Chemistry: analytical and immunological testing – Optical result – Including reagent preparation
Reexamination Certificate
1998-04-13
2001-04-24
Warden, Sr., Robert J. (Department: 1744)
Chemistry: analytical and immunological testing
Optical result
Including reagent preparation
C435S973000
Reexamination Certificate
active
06221674
ABSTRACT:
DESCRIPTION
The invention concerns a process for applying spatially defined reagent areas or reagent spots on a solid phase with a metal or metal oxide surface and also concerns a solid phase binding matrix which comprises different solid phase reactants or/and solid phase reactants at different concentrations in spatially defined reagent spots.
A number of processes are known which can be used to apply small amounts of reagent to different surfaces such as glass or plastic supports. Such processes lead to a microstructuring of the reagents on the surface e.g. in the form of reagent spots that are spatially separated from one another. Of particular interest in this connection are microstructured surfaces in which the reagent spots each have different functionalities, so-called arrays, in which the individual reagent spots for example contain different reactants such as DNA fragments or antibodies. A process is described in WO 92/10 092 which can be used to generate a plurality of different structures on a glass support by means of photoreactive compounds and irradiation using masks. A process is described in U.S. Pat. No. 4,877,745 in which differently functionalized spots can be applied to plastic supports by means of ink-jet.
In contrast to plastic surfaces, metal and metal oxide surfaces have the advantage that they can be coated with an exactly defined matrix layer by self-assembly techniques. A self-assembled monolayer (SAM) is formed for example when organic alkylthiols are adsorbed onto a gold surface, the spontaneous organisation of such a densely packed monolayer being based on strong specific interactions between the support material and the adsorbent (Nuzzo et al., J. Am. Chem. Soc. 105 (1983) 4481). In this manner it is possible to apply an exactly defined monolayer of a binding matrix to the surface of metals such as e.g. gold or silver. Furthermore the specific binding capability of self-assembled solid phases can be further optimized by dilution of the specific solid phase reactants as described in EP-A-0 515 615.
The coating of metal surfaces with microstructures based on self-assembled monolayers is also known. Thus Whitesides et al., Langmuir 10 (1994) 1498-1511 describe a process in which reagents are stamped onto a noble metal surface by means of a special microstructured silicone stamp. This enables microstructured monolayers to be generated with zones that are spatially separated from one another. However, in such a stamping process individual zones are obtained that are all identically functionalized i.e. it is not possible with this technique to obtain a different functionality by a different coating of individual spots as in an array structure.
Furthermore it is known that microstructures of self-assembled monolayers on noble metal surfaces can be formed by irradiation through masks of substrates whose whole area is covered with thiols and subsequent washing (Hemminger et al., Langmuir 10 (1994), 626-628). Spatially separate zones are also formed in this process which are all identically functionalized. A further possibility of producing reagent spots is firstly to apply gold spots to a support that are already spatially separated from one another which are then subsequently coated with reagents. However, it is time-consuming to manufacture such spatially separated gold spots and they have to be generated by vapour-depositing the substrate through masks. Moreover the subsequent coating of the gold spots with reagents for example with micropipettors requires very precise handling which in the case of structures in the &mgr;m range is very difficult to realise and is technically very complicated.
Therefore the object of the invention was to provide a process with which array structures of reagent spots can be applied in a simple manner to metal or metal oxide surfaces.
This object is achieved according to the invention by a process for applying spatially defined reagent areas to a solid phase which is characterized in that a liquid containing an adsorptive binding reagent is contacted with spatially defined areas of a solid phase which comprises an essentially continuous metal or metal oxide surface for an adequate time period to enable the formation of adsorptive bonds between binding reagent and solid phase.
Surprisingly it was possible to eliminate the disadvantages occurring in the state of the art by spotting the reagents used for self-assembly in one solution directly onto a reagent support coated all over with a metal or a metal oxide. The reagent spots can for example be applied by means of ink-jet methods or with an automatic micropipetting device. In this process the reagent solution is applied to the surface in the form of small droplets preferably in the form of microdroplets.
After applying the reagent solution the surface is incubated for a certain time period e.g. 1 to 60 min, preferably 5 to 10 minutes in order to enable adsorptive bonds to form between the binding reagent and metal or metal oxide surface. The incubation period depends on the binding reagent used and on the surface but the incubation is long enough to enable the formation of adsorptive bonds. In this process a self-assembled monolayer of the binding reagent is formed on the metal or metal oxide surface in the form of spatially defined areas. After incubation the surface is rapidly washed with a large excess of solvent in order to prevent smearing of adjacent zones. Aqueous or/and organic solvents can be used as the solvent depending on the binding reagent used.
Surprisingly it is possible to form spatially defined areas in the form of spots by applying adsorptively binding reagents to a continuous metal or metal oxide layer in which a significant smearing of adjacent reagent spots does not occur during the application or subsequently. Despite the merely adsorptive binding between the surface and binding reagent discrete spots are formed since the adsorptive binding reagent is surprisingly stationary. An important advantage of the process is that surface regions can be produced with different coatings or functionalities that can be used for multiparameter assays in detection methods e.g. in immunological or nucleic acid hybridization assays. Furthermore it was found that after completion of the self-assembly the binding reagent is not carried over the support into other reagent spots by the washing process. Furthermore it was found that the individual spatially defined zones are also stable after fabrication of the functionalized surfaces i.e. the adsorbed binding reagents remain at the same site and do not migrate to unoccupied sites on the metal or metal oxide surface.
The areas of the applied reagent spots preferably have a diameter of ≦5 mm, preferably a diameter of ≦1 mm. Reagent spots are most preferably applied in the micrometer range e.g. with a diameter of 50 to 500 &mgr;m. This corresponds to a dropwise added liquid volume of the reagent solution of about 0.1 to 10 nl. Since spatially sharply defined spots are obtained with the process according to the invention it is possible to produce a microarray structure. Different reactants or/and different reactant concentrations e.g. for immunological tests or nucleic acid hybridization tests can be applied to each of the individual spot zones.
A noble metal surface is a preferred surface and particularly preferably a gold or silver surface and most preferably a gold surface. Such a metal surface can for example be formed by vapour-depositing a thin metal layer on a support, for example a glass support. Such a vapour-deposited layer is preferably 10 to 100 nm thick. An SH or SS reagent is preferably used as the adsorptive binding reagent when using a noble metal surface. Such thiol and disulfide reagents are described in detail for example in DE 40 39 677. A compound containing a thiol or disulfide group is particularly preferably used as the adsorptive binding reagent which additionally contains a specifically bindable group such as e.g. an antigen, hapten or biotin group. Biotinylated thiol reagents adsorbed as
Knoll Wolfgang
Sluka Peter
Zizlsperger Manfred
Fulbright & Jaworski LLP
Roche Diagnostics GmbH
Snider Theresa T.
Warden, Sr. Robert J.
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