EP1421217A2

EP1421217A2 - Surface for the immobilisation of nucleic acids

Info

Publication number: EP1421217A2
Application number: EP02769982A
Authority: EP
Inventors: Andreas P. Abel; Markus Ehrat; Eckehard Kauffmann; Dominic Utinger; Vincent Benoit; Susan Margaret De Paul; Marcus Textor; Stephanie Vandevondele; Jeffrey A. Hubbell
Original assignee: Zeptosens AG
Current assignee: Bayer AG
Priority date: 2001-08-27
Filing date: 2002-08-24
Publication date: 2004-05-26
Also published as: WO2003020966A3; WO2003020966A2; US20050009026A1; AU2002338007A1

Abstract

The invention relates to a surface for the immobilisation of one or several first nucleic acids as recognition elements ("Immobilisation Surface"), for the production of a recognition surface for the detection of one or several second nucleic acids in one or more samples which are brought into contact with the recognition surface. The first nucleic acid is applied to a PLL-g-PEG layer as surface for immobilisation, characterised in that the grafting ratio g, in other words the quotient of the number of lysine units and the number of polyethylene glycol side chains (PEG side chains) 7 to 13. The invention also relates to a method for the qualitative and/or quantitative detection of one or more second nucleic acids in one or more samples, characterised in that said samples and optionally further reagents are brought into contact with the above immobilisation surface, upon which one or several first nucleic acids are immobilised as recognition elements for specific binding/hybridisation with said second nucleic acids and changes as a result of the said binding/hybridisation of said second nucleic acid, or further, resulting from applied detection substances introduced for analyte detection are measured by optical or electronic signals.

Description

Surface for immobilization of nucleic acids

The present invention relates to a surface for immobilizing one or more first nucleic acids as recognition elements (“immobilization surface”) for producing a recognition surface for the detection of one or more second nucleic acids in one or more samples brought into contact with the recognition surface, the first nucleic acids on one layer of the graft copolymer poly (L-lysine) -g ~ poly (ethylene glycol) (PLL-g-PEG) are applied as a surface for immobilization, characterized in that the grafting ratio ("grafting ratio") g, i. H. the quotient of the number of lysine units and the number of poly (ethylene glycol) side chains (“PEG” side chains) has an average value between 7 and 13. In the following, the “immobilization surface” defined above is used together with the “recognition surface” the first nucleic acids immobilized thereon.

The invention also relates to a method for the qualitative and / or quantitative detection of one or more second nucleic acids in one or more samples, characterized in that said samples and optionally other reagents are brought into contact with an immobilization surface according to the invention, on which surface one or more first ones Nucleic acids as recognition elements for specific binding / hybridization are immobilized with said second nucleic acids and changes in optical or electronic signals resulting from the binding / hybridization of these second nucleic acids or other detection substances used for analyte detection are measured.

For the purposes of this invention, the term “nucleic acids” should be understood to mean single- and double-stranded compounds from the group consisting of oligonucleotides, polynucleotides, DNA or RNA strands and DNA or RNA analogs, for example with modified bases or "backbones" are formed. This should also include hybrids of DNA and RNA and their analogues. For the detection of one or more analytes from a sample with a complex mixture of a large number of substances, methods are common in which one or more so-called recognition elements, which are biological, biochemical or synthetic, are immobilized on a solid support before they are then immobilized Form are brought into contact with said sample and the analytes contained therein bind to the detection elements specific to them. The solid support can be both a macroscopic type with a surface area of square millimeters to square centimeters and also a microscopic type, for example in the form of so-called beads, ie approximately spherical particles with typical diameters in the micrometer range. The surface of such a solid support with detection elements immobilized thereon will be referred to below as a “detection surface”.

Compared to methods in which the analytes and their recognition elements come together as reaction or binding partners in homogeneous liquid solution, these methods bound to a solid support offer a number of advantages, for example easier separation or differentiation of bound analyte molecules from the sample matrix. These advantages are bought with a restriction of the diffusion-driven mixing between analyte molecules and recognition elements, due to their binding to a solid support.

The nature of these surfaces is of great importance for the production of detection surfaces for the highly efficient and highly selective binding of one or more analytes to be detected in a sample. In order to achieve the lowest possible detection limits, it is desirable to immobilize as many detection elements as possible in a small space in such a way that as many analyte molecules of one type as possible can then be bound in the later detection method. At the same time, it is desirable to maintain the reactivity and biological or biochemical functionality of the recognition elements as high as possible during the immobilization, ie to minimize any signs of denaturation due to immobilization. Another goal is to largely prevent non-specific binding or adsorption of analyte molecules, which in many cases has a limiting effect on the detection limits that can be achieved.

For the analysis of nucleic acids in particular, microarrays with in some cases very high "feature density", i.e. the density of discrete measurement areas with biological or biochemical recognition elements immobilized therein on a common carrier, have been known since about 1990.

For the purposes of the present invention, spatially separated measuring areas, as part of a detection surface, are to be defined by the area that biological or biochemical or synthetic detection elements immobilized there assume for the detection of an analyte from a liquid. These surfaces can have any geometry, for example the shape of points, circles, rectangles, triangles, ellipses or lines. It is possible for up to 1,000,000 measuring ranges to be arranged in a two-dimensional arrangement, with a single measuring range taking up an area of 0.001-6 mm ² . Typically, the density of the measurement areas can be more than 10, preferably more than 100, particularly preferably more than 1000 measurement areas per square centimeter.

In the following, an array is to be referred to as a two-dimensional arrangement of measurement areas on a common carrier. The carrier can have an essentially planar or any other, for example spherical, shape.

For example, US Pat. No. 5,445,934 (Affymax Technologies) describes and claims arrays of oligonucleotides with a density of more than 1000 features per square centimeter. To improve the adhesion and durability of the immobilization of biological or biochemical or synthetic recognition elements, it is often advantageous to first apply a so-called adhesion-promoting layer on the carrier. For example, it can include chemical compounds from the group consisting of silanes, functionalized silanes, epoxies, functionalized, charged or polar polymers and "self-organized passive or functionalized mono- or multilayers". Such adhesion promoter layers and specific requirements for the properties of an adhesion promoter layer that depend on the physical and chemical nature of the support and the respective measuring arrangement are described, for example, in patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT / EP 00/04869 and PCT / EP 01/00605.

U.S. Patent Nos. 5,820,882, 5,232,984, 5,380,556, 6,231,892, 5,462,990 5,627,233 and 5,849,839 describe graft copolymers which contain a charged polyionic backbone and grafted, non-interactive (Adsorption-resistant, uncharged) side chains. The uses of these polymers describe, for example, the production of so-called "biocompatible" surfaces of so-called "microcapsules" to be applied in vivo or of implants. "Biocompatibility" is understood to mean that the surfaces coated in this way have the Accumulation of cells or proteins, which, for example, can lead to immune defense and the final rejection of an implant in a living organism, can be prevented or at least minimized. This property is achieved in that the adhesion is mediated by electrostatic interaction of the charged polymer main chain with an oppositely charged surface of the support to be coated, and the attachment of biomolecules is ensured by the “non-interactive” (uncharged) side chains.

WO 00/65352 describes applications of such polymer coatings in bioanalytics, for example for producing an adhesion-promoting layer for immobilizing biological detection elements on a sensor platform, described. Poly (L-lysine) -g-poly (ethylene glycol), “PLL-g-PEG” is preferred as the graft copolymer. Here, g denotes the grafting ratio, ie the quotient of the number of lysine units and the number of poly (ethylene glycol) side chains (“PEG” side chains).

As mentioned in WO 00/65352, the optimal value of g depends in each case on the size of the PEG side chains and on the particular application. WO 00/65352 describes optimal values of 3 <g <10, preferably 4 <g <7 for PEG chains with a molecular weight of 5000 Da and of 2 <g <8, preferably 3 <g <5, for a PEG molecular weight of 2000 Da named for. These in WO 00/65352 relate to the minimization of non-specific binding in the detection of proteins with sensors on which the analyte-specific recognition elements are immobilized on a surface coated with PLL-g-PEG.

It has now surprisingly been found that for the detection of nucleic acids in nucleic acid hybridization assays, with nucleic acids which are immobilized as recognition elements (here also called "capture probes") on a surface coated with PLL-g-PEG, optimal ratios between specific ones and non-specific binding (or specific and non-specific hybridization) can be achieved with average values of g between 7 and 13.

The first subject of the invention is therefore a surface for immobilizing one or more first nucleic acids as recognition elements for producing a recognition surface for the detection of one or more second nucleic acids in one or more samples brought into contact with the recognition surface, the first nucleic acids on a PLL-g- PEG layer are applied as a surface for immobilization, characterized in that the grafting ratio g has an average value between 7 and 13.

It is preferred that the grafting ratio g has an average value between 8 and 12. At the same time, it is preferred that the molecular weight of the polyethylene glycol side chains ("PEG" side chains) is between 500 Da and 7000 Da. It is particularly preferred that the molecular weight of the PEG side chains is between 1500 Da and 5000 Da.

It is preferred that the surface according to the invention for immobilizing one or more first nucleic acids is applied to a solid support. It is preferably an essentially optically transparent carrier.

The term “essentially optically transparent” should be understood to mean that carriers or layers characterized thereby are at least 95% transparent, at least at the wavelength of a light radiated by an external light source, for its optical path perpendicular to said carrier or layer, provided that In the case of partially reflecting supports or layers, “essentially optically transparent” is understood to mean that the sum of light transmitted, reflected and optionally coupled into a support or into a layer and guided therein at the location of the Impingement of the incident light is at least 95% of the incident light.

Preferably, the essentially optically transparent carrier comprises a material from the group comprising moldable, sprayable or millable plastics, metals, metal oxides, silicates, such as, for. B. glass, quartz or ceramics.

It is also preferred that the immobilization surface itself is essentially optically transparent.

The immobilization surface (as a PLL-g-PEG layer) preferably has a thickness of less than 200 nm, preferably less than 20 nm. Specific embodiments are characterized in that the immobilization surface is applied to a solid support, in the surface of which recesses for producing sample containers are structured. These recesses in the surface of the carrier preferably have a depth of 20 μm to 500 μm, particularly preferably 50 μm to 300 μm.

Embodiments of an immobilization surface according to the invention are preferred, the characteristic of which is that the essentially optically transparent support comprises a continuous optical waveguide or an optical waveguide divided into individual waveguiding regions. The optical waveguide is particularly preferably an optical layer waveguide with a first, essentially optically transparent layer (a) facing the immobilization surface on a second, essentially optically transparent layer (b) with a lower refractive index than layer (a). In addition, it is preferred that said optical layer waveguide is essentially planar.

A characteristic of such an embodiment of an immobilization surface on an optical layer waveguide as a carrier is that, for coupling excitation light into the optically transparent layer (a), this layer is in optical contact with one or more optical coupling elements 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 one end face of the waveguiding layer, preferably cylindrical lenses, and grating couplers are formed.

It is preferred that the excitation light is coupled into the optically transparent layer (a) using one or more grating structures (c) which are formed in the optically transparent layer (a). In addition, it is preferred that the coupling out of light guided in the optically transparent layer (a) takes place with the aid of one or more grating structures (c ') which are in the optically transparent Layer (a) are formed and have the same or different period and lattice depth as lattice structures (c).

Further planar optical layer waveguides suitable as carriers of the immobilization layer according to the invention and their modifications are described, for example, in patent applications WO 95/33197, WO 95/33198, WO 96/35940, WO 98/09156, WO 99/40415, PCT / EP 00/04869 and PCT / EP 01/00605. The content of these patent applications is therefore fully introduced as part of this description.

Those embodiments of an immobilization surface according to the invention in which the nucleic acids immobilized thereon are arranged as recognition elements in discrete (spatially separated) measurement areas are particularly preferred. Up to 1,000,000 measuring ranges can be arranged in a two-dimensional arrangement, and a single measuring range can take up an area of 10 ^" mm - 10 mm. It is preferred that the measuring ranges have a density of more than 10, preferably more than 100, more preferably more than 1000 measuring ranges are arranged per square centimeter.

The discrete (spatially separated) measuring areas on said immobilization surface can be generated by spatially selective application of nucleic acids as recognition elements, preferably using one or more methods from the group of methods, that of "ink jet spotting", mechanical spotting by pen, pen or Capillary, "micro contact printing", fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by their supply in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials as well as photochemical or photolithographic immobilization processes. Another object of the invention is a method for the simultaneous or sequential, qualitative and / or quantitative detection of one or more second nucleic acids in one or more samples, characterized in that said samples and optionally other reagents are brought into contact with an immobilization surface according to one of the embodiments mentioned on which surface one or more first nucleic acids are immobilized as recognition elements for specific binding / hybridization with said second nucleic acids and changes in optical or electronic signals resulting from the binding / hybridization of these second nucleic acids or other detection substances used for analyte detection are measured.

The one or more samples are preferably preincubated with a mixture of the various detection reagents for determining the second nucleic acids to be detected in said samples, and these mixtures are then brought into contact in a single addition step with the first nucleic acids immobilized on an immobilization surface according to the invention. It is preferred that the detection of the one or more second nucleic acids is based on the determination of the change in one or more luminescences.

Various optical excitation configurations are possible for generating luminescence. One possibility is that excitation light for excitation of one or more luminescences is irradiated by one or more light sources in an incident light excitation arrangement.

Another possible configuration is characterized in that excitation light for excitation of one or more luminescences from one or more light sources is radiated in a transmission light excitation arrangement.

Such an embodiment of the method according to the invention is preferred, which is characterized in that the immobilization surface is on a Optical waveguide is arranged, which is preferably essentially planar, that the one or more samples with second nucleic acids to be detected therein and optionally further detection reagents sequentially or after mixing with said detection reagents in a single step with the first nucleic acids immobilized on an immobilization surface according to the invention as detection elements in Be brought into contact and that the excitation light from one or more light sources is coupled into the optical waveguide with the aid of one or more optical coupling elements from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with in front of a face of the waveguiding Layer arranged focusing lenses, preferably cylindrical lenses, and grating couplers is formed

Another preferred embodiment of the method according to the invention is characterized in that the detection of the one or more second nucleic acids on a grating structure (c) or (c ') formed in the layer (a) of an optical layer waveguide on the basis of the second one from the binding / hybridization Nucleic acids or other detection reagents with first nucleic acids immobilized in the area of this lattice structure on an immobilization surface according to the invention as detection elements result in changes in the resonance conditions for coupling an excitation light into the layer (a) of a carrier designed as a layer waveguide or for coupling out light carried in the layer (a) ,

It is particularly preferred that said optical waveguide is designed as an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), that further excitation light using one or more grating structures which are formed in the optically transparent layer (a), are coupled into the optically transparent layer (a) and are guided to the measuring areas (d) located thereon as a guided wave, and that those in the evanescent field continue to be said guided wave generated luminescence of luminescent molecules with one or more detectors and the concentration of one or more nucleic acids to be detected is determined from the intensity of these luminescence signals.

It is possible that (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and coupled out via a grating structure (c) or (c ') luminescence or luminescence of both components (1) and (2 ) can be measured simultaneously.

It is preferred that a luminescent dye or luminescent nanoparticle is used as the luminescent label to generate the luminescence, which can be excited and emits at a wavelength between 300 nm and 1100 nm.

The luminescence label can be on the second nucleic acids to be detected as analytes themselves or in a competitive assay on nucleic acids added to the sample in a known concentration, as competitors, with the same sequence as said second nucleic acids to be detected or in a multi-stage assay on one of the binding partners of the immobilized first nucleic acids as recognition elements or bound to these immobilized first nucleic acids themselves. A multistage assay is understood to mean that not only a single second nucleic acid (as analyte) with an at least partially complementary sequence to the sequence of the respective first nucleic acid is bound or hybridized to the immobilized first nucleic acids, but that, for example, these second nucleic acids are still attached further nucleic acids are bound.

Characteristic of special embodiments of the method according to the invention is that a second or even more luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used. Such embodiments can, by appropriate selection of the spectral Characteristics of the luminescence label used, be configured such that the second or even more luminescence label can be excited at the same wavelength as the first luminescence label, but emit at other wavelengths.

For certain applications, for example for mutually independent measurements with different excitation and detection labels, it is advantageous if the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.

Another special embodiment of the method is characterized in that charge or optical energy transfer from a first luminescence label serving as a donor to a second luminescence label serving as an acceptor is used to detect the second nucleic acids as analytes.

Another special embodiment of the method according to the invention is characterized in that, in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.

It is advantageous if the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength. In particular, it is preferred that the one or more luminescences are measured with a different polarization than that of the excitation light.

The method according to the invention is characterized in that the samples to be examined are aqueous solutions, in particular buffer solutions or naturally occurring body fluids such as blood, serum, plasma, urine or tissue fluids. A sample to be examined can also be an optically cloudy liquid, surface water, a soil or plant extract, a bio or synthesis process broth. The samples to be examined can also be prepared from biological tissue parts or cells. Another object of the invention is the use of an immobilization surface according to the invention and / or a method according to the invention, in each case according to one of the aforementioned embodiments, for quantitative or qualitative analyzes in screening processes in pharmaceutical research, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in the Affinity screening and in research, for qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis and the determination of genomic or proteomic differences in the genome, such as single nucleotide polymorphisms, for measuring protein-DNA interactions, for determining Control mechanisms for mRNA expression and for protein (bio) synthesis, for the preparation of toxicity studies and for the determination of expression profiles, in particular for the determination of biological and chemical M Arcerics such as mRNA, pathogens or bacteria in pharmaceutical product research and development, human and veterinary diagnostics, agrochemical product research and development, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of Pathogens, pollutants and pathogens, especially of salmonella, prions, viruses and bacteria, especially in food and environmental analysis

The invention is exemplarily explained in the following example.

Example:

1. Chemicals 1.1. buffer solutions

The following buffer solutions were used:

Buffer 1:

4X SSC (600 mM NaCl / 60 mM sodium citrate, pH 7.5)

Buffer 2:

4X SSC (600 mM NaCl / 60 mM sodium citrate, pH 7.5) with 50% formamide

Wash buffer 1;

IX SSC (150 mM NaCl / 15 mM sodium citrate, pH 7.5) with 0.1% SDS

Wash buffer 2:

0.1X SSC (15 mM NaCl 1.5 mM sodium citrate, pH 7.5) with 0.1% SDS

Wash buffer 3:

0.1X SSC (15 mM NaCl / 1.5 mM sodium citrate, pH 7.5)

1.2. First nucleic acids to be immobilized

A mouse brain "longmer set", derived from 96 genes (Lion Bioscience, Heidelberg, Germany), which represent weakly to moderately expressed genes from the brain tissue of a mouse, was used. These were oligonucleotides each with a length 70 nucleotides ("Lc-ngmere"), the sequence of which was selected by Operon (Alameda, CA, USA) from the sequences of said genes and which were produced by Operon. 1.3. Second nucleic acids to be detected as analyte

Using the RNeasy kit (QIAGEN, Hilden, Germany), the entire RNA was isolated, starting from the mouse brain. In a further step, the total RNA was isolated from this isolate using the Oligotex kit (QIAGEN, Hilden, Germany), the mRNA. The mRNA isolate was then used as a template for reverse transcription (by means of reverse transcriptase Omniscript, QIAGEN, Hilden, Germany). By using a poly (dT) primer, all mRNA molecules that have a poly (dA) tail were rewritten into cDNA. In this transcription step, nucleotides labeled with Cy5 (Amersham, Arlington Heights, USA) were used, with the result of a fluorescently labeled cDNA.

Depending on the yield of mRNA isolation and the efficiency of reverse transcription, the labeled cDNA represents the entire spectrum of expressed mRNA in the mouse brain used.

1.4. Production of poly (L-lysine) -g-poly (ethylene glycol) (PLL-g-PEG)

materials

Poly (L-lysine) hydrobromide (molecular weight around 20 kDa) was obtained from Sigma-Aldrich (Buchs, Switzerland). The N-hydroxysuccinimidyl ester of methoxy poly (ethylene glycol) propionic acid (MeO-PEG-SPA, molecular weight 2 kDa) was obtained from Shearwater Polymers Inc. (Huntsville, USA). 4- (2-hydroxyethyl) piperazin-1-ethanesulfonic acid (HEPES) and other chemicals for making buffers were purchased from Fluka (Buchs, Switzerland).

All aqueous solutions were prepared with ultrapure water (18 MΩcm) from an "Easy Pure Reverse Osmosis System" (Barnstead Thermolyne, Dubuque, USA). Synthesis of PLL-g-PEG

The synthesis of PLL-g-PEG was described by Sawhney and Hubbell (A. S. Sawhney, J.A. Hubbell, Biomaterials 13 (1992) 863-870). The method used for the investigations on which the present application is based is based on a specification which was developed by Elbert and Hubbell (D.L. Elbert, J.A. Hubbell, /. Biomed. Mater. Res. 42 (1998) 55-65).

N-Hydroxy-succinimidyl esters of poly (ethylene glycol) ("PEG") are reacted with poly (L-lysine) ("PLL") in a stoichiometric ratio to produce the desired product. The details of this synthesis are described below.

The nomenclature chosen below for the designation of the various PLL-g-PEG derivatives includes the molecular weights of the polymer partial chains of the copolymers and the grafting ratio g. Accordingly, “PLL (20) - g / 3.57-PEG (2) "a polymer formed from a main chain made of poly (L-lysine) with a molecular weight of 20 kDa and side chains consisting of poly (ethylene glycol) with a molecular weight of 2 kDa. The grafting ratio of 3.5 means that on average two PEG chains are bound to two out of seven lysine groups. Since all of the polymers mentioned in this example were produced from similar precursors with the same molecular weight, T ^J L (20) -g [3.7] - PEG (2) "the abbreviation" PLL-g / 3.77-PEG "can also be used.

Poly (L-lysine) hydrobromide ("PLL-HBr") is dissolved in 25 ml of sodium tetraborate buffer ("STBB", 50 mM, pH 8.5) per gram of PLL-HBr. The solution is stirred and then filtered (0.22 μm Durapore membrane, sterile Millex GV, Sigma-Aldrich, Buchs, Switzerland) and filled into a sterile culture tube. MeO-PEG-SPA powder is then added in a suitable amount according to the stoichiometric ratio while stirring the solution evenly. After stirring the solution for a further six hours at room temperature, the solution is transferred to a dialysis tube (Spectr / Por dialysis tube, molecular weight limitation (“cut-off”) 6-8 kDa, Sochochim, Lausanne, Switzerland). The dialysis will be 24 hours for one liter of phosphate buffered saline ("PBS", 10mM, pH 7.0) followed by 24 hours of further dialysis in one liter of deionized water. The product is then freeze-dried for 48 hours.

The grafting ratio is checked with the aid of 1H-NMR. 6 different polymers with grafting ratios g of 3.7, 7.4, 8.4, 9.0, 11.8 and 13.0 are produced in the manner described.

2nd carrier

As a carrier of an immobilization surface according to the invention, a planar optical layer waveguide with the outer dimensions 57 mm width (parallel to the grating lines of a grating structure (c) modulated in layer (a) of the layer waveguide) x 14 mm length (perpendicular to the grating structures) x 0.7 mm thickness used. Either directly on the surface of layer (a) or after application of further layers, in particular an immobilization layer according to the invention on layer (a), can be combined with a plate made of polycarbonate with recesses open in the direction of the layer waveguide with the internal dimensions 5 mm width x 7 mm length x 0.15 mm height 6 microflow cells can be generated in the grid of a partial column of a classic microtiter plate (grid 9 mm). The polycarbonate plate can be glued to the carrier in such a way that the recesses are then sealed to one another. This polycarbonate plate is constructed in such a way that it can be joined together with a carrier ("meta carrier") with the basic dimensions of standard microtiter plates in such a way that the grid (sequence in rows or columns) of the inflows of the flow cells corresponds to the grid of the wells of a standard microtiter plate.

The substrate material (optically transparent layer (b) of the planar optical layer waveguide as a carrier consists of AF 45 glass (refractive index n = 1.52 at 633 nm). In the substrate is a pair of coupling-in and coupling-out gratings parallel to the width of the Layer waveguide extending grating lines (318 nm period) of 12 +/- 3 nm grating depth, the grating lines being pronounced over the entire width of the layer waveguide. The distance between the two successive grids is 9 mm, the length of the individual grating structures (parallel to the length of the sensor platform) is 0.5 mm. The distance between the coupling-in and coupling-out gratings of a pair of gratings is selected so that the coupling of the excitation light can take place within the area of the sample containers after combining the layer waveguide with the polycarbonate plate described above, while the coupling-out takes place outside the area of the sample containers. The wave-guiding, optically transparent layer (a) made of Ta ₂ O ₅ on the optically transparent layer (b) has a refractive index of 2.15 at 633 nm (layer thickness 150 nm).

To prepare for the immobilization of the biochemical or biological or synthetic recognition elements, the optical layer waveguide as a carrier is cleaned with organic and inorganic reagents (e.g. propanol and sulfuric acid, with rinsing steps in water in between) in an ultrasound device.

3. Creation of the immobilization surface

A solution of PLL-g-PEG is prepared in PBS buffer (1 mg / ml) and filtered through 0.22 μm Durapore membranes. Instead of PBS buffer, for example, HEPES buffer can also be used. 570 μL of the PLL-g-PEG solution are pipetted into a special incubation chamber for coating a support in accordance with section 2 of this example. The supports are then placed in the incubation chamber in such a way that the surface to be coated, ie the surface of layer (a) as the support, for example a planar optical layer waveguide, comes into contact with the polymer solution. After two hours of incubation at room temperature, the then coated supports are rinsed with ultrapure water and blown dry with nitrogen. 4. Immobilization of the first nucleic acids / generation of discrete measurement areas

The biological recognition elements described in 1.2. described 96 oligonucleotides each with a length of 70 nucleotides in a concentration of 40 μM in 10 mM carbonate buffer (pH 9.2, with the addition of 5% DMSO) applied to the immobilization surface according to the invention, as described above, with a commercial spotter (GMS 417 Arrayer , Affymetrix, Santa Clara, CA, USA) and incubated overnight. The distance between the discrete measurement areas (spots) thus generated is 340 μm. Two spots of the same base sequence are generated in an array, so that a single array comprises 192 spots. Up to 6 identical arrays are produced on a layer waveguide as a carrier, according to section 2.

Arrays of the immobilized first nucleic acids are each generated in the same way on the six supports with immobilization surfaces of different grafting ratios.

The polycarbonate plate described is applied to the carrier coated with the immobilization surface, with the first nucleic acids applied to the immobilization surface, in such a way that the individual sample containers are fluid-tightly sealed off from one another and the “Longmer” arrays produced with the associated coupling-in grid (c) is inside one of the 6 sample containers.

5. Hybridization assay as part of a method according to the invention for the detection of one or more second nucleic acids

The carrier formed as a planar optical layer waveguide with the carrier applied in discrete measurement areas on an immobilization layer according to the invention, joined together with a polycarbonate plate for producing 6 sample containers ("Chambers") according to section 2 of this example is inserted into a "meta carrier". For the purpose of moistening / equilibration, the two-dimensional arrangements of measuring areas (“microarrays”) are filled with 90 μl buffer 1.

For hybridization, a sample of the second nucleic acids to be detected as analyte (“target sample”) is produced from labeled cDNA (according to section 1.3) in an amount corresponding to 25 ng mRNA. For this purpose, the amount of cDNA corresponding to an amount of 25 ng mRNA is prepared in 50 μL of hybridization buffer (buffer 2) are pipetted in. The target sample is heated for 5 minutes to 95 ° C. for denaturation and then stored on ice for 5 minutes. The buffer 1 is sucked out of the chambers and the target sample is pipetted free of air bubbles.

For the hybridization, the “meta carrier” is placed in a thermal cycler (MJ Research PCT-200 with adapter plate), incubated for 35 minutes at 75 ° C. (denaturation step) and then for 18 hours at 42 ° C. (hybridization step).

After completion of the hybridization, the following washing steps are carried out: The chambers are vacuumed by applying a vacuum, then filled with 90 μL buffer 1 and heated to room temperature in the “meta carrier”.

The chambers are then emptied again, filled with 90 μL washing buffer 1 and incubated for 7 minutes at room temperature. The vacuuming and filling are carried out analogously once again with wash buffer 2 and twice with wash buffer 3. Finally, the chambers are sucked empty and filled with buffer 1.

The hybridization assay described is carried out in the same way with all 6 supports with immobilization surfaces with different grafting ratios g. 6. Analytical system and measurement method for the detection of one or more analytes

The excitation light of a laser diode with emission at 635 nm is expanded, using a lens system with a cylindrical lens and an aperture, to form a beam bundle of slit-shaped cross section (perpendicular to the optical axis), the extent of which in the radiation cross section on the planar optical layer waveguide, parallel to the grating lines, corresponds almost completely to the section of the coupling grating located within a sample container.

The angle between the incident, parallel excitation light bundle and the plane of the planar optical layer waveguide is set to the resonance angle for maximum coupling into the waveguiding layer (a) (-10 °), as is the corresponding optimal position of the excitation light to be coupled in on the coupling grating (first grating ). This optimization is carried out in an automated form in that the light coupled out from the second grating located outside the sample containers is directed to a photodiode, the suitably amplified signal of which is optimized to a maximum by appropriate readjustment of the carrier with regard to the coupling angle and lateral position, according to the principle a "feedback loop".

Light emitted by the microarray from the area of the measuring surface within a sample container on the support (visual field approx. 6 mm x 8 mm) designed as a planar optical layer waveguide is collected with a tandem lens and onto a CCD camera with a CCD chip ( active area approx. 5 mm x 7 mm with 512 x 766 pixels, pixel size 9 μm). Depending on the setting of the imaging system, this enables a spatial resolution of 'approx. 10 μm to 20 μm.

Between the two halves of the tandem lens, in an essentially parallel part (ie less than 10 ° divergent or convergent) part of the emission beam path is an interference filter, (670 DF 40, Omega Optical Brattleborough, VT, US) for detecting the light emanating from the array at the fluorescence wavelength of the fluorescence label used (Cy5). After hybridization of the immobilized first nucleic acids with the fluorescence-labeled second nucleic acids supplied as a sample, the emission light from all measurement areas located within a sample container is recorded in a recording with a cooled CCD camera.

7. Processing of the measurement data

The mean signal intensity from the measuring areas (spots) for binding and for the detection of analyte molecules on the basis of a potentially generated fluorescence from luminescent labels (in this example Cy5) is determined using an image analysis software.

The raw data from the individual pixels of the camera represent a two-dimensional matrix of digitized measurement values, with the measured intensity as the measurement value of an individual pixel corresponding to the area on the sensor platform imaged on it. For the evaluation of the data, a two-dimensional (coordinate) network is first placed over the image points (pixel values) in such a way that each spot falls into an individual two-dimensional network element. Each spot is assigned an "evaluation area" (AOI) that can be geometrically adapted as well as possible. These AOIs can have any geometric shape, for example circular. The location of the individual AOIs is individualized as a function of the signal intensity of the individual by the image analysis software Pixels adjusted and optimized, depending on the user specification, the initially set radius of the AOIs can be retained or the shape and size of the fluorescent spots can be readjusted. The mean gross signal intensity of each spot is, for example, as in the present case , which determines the arithmetic mean of the pixel values (signal intensities) within a selected AOI. The background signals are determined from the measured signal intensities between the spots. For this purpose, for example, a group of further circles, which are concentric with a circular spot in question but have a larger radius, can be determined. Of course, the radii of these concentric circles must be chosen smaller than the distances between adjacent spots. For the background determination, the area between the AOI and the first concentric circle can then be discarded, for example, and the area between this first concentric and a second subsequent concentric circle having a larger radius can be defined as the area (AOI) for the background signal determination. It is also possible to define AOIs between adjacent spots, preferably in the middle between them, as AOIs for background signal determination. From this, the mean background signal intensity can then be determined in an analogous manner as above, for example as the arithmetic mean of the pixel values (signal intensities) within an AOI selected for this purpose. The mean net signal intensity can be determined as the difference between the local mean gross and the local mean background signal intensity.

8. Results

The fluorescence signals from the measuring areas (“spots”) of the arrays were measured with all 6 supports with immobilization surfaces with different grafting ratios (g) after the completion of the hybridization assay (according to section 6) in the analytical system according to section 6 of this example. Photographs with the fluorescence signals at 4 different values of g, namely 3.7, 7.4, 9.0 and 11.8, are shown in FIG. To determine the net fluorescence signals, as the difference between the gross fluorescence signals (arithmetic mean of the pixel values in the AOIs) and the background signals, according to section 7 of this example, the signals from two spot pairs (duplicates) were used as examples of the Fluorescence signals after hybridization with strong cDNA (spot group I in Fig. La - d). or less expressed genes (spot group II in Fig. la - d), evaluated (identified in figure la - d). Already the comparison of the images la - ld, which are all shown in the same dynamic range, shows the great influence of the grafting ratio (g) on the signal intensities: A large number of spots are not at all in Figure la (g = 3.7) , weak in Fig. lb (g = 7.4), but clearly visible in Fig. lc (g = 9.0) and Fig. ld (g = 11.8) This applies, for example, to the complete left row of spots.

The determined net fluorescence intensities, as average values of the signals from the two identical spots in each case, are shown in Figure 2 as a function of the grafting ratio g. In the range from g = 3.7 to g = 7.4, for both selected spot Pairs the fluorescence signals relatively low: From g = 8.4 there is a strong increase in the fluorescence signals. With a further increase in g there is an extensive plateau of high signal intensities until the fluorescence signals decrease again from g> 11.8.

It is concluded from these results that for immobilization surfaces of the present type, in order to achieve the highest possible net signals in hybridization assays as described here, the grafting ratio g should have a value between 7 and 13, preferably between 8 and 12 ,

Claims

claims

1. Surface for immobilizing one or more first nucleic acids as recognition elements (“immobilization surface”) for producing a recognition surface for the detection of one or more second nucleic acids in one or more samples brought into contact with the recognition surface, the first nucleic acids on a PLL-g- PEG layer (graft copolymer poly (L-lysine) -g-poly (ethylenegrycol)) are applied as a surface for immobilization, characterized in that the grafting ratio ("grafting ratio") g, ie the quotient of the number of lysine units and the number of poly (ethylene glycol) side chains ("PEG" side chains),. has an average value between 7 and 13.

2. Surface for immobilizing one or more first nucleic acids according to claim 1, characterized in that the grafting ratio (g) has an average value between 8 and 12.

3. Surface for the immobilization of one or more first nucleic acids according to claim 1, characterized in that the molecular weight of the polyethylene glycol side chains ("PEG" side chains) is 500 Da to 7000 Da.

4. Surface for the immobilization of one or more first nucleic acids according to claim 3, characterized in that the molecular weight of the polyethylene glycol side chains ("PEG" side chains) is 1500 Da to 5000 Da.

5. Surface for immobilization of one or more first nucleic acids according to one of claims 1-4, characterized in that this surface is applied for immobilization on a solid support.

6. Surface for the immobilization of one or more first nucleic acids according to claim 5, characterized in that said solid support is an essentially optically transparent support.

7. Surface for the immobilization of one or more first nucleic acids according to claim 6, characterized in that the essentially optically transparent support comprises a material from the group comprising moldable, sprayable or millable plastics, metals, metal oxides, silicates, such as, for. B. glass, quartz or ceramics.

8. Surface for the immobilization of one or more first nucleic acids according to one of claims 1-7, characterized in that this surface is essentially optically transparent.

9. Surface for the immobilization of one or more first nucleic acids according to one of claims 1-8, characterized in that this surface (as a PLL-g-PEG layer) has a thickness of less than 200 nm, preferably less than 20 nm, Has.

10. immobilization surface according to one of claims 6-9, characterized in that this surface for immobilization is applied to a solid support, in the surface of which recesses for the production of sample containers are structured.

11. immobilization surface according to claim 10, characterized in that said recesses in the surface of the carrier have a depth of 20 microns to 500 microns, particularly preferably from 50 microns to 300 microns.

12. immobilization surface according to any one of claims 6 - 11, characterized in that the substantially optically transparent carrier continuous optical waveguide or divided into individual waveguiding areas.

13. immobilization surface according to claim 12, characterized in that the optical waveguide is an optical layer waveguide with a, the immobilization surface facing, first substantially optically transparent layer (a) on a second, substantially optically transparent layer (b) with a lower refractive index than Layer (a).

14. Immobilization surface according to claim 13, characterized in that said optical layer waveguide is essentially planar.

15. immobilization surface according to one of claims 14 - 15, characterized in that, for coupling excitation light into the optically transparent layer (a), this layer is in optical contact with one or more optical coupling elements 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 one end face of the waveguiding layer, preferably cylindrical lenses, and grating couplers are formed.

16. immobilization surface according to claim 15, characterized in that the coupling of excitation light into the optically transparent layer (a) by means of one or more grating structures (c) which are formed in the optically transparent layer (a).

17. immobilization surface according to claim 15, characterized in that the coupling of light guided in the optically transparent layer (a) takes place with the aid of one or more grating structures (c ') which are formed in the optically transparent layer (a) and the like or have different period and lattice depth like lattice structures (c).

18. immobilization surface according to one of claims 1-17, characterized in that the nucleic acids immobilized thereon are arranged as recognition elements in discrete (spatially separated) measurement areas.

19. immobilization surface according to claim 18, characterized in that up to 1,000,000 measuring ranges are arranged in a two-dimensional arrangement and a single measuring range occupies an area of 10 ^"4 mm - 10 mm ² .

20. immobilization surface according to one of claims 18 - 19, characterized in that the measuring areas are arranged in a density of more than 10, preferably more than 100, particularly preferably more than 1000 measuring areas per square centimeter.

21. immobilization surface according to one of claims 18 - 20, characterized in that discrete (spatially separate) measurement areas on said immobilization surface are generated by spatially selective application of nucleic acids as recognition elements, preferably using one or more methods from the group of methods by "InkJet spotting", mechanical spotting using a pen, pen or capillary, "micro contact printing", fluidic contacting of the measuring areas with the biological or biochemical or synthetic recognition elements by supplying them in parallel or crossed microchannels, under the influence of pressure differences or electrical or electromagnetic potentials as well as photochemical or photolithographic immobilization processes.

22. A method for the simultaneous or sequential, qualitative and / or quantitative detection of one or more second nucleic acids in one or more samples, characterized in that said samples and optionally further reagents are brought into contact with an immobilization surface according to one of claims 1-21, on which surface one or more first nucleic acids are immobilized as recognition elements for specific binding / hybridization with said second nucleic acids and from the binding / hybridization of these second nucleic acids or others changes in optical or electronic signals resulting from the detection substances used for analyte detection are measured.

23. The method according to claim 22, characterized in that the one or more samples are preincubated with a mixture of the various detection reagents for determining the second nucleic acids to be detected in said samples and these mixtures are then each in a single addition step with those on an immobilization surface after a of claims 1-21 immobilized first nucleic acids are brought into contact.

24. The method according to any one of claims 22-23, characterized in that the detection of the one or more second nucleic acids is based on the determination of the change in one or more luminescences.

25. The method according to any one of claims 22-24, characterized in that excitation light for excitation of one or more luminescences from one or more light sources is irradiated in an incident light excitation arrangement.

26. The method according to any one of claims 22-24, characterized in that excitation light for excitation of one or more luminescence from one or more light sources is irradiated in a transmission light excitation arrangement.

27. The method according to any one of claims 22-24, characterized in that the immobilization surface is arranged on an optical waveguide. which is preferably essentially planar, that the one or more samples with the second nucleic acids to be detected therein and optionally further detection reagents sequentially or after mixing with said detection reagents in a single step with the first nucleic acids immobilized on an immobilization surface according to one of claims 1 to 21 as detection elements be brought into contact and that the excitation light from one or more light sources is coupled into the optical waveguide with the aid of one or more optical coupling elements from the group consisting of prism couplers, evanescent couplers with matched optical waveguides with overlapping evanescent fields, end face couplers with in front of an end face of the waveguiding layer arranged focusing lenses, preferably cylindrical lenses, and grating couplers is formed.

28. The method according to claim 27, characterized in that the detection of the one or more second nucleic acids on a grating structure (c) or (c ') formed in the layer (a) of an optical layer waveguide on the basis of the second nucleic acids mentioned from the binding / hybridization or further detection reagents with first nucleic acids immobilized in the area of this lattice structure on an immobilization surface according to one of claims 1 to 21 as detection elements resulting in changes in the resonance conditions for coupling an excitation light into the layer (a) of a carrier designed as a layer waveguide or for coupling out in the layer ( a) led light takes place.

29. The method according to claim 27, characterized in that said optical waveguide is designed as an optical layer waveguide with a first optically transparent layer (a) on a second optically transparent layer (b) with a lower refractive index than layer (a), that further using excitation light one or more lattice structures, which are formed in the optically transparent layer (a), into the optically transparent Layer (a) is coupled in and guided to the measuring areas (d) located thereon as a guided wave, and that the luminescence of luminescent molecules generated in the evanescent field of said guided wave is also detected with one or more detectors and the concentration of one or more nucleic acids to be detected from the Intensity of these luminescence signals is determined.

30. The method according to claim 29, characterized in that (1) the isotropically emitted luminescence or (2) coupled into the optically transparent layer (a) and via a lattice structure (c) or (c ') coupled luminescence or luminescence of both components ( 1) and (2) can be measured simultaneously.

31. The method according to any one of claims 29-30, characterized in that a luminescent dye or luminescent nanoparticle is used as luminescent label to generate the luminescence, which can be excited and emits at a wavelength between 300 nm and 1100 nm.

32. The method according to claim 31, characterized in that the luminescence label on the second nucleic acids to be detected as analyte itself or in a competitive assay on in a known concentration, as competitors, nucleic acids added to the sample with the same sequence as said second nucleic acids to be detected or in one multistage assay is bound to one of the binding partners of the immobilized first nucleic acids as recognition elements or to these immobilized first nucleic acids.

33. The method according to any one of claims 31-32, characterized in that a second or even further luminescence label with the same or different excitation wavelength as the first luminescence label and the same or different emission wavelength is used.

34. The method according to claim 33, characterized in that the second or still further luminescence label can be excited at the same wavelength as the first luminescence label, but emit at other wavelengths.

35. The method according to claim 33, characterized in that the excitation spectra and emission spectra of the luminescent dyes used overlap only slightly or not at all.

36. The method according to claim 33, characterized in that charge or optical energy transfer from a first luminescent label serving as a donor to a second luminescent label serving as an acceptor is used to detect the second nucleic acids as analytes.

37. The method according to any one of claims 29-36, characterized in that in addition to the determination of one or more luminescences, changes in the effective refractive index on the measurement areas are determined.

38. The method according to any one of claims 29-37, characterized in that the one or more luminescences and / or determinations of light signals are carried out polarization-selectively at the excitation wavelength.

39. The method according to any one of claims 29-38, characterized in that the one or more luminescences are measured at a different polarization than that of the excitation light.

40. The method according to any one of claims 22-39, characterized in that the samples to be examined are aqueous solutions, in particular buffer solutions or naturally occurring body fluids such as blood, serum, plasma, urine or tissue fluids.

41. The method according to any one of claims 22-39, characterized in that the sample to be examined is an optically cloudy liquid, surface water, a soil or plant extract, a bio or synthesis process broth.

42. The method according to any one of claims 22 -39, characterized in that the samples to be examined are prepared from biological tissue parts or cells.

43. Use of an immobilization surface according to one of claims 1-21 and / or a method according to one of claims 22-42 for quantitative or qualitative analyzes in screening methods in pharmaceutical research, clinical and preclinical development, for real-time binding studies and for determining kinetic parameters in affinity screening and in research, on qualitative and quantitative analyte determinations, in particular for DNA and RNA analysis and the determination of genomic or proteomic differences in the genome, such as for example single nucleotide polymorphisms, for measuring protein-DNA interactions, for determining control mechanisms for mRNA expression and for protein (bio) synthesis, for the preparation of toxicity studies and for the determination of expression profiles, in particular for the determination of biological and chemical marker substances, such as mRNA, pathogens or bacteria in pharmaceutical P Product research and development, human and veterinary diagnostics, agrochemical product research and development, symptomatic and presymptomatic plant diagnostics, for patient stratification in pharmaceutical product development and for therapeutic drug selection, for the detection of pathogens, pollutants and pathogens, especially salmonella, prions , Viruses and bacteria, especially in food and environmental analysis