EP1493031A2 - Active solid support and method for surface immobilization of combinatorial compounds or libraries - Google Patents
Active solid support and method for surface immobilization of combinatorial compounds or librariesInfo
- Publication number
- EP1493031A2 EP1493031A2 EP03745343A EP03745343A EP1493031A2 EP 1493031 A2 EP1493031 A2 EP 1493031A2 EP 03745343 A EP03745343 A EP 03745343A EP 03745343 A EP03745343 A EP 03745343A EP 1493031 A2 EP1493031 A2 EP 1493031A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- group
- active
- protein
- reacted
- solid support
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 125000000217 alkyl group Chemical group 0.000 claims abstract description 6
- 125000003710 aryl alkyl group Chemical group 0.000 claims abstract description 6
- WERYXYBDKMZEQL-UHFFFAOYSA-N butane-1,4-diol Chemical compound OCCCCO WERYXYBDKMZEQL-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229940000406 drug candidate Drugs 0.000 claims abstract description 6
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- 239000002253 acid Substances 0.000 claims abstract description 4
- JHWGFJBTMHEZME-UHFFFAOYSA-N 4-prop-2-enoyloxybutyl prop-2-enoate Chemical compound C=CC(=O)OCCCCOC(=O)C=C JHWGFJBTMHEZME-UHFFFAOYSA-N 0.000 claims abstract description 3
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- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 abstract 1
- 125000003647 acryloyl group Chemical group O=C([*])C([H])=C([H])[H] 0.000 abstract 1
- NHBRUUFBSBSTHM-UHFFFAOYSA-N n'-[2-(3-trimethoxysilylpropylamino)ethyl]ethane-1,2-diamine Chemical compound CO[Si](OC)(OC)CCCNCCNCCN NHBRUUFBSBSTHM-UHFFFAOYSA-N 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
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- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 6
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 6
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 6
- 108020004414 DNA Proteins 0.000 description 5
- NIXOWILDQLNWCW-UHFFFAOYSA-N acrylic acid group Chemical group C(C=C)(=O)O NIXOWILDQLNWCW-UHFFFAOYSA-N 0.000 description 5
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- 125000003396 thiol group Chemical group [H]S* 0.000 description 5
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
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- SHKUUQIDMUMQQK-UHFFFAOYSA-N 2-[4-(oxiran-2-ylmethoxy)butoxymethyl]oxirane Chemical compound C1OC1COCCCCOCC1CO1 SHKUUQIDMUMQQK-UHFFFAOYSA-N 0.000 description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
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- 108091034117 Oligonucleotide Proteins 0.000 description 2
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- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
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- RPFLVLIPBDQGAQ-UHFFFAOYSA-N 1,2-diisothiocyanatobenzene Chemical compound S=C=NC1=CC=CC=C1N=C=S RPFLVLIPBDQGAQ-UHFFFAOYSA-N 0.000 description 1
- WPANETAWYGDRLL-UHFFFAOYSA-N 4-aminobenzenecarboximidamide Chemical compound NC(=N)C1=CC=C(N)C=C1 WPANETAWYGDRLL-UHFFFAOYSA-N 0.000 description 1
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- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
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- 238000013537 high throughput screening Methods 0.000 description 1
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- 239000001509 sodium citrate Substances 0.000 description 1
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- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
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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
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54353—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals with ligand attached to the carrier via a chemical coupling agent
Definitions
- This invention is a new synthetic method of immobilizing drags and drag candidates, as well as generally biologically active molecules, so that their state of biological activation and binding site selectivity remains unaltered.
- this invention provides a method to synthesize a new solid support or support group, which can bind drugs and drug candidate molecules - in scientific terms immobilization or anchoring - namely, through the use of reactive groups placed on linkers with branched structures.
- Our newly invented supports are capable of covalently anchoring small molecules having no linkers and/or molecules that contain linkers of various lengths and reactive groups.
- Such solid supports can be used to create various small molecule arrays, as well as their applications in molecular fields of agrochemisuy, biology, biotechnology and pharmacology.
- Affinity-based methods for identifying and isolating proteins were previously used for immobilizing small molecules on cellulose and various polymers.
- the protein mixture is streamed through the resulting affinity columns.
- the proteins then leave the columns at various speeds, according to their binding affinity toward the immobilized small molecules, and are thus separated from the non-binding or hardly-bound proteins.
- affinity chromatography Collectively known as affinity chromatography, these methods involve time-consuming procedures, require large samples of the small molecules, and do not allow for easy parallel application.
- microarray'' is used in the following description of our invention to denote a miniaturized planar array of small, immobilized molecules.
- the advantage of microarrays over classical high-throughput biological screening is that the anchored compounds are arranged adjacent to each other, so that a large number of measurements under identical conditions can be carried out, allowing for more accurate comparison.
- a further advantage is that the microarray can be combined with high-capacity robotized technologies.
- Small molecules are often equipped with a functional group so that they are able to anchor to the surface of the support.
- a functional group may be a terminal thiol, amino or carboxyl group introduced through a linker arm.
- the modified small molecule may anchor to a surface bearing a disulphide, maleimide, amino, carboxy, esther, epoxy, bromo-cyanide or aldehyde functional group. Binding occurs via the formation of thioether, ether, amide or amine bonds between the small molecule and the surface.
- arrays of peptides, oligonucleotides, small organic molecules, and ligands are produced by immobilizing them on solid supports covered by either glycane or polymers used in solid phase synthesis.
- the bond is via amino, carboxy and/or hydroxy groups of the polymer.
- the drawback of methods using mercapto or epoxy silanized surfaces (USA 5 919 626) is that immobilized molecules lie too close to the surface, which causes poor accessibility for the probe molecules and, consequently, a decrease in number the number of specific bonds formed.
- Developing microarrays for combinatorial chemistry is based on applying solid supports, which can anchor various small molecules. These anchored synthetic molecules have numerous advantageous applications.
- the combinatorial chemical microarrays may potentially become important tools in molecular biology and drug development research.
- this invention consists of a method for producing an active sohd support that is suitable for anchoring drag molecules by covalent bonds, by the following methods: a.) a sohd support, prepared ahead of time, preferably by pre-treatment executed in the presence of strong bases or acids, is reacted with a polyani e-functionahzed alkoxy-silane derivative, preferably 3-[2-(2-annnoethylamino)ethylamino]propyl- trimethoxy-silane, and then washed and dried, bl) the sohd support obtained in the procedure described in Section a.) is reacted with (I)
- Z: bond or a -CO or -CNR1 group, where Rl is an alkyl, aralkyl, or aryl group;
- A: branched or unbranched alkyl or substituted aryl or aralkyl group containing 1-5 carbon atoms;
- the support prepared using the method described in part (a) is reacted with a bi-functional reagent, preferably with 1,4-butanediol dighcidyl-ether or epichlorohydrin in the presence of an organic or inorganic scavenger, which contains terminal epoxy groups and an amino-group reactive functional subgroup.
- glass, polystyrol or polypropylene chips preferably glass microscope slides, are used as the sohd support.
- the invention furthermore consists of the active sohd support produced using the various methods described above.
- a further objective of the invention is to apply the active sohd support described above for the production of both chemical and combinatorial chemical microarrays.
- An additional objective of the invention is to provide a method for immobilizing organic molecules of what is termed "low molecular weight,” having a size of 1500 Da or lower, preferably between 150 and 750 Da. This is achieved by anchoring an unmodified or tethered drug or drag candidate molecule to the active sohd support described above by adding the appropriate functional groups of the drug or drug candidate molecule, preferably executed by a miniaturized robotized binding technique.
- the microarray produced using the active sohd support and the procedure described above, is used for quick execution of agrochemical, biotechnological, biological, or medicinal chemical tests, and especially for studying the interactions between small molecules and proteins.
- Another objective of this invention is to use the microarray, produced using the active solid support and the procedure described above, for identifying new proteins, for rapid analysis of the draggability of proteins, for determining secondary binding sites, for identifying nucleic acids (RNA, DNA) that bind small molecule ligands, for identifying protein-protein, protein-DNA, protein-lipid, protein-nucleotide, protein- carbohydrate, protein-vitamine, and protein-metal ion complexes, or for identifying ligands and/or metal ions.
- RNA nucleic acids
- a further objective consists in the use of the microarray, produced using the active sohd support and the procedure described above, for biological screening assays, in which the microarrays, containing immobilized ligands, and known or unknown proteins or complexes of these proteins formed using biomolecules, are used in either labeled (for example with fluorescent tag) or unlabeled form, or labeled or unlabeled complexes of known or unknown nucleic acids are used, or ions or their complexes are used, or eukaryotic or prokaryotic cells are used, after which the quantity and quality of the bound proteins, nucleic acids, ions, or cells is dete ⁇ nined using suitable analytical technologies.
- Another objective of the invention is to use the microarray, produced using the active sohd support and the procedure described above, for determination of anchoring kinetics, balances and kinetic constants, and balance constants.
- the procedure we have invented may be used to achieve quick and inexpensive immobilization and anchoring of small molecules used as drugs or considered as drag candidates that do or do not contain branched linkers.
- a covalent bond forms between the new sohd support we have invented and the molecules to be anchored.
- the covalent bond results in high stabihty and constant surface concentration.
- the molecules anchored to the surface are called sample molecules, while the compounds that react with those molecules are called probes.
- the method described as part of this invention does not require chemical modifications of the probes to be anchored.
- This approach significantly reduces the costs involved in high throughput production.
- the anchoring does not require further chemical reactions, as opposed to the methods mentioned above such as reduction of the newly formed bonds. Therefore, it is an advantage that the samples containing chemical groups, sensitive to reduction, can be anchored to the sohd structure without damage or modifications to the structure.
- synthetic molecules containing amino, imino, or thiol functional groups or heteroeyclic nitrogen are anchored to a sohd support that has reactive acrylic functional groups on its surface.
- Another variation of our invention involves the anchoring of synthetic molecules containing amino or thiol functional groups to a sohd support that has reactive epoxy functional groups on its surface.
- the functional groups are anchored in high density to the sohd support via branching, non-flexible or conformationally flexible linker arms enabling high-i ⁇ miobihzed molecule concentration on the surface.
- Another advantage of the system is that the linker arms give small molecules better access to the sample and allow for the creation of stable covalent bonds between the active surfaces and the drag or drag candidate molecules.
- a further advantage of our invention is that the hydrophobic properties of the surface can be modulated, which in turn directly affects the density of the microarray produced.
- a hydrophihc surface is used to produce microarrays, larger spots are formed, resulting in a microarray of lower density (100-2000 spots/microarray).
- the hydrophihc property of the surface can, however, be helpful in affinity experiments because the probe molecule does not bind to the surface aspecifically.
- the spots generated using a hydrophobic surface are smaller, hence the density of the microarray is higher (2000-6000 spot microarray), though in certain affinity experiments this may be less desirable.
- the method described herein specifies the use of properly pretreated sohd supports (described below), such as glass, polypropylene or polystyrene slides, preferably glass microscope shdes.
- sohd supports such as glass, polypropylene or polystyrene slides, preferably glass microscope shdes.
- commercially available aldehyde-coated slides were used (SuperAldehyde Cat. No. SMA-25, Arrayit, USA).
- the procedure for pretreating the shdes includes two steps: (i) etching in NaOH and (ii) washing in distilled water.
- Another useful variation of our invention includes derivatization of the pretreated sohd supports in two reaction steps. In the first step, the supports are reacted, preferably with 3-[2-(2-am oethyla ⁇ n o)ethylam
- Free amino functional groups are introduced to the surface during this step.
- the triamino-silanized surface is treated with acryloyl-chloride.
- the result is a molecule having branching carbon chain and amide-bonds, and a structure that includes reactive acryl groups.
- the surface can be modulated to hold active epoxy groups and have either hydrophobic or hydrophihc properties.
- supports are treated with 1,4-butanediol diglycidyl ether or epichlorohydrin, respectively, in the second reaction step. In these cases, epoxy groups are introduced into the positions of the amino groups in the branching structure.
- glass shdes pretreated with NaOH and water are reacted with 3-[2-(2-am oethylammo)ethylamino]propyl- trimethoxysilane in an alcohol solution, preferably a 95% aqueous solution of ethanol or methanol, in the first step of derivatization. Later, the shdes are washed using the same alcohol and water and dried at 100-110°C.
- the shdes are treated with acryloyl-chloride in a non-polar solvent, preferably dichloroethane, dichloromethane or something similar, in the presence of an equivalent amount of a base such as pyridine, dhsopropylethyl-amine, or another similar base. Finally, the shdes are washed using the applied solvent and dried.
- a non-polar solvent preferably dichloroethane, dichloromethane or something similar
- a base such as pyridine, dhsopropylethyl-amine, or another similar base.
- the shdes are treated with 1,4-butanediol-diglycidyl-ether in short carbon chain alcohol containing a dissolved base, preferably in ethanol containing NaOH.
- the shdes may be treated with epichlorohydrin in a non-polar solvent containing a dissolved base, preferably chloroform containing pyridine. Later, the resulting hydrophobic shdes are washed with the particular solvent used and dried.
- sohd supports derivatized using the method described herein allows for immobilization of synthetic molecules at a high density and in an ordered pattern with high stabihty for use in chemical microarray technology.
- the sohd supports produced using the method we have developed are especially suitable for the preparation of chemical microarrays by means of immobihzation of small organic compounds.
- the produced microarrays can be used for investigating interactions between (i) small molecules and proteins, (ii) small molecules and nucleic acids, (hi) small molecules and biomolecules of low molecular weight, or for protein detection based on biochemical interactions, or for high throughput screening of biologically active drug-like molecules.
- the invention is also appropriate for proteomic studies, combinatorial analyses of different nucleic acids, proteins, and molecules of low molecular weight.
- the invention may be used in chemistry, molecular biology, biotechnology, agrochemistry, proteomic studies, and drug development.
- the most generally used method for protein detection involves labeling with fluorescent dyes or the use of fluorescent proteins in high-tliroughput biological screening.
- the method of immobilization we have developed may be applied to fluorescent detection and to other new detection techniques, such as surface plasmon resonance (DE 100 27 397) and planar waveguide (WO-0155691) combined with fluorescent labeling. These techniques detect small changes in surface properties corresponding to biological interactions.
- Figure 1 Chemical arrays prepared by manual pipetting using three developed support surfaces (a: an acrylic surface, b: a hydrophihc epoxy surface, c: a hydrophobic epoxy surface).
- the two lower spots represent samples containing a pH buffer, while the two upper spots represent samples without a pH buffer.
- Figure 2 Arrays prepared by robotic printing using three developed support surfaces (a: an acrylic surface, b: a hydrophihc epoxy surface, c: a hydrophobic epoxy surface). 1-6: concentrations of the apphed biotin solutions.
- Figure 3 Arrays prepared by robotic printing using three developed support surfaces (a: an acrylic surface, b: a hydrophihc epoxy surface, c: a hydrophobic epoxy surface). 1-6: concentrations of the 4-amino-benzamidine solutions used.
- step a Preparation of a triamino silanized surface
- step b Preparation of a reactive acrylic surface
- silanized shdes obtained in step a. were incubated for 2 hours with 30 mmol acryloyl-chloride (ICN Biomedicals Inc., Aurora, Ohio) and 30 mmol dusopropylelhyl-a ine (ICN. Biomedicals Inc., Aurora, Ohio) dissolved in 62.5 mL dicloroethane (DCE) (Merck, Germany). Later, the slides were washed with DCE and dried at room temperature.
- DCE dicloroethane
- Shdes obtained from Example 1, step a.) were incubated for 2 hours with 30 mol epichlorohydrin (Fluka, Germany) and 12mmol (1 mL) pyridine (Fluka, Germany) dissolved in 66.7 mL chloroform (Molar Chemicals Ltd., Hungary, purity >99%). Later, the shdes were washed with chloroform and dried at room temperature.
- Shdes obtained from Example 1, step a.) were incubated for 2 hours with 30 mmol 1,4-butanediol diglycidyl ether (Fluka, Germany) and 5 mmol NaOH dissolved in 63 mL ethanol (Molar Chemicals Ltd., Hungary, purity >99.7%). Afterwards the slides were washed with ethanol and dried at room temperature.
- biotin having an amino linker arm was introduced to the developed surfaces, either manually or using a MicroGrid Total Array System spotting robot (BioRobotics, England).
- the solvents used in both experiments were: dimethyl formamide, dimethyl suffoxide, and water.
- Biotin solutions were introduced in several concentrations in a pH 10 phosphate buffer or without any pH buffer.
- the shdes were incubated for 2 hours at room temperature in a humidity-controlled chamber in order to prevent evaporation of sample droplets.
- the introduced sample volume is 1-0.5 ⁇ L or 1 nL, respectively.
- the shdes After sample immobilization the shdes are washed using an aqueous solution of 1 x SSC (0.1 M NaCl, 15 M tri-sodium-citrate), 1% BSA (bovine serum albumin), and 0.2 w/w% SDS (so(hum-dodecd-sulfate) and then water. Later, the shdes were dried at room temperature and stored at ambient temperature in the dark.
- 1 x SSC 0.1 M NaCl, 15 M tri-sodium-citrate
- BSA bovine serum albumin
- SDS so(hum-dodecd-sulfate
- Arrays prepared in Example 4 were reacted with 10 ⁇ L of Cy3 streptavidine (10 mg L) under a glass coverslip for 1 hour at room temperature. Then the shdes were washed in three steps as follows: Step 1. washing using PBS (phosphate buffer, pH 7.0), Step 2. washing using 0.2 x SSC , Step 3. washing using 2 x SSC. After the shdes had dried, they were scanned using a GSI Lumonics, Scannaray Lite confocal laser scanner. Results are shown in Figures 1 and 2. It was found that immobilization was more effective at higher pH values for all three developed supports. Immobilization is not affected by the solvent used.
- Example 4 As with Example 4, 4'-(6-amino-hexd-ammo)-benzamidine was arrayed, both manually and using a printing robot (MicroGrid Total Array System, BioRobotics, England), onto the developed surfaces in an aqueous solution containing a pH 10 phosphate buffer in six different concentrations. The shdes were incubated for 2 horns at room temperature in a humidity-controlled chamber in order to prevent evaporation of sample droplets.
- a printing robot MicroGrid Total Array System, BioRobotics, England
- the shdes were washed, first with water, then using an aqueous solution of 1 x SSC (0.1 M NaCl, 15 mM to ⁇ -sochum- citrate), 1% BSA (bovine serum albumin) and 0.2 w/w% SDS (sodium-dodecil- sulfate), and then with water again. Later, the slides were dried at room temperature and stored at ambient temperature in the dark.
- 1 x SSC 0.1 M NaCl, 15 mM to ⁇ -sochum- citrate
- BSA bovine serum albumin
- SDS sodium-dodecil- sulfate
- Trypsin was used for a probe molecule, fluorescently labeled with Alexa 647 dye.
- Amino reactive Alexa 647 carboxyhc acid succinimidyl ester (Molecular Probes, USA) was used as a labeling reagent.
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HU0201091A HU226993B1 (en) | 2002-03-29 | 2002-03-29 | Novel active carrier and process for immobilization of combinatorial compounds and compound libraries |
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PCT/HU2003/000025 WO2003083477A2 (en) | 2002-03-29 | 2003-03-28 | Active solid support and method for surface immobilization of combinatorial compounds or libraries |
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US7723126B2 (en) * | 2004-03-24 | 2010-05-25 | Wisconsin Alumni Research Foundation | Plasma-enhanced functionalization of inorganic oxide surfaces |
US20060147943A1 (en) * | 2004-12-30 | 2006-07-06 | Lewis Mark A | Substrates having pendant epoxide groups for binding biomolecules and methods of making and using thereof |
US7396676B2 (en) | 2005-05-31 | 2008-07-08 | Agilent Technologies, Inc. | Evanescent wave sensor with attached ligand |
US20070099180A1 (en) * | 2005-10-31 | 2007-05-03 | Robotti Karla M | Evanescent wave sensor with attached ligand |
HUP0600668A2 (en) | 2006-08-22 | 2008-02-28 | Avicor Kft | Active carrier, process for producing thereof and the use of thereof |
US8029902B2 (en) | 2006-12-11 | 2011-10-04 | Wisconsin Alumni Research Foundation | Plasma-enhanced functionalization of substrate surfaces with quaternary ammonium and quaternary phosphonium groups |
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