DE10128093A1 - Specific-binding method for detecting substances, useful in sandwich immunoassays, based on binding of colloidal particle to a surface through nucleic acid hybridization - Google Patents

Abstract

Specific-binding (M) for detecting a substance (A) in aqueous solution, is new. M comprises: (a) preparing a surface with specific-binding capacity for (A); (b) preparing a binding molecule (B), with specific-binding capacity for (A); (c) linking (B) to a colloidal particle (CP) to impart specific-binding properties; (d) combining surface, test sample and (B)-modified CP so that a surface-bound signal complex (SC) can form; and (e) detecting any CP present in SC. Independent claims are also included for: (1) article for performing M comprising CP with a nucleic acid (NA2) linked to it and one or more (B) containing a nucleic acid (NA1) complementary to NA2, where NA1/2 are linked; and (2) kit for preparing the article.

Description

The introduction of antibody- or receptor-based immunoassays in the 1960s and 1970s led to the development of a large number of highly specific methods for the sensitive and precise detection of low-concentration analyte substances. For example, JR Crowther in ELISA: Theory and Practice, Humana Press Inc., 1995 describes that substances up to the attomol range (1 amol = 1 × 10 ^-18 mol) can be detected with enzyme-enhanced assays or radioimmunoassays. The development of highly effective active ingredients and treatment methods in modern medicine, the appearance of new diseases such as the encephalopathies detectable via the prion protein PrP ^Sc (e.g. bovine spongiform encephalopathy, scrapie, Creutzfeld-Jacob disease, Gerstmann-Straussler-Scheinker syndrome), but However, the increasing research into complex biological processes through proteome research also requires the development of detection methods which, on the one hand, allow the highest possible sensitivity and, on the other hand, the greatest possible parallelism.

One approach to solving this problem is using multiplex analysis perform microstructured test arrays in spatially defined Arrangement contain a large number of binding molecules on their surface. This arrangement of binding molecules can then be used for parallel detection of substances used with one or more Binding molecules on the array enter into specific interactions and thereby bound to the anay. For example, describe MacBeath and Schreiber in Science 2000, 289, 1760 the preparation of a protein array for the high-throughput analysis of protein-protein interactions. Hergenrother et al. describe in J Am Chem Soc 2000, 122, 7849 that low molecular weight Alcohol derivatives are immobilized on glass slides, and that these arrays are used for Detection of protein binding can be used. R. M. de Wildt et al. describe in Nat. Biotechnol. 2000, 18, 989 that test arrays of antibodies for used the high-throughput analysis of antibody-antigen interactions become. All these descriptions have in common that for the detection of the bound analytes always a complex labeling of Bulk sample material is required. This is typically done by Radionuclides, for example by metabolic labeling in the course of the Vivo total protein production will be introduced. Alternatively, the Labeling substances (radionuclides, chromophores, fluorophores or Hapten groups) subsequently introduced (e.g. by chemical modification) become. This also requires a considerable amount of time and material Expenditure. It would therefore be desirable to only add the analyte molecules mark those that are specifically bound to the surface.

This procedure is based on a "two-sided" (sandwich) immune detection enables, as is standard with sandwich ELSIA or sandwich Radioimmunoassay (RIA) is used (see: J. R. Crowther in ELISA: Theory and Practice, Humana Press Inc., 1995). To the problem above multiplex analysis with protein test arrays, however, is only this method limited use, since the diffusion of the substrates in the enzymatic Reinforcement step usually also for unspecific staining of whole Regions leads so that a spatially resolved detection is not possible. The The use of non-diffusible substrates often causes irreversible damage of the test array, which complicate a spatially resolved detection.

In view of the existing disadvantages of the known methods, the first was (Partial) object of the present invention to provide a method that allows the parallel analysis of surface-bound substances in a few Carry out reaction steps with the highest possible sensitivity. Another (partial) task was to do the detection procedure like this indicate that there are as many different demonstrable items as possible Substances such as proteins, peptides, sugars, lipids and the like can be used.

In procedural terms, it was a second (partial) task of the present one Invention to design the method so that the reagents to be used can be produced in the sense of a modular reagent kit. in this connection the components of the reagent kit should have a high physico-chemical Stability especially with regard to thermal and chemical loads have the individual reaction steps.

A third (partial) object of the present invention was an article provide with which the reagent kits can be used to a experimentally easy to carry out detection of substances.

• a) Bereitstellung einer Oberfläche die eine spezifische Bindungsfähigkeit für die nachzuweisende Substanz aufweist;
• b) Bereitstellung eines Bindemoleküls, dass eine spezifische Bindungsfähigkeit für die nachzuweisende Substanz aufweist;
• c) Verknüpfung des Bindemoleküls mit kolloidalen Partikeln, so dass die Partikel eine spezifische Bindungsfähigkeit für die nachzuweisende Substanz erhalten;
• d) Zusammenbringen der Oberfläche, der nachzuweisenden Substanz und der Bindemolekül-verknüpften kolloidalen Partikel, so dass eine Oberflächen- gebundener Signalkomplex gebildet wird;
• e) Nachweis der kolloidalen Partikel, soweit sie in einem Oberflächen- gebundenen Signalkomplex vorliegen

The first (partial) object is achieved by the present invention by carrying out a method with the following steps:

• a) provision of a surface which has a specific binding capacity for the substance to be detected;
• b) provision of a binding molecule which has a specific binding ability for the substance to be detected;
• c) Linking the binding molecule with colloidal particles so that the particles acquire a specific binding ability for the substance to be detected;
• d) bringing together the surface, the substance to be detected and the binding molecule-linked colloidal particles so that a surface-bound signal complex is formed;
• e) Detection of the colloidal particles, insofar as they are present in a surface-bound signal complex

A surface that has a specific binding capacity for the one to be detected Has substance, can each in the sense of the inventive method a binding molecule (see below) connectable surface. In particular can it be a glass, silicon or metal surface or another, act preferably surface suitable for the production of biochips. Under gold or gold-coated surfaces are preferred over the metal surfaces. It can also be plastic surfaces, especially polystyrene or Polycarbonate surfaces act as they are especially in microtiter plate Cavities are present. The surface can also be the surface preferably act magnetic microparticles, such as those for the extraction of Liquid nucleic acids can be used.

The surface can be coated (blocked) with substances that block the Do not disturb process execution. In particular, the surface with Gelatin, milk powder and / or bovine serum albumin (BSA) can be coated. The The surface can also be provided with nucleic acids, insofar as these are Do not interfere with the implementation of the verification procedure.

Binding molecules in the sense of the detection method are those substances or substance mixtures which can preferably bind selectively to the substance to be detected under the selected process conditions. Binding molecules can in particular be receptors such as protein A and enzymes, but also mono- and / or polyclonal immunoglobulins, in particular those of type G, and their fragments. Such fragments are known in particular under the names Fab, F (ab) ₂ , dsFv fragments, scFv fragments and single-chain antibodies. Furthermore, binding molecules can also be low-molecular substances, such as peptides, peptoids, haptens and nucleic acid binding reagents, such as single and double-stranded nucleic acids, ribocynia or aptamers. The use of polyclonal immunoglobulins as binding molecules of the capture and / or detection reagent is preferred because of their often higher stability and often easier availability compared to monoclonal immunoglobulins. If the binding molecule is a substance mixture of several substance variants such as, for example, a polyclonal immunoglobulin, the capture and / or detection reagents each comprise several of the substance variants (for example several of the variants of a polyclonal immunoglobulin) in one molecule or are mixtures of or detection reagents whose molecules differ from one another in their binding molecule variants (for example the individual variants of a polyclonal immunoglobulin).

Colloidal particles in the sense of the detection method are such components or component mixtures consisting of gold, silver, platinum and other metals, Semimetals, metal or non-metal oxides, sulfides or other conductive Materials or semiconductor materials exist and their average Size is in the range of 1-500 nm. Colloids are particularly preferred made of gold with an average diameter of approx. 5-100 nm since they easily and in large quantities by a variety of methods known from the literature can be produced (see for example: G. Schmid, Clusters and Colloids, VCH, Weinheim 1994). There is an advantageous embodiment of the method in using fluorescent semiconductor particles and colloids, their intrinsic fluorescence and semiconductor properties a particularly simple and enables efficient detection.

The detection of the colloidal particles in the sense of the detection method can done by a variety of methods. Here are microscopic Methods such as light and electron microscopy, but also in particular Scanning probe microscopy such as B. the atomic force and Scanning tunneling microscopy preferred. Detection by a Silver development. This is a known one for years Standard method in which metallic nanoparticles and colloids for staining of antibody labels are used, for example, around the perform histological examination of biological samples and tissues (see on this: J. Kreuter, in M. Donbrow (Ed.): Microcapsules and Nanoparticles in Medicine and Pharmacy, CRC Press, Boca Raton, FL 1992).

Specific properties of the colloidal components can also be used to make the detection of the colloids. For example, at Use of fluorescent semiconductor particles made of CdS, CdTe, ZnS or similar materials a direct detection by commercially available fluorescence Scanners or fluorescence microscopes are used. Fluorescent semiconductor particles also enable their detection by photoelectrochemical measurements. in the In the case of gold and silver colloids, it is also preferred to detect the colloidal particles by optical methods such as surface plasmon resonance (see: L. He, M. D. Musick, S. R. Nicewarner, F. G. Salinas, S. J. Benkovic, Natan, M.J., Keating, C.D. Colloidal Au-Enhanced Surface Plasmon Resonance for Ultrasensitive Detection of DNA Hybridization. J Am Chem Soc 2000, 122, 907), reflectometric interference spectroscopy (see: G. Gauglitz. Optical detection methods for combinatorial libraries. Curr Opin Chem Biol 2000, 4, 351), surface-enhanced Raman spectroscopy (surface-enhanced raman spectroscopy, SERS, see: C. D. Keating, K. M. Kovaleski, M. J. Natan. Protein- Colloid Conjugates for Surface Enhanced Raman Scattering: Stability and Control of protein orientation. J Phys Chem B 1998, 102, 9404), by electrical Detection using impedance measurement (see: R. Hintsche, M. Paeschke, A. Uhlig, R. Seitz, in F. W. Scheller, F. Schaubert, J. Fredrowitz (Eds.): Frontiers in Biosensors: Fundamental Aspects, Birkhäuser Verlag 1997, p. 267) or by mass-sensitive methods such as the quartz crystal microbalance (QCM, see: F. Patolsky, K. T. Ranjit, A. Lichtenstein, I. Willner. Dendritic amplification of DNA analysis by oligonucleotide-functionalized Au nanoparticles. Chem. Commun. 2000, 1025) or surface acoustic wave measurements (SAW: Wang et al. (1998) Sensors and Actuators B49, 13).

It has been found that performing silver development is particularly preferred since accelerated by the gold colloids Silver development can detect small amounts of antigen, because of the lack of diffusibility the development of the silver mirror to the spatially resolved Detection of antigens can be used. Thus, the Detect the immune reaction with a commercially available flatbed scanner, whereby elaborate laboratory equipment and expensive detection methods become unnecessary. With that the protein / DNA modified gold colloids especially for reading out Protein microarrays are increasingly being used in immunology Diagnostics and proteome research (see: C. Niemeyer, Chem. Eur. J., in print) can be used (see embodiment 10).

The second (partial) object of the present invention was that To design reagents in such a way that they have a modular structure Reagent kits can be used, in particular a high physical chemical stability of the reagents with regard to thermal and chemical Stresses essential for the successful implementation of the invention Procedure is. The latter point becomes particularly clear when you look at the problems considered when using conventional antibody or Receptor-coated colloidal gold reagents occur. These conventional ones Protein-coated gold particles have a strong tendency to be poorly soluble Form conglomerates in aqueous solutions, so that either strong unspecific reactions occur or the colloids generally no longer for immunological detection methods can be used.

• a) das Bindemolekül mit einer ersten Nucleinsäure verküpft, und
• b) die kolloidalen Partikel mit einer zweiten Nucleinsäure verknüpft werden, die komplementär zur ersten Nucleinsäure ist, und
• c) das Nucleinsäure-modifizierte Bindemolekül mit den Nucleinsäure- modifizierten kolloidalen Partikeln zusammengebracht wird, so dass es zur Hybridisierung der ersten mit der zweiten Nucleinsäure kommt

The second (partial) object is achieved by the present invention by performing step c of the method described above in such a way that

• a) the binding molecule is linked to a first nucleic acid, and
• b) the colloidal particles are linked to a second nucleic acid which is complementary to the first nucleic acid, and
• c) the nucleic acid-modified binding molecule is brought together with the nucleic acid-modified colloidal particles, so that the first nucleic acid hybridizes with the second

It has surprisingly been found that the coupling of Binding molecule and colloidal particle via a hybridization complementary Nucleic acids are not only quick and easy to carry out experimentally, but also leads to the highly efficient functionalization of the colloid with the protein. It is particularly noteworthy that the biological functionality of the Protein in no way by DNA-mediated adsorption on the colloid is affected. The experimental finding was particularly surprising that both the nucleic acid-coupled binding molecules and the nucleic acid coupled colloids an unexpectedly high physicochemical stability demonstrate. The physicochemical stability of the nucleic acid-coupled Components even allowed the proteins adsorbed via DNA hybridization cleave from the colloids or the colloids several times with DNA protein Coating conjugates (cf. embodiment 7).

Nucleic acids in the sense of the detection method are substances that one or several nucleobases (adenine, cytosine, guanine, thymine, uracil) or their functional analogs such as hypoxanthine. The nucleobases are preferably with a sugar, especially with a pentose, Pentopyranose or Pentofuranose linked. Are particularly preferred Ribose and deoxyribose. The sugars, in turn, are preferably over Phosphodiester bonds linked together. The term nucleic acid also includes so-called peptide nucleic acids, in which the sugar phosphate backbone through Amino acids linked by peptide bonds are replaced (PNAs, see, for example, E. Uhlmann, Biol. Chem. 1998, 1045-1052; P. Neilsen, Acc. Chem. Res, 1999 (32), 624-630), and pyranosyl nucleic acids such as p-RNAs (see, for example, M. Bolli, R. Micura, A. Eschenmoser, Chem. Biol. 1997 (4), 309-320).

The nucleic acids are each with the binding molecule or the surface of the colloidal particles connected in such a way that they differ from their respective Binding partners under the chosen process conditions essentially do not solve, that is, that while performing the procedure less than 25% of the bonds break down.

• a) Bereitstellung eines Konjugats, das aus der ersten Nucleinsäure und Streptavidin oder Avidin besteht;
• b) Bereitstellung eines Bindemoleküls, das mit einer Biotin-Gruppe modifiziert ist;
• c) Zusammenbringen des in Schritt i) beschriebenen Konjugates und des in Schritt k) beschriebenen Bindemoleküls, so dass es zur Kupplung dieser Komponenten kommt.

It is particularly preferred if the first nucleic acid and the binding molecule are linked such that step f) is carried out as follows:

• a) providing a conjugate consisting of the first nucleic acid and streptavidin or avidin;
• b) provision of a binding molecule which is modified with a biotin group;
• c) Bringing together the conjugate described in step i) and the binding molecule described in step k) so that these components are coupled.

Here, the first nucleic acid, for example as a biotin derivative, is initially included Streptavidin or avidin, or recombinantly or chemically modified derivatives linked these proteins and the resulting conjugate with the biotinylated Binding molecule mixed, creating a coupling between the first Nucleic acid and the binding molecule is reached. However, it is particularly preferred the use of covalent DNA-streptavidin conjugates (see on Example: Niemeyer, C.M., et al., Nucleic Acids Res 22 (25): 5530-9, 1994), which as Compound molecules can be used to make the biotinylated Linking the binding molecule with the first nucleic acid. Free Biotin Binding sites of streptavidin or avidin should then be added be saturated with biotin to avoid undesirable side reactions.

Instead of a biotin-streptavidin coupling of the substances involved (Binding molecules, nucleic acids) these can also be linked to one another in other ways get connected. In particular, they can have covalent bonds be connected to each other. The use of biotin streptavidin However, compounds is preferred because biotin and streptavidin are in high purity are available at a low price and in large quantities, linking the two Substances with a large number of substances, in particular nucleic acids and Immunoglobulins and proteins is easily possible and because the biotin streptavidin Binding very stable and easy to handle under normal process conditions is. The handling of biotin-streptavidin systems is described, among other things by Diamandis, E.P. and T.K. Christopoulos (1991) ("The biotin (strept) avidin system: prineiples and applications in biotechnology. "Clin Chem 37 (5): 625-36).

The colloidal particles can be linked to the second nucleic acid different ways are done. In particular, amino or thiol derivatized nucleic acids with corresponding ligand groups of the colloidal Components are covalently linked. The chemisorptive bond is preferred Thiol-modified nucleic acids on gold colloids as they become very stable Binding of the nucleic acid to the gold colloids leads and is easy to experiment is accomplished. Colloids from other materials can also be used chemisorption with amino or thiol derivatized nucleic acids functionalize. For example, nanoparticles made from CdTe can also be used initially coupled with an excess of thiolated DNA and then with DNA modified protein are functionalized (see embodiment 2).

The first and second nucleic acids can under the process conditions hybridize with each other. It is preferred if they are among the Process conditions cannot form internal loops, so if there are none there are two sections each within a nucleic acid that are stable with each other can hybridize. It is also preferred if the first and second Nucleic acid not stable under the chosen process conditions Form homodimers so that no two molecules of the first nucleic acid hybridize with each other stably and / or no two molecules of the second Hybridize nucleic acid stably.

The length of the first and second nucleic acids is preferably 5 to 50 Nucleotides, but can also be 50 to 500 nucleotides in length Base pairs. Here are single-stranded nucleic acid molecules particularly preferred, but one of the two nucleic acids can also be used as Double strand can be used, to which the other of the two nucleic acids then attach can bind to a specific sequence by forming a triple helix.

• a) einem oder mehreren Bindungsmolekülen, und dazu passend
• b) eine oder mehrere erste Nucleinsäuren,
• c) einem oder mehreren Arten kolloidaler Partikel, und dazu passend
• d) eine oder mehrere zweite Nucleinsäuren,
• e) Mittel zum Verbinden der Bindungsmoleküle mit jeweils einer der ersten Nucleinsäuren, bzw. Mittel zum Verbinden der kolloidalen Partikel mit jeweils einer der zweiten Nucleinsäuren, aus denen ein oder mehrere, zueinander komplementäre Paare von Bindungsmolekül/erste Nucleinsäure und Kolloidpartikel/zweite Nucleinsäure hergestellt werden können.

Further preferred is a kit for producing the protein functionalized colloidal particles by nucleic acid hybridization, comprising

• a) one or more binding molecules, and matching
• b) one or more first nucleic acids,
• c) one or more types of colloidal particles, and matching
• d) one or more second nucleic acids,
• e) means for connecting the binding molecules to one of the first nucleic acids, or means for connecting the colloidal particles to one of the second nucleic acids, from which one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid are produced can.

• a) eine oder mehrere erste Nucleinsäuren,
• b) einem oder mehreren Arten kolloidaler Partikel, und dazu passend
• c) eine oder mehrere zweite Nucleinsäuren,
• d) Mittel zum Verbinden von Bindungsmolekülen mit jeweils einer der ersten Nucleinsäuren, bzw. Mittel zum Verbinden der kolloidalen Partikel mit jeweils einer der zweiten Nucleinsäuren, aus denen ein oder mehrere, zueinander komplementäre Paare von Bindungsmolekül/erste Nucleinsäure und Kolloidpartikel/zweite Nucleinsäure hergestellt werden können.

Also preferred is a kit for producing the protein functionalized colloidal particles by nucleic acid hybridization

• a) one or more first nucleic acids,
• b) one or more types of colloidal particles, and matching
• c) one or more second nucleic acids,
• d) means for connecting binding molecules to one of the first nucleic acids, or means for connecting the colloidal particles to one of the second nucleic acids, from which one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid are produced can.

• a) eine oder mehrere erste Nucleinsäuren,
• b) eine oder mehrere zweite Nucleinsäuren,
• c) Mittel zum Verbinden von Bindungsmolekülen mit jeweils einer der ersten Nucleinsäuren, bzw. Mittel zum Verbinden kolloidaler Partikel mit jeweils einer der zweiten Nucleinsäuren, so dass ein oder mehrere, zueinander komplementäre Paare von Bindungsmolekül/erste Nucleinsäure und Kolloidpartikel/zweite Nucleinsäure hergestellt werden können.

Also preferred is a kit for producing the protein functionalized colloidal particles by nucleic acid hybridization

• a) one or more first nucleic acids,
• b) one or more second nucleic acids,
• c) means for connecting binding molecules with one of the first nucleic acids, or means for connecting colloidal particles with one of the second nucleic acids, so that one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid can be produced ,

With a compilation of substances in such an inventive Kit DNA-functionalized colloidal particles can be advantageously simple getting produced.

A third (partial) object of the present invention was an article provide with which the reagent kits for the easy-to-perform Detection of substances can be used. This (sub) task will solved in that the surface-bound signal complex in the space between one or more, electrically-conductive, arranged in pairs Electrodes is generated. This can take the form of, for example Microstructured interdigital switching is done at the anode and cathode Are interleaved like a comb, so that no electrical contact between the two poles. The area between the electrodes is now with a capture reagent that the analyte in the electrode Intermediate space immobilized. Then the invention Protein-DNA functionalized colloidal particles on the immobilized Coupled analytes and performed silver development. The secluded Silver mirror causes electrical contact between the electrodes recorded as a signal by means of current, voltage or resistance measurement can be.

The invention is described below with reference to the figures and some Exemplary embodiments described in more detail. They represent:

Fig. 1 shows a schematic representation of the production of protein-DNA-functionalized gold colloids.

Fig. 2 electrophoretic investigation of the DNA-mediated adsorption of proteins to gold colloids.

Fig. 3 is a schematic representation of the use of antibody / DNA-functionalized gold colloids as reagents in a sandwich immunoassay.

Fig. 4 experimental data on the use of antibody-functionalized gold colloids as reagents in a sandwich immunoassay.

Fig. 5 experimental data on the use of antibody-functionalized gold colloids for the analysis of protein microarrays.

Fig. 6 experimental data for use DNA-functionalized cadmium telluride (CdTe) nanoparticles.

Fig. 7 schematic representation of the electrical detection of the use of antibody / DNA-functionalized gold colloids as reagents in a sandwich immunoassay.

Fig. 1 shows schematically the course of the functionalization of colloidal particles (the example of gold colloids) by DNA-mediated adsorption of proteins. Gold colloids are modified with the 5'-thiol-modified oligonucleotide 1 by binding the oligonucleotides to the gold colloids by chemisorption (Au-1). The covalent DNA-streptavidin (STV) conjugates 4 a, b used as modular adapters are synthesized from streptavidin 3 and 5'-thiol-modified oligonucleotides 2 a, b according to known methods (see: Niemeyer et al., (2001 ) Colloid, Polym. Sci. 279, 68). The oligonucleotide 2 a is complementary to the gold colloid-bound oligomer 1 . The DNA-STV conjugate 4 a is coupled with biotinylated binding molecules, for example antibodies against IgG from mouse (aM-IgG) to conjugate 5 or with biotinylated antibodies against IgG from rabbits (aK-IgG) to 6. Antibody-STV-DNA conjugates 5 and 6 bind to 1-modified gold colloids (Au-1) by nucleic acid hybridization. To simplify matters, complementary DNA strands are shown as helices, the 3 'ends of which are marked by arrowheads.

With the help of the DNA-STV conjugates 4 , any antibody-STV-DNA conjugates (e.g. 5 or 6 ) can be produced according to the modular principle. Since the DNA sequence in 4 a (and thus also in 5 or 6 ) is complementary to the gold colloid-bound DNA 1 , any antibodies (or other proteins) can be linked to the colloidal particles. If a second DNA-STV conjugate is used, which was produced, for example, from oligonucleotide 2 b, a second batch of colloidal particles which has been modified with a nucleic acid complementary to 2 b can be coupled to proteins according to the same principle as Au-1 become. This procedure can be carried out with any number of colloidal particles, each modified with an individual DNA sequence, so that any number of particles with any proteins can be specifically functionalized according to the modular principle.

FIG. 2 shows the gel electrophoretic examination of the functionalization of colloidal particles shown in FIG. 1. A non-denaturing 1% agarose gel is shown. The black bands show the mobility of gold colloids with a diameter of 34 nanometers (Au ₃₄ -1 in lane 1 ). In lane 2 Au ₃₄ -1 is coupled with complementary DNA-STV conjugate 4 a (Au ₃₄ -4a), and in lane 3 Au ₃₄ -1 is mixed with non-complementary DNA-STV hybrid 4 b. The identical mobility of the colloids as seen in lane 1 shows that the functionalization of the gold particles is determined solely by the specificity of the nucleic acid hybridization. In lane 4 , Au ₃₄ -1 was coupled with complementary antibody-STV-DNA conjugate 5 . The reduced electrophoretic mobility of the band is caused by the higher molecular weight of the adsorbed antibody. Gold colloids were applied in lane 5 , which were first coupled with a complementary DNA-STV conjugate 4 a, and then the proteins were cleaved off again by treatment with NaOH. The electrophoretic mobility corresponds to that of Au ₃₄ -1 plotted in lane 1 . This experiment, in which no decomposition of the colloidal components is observed, shows the surprisingly high physicochemical stability of the DNA-protein-modified colloids. This stability even makes it possible to regenerate the Au ₃₄ -1 DNA colloids.

Fig. 3 shows the use shows schematically antibody DNA-functionalized colloids as reagents in the sandwich immunoassay. Mouse IgG, represented by black spheres, is immunosorptively bound to the solid phase as a model antigen by surface-immobilized capture antibodies and coupled with Au-5. Silver development is carried out for signal generation. The formation of the silver mirror can be monitored photometrically or by imaging with a camera or a flatbed scanner.

FIG. 4 shows the use of antibody-functionalized colloids, using the gold colloids Au ₃₄ -5 and Au ₁₃ -5 as an example, as reagents in a sandwich immunoassay, which was carried out as outlined in FIG. 3. In Fig. 4a the wells of a microtiter plate are shown which have, depending on the amount of the immobilized antigen, a different highly trained silver mirror. In Fig. 4b the dependence of nm by measuring the absorbance at 490 signal intensities obtained is shown by the amount of the antigen to be detected. The strength of the absorption at 490 nm corresponds to the intensity of the silver level formed. In order to record the absorption, the microtiter plate was measured in the course of silver development on a commercially available microtiter plate reader, with which the absorption of the individual cavities at 490 nm was determined. The black signal sets on the left were obtained with Au ₃₄ -5, the gray histograms on the right with Au ₁₃ -5. Only silver developers without other reagents were measured as controls.

The signal intensities obtained as a function of the amount of antigen show that specific detection of the antigen (mouse IgG) is possible with Au ₃₄ -5. FIG. 4c shows that the controls in which a mixture of Au ₃₄ -1 + 4b-αM-IgG coupled with biotinylated αM-IgG ("correct" antibody but "wrong", ie non-complementary, ie non-binding DNA- STV conjugate) or Au ₃₄ -6 ("wrong" antibody coupled to the gold colloids via STV) was used instead of Au ₃₄ -5, showed no significant silver development. The other controls in which STV-modified gold colloids without antibody (Au ₃₄ -1 + 4a), DNA-modified gold colloids and correct antibodies without STV bridge (Au ₃₄ -1 + αM-IgG), only DNA-modified gold Colloids (Au ₃₄ -1) were used, just like the control in which no antigen was immobilized, only show absorption values that correspond to that of the pure Ag developer reagent.

Analysis of a serial dilution allowed the detection of less than 10 fmol of the antigen. The detection range of the method thus corresponds approximately to that of a conventional enzyme-enhanced immunoassay. The detection sensitivity can be optimized by using colloids of different sizes in the sandwich immunoassay, as shown in FIG. 4b (Au ₁₃ -5, gray histograms). On the one hand, these results show that the modular process for the functionalization of nanoparticles can be transferred directly to other colloids. On the other hand, a significant increase in signal intensities can be achieved with Au ₁₃ -5.

In Fig. 5 experimental data for use of antibody the functionalized gold colloids in the analysis of protein microarrays are shown. The proteins were applied to the chemically activated slide in 5 fields and covalently immobilized. The fields were then overlaid with solutions of Au-5. Unbound Au-5 was removed after incubation by washing the slide several times with wash buffer. Finally, the slide was placed in silver development reagent and, after the silver mirrors had been formed, scanned on a flatbed scanner. The dark spots show the presence of immobilized proteins on the glass slide. The specificity of the detection is shown by staining only the spots in which antigen is immobilized on the glass. The surrounding areas, in which only blocker proteins are bound (see here embodiment 10), on the other hand, remain uncolored.

In FIG. 6 experimental data for use are shown in the analysis of DNA microarrays DNA functionalised cadmium telluride (CdTe) nanoparticles. Catcher oligonucleotides were applied to a chemically activated slide and covalently immobilized. Then the slide was overlaid with solutions of CdTe-1. Unbound CdTe-1 was removed by washing the slide several times and the slide. was then measured in the fluorescence scanner at 532 nm. The light dots show the presence of immobilized CdTe. The specificity of the detection is shown by the fact that light is emitted only from those spots in which binding molecules are immobilized on the glass. The surrounding areas, in which only blocker proteins are bound, show no fluorescence.

Fig. 7 shows schematically the use of antibody-DNA functionalized colloids as reagents for the electrical detection of a sandwich immunoassay. The surface-bound signal complex is generated in the space between a pair of electrically conductive electrodes (a → b). As shown in a), the electrodes can, for example, be in the form of a microstructured interdigital circuit in which the anode and cathode are interleaved in a comb-like manner, so that there is no electrical contact between the two poles. The area between the electrodes is now functionalized with a capture antibody, which immobilizes the analyte in the gap between the electrodes. The protein-DNA-functionalized colloidal particles according to the invention are then coupled to the immobilized antigen (b → c) and silver development is carried out (c → d). The deposited silver mirror in d) causes an electrical contact between the electrodes, which can be recorded as a signal by means of current, voltage or resistance measurement.

embodiments 1. Production of DNA-Coupled Gold Colloids

The synthesis and purification of DNA-coupled gold colloids is carried out according to methods known from the literature, such as e.g. B. in the publication by Storhoff et al. (James J. Storhoff, Robert Elghanian, Robert C. Mucuc, Chad A. Mirkin; (1998), "One-Pot Colorimetric Differentiation of Polynucleotides with Single Base Imperfections Using Gold Nanoparticle Probes" J. Am. Chem. Soc. 120, 1959-1964). Typically, the two gold colloid solutions (ICN Biomedicals GmbH) of different sizes to be used for the DNA coupling are first analyzed by means of transmission electron microscopy in order to determine their actual size. According to this, the average size for the 40 nm colloids was 34.2 ± 2.7 nm (Au ₃₄ ) and 13.2 ± 0.6 nm (Au ₁₃ ) for the 15 nm colloids. For the coupling of the gold colloids Au ₃₄ or Au ₁₃ with DNA molecules to Au _{34 (13)} -1, 2 ml of the colloidal gold solution Au ₃₄ (0.25 nM) or Au ₁₃ (2.01 nM) with 1 ml of a 5'-thiol-modified oligonucleotide solution of the sequence "A" (interactives, 20 μM in dH ₂ O) combined and then purified as described in the above literature. The DNA-coupled colloids Au _{34 (13)} -1 'are quantified photometrically after their purification. The DNA with the designation "A" used for the colloid coupling has the sequence: 5'-thiol-TCC TGT GTG AAA TTG TTA TCC GCT-3 '.

2. Production of DNA-Coupled Cadmium Telluride (CdTe) Particles

For the coupling of the CdTe particles with DNA molecules, 50 µl one CdTe solution (75 µM), stabilized with thioglycolic acid, used and with 750 µl (40 µM in borate buffer, pH = 8.5) of a 5 'thiol-modified Oligonucleotides of sequence "A" mixed. The purification of the DNA coupled CdTe cluster (CdTe-1) was carried out in accordance with the example 1 cited literature. The DNA used for the CdTe coupling with the name "A" has the sequence: 5'-thiol-TCC TGT GTG AAA TTG TTA TCC GCT-3 '.

3. Covalent conjugates 4 from streptavidin and first nucleic acid

To couple the biotinylated antibody with the nucleic acid sequence "A" (see FIG. 1), a covalent conjugate " 4 a" was first prepared from STV and a thiolated nucleic acid "cA" (cf. FIG. 1). The synthesis of this conjugate using a heterobifunctional chemical crosslinker is described in detail in the literature (Niemeyer, CM, T. Sano, CL Smith and CR Cantor (1994), "Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA-streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates. "Nucleic Acids Res 22 (25): 5530-9). In short, a commercially available 5'-thiol-modified oligonucleotide (Interactiva) was linked to a maleimide-activated primary amino group of the STV using sulfo-SMCC (Pierce). The purified product 4 a obtained after subsequent ion exchange chromatography purification on an FPLC (Pharmacia) can usually be stored in TE at 4 ° C. for several months. The sequence of the oligonucleotide (interactives) for the synthesis of the DNA-STV hybrid " 4 a" is: STV-5'-AGC GGA TAA CAA TTT CAC ACA GGA-3 'The DNA sequence of the non-complementary DNA-STV hybrid " 4 b "reads: STV-5'-CCG GGT ACC GAG CTC GAA TTC-3 '.

4. Coupling of the DNA-STV conjugates 4 with biotinylated proteins

To produce DNA-STV-protein conjugates, typically equimolar amounts of DNA-STV hybrids and biotinylated protein are combined. For example, to produce the DNA-STV antibodies, conjugates 5 and 5 b (hybrid-antibody conjugate with wrong sequence) each 150 ul of a 28 nM solution of the DNA-STV hybrid ( 4 a and 4 b) in TETBS buffer mixed with 150 ul of a solution of biotinylated goat anti-mouse IgG (Sigma) in TETBS and the solutions incubated for 10 min. The DNA-STV-antibody conjugate 6 is produced in the same way from biotinylated goat anti-rabbit IgG (Coulter Immunotech) and 4a.

5. Coupling of STV-DNA-antibody conjugates with DNA-modified gold colloids

For coupling the in point 4 . prepared DNA-STV antibodies conjugates 5 and 6 with Au-1 (see FIG. 1) 275 µl of conjugates 5 and 6 (14 nM each) with the same volume of a 0.14 nM solution of Au ₃₄ - 1 or the same volume of a 1.16 nM solution of Au ₁₃ -1 mixed and incubated for one hour. The products Au ₃₄ -5 and Au ₁₃ -5 or Au ₃₄ -6 and Au ₁₃ -6 are used in the sandwich immunoassays without further purification.

6. Gel electrophoresis of protein-DNA modified gold colloids

A 1% agarose gel is produced to characterize the products. Then 10 µl of a solution of Au ₃₄ -5 with a colloid concentration of 0.07 nM are mixed with 2 µl of a gel electrophoresis buffer and applied to the gel. As a control, equal volumes of the solutions Au ₃₄ -1, Au ₃₄ -1 with complementary DNA-STV hybrid 4 a without biotinylated antibody (Au ₃₄ -1 + 4 a) and Au ₃₄ -1 with non-complementary DNA-STV hybrid (Au ₃₄ -1 + 4 b) the same colloid concentration mixed with 2 µl of a gel electrophoresis buffer and applied to the gel. The gel electrophoresis is carried out at 120 volts for 30 min.

7. Regeneration of the protein-DNA-modified gold colloids

To separate the DNA-STV hybrid 4 a from the DNA-coupled colloids Au-1, 80 µl of Au ₃₄ -1 + 4 a (0.07 nM) are mixed with 80 µl of a 100 mM NaOH solution and incubated for 4 hours. The solution is then centrifuged at 14,000 rpm for 3 min. The supernatant is removed and the red pellet is taken up again in 100 nM NaOH solution. This process is repeated twice. After three centrifugation / resuspension steps, the colloids are applied to a gel filtration column (NAP ™ -5, Pharmacia Biotech) and eluted with TETBS buffer. Then the solution (approx. 300 µl) is centrifuged again and the colloids are resuspended with 40 µl TETBS buffer.

8. Use of protein-DNA-modified gold colloids in sandwich immunoassay

To carry out the sandwich immunoassay, microtiter plates are coated with 50 μl of a 20 nM solution of goat anti-mouse IgG (Sigma) and blocked with milk powder against non-specific binding. 50 µl of a solution containing the mouse IgG antigen in different amounts are added to the wells of the plate, incubated for 45 min, and the plate is then washed. Coupling with Au-5 takes place by adding 50 µl of a 0.07 nM solution of Au ₃₄ -5 or a 0.58 nM solution of Au ₁₃ -5 and incubation for 45 minutes. After washing, 50 μl of a silver development kit (BioRad) were added to the cavities and the development of the silver level was monitored photometrically at 490 nm or by imaging on a flatbed scanner.

9. Detection of protein-DNA-modified gold colloids by silver development

To develop silver on the bound gold colloids, the cavities are first washed twice with 100 μl dH ₂ O. Then 100 µl of fixer solution are added (Biorad: 50% ethanol, 10% acetic acid, 10% fixer, 30% dH ₂ O) and incubated for 10 min. After washing twice with dH ₂ O, 50 μl of silver developer (organic wheel) are added to the cavities and incubated with shaking. The microtiter plate is placed in the microtiter plate reader (Wallac) at 5 minute intervals and the absorbance of the silver deposited in the cavities is measured at 490 nm.

10. Use of the protein-DNA-modified gold colloids for the analysis of Protein microarrays

To analyze a protein array, 200 nl of a solution of a goat anti-mouse IgG capture antibody in two different concentrations (5 μM and 0 .mu.m) are first placed on a silanized glass slide which has been chemically activated with phenylene di-isothiocyanate. 5 µM) alternately added to five identical arrays and incubated for two hours. The slide, on which the arrays are framed with a water-repellent immuno-staining pen (G. Kisker Biotechnologie), is placed in a 50 mM aminohexanol solution with 150 mM di-isopropylamine in dimethylformamide (DMF) to erase the amino-reactive surface immersed, and then washed three times with dH ₂ O and three times with TETBS buffer and immersed in a TETBS buffer, each containing 0.1 mg / ml herring sperm DNA and bovine serum albumin and to avoid non-specific antigen or protein DNA - Modified gold colloid binding is used as a blocker. The slide is then washed with TETBS buffer, the arrays are mixed with 1 µmol mouse IgG antigen in 50 µl borate buffer (pH = 9.5) and incubated for 45 min. In the next step, the unbound mouse IgG antigen is first removed from the slide by washing several times and a 0.07 nM solution of Au ₃₄ -5 (50 μl per array) is added. After 45 minutes of incubation, the slide is first washed three times with TETBS buffer, then twice with dH ₂ O and then immersed in a fixing solution (bio wheel) for 10 minutes. The slide is then washed three times for 1 min with dH ₂ O and the silver deposition on the bound gold colloids on the slide is started by adding 50 µl of silver developer (Biorad). After a clear silver deposition can be seen, the silver developer is discarded, the slide is first washed three times for 1 min with dH ₂ O and then air-dried. The slide is then scanned on a flatbed scanner.

Claims (12)

1. A method for the detection of a substance in aqueous solution, comprising the following steps: a) provision of a surface which has a specific binding capacity for the substance to be detected; b) provision of a binding molecule which has a specific binding ability for the substance to be detected; c) Linking the binding molecule with colloidal particles so that the particles have a specific binding ability for the substance to be detected; d) bringing together the surface, the substance to be detected and the binding molecule-linked colloidal particles so that a surface-bound signal complex is formed; e) detection of the colloidal particles, insofar as they are present in a surface-bound signal complex. 2. The method according to claim 1, in which step c) is carried out as follows: a) Linking the binding molecule with a first nucleic acid; b) linking the colloidal particles with a second nucleic acid which is complementary to the first nucleic acid; c) bringing the nucleic acid-modified binding molecule together with the nucleic acid-modified colloidal particles so that the first nucleic acid hybridizes with the second. 3. The method according to claim 1-2, in which step f) is carried out as follows: a) providing a conjugate consisting of the first nucleic acid and streptavidin or avidin; b) provision of a binding molecule which is modified with a biotin group; c) Bringing together the conjugate described in step i) and the binding molecule described in step k) so that these components are coupled. 4. The method according to any one of the preceding claims, characterized in that that to detect the colloidal particles this by silver development be made visible. 5. The method according to any one of the preceding claims, characterized in that it is the surface provided under step a)
a sensor or sensor element,
an array such as B. a DNA (micro) array, a protein (micro) array,
a test array carrying peptides, peptoids or small molecules such as pharmacophores,
a component of a (micro) reaction vessel or
is an optically or electronically active element. 6. The method according to any one of the preceding claims, characterized in that the surface-bound signal complex in the space between one or several pairs of electrically conductive electrodes is built up so that to detect the colloidal particles, they are subjected to silver development be that leads to the deposition of a silver mirror and thus an electrical Contact between the electrodes is established, which is caused by current, voltage or resistance measurement can be recorded as a signal. 7. Articles which are used to carry out the method according to one of the preceding claims as reagents, characterized in that a) colloidal particles are linked to a second nucleic acid, b) one or more binding molecules are linked to a first nucleic acid complementary to the second nucleic acid, c) the first and the second nucleic acid are linked together 8. Article according to claim 7, characterized in that the first Nucleic acid via biotin and streptavidin or avidin with the binding molecule is linked. 9. Kit for producing the article according to claim 7 to 8, consisting of a) one or more binding molecules, and matching b) one or more first nucleic acids, c) one or more types of colloidal particles, and matching d) one or more second nucleic acids, e) means for connecting the binding molecules to one of the first nucleic acids, or means for connecting the colloidal particles to one of the second nucleic acids, from which one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid are produced can. 10. Kit for producing the article according to claim 7 to 8, consisting of a) one or more first nucleic acids, b) one or more types of colloidal particles, and matching c) one or more second nucleic acids, d) means for connecting binding molecules with one of the first nucleic acids, or means for connecting the colloidal particles with one of the second nucleic acids, from which one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid are produced can. 11. Kit for producing the article according to claim 7 to 8, consisting of a) one or more first nucleic acids, b) one or more second nucleic acids, c) means for connecting binding molecules with one of the first nucleic acids, or means for connecting colloidal particles with one of the second nucleic acids, so that one or more mutually complementary pairs of binding molecule / first nucleic acid and colloidal particles / second nucleic acid can be produced , 12. Use of the article according to claim 7 to 8, in analytical processes based on the detection of colloids by surface plasmon resonance, Reflectometric interference spectroscopy, surface-enhanced Raman spectroscopy, electrical detection using impedance measurement, or mass-sensitive methods such as the quartz crystal microbalance or the Surface acoustic wave measurement based.