EP1877773A1

EP1877773A1 - Novel equipment and method for coating substrates for analyte detection by way of an affinity assay method

Info

Publication number: EP1877773A1
Application number: EP06724515A
Authority: EP
Inventors: Eginhard Schick; Dominic Utinger; Claudio Calonder
Original assignee: Bayer Technology Services GmbH
Current assignee: Bayer Intellectual Property GmbH
Priority date: 2005-04-26
Filing date: 2006-04-22
Publication date: 2008-01-16
Anticipated expiration: 2026-04-22
Also published as: JP5002584B2; CA2605750A1; CN101208599A; EP1877773B1; JP2008539399A; US20090311773A1; US9050615B2; ES2525324T3; WO2006114249A1; CN101208599B; AU2006239534B2; DK1877773T3; AU2006239534A1

Abstract

The invention relates to several embodiments of equipment for coating substrates for detecting one or more analytes by way of an affinity assay method, comprising: a receptacle receiving a liquid to be atomized ('liquid receptacle') with substances (compounds) to be deposited onto at least one surface of said substrates and an atomized volume produced by the liquid in the operating state; an actuator for triggering the atomization process and; a fixture for receiving and storing the substrates during the coating process. The invention is characterized in that the substrates are not in contact with the surface of the liquid to be atomized. The invention also relates to several embodiments of methods for coating substrates with coupler and/or passivation layers for use in the detection of one or more analytes by way of an affinity assay method.

Description

New apparatus and method for coating carrier substrates for analyte detection by affinity detection method

Technical background of the invention

For many applications, it is necessary to determine a variety of analytes in a sample of possibly complex composition and nature, for example in diagnostic procedures for determining the health of an individual or in drug discovery and development for determining the influence of an organism and its complex functioning by supplying biologically active compounds.

While known analytical separation methods are generally optimized to separate within the shortest possible time the largest possible number of compounds contained in a given sample according to a given physicochemical parameter, such as the molecular weight or the quotient of molecular charge and mass, based bioaffinity Detection method to recognize each with a biological or biochemical or synthetic recognition element high specificity, hereinafter also referred to as "binding partner" or "specific binding partner", the corresponding (single) analyte highly selective in a complex composite sample and bind. The detection of a large number of different compounds thus requires the use of a corresponding number of different specific recognition elements.

A detection method based on bioaffinity reactions can be carried out both in a homogeneous solution and on the surface of a solid support ("carrier substrate"). Depending on the specific structure of the method, after binding of the analytes to the corresponding recognition elements and optionally further detection substances and optionally between different Each step of the washing steps is necessary in order to separate the complexes formed from the recognition elements and the analytes to be detected and optionally further detection substances from the remainder of the sample and any additional reagents used. For determining a plurality of analytes or examining a plurality of samples, especially in industrial analytical laboratories, methods are used in which the detection of different analytes in discrete sample containers or "wells" of these plates takes place in so-called microtiter plates. Most widely used are plates with a grid of 8 x 12 wells on a base of typically about 8 cm x 12 cm, wherein the filling of a single well a volume of a few hundred microliters is required. For many applications, however, it would be desirable to simultaneously assay multiple analytes in a single sample container using as small a sample volume as possible.

In WO 84/01031, US Pat. No. 5,807,755, US Pat. No. 5,837,551 and US Pat. No. 5,432,099, the immobilization for the analyte of specific recognition elements in the form of small "spots" as discrete measurement areas, sometimes well below 1 mm ² area proposed on a common carrier substrate in order to make by binding a small portion of existing analyte molecules only dependent on the incubation time, but - in the absence of a continuous flow - to make substantially independent of the absolute sample volume concentration determination of the analyte. A multiplicity of such "spots" as measuring regions represents in a two-dimensional arrangement on a common carrier substrate a so-called "microarray".

Meanwhile, methods for the simultaneous detection of a multiplicity of different nucleic acids in a sample by means of corresponding complementary nucleic acids immobilized on a carrier substrate in discrete, spatially separated measuring regions as recognition elements are relatively widespread. For example, based on simple glass or microscope slides as carrier substrates, arrays of oligonucleotides are known as recognition elements with a very high feature density (density of measurement areas on a common solid support). For example, in US Pat. 5,445,934 (Affymax Technologies) describes and claims arrays of oligonucleotides with a density of more than 1000 features per square centimeter.

Recently, descriptions of arrays and similar detection methods carried out therewith for the simultaneous determination of a plurality of proteins, for example in US Pat. 6,365,418 bl. The simplest form of immobilization of the binding partners for analyte detection is physical adsorption, for example due to hydrophobic interactions between the binding partners and the carrier substrate. However, these interactions can be greatly altered by the composition of the medium and its physicochemical properties, such as polarity and ionic strength. In particular, in the case of sequential addition of various reagents in a multi-step assay, the adhesion of the binding partner after purely adsorptive immobilization on the surface is often insufficient.

It is therefore often preferred to immobilize the binding partners on an adhesion-promoting layer applied to the carrier substrate. As suitable for the preparation of the primer layer, a variety of materials are known, for example, unfunctionalized or functionalized silanes, epoxides, functionalized, charged or polar polymers and "self-assembled passive or functionalized mono- or multilayers", alkyl phosphates and phosphonates, multifunctional block copolymers , such as poly (L) lysine / polyethylene glycols.

For example, in WO 00/65352, coatings of bioanalytical sensor platforms or implants for medical applications are used as carrier substrates with graft copolymers as adhesion-promoting layer, with a polyionic main chain, for example binding to a carrier substrate (electrostatically), and non-interactive "(adsorption-resistant) side chains.

In order to minimize nonspecific binding of analytes or their detection substances or other binding partners, in particular in the (uncovered) areas between the measurement areas (spots) generated by locally addressed task for analyte detection, it is often preferred to "passivate" these areas spatially separated measurement areas on the carrier substrates compounds which are "chemically neutral" to the analyte or against their detection substances or other binding partners, d. h.-non-binding, are.

Said against the analyte or its detection substances or other binding partners "chemically neutral", ie, these non-binding components (hereinafter also referred to as "Passivierungsverbindungen") may be known be selected from the groups of albumins, in particular bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, non-specific or empirical for the analyte or antibodies to be detected non-specific antibodies (especially for immunoassays), detergents - such as Tween 20 -, not with to be analyzed polynucleotides hybridizing, fragmented natural or synthetic DNA, such as an extract of herring or salmon sperm (especially for polynucleotide hybridization assays), or uncharged, but hydrophilic polymers, such as polyethylene glycols or dextranes.

In order to enable the generation of reliably quantifiable data from the binding signals, for example by means of fluorescence detection, from different measurement areas (spots) of a microarray, it is necessary to ensure a uniform surface density and binding activity of immobilized binding partners in measurement ranges to be compared. To fulfill this requirement, a high spatial homogeneity of the adhesion-promoting layer on which the discrete measurement areas (spots) are generated is an essential prerequisite. Similar demands on the spatial homogeneity exist for the applied passivation layer, to ensure a uniform, as low as possible signal background between the designated measurement areas (spots).

Depending on the molecular nature of the components of the adhesion-promoting layer to be produced and, of course, the thermal and chemical stability of the carrier substrates to be coated, various methods can be used for the application of the adhesion-promoting layer on the carrier substrate. Silanizations can be carried out, for example, both in the gas and the liquid phase, for example by means of dipping processes. While the coating processes in the gas phase, with sufficiently large sized reaction vessels (in comparison to the size of the carrier substrates to be coated), generally lead to good homogeneity of the deposited layer, layers deposited from the liquid phase often show strong spatial inhomogeneities, for example trace traces Application of dipping method.

Since the gas phase deposits typically require elevated process temperatures, the step of applying passivation layers occurs after application of the In most cases non-heat-resistant detection elements for analyte detection, usually from the liquid phase. For the passivation step, a dipping process is typically used. In this case, the carrier substrate is dropped into a vessel which is filled with a solution of the analytes or their detection substances or other binding partners "chemically neutral", ie this non-binding compounds ("passivation") to the entire surface of the carrier substrate as possible to wet quickly and simultaneously with the passivation solution. Subsequently, the carrier substrate is left in the passivation solution for a predetermined period of time ("incubated"), so that the compounds used for surface passivation can be adsorbed on the substrate surface.

An advantage of this conventional method is that it can be carried out without any further aids and does not require special requirements for the abilities of the laboratory personnel.

However, a disadvantageous feature of this method is a relatively high risk of "blurring" of spots during the moment of immersion of the support substrate by passivation solution flowing along the substrate surface, thereby desorbing and sweeping away material from the discrete measurement areas (immobilized specific binding partners) and can occur in the Environment in the direction of the relative flow direction of the passivation solution (based on the substrate surface) are not adsorbed again in not completely covered with Passivierungsverbindungen areas.

The extent of "smearing" of spots depends, among other things, on the surface density of the immobilized specific binding partners in the discrete measurement areas and the composition of the passivation solution, in particular on the solubility of the specific binding partners in this passivation solution Feature density, ie, with a small distance between adjacent spots, the quantitative analysis of the assay signals from an array of measurement areas strongly affect or even exclude, due to the resulting inhomogeneous distribution of the background signals from the areas between the discrete measurement areas. In particular, if immobilized material is even transported from one spot to an adjacent spot, this undesirable effect can lead to a meaningful analysis of the assay signals is no longer possible. Another disadvantage of this method is the inherent need for relatively large volumes of passivation solution and associated relatively high costs.

In order to avoid the described effect of "smearing", the use of spraying methods is known, for example with the aid of atomizers, in which the passivation solution is applied in the form of small liquid droplets on the substrate surface until a continuous liquid film has formed on this surface a saturated atmosphere of the liquid vapor (ie at 100% luchtfeuchtigkeit in the case of an aqueous passivation solution) for a predetermined period of time, in turn so that the compounds used for surface passivation can be adsorbed on the substrate surface.To avoid trace traces, the support substrates during the implementation of this passivation process are horizontal (relative to the substrate surface to be coated) stored.

If this procedure is carried out properly, it is possible to largely avoid "smearing" of the spots.Also advantageous is the need for a passivation solution, which is typically a factor of 10, in comparison to the previously described dipping method.

However, the required uniform wetting and quite accurate dosage of the amount of liquid applied, which are required to form a homogeneous liquid film on the substrate surface, represent a procedural inherent difficulty associated with increased demands on the operator. In the case of an excess of applied passivation, for example, again A modular system based on this coating method for producing microarrays with nucleic acids, proteins or other chemical or biological compounds as specific binding partners immobilized in discrete measurement ranges has been described, for example, in International Patent Application WO 01/57254.

Despite clear advantages compared to the dipping process, the results of the spraying process are also not optimal. Due to the ejection of the droplets over one Nozzle or an atomizer, the droplets have a momentum more or less strong against this surface impulse at the moment of impact on the surface to be coated. This is associated with the risk of splashing into even smaller droplets when hitting the surface, so that the edges of the measuring areas (spots) to be generated are generally not generated well-defined. In addition, spray processes generally produce relatively large droplets with a large variation in droplet size.

task

There is therefore a need for the development of a coating method from the liquid phase and an apparatus for performing this coating method, whereby a similar high homogeneity of the layers produced as in a deposition from the gas phase can be achieved and for which the use of the smallest possible amount of liquid is required. In addition, it is desirable that a corresponding novel coating method and a coating apparatus to be used therefor are suitable both for the application of an adhesion-promoting layer and a passivation layer. From the point of view of cost-effectiveness, the aim is to achieve the most cost-effective solution possible, i. keep the intrumentelle effort as low as possible. A corresponding new coating apparatus should be easy to operate and a coating process to be carried out easily be automated.

Brief description of the invention

It has now surprisingly been found that the stated requirements with the inventive method described below, based on a nebulization of solution which contains the compounds to be applied to the carrier substrates and wherein no significant pulse from the mist on the carrier substrates to be deposited droplets in the direction of the carrier substrates required is, can be fulfilled. The coating apparatus according to the invention developed for carrying out this method is characterized by a very simple construction, in which cost-effective commercially available components can be used, and also simple operability. The method according to the invention, to be carried out with a coating apparatus according to the invention according to one of the embodiments described below, is suitable for applying both adhesion-promoting and passivation layers on any but preferably planar carrier substrates for the detection of analytes in affinity detection methods.

The inventive method represents a further development of the spraying method described above, wherein the production of very fine liquid droplets for the inventive method in a preferred embodiment by ultrasound treatment. The coating apparatus used for this method comprises in a preferred embodiment a closed container with a holder for horizontal storage of the carrier substrates (based on the surface of the liquid to be atomized) and an underlying ultrasonic generator, which is immersed in the liquid to be atomized. The inventive method is characterized in that the droplets produced are substantially smaller than in the case of the spray process. In operation, a very dense fog is generated over the liquid to be atomized, which is evenly distributed in a preferred embodiment by turbulence using a weak additional nitrogen stream in the container, the container is preferably closed except for gas inlets and gas outlets. Since there is no flow of the coating solution with respect to the surfaces of the substrates to be coated, smearing of the spots as described for the dipping method is prevented in the process of the present invention Accordingly, there are hardly any procedural constraints on the selection of the composition of both Passivation solution and a "spotting solution" to be used in a preceding step for immobilizing the specific binding partner in discrete measuring ranges and the concentration of this solution to specific binding partners and thus the resulting surface density of immobilized specific binding partner in the measuring ranges. In particular, due to the absence of flows along the surface of the carrier substrates during the surface passivation step, there is no risk of "smearing" between adjacent spots, so that their producible density in an array of measuring regions can only be determined by the dosing fineness and positioning accuracy of the apparatus to be used for generating the discrete measuring regions ( "Spotter") is limited. The inventive method is also characterized by the possibility of simultaneous coating of a large number of carrier substrates in a common, appropriately sized container and ease of automation and is easily feasible even for untrained personnel. The liquid volumes to be used for coating the carrier substrates are of similar order of magnitude as in the case of the spray method.

Brief description of the pictures

Fig. 1 shows schematically a coating apparatus according to the invention.

Fig. 2 shows the geometry of an array of measurement areas with 12 different applied samples in a two-dimensional array ("microarray") and a linear array of 6 arrays on a common carrier substrate.

3A-3C show the fluorescence signals of microarrays, wherein the free surfaces of the associated carrier substrates were passivated by means of different coating methods, in each case with enlargements of the marked image sections underneath (A: dipping method, B: spraying method, C: nebulizing method according to the invention).

4A shows the mean values and standard deviations of the background signal intensities, which were respectively determined between all spots of the microarrays, wherein the free surfaces of the associated carrier substrates were passivated by means of different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention).

4B shows the mean values and standard deviations of the fluorescence intensities of all reference spots (for explanation, see in the exemplary embodiment) of the microarrays, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention). 5A shows the averaged intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulisation method according to the invention) and the microarrays afterwards with solutions of the antibody Al (anti-p53) and then each incubated for detection by fluorescence detection with Alexa 647 fluorine anti-rabbit Fab fragments.

5B shows the average intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, wherein the free surfaces of the associated carrier substrates were passivated using different coating methods (A: dipping method, B: spraying method, C: nebulizing method according to the invention) and the microarrays afterwards with solutions of antibody A2 (anti-phospho-p53) and then each incubated for detection by fluorescence detection with Alexa 647 fluorine anti-rabbit Fab fragments.

Detailed description of the invention

The first object of the present invention is an apparatus for coating carrier substrates for detecting one or more analytes in an affinity detection method, comprising:

- A container for receiving liquid to be atomized ("liquid container") contained therein, on at least one surface of said carrier substrates to be deposited substances (compounds) and a generated above the liquid in the operating condition fog volume, an actuator for causing the misting process and

a holder for receiving and supporting the carrier substrates during the coating process, characterized in that the carrier substrates are not in contact with the surface of the liquid to be atomized. The term "liquid to be atomized" is to be understood as the total amount of liquid within the coating apparatus according to the invention, to which the pulses of the actuator act to atomize the liquid, with the consequence of the conversion of a part of this liquid into mist.

Preferably, the generation of the mist above the liquid to be atomized is effected by the action of ultrasound within that liquid. Accordingly, it is preferred that said actuator serves to generate ultrasound.

Various technical methods for generating ultrasound are known, for example using piezoelectric crystals, oscillating membranes etc. It is preferred that said actuator comprises the membrane of an ultrasonic generator.

In addition, it is preferred that said actuator is immersed in the operating state in liquid to be atomized. Preferably, the actuator is completely within the liquid to be atomized.

Furthermore, it is advantageous if the intensity and frequency of the ultrasound acting on the liquid to be atomized can be regulated and / or measured by means of suitable precautions.

As mentioned in the requirements for a new coating process, the uniformity and high homogeneity of the layer to be produced is of utmost importance. In order to be able to guarantee this, it is desirable to have as small as possible a size distribution of the smallest possible droplets of a mist to be deposited. In the case of simple, commercial nebulizers, such as those used primarily in terrarium, but must also be expected with the occurrence of large drops or even splashes from the liquid to be atomized.

It is therefore preferred that the coating apparatus according to the invention comprises a droplet separator. This droplet separator is to be arranged in the volume of space between the surface of the liquid to be atomized and the holder on which the carrier substrates to be coated are stored during the coating process. There are different Ausftihrungsformen suitable for pot smoke. In principle, a mist eliminator to be used may be impermeable to vapor and mist (for example, if the mist eliminator is a closed solid). It may be advantageous if the mist eliminator has the geometric shape of a concave mirror. For example, a watch glass (having a concave surface) may be used as a mist eliminator.

A drop separator to be used may also be permeable to drops up to a defined size, for example with a diameter of less than 200 μm. This can be technically realized, for example, by virtue of the fact that the mist eliminator comprises a fine-meshed net, with the mesh spacing of which the maximum size of drops to be passed is determined.

It is preferred that the carrier substrates are coated during storage in the holder during the coating process on its side facing away from the surface of the liquid to be atomized / surface, wherein a coating on other surfaces must not be excluded.

Using masks to be applied to the carrier substrates to be coated, it is also possible to use a coating apparatus according to the invention in a coating method according to the invention to produce geometrically structured coatings by optionally sequential atomization of one or more optionally different liquids. The prerequisite for producing reproducible coated regions on the carrier substrates in their geometry is that areas of the carrier substrate that are not to be coated in each case are covered fluidically by a corresponding suitable mask, so that no mist droplets reach the areas not to be coated.

In order to be able to fulfill the primary objective of producing a uniform and homogeneous coating of the carrier substrates, it is furthermore advantageous if the coating apparatus according to the invention additionally comprises provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the surroundings of said carrier substrates , For example, it may be helpful if a gas is admitted into the container of the apparatus (ie into the air or gas or mist space), which mixes with the generated mist and / or swirls with it.

Therefore, it is advantageous if the coating apparatus additionally comprises at least one gas inlet. The apparatus may additionally include one or more outlets for venting gas and / or mist.

It may also be advantageous if said provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the vicinity of said carrier substrates comprise a ventilator with which the generated mist and, if appropriate, additional gases introduced into the container of the apparatus are fluidized to achieve a better mixing and thus elimination of inhomogeneities of the mist distribution.

To ensure constant and well-defined conditions during the coating process, it may further be advantageous if the coating apparatus according to the invention additionally comprises provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or one or all walls of the liquid container. It is also preferred that the holder of the coating apparatus for receiving and / or storing the carrier substrates during the coating process is thermostatable.

For the same reason, it may also be advantageous if the coating apparatus additionally comprises provisions for controlling and / or regulating the pressure within the liquid container during the coating process.

In order to ensure the uniformity and homogeneity of the coating of the carrier substrates, in particular to exclude an influence possibly despite appropriate precautions still existing inhomogeneities of the fog to be generated in the container of the apparatus according to the invention, it may also be advantageous if the coating apparatus additionally provides for the rotation of the carrier substrates around an axis perpendicular to the mounting plane. Due to an inherent property of the inventive method, namely a substantially spatially non-directional deposition, the droplet formation and thus application of the compounds contained for surface coating takes place not only on the free surfaces of the carrier substrates, but for example on the walls of the liquid container of the inventive coating apparatus. Since the compounds to be applied to the carrier substrates can be very special substances in highly pure form, which consequently may be relatively expensive, it is preferred that the coating apparatus according to the invention additionally nebulizes provisions for collecting and recycling / recycling on the walls of the liquid container Liquid includes.

In addition, it is advantageous if the coating apparatus according to the invention additionally comprises provisions to facilitate the cleaning of the liquid container. For example, the provisions may include a hydrophobic coating on the surface of said container walls, both for recirculation to the inner walls of the liquid container and liquid to be atomized, and for ease of cleaning. Such provisions may also relate to the geometric shape, for example, by corners are avoided in which liquid can collect and is difficult to remove again, or at least rounded.

Preferably, the carrier substrates to be coated are stored substantially horizontally in the holder of the coating apparatus. By the term "essentially horizontal" deviations of up to +/- 10 ° from a horizontal bearing should be included.

It is also advantageous if the coating apparatus additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and surfaces of the carrier substrates to be coated.

Preferably, the liquid container is closed except for optional gas inlet and optional additional gas and / or mist outlets.

Preferably, the liquids to be atomized are low-viscosity liquids having a viscosity of less than 3 cP. In particular, this may be act aqueous solutions. However, the liquids to be atomized may also be organic, in particular alcoholic solutions.

In addition, it is preferred that the carrier substrates to be coated are substantially planar. The term "essentially planar" should be understood to mean that said carrier substrates comprise a plane in which, apart from a possibly existing three-dimensional structure (such as side walls of sample containers to be provided on the carrier substrate surface), the surface to be coated is located to substantially parallel second plane in which the opposite surface of the carrier substrates is located, wherein under the name "substantially parallel" deviations of up to +/- 10 ° of parallelism are included. As "substantially planar" carrier substrates with both smooth and rough surfaces to be coated are to be understood.

The carrier substrates to be coated may consist of a single (self-supporting) layer, such as. As glass slides, or even of several layers.

It is preferred that at least one layer of the carrier substrates to be coated in the propagation direction of an irradiated excitation light or measurement light is substantially optically transparent.

The term "optical transparency" of a material or of a carrier substrate should be understood to mean that the run length of a light propagating in said material or in said carrier substrate or in the (high-index) waveguiding film of a carrier substrate (see below) as optical waveguide is at least is a portion of the visible spectrum (between 400 nm and 750 nm) greater than 2 mm, provided that this run length is not limited by structures for changing the propagation direction of said light, for example, the run length of optically visible light, ie the distance on the path of the Light in the corresponding material, until the light intensity decreases to a value 1 / e of the intensity of origin when the light enters this material, on the order of several centimeters (eg in thin-film waveguides, see below) up to meters or kilometers (in Trap of optical fiber n for the optical signal transmission). In the case of a grating waveguide structure based on a thin film waveguide, the propagation length of an in of the waveguiding layer guided light by a decoupling diffractive grating (formed in the waveguiding layer) are limited to a few microns. However, this limitation of the run length is due to the structuring, and not by the material properties of the structure. For the purposes of the present invention, such a lattice waveguide structure is to be referred to as "optically transparent." Within the scope of the present invention, "support structures" or "layers that are substantially optically transparent" are to be understood as meaning the intensity of a support substrate or layers transmitted light by less than 50%.

The at least one in the propagation direction of an irradiated excitation light or measurement light substantially optically transparent layer of carrier substrates to be coated, for example, comprise a material which is selected from the group consisting of silicates, for. As glass or quartz, transparent thermoplastic molding, sprayable or millable plastics, such as polycarbonates, polyimides, acrylates, in particular polymethyl methacrylates, polystyrenes, cyclo-Olefϊnpolymere and cyclo-olefin copolymers.

In a specific embodiment of a coating apparatus according to the invention, the support substrates to be coated comprise a thin metal layer, preferably of gold or silver, optionally on an underlying intermediate layer with refractive index preferably <1.5, wherein the thickness of the metal layer and the possible intermediate layer are selected such that a Oberflächenplasmon at the wavelength of an incident excitation light and / or at the wavelength of a generated luminescence can be excited. The thickness of the metal layer is preferably between 10 nm and 1000 nm, more preferably between 30 nm and 200 nm.

The conditions for generating a surface plasmon resonance, as well as for combination with luminescence measurements, as well as waveguiding structures are widely described in the literature.

The term "luminescence" in this application refers to the spontaneous emission of photons in the ultraviolet to infrared range after optical or non-optical, such as electrical or chemical or biochemical or thermal excitation. For example, chemiluminescence, bioluminescence, electroluminescence and in particular fluorescence and phosphorescence are included under the term "luminescence". Fluorescence and phosphorescence are particularly preferred forms of luminescence.

It is preferred that the carrier substrates to be coated comprise optical waveguides comprising one or more layers. In this case, the carrier substrates may be formed throughout as optical waveguides or comprise discrete waveguiding regions.

Corresponding waveguiding regions are to be understood as "continuous waveguiding regions" which extend over substantially the entire region of the part of the surface of a carrier substrate used for analyte detection without an interruption of the high-refractive, waveguiding layer.

Optical waveguides are particularly well suited as a carrier substrate for analyte detection in an affinity detection method, since waveguiding combines the formation of a so-called "evanescent" field at the interfaces of the high-refractive-wave waveguiding layer with the adjacent layers (which may also be air) with lower refractive index The penetration depth of this evanescent field into the environment is limited to dimensions less than the wavelength of the guided light (eg, 200 nm to 400 nm) such that interactions of analyte molecules or detection molecules or molecular moieties (such as fluorescent labels ) with this evanescent field excited and observed spatially highly selectively on a surface of the waveguide and interference signals from the far field, eg., From the depth of a sample medium, can be largely excluded.

Therefore, it is generally preferred that the continuous or discrete waveguiding regions to be coated on the carrier substrates comprise a surface of the carrier substrates to be coated.

It is particularly preferred that the carrier substrates to be coated planar waveguide optical waveguide having a substantially optically transparent, waveguiding layer (a) on a second, also substantially optically transparent layer (b) having a lower refractive index than layer (a) and optionally also in Substantially optically transparent intermediate layer (b ') between layer (a) and layer (b) also with a lower refractive index than layer (a).

Given the material of layer (a) and given refractive index, the lower the layer thickness, the greater the sensitivity up to a lower limit of the layer thickness. The lower limit value is determined by the termination of the light pipe when the value falls below a value which depends on the wavelength of the light to be led, as well as by an increase in the propagation losses in the case of very thin layers with further layer thickness decrease. It is preferred that the product of the thickness of the layer (a) and its refractive index is one tenth to one whole, preferably one third to two thirds, of the wavelength of an excitation light or measurement light to be coupled into the layer (a).

For the coupling of excitation light or measuring light in an optical waveguide a variety of methods are known. In the case of a relatively thick waveguiding layer up to a self-supporting waveguide, it is possible to focus the light into an end face of the waveguide using lenses of suitable numerical aperture such that it is guided by total internal reflection. In the case of waveguides with a larger forehead width than waveguide layer thickness cylinder lenses are preferably used for it. In this case, the lenses can be arranged both physically remote from the waveguide and also connected directly to it. In the case of lower waveguide layer thicknesses, this form of end face coupling is less suitable. It is then better to use the coupling via prisms, which are preferably added to the waveguide without interstice or connected to the waveguide via a refractive index-matching liquid. It is also possible to introduce the excitation light via an optical fiber to the optical waveguide and to couple in via an end face or to couple the light coupled into another waveguide into the waveguide by bringing both waveguides close to each other such that their evanescent fields overlap and therewith Energy transfer can take place.

It is therefore preferred that the discrete or continuous waveguiding regions of the carrier substrates to be coated during the detection step of an affinity detection method with said carrier substrates in optical interaction with one or more optical coupling elements for coupling excitation or measuring light from one or more light sources, said optical coupling elements being selected from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with focusing lenses arranged in front of a waveguiding layer of the carrier substrates, preferably cylindrical lenses, and grating coupler.

It is particularly preferred that the discrete or continuous waveguiding regions of the carrier substrates to be coated are in contact with one or more grating structures (c), which enable the coupling of excitation light or measuring light into waveguiding layers of said carrier substrates, and / or to one or more grating structures (c '), which enable the decoupling of excitation light or measuring light from waveguiding layers of said carrier substrates are, wherein in the case of simultaneously present on a carrier substrate grating structures (c) and (c') may have the same or different grating periods.

Said lattice structures are preferably relief gratings of any desired profile, for example of rectangular, triangular, sawtooth, semicircular or sinusoidal profile, or of phase or volume gratings with a periodic modulation of the refractive index in the essentially planar layer (a ). Preferably, grating structures (c) are formed as surface relief gratings.

The grating structures (c) and / or (c ') may be mono- or multi-diffractive and have a depth of 2 nm-100 nm, preferably 10 nm-30 nm, and a period of 200 nm-1000 nm, preferably 300 nm - 700 nm, have. The ratio of the land width of the grid lines to the grating period may be between 0.01 and 0.99, with a ratio between 0.2 and 0.8 being preferred.

It is preferable that the refractive index of the first optically transparent layer (a) is larger than 1.8. It is also preferable that the first optically transparent layer (a) comprises a material selected from the group consisting of silicon nitride, TiO ₂ , ZnO, Nb ₂ O _5, Ta ₂ O ₅ , HfO ₂ , and ZrO ₂ , particularly preferably TiO ₂ , Ta ₂ O ₅ or Nb ₂ O ₅ . It is also preferred that the second optically transparent layer (b) of the carrier substrates to be coated comprises a material from the group consisting of silicates, for. Glass or quartz, transparent thermoplastic moldable or millable plastics, for example polycarbonates, polyimides, acrylates, especially polymethyl methacrylates, polystyrenes, cyclo-olefin polymers and cyclo-olefin copolymers.

Various embodiments of planar optical thin-film waveguides which are suitable as carrier substrates are described, for example, in international patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 01/79821, WO 01/88511, WO 01/55691 and WO 02/79765. The embodiments described in these patent applications of special carrier substrates, usually referred to as sensor platforms, and methods to be carried out therewith for analyte detection, as well as the content of this application are hereby incorporated in their entirety as part of the present invention.

Characteristic of a preferred group of embodiments of coating apparatuses according to the invention is that the carrier substrates to be coated enable the detection of one or more analytes in an affinity detection method by means of medium detection of one or more excited luminescences.

It is characteristic of a further group of embodiments that the carrier substrates to be coated enable detection of one or more analytes in an affinity detection method by means of detection of changes in the effective refractive index in the near field (evanescent field) on a surface of said carrier substrates.

Another object of the present invention is a method for coating carrier substrates for the detection of one or more analytes in an affinity detection method, characterized in that said to be coated carrier substrates in a holder of a coating apparatus according to the invention according to one of

Embodiments are inserted, - contained in the liquid container of said coating apparatus

Liquid is fogged up and - From the generated mist, a deposition of substances contained in the nebulised liquid (compounds) on the carrier substrates to be coated takes place, wherein the carrier substrates in any contact with the surface to be nebulized

Liquid are located.

Various technical methods for generating ultrasound are known, for example using piezoelectric crystals, vibrating membranes, etc. It is preferred that said actuator comprises the membrane of an ultrasonic generator and the atomization of liquid occurs by means of ultrasonic waves generated therein.

In addition, it is preferred that the coating apparatus comprises a droplet separator which prevents the contact of splashes and large droplets from the liquid to be atomized with the carrier substrates to be coated. A "large" drop is to be understood as meaning a drop with a diameter of more than 200 μm, whereby the mist eliminator may be impermeable to gas and mist, for example, the mist eliminator may be a closed solid For example, a watch glass (having a concave surface) may be used as a mist eliminator. A mist eliminator to be used may also be permeable to drops up to a defined size. This can be technically realized, for example, by virtue of the fact that the mist eliminator comprises a fine-meshed net, with the mesh spacing of which the maximum size of drops to be passed is determined.

In order to be able to fulfill the primary objective of producing a uniform and homogeneous coating of the carrier substrates, it is furthermore advantageous if the coating apparatus according to the invention comprises provisions for producing a uniform distribution of the generated mist to be deposited on the carrier substrates in the surroundings of said carrier substrates.

For example, it may be helpful if the coating apparatus additionally comprises at least one gas inlet via which a gas is introduced into the liquid container, which gas mixes with the generated mist. The apparatus may additionally include one or more outlets for venting gas and / or mist.

It can also be advantageous for the uniformity and homogeneity of the coating if a uniform distribution of the generated mist to be deposited on the carrier substrates in the surroundings of said carrier substrates is produced by means of a fan.

To ensure constant and well-defined conditions during the coating process, it may also be advantageous if the coating apparatus according to the invention additionally comprises provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or individual or all walls of the liquid container and the temperature of the nebulizing Liquid and / or single or all walls of the liquid container is controlled and / or varied during the coating process. It is also preferred that the holder of the coating apparatus for receiving and / or storing the carrier substrates is thermostated during the coating process.

For the same reason, it may also be advantageous if the coating apparatus additionally includes provisions for controlling and / or regulating the pressure within the Liquid container during the coating process and the pressure during the coating process is controlled and / or varied.

In order to ensure the uniformity and homogeneity of the coating of the carrier substrates, in particular to exclude an influence possibly despite appropriate precautions still existing inhomogeneities of the fog to be generated in the container of erfϊndungsgemässen apparatus, it may also be advantageous if the carrier substrates during the coating process about an axis be rotated perpendicular to the mounting plane.

A particular variant of the method according to the invention is characterized in that geometrically structured coatings are produced by optionally sequential atomization of one or more optionally different liquids using masks to be applied to the carrier substrates to be coated with a coating apparatus according to the invention. The prerequisite for the production of coated regions reproducible in their geometry on the carrier substrates is that areas of the carrier substrate which are not to be coated are covered in a fluid-tight manner by a suitable mask, so that no mist droplets reach the areas not to be coated.

Preferably, the carrier substrates to be coated are stored substantially horizontally during the coating process in the holder of the coating apparatus.

It is also advantageous if the coating apparatus additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and surfaces of the carrier substrates to be coated, and so that a well-defined distance between said liquid and the liquid surfaces to be coated for the period of the coating process is set.

To reduce the consumption of liquid to be atomized, it is also preferred that liquid deposited on the walls of the liquid container be collected and returned to the liquid to be atomized.

Preferably, the liquid container of the coating apparatus is closed except for optional gas inlet and optional additional gas and / or mist outlets.

Preferably, the liquids to be atomized are low-viscosity liquids having a viscosity of less than 3 cP. In particular, these may be aqueous solutions. However, the liquids to be atomized may also be organic, in particular alcoholic solutions.

In addition, it is preferred that the carrier substrates to be coated are substantially planar.

The at least one in the propagation direction of an irradiated excitation light or measurement light substantially optically transparent layer of carrier substrates to be coated, for example, comprise a material which is selected from the group consisting of silicates, for. As glass or quartz, transparent thermoplastic molding, sprayable or millable plastics, such as polycarbonates, polyimides, acrylates, in particular polymethyl methacrylates, polystyrenes, cyclo-olefin polymers and cyclo-olefin copolymers. In a special embodiment of a coating apparatus according to the invention, the carrier substrates to be coated comprise a thin metal layer, preferably of gold or silver, optionally on an underlying intermediate layer with refractive index preferably <1.5, wherein the thickness of the metal layer and the possible intermediate layer are selected such that a Oberflächenplasmon at the wavelength of an incident excitation light and / or at the wavelength of a generated luminescence can be excited.

In this case, it is generally preferred that the continuous or discrete waveguiding regions to be coated on the carrier substrates comprise a surface of the carrier substrates to be coated.

In this case, it is preferred that the discrete or continuous waveguiding regions of the carrier substrates to be coated can be brought into optical interaction with one or more optical coupling elements for coupling excitation or measuring light from one or more light sources during the detection step of an affinity detection method with said carrier substrates, wherein said optical coupling elements are selected from the group comprising prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, face couplers with focusing lenses arranged in front of one waveguide layer of the carrier substrates, preferably cylindrical lenses, and grating couplers. It is particularly preferred that the discrete or continuous waveguiding regions of the carrier substrates to be coated are in contact with one or more grating structures (c), which enable the coupling of excitation light or measuring light into waveguiding layers of said carrier substrates, and / or to one or more grating structures (c '), which enable the decoupling of excitation light or measuring light from waveguiding layers of said carrier substrates are, wherein in the case of simultaneously present on a carrier substrate grating structures (c) and (c') may have the same or different grating periods.

It is preferable that the refractive index of the first optically transparent layer (a) is larger than 1.8. It is also preferable that the first optically transparent layer (a) comprises a material selected from the group consisting of silicon nitride, TiO ₂ , ZnO, Nb ₂ O ₅ , Ta ₂ O ₅ , HfO ₂ , and ZrO ₂ , particularly preferably TiO ₂ , Ta ₂ O ₅ or Nb ₂ O ₅ .

It is also preferred that the second optically transparent layer (b) of the carrier substrates to be coated comprises a material from the group consisting of silicates, for. As glass or quartz, transparent thermoplastic molding, sprayable or millable plastics, such as polycarbonates, polyimides, acrylates, in particular polymethyl methacrylates, polystyrenes, cyclo-olefin polymers and cyclo-olefin copolymers.

Characteristic of a preferred group of embodiments of the coating method according to the invention is that the carrier substrates to be coated enable the detection of one or more analytes in an affinity detection method by means of detection of one or more excited luminescences.

It is characteristic of a further group of embodiments that the carrier substrates to be coated enable the detection of one or more analytes in an affinity detection method by means of detection of changes in the effective refractive index in the near field (evanescent field) on a surface of said carrier substrates. A group of embodiments of the method according to the invention is characterized in that the layer to be deposited on the carrier substrates is an adhesion-promoting layer.

It is preferred that said adhesion promoting layer has a thickness of less than 200 nm, more preferably less than 20 nm.

A large number of compounds are suitable for producing the adhesion-promoting layer in a coating process according to the invention. For example, the primer layer may comprise a chemical compound from the groups comprising silanes, functionalized silanes, epoxides, functionalized charged or polar polymers, and "self-assembled passive or functionalized monolayers or multilayers", thiols, alkyl phosphates and phosphonates, multifunctional block copolymers, such as poly (L) lysine / polyethylene glycols.

The method according to the invention is characterized in that one or more specific binding partners are immobilized on the surface of the carrier substrates for the detection of one or more analytes in an affinity detection method (binding the binding partner from a supplied solution to the immobilized binding partner).

These specific binding partners can be applied to an adhesion-promoting layer applied by means of the coating method according to the invention or else directly to the uncoated surface of the carrier substrates, with areas of the surface remaining free of specific binding partners preferably being provided with a passivation layer in a subsequent coating step according to the method according to the invention below).

In a broadly applicable embodiment of the method according to the invention, the specific binding partners immobilized on the surface of said carrier substrates are biological or biochemical or synthetic recognition elements for the specific recognition of one or more analytes present in a sample supplied. In this case, different such specific recognition elements are present in each case in as highly pure a form as possible, generally in different discrete measurement ranges, so that different analytes from the sample generally bind to measurement regions with different recognition elements. Such arrays of measurement areas are also referred to as "capture arrays".

Since different recognition elements differ more or less strongly in their physicochemical properties (eg in their polarity), there are also corresponding differences in the conditions for optimal immobilization of these recognition elements, for example by adsorption or covalent bonding, in discrete measurement ranges on one common solid support, optionally on a primer layer applied thereon. Consequently, the immobilization conditions (such as the type of adhesion-promoting layer) chosen for immobilizing a multiplicity of different recognition elements hardly at the same time represent an optimum for all recognition elements, but merely a compromise between the immobilization properties of the different recognition elements.

It is also disadvantageous in this type of assay that the detection of analytes in a large number of different samples generally requires the provision of a corresponding number of discrete arrays of recognition elements to which the different samples are supplied on common or discrete carriers. To study a variety of different samples this means the need for a large number of discrete arrays, the production of which is relatively expensive.

In International Patent Applications PCT / EP 03/09561 and PCT / EP 03/09562, the contents of which are hereby incorporated in their entirety as part of the present invention, a novel assay structure is proposed, which makes it possible to have a plurality of samples in an array simultaneously examine a common carrier for analytes contained in the samples. For this purpose, not the different specific recognition elements but the samples to be examined are themselves untreated or, after as few preparation steps as possible, are applied in discrete measurement areas in an array on a carrier substrate. Such an assay construction is referred to in the two referenced applications as an "inverted assay architecture". Characteristic of a further widely applicable embodiment of the method according to the invention is therefore that the specific binding partners immobilized on the surface of said carrier substrates are the one or more analytes themselves, which are embedded in a native sample matrix or modified with one or more processing steps Form of the sample matrix are immobilized.

Said binding partners, i. the self-immobilized analyte to be detected or detected in a supplied sample and / or their immobilized or supplied in a supplied detection reagent biological or biochemical or synthetic Erkenuungselemente can be selected from the group which proteins, such as monoclonal or polyclonal antibodies and antibody fragments, peptides, enzymes , Glycopeptides, oligosaccharides, lectins, antigens for antibodies, proteins functionalized with additional binding sites ("tag proteins", such as "histidine tag proteins") and nucleic acids (for example DNA, RNA, oligonucleotides) and nucleic acid analogs (eg. PNA), aptamers, membrane-bound and isolated receptors and their ligands, cavities produced by chemical synthesis for incorporation of molecular imprints, natural and artificial polymers, etc.

In this case, said specific binding partners applied to the surface of the carrier substrates can be immobilized in discrete measuring areas (spots) which can have any geometry, for example circular, oval, triangular, rectangular, polygonal, etc., wherein a single measuring area has identical or different specific binding partners may contain.

It is preferred that discrete measurement ranges be created by spatially selective application of specific binding partners on said carrier substrates, preferably using one or more of the group of methods which include "ink jet spotting", mechanical spotting, "micro contact printing", fluidic Contacting of the areas for the measuring ranges to be created with the compounds to be immobilized by their supply in parallel or crossed microchannels, under the effect of pressure differences or electrical or electromagnetic potentials, as well as photochemical and photolithographic immobilization processes. As already mentioned above, in order to minimize nonspecific binding of analyte molecules or their detection reagents in areas free of immobilized specific binding partners of the carrier substrate surfaces, it is preferred that between the spatially separated measurement areas or in unoccupied partial areas within these measurement areas towards the analytes and / or towards its binding partners "chemically neutral" compounds are applied. These are preferably "chemically neutral" compounds relative to the analytes and / or their binding partners, for example selected from the groups which include albumins, in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, alien or empirical for the analyte (s) to be detected and / or their Binding partners non-specific antibodies (especially for immunoassays), detergents - such as Tween 20 -, fragmented natural or synthetic DNA not hybridizing with polynucleotides to be analyzed, such as extracts of herring or salmon spermatozoa (especially for polynucleotide hybridization assays), or uncharged ones; but hydrophilic polymers such as polyethylene glycols or dextranes.

The present invention therefore relates to a method according to the invention according to one of the aforementioned embodiments, which is characterized in that the layer deposited on the carrier substrates is a passivation layer which is located between the spatially separated measuring areas or in unoccupied partial areas within these measuring areas Analytes and / or to its binding partners "chemically neutral" compounds after the generation of these measuring ranges is applied and preferably, for example, comprises compounds from the groups which albumin, especially bovine serum albumin or human serum albumin, casein, unspecific, polyclonal or monoclonal, alien or empirical for the or the analytes to be detected and their binding partners nonspecific antibodies (especially for immunoassays), detergents - such as Tween 20 -, do not hybridize with polynucleotides to be analyzed end, fragmented natural or synthetic DNA, such as extracts of herring or salmon sperm (especially for polynucleotide hybridization assays), or even uncharged but hydrophilic polymers, such as polyethylene glycols or dextrans. A further subject of the present invention is a carrier substrate for the detection of one or more analytes in an affinity detection method comprising an adhesion-promoting layer, characterized in that said adhesion-promoting layer is produced by a coating method according to the invention according to one of said embodiments.

Likewise provided by the present invention is a carrier substrate for the detection of one or more analytes in an affinity detection method comprising a passivation layer covering the carrier substrate at least in partial regions, characterized in that said passivation layer is produced by a coating method according to the invention according to one of said embodiments.

Another object of the present invention is a carrier substrate according to one of the aforementioned embodiments for use in human and / or animal diagnostics.

The present invention will be explained in more detail by way of example in an exemplary embodiment.

Examples:

1. Coating apparatus and coating method according to the invention

A schematic representation of a coating apparatus according to the invention is shown in FIG. 1. In the present example, with the apparatus according to the invention, the areas of unprotected areas of specific binding partners are to be "passivated" by carrier substrates prepared for an affinity detection method, i.e. a "passivation layer" is applied in these areas. The inventive apparatus in this exemplary embodiment comprises a desiccator (1) with a volume of about 2 1 as a container for the liquid to be atomized and the volume of mist to be generated over the liquid, a holder (2) for receiving the carrier substrates to be coated Ultrasonic atomizer ("Lucky Reptile Mini-Nebulizer", Reptilica, D-90431 Nuremberg, Germany) as an actuator (3) for liquid atomization, a watch glass as a droplet separator (4) and a gas inlet (5) and an outlet (6) for gas and / or generated fog.

In the operating state of the ultrasonic generator is immersed in the liquid to be atomized (7). In order to minimize the required volume of liquid, in the embodiment of the present example, the ultrasonic generator mounted on the bottom of the desiccator was poured into polydimethylsiloxane (PDMS) just below the vibrating vibrating diaphragm, requiring only the application of a thin layer of fluid to be atomized is.

The finest droplets produced by the action of ultrasound and rising above the liquid level are additionally swirled by means of a small stream of nitrogen, which is introduced into the container via the inlet (5), in order to distribute the resulting mist as homogeneously as possible throughout the entire container produce.

To be coated planar optical thin-film waveguides as carrier substrates, with the external dimensions of 14 mm wide x 57 mm long x 0.7 mm thick (for more details see below), in the holder (2) during the coating process at a distance of about 8 cm the liquid surface stored horizontally (with respect to the liquid surface). The holder is formed in the present example as a perforated carrier made of plastic, so that through these holes Excess liquid separated from the mist can flow off. In the present exemplary embodiment, the holder can accommodate ten thin-film waveguides as carrier substrates with the dimensions mentioned.

The watch glass as a droplet is glued in the present example to the underside of the holder (2) and shields the substrates to be coated against splashes from the nebulizing solution (coating solution).

The generated, very homogeneously distributed mist deposits on the carrier substrates, with in the present example on the top (with respect to the storage in the coating apparatus) arranged high-refractive wave-guiding layer (a), in the form of very small droplets, and already within 5 minutes to 10 Minutes forms on the tops of these substrates a thin, continuous liquid film. After a total incubation time of 30 minutes, the carrier substrates are removed from the coating apparatus, thoroughly rinsed with flowing ultrapure water (Millipore) and finally dried in a stream of nitrogen.

In the present example, a volume of about 2 ml of passivation solution (liquid to be atomized) is required for a thin-film waveguide of the stated dimensions as the carrier substrate.

2. Carrying out conventional coating processes 2.1. carrier substrates

In the present examples (as already mentioned under 1.), the carrier substrates used for an affinity detection method to be carried out later are planar optical thin-film waveguides, each with the outer dimensions 14 mm wide x 57 mm long x 0.7 mm thick. These support substrates each comprise a glass substrate (AF 45) and a 150 nm thin, high refractive layer of tantalum pentoxide applied thereon. In the glass substrate, parallel to the length, two surface relief gratings (grating period: 318 nm, grating depth: (12 +/- 2) nm) are modulated at a distance of 9 mm, which are to serve as diffractive gratings of the light coupling into the high refractive layer. On the surface of the metal oxide layer of this carrier substrate, a monolayer of mono-dodecyl phosphate (DDP) formed as an adhesion-promoting layer by spontaneous self-assembly ("soap assembly") is applied hydrophobic bonding layer provided carrier substrates are each 6 identical microarrays of 144 discrete measuring areas (spots), in turn, in an array of 16 rows and 9 columns, with an inkjet spotter (model NP 1.2, GeSiM, Grosserkmannsdorf, Germany) applied is created by applying a single droplet of approximately 350 pL volume to the chip surface.

2.2. Reagents and generation of arrays of measurement areas on the carrier substrates

In the present example, the analytes to be detected themselves are to be immobilized in a subsequent affinity detection method on the prepared carrier substrates, embedded in a native sample matrix or in a form of sample matrix (cell lysate) prepared with a few sample preparation steps. These forms of the samples are also referred to below as "nature-identical samples." The detection step should then take place after addition of further detection reagents.

For the detection of biologically relevant protein analytes in the "nature-identical" samples, a human colon cancer cell line (HT29) is used These adherent cells are incubated in modified McCoy's 5A medium at 37 ° C in conventional culture plastic bottles (Greiner Bio-One). St. Gallen, Switzerland, Cat No: 658170) Similar cell cultures of different culture flasks are then either exposed to UV light for 10 minutes or treated with 10 μM doxorubicin.As a comparative sample to these treated cell cultures, an otherwise similar cell culture is used remains untreated and will serve as a negative control in the analytical detection method. After treatment, the different cell cultures are washed with 10 ml PBS (phosphate buffered saline, cooled to 4 ° C).

Thereafter, the cells are detached from the bottom of the culture flasks by adding lysis buffer containing 7 M urea, 2 M thiourea and complete (protease inhibitor, Roche AG, 1 tablet / 50 ml) and at the same time completely lysed, whereby all protein-containing cell constituents are spontaneously denatured and solubilized , The cell lysate thus obtained is centrifuged for 5 minutes at 13,000xg in a benchtop centrifuge (Eppendorf, Hamburg, Germany) to separate insoluble cell components (e.g., DNA and cell membrane fragments). The supernatant is collected and used for the following measurements, the total protein concentration typically being between 5 mg / ml and 10 mg / ml.

The described treatments of the HT29 cell cultures lead to damage of the DNA, namely UV irradiation and the like. by chain breaking or by formation of thymine dimers and by doxorubicin addition by its intercalation between adjacent bases of the DNA. This has the consequence that within damaged cells certain signal paths are activated or deactivated, e.g. may result in programmed cell death (apoptosis). Responsible for activating or deactivating signaling pathways are certain key proteins (called "marker proteins") that regulate one or more signaling pathways through phosphorylation at one or more different sites.

An example of the regulation of a signaling pathway via a marker protein is the tumor suppressor protein p53, which via its degree of phosphorylation controls cell division, apoptosis and certain repair mechanisms for damaged DNA. These signaling pathways are often disturbed in cancer cells at one or more sites by mutations or lack of one or more marker proteins in their regulation, which may ultimately be responsible for uncontrolled growth.

The detection and determination of the relative levels of p53 and P-p53 (phosphorylated form of p53) is performed by using highly specific antibodies that bind to these proteins, which directly act as analytes in the recovered and treated cell lysates the carrier substrates (preferably after application of a primer layer as described above) to be immobilized.

The cell lysates obtained are diluted by a factor of 10 to 20 to a total protein concentration of about 0.4 mg / ml and then applied in discrete measurement areas to produce an array of measurement areas on the metal oxide surface of the thin-film waveguides provided with the adhesion-promoting layer as carrier substrates. In addition to the measurement areas with cell lysates applied therein, each microarray contains further measurement areas with Cy5 fluorescently labeled bovine serum albumin (Cy5-BSA) immobilized therein, which are used to refer to local differences and / or temporal variations of the excitation light intensity during the measurement ("reference spots"). Cy5-BSA is applied in a concentration of 0.5 nM in 7 M urea, 2 M thiourea (labeling rate: about 3 Cy5 molecules per BSA molecule).

The geometry of the arrangement of the measurement areas in a two-dimensional array and a linear arrangement of six (identical) arrays on a carrier substrate are shown in FIG. 2. The diameter of the spots, with a center-to-center distance of 300 μm, is about 120 μm. An array of measuring ranges for these examples comprises in each case an arrangement of measuring regions with 12 different samples applied in 4 replicates, the 4 identical measuring regions each being arranged in a common column perpendicular to the direction of propagation of the light guided during the detection step in the waveguiding layer of these carrier substrates , The reproducibility of the measuring signals within the array of measuring ranges is to be determined with the help of the 4 identical measuring ranges. Between and adjacent to the columns of measurement areas with samples to be analyzed applied therein are columns of measurement areas with Cy5-BSA applied thereto (for referencing purposes). The analytical platform according to the invention in this example comprises 6 similar arrays of measuring ranges, as shown in FIG. 2.3. Passivation of the free areas between and within the measuring ranges

After application of the "naturally identical" samples and Cy5-BSA, the support substrates are dried in dust-free room air before the free, uncovered hydrophobic surface areas of the support substrates in a further step to minimize undesired non-specific binding of detection reagents, in this case antibodies and / or fluorescently labeled molecules, are saturated (passivated) with bovine serum albumin (BSA).

Two further methods (2.3.1 immersion method and 2.3.2 spray method) are compared with the surface passivation method according to the invention already described under 1., in which case freshly filtered passivation solution (50 mM imidazole, 10 mM NaCl, 3% BSA (w / v) pH 7.4). After passivation of the free surface by the methods described under 1. or below, the support substrates are stored at 4 ° C in closed polystyrene tubes until measurement in the context of the affinity detection method to be carried out below.

2.3.1. immersion method

The planar optical thin-film waveguides as carrier substrates are dropped vertically into a vessel filled with passivation solution (polystyrene tube), so that the entire surface of the carrier substrates is wetted as quickly and simultaneously as possible. After one hour of incubation at room temperature, the carrier substrates are thoroughly rinsed under running ultrapure water (Millipore) and then dried in a stream of nitrogen (quality 50). For each thin-film waveguide of the dimensions mentioned as the carrier substrate, a volume of about 25 ml of passivation solution is required.

2.3.2. spraying

The passivation solution is sprayed onto the carrier substrates here by means of a chromatography atomizer (Glas Keller Cat. No. 12.159.603, Basel, Switzerland) and a pressure of approximately 3.5 bar, until a continuous layer is obtained on the surface to be coated Liquid film has formed. The distance between the exit nozzle of the atomizer and the carrier substrate surface is approximately 30 cm. Subsequently, the carrier substrates thus treated are incubated in a closed container at 100% humidity for one hour at room temperature, then thoroughly rinsed under running ultrapure water (Millipore) and finally dried in a stream of nitrogen (quality 50). Per carrier substrate of the embodiment mentioned in these examples, a volume of about 3 ml passivation solution is needed.

3. Affinity demonstration 3.1. Assay architecture

The detection of certain proteins in general (ie, for example, with or without phosphorylation) or certain proteins especially in activated (eg phosphorylated) form in the immobilized in discrete measurement areas applied cell lysates by sequential addition of appropriate detection reagents prior to measuring the resulting fluorescence signals: To prepare for a first assay step is polyclonal analyte-specific rabbit antibodies (antibody Al (# 9282): anti-p53; antibody A2 (# 9284): anti-phospho-p53 (Serl5); both antibodies purchased from Cell Signaling Technology, INC., Beverly, MA) diluted 1: 500 in assay buffer (50 mM imidazole, 100 mM NaCl, 5% BSA, 0.1% Tween 20 pH 7.4). Of these different antibody solutions, 30 μl each are applied to each of the 6 identical arrays of measurement areas, followed by incubation at room temperature overnight (first assay step). Excess non-specifically bound antibodies are removed by washing each array with assay buffer (2 x 200 μl).

For the detection of bound analyte-specific antibodies contained in the immobilized cell lysates in discrete measurement ranges, a second assay step is carried out using an Alexa Fluor 647-labeled anti-rabbit Fab fragment (Molecular Probes, cat no Z-25308 , Leiden, The Netherlands), which binds to the aforementioned antibodies Al and A2. This fluorescently labeled Fab fragment is, starting from the commercially available stock solution, in a dilution of 1: 500 in assay buffer applied to the arrays (each 30 ul) and then for 1 hour at Room temperature incubated in the dark. Subsequently, the arrays are washed with assay buffer (two times each with 200 μl) to remove non-specifically bound fluorescently labeled Fab fragments. Thereafter, the prepared analytical platforms are stored in the ZeptoREADER ™ (see below) until the detection step by excitation and detection of resulting fluorescence signals.

3.2. Determination of the fluorescence signals from the arrays of measuring ranges

The fluorescence signals from the various arrays of measurement ranges are sequentially automatically measured with a ZeptoREADER ™ (Zeptosens AG, CH-4108 Witterswil, Switzerland). For each array of measuring ranges, the planar optical thin-film waveguide is adjusted as a carrier substrate (according to 2.1.) To fulfill the resonance condition for the light coupling via a grating structure (c) in the waveguiding tantalum pentoxide layer and to maximize the available in the measuring ranges excitation light. Subsequently, a user-selectable number of images of the fluorescence signals from the respective array is generated by each array, wherein different exposure times can be selected. The excitation wavelength in the measurements for the present example is 635 nm, the detection of the fluorescent light with a cooled camera at the fluorescence wavelength of Cy5, using an interference filter (transmission (675 ± 20) nm) for the suppression of scattered light at the excitation wavelength is positioned in front of the lens of the camera. The generated fluorescence images are automatically stored on the disk of the control computer. Further details of the optical system (ZeptoREADER ™) are described in International Patent Application PCT / EP01 / 10012, which is hereby incorporated in its entirety as part of this application.

3.3. Evaluation and referencing

The mean signal intensity from the measurement areas (spots) is determined using image analysis software (ZeptoVIEW ™, Zeptosens AG, CH-4108 Witterswil) allows to semi-automatically evaluate the fluorescence images of a large number of arrays of measurement areas.

The raw data of the individual pixels of the camera represent a two-dimensional matrix of digitized measured values, with the measured intensity as the measured value of a single pixel corresponding to the area imaged on it on the sensor platform. For the evaluation of the data, first of all, a two-dimensional (coordinate) network is placed over the pixels (pixel values) in such a way that the partial image of each spot falls into an individual two-dimensional network element. Within this network element, each spot is assigned a circular area of interest (AOI) with a user-definable radius (typically 120 μm), which can be adjusted as well as the location of the individual AOIs individually as a function of the signal intensity of the individual The average radius signal intensity of each spot determines the arithmetic mean of the pixel values (signal intensities) within a selected evaluation range.

The background signals are determined from the measured signal intensities between the spots. For this purpose, four further circular areas (typically taken together with the same radius as for the evaluation areas of the spots) are defined per spot as evaluation areas for background signal determination, which are preferably arranged in the middle between between adjacent spots. From these four circular areas, the average background signal intensity is determined, for example, as the arithmetic mean of the pixel values (signal intensities) within an AOI selected for this purpose. The average net signal intensity from the measurement areas (spots) is then calculated as the difference between the local average gross and local mean background signal intensity of the respective spot.

The referencing of the net signal intensity of all spots is done with the help of reference spots (Cy5-BSA) of each array of measuring ranges. For this purpose, the net signal intensity of each spot is divided by the average of the net signal intensities of the adjacent reference spots of the same row (arranged parallel to the propagation direction of the light guided in the evanescent field sensor platform). Through this referencing, the local differences of the available excitation light intensity become orthogonal to the light propagation direction both within each microarray and between different microarrays.

3.4. Results

3A shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the measurement areas were passivated by means of the dipping method (according to 2.3.1.). The signal intensity within each individual reference spot and between different reference spots (Cy5-BSA) is distributed very evenly and homogeneously, and the edge of the almost perfectly circular spots is sharply delimited from the background (see detail image). In contrast, the measuring ranges of the immobilized cell lysates are characterized by tail-like "smears", which is particularly noticeable at high signal intensities. As described above, these "smears" are caused during the moment of immersion of the carrier substrates provided with the spots into the passivation solution. and by non-firmly adsorbed sample components dissolved from the measurement areas by the passivation solution, which are adsorbed along the flow in the opposite direction in the immediate vicinity of such a measurement area on the free, not yet passivated carrier substrate surface, before they even contain the BSA contained in the passivation solution can be passivated. Since these sample portions, which have been removed from the measurement areas and resorbed in the neighborhood, always contain a certain content of analyte to be detected, a corresponding fluorescence signal is visible on reading at said locations.

3B shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the spots were passivated by means of the spray method (according to 2.3.2.). The signals from the reference spots are comparable in terms of their shape and uniformity or homogeneity as well as their intensity with those of a microarray after use of the dipping method. The signals from the measurement areas with immobilized cell lysates are also comparable in their intensity to the corresponding measurement signals from the microarrays which had been subjected to the immersion process. Due to the spray process, in contrast However, for the dipping method, negligible flows of passivation solution on the carrier substrate surface, the cell lysate spots do not show the "smudges" described above, but only smaller "outgrowths" of lesser fluorescence intensity, which are evidently arranged approximately randomly around the intended spots. These are most likely caused by the local dissolution and outflow of non-bound cell lysate at the edges of the measurement areas, since the impinging small spray droplets of the passivation solution on impact with the surface have a non-negligible impulse perpendicular to the coating surface, which can lead to the generation of splashes.

3C shows a typical image of the fluorescence signals of a microarray according to an assay for the detection of p53, in which free areas between the measurement areas were passivated by means of the method according to the invention by fogging passivation solution, as described under 1. Striking here, compared to the passivated with the other methods described microarray, the high quality with comparably good homogeneity and shape of reference spots and Zellysatspots. "Smudges" or "outgrowths" of the cell lysate spots can be avoided here due to the fact, apart from the influence of gravity, essentially undirected and pulse-free application of the passivation in the form of fine mist droplets, the size of which is well below that produced by spraying droplets.

The efficiency of passivation of the surface free of components from the immobilized sample, i. the degree of suppression of nonspecific binding by means of the BSA contained in the passivation solution can be determined semiquantitatively from the signal intensity measured in the areas free of spots (between the spots, "background signals") nonspecific binding of the fluorescently labeled detection reagents (Alexa 647 anti-rabbit Fab) used in the assay to the BS A-free surface give a higher signal than a surface continuously coated with BSA.

Figure 4A shows the averages and standard deviations of the background signal intensities determined between all spots of the free carrier substrate surfaces passivated by the three different methods with the microarrays generated thereon. The Designations A, B and C, as well as in FIGS. 4B, 5A and 5B, refer to passivation by means of the dipping method (A), spraying method (B) or method by misting of the passivating solution (C). It is surprisingly evident from the measured (lower) background signal intensities that the passivation efficiency after treatment with the spray method and the nebulization method according to the invention is significantly higher than after application of the dipping method to surface passivation. In addition, the standard deviation of the background signal intensities after application of the spray method or the nebulization method according to the invention is in each case significantly lower than after use of the dipping method for surface passivation, which led to a standard deviation of the background signal intensities of 34%. It is concluded that the uniformity or homogeneity of the coating after application of the spraying or misting process is higher than after use of the dipping process.

4B shows the mean values of the fluorescence intensities of all reference spots of the microarrays, wherein the free surfaces of the associated carrier substrates were in turn treated with the three different coating methods. Surprisingly, it emerges from the comparison that the signal intensity after application of the spraying process is increased slightly and significantly after the application of the nebulization method (namely by about 60%), in comparison to the signals after application of the dipping method. These differences are attributed to the reduction in the volume of applied passivating solution, which is likely to partially relieve the referenced Cy5-BSA compounds, as well as the nearly pulse-free application of the passivating solution in the case of the fogging process.

5 A shows the average intensities and standard deviations of the fluorescence signals from the measuring ranges of the microarrays intended for analyte detection, the carrier substrate surfaces of which were each treated with the different passivation methods and which are subsequently treated with solutions of the antibody Al (anti-p53) (FIG. 5 A, top ) and A2 (anti-phospho-p53) (Figure 5A, bottom) and then each incubated for detection by fluorescence detection with Alexa 647 Fluor anti-rabbit F _ab fragments. The measured fluorescence signal intensities correlate with the respective relative content of analytes contained in a cell lysate (corresponding to Zellysatkonzentration; higher signal corresponding to a higher analyte concentration, the correlation obviously being nonlinear in nature).

Compared to the control sample without pretreatment (each marked in Fig. 5A and Fig. 5B as "Control") show the lysate with UV light (each marked in Fig. 5A and Fig. 5B with "+ UV") and in particular the doxorubicin-treated HT29 cell culture (each labeled in Fig. 5A and Fig. 5B with "+ Dx") a significantly increased level of p53, caused by an increase in the expression of this protein in the cells in question.

In contrast, Fig. 5B shows that the content of phospho-p53 in the UV-light-treated sample is also markedly increased compared to the control sample, while the content of phospho-p53 in the doxorubicin-treated sample, despite a massively increased total concentration of p53 only slightly above (in the case of lysate concentrations from 0.2 mg / ml to 0.4 mg / ml) or even below (in the case of the lysate concentration of 0.1 mg / ml) that of the control sample. This demonstrates that the DNA damage-induced signaling pathway, in which phospho-p53 acts as a key regulatory protein, clearly responds to treatment with UV light in this cell line, but apparently only weakly to doxorubicin treatment.

With regard to the influence of the different methods used for passivating the free carrier substrate surfaces, it is essential that the measured signal intensities from the analyte detection ranges do not differ statistically significantly, taking into account the experimentally induced variations (error bars). regardless of the coating method used for surface passivation. This means that - obviously in contrast to the effects on the Cy5 -BS A compounds used for referencing - the different passivation methods do not differ in their influence on the cell lysates adsorbed on the carrier substrates.

In summary, these results show that the method according to the invention for applying the passivation solution to the carrier substrate surface by means of atomization, in contrast to the conventional dipping method and also to the spraying method, offers distinct advantages and completely fulfills the requirements set. For the expert is it can be seen that the method for the surface-passivation carrier substrate coating illustrated in the preceding exemplary embodiments can be applied directly to coatings with suitable adhesion-promoting layers and can be generalized in this way.

Claims

Claims:

An apparatus for coating carrier substrates for detecting one or more analytes in an affinity detection method, comprising: a container for receiving liquid to be atomized ("liquid container") with substances (compounds) to be deposited on at least one surface of said carrier substrates, and one above the liquid in the operating condition produced mist volume,

an actuator for causing the misting process and

a holder for receiving and supporting the carrier substrates during the coating process, characterized in that the carrier substrates are not in contact with the surface of the liquid to be atomized.

2. Apparatus according to claim 1, characterized in that said actuator is used to generate ultrasound.

3. Apparatus according to any one of claims 1 - 2, characterized in that said actuator comprises the membrane of an ultrasonic generator.

4. Apparatus according to any one of claims 1-3, characterized in that said actuator is immersed in the operating state in liquid to be atomized.

5. Apparatus according to any one of claims 1-4, characterized in that it additionally comprises a mist eliminator.

6. Apparatus according to claim 5, characterized in that the mist eliminator for vapor and mist is impermeable.

7. Apparatus according to claim 5, characterized in that the mist eliminator is permeable to drops up to a defined size.

8. Apparatus according to claim 6, characterized in that the mist eliminator has the shape of a concave mirror.

9. Apparatus according to claim 7, characterized in that the mist eliminator comprises a fine-meshed network, with the mesh spacing, the maximum size to be passed through drops is determined.

10. Apparatus according to any one of claims 1-9, characterized in that it additionally comprises at least one gas inlet.

11. An apparatus according to any one of claims 1-10, characterized in that it additionally comprises provisions for generating a uniform distribution of the deposited and deposited on the carrier substrates mist in the environment of said carrier substrates.

12. An apparatus according to claim 11, characterized in that said provisions for generating a uniform distribution of the generated and to be deposited on the carrier substrates fog in the vicinity of said carrier substrates comprise a fan.

13. Apparatus according to any one of claims 1-12, characterized in that it additionally comprises provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or individual or all walls of the liquid container.

14. Apparatus according to any one of claims 1 -13, characterized in that the holder of the coating apparatus for receiving and / or storage of the carrier substrates during the coating process is thermostatable.

15. Apparatus according to any one of claims 1-14, characterized in that it additionally comprises provisions for controlling and / or regulating the pressure within the liquid container during the coating process.

16. Apparatus according to any one of claims 1-15, characterized in that it additionally comprises provisions for the rotation of the carrier substrates about an axis perpendicular to the mounting plane.

17. An apparatus according to any one of claims 1-16, characterized in that it additionally comprises provisions for collecting and for recirculation / recovery on the walls of the liquid container separated atomized liquid.

18. Apparatus according to any one of claims 1-17, characterized in that it additionally comprises provisions to facilitate the cleaning of the liquid container.

19. An apparatus according to any one of claims 1 -18, characterized in that it additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and surfaces to be coated of the carrier substrates.

20. Apparatus according to any one of claims 1 -18, characterized in that the carrier substrates are mounted in the holder substantially horizontally.

21. Apparatus according to any one of claims 1-20, characterized in that the liquid container, except for optional gas inlet and optional additional outlets for gas and / or mist, is closed.

22. An apparatus according to any one of claims 1 -21, characterized in that it is low-viscosity liquids with a viscosity of less than 3 cP in the liquids to be atomized.

23. Apparatus according to any one of claims 1 -22, characterized in that it is aqueous solutions to be atomized liquids.

24. Apparatus according to any one of claims 1 -22, characterized in that it is organic solutions in the liquids to be atomized.

25. Apparatus according to claim 24, characterized in that it is to be atomized liquids are alcoholic solutions.

26. Apparatus according to any one of claims 1 -25, characterized in that the carrier substrates to be coated are substantially planar.

27. Apparatus according to any one of claims 1 to 26, characterized in that the carrier substrates to be coated consist of one or more layers.

28. A method for coating carrier substrates for detecting one or more analytes in an affinity detection method, characterized in that said carrier substrates to be coated are placed in a holder of a coating apparatus according to any one of claims 1-40, nebulized liquid contained in the liquid container of said coating apparatus is and from the generated mist, a deposition of substances contained in the nebulised liquid (compounds) on the carrier substrates to be coated, wherein the carrier substrates in any contact with the surface to be nebulized

Liquid are located.

A method according to claim 28, characterized in that said actuator is for the generation of ultrasound.

30. The method according to any one of claims 28 - 29, characterized in that the actuator of said coating apparatus comprises the membrane of an ultrasonic generator and the atomization of liquid by means of ultrasonic waves generated therein.

31. The method according to any one of claims 28 - 30, characterized in that the actuator of said coating apparatus is immersed in the operating state in liquid to be atomized.

32. The method according to any one of claims 28 - 31, characterized in that the coating apparatus additionally comprises a mist eliminator, which the Contact of splashes and large drops of the liquid to be atomized with the carrier substrates to be coated prevented.

33. The method according to claim 32, characterized in that the mist eliminator of said coating apparatus is impermeable to vapor and mist.

34. The method according to claim 32, characterized in that the droplet separator of said coating apparatus is permeable to droplets up to a defined size.

35. The method according to claim 33, characterized in that the mist eliminator of said coating apparatus has the shape of a concave mirror.

36. The method according to claim 34, characterized in that the droplet separator of said coating apparatus comprises a fine-meshed mesh, with the mesh spacing of which the maximum size of droplets to be passed is determined.

37. The method according to any one of claims 28-36, characterized in that the coating apparatus additionally comprises at least one gas inlet, via which a gas is introduced into the liquid container, which gas mixes with the generated mist.

38. The method according to any one of claims 28 - 37, characterized in that the coating apparatus additionally comprises provisions for generating a uniform distribution of the generated and deposited on the carrier substrates mist in the environment of said carrier substrates.

39. The method according to claim 38, wherein a uniform distribution of the generated mist to be deposited on the carrier substrates in the vicinity of said carrier substrates is produced by means of a fan.

40. The method according to any one of claims 28-39, characterized in that the coating apparatus additionally provisions for controlling and / or regulating the temperature of the liquid to be atomized and / or single or all walls the liquid container and the temperature of the liquid to be atomized and / or individual or all walls of the liquid container is controlled and / or varied during the coating process.

41. The method according to any one of claims 28 -40, characterized in that the holder of the coating apparatus for receiving and / or storage of the carrier substrates is thermostated during the coating process.

42. The method according to any one of claims 28 - 41, characterized in that the coating apparatus additionally comprises provisions for controlling and / or regulating the pressure within the liquid container during the coating process and the pressure is controlled and / or varied during the coating process.

43. The method according to any one of claims 28 -42, characterized in that the carrier substrates are rotated during the coating process about an axis perpendicular to the mounting plane.

44. The method according to any one of claims 28-43, characterized in that collected on the walls of the liquid container separated liquid and is returned to the liquid to be atomized.

45. The method according to any one of claims 28-44, characterized in that the coating apparatus additionally comprises provisions for the controlled adjustment and / or variation of the distance between the surface of the liquid to be atomized and to be coated surfaces of the carrier substrates and thus a well-defined distance between said liquid and the liquid surfaces to be coated for the period of the coating process is set.

46. The method according to any one of claims 28 - 45, characterized in that the carrier substrates of the coating apparatus are mounted in the holder substantially horizontally.

47. The method according to any one of claims 28 - 46, characterized in that the liquid container of the coating apparatus, except for optional gas inlet and optional additional outlets for gas and / or mist, is closed.

48. The method according to any one of claims 28 - 47, characterized in that it is the liquids to be atomized low viscosity liquids having a viscosity of less than 3 cP.

49. The method according to any one of claims 28 - 48, characterized in that it is aqueous solutions to be atomized liquids.

50. The method according to any one of claims 28 - 49, characterized in that it is organic solutions in the liquids to be atomized.

51. The method according to claim 50, characterized in that it is to be atomized liquids are alcoholic solutions.

52. The method according to any one of claims 28 - 51, characterized in that the coating of the carrier substrates is geometrically structured using masks applied to the carrier substrates to be coated.

53. The method according to any one of claims 28-52, characterized in that the carrier substrates to be coated are substantially planar.

54. The method according to any one of claims 28 - 53, characterized in that the carrier substrates to be coated consist of one or more layers.

55. The method according to claim 54, wherein at least one layer of the carrier substrates to be coated is substantially optically transparent in the propagation direction of an irradiated excitation light or measurement light.

56. The method according to any one of claims 28-55, characterized in that the carrier substrates to be coated, the detection of one or more analytes in one Enable affinity detection methods by detecting one or more excited luminescences.

57. Method according to claim 28, characterized in that the layer deposited on the carrier substrates is an adhesion-promoting layer.

A method according to claim 57, characterized in that said primer layer has a thickness of less than 200 nm, preferably less than 20 nm.

59. The method according to any one of claims 57-58, characterized in that said adhesion-promoting layer comprises a chemical compound from the groups comprising silanes, functionalized silanes, epoxides, functionalized, charged or polar polymers and "self-assembled passive or functionalized monolayers or multilayers" , Thiols, alkyl phosphates and phosphonates, multifunctional block copolymers such as poly (L) lysine / polyethylene glycols.

60. The method according to any one of claims 28-59, characterized in that on the surface of the carrier substrates one or more specific binding partner for the detection of one or more analytes in an affinity detection method (binding of the binding partner from a supplied solution to the immobilized binding partner) are immobilized.

61. The method according to claim 60, characterized in that the specific binding partners immobilized on the surface of said carrier substrates are the one or more analytes themselves, which are embedded in a native sample matrix or in a modified form of the one or more processing steps Sample matrix are immobilized.

62. The method according to claim 60, characterized in that the specific binding partners immobilized on the surface of said carrier substrates are biological or biochemical or synthetic recognition elements for the specific recognition of one or more analytes present in a sample supplied.

63. Method according to one of claims 60-62, characterized in that said binding partners, i. the self-immobilized analyte to be detected or detected in a supplied sample and / or their biological or biochemical or synthetic recognition elements immobilized or supplied in a supplied detection reagent are selected from the group consisting of proteins, for example mono- or polyclonal antibodies and antibody fragments, peptides, enzymes, Glycopeptides, oligosaccharides, lectins, antigens for antibodies, proteins functionalized with additional binding sites ("tag proteins", such as "histidine tag proteins"), and nucleic acids (eg, DNA, RNA, oligonucleotides) and nucleic acid analogs (eg, PNA ), Aptamers, membrane-bound and isolated receptors and their ligands, cavities generated by chemical synthesis for incorporation of molecular imprints, natural and artificial polymers, etc.

64. The method according to any one of claims 60-63, characterized in that on the surface of said carrier substrates applied specific binding partner in discrete measuring areas (spots) are immobilized, which any geometry, such as circular, oval, triangular, rectangular, polygonal shape, etc. may have, where a single measurement range may contain similar or different specific binding partners.

65. Method according to claim 64, characterized in that "chemically neutral" compounds are applied between the spatially separated measurement areas or in unoccupied partial areas within these measurement areas relative to the analytes and / or to its binding partners, preferably for example consisting of the groups which albumin, in particular bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal antibodies which are non-specific or empirically non-specific for the analyte (s) and their binding partner (in particular for immunoassays), detergents - such as Tween 20 -, fragmented natural which does not hybridize with polynucleotides to be analyzed or synthetic DNA, such as extracts of herring or salmon sperm (in particular for Polynucleotide hybridization assays) or uncharged but hydrophilic polymers such as polyethylene glycols or dextrans.

66. Method according to claim 28, characterized in that the layer deposited on the carrier substrates is a passivation layer which is located between the spatially separated measuring areas or in unoccupied partial areas within these measuring areas opposite the analytes and / or opposite to the latter Binders "chemically neutral" compounds after the generation of these ranges is applied and preferably, for example, comprises compounds from the groups which albumins, especially bovine serum albumin or human serum albumin, casein, nonspecific, polyclonal or monoclonal, alien or empirical for the one or more to be detected and their binding partner non-specific antibodies (especially for immunoassays), detergents - such as Tween 20 -, fragmented natural or synthetic DNA not hybridizing with polynucleotides to be analyzed, such as extracts of H ering or salmon sperm (especially for polynucleotide hybridization assays), or also uncharged but hydrophilic polymers such as polyethylene glycols or dextrans.

67. Carrier substrate for the detection of one or more analytes in a affinity-detection method comprising an adhesion-promoting layer, characterized in that said adhesion-promoting layer is produced by a coating method according to one of claims 28-66.

68. Carrier substrate for detecting one or more analytes in an affinity detection method comprising a passivation layer covering the carrier substrate at least in partial areas, characterized in that said passivation layer is produced by a coating method according to one of claims 28-66.

69. Carrier substrate according to one of claims 67 - 68 for use in human and / or animal diagnostics.