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WO2003066884A2 - Procede de marquage d'articles - Google Patents

Procede de marquage d'articles Download PDF

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Publication number
WO2003066884A2
WO2003066884A2 PCT/EP2003/001186 EP0301186W WO03066884A2 WO 2003066884 A2 WO2003066884 A2 WO 2003066884A2 EP 0301186 W EP0301186 W EP 0301186W WO 03066884 A2 WO03066884 A2 WO 03066884A2
Authority
WO
WIPO (PCT)
Prior art keywords
partners
binding
bound
partner
complementary
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/EP2003/001186
Other languages
German (de)
English (en)
Other versions
WO2003066884A3 (fr
Inventor
Filipp Oesterhelt
Thao Mai
Katrin Spinnler
Kerstin Blank
Stefan Müllner
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
NANOTYPE GmbH
Original Assignee
NANOTYPE GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by NANOTYPE GmbH filed Critical NANOTYPE GmbH
Priority to AU2003208808A priority Critical patent/AU2003208808A1/en
Publication of WO2003066884A2 publication Critical patent/WO2003066884A2/fr
Publication of WO2003066884A3 publication Critical patent/WO2003066884A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase

Definitions

  • the invention relates to a method for verifying the authenticity of an object.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the ability of biological molecules to form specific bonds with specific binding partners is used to solve this problem.
  • the possibilities that a differential force test offers, which can compare and evaluate binding forces, are used.
  • a method for proving the authenticity of an 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.
  • 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.
  • a counterfeit-proof system is thus made available.
  • 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.
  • Objects that are to be protected against imitation 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.
  • 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.
  • 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.
  • the first surface is formed on the article itself.
  • a layer or coating to the object.
  • 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.
  • one of the two surfaces can carry such a coating that diffusion of binding partners is possible, areas being separated in a grid-like manner.
  • 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.
  • 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.
  • any analytically detectable group or substance can be used as a marker for the method according to 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.
  • 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.
  • 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.
  • markings per specific binding pair 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a specific partner such as, for example, antigen-associated antibodies or biotin-avidin / streptavidin
  • a group or class of compounds eg antibody-protein A.
  • 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.
  • antibodies and a substance with an epitope recognized by it are used as the binding pair.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the magnitude of the force can depend on the pulling rate.
  • 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.
  • the pull rate should be taken into account.
  • the pull rate is not important under the conditions described.
  • 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.
  • the partner immobilized via a detector bond when he meets his binding partner, moves with him with and onto the other surface.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • one surface is continuously covered with binding partners, while the second surface has binding partners only in certain sections.
  • 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.
  • 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.
  • 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.
  • the 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • bases A, T, G, C and U 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.
  • 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.
  • 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.
  • a further stage is preceded.
  • This embodiment is intended for containers which contain high-quality liquids, for example perfumes.
  • 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.
  • 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.
  • 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.
  • 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.
  • one of the ingredients of the liquid is provided as a partner of a specific binding pair.
  • 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.
  • some of the liquid is then applied to the identification surface on the container and allowed to react.
  • the stamp 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • various antibodies or straptavidin top left streptavidin, top right anti- Digoxygenin, bottom left anti-antitrypsin, bottom right anti-biotin
  • 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).
  • ⁇ M stands for the unit ⁇ mol / l.
  • 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.
  • Glass supports with aldehyde groups on the surface 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 .
  • oligonucleotides were bound to the PEG-coated support, which should establish the connection between the binding partner and the glass support.
  • a first detector binding oligo designated as oligo 61, with an amino group at the 5 'end was used.
  • Oligo 61 A 25 ⁇ mol dilution of Oligo 61 in PBS plus 7.5 mg / ml EDC was used. 0.6 ⁇ l 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 .
  • 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):
  • 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):
  • a microstructured stamp was made that consisted of 1 mm thick PDMS (polydimethylsiloxane), which had a grid with squares of 100 ⁇ 100 ⁇ m 2 , which were separated by depressions 25 ⁇ m wide and 1 ⁇ m deep.
  • PDMS polydimethylsiloxane
  • 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 ⁇ l / 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 ⁇ l 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 .
  • EDC / NHS The functional groups were then activated with EDC / NHS.
  • 10 mg / ml EDC and 10 mg / ml NHS in PBS pH 7.4 were prepared.
  • the stamps were incubated with 30 ⁇ l 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 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.
  • the PDMS was washed with PBS for 2 to 3 minutes, rinsed with water and dried with N 2 .
  • 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.
  • 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 ⁇ M or 8 ⁇ M. 10 ⁇ l 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 .
  • a PDMS piece with four spots from different binding partners 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.
  • a PDMS piece with four spots from different binding partners 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.
  • a PDMS piece with four spots from different binding partners 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.
  • 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.

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  • Chemical & Material Sciences (AREA)
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  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
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  • Physics & Mathematics (AREA)
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Abstract

L'invention concerne un procédé destiné à prouver l'authenticité d'un article, ce dernier présentant sur l'une de ses surfaces au moins une section, sur laquelle des partenaires d'une paire se liant spécifiquement sont liés selon un agencement prédéterminé. Ladite surface est en contact avec une seconde surface, qui présente des sections correspondantes sur lesquelles, au moins dans des zones sélectionnées, des partenaires de liaison complémentaires sont disposés, de façon à réagir les uns avec les autres, au moins une partie des partenaires portant un marquage détectable. Lesdites sections sont alors séparées et l'agencement des marquages ainsi formé sur au moins une des deux sections est détecté. Après la séparation, ledit agencement de partenaires et/ou de marquages, formé sur au moins une des deux surfaces, se différencie de l'agencement d'origine des partenaires liés et/ou des marquages sur la surface de l'article.
PCT/EP2003/001186 2002-02-09 2003-02-06 Procede de marquage d'articles Ceased WO2003066884A2 (fr)

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AU2003208808A AU2003208808A1 (en) 2002-02-09 2003-02-06 Method for labeling articles

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DE10205506A DE10205506C1 (de) 2002-02-09 2002-02-09 Verfahren zur Markierung von Gegenständen
DE10205506.8 2002-02-09

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053161A1 (fr) * 2002-12-12 2004-06-24 Hapemo Da Procede d'identification et/ou d'authentification d'articles
WO2005021755A1 (fr) * 2003-08-29 2005-03-10 Applera Corporation Compositions, procedes, et trousses pour la fabrication d'etiquettes moleculaires codees
US7198900B2 (en) 2003-08-29 2007-04-03 Applera Corporation Multiplex detection compositions, methods, and kits

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
NL1028064C2 (nl) * 2005-01-18 2006-07-19 Vhp Ugchelen Bv Authenticatiewerkwijze en -systeem voor het authenticeren van veiligheidsdocumenten en veiligheidsdocument.
US8703068B1 (en) 2009-03-04 2014-04-22 The Procter & Gamble Company Counterfeit detection kit

Family Cites Families (4)

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Publication number Priority date Publication date Assignee Title
US5445970A (en) * 1992-03-20 1995-08-29 Abbott Laboratories Magnetically assisted binding assays using magnetically labeled binding members
CA2217209A1 (fr) * 1997-12-01 1999-06-01 Jia Bei Zhu Authentification immuno-chimique a une etape
DE19927051C2 (de) * 1999-06-14 2002-11-07 November Ag Molekulare Medizin Verfahren und Vorrichtung zur Identifizierung einer Nukleotidsequenz
DE10000629C5 (de) * 2000-01-10 2010-06-02 november Aktiengesellschaft, Gesellschaft für Molekulare Medizin Verfahren zur Identifizierung einer auf einen festen Körper aufgebrachten Markierung

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004053161A1 (fr) * 2002-12-12 2004-06-24 Hapemo Da Procede d'identification et/ou d'authentification d'articles
WO2005021755A1 (fr) * 2003-08-29 2005-03-10 Applera Corporation Compositions, procedes, et trousses pour la fabrication d'etiquettes moleculaires codees
WO2005024021A1 (fr) * 2003-08-29 2005-03-17 Applera Corporation Compositions, methodes et kits d'assemblage de sondes comprenant des etiquettes moleculaires codees
US7198900B2 (en) 2003-08-29 2007-04-03 Applera Corporation Multiplex detection compositions, methods, and kits

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AU2003208808A1 (en) 2003-09-02
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DE10205506C1 (de) 2003-06-18

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