US20040235146A9 - Surface for the immobilization of ligands - Google Patents
Surface for the immobilization of ligands Download PDFInfo
- Publication number
- US20040235146A9 US20040235146A9 US10/297,244 US29724403A US2004235146A9 US 20040235146 A9 US20040235146 A9 US 20040235146A9 US 29724403 A US29724403 A US 29724403A US 2004235146 A9 US2004235146 A9 US 2004235146A9
- Authority
- US
- United States
- Prior art keywords
- binding
- anchor
- interaction
- measuring
- molecules
- 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.)
- Abandoned
Links
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- 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
- the present invention relates to binding surfaces for the immobilization of ligands, ligand surfaces and structured surface arrays which present a plurality of identical or different ligands.
- the invention further relates to a process for the production and the use of such surfaces and to specific binder molecules which can be used for the preparation thereof.
- receptor-ligand systems wherein the receptor is usually a biomacromolecule (e.g. a protein or single-stranded DNA) and the ligand is a “probe”, a molecule usually of low molecular weight and of biological or synthetic origin (peptides, oligonucleotides or what are referred to as small organic molecules).
- a biomacromolecule e.g. a protein or single-stranded DNA
- the ligand is a “probe”, a molecule usually of low molecular weight and of biological or synthetic origin (peptides, oligonucleotides or what are referred to as small organic molecules).
- ligands exhibit highly specific structural properties which can interact with a receptor provided it exhibits corresponding structures.
- the binding to the receptor can take place via one or several ligands.
- systems are studied wherein both interaction partners are macromolecules (e.g. protein-protein interaction).
- Interaction analysis is used in the pharmaceutical and agrochemical industries for researching active substances. The goal is to analyze as large a number of different samples as possible in as short a time period as possible. There is a growing interest to develop testing systems that are able to simultaneously subject a large number of both identical and different molecules to a certain test method for detecting their biospecific binding behavior in order to identify molecules with a specific effect (High-Throughput Screening, HTS). Such a parallelization is closely linked to a simultaneous miniaturization of the testing arrangement and automation of the testing procedure. Furthermore, interaction analysis is used in the research of the genome (polymorphisms (SNP) analysis or expression pattern analysis) and also in food analysis.
- SNP polymorphisms
- the bond is preferably a covalent bond or a bond formed by adsorption.
- the immobilization of an interaction partner facilitates the process, such as for example carrying out washing steps, and in combination with a suitable, most of the time optical, detection process (e.g. fluorescence measurement) it may provide information on the presence and degree of interaction between the interaction partners on a molecular level. Comparability and reproducibility of the results are important prerequisites for the interpretation of such data stemming from the interaction between receptor and ligand.
- Bioactive surfaces can be generated on different organic and inorganic carriers, such as for example cellulose, silicates or noble metal surfaces.
- these carriers can be components of a sensor system.
- films consisting of organic monolayers (Bain and Whitesides, Angew. Chem. 101 [Applied chemistry 101] (1989) 522-8; Zhong and Porter, Anal. Chem. [Analytical chemistry] (1995) 709A-715A) are especially advantageous (physicochemical stability, structural uniformity). Processes are known for preparing such films, wherein for example in a first step alkyl thiols are chemisorbed onto gold.
- the long-chain molecules assemble on the solid phase as a highly ordered monolayer (self-assembled monolayer, SAM), whereby the gold atoms are complexed by the sulfur functions.
- SAM self-assembled monolayer
- Short-chain alkyl thiols however, such as butane thiol, do not form such highly ordered monolayers (R. Berger, et al., Science 27 (1997), 2021-2024).
- SAMs which in the above-mentioned case do not yet exhibit any specific bioactive structures, are known in the prior art and have been appropriately characterized by means of many physical methods. In Poirier and Pylant, Science, 272 (1996) 1145-8, scanning tunneling microscopic photographs of such monolayers on gold can be found. Apart from gold, other metals as well can serve as the solid phase for such monolayers.
- anchors The immobilization of an interaction partner imparting bioactivity to the surface, in particular to a sensor surface, can be effected by so-called anchors.
- a suitable anchor molecule comprises at least two functional units present at opposite ends of the anchor and allowing binding to the solid phase surface on the one hand and binding to the interaction partner to be immobilized on the other hand. The latter will be referred to in the following as “head group”.
- head group The nature of the structural element connectiong these functional groups should preferably be such that it allows at least the formation of a highly ordered monolayer on the basis of the anchor molecules.
- the generation of a bioactive surface using such anchors can be carried out in one or several steps.
- the interaction partner is bonded to the head group of the anchor prior to its immobilization.
- Suitable anchors for the single-step process are described in DE 199 24 606.8.
- LAC ligand-anchor conjugate
- a binding surface in the form of an organic boundary layer is generated which does not yet exhibit the desired specific structural features, however, which is suitable for binding or can be activated to bind the interaction partners to be immobilized, e.g. ligands.
- immobilization of an interaction partner takes place after the generation of a first boundary layer that is not bond-specific by means of a covalent, ionic or complex bond via the head group of the binding component.
- the head group can be used directly or after it has been activated. Examples of such head groups are described in EP 485 874 A2.
- An important advantage of the multi-step process is the fact that the same surface can be used for every interaction partner to be immobilized. Therefore, the resulting concentration of the interaction partners to be immobilized is the same on the surface as well, which is not or not always a given in the single-step process. This is particularly important for the comparability and reproducibility of the different measuring results.
- the chemical homogeneity of the non-biospecific binding surface decreases again as the number of potentially necessary activation steps increases.
- a step-by-step formation of a diluted binding layer is described in WO 98/40739 A1, wherein in a first step cystamine is applied onto a gold surface and the provision of maleimide as head group is realized in a second chemical reaction.
- the disadvantage of this process is that another activation step is required for obtaining a binding surface.
- reaction partner is often a macromolecule carrying a maleimidyl group.
- the product of the reaction of the thiol and the maleimidyl group is a thio succinimide (Michael addition) (G. T. Hermanson, Bioconjugate Techniques, Academic Press 1996, 229-252; Suda et al. Biochem. Biophys. Res. Comm. (1999), 261, 276-282).
- a number of reaction partners for the thiol group are known from the prior art (referred to in the following as mercaptophiles), which also allow its selective and quantitative reaction.
- maleimides include the following compounds:
- the thiol/mercaptophile system offers the essential advantage that the binding of the interaction partner to be immobilized can be carried out under gentle reaction conditions (ambient temperature, neutral pH, buffer solutions can be used). This is of particular importance when unstable compounds or proteins that denature easily are used. Another advantage is that compared to carboxylic acid, amine or amide groups, a thiol functionality hardly ever occurs in active substances. Thus, an undesired reaction of mercaptophiles that may remain after the immobilization of one interaction partner and the target can largely be avoided.
- the thiol/maleimide system is furthermore particularly known for its high degree of selectivity compared to other functionalities such as hydroxyl, amine, carboxyl or hydroxylamine groups.
- the formation of the covalent bond is characterized by a high reaction rate (cf. Schelté et al., Bioconj. Chem. (2000), 11, 118-123).
- anchors comprising both a thiol group for binding to the solid phase surface and a mercaptophilic head group for immobilizing an interaction partner are not disclosed in the prior art since both during synthesis and during the generation of the binding surface both intra- and intermolecular side reactions, and even polymerization, may occur (with respect to the purposeful polymerization of oligothiols with bis-maleimides, cf. L. R. Dix et al., Eur. Polym. J. 31 (1995), 653-658).
- EP 485 874 A2 refers to this problem, which is avoided by the exclusive use of disulfide and sulfide groups in the anchor, in order to use for example maleimide as head group for the immobilization of proteins (reaction with —SH group of a Fab′ fragment).
- EP 698 787 A1 uses short-chain, maleimide-carrying disulfide anchors for the immobilization of proteins.
- anchors based on disulfide and sulfide groups show the disadvantage that an undesired spatial proximity of the head groups is generated. In this case, the head groups can interfere with each other in the immobilization reaction.
- EP 485 874 A2 and EP 698 787 A1 do not only apply anchor molecules onto the solid phase surface. Rather, the anchor molecules are “diluted”. This is due to the fact that if one partner, for example a ligand is bound to a solid phase carrier for interaction analysis, adjacent ligands on the surface can interfere with each other or with the interaction between the adjacent ligand and the interaction partner to be detected.
- mixed surfaces as shown in FIG. 1—can be applied, which are composed of ligand-carrying anchor molecules and so-called diluting molecules that do not carry any ligands and thus dilute the measuring surface.
- the structural nature of the diluting component has to meet the prerequisite that it will not influence the interaction of the immobilized interaction partner and the free interaction partner. In particular, if possible, no specific or unspecific binding between the free interaction partner and the diluting component should occur (e.g. diluant with as high a resistance to protein adsorption as possible). Furthermore, the anchor molecule and the diluting component should be as structurally similar as possible to ensure that their mixing behavior on the solid phase surface is as homogeneous as possible.
- binding surfaces presenting reactive head groups which are capable of entering into selective and highly quantitative reactions with interaction partners to be immobilized.
- the present invention relates to novel anchor molecules comprising these head groups which can be used, together with diluting molecules, to provide such binding surfaces.
- the invention further relates to sensor or measuring surfaces obtainable by immobilizing specific interaction partners on the binding surfaces of the present invention, as well as to surface arrays comprising a plurality of such identical or different measuring surfaces.
- a binding surface according to the present invention comprises a solid phase carrier on which an organic monolayer can assemble spontaneously, preferably comprising at least two components, a binding component and a diluting component (anchor and diluant).
- the solid phase carrier consists of a substrate formed by a metal, preferably a noble metal, especially preferred gold, or carries a layer of such a metal.
- the metal layer can be applied with the help of an intermediate layer which serves as a primer.
- the material used for the substrate depends on measuring method employed. If optical reflection processes such as surface plasmone resonance (SPR) are used, the substrate preferably consists of glass or a plastic material. Due to the use of sulfur-containing compounds for the immobilization of ligands, a gold layer and a chromium layer as a primer are preferably applied onto such substrates.
- the spatial design of the solid phase carrier to be used in accordance with the present invention is not restricted, although planar, two-dimensional structures are preferably used for sensor applications. However, depending on the field of application, three-dimensional shapes such as spheres or hollow bodies may be used as well.
- Processes for the Generation of the binding layers according to the present invention start from a solution of the anchor molecules which is brought into contact with the solid phase carrier.
- Preferred diluted measuring surfaces are obtained when a solution is used that contains a mixture of anchor molecules and diluting components in a certain molar ratio.
- the solution has a molar ratio of anchor/diluant between 1:2 and 1:10,000.
- Especially preferred dilution ratios range from 1:10 to 1:100.
- the total concentration of anchor and diluting components preferably lies in the range of from 0.001 to 100 mmol/l, especially preferred about 0.1 to 1 mmol/l.
- the entire surface of the solid phase carrier is coated, for example by immersing in a tub, with the (diluted) binding layer.
- the solution for applying the solution in a spatially defined manner, commercially available pipetting or spotting devices, as well as micropipetting devices or ink-jet processes can be used.
- a spatial structure of the measuring surface is achieved by the selective application of the interaction partners to be immobilized onto the large-area binding surface.
- Suitable solvents include aqueous (e.g. buffer solutions) or organic solvents (e.g. methanol, ethanol, acetonitrile. N,N-dimethylformamide, ethylene glycol) or mixtures thereof.
- the anchor molecule solutions preferably comprise an acid such as trifluoroacetic acid, hydrochloric acid or phosphoric acid.
- Anchor molecules suitable for preparing the binding surface according to the present invention comprise at least two functional units present at opposite ends of the anchor. As a group allowing the bonding of the anchor with the surface of a carrier, these anchors comprise a thiol group. For binding the interaction partner to be immobilized to the anchor, the anchor comprises a mercaptophilic head group M as a second functional unit.
- the anchors according to the present invention have the following general structure:
- Suitable structural elements R that both promote the formation of a monolayer and optionally also allow the adjustment of suitable distances between the head groups and the carrier surface with the help of spacer groups are described with respect to the anchor molecules in the German patent application DE 199 24 606.8, whose entire corresponding disclosure content is hereby incorporated by reference.
- the moiety R is a branched or straight-chain, optionally substituted, saturated or unsaturated hydrocarbon chain which may be interrupted by heteroatoms, aromatic and heterocyclic compounds and comprises 5 to 2,000 atoms, including heteroatoms.
- the anchor should comprise structures that impede or prevent a passive adsorption of the free interaction partner both on the anchor structure and the measuring surface.
- the anchor comprises a spacer group which allows adjustment of the total chain length and of the flexibility of the LAC.
- R a causes the formation of an SAM and for this purpose, it should essentially be hydrophobic. It comprises a branched or straight hydrocarbon chain with 5 to 50 carbon atoms, which may be saturated or partially unsaturated and which may be interrupted by aromatic or heterocyclic groups or heteroatoms. A fully saturated hydrocarbon chain without heteroatoms is preferred. In a preferred embodiment, it has the general formula —(CH 2 ) n 13 , wherein n is an integer of 5 to 50, preferably 5 to 25, especially preferred 5 to 18 and most preferred 8 to 12.
- R b is preferably a spacer that allows adjustment of the total chain length and of the flexibility of the ligand-anchor conjugate.
- R b is a hydrocarbon chain which is interrupted by heteroatoms and therefore hydrophilic and impedes a passive adsorption of the receptor.
- the chain comprises 2 to 1,000 atoms, including heteroatoms; chain lengths of 5 to 500 are preferred, and especially preferred are chain lengths between 10 and 100 atoms.
- R b is an oligoether of the general formula —(OAlk) y — wherein y is a natural number and Alk represents an alkylene group.
- y is a natural number and Alk represents an alkylene group.
- Alk represents an alkylene group.
- the Alk group preferably comprises 1 to 20, more preferably 2 to 10 and particularly preferred 2 to 5 carbon atoms. Especially preferred is —(OC 2 H 4 ) y —.
- R b is an oligoamide consisting of dicarboxylic acids and diamines and/or aminocarboxylic acids, wherein the amines independently preferably comprise 1 to 20, more preferably 1 to 10 carbon atoms, and may also be interrupted by further heteroatoms, in particular oxygen atoms.
- the carboxylic acid monomers independently preferably comprise 1 to 20, more preferably 1 to 10 carbon atoms and may also be interrupted by further heteroatoms, in particular oxygen atoms.
- Suitable compounds for the introduction of R b in an especially preferred embodiment include commercially available compounds such as in particular glycol ethers such as e.g. triethylene glycol, triethylene glycol monomethylether, tetraethylene glycol, ⁇ -diamines such as ethylene-, propylene-, butylene-, or pentylenediamine or 1,8-diamino-3,6-dioxaoctane, but also dicarboxylic acids such as succinic acid, 1,13-diamino-4,7,10-triooxatridecane, 3,6,9-trioxaundecanedioic acid, 8-amino-3,6-dioxaoctanoic acid or 4-aminobenzoic acid as well as their derivatives or combinations of identical components (such as e.g.
- glycol ethers such as e.g. triethylene glycol, triethylene glycol monomethylether, tetraethylene glycol,
- 4-aminobenzoic acid in the case of 8-amino-3,6-dioxaoctanoic acid or 4-aminobenzoic acid) or combinations of different components (such as e.g. 1,13-diamino-4,7,10-triooxatridecane and 3,6,9-trioxaundecanedioic acid in an alternating sequence).
- 4-aminobenzoic acid is the fact that it can easily be detected spectroscopically, for example by means of a UV spectroscopy.
- Q 1 ,Q 5 are —NH—C(O)—, —C(O)—NH— or a bond
- Q 2 ,Q 3 ,Q 4 are —NH—C(O)— or —C(O)—NH—
- a is 5 to 20, preferably 8 to 12, particularly preferred 10;
- b is 0 to 5, preferably 0 if Q 1 is a bond and 1 to 10, preferably 2 to 7, particularly preferred 3 to 5 in all other cases;
- c, c′ are 1 to 5, preferably 1 to 3, particularly preferred 1;
- d, d′ are 1 to 5, preferably 1 to 3, particularly preferred 2;
- e, e′ are 1 to 5, preferably 1 to 3, particularly preferred 2;
- f, f′ are 1 to 5, preferably 1 to 3, particularly preferred 1;
- i is 1 to 3, preferably 1 to 2, particularly preferred 1;
- j is 0 to 5, preferably 1 to 3, particularly preferred 2;
- k is 0 to 5.
- Mercaptophilic head groups M are for example iodo- or bromoacetamide, pyridylthio compounds, Michael acceptors in general, acrylic acid derivatives such as its esters, amides, lactones or lactames, methylene-gem-difluorocyclopropanes, ⁇ , ⁇ -unsaturated aldehydes and ketones as well as ⁇ , ⁇ -unsaturated sulfones and sulfonamides.
- Preferred head groups M are those of the general formula
- R 1 and R 2 are independently hydrogen or C 1 -C 5 alkyl, preferably methyl, ethyl or n-propyl,
- R 3 and R 4 are independently hydrogen or C 1 -C 5 alkyl, preferably methyl, ethyl or n-propyl, or R 3 and R 4 together form ⁇ O and
- R 3 and R 4 together form ⁇ O, and most preferred, the head group is a maleimidyl group.
- Processes for the synthesis of an anchor molecule can be carried out in a solid phase or in solution.
- the thiol group is preferably provided with a protective group. Suitable protective groups are described in T. W. Greene, P. G. M. Wuts, Protective Groups in Organic Synthesis, Wiley 1999 3 , Chapter 6 Protection for the Thiol Group.
- the coupling to the resin preferably takes place via the thiol group so that the thiol group remains masked during synthesis.
- the cleaving from 25 the solid phase or the protective group is carried out in an acidic environment (for example 1% TFA in dichloromethane). It has been found that this way the side reactions of the thiol and the mercaptophilic compound described in EP 485 874 A2 can be prevented.
- the diluting component comprises diluting molecules of the general formula
- the diluting component as well includes a preferred moiety R comprising the subunits R a and R b and, in a preferred embodiment, corresponding to the structural formula given above.
- the diluting molecules must not influence the interaction between the immobilized interaction partner and the free interaction partner. In particular, there should be no specific or unspecific binding between the free interaction partner and the diluting molecule.
- the anchor molecule and the diluting molecule should be as structurally similar as possible in order to ensure that their chemical behavior on the solid phase surface is similar as well (homogeneous miscibility on the solid phase surface). It is advantageous if the total chain length of the diluting molecule is shorter than that of the anchor molecule, for example by one structural unit, such as an ethylene glycol unit. This can for example be achieved by leaving out the last component in the diluant structure when forming the anchor and the diluant from commercially available synthesis components.
- the head group of the diluting molecule should be different from that of the anchor molecule.
- Preferred head groups for the diluting molecule include methoxy and acylamide groups. Acetamide is especially preferred, in particular if maleimidyl is used as the anchor head group.
- a (diluted) measuring surface according to the present invention is generated by a chemical reaction of the interaction partner to be immobilized and the mercaptophilic head group M of the anchor.
- the interaction partner to be immobilized carries a thiol group which reacts in as selective and quantitative a manner as possible while forming a covalent bond with the head group of the anchor (cf. C. Boeckler et al., J. Immun. Meth. (1996) 191, 1-10). If no suitable functionality is present, it can be generated by converting available functional groups in the interaction partner to be immobilized, e.g. by reductively cleaving a disulfide bond to form a thiol (P. Parham, J. Immunol.
- the additional molecule can furthermore function as a spacer between the head group of the anchor and the interaction partner to be immobilized, wherein the latter is kept away from the head group and thus the electronic and steric properties of the head group do not affect the binding properties of the interaction partner.
- Interaction partners to be immobilized/immobilized interaction partners or ligands in the present invention are chemically unmodified or modified, i.e. provided with a thiol functionality, interaction partners or ligands.
- immobilized interaction partners generally refers to structural elements which due to their structural characteristics can enter into specific interaction with test substances or subunits thereof. With the help of such immobilized structures, targets can be bound. e.g. in screening processes, which exhibit corresponding compatible structural units. Knowledge of the structure of the ligand then inter alia allows conclusions as to the possible structure or specific structural elements of the receptor.
- ligand is not used consistently in the prior art literature. It should therefore be emphasized that in the present invention the term “ligand” is used in particular for so-called “small molecules” which are preferably bound covalently to the binding surface.
- ligands include oligomers or small organic molecules, such as peptides, oligonucleotides, carbohydrates (glycosides), isoprenoids or lipid structures.
- the molecular weight is usually the basis for the definition of such small molecules.
- the mercaptophilic head group M should preferably serve to immobilize such a ligand, for example a so-called “small molecule” or “low molecular weight molecule”.
- WO 89/03041 and WO 89/03042 describe molecules with molecular masses of up to 7,000 g/mol as small molecules. Usually, however, molecular masses between 50 and 3,000 g/mol, more frequently between 75 and 2,000 g/mol and most of the time in the range of 100 to 1000 g/mol are mentioned.
- the molecular weight of a lioand/small molecule should be between 50 and 3,000 g/mol, preferably between 75 and 1,500 g/mol.
- ligands can optionally specifically bind to the receptors. They can be synthesized using reactions known in the prior art (cf. e.g. J. S. Complementary aromatic compound, a terpene, an organophosphorous compound, a chelate complex, a neurotransmitter, a substituted amine, an alcohol, an ester, an ether or a carboxylic acid and its derivatives and combinations thereof. They can be synthesized using reactions known in the prior art (cf. e.g. J. S. Computel, G. Jung, Angew. Chem. Int. Ed. 35, (1996) 17-42).
- ligands may have to be chemically modified such that they are able to react with the mercaptophilic head group of the anchors according to the present invention.
- a list of exemplary compounds that can be bound as ligands, optionally after modification of a functional group or addition of an additional molecule, to the binding surfaces of the present invention can be found in WO 00/73796 A2, which was filed on May 26, 2000 with the German Patent & Trademark Office and whose corresponding disclosure content is hereby incorporated by reference.
- the terms “immobilized interaction partner” or “interaction partner to be immobilized” also encompass macromolecules, preferably biomolecules or receptors such as proteins, oligo- or polypeptides, DNA, RNA, oligo- or polynucleotides, prosthetic groups, vitamins, lipids, mono-, oligo- and polysaccarides, as well as their modifications, and synthetic molecules such as e.g. fusion proteins and synthesized primers.
- These macromolecules as well may have to be chemically modified for use in the present invention in order to guarantee their covalent binding to the mercaptophilic head group of the anchor molecules.
- Molecules acting as free interaction partners are preferably molecules occurring in biological systems or molecules interacting with such molecules, in particular receptors such as proteins, DNA, RNA, oligonucleotides, prosthetic groups, vitamins, lipids, mono-, oligo- and polysaccarides, as well as synthetic molecules such as e.g. fusion proteins and synthesized primers.
- receptors such as proteins, DNA, RNA, oligonucleotides, prosthetic groups, vitamins, lipids, mono-, oligo- and polysaccarides, as well as synthetic molecules such as e.g. fusion proteins and synthesized primers.
- a solution containing the interaction partner to be immobilized is applied onto the binding surface. It is an advantage of the present invention that the concentration of the ligand (if one is used) on the surface is only determined by the dilution of the binding surface and not by the concentration of the ligand in the solution to be applied. This is in particular advantageous if many interaction partners to be immobilized of which only an approximate concentration is known are processed simultaneously, which is often the case with ligands obtained from combinatorial synthesis. This increases the reproducibility and comparability of different test measurements.
- the application of the interaction partner to be immobilized is not restricted to specific processes; however, conventional pipetting or spotting devices, but also stamping or ink-jet processes can be employed for a more precise localization of the active areas on the binding surface.
- Another aspect of the present invention relates to the provision of an array comprising a plurality of fields on a planar solid phase carrier.
- Each individual field can be used as a separate measuring surface.
- these measuring surfaces differ in the type of interaction partner immobilized on each one, whereby a single measuring surface can both represent a single type of interaction partner and a mixture of different interaction partners.
- a variety of interaction partners to be immobilized are applied onto the binding surface and subjected to a measuring process.
- the spatial structure of the resulting array can be predetermined by mechanical structuring the carrier.
- structured carrier plates When structured carrier plates are used as sensor surfaces in the present invention, they preferably comprise a plurality of evenly positioned fields with addressable positions for the generation of binding surfaces wherein these fields are located in low-depth cavities. This provides a barrier for the liquid while at the same time the surface is kept as small as possible in order to minimize possible unspecific adsorption phenomena.
- these fields comprise a layer of the material allowing the binding of the thiol-functionalized anchors.
- the cavities Preferably, the cavities have a depth of 20 to 100 ⁇ m and the anchors are immobilized at their bottom which is formed for example by a metal or metal oxide, preferably a noble metal such as gold.
- FIGS. 7 to 9 each show a schematic detail of a cross-section of a preferred carrier plate.
- a copper-clad base material ( 4 ) can be used which preferably already carries a metal layer ( 3 ) such as copper and which is provided with said carrier layer ( 2 ) by means of a galvanic deposition process.
- the thickness of the carrier layer is only a few micrometers, the exact thickness necessary for generating a closed layer.
- an UV-exposable protective layer ( 1 ) For this purpose, either photoresists common in semiconductor production or other protective paints that are UV-exposable and can therefore be structured can be used.
- the paint layers preferably have a thickness of 20 ⁇ m to 100 ⁇ m.
- an image of a mask is projected onto the protective layer.
- the mask preferably exhibits round or rectangular/square patterns.
- defined openings are formed in the protective layer which expose the carrier layer underneath.
- the protective layer that can be photostructured also forms the walls of the cavities ( 6 ) and thus the form of the cavity ( 6 ) and its opening.
- FIG. 8 shows a carrier plate ( 5 ) with deeper cavities ( 6 ) wherein, however, the portion of unprotected wall surface is not increased.
- This carrier plate preferably has a basis material ( 4 ) on whose surface a metallic coating ( 3 ) is provided, which in turn is provided with a protective layer ( 1 ).
- At least one cavity ( 6 ) is provided in the protective layer ( 1 ) and the metal layer ( 3 ), which in the area of the metal layer ( 3 ) well-shaped and is provided with a carrier layer ( 2 ) and which in the area of the protective layer ( 1 ) tapers towards the depression, wherein the lower rim of the cavity portion provided in the protective layer ( 1 ) has a smaller radius than the upper rim of the cavity portion formed in the metal layer ( 3 ).
- the production of a carrier plate as shown in FIG. 9 also starts from a coated plate.
- the thickness of the layer already present ( 3 ) is preferably 100 ⁇ m to 150 ⁇ m.
- the layer ( 3 ) is structured such that it already exhibits wells.
- the plate is coated with the carrier layer ( 2 ) by means of a galvanic process.
- a protective layer ( 1 ) is structured such that a structure is formed in the protective layer ( 1 ) above the cavities ( 6 ) etched into the layer ( 3 ).
- the depth of the thus formed cavity ( 6 ) is the sum of the depth of the etched structure and the thickness of the protective layer ( 1 ).
- the cavities are arranged such that a regular, preferably Cartesian, grid of columns and lines is formed on the carrier plate.
- the size and shape of the carrier plate can be selected as desired and can easily be adapted to the detection system used. If spotting robots are used for immobilizing the anchors or interaction partners, or if the binding surface is present on microtitration plates, the distance between the fields should preferably be adjusted to the microtitration format or the spotting device used.
- the number of fields can also exceed the number of subunits of the microtitration plate, i.e. multiple fields can be used per surface.
- a square carrier plate of about 12 ⁇ 12 cm may comprise a total of 9216 fields which can be covered by means of a pipetting robot from six conventional 1536 microtitration plates.
- a spatially structured presentation of identical or different immobilized to interaction partners can also be achieved by covalently binding the interaction partner to a predetermined portion of the binding surface after an amount of liquid has selectively been applied, without it being necessary to physically divide the surface into individual compartments.
- techniques for applying reagent spots onto metal or metal oxide surfaces as described in EP-A-0 872 735 can analogously be applied to the binding surfaces of the present invention. If solutions of the interaction partners/ligands are applied onto a homogeneous binding surface, the concentration of the ligand/interaction partner on the finished measuring surface is determined by the dilution of the binding surface. Consequently, there is the additional advantage compared to EP 872 735 A1 that precisely defined amounts of liquid only have to be applied to the solid phase carrier surface once to generate a measuring array (namely when the binding layer is generated).
- the present invention can be employed in HTS, in the research of active substances or in medical diagnostics.
- Suitable measuring methods for detecting interaction between an immobilized (surface-bound) interaction partner/ligand and a free interaction partner/receptor wherein the solid phase carrier only serves for the immobilization of one interaction partner are based on the verification of the specific binding reaction by means of electrochemical (electro immunoassays), radiochemical (e.g. radioimmunoassay), mass-sensitive or optical processes such as fluorescence or luminescence measurements, in particular enzyme assays.
- electrochemical electrochemical
- radiochemical e.g. radioimmunoassay
- mass-sensitive or optical processes such as fluorescence or luminescence measurements
- enzyme assays in particular enzyme assays.
- the so-called ELISA technique enzyme-linked immunosorbent assay, immunoassays using solid-phase technique
- Reflecto-optical processes such as surface plasmone resonance are suitable for a marker-free detection of the interactions.
- the solid phase carrier is part of the sensor system.
- the surfaces of the present invention can also be used in classic processes such as affinity chromatography.
- the resin was washed once with N,N-dimethylformamide (DMF), once with 5% water in DMF, four times with DMF, three times with dichloromethane and twice with hexane and dried under vacuum.
- N,N-dimethylformamide DMF
- the loading of the resin with N-(N 5 -Fmoc-5-aminopentyl)-11-mercaptoundecaneamide was determined by means of Fmoc analysis (G. B. Fields, R. L. Noble, Int. J. Peptide Protein Res. 1990, 35, 161-214) to be 0.35 mmol/g (yield: 60% of the theoretical value).
- Fmoc-8-amino-3,6-dioxa-octanoic acid was coupled to 500 mg resin (0.175 mmol) from b) and then the Fmoc protective group was cleaved off as described in b). Subsequently, the free amino groups were acetylated by incubating the resin for 30 min with 10 ml 1/1/2 (v/v/v) acetic acid anhydride/pyridine/DMF. Then the resin was washed five times with DMF and three times with dichloromethane.
- the binding surface consists of the anchor component from Example 2 and the diluting component from Example 1.
- stock solutions of the individual components are prepared by dissolving the solids in ethylene glycol/0.1% TFA with the desired molar concentration which is verified by means of Ellman's test (G. L. Ellman, Arch. Biochem. Biophys. 82 (1959), 70-77) or based on the extinction coefficient at 295 nm. Then the stock solutions of anchor and diluting components are mixed in the desired ratio (1:10, v/v) and the gold surface is incubated with this 100 ⁇ M to 1 mM solution for 1 hour at room temperature. It is then washed with methanol/0.1% TFA and several times with water/acetic acid (2 ppm) and dried in a nitrogen stream.
- the preparation of the protein surface is carried out by incubating the binding surface from Example 3 (1:10 dilution) with a 17 ⁇ M solution of caspase 7T7 (modified by means of a T7 and an oligo-His tag) in 0.2 M NaH 2 PO 4 /Na 2 HPO 4 , pH 7, (20 min, room temperature). Then the chip is washed with acetic acid/water (1 ⁇ l/500 ml), treated for 5 min with 10 mM 2-mercaptoethanol in 0.2 M NaH 2 PO 4 /Na 2 HPO 4 , pH 7, washed with acetic acid/water (see above) and dried in a nitrogen stream.
- the change in the SPR signal (in RU) after spraying on 100 ⁇ l antibody solution was as follows: antibody RU anti-T7 antibody, 6.7 nM 206 anti-oligo-His antibody, 11 nM 1067
- the goal of this measuring method is the vibrational spectroscopy of a thin layer located on a metal surface.
- the IR light at a grazing incidence is reflected off the metal surface.
- Measurements are only carried out with IR light polarized in the incidence plane since light with a perpendicular polarization direction cannot contribute to the measurement.
- transition dipole moments which comprise portions at least parallel to the E-field of the radiation contribute to the absorption.
- the upper plot in FIG. 2 shows the FTIR spectrum of the pure diluting component (A).
- the CH 2 bands can be seen in the area of 2.900 cm ⁇ 1 , the CONH bands in the area of 1.500 to 1,700 cm ⁇ 1 and the C—O—C band at 1.122 cm ⁇ 1 .
- the bottom-most plot shows the spectrum of the pure anchor component with ligand (B). Compared to the spectrum of the diluant, the band at 1.716 cm ⁇ 1 stands out. This band can be assigned to the imide group of the anchor component. When surfaces are prepared with an increasing anchor component concentration (spectra from top to bottom), a systematic increase in the imide band can be observed.
- the area of the peak at 1.716 cm ⁇ 1 was determined and plotted against the concentration of the anchor component (cf FIG. 3).
- a linear fit to the measuring data clearly shows the connection and thus the conformity of the anchor molecule concentration on the gold surface and the concentration in the solution during gold coating.
- XPS X-ray photoelectron spectroscopy
- the layer thicknesses obtained from XPS measurements and ellipsometric measurements correspond to the length of the diluting molecule from Example 1.
- the molecular length of the diluting component is 44 ⁇ . Since the molecules do not protrude at a right angle from the surface but are usually bound at a specific angle, the somewhat shorter measured layer thickness (34 ⁇ ) indicates that these surfaces are SAiMs. A bond angle of the molecules of about 50° with respect to the surface is therefore conceivable.
- the protein adsorption resistance was determined by means of surface plasmone resonance on Biacore® 3000 with the following proteins: ribonuclease A (bovine) 0.1 mg/ml lysozyme (chicken) 0.1 mg/ml egg albumin (chicken) 0.1 mg/ml D-amino-acid oxidase (procine) 0.1 mg/ml pyruvate kinase (rabbit) 0.1 mg/ml fibriogen (human) 1 mg/ml glutathione reductase (wheat germ) 1 mg/ml
- the values shown in FIG. 4 are average values from 4 measurements.
- a short-chain disulfide (compound A of EP 698 787), the diluting molecule from Example 1 and 12,15,18-trioxa-20-hydroxyicosan-1-thiol (Whitesides et al. J.Am.Chem.Soc. 1995, 117, 12009-10) were used as a diluants.
- the chain length of the molecule influences the unspecific adsorption resistance, which increasingly occurs in short-chain diluting molecules.
- Example 4 Analogously to Example 4, pY with cysteine as additional molecule is used to bind pY to the binding surface from Example 3 (“pY tag”).
- a gold chip was incubated with a 1:10 mixture of the anchor component from Example 2 and the diluting component from Example 1 in ethylene glycol and 1% TFA at a total concentration of 0.5 mM. Then the chip was washed several times in methanol/0.1% TFA and then in water pH 7.0. Chips pretreated in this manner were dried in a nitrogen stream.
- Such chips were then incubated overnight in BSA blocking solution (50 mM Tris/HCl, 150 mM NaCl, 5 g/l BSA, 0.05% (v/v) Tween-20®, pH 7.3 ).
- BSA blocking solution 50 mM Tris/HCl, 150 mM NaCl, 5 g/l BSA, 0.05% (v/v) Tween-20®, pH 7.3 ).
- the verification of the pY groups on the surface of the chip was carried out by means of an immunoassay wherein the chip was first incubated with a 1:5,000 a-pY antibody in blocking solution and then with 1:5,000 anti-mouse-Fab-POD. Between the incubation steps, the blocking solution was washed (2 ⁇ 1 min). Prior to the detection of the luminescence signal with the Super-Signal-Plus Substrate from Roche Diagnostics, it was again washed in TBS buffer. The luminescence reaction was observed in a Lumi-Imager (Roche
- a 12 ⁇ 12 cm gold chip (40 nm gold/1 nm chromium on glass) was incubated with a 1:10 mixture (dilution) of anchor component from Example 2 and diluting component from Example 1 in ethylene glycol and 1% trifluoroacetic acid (TFA) at a total concentration of 1.0 mM. Then the chip was washed several times in methanol/1% TFA and then in water pH 7.0. Chips pretreated in this manner were dried in a nitrogen stream.
- TFA trifluoroacetic acid
- a library of 9216 thiol-containing ligands were deposited on these chips by means of a spotting device in an arrangement of 96 ⁇ 96 spots with a distance of 1.125 mm between the spots.
- the thiol-containing ligands applied onto the surface are dissolved in a 40 ⁇ M solution of 0.2 M Pi, 5 mM EDTA and 10% (v/v) ethylene glycol pH 7.0.
- the spotting device releases about 10 nl per spot so that in each spot a high excess of the thiol-containing ligand compared to the surface-bound maleimide group is guaranteed and a complete reaction of the maleimide groups can be achieved.
- the maleimide groups were then saturated by incubating the chip in 0.2 M phosphate buffer pH 7.0, 10 mM mercaptoethanol for 30 min.
- Such chips were then incubated overnight in BSA blocking solution (50 mM Tris/HCl, 150 mM NaCl, 5 g/1 BSA, 0.05% (v/v) Tween-20®, pH 7.3).
- BSA blocking solution 50 mM Tris/HCl, 150 mM NaCl, 5 g/1 BSA, 0.05% (v/v) Tween-20®, pH 7.3.
- the analysis of potential binding partners of the target protein thrombin was carried out by means of an immunoassay.
- the chip was incubated for 4 hours in 10 nM thrombin in blocking solution. After washing twice for two minutes in blocking solution, a 1:1,000 dilution of a polyclonal anti-thrombin antibody was incubated with the chip for 2 hours. After again washing twice in blocking solution, an anti-rabbit antibody POD conjugate was incubated with the chip for 2 hours in order to determine the binding activity. Finally, the chip was washed twice for 2 min in TBST. The chemical luminescence reaction was verified by reacting the Lumi-Light Plus Substrate in the Lumi Imager (Roche Diagnostics, Germany).
- FIG. 6 shows a photograph of the chemical luminescence reaction (10 nM thrombin). Black spots (represented in the Imager as light spots) indicate binding of the thrombin. Discrete intensities can be seen as spots in certain positions. Since every compound on the array has certain spatial coordinates, specific chemical structures can be assigned to the spots.
- the products were dissolved in ethyl acetate and were each washed three times with 10% sodium carbonate solution, twice with a saturated sodium chloride solution and twice with distilled water. The solutions were dried over sodium sulfate and concentrated in a rotary evaporator until dry.
- the mixtures were stirred for 3 hours at room temperature, then the urea was filtered off and the solvent was concentrated until dry.
- the products (4), (5) and (6) were dissolved in ethyl acetate and were each washed three times with 0.1 M HCl, twice with a saturated sodium chloride solution and twice with distilled water. The solutions were then dried over sodium sulfate and concentrated until dry.
- the products (7) and (8) were dissolved in ethyl acetate (EE) and were each washed three times with 10% sodium carbonate solution, twice with a saturated sodium chloride solution and twice with distilled water. The solutions were dried over sodium sulfate and concentrated until dry.
- EE ethyl acetate
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DE19924606A1 (de) * | 1999-05-28 | 2000-11-30 | Graffinity Pharm Design Gmbh | Ligand-Anker-Konjugate |
US20040082079A1 (en) * | 2001-02-05 | 2004-04-29 | Ralf Besenbruch | Low affinity screening method |
US20040248111A1 (en) * | 2001-02-07 | 2004-12-09 | Gunther Metz | Screening method using solid supports modified with self-assembled monolayers |
DE10245651A1 (de) * | 2002-09-30 | 2004-04-08 | Graffinity Pharmaceuticals Ag | Qualitätskontrolle von Mikroarray Sensorfeldern |
EP1439394A1 (de) * | 2002-12-20 | 2004-07-21 | Graffinity Pharmaceuticals Aktiengesellschaft | Verfahren und Vorrichtung zum Erstellen von ADME Profilen in der frühen Wirkstoffentwicklung |
DE10308894A1 (de) * | 2003-02-28 | 2004-09-09 | Graffinity Pharmaceuticals Ag | Qualitätskontrolle von Bindungsmessungen auf Mikroarrays |
WO2006002141A2 (en) * | 2004-06-18 | 2006-01-05 | Wisconsin Alumni Research Foundation | Detection of post-translationally modified peptides with liquid crystals |
WO2006132326A1 (ja) * | 2005-06-09 | 2006-12-14 | Hiroshima University | 生細胞固定化法及び生細胞活性化機能測定センサー |
HUP0600668A2 (en) | 2006-08-22 | 2008-02-28 | Avicor Kft | Active carrier, process for producing thereof and the use of thereof |
US7759677B2 (en) * | 2006-09-05 | 2010-07-20 | Electronics And Telecommunications Research Institute | Molecular electronic device including organic dielectric thin film and method of fabricating the same |
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US5156989A (en) * | 1988-11-08 | 1992-10-20 | Siliconix, Incorporated | Complementary, isolated DMOS IC technology |
AU4343589A (en) * | 1988-11-23 | 1990-06-12 | California Medical Products, Inc. | A head immobilizer |
US5387555A (en) * | 1992-09-03 | 1995-02-07 | Harris Corporation | Bonded wafer processing with metal silicidation |
EP0485874B1 (de) * | 1990-11-14 | 1995-09-20 | F. Hoffmann-La Roche Ag | Immunsensorischer Wandler und Verfahren zu seiner Herstellung |
DE4219159A1 (de) * | 1992-06-11 | 1993-12-16 | Boehringer Mannheim Gmbh | Selbst assemblierende Monoschicht mit kurzkettigen Linkern |
US5763191A (en) * | 1990-12-12 | 1998-06-09 | Boehringer Mannheim Gmbh | Universal binding film |
US5386136A (en) * | 1991-05-06 | 1995-01-31 | Siliconix Incorporated | Lightly-doped drain MOSFET with improved breakdown characteristics |
KR950013435B1 (ko) * | 1992-05-22 | 1995-11-08 | 삼성전자주식회사 | 정상상 및 거울상을 위한 고체촬영소자 |
US5439842A (en) * | 1992-09-21 | 1995-08-08 | Siliconix Incorporated | Low temperature oxide layer over field implant mask |
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DE4430023A1 (de) * | 1994-08-24 | 1996-02-29 | Boehringer Mannheim Gmbh | Elektrochemischer Sensor |
US5545909A (en) * | 1994-10-19 | 1996-08-13 | Siliconix Incorporated | Electrostatic discharge protection device for integrated circuit |
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US5798295A (en) * | 1997-06-09 | 1998-08-25 | Motorola, Inc. | Method for forming a buried contact on a semiconductor substrate |
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US20080167196A1 (en) | 2008-07-10 |
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US20040023366A1 (en) | 2004-02-05 |
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