WO2003066884A2 - Method for labeling articles - Google Patents

Abstract

The invention relates to a method for proving the authenticity of an article. The article, on one of its surfaces, has a section on which partners of a specifically binding pair are bound in a predetermined arrangement. The surface is contacted with a second surface that has corresponding sections on which, in at least selected areas, complementary binding partners are disposed, in such a manner that the partners react with one another, at least a part of the partners carrying a detectable label. The surfaces are then separated and the arrangement of labels so produced on at least one of the two surfaces is detected. After separation, the arrangement of partners and/or labels produced on at least one of the two surfaces differs from the original arrangement of the bound partners and/or of the labels on the surface of the article.

Description

 Process for marking objects

description

The invention relates to a method for verifying the authenticity of an object.

In many areas it is necessary or desirable to determine the authenticity of an item or to follow its path. There is an interest in identifying high-quality or safety-relevant products in order to distinguish them from imitations. Many methods are known for this, for example serial numbers, barcodes, magnetic strips, stamps, reflective foils or threads, holographies and similar identifying means. On the other hand, it is necessary to make documents such as banknotes, ID cards, cards etc. tamper-proof. There is also an interest in tracking the path of products or pollutants through distribution or sales networks. In all of these cases, it is necessary to put a mark on the products that clearly shows whether it is an original product or an imitation or counterfeit. In general, the product used for product labeling must offer the security ^" that it cannot be counterfeited.

A wide range of options have already been proposed for product labeling, which also use the specific binding of biological molecules.

For example, WO87 / 06383A1 discloses a method for marking objects, in which a macromolecular compound referred to as a signal compound is applied to the object without revealing its identity, and the presence of this signal compound is then detected. A DNA strand from a bacterium that is placed on or in the object is proposed as the signal connection. A hybridization with a complementary DNA strand is then carried out for detection, and the successful hybridization confirms the authenticity of the object. The teaching of WO01 / 51652A2 follows a similar approach, according to which a biopolymer is immobilized on an object and then made visible in a one-step detection process by reaction of the biopolymer with a second biopolymer capable of binding with it.

Furthermore, WO99 / 47702A2 describes a method for identifying a solid, in which a nucleotide sequence is immobilized on a solid and is hybridized for detection with a second nucleotide sequence, wherein when the second nucleotide sequence binds to the first nucleotide sequence, one bound to the second nucleotide sequence fluorophoric group is either amplified or deleted, resulting in a detectable change in fluorescence upon binding.

Furthermore, EP 0 409 842B1 proposes a method for identifying the origin of a chemical or chemical composition, in which a marker compound is added to the chemical or composition and the presence of the marker compound in this chemical or composition is then detected. According to EP 0 409 842B1, several marker compounds can also be used, the analysis being carried out in that the marker compounds are then extracted from or from the chemical and e.g. be separated on a chromatography column.

A similar approach is also followed in WO98 / 33162A1, where a marker is used which is only physically detectable in certain concentrations. This marker is added to the product in undetectable concentration and then separated, enriched and detected using an affinity column.

The teaching of document WO98 / 28728A1 deals with the problem of proving whether a container which is provided with a seal label is still closed in the original or has already been opened. For this purpose, a sealing label is provided, which is designed such that predetermined parts become detached when opened, while other places remain adhering, as a result of which a lower colored one is attached Layer changed their appearance. This means that the parts of the label can no longer be restored to their original state.

The inventor of WO00 / 77496A1 takes a different path. Here, a method for identifying an object is proposed, in which the reflection of electromagnetic waves is used as a means of detection, the reflection being influenced by the reaction of two polymers with one another, the one polymer being bound via metallic clusters.

Furthermore, US Pat. No. 5,445,970A discloses a method for carrying out binding assays, in which a magnetic material is used as the detectable marking. The degree of association of interaction partners, e.g. Antibodies can be measured by an external tensile force.

Many of these known methods have in common that a specifically bindable substance is attached to or added to the object to be identified, and later the presence of this substance is then detected. The disadvantage of the known methods, however, is that the possibilities for variation are limited and that as soon as the substance used is known, the product marking can be imitated. In the cases in which the use of several marker molecules is proposed, their detection is complex since the labels have to be separated using chromatography columns.

The object of the invention was therefore to provide a method with which objects can be marked securely, a large variety of codes being possible, so that protection against counterfeiting is achieved in practice. In addition, it was an object of the invention to provide a method using several or many different markings and / or codes, without it being necessary to use chromatography columns or other devices for the separation. The ability of biological molecules to form specific bonds with specific binding partners is used to solve this problem. In preferred embodiments, the possibilities that a differential force test offers, which can compare and evaluate binding forces, are used.

According to the invention, a method for proving the authenticity of an object is provided, the object having at least one surface section on a surface on which partners of a specifically binding pair are bound in a predetermined arrangement, the surface with a second surface which has corresponding surface sections , on which complementary binding partners are present, at least in selected areas, is brought into contact in such a way that the partners can react with one another, at least one of the two partners bearing a detectable marking, the surfaces then being separated and the resulting arrangement of the Markings is detected, the arrangement of the markings that has arisen after separation on at least one of the two surfaces from the original arrangement of the bound partner and / or markings on the surface of the object ands differs.

With the method according to the invention, it is possible to identify objects by generating detectable patterns which only arise when two different surfaces, one of them on the object to be identified or connected to it, are brought into contact with one another and then separated. The pattern is generated by binding corresponding partners of a specific binding pair on the two surfaces, at least one of which is provided with a detectable marking.

According to the invention, a counterfeit-proof system is thus made available. In general, it is almost impossible to prove the pattern provided on the object to be authenticated, since very little material is available for an analysis and it is not known which binding partners are used. Even if the pattern present on the object could be detected, this would not lead to knowledge of the pattern created after the separation.

Essential for the method according to the invention are therefore two specifically binding partners, at least one of which bears a marking, and the arrangement of the respective partners on the surface. It is also essential that at least some of the binding partners and / or their marking or their position change due to the binding with the respective complementary partner, i.e. due to the bond with the complementary partner, some of the binding partners or parts thereof have to change location or the marking has to change demonstrably.

For authentication, all objects are suitable which have a surface on which either a marking can be applied or on which a marking can be applied. Packaging is also suitable for authentication. Objects that are to be protected against imitation, as stated above, include expensive products such as watches, security-related products such as spare parts for airplanes, documents such as ID cards and credit cards. It is only important for the method according to the invention that a surface section is available on the object on which the binding partners can be applied. It should preferably be a flat surface section, although non-flat surfaces are also suitable for the method.

Two surfaces are necessary for carrying out the method according to the invention, on or on which partners are located. One surface is on the object whose authenticity is to be proven. Depending on the object to be identified, the surface can be formed directly by this object or, for example, as a layer, coating, Sticker or label to be applied to the item. The second surface is formed on a carrier separately from the first surface. Both surfaces must be designed in such a way that they can accommodate and hold the partners of the specific binding pair. In addition, the surfaces must be such that the binding partners can be brought into close contact at least in one surface section.

The two surfaces that are to be brought into contact need not necessarily be of the same area. The only requirement for carrying out the method according to the invention is that there is at least one surface section on each of the two surfaces in which binding partners can meet. Surface sections that can meet and have binding partners that can react with each other are referred to here as "corresponding surface sections". These surface sections can have any possible geometry, i.e. it can be flat triangles, squares, rectangles, circles, other flat surfaces or also curved or three-dimensionally shaped surfaces which have the same contours in at least one section or are suitable by their nature to be brought into contact with one another. So one of the two surfaces or both surfaces can be of such a material or have a shape that they can adapt, e.g. one of the surfaces may be in the form of a roller or a film, or the material for the surface may be soft or rubbery. It can also be a gel-like coating.

For example, there may be a surface section with specifically bindable partners arranged in a pattern on the object whose authenticity is to be proven and the second surface only cover a small section thereof. Even if the pattern is visible or detectable on the first surface, the area in which the second surface provides binding partners cannot be predicted. This also increases the security against imitation. The material from which the two surfaces are made can be the same or different and one or both of the surfaces can be coated in a suitable manner. Either one surface is preferably made of a rigid material and the other one is made of an elastic material, or both surfaces are made of elastic material, so that the respective surface sections of the first and second surfaces can adapt exactly to one another when they are brought into contact, and thus optimal contact of the individual binding partners is possible is. The surfaces can be formed, for example, from glass, polydimethylsiloxane (PDMS), nylon, polystyrene or other plastics. At least one of the two surfaces is preferably produced from an elastic material, preferably an elastic plastic. At least one surface is particularly preferably formed from a siloxane, in particular polydimethylsiloxane. Various other flexible materials or mixtures thereof are possible. Another possible material is polyacrylic amide gel, the elastic properties of which can be adapted to the experimental requirements by means of the molecular weight and the degree of crosslinking. The surface can be constructed from a single material, a mixture of materials or also a system of elements from one or different materials.

In one embodiment, the first surface is formed on the article itself. However, it is also possible to apply a layer or coating to the object. It is also possible to provide a solid phase made of glass that is derivatized to provide binding groups for the immobilization of a partner. Silanized glass can be mentioned here as an example. This solid phase can then e.g. be placed on the packaging of the item.

Binding partners are bound on the two surfaces in predetermined arrangements. The arrangement of the binding partners on their respective surfaces can be the same or different. So both surfaces can only have corresponding surface sections or not each other have congruently covering surface sections in which binding partners are bound in each case. The arrangement of the binding partners does not have to be congruent in the congruent surface sections, but can be different. It may be that, for example, a binding partner is not opposed to a binding partner or a partner that is not specifically binding.

In a further embodiment, one of the two surfaces, as a rule the surface that is not on the object, can carry such a coating that diffusion of binding partners is possible, areas being separated in a grid-like manner. In this case, the contact of the specifically bindable partners is made possible by bringing the two surfaces together so that the binding partner present in the surface layer can migrate to the other surface and meet the complementary binding partner there. For this embodiment, one of the two surfaces is preferably coated with a gel.

At least some of the binding partners have at least one of the two binding partners provided with a label. These markings in turn form patterns. The patterns formed by the markings can be varied, there are no limits to the imagination here. It is only essential to the invention that the pattern present on the respective surfaces before the surfaces are brought into contact and the pattern resulting on the surfaces after the bringing into contact and separation are different from one another. Pattern is not understood to mean the arrangement of the binding partners, but the arrangement of the markings. The arrangement of the markings offers many variation options.

Further options for variation also result from the selection of the marking. Any analytically detectable group or substance can be used as a marker for the method according to the invention. In the context of the invention, labeling is understood to mean a property by which some binding partners differ from others and which is detectable. Physically detectable parameters are preferably used. In particular, radioactive atoms or groups which bring about a change in optical or electrical properties come into consideration for the marking. All reporter groups known to the person skilled in the art are suitable here. Examples are radioactive markers such as ³ H, ¹⁴ C, ³⁵ S, fluorescent, luminescent, chromophore groups or dyes, metals or conductive groups, but also substrates of enzymes or reporter enzymes. Fluorescent or chromophoric groups are preferably used as the label. When proteins are used as binding partners, it is also possible to stain the protein substance bound to the surface, for example with silver or with Coomas-blue.

At least one of the two specifically binding partners has a label. The label is preferably a fluorescent dye, for example fluorescein isothiocyanate (FITC), fluorescein, rhodamine, tetramethylrhodamine 5 (and 6) isothiocyanate (TRITC), Texas Red, cyanine dyes (CY3 or CY5), etc. Fluorescent dyes are advantageous because they can be detected in very small amounts. As a rule, the label is covalently linked to the binding partner.

It is also possible to use several markings per specific binding pair and different markings can be used for the same binding partner, for example partners of different binding pairs or eg the two partners of a pair can be marked differently. In the latter case, for example, a pattern could be generated for the case of fluorescent markings, a "mixed color" being formed at locations where specific binding has taken place, while the pure color is retained at locations where there is only one binding partner. This also extends the possibilities for variations. When using different fluorescent markings, those can be used which are each excited by light of the same wavelength or by radiation of different wavelengths. It is essential for the invention that the arrangement of the markings changes as a result of bringing the specifically binding partners into contact and their binding. This can be done, for example, by changing the marking during the binding, by at least changing the location of the parts of a binding partner that carry the marking, or by the specific binding of the binding partner leaving the surface on which it is immobilized. The arrangement of the markings preferably changes in that some of the binding partners are released from their immobilization site by entering into the specific binding.

In a particularly preferred embodiment of the present invention, a comparison of the binding forces of different bonds is used, as described in the applicant's application, PCT / EP01 / 09206, entitled "Method and device for characterizing and / or detecting a binding complex" in order to remove one of two binding partners of a specific binding pair from the surface to which it is initially bound. This can be both the binding partner immobilized on the one to be authenticated and on the second surface.

A wide variety of partners capable of binding to one another can be considered as specifically bindable pairs, the specificity of the binding being able to relate both to a specific partner, such as, for example, antigen-associated antibodies or biotin-avidin / streptavidin, and to a group or class of compounds, eg antibody-protein A. Examples of specifically binding partners are: antibodies, peptides, other scaffold proteins, DNA, RNA, RNA aptamers and thus the pairs of antigen-antibody, hapten-antibody, anti-idiotype-antibody-antibody, DNA -DNA, RNA-RNA, DNA-RNA, RNA aptamer peptide, receptor ligand, lectin sugar, zinc finger protein DNA, enzyme substrate, biotin-avidin / streptavidin etc., as well as the respective derivatives or analogues. So instead of antibodies their bindable fragments and instead of DNA nucleic acid derivatives etc. can be used. In one embodiment, antibodies and a substance with an epitope recognized by it, such as antigens, haptens or anti-idiotype antibodies, are used as the binding pair. In the present description, the term “antibody” is also understood to mean antibody fragments or antibody derivatives, as well as functional fragments of antibodies or derivatives thereof, which can recognize and bind the epitope. The antibodies can be polyclonal or monoclonal antibodies, but monoclonal antibodies are preferred. Known fragments and derivatives are Fv, Fab, Fab 'or F (ab') ₂ fragments, "single-chain antibody fragments", bispecific antibodies, chimeric antibodies, humanized antibodies and fragments which determine CDRs (complementarity) regions) that recognize an epitope of the binding partner.

In a particularly preferred embodiment, nucleic acids such as DNA (deoxyribonucleic acid) or RNA (ribonucleic acid) and artificial nucleic acids such as LNA (Locked Nucleic Acid) and PNA (peptide nucleic acid) and three-dimensional structures of nucleic acids, preferably aptamers, are used as partners of the specifically binding pair ,

The binding partners, for example the antibodies, fragments or derivatives thereof or nucleic acids, can be immobilized in a manner known per se, both covalent and non-covalent bonds and bonds directly to the surface or via bridge molecules are considered. One of the two partners of the specific binding pair is preferably permanently immobilized and the other is bound in such a way that the binding to the surface opens as soon as the binding to the binding partner takes place. In the context of the present invention, a permanent bond is understood to mean a bond which remains essentially stable before the surfaces are brought into contact and does not become detached even when the binding partner binds to its specific counterpart. Permanent bonding can take place, for example, via functional groups which the binding partner has or which have been introduced into the molecule to functional groups which are provided by the surface. According to the present invention, it is preferred that at least one of the two partners of the specifically bindable pair does not bind directly to the surface, but rather either via bridge molecules which are immobilized on the surface or via a further specifically binding pair.

A preferred method is to biotinylate a partner and to connect it to the likewise biotinylated surface via a streptavidin molecule. Monoclonal antibodies can be activated chemically by oxidizing certain groups of their glycosylations to aldehyde groups. These aldehyde groups can in turn bind amino groups or hydrazide groups on a modified surface (see Solomon et al., Journal of Chromatographie, 1990, Vol. 510, 321-329). Another method which is known to the person skilled in the art is the conjugation of amino groups of the antibody with carboxy groups of a surface by using ethyl- (dimethylamino) -carbodiimide / N-hydroxy-succinimide.

In a preferred embodiment of the method according to the invention for verifying the authenticity of an object, one of the partners is bound to the surface via an adhesive bond and the other is bound via a detector bond, the binding force of the detector bond being less than the binding force of the specific one when the tensile force is applied from the outside Binding of the two partners and less than the binding force of the adhesive bond.

In this embodiment, the two partners are bound in such a way that only one of the two partners is bound to a surface via a bond referred to here as adhesive bond, while the other associated partner is bound to the other surface via a bond referred to here as detector binding.

"Adhesive bond" is a form of permanent bond and means that the bond of the partner to the surface is so strong that with a pull exerted on both partners connected via the specific bond, the adhesive bond bond is not opened and the partner remains immobilized, that instead the detector bond loosens and that the adhesive bond is not easily released under the application conditions.

"Detector binding" is understood here to mean a binding whose binding affinity is large enough to keep the other partner of the specific binding pair bound to the surface until contact is made with the other partner, but whose binding force is caused by an externally applied pull is weaker than the force of the specific bond between the partners and the force of the adhesive bond with which the other partner is bound, so that if a pull is exerted on both partners connected via the specific bond, the detector bond is released and thus the partner bound via the detector bond remains attached to the partner bound via the adhesive bond and is detached from the surface. The detector binding is preferably designed in such a way that under the test conditions it is stronger under an applied force than the non-specific binding to a corresponding surface, which may be suitably prepared, so that the partner immobilized via the detector binding remains bound to "its" surface, if he does not meet a specifically bindable partner.

In the context of the invention, “binding partner” or partner of a specifically binding couple is generally understood to mean a single partner of a couple. Many identical partners, a number of both partners of a binding pair, or different partners of different pairs can be bound on a surface section.

In the case of a force comparison, as is used in this embodiment, the magnitude of the force can depend on the pulling rate. In this case, the rate of the tensile force applied can be an important parameter under certain circumstances, since the separating forces can vary at different force rates. In these cases, in order to be able to compare the forces, the pull rate should be taken into account. In other embodiments, for example the one described below In contrast, the "Unzip" systems, the pull rate is not important under the conditions described.

Thus, when two surfaces are brought into contact, e.g. Partners of a specific binding pair are immobilized on one surface by adhesive bonding, while partners on the other surface are bound by detector binding, and the partners have the opportunity to form the specific bond with one another, which is made possible by the contacting, so occurs when the surfaces are separated the three existing bonds - adhesive bond, specific bond, detector bond - one pull and the weakest of the three bonds under the applied force - the detector bond - is separated. The adhesive bond and the specific bond remain. As a result, the partner immobilized via a detector bond, when he meets his binding partner, moves with him with and onto the other surface.

Since at least one of the two partners of a specific binding pair usually bears a label, after separating the two surfaces for couples that have been subjected to the force test, the marking on the surface to which a partner is bonded via the adhesive bond is during on the surface to which a partner was bound by a detector bond and this partner met a complementary partner, the marking has disappeared. Both cases can be evaluated for the method according to the invention, i.e. the marking can be determined as well as its absence or its change can be determined.

In this way it is possible to generate a large number of patterns, which can also be varied in a simple manner, so that it is impossible to imitate the patterns. Since the nature of the binding partners as well as their position and number as well as the nature of their respective binding can be varied, it is impossible to find out what the pattern created after contacting and separating the two surfaces will look like, even if the pattern is on one of the two surfaces is known. As well as the type of marking the type of binding is essential for the resulting pattern cannot be derived from the knowledge of the partners bound on one surface, how this pattern is changed and / or supplemented by contact with the second surface and the subsequent separation of the two. In addition, not only can the type, number, and position of the binding partner and marker and the type of binding be varied as desired, but it is also possible to provide different binding partners for a binding partner of a binding pair, for example different antibodies on the one hand and on the other hand Protein A etc. The distribution of the label (s) among the binding partners can also be varied.

In the method according to the invention, it is possible to vary the type, position and number of markings as well as the position and number of binding partners as well as the type of binding as well as the presence or not of a binding partner. Different patterns are therefore obtained, depending on whether or where the adhesive bond is provided on the first or second surface for a binding pair, depending on which marking is used at which location or on which binding partner, depending on how many different partners on which Depending on how many different markings are used and whether there is a binding partner for a partner at the corresponding position on the other surface. For example, a pattern can be formed on the object to be authenticated, which is then changed or supplemented after it has been brought into contact and then separated. It is also possible that the surface section which is used to identify the object is uniformly provided with partners of a specifically binding pair, who either all have a marking or which are all unmarked, and a pattern only results after contacting and separation. A pattern can be created on one of the two surfaces or on both. The resulting pattern can be, for example, a logo, a trademark, a note, etc.

In a preferred embodiment, which is explained in more detail below, one of the two binding partners is connected via a further specific binding pair, particularly preferably immobilized on a surface via complementary nucleic acid strands.

According to the invention, it is preferred that not only one type of partner of a binding pair, but either both types of partners or even partners of different binding pairs are bound to both surfaces, in particular the surface section arranged on the object.

In a preferred embodiment, the partners of the specifically binding pair are arranged in predetermined areas, the areas being arranged in a grid-like manner. Individual areas are referred to as "spots". There are various options for arranging the partners.

An advantage of the invention is that the selection of the partners to be bound can be made very flexible. One, a few, or many different types of partners can be arranged on both surfaces. For example, many different partners or partners of many different binding pairs are specifically bound in selected spots on the surface section.

The two surfaces are brought into contact so that the binding partners can bind with sufficient efficiency.

In order to minimize non-specific interactions of the binding partners with the surface, the surface can either be bound by protein substances or polymers, e.g. passivated by the binding of polyethylene glycol and / or polymers can be used to bind the binding partners to the surface.

The partners are each immobilized on a predetermined area, which area can be laid out continuously, in a grid pattern, in any pattern. The design of the areas is arbitrary, provided that when the two surfaces are brought into contact there are surface sections in which able partners to meet each other. According to the invention, it is entirely possible that not every binding partner encounters a corresponding complementary partner in a surface section, but that some partners do not have a partner or a partner who is not capable of binding. All of this serves to keep the variability of the resulting patterns large. In one embodiment, one surface is continuously covered with binding partners, while the second surface has binding partners only in certain sections. In other embodiments, binding partners are present on both surfaces in predetermined areas, the areas which lie opposite one another being congruent or being able to overlap one another as required. It is also possible that different patterns of bound partners are provided on each of the two surfaces, only partial areas of which come into contact with one another when the surface is brought into contact, while other partial areas are in contact with no opposite partner or at least without a partner capable of binding.

The partners can be bound to the surfaces in different ways. In order to optimally ensure the accessibility of the region responsible for the specific binding, in a preferred embodiment the binding to the surface takes place via bridge-like or rake-like molecules, some of which bind to the surface, e.g. via specially provided functional groups, and the other part of which is available for binding the partner.

The partners can be bound to the two surfaces by the same or different molecules. The bridge molecules can be tailored to the respective binding partner. Both short-chain bridge molecules and long-chain polymers can be used to bind the binding partners. In the case of short-chain partners, the length of the chain is generally in the range from 2 to 20 atoms, the chain atoms generally being carbon atoms, nitrogen atoms, possibly in combination with oxygen, and bridging atoms can carry side groups. Even the use of Metal complexes are being considered. If polymers are used to bind the binding partners, the polymers known for this purpose come into consideration here. The chain length should be set so that the partner to be bound is at a suitable distance from the surface and is accessible to the other binding partner. The polymer can also serve to shield the surface in order to suppress non-specific bonds.

The partners of the specifically bindable pair, which are bound to the surface in an adhesive bond, can, as described above, be bound via a bridge molecule and / or via a further specifically binding pair. In this case, the further specifically binding pair should have such a binding force that the binding does not open under the tension exerted on the bonds that form and keeps the binding partner on the surface. Examples of this are e.g. biotinylated antibodies and a surface coated with streptavidin etc.

If a further specifically binding pair is used to immobilize a binding partner, which is referred to below as a detector pair, then this provides the detector binding and has a sufficiently high affinity or a sufficiently low dissociation rate. This prevents the binding partner from detaching or dissociating from the surface before it could come into contact with the other binding partner. A high affinity or a low dissociation rate with simultaneously low stability under an externally applied tensile force is therefore desired for the detector binding. If the bond width increases with the same activation energy, the force sufficient to separate the bond is reduced. The stability of the detector bond is set so that it is weaker than the adhesive bond under an external tensile force.

Two complementary strands of nucleic acid are preferably used as the pair of detectors for immobilizing a binding partner on the surface. The complementary nucleic acid strands used for the immobilization can each because they are bound via both the 3 'and 5' ends. It is now advantageous for the method according to the invention if the binding is carried out in such a way that when a tensile force is exerted on the hybridized strands, the bonds can open like a zipper. In one embodiment, the binding partner is bound to the complementary strand in such a way that a zipper-like separation also occurs when a tensile force is applied. This is accomplished as follows. If the immobilized first strand is fixed at the 3'-end to the surface, the 5'-end remaining unfixed, the binding partner is bound to the complementary second strand at the 5'-end, so that when the binding partner pulls is exerted, the force acts on the 5 'end of the complementary second strand and thus only acts on one base pair, which can open one after the other, so that the strands are separated like a zipper. If the binding partner is bound at the 3 'end in this case, the force required to separate the strands is much greater, since it acts on all base pairs at the same time. If the immobilized first strand is immobilized with the 5 'end, the binding partner must be bound accordingly at the 3' end of the second strand in order to effect the zipper-like separation.

The binding partner should be bound to the complementary nucleic acid strand in such a way that neither the hybridization of the complementary nucleic acid strands nor the binding of the binding partner with the other partner is hindered.

In a further embodiment, a binding partner is a polypeptide, to the C-terminal end of which a nucleic acid is covalently coupled. Such connections can be made, for example, by the methods disclosed in WO 01/04265. The method works with mRNA molecules that are modified with a puromycin tag and that bind to the C-terminus of their polypeptide after they have been expressed in vitro.

Possibilities for immobilizing nucleic acid strands are known to the person skilled in the art and do not require any further explanation here. An advantage of nucleic acid duplexes as a pair of detectors for "suspension" or immobilization of binding partners is that the high binding affinity between nucleic acid strands with a sufficient number of base pairs ensures that the complex does not dissociate from the surface at an early stage without an interaction between the partners of the specific binding Couple was possible and / or without an external force was applied. Another advantage is that by varying the bases, in particular the GC content, the separating power of the duplex can be set precisely. The person skilled in the art is aware that in addition to the bases A, T, G, C and U, other bases such as, for example, inosine or artificial nucleic acids such as PNA and / or LNA can also be used and the separation force can thus be varied further. The separation force can also be varied by modifying bases. This allows the person skilled in the art to "fine-tune" the separating force.

It is usually not necessary to know the exact separation forces of the detector pair or detector bond and the binding of the specifically binding partners. It is usually sufficient to know the order of magnitude if the separation forces of the two complexes differ greatly. For example, the separation force between antigen and antibody is generally significantly higher than the force required to separate a nucleic acid duplex in the manner described above in a "zipper-like" manner.

In the event that the separation forces are to be set very precisely, it is possible to measure the separation force between two molecules directly with a force microscope. This technique has been described in detail in various scientific publications. Antibody-antigen separation forces are in the range from 50 pN to 150 pN (Schwesinger et al., PNAS, 2000, Vol. 97, 18, 9972-9977); In order to separate a nucleic acid duplex like a zipper, 9 ± 3 pN must be used for a pure A / T sequence and 20 ± 3 pN for a pure C / G sequence (Rief et al., Nature structural biology, 1999, Vol. 6, 4, 346-349) to separate a nucleic acid duplex by applying a force to the 5 'end of the first and the 5' end of the second Strands or on the 3 'ends, depending on the number of base pairs involved, 30 to 150 pN must be applied. Biotin forms a particularly stable bond under external force to avidin. To separate this bond, 160 ± 20 pN must be applied (Florin et al., Science, 1994, Vol. 264, 415-417).

In a further embodiment of the present invention, a further stage is preceded. This embodiment is intended for containers which contain high-quality liquids, for example perfumes. In this embodiment, a first surface is applied to the outside of the container, which has partners of a specific binding pair in at least one surface section. Complementary partners of the specific binding pair are added to the liquid in the container in an amount so small that it cannot easily be analyzed in the liquid. Furthermore, a second surface is provided, to which partners are bound who are capable of binding with the specifically binding partner present in the liquid. If the authenticity of the product is to be verified, the liquid contained in the container is applied to the surface on the outside of the container and then the second surface is brought into contact with this surface and then separated. The pattern identifying the object is then made visible on one of the two surfaces or on both surfaces. For example, a surface section on which antibodies against an antigen are covalently bound can be arranged on a perfume bottle. The perfume contained in the perfume bottle may contain small amounts of an antigen. In the event that it is to be proven that it is an original product, some of the perfume is applied to the surface section with the antibodies and allowed to react and then rinsed to remove unbound components. A second surface, which has antibodies against another epitope of the antigen, is then applied to the first surface in such a manner that contact between the partners bound to both surfaces is possible. After a predetermined contact time, the two surfaces are separated again. Then it is determined where the marker is located. If the intended pattern results, if it is an original product, if the pattern is not found, it is a forgery, whereby both the contents of the container and the container itself can be counterfeited.

In a particularly preferred embodiment of the latter variant, one of the ingredients of the liquid is provided as a partner of a specific binding pair. In this case, specifically binding partners of one or more constituents of the liquid are immobilized on a surface section of the container, specifically with detector binding and / or adhesive binding in a predetermined pattern. A stamp-like substrate is provided as the counter surface, for example, a solid body being provided with an elastic support which serves as a stamp surface. Partners that are specifically bindable are in turn immobilized on this stamp surface with substances from the liquid present in the container, these partners again preferably being bound in a predetermined pattern with detector binding and / or adhesive binding. For authentication, some of the liquid is then applied to the identification surface on the container and allowed to react. After rinsing the surface in order to remove unbound and excess components, the stamp is then placed on this surface section in such a way that there is contact between the two surfaces and the corresponding binding partners can react. Then the two surfaces are separated. At the points where a suitable binding partner was available, a marker was also transferred so that the newly created pattern of the binding partner or the bound marker is now visible.

A further improvement of this method variant is achieved if the binding partner is bound in either predetermined sections with either adhesive binding or detector binding. The binding partners, which are bound to their surfaces by means of a detector bond, are carried along by the specific binding partners, while the binding partners, which are bound by adhesive bonding, stick to the respective surfaces. This creates a pattern, whereby the partners, who are bound by adhesion to the Surface bound, remain and bear both the partners who are capable of binding with them and those who are in turn capable of binding with these partners. If one of these three partners carries a marking, this marking is thus on the partner bound via the adhesive bond and thus on this surface. Depending on which of the bound partners the mark (s) is / are sitting, the partners bound via adhesive bonding form a pattern alone and / or with binding partners from the liquid and / or with binding partners from the other surface. Since knowledge of the ingredients of the liquid alone cannot be used to determine where which bonds might take place, and even knowing what is immobilized on the surface as a binding partner, it is not possible to determine which pattern will result, thus becoming a counterfeit-proof means of identification provided.

Since the binding partners immobilized on a surface section are biological molecules which are exposed to the environment and can thereby be changed, it is provided in a preferred embodiment that the surface section which carries the binding partners is covered with a protective coating until the Authentication takes place. Protection can be brought about by sticking on a film which does not bind and does not change the binding partners, by means of a coating layer which is inert to the binding partners, or similar means.

Description of the figures:

Figure 1 shows schematically the structure of an experimental implementation. FIG. 2 shows a fluorescence scan image of a surface on which various antibodies or straptavidin (streptavidin on the top left, anti-digoxygenin on the top right, anti-antitrypsin on the bottom left, anti-biotin on the bottom right) were immobilized on and with a surface the detector complex was immobilized with biotin.

FIG. 3 shows a fluorescence scan image from a surface on which various antibodies or straptavidin (top left streptavidin, top right anti- Digoxygenin, bottom left anti-antitrypsin, bottom right anti-biotin) and which was brought into contact with a surface on which the detector complex was immobilized with digoxygenin.

Figure 4 shows a diagram summarizing the results obtained with an unzip oligo with and without hapten on surfaces coated with four different proteins (anti-biotin antibody, anti-digoxygenin antibody, anti -Antitrypsin antibodies and streptavidin).

The invention is illustrated by the following examples. The abbreviation μM stands for the unit μmol / l.

Examples

For all experiments, ultrapure water was used for washing and the preparation of the buffers, and oil-free N ₂ gas was used for drying. All chemicals were analytical grade.

Experiments were carried out in which fluorescent-labeled binding partners were immobilized on a glass support and the complementary binding partners or non-specifically binding binders were immobilized on a stamp.

This example serves to show that in a preferred embodiment of the method according to the invention, when two surfaces are brought into contact, on each of which specific binding partners are immobilized, a binding partner is only transferred when it meets its specific binding partner, however, that a transfer does not take place if there is a non-specifically binding partner on the other surface. It also shows that when force is exerted on a chain of three different bonds - adhesive bond, specific bond, detector bond - the zip-like bond used as the detector bond breaks between two DNA chains and not the bond between the two selected specific binding partners or immobilization on the surfaces, such as covalent binding, which each have a stronger binding force.

a) Preparation of the glass slides

Glass supports with aldehyde groups on the surface (type CSS, Genpak, Brighton, GB) were used. These carriers were coated with 20 mg / ml HCl-NH ₂ -PEG-COOH (Shearwater, Huntsville, USA) in PBS pH 7.4. 150 ul solution were used per vehicle; the slides were incubated overnight in a humid atmosphere at room temperature, covered with a 24 x 60 mm ² coverslip. The supports were then rinsed with water and dried with N ₂ .

The Schiff bases formed were then reduced with 1% by weight NaBH in water for 30 minutes at room temperature. It was then rinsed again with water and dried with N ₂ .

In a further step, oligonucleotides were bound to the PEG-coated support, which should establish the connection between the binding partner and the glass support. For this purpose, a first detector binding oligo, designated as oligo 61, with an amino group at the 5 'end was used. A 25 μmol dilution of Oligo 61 in PBS plus 7.5 mg / ml EDC was used. 0.6 μl of this solution were spotted on and incubated in a moist atmosphere at 40 ° C. for 2 hours. It was then rinsed with water and dried with N ₂ .

The sequences of the oligos used are given below: Oligo 61 (first detector complex oligo): 5'-NH ₂ -AAA AAA AAA ATC TCC GGC TTT ACG GCG TAT-3 'Oligo 78 (unzip, without hapten):

5'-Cy3-ATA CGC CGT AAA GCC GGA GAC AGA TAA GAC GCT ACA TGA AAA AAA AAA AA-3 '

Oligo 79 (unzip, with biotin): 5'-Cy3-ATA CGC CGT AAA GCC GGA GAC AGA TAA GAC GCT ACA TGA AAA AAA AAA AA-biotin-3 '

Oligo 82 (unzip, with digoxygenin):

5'-Cy3-ATA CGC CGT AAA GCC GGA GAC AGA TAA GAC GCT ACA TGA AAA AAA AAA AA-digoxygenin-3 '

b) Preparation and production of the PDMS stamp

A microstructured stamp was made that consisted of 1 mm thick PDMS (polydimethylsiloxane), which had a grid with squares of 100 × 100 μm ² , which were separated by depressions 25 μm wide and 1 μm deep.

To produce the PDMS stamp, a mixture of crosslinking reagent and silicone elastomer (Sylgard 184, Dow Corning) (1:10) was poured after intensive degassing between a structured silicon wafer and a flat PMMA (polymethyl methacrylate) plate. This assembly was stored vertically at room temperature for 24 hours for polymerization. The polymerized PDMS was cut into stamps in a size of 1 cm.

The structured PDMS was functionalized in a plasma cleaner for 60 seconds in the presence of ice. The pressure was reduced to about 2 mbar.

The plasma treated PDMS stamps were then silanized. For this purpose, 2% aldehyde-ethoxysilane, 88% ethanol and 10% water were mixed and 50 μl / cm_ of this mixture were incubated with the PDMS in a moist atmosphere for 30 minutes at room temperature. The stamps were then washed twice with ethanol, three times with water and dried with N ₂ .

The pretreated silanized stamps were then coated with PEG. 20 mg / ml HCl-NH ₂ -PEG-COOH (Shearwater, Huntsville, USA) in PBS pH 7.4 was used. For this purpose, 150 to 200 μl solution per PDMS was Stamp (1 cm ² ) incubated in a humid atmosphere at room temperature overnight. It was rinsed with water and dried with N ₂ .

The Schiff bases formed were then reduced with 1% by weight NaBH ₄ in water for 30 minutes at room temperature. It was then rinsed with water and dried with N ₂ .

The functional groups were then activated with EDC / NHS. For this purpose 10 mg / ml EDC and 10 mg / ml NHS in PBS pH 7.4 were prepared. The stamps were incubated with 30 μl each for 30 minutes at room temperature under a cover glass with a diameter of 12 mm. It was then rinsed again with water and dried with N ₂ .

The specifically binding binding partners were then bound to the stamps activated in this way. Antibiotin antibodies, antidigoxygenin antibodies, anti-antitrypsin antibodies and streptavidin were used as binding partners. Solutions of 5 mg / ml polyclonal antibiotin antibody, 5 mg / ml polyclonal antidigoxygenin antibody, 50 μg / ml monoclonal anti-antitrypsin antibody or 100 μg / ml streptavidin in PBS with 40% glycerol were prepared. In each case 4 μl were spotted on a PDMS stamp and incubated for 1 hour at room temperature in a humid atmosphere. Then was washed first with PBS with 0.05% Tween 20 and 1% BSA for 1 minute and then with PBS with 0.05% Tween 20 for 2 to 3 minutes.

The still free functional groups on the PDMS were then blocked with 2% BSA for at least 2 hours at room temperature. The stamps could also be stored in this blocking solution. Before incubation with the detector complex on the glass slide, the PDMS was washed with PBS for 2 to 3 minutes, rinsed with water and dried with N ₂ .

In order to transfer the binding partners bound via the detector binding to the binding partners which were bound to the PDMS, the PDMS was applied to the glass support in 1 x SSC for 30 minutes at room temperature. suppressed. The two surfaces were then carefully separated, rinsed with water and dried with N ₂ .

The fluorophores transferred during this transfer were measured with a GenePix 4000 B fluorescence microarray scanner (Axon Instruments Inc., USA). Both surfaces were measured with the 532 nm laser (PDMS: PMT 600 V, 100% energy, focus 100; glass support: PMT 550 V, 33% energy, focus 0). The background fluorescence of the PDMS (before stamping outside of the spots) is about 200 and the background fluorescence of the glass slide (outside of the bound oligos) is about 100. The intensities of the spots were measured with the software that comes with the instrument by adding a Average in a circular area was calculated. The grid-like structure in the spots is neglected by this measurement.

As a control, an unzip oligo was bound from the solution onto the blocked antibody spots. One type of oligo (with biotin, digoxygenin or without hapten) was diluted in PBS with 10% glycerin and 0.05% Tween 20 for some experiments (see Table 7). The final concentration of the oligo was 2 μM or 8 μM. 10 μl of this solution were incubated on the four antibody spots for 30 minutes at room temperature under a cover glass with a diameter of 12 mm. In the case where the oligo was dissolved in PBS without Tween 20, the glass slide was only rinsed with water and dried. Otherwise, the glass slide was washed with PBS with 0.05% Tween 20 for 2 to 3 minutes, rinsed with water and dried with N ₂ .

Results

A PDMS piece with four spots from different binding partners (antibodies or streptavidin) was pressed onto a glass slide on which Unzip-Oligo was hybridized with biotin to the first detector binding oligo. A PDMS section was then scanned for transferred oligos using the fluorescence scanner. The experiment was repeated several times and the results are summarized in Table 1. The respective glass supports were before and after Stamp scanned and the results summarized in Table 2. 2 shows an example of a fluorescence scan of a PDMS section after stamping the unzip oligo with biotin. Streptavidin was attached on the top left, anti-digoxygenin on the top right, anti-antitrypsin on the bottom left, anti-biotin on the bottom right. The light spots are the antibiotic antibodies and streptavidin marked with Unzip-Oligo. The darker areas of non-specific transfer of anti-digoxygenin and anti-antitrypsin antibodies can also be seen.

Table 1

Fluorescence intensity on the PDMS after stamping with Unzip-Oligo with Biotin

Table 2

Fluorescence intensity on the glass slide before and after stamping with unzip oligo with biotin

A PDMS piece with four spots from different binding partners (antibodies or streptavidin) was pressed onto a glass slide on which Unzip oligo was hybridized with digoxygenin to the first detector binding oligo. After that, a PDMS section was transferred to transferred oligos using the fluorescence scanner scanned. The experiment was repeated several times and the results are summarized in Table 3. The respective glass slides were scanned before and after stamping. The results are summarized in Table 4. An example of a fluorescence scan of a PDMS stamped with an unzip oligo with digoxygenin is shown in FIG. 3. Streptavidin was attached at the top left, anti-digoxygenin at top right, anti-antitrypsin at bottom left, anti-biotin at bottom right. The only bright area is the one where anti-digoxygenin antibody was immobilized, all other spots are very weak.

Table 3

Fluorescence intensity on the PDMS after stamping Unzip-Oligo with

digoxygenin

Table 4

Fluorescence intensity on the glass substrate before and after stamping with

Unzip oligo with digoxygenin

A PDMS piece with four spots from different binding partners (antibodies or streptavidin) was pressed onto a glass slide on which Unzip oligo was hybridized to the first detector binding oligo without hapten. A PDMS section was then scanned for transferred oligos using the fluorescence scanner. The result is summarized in Table 5. The respective glass carrier was scanned before and after stamping and the result is summarized in Table 6.

Table 5

Fluorescence intensity on the PDMS after stamping Unzip-Oligo without hapten

Tabelle 6

Table 6

Fluorescence intensity on the glass substrate before and after stamping

Unzip oligo without hapten

As a control, the oligos with and without hapten were also bound to protein spots from the solution. The resulting fluorescence intensities are summarized in Table 7.

Table 7

Fluorescence intensity on the spots after binding of the oligos from the solution (oligo 78 = unzip oligo without hapten, oligo 79 = unzip oligo with biotin, oligo 82 = unzip oligo with digoxygenin, PBST = PBS with 0.05% Tween 20)

The results obtained on stamping, which are summarized in FIG. 4, show a highly specific transfer of the unzip oligo to the corresponding protein spot (fluorescence intensities between 20,000 and 30,000), while the nonspecific transfer to the other protein spots quite is low (about 1600) and is independent of the oligo used. The ratio of specific to unspecific transfer is greater than 10: 1.

FIG. 4 shows a summary of the results obtained when the unzip oligo was stamped with and without hapten on four different protein spots. The binding of the oligo from the solution provides less intense spots in the case of a specific binding (approximately 5800), but also a lower non-specific binding (approximately 300). This leads to a similar ratio of specific to non-specific binding. The higher intensities of the spots, which are achieved by stamping, are based on a higher local concentration. Neither the presence of Tween 20 in the binding buffer nor different washing stages influence the antigen binding in any particular way.

Claims

claims 1 . Method for proving the authenticity of an object, the object having at least one surface section on a surface on which partners of a specifically binding pair are bound in a predetermined arrangement, the surface with a second surface having corresponding surface sections on which at least in selected Areas of complementary binding partners are present in such a way that the partners can react with one another, whereby at least some of the partners have a detectable marking, the areas are then separated and the resulting arrangement of the markings is detected on at least one of the two areas, whereby the arrangement of the bound partners and / or markings created after separation on at least one of the two surfaces differ from the original arrangement of the bound partners and / or the markings on the surface of the object et. 2. The method according to claim 1, characterized in that the complementary partners are either covalently or non-covalently bound to selected surface sections of the second surface. 3. The method according to claim 2, characterized in that in each case complementary partners of a specific binding pair are bound to the surfaces of the corresponding surface sections in predetermined areas, at least one of which bears a marking, wherein each of the partners has an adhesive bond to the surface is bound and the other is bound via a detector bond, the binding force of the detector bond being less than the binding force of the specific binding of the two partners and less than the binding force of the adhesive bond under an externally applied tensile force. 4. The method according to any one of the preceding claims, characterized in that at least one of the two surfaces consists of an elastic material or is coated with an elastic material. 5. The method according to any one of the preceding claims, characterized in that at least one of the two surfaces is coated with polydimethylsiloxane or a derivative thereof. 6. The method according to any one of the preceding claims, characterized in that one of the two surfaces, which is arranged on the object to be identified, as a coating made of an elastic material on a carrier material, which can be paper, a plastic film or the surface of the object, is trained. 7. The method according to any one of the preceding claims, characterized in that one of the partners of the specific binding pair is immobilized either directly covalently, via a bridge molecule and / or via a second specific binding pair on one of the two surfaces with an adhesive bond. 8. The method according to any one of the preceding claims, characterized in that the detector binding takes place via a specifically binding pair of detectors, the binding having a high affinity but low binding force. 9. The method according to claim 8, characterized in that the detector binding is carried out by two complementary strands of nucleic acid. 10. The method according to claim 9, characterized in that the complementary nucleic strands are DNA, RNA, LNA and / or PNA strands. 1 1. The method according to any one of the preceding claims, characterized in that the two partners of the specifically binding pair of antigen-antibody, hapten-antibody, DNA-DNA, RNA-RNA, DNA-RNA, aptamer peptide, aptamer protein, receptor Ligand, lectin sugar, zinc finger protein, DNA, enzyme substrate, biotin avidin / streptavidin or the respective derivatives or analogues thereof. 12. The method according to any one of the preceding claims, characterized in that the partners of the specifically binding pair are biotin and streptavidin or antibiotin antibodies. 13. The method according to any one of the preceding claims, characterized in that the binding of the respective partner of the specific binding pair on each surface takes place partly via adhesive binding and partly via detector binding. 14. The method according to any one of the preceding claims, characterized in that different partners of specifically binding pairs are immobilized on a surface. 15. The method according to any one of the preceding claims, characterized in that in each case only a predetermined proportion of the partners of a specific binding pair is opposite a corresponding partner of the specific binding pair on corresponding surface sections of the two surfaces. 16. The method according to any one of the preceding claims, characterized in that a group is used as the marking, which is optically and / or electrically detectable. 17. The method according to any one of the preceding claims, characterized in that a radioactive, fluorescent, luminescent, chromophoric label or a dye or a conductive group is used as the label. 18. The method according to claim 17, characterized in that a fluorescent group is used as the label. 19. The method according to claim 18, characterized in that the label used is fluorescein isothiocyanate, fluorescein, rhodamine, tetramethylrhodamine 5- (and-6) -isothiocyanate, Texas Red or a cyanine dye. 20. The method according to any one of the preceding claims, characterized in that several markings are used, which are bound to different partners of specific binding pairs. 21. The method according to claim 1, characterized in that the second surface carries a coating which is divided into regions, complementary binding partners being present in predetermined regions, which can migrate to the respective partners upon contact with the first surface, the contact of the bindable partner by diffusion of complementary partners. 22. The method according to claim 21, characterized in that the coating of the second surface allows diffusion of the complementary binding partners. 23. The method according to claim 22, characterized in that the coating of the second surface is a gel. 4. The device for verifying the authenticity of an object in a method according to claim 1, comprising a surface on which at least in a surface section partners of a specific binding pair that carry a label are bound, at least some of the partners being bound via a detector binding , wherein the detector bond is designed such that its binding force is weaker than the binding force between the specific binding partners, only by bringing it into contact with a second surface which has complementary partners of the specific binding pair and separating the on both surfaces the pattern confirming the authenticity is created.