WO2007090511A1

WO2007090511A1 - Polyelectrolyte monolayers and multilayers for optical signal converters

Info

Publication number: WO2007090511A1
Application number: PCT/EP2007/000448
Authority: WO
Inventors: Karlheinz Hildenbrand
Original assignee: Siemens Medical Solutions Diagnostics Gmbh
Priority date: 2006-02-03
Filing date: 2007-01-19
Publication date: 2007-08-16

Abstract

The invention relates to a method for coating dielectric materials with polyelectrolyte monolayers and multilayers as well as to an optical signal converter having a coating of these polyelectrolyte layers and to the use thereof.

Description

Polyelectrolyte monolayers and multilayers for optical signal transducers

The invention relates to methods for coating dielectric materials with polyelectrolyte mono- and multilayers and to an optical signal converter having a coating of these polyelectrolyte layers and to the use thereof.

Dielectric materials are modified with polyelectrolyte monolayers or multilayers for bio- and chemofunctionalization, with the aim of producing chemical and / or biochemical (biochemical) recognition elements, e.g. Receptors, antibodies, DNA, etc., to be able to immobilize on the surface. Such coated dielectric materials, e.g. Coated optical waveguides are used as signal transducers (transducers), such as those used in sensor technology in biosensors or chemosensors.

Bio or chemosensors are devices that can qualitatively or quantitatively detect an analyte with the aid of a signal converter and a recognition reaction. The recognition reaction is generally referred to as the specific binding or reaction of a so-called analyte with a so-called recognition element. Examples of recognition reactions are the binding of ligands to complexes, the complexation of ions, the binding of ligands to (biological) receptors, membrane receptors or ion channels, of antigens or haptens to antibodies, of substrates to enzymes, of DNA or RNA to specific proteins, the hybridization of DNA / RNA / PNA or the processing of substrates by enzymes. Analytes may be: ions, proteins, natural or artificial antigens or haptens, hormones, cytokines, mono- and oligosaccharides, metabolites, or other biochemical markers used in diagnostics, enzyme substrates, DNA, RNA, PNA, potential drugs , Medicines, cells, viruses. Examples of recognition elements are: complexing agents for metals / metal ions, cyclodextrins, crown ethers, antibodies, antibody fragments, anticalins, enzymes, DNA, RNA, PNA, DNA / RNA-binding proteins, enzymes, receptors, membrane receptors, ion channels, cell adhesion proteins, Gangliosides, mono- or oligosaccharides.

These bio- or chemosensors can be used in environmental analysis, the food industry, human and veterinary diagnostics and crop protection to qualitatively and / or quantitatively determine analytes. The specificity of the recognition reaction also makes it possible to qualitatively or quantitatively determine analytes in complex samples such as ambient air, contaminated water or body fluids without or with little previous purification. In addition, bio- or chemosensors can also be used in (bio-) chemical research and drug discovery to study the interaction between two different substances (eg between proteins, DNA, RNA, or biologically active substances and proteins, DNA, RNA, etc.). ). Integration of the recognition reaction with the signal transducer into a biosensor or chemosensor can be accomplished by immobilizing the recognition element or analyte on the surface of the transducer. By the recognition reaction, ie the binding or the reaction of the analyte with the recognition element, the optical properties of the medium change directly on the surface of the signal transducer (eg change in the optical refractive index, absorption, fluorescence, phosphorescence, luminescence, etc.) , which is translated by the signal converter into a measurement signal.

Optical waveguides are a class of signal transducers that can detect the change in optical properties of a medium that is adjacent to a waveguiding layer, typically a dielectric. If light is transported as a guided mode in the waveguiding layer, the light field at the interface medium / waveguide does not drop abruptly, but decays exponentially in the so-called detection medium adjoining the waveguide. This exponentially decaying light field is called an evanescent field. If very thin waveguides are used whose refractive index differs as strongly as possible from that of the adjacent medium, decay lengths of the evanescent field (intensity decreases to the value 1 / e) of <200 nm are achieved. If the optical properties of the medium adjacent to the waveguide (eg change in the optical refractive index, luminescence, etc.) change within the evanescent field, this can be detected by means of a suitable measurement setup. or chemosensors is that the change in the optical properties of the medium is detected only very close to the surface of the waveguide, namely, if the recognition element or analyte is immobilized at the waveguide interface, binding to the recognition element or reaction of the waveguide may occur Detection element surface-sensitively detected, if this change the optical properties of the detection medium (liquid, solid, gaseous) at the interface to the waveguide.

When optical waveguides are used as biosensors or chemosensors, high demands are placed on the waveguide to detection medium interface:

• Under the reaction conditions of the recognition reaction, the waveguide / detection medium interface must be stable.

• The recognition elements must be immobilized within the range of the evanescent field of the waveguide.

• Under the reaction conditions of the recognition reaction, the immobilization of the recognition element must be stable. • The functionality of the recognition elements must still be present after immobilization.

• In order to detect only the specific recognition reaction by the signal transducer, any kind of non-specific binding to the waveguide / detection medium interface must be suppressed.

On the surface of waveguides can be immobilized in various ways recognition elements. This can be done, for example, by physisorption of the recognition elements on the transducer surface. Clerc and Lukosz describe the physisorption of avidin on SiO ₂ -TiO ₂ waveguide surfaces. In a second step, biotinylated antibodies can be immobilized on the thus-applied avidin layers by utilizing the high-affinity avidin-biotin binding. A disadvantage of this immobilization method of waveguide surface recognition elements is the instability of the physisorbed avidin layer. A change in the reaction conditions, such as temperature changes, pH changes, addition of detergents, etc., can lead to a desorption of the avidin layer and thus also of the antibody.

G. Gao describes in Surface & Coating Technology (2005) 244-250 an "oxygen plasma" method that allows adsorptive binding of DNA to silica wafers.

However, it is known that activated surfaces produced by plasma treatment do not have good long term stability and are therefore to be critically evaluated for reproducibility.

The recognition elements, also known as "capture" molecules, can also be covalently bound to the surface of a waveguide, with bifunctional silanes forming a covalent bond with the waveguide surface, as well as a second functional group in this silane These covalent bonds to the waveguide surface must be operated under absolutely dry reaction conditions to prevent hydrolysis of the reactive silane waveguide surfaces are stable under acidic, neutral, and slightly basic conditions, but hydrolysis of the silane may occur at pH values above 9, which may result in desorption of the recognition elements from the surface, a further disadvantage of this immobilization method gt in the relatively high unspecific adsorption of proteins such as albumin to the thus functionalized waveguide surfaces. The unspecific binding to these waveguide surfaces can be reduced by post bonding In a second step, the recognition elements are bound to the surface of blocking agents such as polyethylene glycols.

Alternatively, the binding of hydrophilic polymers, e.g. Polyacrylamides, dextrans, polyethylene glycols, etc. described on previously silanized waveguide surfaces. These polymers have the task of minimizing the non-specific binding of proteins etc. to the surface. The recognition elements are then covalently bonded to these polymers in a further step. The problem with this surface functionalization is that several steps have to be carried out to immobilize the recognition elements on the surface and the instability of the bond to the waveguide surfaces at pH> 9.

The recognition elements can also be bound to polymers which are applied directly to the waveguide layers without prior silanization.

Polymer as interface between waveguide surface and recognition element is content of the present description.

Taking into account the o.g. Interface Requirements Waveguide / detection medium must allow the polymer interface to irreversibly bond to both the substrate and the sensing element under the reaction conditions, and must be as thin as possible, due to the intensity decreasing with 1 / e.

Therefore, it would be advantageous to use polymer monolayers which, in addition, after the coupling of the punctually arranged recognition elements, enable blocking against nonspecific adsorption of interfering components in the simplest possible method step.

Since the surfaces of waveguide layers, or of substrates in general, are not exactly different depending on the field of application and are generally not known in terms of surface chemistry, the provision of suitable polymers is a great challenge for producing efficient biosensors or chemosensors.

There has therefore been no lack of attempts to produce by synthesis tailor-made polymers for certain substrate surfaces.

With regard to coating substrates with metal oxide surfaces, such as TiO _2, Nb ₂ Os or Ta ₂ Os, for example, ionic polymers are also described.

In WO 03/020966 poly (L-lysine) -g-poly (ethylene glycol) graft copolymers (PLL-g-PEG) are described. In this case, "g" denotes the grafting ratio, ie the quotient from the

Number of lysine units and the number of polyethylene glycol chain chains. It has been shown that good results can only be achieved with these PLL-g-PEG graft copolymers if the graft ratio "g" is within the range of 8 to 12 and the PEG side chains in the molecular weight range of 1500 to 5000 g / mol.

This complex and narrowly defined special polymer meets the demands made regarding good availability, consistent quality and universal applicability for different substrate surfaces only insufficient.

Another disadvantage of this method is the instability of these layers to pH values of less than 3 and greater than 9, as well as high salt concentrations, since under these conditions, the electrostatically bonded polymer desorbs from the surface.

WO 02/068481 describes phosphorus-containing polymers for coating dielectric materials and their use in optical signal converters. These are water-soluble polymers which are prepared, for example, by reacting polyallylamine hydrochloride with formaldehyde and phosphorous acid, according to the so-called Mannich Mödritzer reaction.

These polyphosphonamides, in addition to cationic ones, also have anionic groups which are rather counterproductive to coupling of recognition molecules, for example DNA, by electrostatic means. In addition, experience shows that the o.g. Mannich Mödritzer Implementation of a process that forms other products in addition to the desired Polyphosphonamiden because of side reactions.

On the other hand, gene chip concepts based on polyelectrolyte bonding are particularly preferred embodiments in the coating of biophore and co-receptors because of their simple production method.

B. Laguitton describes in US 6,689,478 the coupling of DNA recognition molecules to polyelectrolyte (PEL) multilayers, wherein the uppermost binding layer is a cationic polymer (cationic PEL). Polyelectrolytes are polymers that carry ionic or ionizable groups in their repeating unit. Examples of cationic polyelectrolytes are polyamines, such as polyethylenimine (PEI) or polyammonium compounds, such as polyallylamine hydrochloride or polydiallyldimethylammonium chloride (P DADMAC). Examples of anionic polymers are the salts of polyacrylic acids (PAS), polystyrene sulfonic acids or dextran sulfonic acid. Polyelectrolyte multilayers consist of an alternating structure of oppositely charged polyelectrolytes, as described, for example, in "Multilayer Thin Films" G. Decher, Wiley-VCH, 2003. While the binding of nucleic acids to PEL multilayers is also state of the art ( B. Sukhoukov, "Multilayer Films Containing Immobilized Nucleic Acids" Biosensors & Bioelectronics, vol. 11, no. 9, 913-922, 1966), the challenge, as described in US Pat. No. 6,689,478, is to bind the first polymer layer to a cationic polymer to the glass substrate. This challenge is solved by first treating the glass supports in a complex manner with hydrogen peroxide and then with sulfuric acid, whereby the glass surface is anionically modified. In contrast, B. Languitton in US Pat. No. 6,689,478 argues that with monolayer polymer layers, the requirements for reliable substrate coating can not be achieved.

It has now surprisingly been found that polymer modification of substrate surfaces in polymer monolayers is nevertheless possible if the aforementioned polyelectrolyte polymers which are readily available in high quality have a particularly high molecular weight.

Starting from a waveguide with a high refractive Ta ₂ O ₅ surface, it was initially found in a screening process with various cationic and anionic polyelectrolytes that this surface showed no affinity for anionic polyelectrolytes.

For example, within the group of cationic polyelectrolytes, that is, polymers carrying ammonium structures in their repeating unit, it has been found that polyvinylamines of molecular weight (MW) of 50,000 g / mol did not exhibit sufficient affinity for the Ta ₂ O ₅ surface Polyvinylamines with MW 340 000 g / mol met all the requirements.

Polyvinylamines are polymers formed by acidic or alkaline hydrolysis of poly (N-vinylformamides) as described in J. Appl. Pole. See VoI 86, 3412-3419 (2002). The corresponding products are manufactured by BASF AG under the trade name "Lupamin" in various molecular weights.These products are used on a large scale, for example as paper chemicals, in the personal care sector, as superabsorbent or dispersant.In the Lupamin products of sale are those from the hydrolysis For the field of application described, the modification of waveguide surfaces, both the salt-containing and the desalted form can be used, for example desalination by ultrafiltration.

The same was found with linear polyethylenimines (IPEI). While IPEI with MW 25 000 g / mol (Polyscience Inc.) did not meet the requirements, the very high molecular weight IPEI 500 000 g / mol again met all requirements. Polyamines with uncharged N in their repeating unit also count as aqueous cationic polyelectrolytes, since they are at least partially protonated under these conditions. Another cationic polyelectrolyte having a high molecular weight is poly (diallyldimethylammonium chloride), which is readily available as an aqueous solution in the molecular weight range of 400,000 to 500,000 g / mol. In contrast to the previously mentioned cationic polyelectrolytes, these are quaternary ammonium groups, ie the state of charge is independent of the pH.

In the following, the method according to the invention for the production of a planar waveguide (PWG: planar wave guide) with DNA as recognition molecule (capture DNA) will be described on the example Lupamin 9095 (polyvinylamine PVAm 340 000 g / mol).

1. Loading the substrate with Lupamin 9095

The substrate (PWG made of quartz glass with Ta ₂ O ₅ surface) is immersed for 30 min in a highly diluted Lupamin 9095 solution (0.005% in water) and then immersed in water for 30 min and dried.

2. Apply the capture DNA spots

By means of a micropipette onto the polymer coated Ta ₂ O ₅ side of the dye-labeled DNA solution was pipetted (about 10 ^-10 m) and 20 min. At RT

(ditched).

3. Block the unpaved surface

The PWG provided with the capture DNA spot is immersed in an aqueous dextran sulfate (DexS) solution (MW: 500,000 g / mol, 0.05% in water) for 30 min and briefly washed with water.

The PWG thus provided with capture DNA spots can be used directly for hybridization experiments.

The method described here is a process that is hard to beat in terms of simplicity and reproducibility.

In addition to the good availability of the polymers v. a. to mention the number and the simplicity of the individual process steps.

For example, in Laguitton in US Pat. No. 6,689,478, where capture DNA is bound to polyelectrolyte multilayers (alternating sequence of cationic and anionic polyelectrolyte monolayers), the following, sometimes quite complex, individual steps are contained: Functionalization of the substrate surface with H ₂ O ₂ and sulfuric acid, multiple application of different polyelectrolytes, pipetting of the capture DNA, washing off the capture DNA excess, blocking of the non-spotted surface with a bifunctional reagent and washing off of the reagent excess.

In this context, reference should be made in particular to the step 3. (blocking) of the method according to the invention.

When the PWG is immersed with the capture DNA spot (s) in the dextran sulfate solution, the capture DNA excess is washed off simultaneously and the polyelectrolyte binding of dextran sulfate to the unspotted lupamine surface blocks the nonspecific adsorption of DNA. Dextran sulfate as a blocking agent in DNA chips is also advantageous in that in the subsequent recognition reaction (hybridization) is often also dextran sulfate used as an aid in the corresponding hybridization buffer.

Although the previously mentioned polymers, Lupamin 9095 and dextran sulfate are preferably used, it is possible in principle to use alternative polyelectrolyte combinations.

Concerning. cationic polyelectrolytes which bind to the substrate surface in high affinity and permit ionic or covalent attachment of the capture DNA, for example, all known cationic polyelectrolytes in question, provided they have a very high molecular weight, vzgw. higher than 100,000, more preferably higher than 250,000 g / mol. Examples are polyallylamine, polydimethyldiallylammonium chloride, polyvinylpyridine, cationically modified polyacrylates and polyethyleneimine (PEI), which may be branched or linear, depending on the method of preparation. Linear, very high molecular weight PEI can be readily prepared from the readily available poly (2-ethyl-2-oxazoline) (MW: 500,000 g / mol) by acid hydrolysis.

The mentioned cationic polyelectrolytes can be used both as hydrochloride and in their aminic form. Namely, the aminic form is partially protonated in aqueous solution and thus also has cationic charge properties in this form.

Concerning. anionic polyelectrolytes which are used by interaction with the cationic surface for anionic blocking against non-specific DNA, in addition to dextran sulfate all known anionic polyelectrolytes, for example, the Na salts of polystyrenesulfonic acid, polyacrylic acid, or Polyacrylsärecopolymere or polymaleic acid and their copolymers in question. While in the above-mentioned cationic polyelectrolytes for the process of the present invention, as mentioned above, a very high molecular weight is required, the anionic polyelectrolytes may also be used in their low-molecular form.

For example. were obtained with dextran sulfate both MW 5000 and 500 000 g / mol excellent blocking properties against nonspecific DNA adsorption.

Although the test elements based on physisorption (polyelectrolyte interaction) are preferred with regard to DNA chips in the method according to the invention, the substrates modified with cationic polyelectrolytes in their aminic form can also be modified via covalent bonding mechanisms. Thus, it is known that biomolecules can be coupled to aminic surfaces via bifunctional reagents, such as glutaric dialdehyde or bis-N-hydroxysuccinimides, for example disuccinimidylsuberate (DSS) or sulfo-DSS.

In an analogous manner, the blocking step can also take place via covalent bonding mechanisms. For example. For non-specific protein adsorption via bifunctional reagents, polyethylene glycols, amine-modified polyethylene glycols or hyperbranched polyglycerol (hyperpolymers GmbH) can be coupled in. Polyethylene glycols can also be, for example, isocyanate-functionalized polyethylene glycols, for example polyethylene glycol monomethyl ether or succinimidyl ester derivatized polyethylene glycols (Shearwater Polymers). be coupled.

The described, particularly preferred method, based on high molecular weight cationic polyelectrolytes as a polymer interface and anionic polyelectrolytes as a blocking layer against nonspecific DNA adsorption, refers to substrates with metal oxide, vzgw. Ta ₂ Os surfaces.

Accordingly, for other substrate surfaces which, for example, are partially positively charged, these ratios can be reversed so that anionic high molecular weight polyelectrolytes can serve as polymer interface.

The high molecular weight cationic polyelectrolytes are preferably suitable for anchoring the polymer on waveguides made of materials such as TiO ₂ , Ta ₂ O ₅ , ZrO ₂ , HfO ₂ , Al ₂ O ₃ , SiO ₂ (Si (Ti) O ₂ ), In ₂ O ₃ / SnO ₂ (TTO), aluminosilicates, Nb ₂ O ₅ , vanadium oxides, or mixtures of these materials. However, the waveguide materials can also be oxides or hydroxides of the following elements which can form oxides or hydroxides: Sc, Y, Ti, Zr, Hf, V, Nb, Ta, Cr, Mo, W, Mn, Tc, Re, Fe, Ru , Os, Co, Rh, Ir, Ni, PD, Pt, Cu, Ag, Au, Zn, Cd, Hg, B, Al, Ga, In, Tl, Ge, Sn, Pb, As, Sb, Bi, lanthanides, actinides and mixtures thereof, as well as mixtures of group IIa (Be, Mg, Ca, Sr, Ba, Ra) and VIb (Se, Te, Po)).

The polymer is applied to the waveguide surfaces from organic or preferably aqueous solution. This can be done by incubation in the solution, such as dipping, spraying, spotting, spin coating or similar conventional methods. Typically, solutions between 0.1 to 0.0001 wt%, in particular between 0.05 and 0.001 wt%, used and the waveguide surfaces at temperatures between 0 and 200 ⁰ C, in particular between 20 and 30 ⁰ C coated. The incubation time of the white materials with the polymer solutions may be between 10 seconds and 48 hours, typically between 10 minutes and 24 hours. After incubation, the waveguides are rinsed with organic solvents or aqueous solutions and optionally further derivatized.

Chemisorbieren polyelectrolytes for bio- and chemofunctionalization from organic or aqueous solution on waveguide surfaces. They form a stable layer on the waveguide due to the specific polyelectrolyte bonding to waveguide materials. The binding is stable over a wide pH range (pH = 1 to pH = 14), temperature range (0 ^° C. to 100 ^° C.) as well as to high salt concentrations (III). Also, the presence of detergents in the reaction solution does not result in desorption of the polymer from the waveguide surface. Since, after a polyelectrolyte coating of the substrate surface with a monomolecular layer, a further adsorption of polymers charged in the same direction is prevented, only a monolayer of polymer can be applied to the surface in the sense of chemisorption. This can ensure that the recognition elements are immobilized within the evanescent light field and thus in the sensitive detection area of the signal converter.

The waveguide chips coated in the manner described can be used for any type of qualitative, semi-quantitative or quantitative analytical assays.

Recognition elements can be immobilized covalently, coordinatively or via another linkage to the functional groups of the polymer and thus to the surface of the biosensor or chemosensor, directly or with the aid of a crosslinker. The direct coupling of the detection elements can take place before the coating of the waveguides with the polymer or thereafter. The recognition elements can be obtained by their own functional groups such as carboxylic acid, carboxylic acid ester, carboxylic acid chloride, carboxylic acid anhydride, carboxylic acid nitrophenyl ester, carboxylic acid N-hydroxysucinimide, carboxylic acid imidazolide, carboxylic acid pentafluorophenyl ester, hydroxy, toulo-sulfonyl, trifluoromethylsulfonyl, epoxy, aldehyde, ketone, β-dicarbonyl, isocyanate, Thioisocyanate, nitrile, amine, aziridine, hydrazine, hydrazide, nitro, thiol, disulphide, thiosulphite, halogen, iodoacetamide, bromoacetamide, chloroacetamide, boric acid esters, maleimide, α, β-unsaturated carbonyls, Phosphate, phosphonate, hydroxymethylamide, alkoxymethylamide, benzophenone, azide, triazene, acylphosphine, to which functional groups of the polymer are covalently bonded. The combination which functional group of the recognition element reacts with which functional group of the polymer results from the reaction possibilities known to the chemist between the functional groups.

Proteins as recognition elements can e.g. be immobilized on the polymer via its amino acid side chains. Specifically, amino acids such as e.g. Lysines, cysteines, serines, tyrosines, histides, glutamates, aspartates located on the surface of a protein have functional groups in their side chains that can form a covalent bond with the functional groups of the polymer. Functional groups can also be generated in the recognition elements by derivatization (phosphorylation of tyrosines), oxidation (e.g., oxidation of diol units of glycosylated proteins to aldehyde groups), reduction (e.g., of disulfide bonds to thiols), or coupling of a crosslinker.

In addition to the covalent immobilization of the recognition elements to the polymer, the recognition elements can also be bound coordinately to the polymer. With methods of molecular biology, e.g. Proteins such as enzymes, antibody fragments and receptors with specific affinity sequences such as e.g. the 6x histidine tag. These affinity sequences have a high affinity and specificity to metal ion complexes, e.g. Nickel nitrilotriacetic acid or copper iminodiacetic acid, which may be incorporated as a functional group F in the polymer.

Alternatively, biochemical recognition reactions can also be used to immobilize recognition elements to the polymer. The very specific and high affinity binding of biotin to streptavidin can be used to immobilize recognition elements to the polymer. For this purpose, the streptavidin must first be immobilized. The recognition element is then functionalized with biotin and can be indirectly bound to the polymer via the streptavidin-biotin interaction. Alternatively, the recognition element can be molecularly biological or chemically provided with a short amino acid sequence, the so-called StrepTag ²⁴ , which also has a high specificity and affinity for streptavidin.

The signal transducers according to the invention, characterized in that the recognition elements are bound to polyelectrolyte monolayers, are indeed preferred content of the present application. Nevertheless, it may sometimes be advantageous if the recognition elements are bound to polyelectrolyte multilayers. Although a multilayer structure induces a greater distance of the detection element from the waveguide surface - and thus a reduced Sensitivity - on the other hand, defects in the multi-layer process can be compensated by so-called "bridge effects".

However, it is essential for the substrate coating according to the invention that the polyelectrolyte used for the first substrate coating has a very high molecular weight, vzgw. greater than 100,000, more preferably greater than 250,000 g / mol. For the following layers also lower molecular weight polyelectrolytes can be used.

One embodiment of the immobilization surfaces according to the invention is the use on optically transparent supports, which are characterized in that they comprise continuous or individual waveguiding regions (optical waveguides). The optical waveguide is particularly preferably an optical waveguide with a first substantially optically transparent layer (a) facing the imaged surface on a second substantially transparent layer (b) with a lower refractive index than layer (a). It is also preferred that said optical waveguide be substantially planar.

Characteristic of such an embodiment of an immobilization surface according to the invention on an optical slab waveguide as 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 learning of prism couplers , evanescent fields, end face couplers with arranged in front of an end face of the waveguiding layer focused lenses and grating couplers is formed.

In this case, it is preferred that the coupling of excitation light into the optically transparent layer (a) takes place by means of one or more grating structures (c) which are formed in the optically transparent layer (a).

An object of the invention is a surface for the immobilization of one or more first nucleic acids as Erkennungselemeπte for producing a recognition surface for the detection of one or more second nucleic acids in one or more contacted with the detection surface in contact samples, wherein the first nucleic acids on a cationic high molecular weight polyelectrolyte layer as Surface are applied for immobilization.

Such 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 regions, are particularly preferred. In a two-dimensional arrangement up to 2,000,000 measuring ranges can be arranged, and a single measuring range can occupy an area of 10- ⁵ mm ² to 10 mm ² . It is preferred that the measuring ranges are arranged in a density of more than 100, preferably more than 1000 measuring ranges per square centimeter.

The discrete (spatially separated) measurement areas on said immobilization surface may be generated by spatially selective application of nucleic acids as recognition elements, preferably using one or more of the group of methods known from "inkjet spotting", mechanical spotting by stylus, Spring or capillary, "Micro contact spotting", fluidic contacting of the measuring range 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 and photochemical or photolithographic immobilization is formed.

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 further reagents with one of the immobilization surfaces according to the invention according to one of the embodiments in Are contacted, on which one or more nucleic acids are bound as recognition elements for specific binding / hybridization with second nucleic acids and from the binding / hybridization of this second nucleic acid or further for analyte detection of inserted detection substances resulting change of optical or e- lektrischen signals are measured.

It is preferred that the detection of one or more second nucleic acids based on the determination of the change of one or more luminescences. For luminescence generation various optical excitation configurations are possible. One possibility is that excitation light for exciting one or more luminescences from one or more light sources is irradiated in an incident light excitation arrangement.

Another possible configuration is characterized in that excitation light for excitation of one or more lumine lengths of one or more light sources is irradiated in a transmission light excitation device.

Such an embodiment of the immobilization surfaces and / or methods according to the invention is preferably characterized in that the surface is arranged on an optical waveguide such 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 are brought into contact with the bound first nucleic acids as recognition elements and the excitation light from one or more light sources is coupled into the optical waveguide with the help of one or more optical coupling elements from the group of prism couplers, evanescent FeI- , Endflächenkopplern with arranged in front of an end face of the waveguiding layer fo- kussierten lenses and grating couplers is formed.

It is preferred that the light wave traveling in the waveguide produces luminescence from molecules capable of luminescence, and that this luminescence is detected by one or more detectors. From the intensity of the luminescence signal, the concentration of one or more subsequent nucleic acids to be detected can be determined.

The luminescent label can be coupled to the second nucleic acid to be detected itself, or bound in a competitive approach to molecules of known concentration and sequence, as competitor, and thus added to the sample. The Lutninszenz label can also be introduced by a third binding partner in the analysis mixture.

It is preferred that luminescent dyes or luminescent nanoparticles are used as luminescent labels for generating the luminescence, which are excited and emit at a wavelength of between 300 nm and 1100 nm.

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. The samples to be examined can also be prepared from biological tissue parts or cells.

The invention further relates to the use of the immobilization surfaces and / or methods according to the invention in qualitative and quantitative DNA and RNA analysis, for example the determination of genomic differences such as the single nucleotide polymorphisms (SNPs) or DNA amplifications or DNA deletions or DNA methylation or for the detection and quantification of mRNA (expression profiling).

In the following examples, waveguides with polymeric monolayers (polyvinylamine hydrochloride) and with a three-layer structure (polyvinylamine hydrochloride / dextran sulfate / polyvinylamine hydrochloride) as immobilization surfaces are described and their application in nucleic acid analysis. Examples

1. Modification of a planar waveguide with a monolayer of polyvinylamine hydrochloride, subsequent doping with capture DNA spots and blocking with dextran sulfate

A planar waveguide (Unaxis Balzers, Liechtenstein) in the dimensions 2x1 cm, consisting of AF 45 glass with a waveguiding, optically transparent, high refractive Ta ₂ O ₅ layer (refractive index of 2.10 at 633 nm, layer thickness 185 nm) and parallel to the width extending grating lines (318 nm period with 32 +/- 3 nm lattice depth)

- Dipped in water for 30 min in a 0.005% solution of Lupamin 9095 (BASF).

The lupamine-modified waveguide was washed in pure water for 30 min

Excess water residues were dried with a dry N ₂ stream

Using a micropipette, 5 μl of a fluorescence-labeled DNA sequence (Cy5 - 50 mer: 5'-Cy5-CAA CAG TGC AAC CTT GGA AGC AGA TGT AGA TGT TGT TGT GTC ACC TCC AT 3 ', from BioTeZ Berlin) were applied to the high-index, Lupamin coated surface pipetted. In the same way, analogue capture oligo spots were applied at two other sites.

After incubation for 30 min. At RT, the spotted waveguide was immersed in an aqueous dextran sulfate solution (MW: 500,000 g / mol, from Fluka) for 30 min and washed briefly with water.

The functional test was carried out by coupling a laser light beam of wavelength 635 nm.

At the sites where the capture DNA spots were pipetted on, caused by the emission of the Cy5 fluorescent dye (Amersham), bright spots were observed.

For comparison, the above-mentioned Cy5 50 mer capture solution was pipetted to a site blocked with dextran sulfate against DNA adsorption and washed with water.

During the subsequent optical function test, no color signal was found at the corresponding points. 2. Modification of a pianar waveguide with a polyelectrolyte triplicate layer of polyvinylamine hydrochloride / dextran sulfate / polyvinylamine hydrochloride, subsequent doping with capture DNA spots and blocking with dextran sulfate

A planar waveguide (Unaxis, Balzers, Liechtenstein) in the dimensions 2x1 cm, consisting of AP 45 glass with a waveguiding, optically transparent, high refractive Ta _Σ Os layer (refractive index of 2.15 at 633 nm, layer thickness 185 nm) and parallel to the width Grid lines (318 nm period with 32 +/- 3 nm lattice depth)

1. Dipped in water for 30 minutes in a 0.005% solution of Lupamin 9095 (BASF).

2. The lupamine-modified waveguide was washed in pure water for 30 min.

3. The waveguide with the lupamine monolayer was immersed in a 0.05% aqueous dextran sulfate solution for 30 min.

4. Subsequently, it was washed in pure water for 30 min.

5. Repeat step 1.

6. The waveguide with the polyelectrolyte triple layer was washed in water for 30 min.

7. Excess water residues were dried with a dry N ₂ stream.

Using a micropipette, 5 μl of a fluorescence-labeled DNA sequence (Cy5 - 50 mer: 5'-Cy5-CAA CAG TGC AAC CTT GGA AGC AGA TGT AGA TGT TGT TGT GTC ACC TCC AT 3 ', from BioTeZ Berlin) pipetted onto the high refractive, lupamin coated surface. In the same way, analogue capture oligo spots were applied at two other sites.

9. After incubation for 30 min at RT, the spotted waveguide was immersed in an aqueous dextran sulfate solution (MW: 500,000 g / mol, from Fluka) for 30 min and washed briefly with water.

At the sites where the capture DNA spots were pipetted on, caused by the emission of the Cy5 fluorescent dye (Amersham), bright spots were observed. For comparison, the above-mentioned Cy5 50 mer capture solution was pipetted to a site blocked with dextran sulfate against DNA adsorption and washed with water. During the subsequent optical function test, no color signal was found at the corresponding points.

Example 3

Hybridization of bacterial cDNA on lupamine coated planar waveguide chips

A planar waveguide chip (SensiChip, Zeptosens, Bayer Switzerland AG, Witterswil, Switzerland) of 14mm x 57mm consisting of AF 45 glass with a waveguiding, optically transparent, high refractive Ta ₂ O ₅ layer (refractive index of 2.12 at 535 nm, layer thickness 145 nm) and parallel to the width-extending grating lines (318 nm period with 13 +/- 2 nm grating depth)

60 min. In a 0.01% solution of Lupamin 9095 (BASF) immersed in water

then the chips were washed for 30 min in Miüipore water and placed for a further 4 hours in a fresh Millipore water.

Excess water residues were removed with a dry N ₂ stream.

- The chips were placed in a contact spotter and spotted with different oligonucleotides solutions in a concentration of 10 ^'5 M. The oligonucleotide was synthesized by the company Operon (Cologne, Germany) and had a length of 70 bases. Four of the oligonucleotides had a sequence complementary to the bacterial control cDNA used in the later hybridization (B.subtilis sequences, ATCC 87482 and 87483, reference sequences X17013 and M24537, respectively, The American Type Culture Collection, www -atccorg)

After an incubation time of 60 min at RT, the spotted waveguide was placed in an aqueous blocking solution for 5 min (5% dextran sulfate (MW: 500,000 g / mol, 1% Tween, 10 mM Tris, 30% formamide, pH 8.5). dipped in water and then washed with water for 1 min.

Excess water residues were removed with a dry N ₂ stream.

The bespotteten and blocked chips were inserted into the hybridization chambers of the SensiChip system of Zeptosens (Bayer Switzerland AG, Witterswil, Switzerland) according to the system distributor. The chips were incubated according to system instruction first in SB buffer and then in PHB buffer. Containing the Cy5-labeled cDNA- The hybridization mixture was also prepared according to the specifications of the SensiChip system. The cDNA had previously been synthesized in a labeling reaction using the LabelStar kit from Qiagen Hilden, Germany) with the fluorescent dye Cy5 (Amersham, Arlington Heights, USA) from the bacterial mRNA. The hybridization mixture was heated at 95 ° for 5 min just prior to filling in the hybridization chambers. The cDNA was at concentrations of 5x10 ^"14 M - to 10 ^'12 M based on the starting RNA quantity employed since hybridization volume was 40 .mu.l After an overnight incubation at 42 ° C, the chips were washed according to the system distributor with the system wash buffers.. The chips were read in the Zepto-Reader (PWG system, CCD camera based) with different exposure times The intensities determined as a function of the different sample concentrations are shown in FIG.

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Claims

Patentansprücbe

1. Polyelectrolyte mono- and multilayers for optical signal transducers, characterized in that the polyelectrolyte polymers have a particularly high molecular weight.

2. polyelectrolyte mono- and multilayers for optical signal transducers according to claim 1, characterized in that the polyelectrolyte polymers have a molecular weight of at least 300,000 g / mol.

3. polyelectrolyte mono- and multilayers for optical signal transducers according to claim 1, characterized in that the polyelectrolyte polymers have a molecular weight of at least 400,000 g / mol.

4. polyelectrolyte mono- and multilayers for optical signal transducers according to claim 1, characterized in that the polyelectrolyte polymers have a molecular weight of at least 500,000 g / mol.

5. polyelectrolyte mono- and multilayers for optical signal transducers according to claims 1 to 4, characterized in that the polyelectrolyte polymers are selected from the group: polyvinylamine, polyetylenimine and poly (diallyl dimethylammonium chloride).