Process for the application of reagent spots

A process is described 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 the binding reagent and the solid phase.

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 .mu.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
 .ltoreq.5 mm, preferably a diameter of .ltoreq.1 mm. Reagent spots are
 most preferably applied in the micrometer range e.g. with a diameter of 50
 to 500 .mu.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 an SAM to a surface
 can subsequently be coated with streptavidin, a different biotin content
 in different spots leading to different streptavidin layers.
 However, it is also possible to use a metal oxide as the surface. Examples
 of suitable metal oxide surfaces are SiO.sub.2, TiO.sub.2, Al.sub.2
 O.sub.3, Sn.sub.2 O.sub.3, Ag.sub.2 O, La.sub.2 O.sub.3, Ta.sub.2 O.sub.5
 and mixtures thereof. A metal oxide surface composed of SiO.sub.2 and/or
 TiO.sub.2 is preferably used. If a metal oxide surface is used a compound
 containing a silane group is preferably used as the adsorptive binding
 reagent. Such compounds are described for example in EP-A-0 664 452. When
 compounds containing silane groups are adsorbed to metal oxide surfaces a
 covalent cross-linking for example by heating takes place after the
 adsorption.
 The adsorptive binding reagents used according to the invention preferably
 contain, in addition to the respective adsorptive groups such as an SH, SS
 or silane group, at least one specifically bindable group such as biotin,
 a biotin derivative, streptavidin, a hapten, an antigen and/or a nucleic
 acid sequence which specifically bind to a binding partner e.g. to an
 analyte to be determined. The binding of analytes to a functionalized
 solid phase matrix according to the invention can for example be detected
 by confocal scanner fluorescence microscopy or by plasmon resonance
 spectroscopy. The detection is preferably carried out by means of plasmon
 resonance microscopy (B. Ruthenhausler et al., Nature, vol. 332 (1988)
 615-617). The binding of the analyte can be detected with surface plasmon
 resonance without using labelling reagents. The simultaneous determination
 of plasmon resonance and fluorescence detection is particularly preferably
 used to detect the binding of an analyte. In this case the plasmon
 resonance signal and the fluorescence signal are obtained independently of
 one another which also enables the determination of reaction kinetics in
 addition to a high sensitivity (10.sup.-14 mol/l).
 A further subject matter of the invention is a solid phase matrix
 comprising a support which is essentially continuously coated with a metal
 or metal oxide layer on which spatially defined reagent areas are arranged
 which (a) contain different solid phase reactants or/and (b) contain a
 solid phase reactant at different concentrations. Such a solid phase
 binding matrix preferably comprises at least three differently
 functionalized zones on a surface and particularly preferably at least 5
 differently functionalized zones. The diameter of the reagent spots is
 preferably .ltoreq.5 mm, more preferably .ltoreq.1 mm and most preferably
 .ltoreq.500 .mu.m. Such a solid phase matrix with reagent spots in the
 .mu.m range can also be called a microarray.
 A further subject matter of the invention is a method for the detection of
 a binding or/and interaction of analytes in which a solid phase matrix
 according to the invention is used. If plasmon resonance spectroscopy
 or/and plasmon resonance microscopy is used as the detection method, a
 real time detection is possible and a label is not required since the
 layer thickness can be measured. Such a method can be used in molecular
 diagnostics to determine allergies, in immunology as well as for nucleic
 acid hybridization. A combination of plasmon resonance spectroscopy and
 fluorescence detection or a combination of plasmon resonance microscopy
 and fluorescence detection is particularly preferably used to detect the
 binding or/and interaction of the analyte.

EXAMPLE 1
 Application of Reagent Spots to a Surface
 The principle of the process according to the invention is elucidated by a
 preferred embodiment with reference to FIG. 1. Firstly a ca. 50 nm thick
 gold layer is vapour-deposited on a glass support made of LASFN9 high
 index glass in a high vacuum (ca. 10.sup.-7 mbar) with the aid of a high
 vacuum coating apparatus (Leibold Co.). Immediately after
 vapour-depositing the gold layer the support (10) is clamped on an XYZ
 table (Isel-EP 1090/4) (2) which is equipped with a silicon piezo pump
 (GeSIM, Dresden) (4). A microtitre plate (6) is also attached to the
 working surface of the XYZ table (2) which contains in the respective
 different wells the various adsorptive binding reagents to be applied
 dissolved in a solvent as well as the pure solvent. As substances for the
 adsorption a water-soluble thiol system composed of HS-Prop-DADOO-X-biotin
 (bindable reagent) (FIG. 2a) and HS-C2-aminoethoxy ethanol (diluent
 component) (FIG. 2b) was dissolved at a concentration of 5.times.10.sup.-4
 molar in water and an ethanol-soluble thiol system composed of
 HS-C12-DADOO-biotin (bindable biotin component) (FIG. 2c) and
 mercaptoundecanol (diluent component) (FIG. 2d) was dissolved at a
 concentration of 5.times.10.sup.-4 molar in ethanol. These systems were
 placed in different wells of the microtitre plate at different mixing
 ratios with a proportion of biotin component of 0%, 10%, 50% and 100%. In
 order to apply reagent spots to the gold surface the tip of the pump (4)
 was now dipped into the corresponding liquid and then driven to the
 programmed position on the gold coated surface where a drop with a volume
 of ca. 1 nl (8) is deposited. Afterwards the pump is rinsed twice with
 pure solvent and the process is repeated with the next solution, the next
 spot being deposited at a distance of ca. 600 .mu.m from the previous
 spot. This process can be repeated as often as desired.
 Subsequently the gold surface on which the reagent spots have been applied
 is allowed to set for ca. 10 min during which a self-assembled monolayer
 forms on the surface covered with the drop. Subsequently the gold surface
 is rinsed over its entire area with a pure solvent in order to wash away
 non-adsorbed molecules. In this manner it is possible to produce surfaces
 with spatially defined reaction spots within a short period. The diameter
 of the spots is ca. 200 .mu.m.
 EXAMPLE 2
 Plasmon Microscopic Examination of the Reagent Spots Applied to a Gold
 Surface
 In order to determine the optical layer thicknesses of the prepared biotin
 monolayers and in order to examine the binding property towards
 streptavidin, the gold surface prepared with reagent spots was examined
 with the aid of surface plasmon microscopy in the Kretschmann
 configuration (prism coupled). In this process, as shown in FIG. 3 surface
 plasmons are excited through a prism (16) made of high-refraction glass at
 a specific angle of incidence on the metal layer (18) by widened laser
 light (red He--Ne laser) (14) that is p-polarized through a polarizer
 (12). The reflected light is recorded by a CCD camera (20) in relation to
 the angle of incidence. In addition the construction shown in FIG. 3
 includes a goniometer (30) as well as two lenses (28, 32) which form the
 microscope optics. Different layer thicknesses on the reagent spots result
 in different light-dark contrasts on the recorded picture. With the aid of
 image analysis systems (22) comprising a computer it is possible to
 determine the absolute increase in the layer thickness as well as its time
 course with an accuracy of ca. 0.1 nm. FIG. 4 shows the pictures obtained
 by means of the CCD camera of the spots coated with thiol before and after
 streptavidin binding. FIG. 5 shows the time course of the intensities
 obtained by image analysis of streptavidin binding to differently coated
 spots. Six pictures are shown in FIG. 4a which each show 12 identically
 functionalized thiol spots at different angles of incidence of the laser
 light. A coated support with 16 reagent spots is shown in FIG. 4b in which
 case sets of 4 spots have the same functionalization, the left picture
 having been taken before the binding of streptavidin, the right-hand
 picture having been taken after the binding of streptavidin. As can be
 clearly seen the process according to the invention enables spatially
 separate, differently functionalized reagent spots to be applied to a
 continuous surface.
 FIG. 5 shows the time course of the adsorption of streptavidin to a surface
 coated with four different hydrophilic thiol mixtures obtained by
 analysing the reflection intensity of the plasmon resonance microscopy. K1
 represents a reagent spot on which a pure solvent solution was incubated.
 This spot was subsequently treated with streptavidin. As shown in FIG. 5
 there is only a very slight binding of streptavidin to the reagent spot.
 K2 represents a reagent spot which was incubated with a solution composed
 of 1% biotinylated thiol and 99% diluent thiol and subsequently treated
 with streptavidin. It can be seen that there is a considerably higher
 streptavidin binding than in curve K1. K3 represents a reagent spot that
 was incubated with 100% biotinylated thiol, i.e. without diluent
 molecules, and subsequently treated with streptavidin. There is a
 considerable increase in the layer thickness to 33.+-.3 .ANG. compared to
 K2 where the layer thickness was 22.+-.3 .ANG.. Finally K4 represents a
 reagent spot which was treated with 10% biotinylated thiol and 90% diluent
 molecules. In this case a dilute homogeneous binding layer is formed which
 has the highest reflection after treatment with streptavidin from which a
 layer thickness of 43.+-.3 Angstrom can be calculated. As shown in FIG. 6
 reagent spots with different coatings can be applied to a single flat gold
 support resulting in the formation of an array system. In addition it is
 possible using such an array to detect bindings or/and interactions of
 analytes in which the layer thickness (coating amount) as well as the
 reaction kinetics (adsorption rate) can be determined.