
- 239000006185 dispersion Substances 0.000 claims abstract description 99
- 239000000126 substance Substances 0.000 claims abstract description 76
- 229920000620 organic polymer Polymers 0.000 claims abstract description 65
- 101710120037 Toxin CcdB Proteins 0.000 claims abstract description 18
- 238000005538 encapsulation Methods 0.000 claims abstract description 17
- 238000001493 electron microscopy Methods 0.000 claims abstract description 9
- 239000000427 antigen Substances 0.000 claims abstract description 7
- 108091007433 antigens Proteins 0.000 claims abstract description 7
- 102000036639 antigens Human genes 0.000 claims abstract description 7
- 239000000243 solution Substances 0.000 claims description 96
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims description 60
- 229910052737 gold Inorganic materials 0.000 claims description 57
- 239000010931 gold Substances 0.000 claims description 57
- 238000011282 treatment Methods 0.000 claims description 31
- 108010010803 Gelatin Proteins 0.000 claims description 26
- 229920000159 gelatin Polymers 0.000 claims description 26
- 235000019322 gelatine Nutrition 0.000 claims description 26
- 235000011852 gelatine desserts Nutrition 0.000 claims description 26
- 239000008273 gelatin Substances 0.000 claims description 22
- 239000004971 Cross linker Substances 0.000 claims description 14
- 230000001588 bifunctional effect Effects 0.000 claims description 14
- 239000000834 fixative Substances 0.000 claims description 14
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 11
- 239000007864 aqueous solution Substances 0.000 claims description 11
- 230000015572 biosynthetic process Effects 0.000 claims description 11
- 229960000587 glutaral Drugs 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 claims description 8
- 108010046516 Wheat Germ Agglutinins Proteins 0.000 claims description 8
- 108090000623 proteins and genes Proteins 0.000 claims description 8
- 102000004169 proteins and genes Human genes 0.000 claims description 8
- 238000010186 staining Methods 0.000 claims description 7
- KDLHZDBZIXYQEI-UHFFFAOYSA-N Palladium Chemical compound [Pd] KDLHZDBZIXYQEI-UHFFFAOYSA-N 0.000 claims description 6
- AFOSIXZFDONLBT-UHFFFAOYSA-N divinyl sulfone Chemical compound C=CS(=O)(=O)C=C AFOSIXZFDONLBT-UHFFFAOYSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 6
- 239000012620 biological material Substances 0.000 claims description 5
- 238000001514 detection method Methods 0.000 claims description 5
- 108010088751 Albumins Proteins 0.000 claims description 4
- 102000009027 Albumins Human genes 0.000 claims description 4
- 108090001090 Lectins Proteins 0.000 claims description 4
- 102000004856 Lectins Human genes 0.000 claims description 4
- PCSMJKASWLYICJ-UHFFFAOYSA-N Succinic aldehyde Chemical compound O=CCCC=O PCSMJKASWLYICJ-UHFFFAOYSA-N 0.000 claims description 4
- 125000001931 aliphatic group Chemical group 0.000 claims description 4
- ZWIBGKZDAWNIFC-UHFFFAOYSA-N disuccinimidyl suberate Chemical compound O=C1CCC(=O)N1OC(=O)CCCCCCC(=O)ON1C(=O)CCC1=O ZWIBGKZDAWNIFC-UHFFFAOYSA-N 0.000 claims description 4
- 150000004676 glycans Chemical class 0.000 claims description 4
- 239000002523 lectin Substances 0.000 claims description 4
- 230000004807 localization Effects 0.000 claims description 4
- 239000005017 polysaccharide Substances 0.000 claims description 4
- 238000000746 purification Methods 0.000 claims description 4
- -1 avidi Proteins 0.000 claims description 3
- 229920001817 Agar Polymers 0.000 claims description 3
- 108010062580 Concanavalin A Proteins 0.000 claims description 3
- 108090000288 Glycoproteins Proteins 0.000 claims description 3
- 102000003886 Glycoproteins Human genes 0.000 claims description 3
- 229920000881 Modified starch Polymers 0.000 claims description 3
- 108010033737 Pokeweed Mitogens Proteins 0.000 claims description 3
- KJTLSVCANCCWHF-UHFFFAOYSA-N Ruthenium Chemical compound [Ru] KJTLSVCANCCWHF-UHFFFAOYSA-N 0.000 claims description 3
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 claims description 3
- 229920002472 Starch Polymers 0.000 claims description 3
- 239000008272 agar Substances 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 3
- 229910052741 iridium Inorganic materials 0.000 claims description 3
- GKOZUEZYRPOHIO-UHFFFAOYSA-N iridium atom Chemical compound [Ir] GKOZUEZYRPOHIO-UHFFFAOYSA-N 0.000 claims description 3
- 238000002372 labelling Methods 0.000 claims description 3
- 238000000386 microscopy Methods 0.000 claims description 3
- 235000019426 modified starch Nutrition 0.000 claims description 3
- 239000011368 organic material Substances 0.000 claims description 3
- 229910052762 osmium Inorganic materials 0.000 claims description 3
- SYQBFIAQOQZEGI-UHFFFAOYSA-N osmium atom Chemical compound [Os] SYQBFIAQOQZEGI-UHFFFAOYSA-N 0.000 claims description 3
- 229910052763 palladium Inorganic materials 0.000 claims description 3
- 229910052697 platinum Inorganic materials 0.000 claims description 3
- 229920001184 polypeptide Polymers 0.000 claims description 3
- 229920001282 polysaccharide Polymers 0.000 claims description 3
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 3
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 3
- 229910052703 rhodium Inorganic materials 0.000 claims description 3
- 239000010948 rhodium Substances 0.000 claims description 3
- MHOVAHRLVXNVSD-UHFFFAOYSA-N rhodium atom Chemical compound [Rh] MHOVAHRLVXNVSD-UHFFFAOYSA-N 0.000 claims description 3
- 229910052707 ruthenium Inorganic materials 0.000 claims description 3
- 210000002966 serum Anatomy 0.000 claims description 3
- 229910052709 silver Inorganic materials 0.000 claims description 3
- 239000004332 silver Substances 0.000 claims description 3
- 235000019698 starch Nutrition 0.000 claims description 3
- 230000002186 photoactivation Effects 0.000 claims description 2
- 238000005199 ultracentrifugation Methods 0.000 claims description 2
- 239000000523 sample Substances 0.000 abstract description 8
- 238000006243 chemical reaction Methods 0.000 abstract description 4
- 230000027455 binding Effects 0.000 description 23
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 21
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 12
- 102000018711 Facilitative Glucose Transport Proteins Human genes 0.000 description 7
- 108091052347 Glucose transporter family Proteins 0.000 description 7
- 241000283973 Oryctolagus cuniculus Species 0.000 description 7
- 210000003617 erythrocyte membrane Anatomy 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 6
- 239000011248 coating agent Substances 0.000 description 6
- 238000000576 coating method Methods 0.000 description 6
- 238000001246 colloidal dispersion Methods 0.000 description 6
- 239000012528 membrane Substances 0.000 description 6
- 229910052708 sodium Inorganic materials 0.000 description 6
- 239000011734 sodium Substances 0.000 description 6
- 239000011780 sodium chloride Substances 0.000 description 6
- 241000894007 species Species 0.000 description 6
- 239000000872 buffer Substances 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 229940126619 mouse monoclonal antibody Drugs 0.000 description 5
- 238000006722 reduction reaction Methods 0.000 description 5
- 238000001179 sorption measurement Methods 0.000 description 5
- 230000006641 stabilisation Effects 0.000 description 5
- 238000004627 transmission electron microscopy Methods 0.000 description 5
- 229920005654 Sephadex Polymers 0.000 description 4
- 239000012507 Sephadex™ Substances 0.000 description 4
- 210000003743 erythrocyte Anatomy 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- 241000283707 Capra Species 0.000 description 3
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000001042 affinity chromatography Methods 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000010494 dissociation reaction Methods 0.000 description 3
- 230000005593 dissociations Effects 0.000 description 3
- 239000003480 eluent Substances 0.000 description 3
- 230000000925 erythroid effect Effects 0.000 description 3
- 238000005227 gel permeation chromatography Methods 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 229950006780 n-acetylglucosamine Drugs 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 230000009870 specific binding Effects 0.000 description 3
- 230000003019 stabilising effect Effects 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 108090001008 Avidin Proteins 0.000 description 2
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 2
- 239000000020 Nitrocellulose Substances 0.000 description 2
- 230000004931 aggregating effect Effects 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 239000003638 chemical reducing agent Substances 0.000 description 2
- 239000012141 concentrate Substances 0.000 description 2
- FDWREHZXQUYJFJ-UHFFFAOYSA-M gold monochloride Chemical compound [Cl-].[Au+] FDWREHZXQUYJFJ-UHFFFAOYSA-M 0.000 description 2
- 230000003993 interaction Effects 0.000 description 2
- 229920001220 nitrocellulos Polymers 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000001488 sodium phosphate Substances 0.000 description 2
- 229910000162 sodium phosphate Inorganic materials 0.000 description 2
- 238000011105 stabilization Methods 0.000 description 2
- 239000012128 staining reagent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- RYFMWSXOAZQYPI-UHFFFAOYSA-K trisodium phosphate Chemical compound [Na+].[Na+].[Na+].[O-]P([O-])([O-])=O RYFMWSXOAZQYPI-UHFFFAOYSA-K 0.000 description 2
- BFYCFODZOFWWAA-UHFFFAOYSA-N 2,4,6-trimethylpyridine-3-carbaldehyde Chemical compound CC1=CC(C)=C(C=O)C(C)=N1 BFYCFODZOFWWAA-UHFFFAOYSA-N 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 108010001478 Bacitracin Proteins 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108010078791 Carrier Proteins Proteins 0.000 description 1
- OVRNDRQMDRJTHS-UHFFFAOYSA-N N-acelyl-D-glucosamine Natural products CC(=O)NC1C(O)OC(CO)C(O)C1O OVRNDRQMDRJTHS-UHFFFAOYSA-N 0.000 description 1
- OVRNDRQMDRJTHS-FMDGEEDCSA-N N-acetyl-beta-D-glucosamine Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O OVRNDRQMDRJTHS-FMDGEEDCSA-N 0.000 description 1
- MBLBDJOUHNCFQT-LXGUWJNJSA-N N-acetylglucosamine Natural products CC(=O)N[C@@H](C=O)[C@@H](O)[C@H](O)[C@H](O)CO MBLBDJOUHNCFQT-LXGUWJNJSA-N 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 1
- 239000002250 absorbent Substances 0.000 description 1
- 230000002745 absorbent Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 229960003071 bacitracin Drugs 0.000 description 1
- 229930184125 bacitracin Natural products 0.000 description 1
- CLKOFPXJLQSYAH-ABRJDSQDSA-N bacitracin A Chemical compound C1SC([C@@H](N)[C@@H](C)CC)=N[C@@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]1C(=O)N[C@H](CCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2N=CNC=2)C(=O)N[C@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)NCCCC1 CLKOFPXJLQSYAH-ABRJDSQDSA-N 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 239000004202 carbamide Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000002860 competitive effect Effects 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000003431 cross linking reagent Substances 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000001747 exhibiting effect Effects 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002344 gold compounds Chemical class 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 150000004804 polysaccharides Polymers 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 239000012429 reaction media Substances 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000012279 sodium borohydride Substances 0.000 description 1
- 229910000033 sodium borohydride Inorganic materials 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000012064 sodium phosphate buffer Substances 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 210000001768 subcellular fraction Anatomy 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- XYYVDQWGDNRQDA-UHFFFAOYSA-K trichlorogold;trihydrate;hydrochloride Chemical compound O.O.O.Cl.Cl[Au](Cl)Cl XYYVDQWGDNRQDA-UHFFFAOYSA-K 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000012800 visualization Methods 0.000 description 1
- 239000011800 void material Substances 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/551—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being inorganic
- G01N33/553—Metal or metal coated
Definitions
- the present invention relates to colloidal solutions or dispersions of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble.
- These attached, target-specific species can then react with the appropriate target molecules or sites of an organic material of interest. Properties such as the colour, the high electron- stopping power and high density of the central noble metal particles may then be exploited, e.g., in the visualization of the sites to which the particles of the invention have become attached or in the purification or fractionation of preparations of biological material, e.g. preparations of cell vesicles.
- colloidal solutions or dispersions also called sols
- sols colloidal solutions or dispersions of noble metals in water
- the stabilisation is presumably due to adsorption of the substances in question to the surface of the noble metal particles, thereby preventing the latter from aggregating, possibly by providing the particles with the same charge or by preventing hydrophobic interaction between "naked" particles.
- the free stabilising substance with the specific properties must clearly not be present in significant concentrations in the colloidal solution or dispersion, since it will compete with the binding of the gold probe to the substance which is to be labelled. If the free substance is removed from the solution, the substance bound to the gold particles will gradually be released until equilibrium between the free and the bound form is again attained. This leads to unspecific binding of partially exposed noble metal particles, partly to the preparation and partly to other particles, and it also leads to blocking of molecules or side-chains in the preparation by the free (i.e.
- the present invention provides colloidal solutions of noble metal particles which are stably encapsulated by an outer layer or coating comprising cross-linked molecules of a relatively inert organic polymeric substance. This interlocking of the individual molecules of the substance surrounding the noble metal particles prevents the dissociation or displacement of the central noble metal particles and can thus substantially eliminate the aggregation and unspecific adsorption phenomena often encountered (vide supra) .
- the encapsulating layer of polymer also makes it possible, by means of one or more chemical reactions, to covalently attach a chosen substance having special affinity for a substance or site which it is desired to detect or label.
- the latter attached chosen substance which cannot be attached directly to the "naked" noble metal particle by covalent bonding, is chosen so as to have a known natural affinity only for the substance or the type of side-chain or binding site which it is desired to detect or label.
- This outermost, attached substance thus imparts to the noble metal particles the ability to bind specifically to a particular substance or a particular side- chain or site in a preparation, e.g. a preparation of biological nmaterial. Since both the encapsulating polymer layer and the specifically binding substance are held in place by means of stable covalent bonds, any free excess of these substances in the colloidal solution or dispersion can be removed completely without jeopardising the stability of the solution.
- Fig. 1 Dot blots on nitrocellulose paper.
- the reagents applied to the regions A-F of the strips were as follows:
- Example 6 (b) After incubation for 30 min in "Buffer A” (see Example 6 (b) ) a gold- containing sol stabilized directly with protein G was applied to the left-hand strip, and the gold-containing dispersion prepared as in Example 5 was applied to the right-hand strip. After 1 hour the strips were washed with Buffer A.
- Fig. 2 A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with a mouse monoclonal antibody directed against the C-terminus of the HepG2/erythroid glucose transporter, then with a rabbit antibody against mouse 7-globulins, and finally with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. The localized grouping of the gold-containing particles (visible as roughly circular black dots) due to specific binding is clearly visible.
- Fig. 3 A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated only with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. The almost complete lack of gold-containing particles is apparent.
- Fig.4 A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with the rabbit antibody against mouse ⁇ - lobulins referred to in connection with Fig. 2, and then with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. As with Fig. 3, the almost complete lack of gold-containing particles is apparent.
- Fig. 5 A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with mouse monoclonal antibody directed against the C-terminus of the HepG2/erythroid glucose transporter, then with the gold-containing colloidal dispersion prepared as described in Example 9. Negative staining with sodium silicotungstate. Magnification x 184,800. The localized grouping of the gold-containing particles (visible as roughly circular black dots) due to specific binding is clearly visible.
- Fig. 6 As for Fig. 5, but showing the edge of the erythrocyte membrane and the lack of bound gold-containing particles outside the membrane (i.e. in the lighter coloured region to the left in the photograph) .
- Fig. 7 A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated only with the gold-containing colloidal dispersion prepared as described in Example 9. Negative staining with sodium silicotungstate. Magnification x 184,800. The almost complete lack of gold-containing particles is apparent.
- One aspect of the present invention provides a method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
- step (c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) .
- the meaning of the term “substantially permanently encapsulated” is that the encapsulated noble metal particles are not released or displaced to any significant extent from the encapsulating layer in the course of the reaction sequences, reagent treatments, washing procedures etc. to which the colloidal particle dispersion may subsequently be subjected.
- relatively inert is intended to imply that the organic polymer used to form the encapsulating layer may react with appropriately chosen, relatively highly reactive chemical reagents, such as bifunctional cross-linkers or fixatives (vide infra) , but in general does not readily react with buffer media, solvents or (directly) with a substance of specific affinity for target molecules which may to be subsequently attached (vide infra) to the encapsulated particles.
- Water solubility of the free form of the organic polymer used to encapsulate the noble metal particles is, of course, important to ensure adequate homogeneity of the reaction medium during the attachment of the organic polymer molecules to the colloidal noble metal particles, and thus ensure satisfactory uniformity of the extent of encapsulation and the thickness of the encapsulating layer.
- a further aspect of the invention provides a method for the preparation of an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water- soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
- step (c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) and, optionally,
- step (d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer.
- Yet another aspect of the invention provides a method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are functionalities or side-chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
- step (c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) and, optionally, (ii) to the establishment of chemically reactive functionalities or side-chains on the outer surface of said layer, said functionalities or side-chains being capable of subsequently reacting with said chosen substance,
- step (d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer,
- the noble metal particles within the context of the present invention are particles of a noble metal selected from the group consisting of gold, silver, rhodium, iridium, platinum, palladium, ruthenium and osmium.
- the collidal noble metal particles used in preferred embodiments of methods according to the invention are colloidal gold particles which have been formed by chemical reduction with a reducing agent such as a borohydride or citrate, and preliminary experiments indicate that the size range of the colloidal gold particles formed may be varied to some extent by varying the concentration of the water-soluble gold compound used as starting material and/or by varying the temperature at which reduction is performed.
- the size of the noble metal particles in the context of the present invention is in the range of about 1-20 nm, preferably about 1-15 nm, more preferably about 1-10 nm, most preferably about 1-5 nm. It is particularly preferred that the size of the noble metal particles is predominantly in the range of 1-4 nm or predominantly in the range of 10-15 nm.
- the organic polymer from which the encapsulating layer is formed is selected from the group consisting of: natural and synthetic polypeptides and proteins, including antigens, antibodies and glycoproteins, and polysacchari- des.
- Other polymeric substances which may be of value in this connection are polyethylene glycols and polynucleotides.
- the organic polymer is selected from the group consisting of: gelatins of human or animal origin, serum albumins of human or animal origin, agar, and soluble starches, including modified starches.
- the organic polymer used is a gelatin of human or animal origin, the aqueous solution of said gelatin used in step (b) of the method having a pH in the region of about 11-12, preferably a pH of about 11.5.
- the thickness of the covalently linked layer of organic polymer on the noble metal particles is in the range of about 1-2 nm, and is preferably about 1 nm.
- the treatment carried out in step (c) of a method of the invention and leading to the formation of covalent linkages between individual molecules of the organic polymer is suitably either a treatment with a bifunctional cross-linker or fixative, or a photoactivation treatment.
- a treatment with a bifunctional cross-linker or fixative is employed, and the bifunctional cross-linker or fixative is preferably selected from the group consisting of: aliphatic dialdehydes, including succinic dialdehyde and glutaric dialdehyde,
- Dialdehydes such as glutaric dialdehyde (glutaraldehyde) are particularly well suited for use as a cross-linking reagent in the case of encapsulating organic polymers which are proteins (e.g. gelatin or albumin) .
- Divinyl sulphone will be particularly suited when the encapsulating organic polymer has suitable free hydroxy groups, as is the case with, e.g., numerous polysaccharides.
- the treatment carried out in step (c) or (d) of such a method and leading to the establishment of the chemically reactive functionalities or side-chains is preferably also a treatment with a bifunctional cross-linker or fixative, particularly a bifunctional cross-linker or fixative selected from those mentioned above.
- the chosen substance which becomes covalently attached via functionalities or side-chains on the outer surface of the polymer layer, or with which functionalities or side-chains established on the outer surface of the polymer layer may subsequently react to form a covalent bond thereto is preferably an organic substance with the ability, when covalently attached via said functionalities or side- chains, to bind specifically and non-covalently to a further organic substance or material. It is particularly preferred that this chosen substance is selected from the group consisting of:
- antigens and antibodies including monoclonal and polyclonal antibodies,
- lectins including wheatgerm agglutinin, Concanavalin A, Phytohaemagglutinin and Pokeweed mitogen, avidin,
- the present invention also concerns an aqueous colloidal solution or dispersion of particles prepared by any method according to the invention.
- the invention further provides the following aqueous colloidal solutions or dispersions of particles:
- an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer;
- the functionalities or side-chains are preferably derived from a bifunctional cross-linker or fixative, in particular a bifunctional cross-linker or fixative chosen from those mentioned above.
- the covalently attached chosen substance is an organic substance with the ability, while covalently attached via said functionalities or side-chains, to bind specifically and non-covalently to a further organic substance or material.
- This chosen substance is preferably selected from those mentioned above.
- FIG. 1 Further aspects of the invention relate (i) to the use of an aqueous colloidal solution or dispersion according to the invention for the labelling, staining, detection, identification or localization of organic substances or particular regions or moieties of organic materials, e.g. in preparations of biological material, particularly in connection with electron microscopy, light microscopy, electrophoretic separation techniques or ultracentrifugation; and (ii) to the use of an aqueous colloidal solution or dispersion according to the invention for the purification or fractionation of preparations of biological material.
- Aqueous colloidal solutions or dispersions according to the invention may be used for the detection, identification or localisation of any substance, side-chain or binding site which occurs immobilised in such way that it can be brought into contact with an appropriate aqueous colloidal solution or dispersion according to the invention, provided that another substance is available that binds specifically to the substance or the side-chain which is to be identified and that this other substance can be coupled covalently to the colloidal particles of a colloidal solution or dispersion according to the invention.
- Detection takes place by soaking or otherwise incubating the preparation in which it is desired to detect a particular species or binding site in the aqueous colloidal solution or dispersion, the particles of which bear the relevant chosen, specifically binding substance, after which unbound colloidal particles are removed by washing.
- the specificity of the reaction can be checked by prior treatment of the preparation with the specifically reacting substance in free form, as a result of which the ability of the preparation to bind the colloidal particles in question will be abolished.
- Aqueous colloidal solutions or dispersions according to the invention can also be used for purification of e.g. vesicles in subcellular fractions, in microorganisms or in other cells in connection with (ultra)centrifugation in a density gradient, the binding of the colloidal particles (with their dense, encapsulated noble metal particles) to vesicles or cells leading to the formation of a complex of high density.
- a drop of the latter gold sol was transferred to a standard carbon- coated electron microscopy (EM) grid, and the grid was then washed with a negative-staining reagent in the form of a 2% (w/v) aqueous solution of sodium silicotungstate. After careful removal of the bulk of the liquid with absorbent tissue, the grid was dried at 30°C for 15 min in a drying oven and examined in a JEOL 100 CX transmission electron microscope, revealing gold particles of size predominantly in the range of 1-4 nm.
- EM electron microscopy
- Example 1 Examination of the gold particles in this sol by transmissiom electron microscopy (TEM) as in Example 1 revealed gold particles of size predominantly in the range of 10-15 nm.
- TEM transmissiom electron microscopy
- Example 2 An entire portion (i.e. ca. 210 ml) of gold sol prepared as in Example 1 was added with stirring to the alkaline gelatin solution prepared in Example 3 (a) , above, and the resulting solution was left to stand at room temperature for 10 min. Examination of the particles in the thus-stabilized sol at this point by TEM as described in Example 1 showed that the gold particles were covered with an essentially uniform coating of gelatin with a thickness of ca. 1 nm.
- the latter particle concentrate was subjected to gel chromatography by passage through a column (typical dimensions: diam. ca. 3 cm x length ca. 22 cm) of Sephadex® G-25 (Pharmacia, Sweden) which had been pre-- equilibrated with aqueous sodium chloride/sodium phosphate (200 mM/200mM, pH 7.5), and the gold-containing particle dispersion (which was clearly visible on the column owing to its characteristic yellowish red colour) eluting in the void volume (about 40 ml) was collected.
- the particle dispersion prepared according to Example 5 can be used to label the Fc part of 7-globulins.
- Dot blots on nitrocellulose paper revealed a much higher specificity of 7- lobulin labelling than with a particle dispersion obtained by simple (non- covalent) attachment of protein G to the gold particles of a sol prepared as described in Example 1.
- the particle dispersion prepared according to Example 5 can be used to determine the abundance (i.e. number) of these transporter proteins by TEM.
- a mouse monoclonal antibody against the C-terminus of the HepG2/erythroid glucose transporter was employed, the procedure being as follows:
- Intact human erythrocytes were allowed to sediment onto a polylysine- coated, standard carbon-coated EM grid and the erythrocytes adhering to the grid were ruptured by washing with distilled water at ⁇ 4°C, the inner surface of the erythrocyte membrane thereby being exposed.
- the grid bearing the thus-ruptured erythrocytes was then incubated for 30 min in a solution [ca.
- the grid was then incubated for ca. 1-2 min in the particle dispersion prepared as in Example 5, washed thoroughly with Buffer A, and then treated with a 2% (v/v) solution of glutaric dialdehyde in aqueous sodium chloride/sodium phosphate (200 mM/200mM, pH 7.5) (this treatment renders the binding of the particle-bound protein G to the membrane-bound antibodies more permanent, presumably via the formation of bridging covalent linkages between the protein G moieties and the antibodies). Finally, the grid was washed with negative-staining reagent and dried as described in Example 1.
- TEM (see Fig. 2) clearly showed the expected pattern of localized distribution of the gold-containing particles attached specifically to the antibody-bearing glucose transporter structures of the membrane; a count of the number of gold-containing particles bound per unit area indicates, in fact, that there is essentially quantitative binding of these gold-containing particles to the glucose transporter proteins, i.e. in 1:1 ratio.
- Example 8 Using the same procedure as in Example 5, but substituting wheatgerm agglutinin for protein G, a particle dispersion exhibiting binding specificity towards certain carbohydrates is obtained (see Example 8).
- EXAMPLE 8 Affinity chromatography of the particle dispersion bearing covalently attached wheatgerm agglutinin A particle dispersion prepared according to Example 7 was subjected to affinity chromatography on a column (typical dimensions: diam. ca. 1 cm x length ca. 3 cm) containing N -acetylglueosamine coupled to divinyl sulphone activated agarose (Kem-En-Tec Ltd ApS, Copenhagen, Denmark) .
- the particles of the dispersion (clearly visible on the column owing to the characteristic red colour) exhibited strong binding affinity for the column material, but could be eluted satisfactorily, in dispersed form, from the column using a 0.3 M solution of W-acetylglucosamine in aqueous Tris buffer (20 mM, pH 7.4) as competitive eluent.
- the stability of the dispersed particles in question towards this affinity chromatography procedure was demonstrated further by the fact that the dispersion eluted from the column could, after removal of the W-acetylglucosamine eluent by gel chromatography on Sephadex® G-25, be recyled repeatedly through the affinity column, i.e. repeatedly bound and re-eluted.
- Example 5 Using the same procedure as in Example 5, but employing affinity-purified goat antibodies directed against mouse 7-globulin (Z 420, Dakopatts a/s, Glostrup, Denmark) instead of protein G, a particle dispersion suitable for determination of the glucose transporter number in a manner similar to that described in Example 6 (b) was obtained (see Figs. 5, 6 and 7).
- the specifically binding species in question i.e. protein G, wheatgerm agglutinin and goat anti-mouse 7-globulin antibodies
- protein G protein G
- wheatgerm agglutinin and goat anti-mouse 7-globulin antibodies were coupled to freshly prepared, activated gelatin-coated gold particles.
- glutaric dialdehyde-activated particle dispersions prepared as described show no major loss of ability to bind proteinaceous species of the above types within a period of at least two weeks or more at ambient temperature; at lower temperatures their "shelf-life" may well be prolonged considerably.
- the sale of "ready-made" activated particle dispersions of the present type to which chosen target-specific species may subsequently be covalently attached by the user is thus a realistic commercial possibility.
Abstract
Methods are provided for the preparation of aqueous colloidal solutions or dispersions of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer. The colloidal particles of the prepared solutions or dispersions may bear chemically reactive functionalities or side-chains covalently attached to the outer surface of the polymer layer. These functionalities or side-chains are capable of reaction with a chosen substance so as to form a covalent bond thereto, the chosen substance being, e.g., a substance such as an antigen, an antibody or protein G which while attached to the colloidal particles can bind non-covalently to other species. Aqueous colloidal solutions or dispersions of the above types are useful, e.g., as probes in electron microscopy.
Description
POLYMER-COATED NOBLE METAL PARTICLES AND A METHOD FOR THEIR PREPARATION
FIELD OF THE INVENTION
The present invention relates to colloidal solutions or dispersions of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble. On the outer surface of these particles there may be established chemically reactive functionalities or side-chains via which certain "target-specific" organic substances may be covalently attached to the particles. These attached, target-specific species can then react with the appropriate target molecules or sites of an organic material of interest. Properties such as the colour, the high electron- stopping power and high density of the central noble metal particles may then be exploited, e.g., in the visualization of the sites to which the particles of the invention have become attached or in the purification or fractionation of preparations of biological material, e.g. preparations of cell vesicles.
BACKGROUND OF THE INVENTION
It is well known that colloidal solutions or dispersions (also called sols) of noble metals in water can be stabilised to some extent by the addition of various water-soluble organic substances. The stabilisation is presumably due to adsorption of the substances in question to the surface of the noble metal particles, thereby preventing the latter from aggregating, possibly by providing the particles with the same charge or by preventing hydrophobic interaction between "naked" particles.
Previously, considerable effort has been devoted to attempts to stabilise aqueous colloidal solutions or dispersions of gold particles by adding substances whose interaction with (binding to) other substances is well charactised, e.g. monoclonal antibodies, lectins, avidin, protein A or protein G, and using this approach
so-called specific gold probes have been prepared for the detection of the localisation of a particular species or site in a preparation, advantage been taking of the specificity of the stabilising substance and also of the fact that the gold particles are easily recognisable, either by their colour or (in electron microscopy) by their shape and their ability to stop electrons. Such gold probes are available commercially and are used as labels for a particular substance or group of substances in connection with electron microscopy, light microscopy or various types of electrophoretic separation.
It has, however, been found for several of the stabilising substances which provide the gold probe with its specificity that the adsorption to the noble metal particles is reversible to an unfavorable extent. If the probe is to function as intended, the free stabilising substance with the specific properties must clearly not be present in significant concentrations in the colloidal solution or dispersion, since it will compete with the binding of the gold probe to the substance which is to be labelled. If the free substance is removed from the solution, the substance bound to the gold particles will gradually be released until equilibrium between the free and the bound form is again attained. This leads to unspecific binding of partially exposed noble metal particles, partly to the preparation and partly to other particles, and it also leads to blocking of molecules or side-chains in the preparation by the free (i.e. not particle-bound) specifically reactive substance. This problem is of variable severity, depending on the nature of the substance used for stabilisation. In certain cases it may take a couple of days after removal of free (dissolved) specifically reactive substance from the solution before significant amounts are released from the gold particles and are again present in the free state in the solution, while in other cases this process takes place so fast that a specific probe cannot be prepared. Attempts have been made to add an inert substance to the colloidal solution or dispersion of noble metal particles after the.colloidal solution has been primarily stabilised with the substance which is to provide the specificity. This leads to further stabilisation of the colloidal solution, but since, in general, the inert substance also binds reversibly to the surface of the noble metal particles, such an addition is of limited value and
can in unfavourable cases accelerate the dissociation of the specifically reactive substance from the noble metal particles; it can also mask the fact that this dissociation has in fact taken place, since the presence of the inert substance prevents the particles from aggregating. Furthermore, the presence of an inert substance can, on applying the probe to the preparation of interest, suppress most of the unspecific binding of the noble metal particles to molecules or side-chains in the preparation. This "contrast enhancement" in the unspecific binding, can lead to a pattern of binding which disappointingly resembles specific binding and which can only be revealed as being unspecific by comparison with control preparations on which the target binding sites have previously been blocked using the specifically binding substance.
BRIEF DISCLOSURE OF THE INVENTION
The present invention provides colloidal solutions of noble metal particles which are stably encapsulated by an outer layer or coating comprising cross-linked molecules of a relatively inert organic polymeric substance. This interlocking of the individual molecules of the substance surrounding the noble metal particles prevents the dissociation or displacement of the central noble metal particles and can thus substantially eliminate the aggregation and unspecific adsorption phenomena often encountered (vide supra) .
The encapsulating layer of polymer also makes it possible, by means of one or more chemical reactions, to covalently attach a chosen substance having special affinity for a substance or site which it is desired to detect or label. The latter attached chosen substance, which cannot be attached directly to the "naked" noble metal particle by covalent bonding, is chosen so as to have a known natural affinity only for the substance or the type of side-chain or binding site which it is desired to detect or label. This outermost, attached substance thus imparts to the noble metal particles the ability to bind specifically to a particular substance or a particular side- chain or site in a preparation, e.g. a preparation of biological nmaterial. Since both the encapsulating polymer layer and the specifically binding substance are held in place by means of stable
covalent bonds, any free excess of these substances in the colloidal solution or dispersion can be removed completely without jeopardising the stability of the solution.
DESCRIPTION OF THE FIGURES
Fig. 1: Dot blots on nitrocellulose paper. The reagents applied to the regions A-F of the strips were as follows:
A: poly(Glu-Tyr) (Sigma; 2 μl, 10 mg/ml)
B: gelatin (G2500, Sigma; 2μl, 10 mg/ml)
C: "Molecular Weight Standard, High" (Bio-Rad) D: Bacitracin (Sigma; 2 μl, 10 mg/ml)
E: rabbit 7-globulin (Dakopatts a/s)
F: polylysine (Sigma; 2 μl, 0.1 mg/ml) (G: blank)
After incubation for 30 min in "Buffer A" (see Example 6 (b) ) a gold- containing sol stabilized directly with protein G was applied to the left-hand strip, and the gold-containing dispersion prepared as in Example 5 was applied to the right-hand strip. After 1 hour the strips were washed with Buffer A.
It is clear from a comparison of the two strips that the gold- containing colloidal dispersion of the invention (with covalently attached protein G) binds quite specifically to rabbit 7-globulin, whereas the gold sol stabilized by simple adsorption of protein G exhibits a broad spectrum of binding affinity.
Fig. 2: A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with a mouse monoclonal antibody directed against the C-terminus of the HepG2/erythroid glucose transporter, then with a rabbit antibody against mouse 7-globulins, and finally with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. The localized grouping of the gold-containing particles (visible as
roughly circular black dots) due to specific binding is clearly visible.
Fig. 3: A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated only with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. The almost complete lack of gold-containing particles is apparent.
Fig.4: A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with the rabbit antibody against mouse γ- lobulins referred to in connection with Fig. 2, and then with the gold-containing colloidal dispersion prepared as described in Example 5. Negative staining with sodium silicotungstate. Magnification x 184,800. As with Fig. 3, the almost complete lack of gold-containing particles is apparent.
Fig. 5: A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated first with mouse monoclonal antibody directed against the C-terminus of the HepG2/erythroid glucose transporter, then with the gold-containing colloidal dispersion prepared as described in Example 9. Negative staining with sodium silicotungstate. Magnification x 184,800. The localized grouping of the gold-containing particles (visible as roughly circular black dots) due to specific binding is clearly visible.
Fig. 6: As for Fig. 5, but showing the edge of the erythrocyte membrane and the lack of bound gold-containing particles outside the membrane (i.e. in the lighter coloured region to the left in the photograph) .
Fig. 7: A transmission electron microscopy photograph of a human erythrocyte membrane fragment which had been incubated only with the gold-containing colloidal dispersion prepared as described in Example 9. Negative staining with sodium silicotungstate. Magnification x
184,800. The almost complete lack of gold-containing particles is apparent.
DETAILED DESCRIPTION OF THE INVENTION
One aspect of the present invention provides a method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer, and
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) .
As already implied in the discussion above, the meaning of the term "substantially permanently encapsulated" is that the encapsulated noble metal particles are not released or displaced to any significant extent from the encapsulating layer in the course of the reaction sequences, reagent treatments, washing procedures etc. to which the colloidal particle dispersion may subsequently be subjected. The term "relatively inert" is intended to imply that the organic polymer used to form the encapsulating layer may react with appropriately chosen, relatively highly reactive chemical reagents,
such as bifunctional cross-linkers or fixatives (vide infra) , but in general does not readily react with buffer media, solvents or (directly) with a substance of specific affinity for target molecules which may to be subsequently attached (vide infra) to the encapsulated particles.
Water solubility of the free form of the organic polymer used to encapsulate the noble metal particles is, of course, important to ensure adequate homogeneity of the reaction medium during the attachment of the organic polymer molecules to the colloidal noble metal particles, and thus ensure satisfactory uniformity of the extent of encapsulation and the thickness of the encapsulating layer.
A further aspect of the invention provides a method for the preparation of an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water- soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer,
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal
particles following the treatment in step (b) and, optionally,
(ii) to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer, and
(d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer.
Yet another aspect of the invention provides a method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are functionalities or side-chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer,
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) and, optionally, (ii) to the establishment of chemically reactive
functionalities or side-chains on the outer surface of said layer, said functionalities or side-chains being capable of subsequently reacting with said chosen substance,
(d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer,
(e) allowing said chosen substance to react with said established, chemically reactive functionalities or side-chains so as to form a covalent bond thereto.
The noble metal particles within the context of the present invention are particles of a noble metal selected from the group consisting of gold, silver, rhodium, iridium, platinum, palladium, ruthenium and osmium. The collidal noble metal particles used in preferred embodiments of methods according to the invention are colloidal gold particles which have been formed by chemical reduction with a reducing agent such as a borohydride or citrate, and preliminary experiments indicate that the size range of the colloidal gold particles formed may be varied to some extent by varying the concentration of the water-soluble gold compound used as starting material and/or by varying the temperature at which reduction is performed.
The size of the noble metal particles in the context of the present invention is in the range of about 1-20 nm, preferably about 1-15 nm, more preferably about 1-10 nm, most preferably about 1-5 nm. It is particularly preferred that the size of the noble metal particles is predominantly in the range of 1-4 nm or predominantly in the range of 10-15 nm.
In another aspect of the invention, the organic polymer from which the encapsulating layer is formed is selected from the group
consisting of: natural and synthetic polypeptides and proteins, including antigens, antibodies and glycoproteins, and polysacchari- des. Other polymeric substances which may be of value in this connection are polyethylene glycols and polynucleotides.
In a further aspect, the organic polymer is selected from the group consisting of: gelatins of human or animal origin, serum albumins of human or animal origin, agar, and soluble starches, including modified starches.
In a particularly preferred aspect of a method according to the invention, the organic polymer used is a gelatin of human or animal origin, the aqueous solution of said gelatin used in step (b) of the method having a pH in the region of about 11-12, preferably a pH of about 11.5.
Preliminary experiments carried out using a range of pH values have shown that the adsorption of the gelatin to, e.g., gold sols which takes place upon addition of the gelatin solution to the sol does not lead to satisfactorily uniform envelopment of the noble metal particles by a coating or layer of gelatin molecules unless the pH is rather high. Furthermore, there is also some evidence to indicate that gelatins with a "Bloom number" (a measure of the strength of a aqueous gel produced by the gelatin in question) in the range 10- 100 are particularly suitable.
In another aspect of the invention, the thickness of the covalently linked layer of organic polymer on the noble metal particles is in the range of about 1-2 nm, and is preferably about 1 nm.
The treatment carried out in step (c) of a method of the invention and leading to the formation of covalent linkages between individual molecules of the organic polymer is suitably either a treatment with a bifunctional cross-linker or fixative, or a photoactivation treatment. In preferred embodiments of methods according to the invention a treatment with a bifunctional cross-linker or fixative is employed, and the bifunctional cross-linker or fixative is preferably selected from the group consisting of:
aliphatic dialdehydes, including succinic dialdehyde and glutaric dialdehyde,
divinyl sulphone, and
disuccinimidyl suberate.
Dialdehydes such as glutaric dialdehyde (glutaraldehyde) are particularly well suited for use as a cross-linking reagent in the case of encapsulating organic polymers which are proteins (e.g. gelatin or albumin) . Divinyl sulphone will be particularly suited when the encapsulating organic polymer has suitable free hydroxy groups, as is the case with, e.g., numerous polysaccharides.
In methods according to the invention in which chemically reactive functionalities or side-chains are established on the outer surface of the encapsulating polymer layer, the treatment carried out in step (c) or (d) of such a method and leading to the establishment of the chemically reactive functionalities or side-chains is preferably also a treatment with a bifunctional cross-linker or fixative, particularly a bifunctional cross-linker or fixative selected from those mentioned above. Furthermore, in methods of the latter category, the chosen substance which becomes covalently attached via functionalities or side-chains on the outer surface of the polymer layer, or with which functionalities or side-chains established on the outer surface of the polymer layer may subsequently react to form a covalent bond thereto is preferably an organic substance with the ability, when covalently attached via said functionalities or side- chains, to bind specifically and non-covalently to a further organic substance or material. It is particularly preferred that this chosen substance is selected from the group consisting of:
antigens and antibodies, including monoclonal and polyclonal antibodies,
lectins, including wheatgerm agglutinin, Concanavalin A, Phytohaemagglutinin and Pokeweed mitogen,
avidin,
protein A,
protein G,
RNA, and
DNA.
The present invention also concerns an aqueous colloidal solution or dispersion of particles prepared by any method according to the invention.
The invention further provides the following aqueous colloidal solutions or dispersions of particles:
(A) an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer;
(B) an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer;
and
(C) an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are functionalities or side- chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
In aqueous colloidal solutions or dispersions of category (B) or (C) , above, the functionalities or side-chains are preferably derived from a bifunctional cross-linker or fixative, in particular a bifunctional cross-linker or fixative chosen from those mentioned above.
In a further aspect relating to an aqueous colloidal solution or dispersion of category (C) , above, the covalently attached chosen substance is an organic substance with the ability, while covalently attached via said functionalities or side-chains, to bind specifically and non-covalently to a further organic substance or material. This chosen substance is preferably selected from those mentioned above.
Further aspects of the invention relate (i) to the use of an aqueous colloidal solution or dispersion according to the invention for the labelling, staining, detection, identification or localization of organic substances or particular regions or moieties of organic materials, e.g. in preparations of biological material, particularly in connection with electron microscopy, light microscopy, electrophoretic separation techniques or ultracentrifugation; and (ii) to the use of an aqueous colloidal solution or dispersion according to the invention for the purification or fractionation of preparations of biological material.
Aqueous colloidal solutions or dispersions according to the invention may be used for the detection, identification or localisation of any substance, side-chain or binding site which occurs immobilised in such way that it can be brought into contact with an appropriate aqueous colloidal solution or dispersion according to the invention, provided that another substance is available that binds specifically to the substance or the side-chain which is to be identified and that this other substance can be coupled covalently to the colloidal particles of a colloidal solution or dispersion according to the invention. Detection takes place by soaking or otherwise incubating the preparation in which it is desired to detect a particular species or binding site in the aqueous colloidal solution or dispersion, the particles of which bear the relevant chosen, specifically binding substance, after which unbound colloidal particles are removed by washing. The specificity of the reaction can be checked by prior treatment of the preparation with the specifically reacting substance in free form, as a result of which the ability of the preparation to bind the colloidal particles in question will be abolished.
Aqueous colloidal solutions or dispersions according to the invention can also be used for purification of e.g. vesicles in subcellular fractions, in microorganisms or in other cells in connection with (ultra)centrifugation in a density gradient, the binding of the colloidal particles (with their dense, encapsulated noble metal particles) to vesicles or cells leading to the formation of a complex of high density.
EXPERIMENTAL SECTION
EXAMPLE 1. Gold sol preparation by reduction with borohydride
20 mg of "gold chloride" (hydrated tetrachloroauric acid; DEGUSSA, West Germany) was dissolved in 200 ml of triply distilled water. To this solution was added, at ambient temperature and with stirring, a freshly prepared solution of 57 mg of sodium borohydride in 10 ml of distilled water. The solution became yellow-red in colour owing to formation of colloidal gold particles.
A drop of the latter gold sol was transferred to a standard carbon- coated electron microscopy (EM) grid, and the grid was then washed with a negative-staining reagent in the form of a 2% (w/v) aqueous solution of sodium silicotungstate. After careful removal of the bulk of the liquid with absorbent tissue, the grid was dried at 30°C for 15 min in a drying oven and examined in a JEOL 100 CX transmission electron microscope, revealing gold particles of size predominantly in the range of 1-4 nm.
EXAMPLE 2. Gold sol preparation by reduction with citrate
20 mg of "gold chloride" (tetrachloroauric acid trihydrate; DEGUSSA, West Germany) was dissolved in 200 ml of triply distilled water. The solution was heated to boiling, and 7.5 ml of a 1% w/v solution of trisodium citrate dihydrate (Merck, West Germany) was then added and the mixture boiled for 30 min. During this time the mixture became red in colour.
Examination of the gold particles in this sol by transmissiom electron microscopy (TEM) as in Example 1 revealed gold particles of size predominantly in the range of 10-15 nm.
EXAMPLE 3. Stabilization of the gold sols with gelatin
(a) Preparation of gelatin solution for sol stabilization
A 0.1% (w/v) aqueous solution of porcine skin gelatin (G6144, Sigma, St. Louis, USA) was boiled for 30 min, cooled to room temperature, and centrifuged for 2 hours at 40,000 rpm (Beckman Instruments Ti 70 rotor) . 3.2 ml of the resulting supernatant was heated to boiling and then mixed with 3.2 ml of 0.125 M NaOH in a 250 ml glass conical flask. The mixture was kept at 37°C for 10 min.
(b) A gold sol prepared according to Example 1 was stabilized with gelatin as follows:
An entire portion (i.e. ca. 210 ml) of gold sol prepared as in Example 1 was added with stirring to the alkaline gelatin solution prepared in Example 3 (a) , above, and the resulting solution was left to stand at room temperature for 10 min. Examination of the particles in the thus-stabilized sol at this point by TEM as described in Example 1 showed that the gold particles were covered with an essentially uniform coating of gelatin with a thickness of ca. 1 nm.
(c) A gold sol prepared according to Example 2 was stabilized with gelatin in the same manner as described in Example 3 (b) , above, and examination by TEM as before showed that these relatively larger gold particles were also coated with a ca. 1 nm layer of gelatin.
EXAMPLE 4. Cross- linking and activation of the gelatin coating of the particles in the gelatin- stabilized gold sols
(a) To the gelatin-stabilized gold sol prepared according to Example 3 (b) was added, at ambient temperature, 25 ml of a 5% (v/v) aqueous solution of glutaric dialdehyde in 160 mM aqueous sodium phosphate buffer (pH 7.0). The mixture was allowed to stand for 1 hour, and it was then layered carefully onto a "density medium" consisting of a 90% (v/v) solution of glycerol in 0.9 % (w/v) aqueous NaCl and centrifuged for 3 hours at 25,000 rpm (Beckman Instruments SW28 rotor) . The gold-containing particle dispersion fraction retained in the uppermost part of the layer of density medium was collected. In order to remove, inter alia , excess of glutaric dialdehyde, the latter particle concentrate was subjected to gel chromatography by passage through a column (typical dimensions: diam. ca. 3 cm x length
ca. 22 cm) of Sephadex® G-25 (Pharmacia, Sweden) which had been pre-- equilibrated with aqueous sodium chloride/sodium phosphate (200 mM/200mM, pH 7.5), and the gold-containing particle dispersion (which was clearly visible on the column owing to its characteristic yellowish red colour) eluting in the void volume (about 40 ml) was collected.
TEM examination (as described in Example 1) of the activated particles in the latter dispersion showed them to be very uniformly dispersed, with a mean gold particle diameter of ca. 3 nm and with an essentially uniform gelatin coating with a thickness of ca.l nm.
(b) The gelatin-stabilized gold sol prepared according to Example 3 (c) was treated with glutaric dialdehyde as described in Example (a) . After being allowed to stand for 1 hour, the mixture was carefully layered onto a "two-stage density medium" consisting of an upper layer of a 40% (v/v) solution of glycerol in 0.9% (w/v) aqueous NaCl and a lower layer of pure glycerol. After centrifugation for 30 min at 15,000 rpm (Beckman Instruments SW28 rotor) , the gold-containing particle dispersion retained at the 40% glycerol/100% glycerol interface was collected and the particle concentrate was subjected to gel chromatography on a column of Sephadex® G-25 as described in Example 4(a.).
TEM examination of the gold-containing particles of the eluted particle dispersion again revealed very uniform dispersion of the particles, the mean gold particle diameter being ca. 12 nm with an essentially uniform gelatin coating of thickness ca. 1 nm.
EXAMPLE 5. Covalent attachment of protein G to activated, gelatin- coated gold particles
To 20 ml of Sephadex® G-25-purified, activated particle dispersion prepared according to Example (a) was added 0.5 mg of protein G
(Pharmacia, Sweden) , and the solution was left to stand overnight at room temperature. A solution of 300 mg of urea in 1 ml of triply distilled water was then added and the solution was allowed to stand at room temperature for 2 hours. The resulting particle dispersion
was then centrifuged for 2 hours at 41,000 rpm (Kontron SW41.14 rotor) after layering on a two-step glycerol density medium [40% (upper layer) and 90% (lower layer), respectively, in 0.9% (w/v) aqueous NaCl] , and the particle dispersion fraction retained in the region around the interface between the two glycerol concentrations was collected.
EXAMPLE 6. Binding specificity of the dispersed particles bearing covalently attached protein G
(a) The particle dispersion prepared according to Example 5 can be used to label the Fc part of 7-globulins. Dot blots on nitrocellulose paper (see Fig. 1) revealed a much higher specificity of 7- lobulin labelling than with a particle dispersion obtained by simple (non- covalent) attachment of protein G to the gold particles of a sol prepared as described in Example 1.
(b) Using a suitable antibody directed against glucose transporter proteins in human erythrocytes, the particle dispersion prepared according to Example 5 can be used to determine the abundance (i.e. number) of these transporter proteins by TEM. In the present example a mouse monoclonal antibody against the C-terminus of the HepG2/erythroid glucose transporter was employed, the procedure being as follows:
Intact human erythrocytes were allowed to sediment onto a polylysine- coated, standard carbon-coated EM grid and the erythrocytes adhering to the grid were ruptured by washing with distilled water at < 4°C, the inner surface of the erythrocyte membrane thereby being exposed. The grid bearing the thus-ruptured erythrocytes was then incubated for 30 min in a solution [ca. 10 μg/ml; in a buffer of 20 mM phosphate in 0.9% (w/v) aqueous NaCl, pH 7.4, and containing 1% bovine serum albumin (hereafter denoted "Buffer A"] of the above- mentioned mouse monoclonal antibody and then washed five times with Buffer A. The washed grid was then incubated in a completely analogous manner with affinity purified Rabbit antibodies against mouse 7-globulin (Z 412, Dakopatts a/s, Glostrup, Denmark) and washed as before (it may be mentioned that subsequent experiments indicate
that this latter incubation with a second antibody may be omitted with only slight reduction in binding efficiency of particle-bound protein G) . The grid was then incubated for ca. 1-2 min in the particle dispersion prepared as in Example 5, washed thoroughly with Buffer A, and then treated with a 2% (v/v) solution of glutaric dialdehyde in aqueous sodium chloride/sodium phosphate (200 mM/200mM, pH 7.5) (this treatment renders the binding of the particle-bound protein G to the membrane-bound antibodies more permanent, presumably via the formation of bridging covalent linkages between the protein G moieties and the antibodies). Finally, the grid was washed with negative-staining reagent and dried as described in Example 1.
TEM (see Fig. 2) clearly showed the expected pattern of localized distribution of the gold-containing particles attached specifically to the antibody-bearing glucose transporter structures of the membrane; a count of the number of gold-containing particles bound per unit area indicates, in fact, that there is essentially quantitative binding of these gold-containing particles to the glucose transporter proteins, i.e. in 1:1 ratio. In contrast, when the incubation of the ruptured membranes with both the mouse monoclonal antibody and the rabbit antibody against mouse
7-globulins was omitted (see Fig. 3), or when the ruptured membranes were incubated only with the rabbit antibody against mouse 7-globulins (see Fig. 4), very little binding of the gold-containing particles to the membrane was visible
EXAMPLE 7. Covalent attachment of wheatgerm agglutinin to activated , gelatin- coated gold particles
Using the same procedure as in Example 5, but substituting wheatgerm agglutinin for protein G, a particle dispersion exhibiting binding specificity towards certain carbohydrates is obtained (see Example 8).
EXAMPLE 8. Affinity chromatography of the particle dispersion bearing covalently attached wheatgerm agglutinin
A particle dispersion prepared according to Example 7 was subjected to affinity chromatography on a column (typical dimensions: diam. ca. 1 cm x length ca. 3 cm) containing N -acetylglueosamine coupled to divinyl sulphone activated agarose (Kem-En-Tec Ltd ApS, Copenhagen, Denmark) . The particles of the dispersion (clearly visible on the column owing to the characteristic red colour) exhibited strong binding affinity for the column material, but could be eluted satisfactorily, in dispersed form, from the column using a 0.3 M solution of W-acetylglucosamine in aqueous Tris buffer (20 mM, pH 7.4) as competitive eluent. The stability of the dispersed particles in question towards this affinity chromatography procedure was demonstrated further by the fact that the dispersion eluted from the column could, after removal of the W-acetylglucosamine eluent by gel chromatography on Sephadex® G-25, be recyled repeatedly through the affinity column, i.e. repeatedly bound and re-eluted.
By way of comparison, when a dispersion prepared by simple (non-covalent) attachment of wheatgerm agglutinin to the gold particles of a sol prepared as described in Example 1 was subjected to the same treatment, no gold-containing material could be eluted from the affinity column using the N-acetylglucosamine eluent, but remained unspecifically adsorbed in the column.
EXAMPLE 9. Covalent attachment of goat anti-mouse -y-globulin to activated , gelatin- coated gold particles
Using the same procedure as in Example 5, but employing affinity-purified goat antibodies directed against mouse 7-globulin (Z 420, Dakopatts a/s, Glostrup, Denmark) instead of protein G, a particle dispersion suitable for determination of the glucose transporter number in a manner similar to that described in Example 6 (b) was obtained (see Figs. 5, 6 and 7).
In the above-described Examples 5, 7 and 9, the specifically binding species in question (i.e. protein G, wheatgerm agglutinin and goat anti-mouse 7-globulin antibodies) were coupled to freshly prepared, activated gelatin-coated gold particles. However, preliminary
experiments indicate that the glutaric dialdehyde-activated particle dispersions prepared as described show no major loss of ability to bind proteinaceous species of the above types within a period of at least two weeks or more at ambient temperature; at lower temperatures their "shelf-life" may well be prolonged considerably. The sale of "ready-made" activated particle dispersions of the present type to which chosen target-specific species may subsequently be covalently attached by the user is thus a realistic commercial possibility.
Claims
1. A method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water- soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer, and
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) .
2. A method for the preparation of an aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are established chemically reactive functionalities or side- chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer,
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) and, optionally, (ii) to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer, and
(d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer.
3. A method for the preparation of an aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water- soluble, on the outer surface of which layer there are functionalities or side-chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer, the method comprising
(a) providing an aqueous colloidal solution or dispersion comprising colloidal particles of a noble metal,
(b) treating said aqueous colloidal solution or dispersion comprising colloidal noble metal particles with an aqueous solution comprising said organic polymer,
(c) subjecting the colloidal solution or dispersion resulting from step (b) to a treatment leading (i) to the formation of covalent linkages between individual molecules of said organic polymer which have become adsorbed to the colloidal noble metal particles following the treatment in step (b) and, optionally, (ii) to the establishment of chemically reactive functionalities or side-chains on the outer surface of said layer, said functionalities or side-chains being capable of subsequently reacting with said chosen substance,
(d) in those cases where establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer has not been effected in step (c) , subjecting the colloidal solution or dispersion resulting from step (c) to a further treatment leading to the establishment of said chemically reactive functionalities or side-chains on the outer surface of said layer,
(e) allowing said chosen substance to react with said established, chemically reactive functionalities or side-chains so as to form a covalent bond thereto.
4. A method according to any of the preceding claims, wherein the colloidal noble metal particles of the aqueous colloidal solution or dispersion provided in step (a) are colloidal particles of a noble metal selected from the group consisting of gold, silver, rhodium, iridium, platinum, palladium, ruthenium and osmium.
5. A method according to any of the preceding claims, wherein the organic polymer from which the encapsulating layer is formed is selected from the group consisting of:
natural and synthetic polypeptides and proteins, including antigens, antibodies and glycoproteins, and
polysaccharides.
6. A method according to claim 5, wherein the organic polymer is selected from the group consisting of:
gelatins of human or animal origin,
serum albumins of human or animal origin,
agar, and
soluble starches, including modified starches
7. A method according to claim 6, wherein the organic polymer is a gelatin of human or animal origin, the aqueous solution of said gelatin used in step (b) having a pH in the region of about 11-12, preferably a pH of about 11.5.
8. A method according to any of the preceding claims, wherein the size of the colloidal noble metal particles in the aqueous colloidal solution or dispersion provided in step (a) is in the range of about 1-20 nm, preferably about 1-15 nm, more preferably about 1-10 nm, most preferably about 1-5 nm.
9. A method according to any of the preceding claims, wherein the size of the colloidal noble metal particles in the aqueous colloidal solution or dispersion provided in step (a) is predominantly in the range of 1-4 nm.
10. A method according to any of claims 1-8, wherein the size of the colloidal noble metal particles in the aqueous colloidal solution or dispersion provided in step (a) is predominantly in the range of 10-15 nm.
11. A method according to any of the preceding claims, wherein the thickness of the covalently linked layer of organic polymer formed
on the colloidal noble metal particles is in the range of about 1-2 nm.
12. A method according to any of the preceding claims, wherein the thickness of the covalently linked layer of organic polymer formed on the colloidal noble metal particles is about 1 nm.
13. A method according to any of the preceding claims, wherein the treatment in step (c) leading to the formation of covalent linkages between individual molecules of said organic polymer is selected from:
a treatment with a bifunctional cross-linker or fixative, and
a photoactivation treatment.
14. A method according to claim 13, wherein the bifunctional cross- linker or fixative is selected from the group consisting of:
aliphatic dialdehydes, including succinic dialdehyde and glutaric dialdehyde,
divinyl sulphone, and
disuccinimidyl suberate.
15. A method according to any of claims 2-14, wherein the treatment in step (c) or (d) leading to the establishment of chemically reactive functionalities or side-chains on the outer surface of said layer is a treatment with a bifunctional cross-linker or fixative.
16. A method according to claim 15, wherein the bifunctional cross- linker or fixative is selected from the group consisting of:
aliphatic dialdehydes, including succinic dialdehyde and glutaric dialdehyde,
divinyl sulphone, and
disuccinimidyl suberate.
17. A method according to any claims 2-16, wherein said chosen substance is an organic substance with the ability, when covalently attached via said functionalities or side-chains, to bind specifically and non-covalently to a further organic substance or material.
18. A method according to claim 17, wherein said chosen substance is selected from the group consisting of:
antigens and antibodies, including monoclonal and polyclonal antibodies,
lectins, including wheatgerm agglutinin, Concanavalin A, Phytohaemagglutinin and Pokeweed mitogen,
avidi ,
protein A,
protein G,
RNA, and
DNA.
19. An aqueous colloidal solution or dispersion of particles prepared by a method according to any of claims 1-14 and comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
20. An aqueous colloidal solution or dispersion of chemically reactive particles prepared by a method according to any of claims 1-18 and comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
21. An aqueous colloidal solution or dispersion of particles prepared by a method according to any of claims 1-18 and comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are functionalities or side-chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
22. An aqueous colloidal solution or dispersion of particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
23. An aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are established chemically reactive functionalities or side-chains capable of subsequently
reacting with a chosen substance so as to form a covalent bond thereto, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
24. An aqueous colloidal solution or dispersion of chemically reactive particles comprising noble metal particles substantially permanently encapsulated by a layer of a relatively inert organic polymer which in its free state is substantially water-soluble, on the outer surface of which layer there are functionalities or side- chains via which a chosen substance is covalently attached, the substantial permanence of the encapsulation of the noble metal particles by said layer being due to the presence of covalent linkages formed between individual encapsulating molecules of said organic polymer.
25. An aqueous colloidal solution or dispersion according to any of claims 22-24, said noble metal particles being particles of a noble metal selected from the group consisting of gold, silver, rhodium, iridium, platinum, palladium, ruthenium and osmium.
26. An aqueous colloidal solution or dispersion according to any of claims 22-25, said organic polymer from which the encapsulating layer is formed being a member of the group consisting of:
natural and synthetic polypeptides and proteins, including antigens, antibodies and glycoproteins, and
polysaccharides,
27. An aqueous colloidal solution or dispersion according to claim 26, said organic polymer being a member of the group consisting of:
gelatins of human or animal origin,
serum albumins of human or animal origin,
agar,
soluble starches, including modified starches
28. An aqueous colloidal solution or dispersion according to any of claims 22-27, the size of the noble metal particles being in the range of about 1-20 nm, preferably about 1-15 nm, more preferably about 1-10 nm, most preferably about 1-5 nm.
29. An aqueous colloidal solution or dispersion according to any of claims 22-28, the size of the noble metal particles being predominantly in the range of 1-4 nm.
30. An aqueous colloidal solution or dispersion according to any of claims 22-28, the size of the noble metal particles being predominantly in the range of 10-15 nm.
31. An aqueous colloidal solution or dispersion according to any of claims 22-30, the thickness of the covalently linked layer of organic polymer on the noble metal particles being in the range of about 1-2 nm.
32. An aqueous colloidal solution or dispersion according to any of claims 22-31, the thickness of the covalently linked layer of organic polymer on the noble metal particles being about 1 nm.
33. An aqueous colloidal solution or dispersion according to any of claims 23-32, said functionalities or side-chains being derived from a bifunctional cross-linker or fixative.
34. An aqueous colloidal solution or dispersion according to claim
33. said bifunctional cross-linker or fixative being a member of the group consisting of:
aliphatic dialdehydes, including succinic dialdehyde and glutaric dialdehyde,
divinyl sulphone, and
disuccinimidyl suberate.
35. An aqueous colloidal solution or dispersion according to any of claims 24-34, said covalently attached chosen substance being an organic substance with the ability, while covalently attached via said functionalities or side-chains, to bind specifically and non- covalently to a further organic substance or material.
36. An aqueous colloidal solution or dispersion according to claim 35, said covalently attached chosen substance being a member of the group consisting of:
antigens and antibodies, including monoclonal and polyclonal antibodies,
lectins, including wheatgerm agglutinin, Concanavalin A, Phytohaemagglutinin and Pokeweed mitogen,
avidi ,
protein A,
protein G, .
RNA, and
DNA.
37. The use of an aqueous colloidal solution or dispersion according to any of claims 19-36 for the labelling, staining, detection, identification or localization of organic substances or particular regions or moieties of organic materials, e.g. in preparations of biological material.
38. The use according to claim 37 in connection with electron microscopy, light microscopy, electrophoretic techniques or ultracentrifugation.
39. The use of an aqueous colloidal solution or dispersion according to any of claims 19-36 for the purification or fractionation of preparations of biological material.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
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DK1420/89 | 1989-03-22 | ||
DK142089A DK142089D0 (en) | 1989-03-22 | 1989-03-22 | Precious metal particles with cross-linked, inert coating for detection of biomolecules |
Publications (1)
Publication Number | Publication Date |
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WO1990011128A1 true WO1990011128A1 (en) | 1990-10-04 |
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PCT/DK1990/000080 WO1990011128A1 (en) | 1989-03-22 | 1990-03-22 | Polymer-coated noble metal particles and a method for their preparation |
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AU (1) | AU5350090A (en) |
DK (1) | DK142089D0 (en) |
WO (1) | WO1990011128A1 (en) |
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WO1997034150A1 (en) * | 1996-03-14 | 1997-09-18 | Abbott Laboratories | Binding members extending from particles for immunoassay |
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---|---|---|---|---|
EP0428412A2 (en) * | 1989-11-16 | 1991-05-22 | Ortho Diagnostic Systems Inc. | Method of producing a metal sol reagent containing colloidal metal particles of a preselected size |
EP0428412A3 (en) * | 1989-11-16 | 1992-02-26 | Ortho Diagnostic Systems Inc. | Method of producing a metal sol reagent containing colloidal metal particles of a preselected size |
GR900100804A (en) * | 1989-11-16 | 1992-04-17 | Ortho Diagnostic Systems Inc | Method of producing a metal for reagent containingmethod of producing a metal for reagent containing colloidal metal particles of a preselected size colloidal metal particles of a preselected size |
WO1997034150A1 (en) * | 1996-03-14 | 1997-09-18 | Abbott Laboratories | Binding members extending from particles for immunoassay |
EP0811848A2 (en) * | 1996-06-03 | 1997-12-10 | Bayer Corporation | Sol particle decay protection immunoassay |
EP0811848A3 (en) * | 1996-06-03 | 1998-05-27 | Bayer Corporation | Sol particle decay protection immunoassay |
Also Published As
Publication number | Publication date |
---|---|
DK142089D0 (en) | 1989-03-22 |
AU5350090A (en) | 1990-10-22 |
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