WO2002093504A2

WO2002093504A2 - Method for the forgery-proof marking and identification of objects

Info

Publication number: WO2002093504A2
Application number: PCT/EP2002/005079
Authority: WO
Inventors: Hans Kosak; André JOSTEN
Original assignee: november Aktiengesellschaft Gesellschaft für Molekulare Medizin
Priority date: 2001-05-11
Filing date: 2002-05-08
Publication date: 2002-11-21
Also published as: AU2002312885A1; DE10122836A1; WO2002093504A3; US20050214532A1; DE10292112D2

Abstract

The invention relates to a security thread for the forgery-proof marking of objects, comprising at least one fibre (F) with nucleic acid molecules (N) bonded with the one end thereof to a fibre surface of the fibre (F). The other end of the nucleic acid molecule (N) is free such that complementary nucleic acid molecules (N') may bind to the nucleic acid molecule (N).

Description

Security thread for forgery-proof marking of objects

The invention relates to a security thread for counterfeit-proof marking of objects and a method for counterfeit-proof marking and identification of objects.

It is known to secure objects with markings that can only be detected using a specific detection material. The purpose of these markings is to determine the authenticity of an object in an irrefutable manner. The prerequisite for this is that the marking cannot be changed, decrypted, imitated or removed by third parties.

A method for concealed security marking of objects is known from EP 0 408 424 B1, in which a chemical compound is applied to the object. A nucleic acid with a selected sequence, which is applied to the object in solution, is proposed as the chemical compound. The nucleic acid can then be detected using a suitable detection means, whereby the object is identified. However, this method has the disadvantage that the applied nucleic acids are incorporated into the object, which is to be done in particular by impregnating the object with the nucleic acid-containing solution. However, this presupposes that the object can absorb the nucleic acid. Alternatively, it is proposed that the nucleic acid be carried on a suitable carrier

Apply material and then incorporate this carrier into the object. However, this has the disadvantage that it is comparatively easy to use the impregnated support and separate the object from each other, thereby preventing identification of the object. The basic disadvantage of both variants is that a nucleic acid applied by impregnation can be removed, for example, by solvents. Another disadvantage of the method known from EP 0 408 424 B1 is based on the fact that there are undefined nucleic acids bound to the carrier. Such an undefined bond is relatively weak. Because of the undefined binding, nucleic acids complementary to the nucleic acid can only bind to a small extent. It can also lead to non-specific bindings. In this case, only a very small amount of nucleic acids of a sequence-specific binding is accessible. This reduces the specificity and sensitivity of the known method.

WO 01/09607 AI describes a microarray in which, for the detection of substances possibly contained in a solution, a multiplicity of fibers are held in a predetermined fixed position relative to one another on a carrier. Each of the fibers is provided with a special chemical detection reagent on its surface. For the detection, a solution possibly containing the substances sought is brought into contact with the microarray and a check is carried out to determine whether and if so on which fibers a reaction takes place. From this it can be concluded whether and if so which of the substances searched for are contained in the solution. - Such microarrays are unsuitable for forgery-proof marking of objects. The identification of a detection reaction taking place on the fibers requires a lot of equipment. It cannot be carried out on site. DE 197 38 816 AI describes a method for marking solid, liquid and gaseous substances. A synthetically produced nucleic acid sequence formed from several sequence sections is detachably connected to the object to be marked. For identification, the nucleic acid sequence is removed from the object and detected by means of PCR using predetermined primers. The known method is complex because of the need to separate the nucleic acids and to carry out a PCR required for identification. Identification cannot be carried out on site.

The object of the invention is to eliminate the disadvantages of the prior art. In particular, a simple possibility for counterfeit-proof marking of objects is to be specified. The marking should be as simple and inexpensive to produce as possible and accessible to conventional textile processing methods. According to a further object of the invention, an identification method that can be carried out easily and with which the tamper-proof marking can be identified on site is to be specified.

This object is solved by the features of claims 1 and 31. Appropriate configurations of the inventions result from the features of claims 2 to 30 and 32 to 43.

According to the invention, a security thread is provided for counterfeit-proof marking of objects with at least one fiber, nucleic acid molecules being bound to a fiber surface of the fiber, each having one end thereof and the other end of the nucleic acid molecules le is free in each case, so that complementary nucleic acid molecules can be bound to the nucleic acid molecules.

Nucleic acid molecules in the sense of the present invention are understood to mean organic molecules which have a specific affinity for organic molecules which are complementary thereto. The specific affinity causes specific binding of such molecules. For example, is a strand of DNA that hybridizes with a complementary counter strand. Other suitable nucleic acid molecules are e.g. RNA, PNA, proteins, peptides, synthetic oligonucleotides and the like.

The proposed security thread is suitable for reliable labeling and identification because of the nucleic acid molecules N bound to it. The nucleic acid molecules N can be bound to the fiber surface in defined positions. Binding is preferably carried out where the fiber surface has a corresponding functional group. The nucleic acid molecules N bound to the fiber surface can be specifically detected by means of complementary nucleic acid molecules N ^λ .

The fiber can basically be formed from any fibrous material. The fiber is advantageously formed from a natural or synthetic polymer. The natural polymer is expediently selected from the following group: cellulose, chitin, silk, wool, cotton, hemp, flax or derivatives of these polymers. The synthetic polymer is expediently selected from the following group: polyamide, polyacrylonitrile, nylon, polypropylene, polyvinylidene fluoride, polycarbonate, polystyrene or derivatives of these polymers. In addition, the fiber can also consist of inorganic materials such as glass, quartz or a metal, in particular gold or platinum.

The type of binding of the nucleic acid molecules N to the fiber surface depends on the chemical nature of the fiber material and the intended use of the fiber. The nucleic acid molecules N are preferably connected to the fiber via a defined bond. In this context, a defined bond is understood to mean a known chemical bond. In contrast, undefined bonds, such as those found in UV-cross-linked DNA on nylon, are bonds in which it is not possible to specify the atoms of the nucleic acid molecules from which the binding to the fiber takes place. In addition, the number of bonds with which a nucleic acid molecule N is bound to the fiber is unknown. The binding of the nucleic acid molecules N to the fiber via defined bonds offers the advantage that the type of binding of all nucleic acid molecules N to the fiber is largely identical. The nucleic acid molecules N can be linked to the fiber at defined positions, so that the change in the activity and the accessibility of the nucleic acid molecules N caused by the binding is the same and known.

The nucleic acid molecule N can be bound to the fiber surface via a covalent bond. The high binding coefficient of a covalent bond prevents simple removal of the nucleic acid molecules N from the fiber surface, for example by using a solvent. The nucleic acid molecule N is preferably bound to the fiber surface via a carboxy, phosphate, amino, thiol, psoralen, cholesteryl or digoxigenin group. The nucleic acid molecule is expediently bound to the fiber surface via an intermediate layer, preferably containing streptavidin. Such binding is particularly preferred because of its high affinity constant. This bond cannot be broken even using a strong base such as caustic soda. However, the intermediate layer can also be a functionalized silane layer. For example, the nucleic acid molecules N can be bound to quartz / glass fibers via derivatized silanes. For this purpose, the fiber surface is silylated. Nucleic acid molecules containing SH groups can bind to gold fibers.

Not all surface groups of the fiber suitable for binding with a nucleic acid molecule N have to be saturated with a nucleic acid molecule. The free functional groups remaining after the attachment of the nucleic acid molecules to the fiber surface can remain in this state or can be saturated by suitable reactions. Free thiol groups can, for example, be oxidized to disulfides or reacted with low molecular weight substances such as iodoacetamide.

Nucleic acid molecules N with the same specific sequence in each case or different nucleic acid molecules N1, N2, that is to say nucleic acid molecules with a different sequence, can be bound to the fiber surface. In addition, further nucleic acid molecules with a non-specific sequence can be bound to the fiber. If fibers are used whose surface has nucleic acid molecules bound with different sequences, the nucleic acid molecules N are preferably bound to defined areas of the fiber surface. The diameter of the fibers can be 100 nm to 100 μm. Such fibers can be used to produce security threads using known methods. However, it is also possible for the nucleic acid molecules N to be bound to the fiber only after the thread has been produced. This can conveniently be done by chemical synthesis.

The security thread can comprise at least one further fiber. The fibers of a security thread are held together by the geometric arrangement of the fibers, for example twisting, or by chemical crosslinking of the fibers with one another. Several physical properties of a security thread, such as length and tensile strength, are greater than those of a fiber. By appropriate selection of certain parameters, such as the number ^'of fibers per yarn, the type of twist and / or the use of various fibers, the properties of the security thread can be tailored. The security thread according to the invention can be formed from fibers of different materials. In addition to fibers not modified with nucleic acid molecules, fibers modified with different nucleic acid molecules N, Nl, N2 can also be used. The diameter of the is expediently

Security thread 1 μm to 1 mm.

The security thread according to the invention is expediently incorporated into textiles. The textiles can have security threads modified with different nucleic acid molecules. Known processes such as spinning, weaving, knitting, crocheting, knots,

Knotting, sewing or embroidery are used. The nucleic acid-modified security threads can form a pattern in the textile that can be created using the complementary nucleic acid Molecular N ^{v is} detectable. This pattern can be implemented, for example, as a geometric pattern in the form of a symbol or a bar code.

According to a further provision of the invention, a label, in particular for textiles, is provided which contains at least one security thread according to the invention. The label can of course also contain security threads that have been modified with different nucleic acids. Such a label can, for example, be sewn into textiles, shoes or headgear.

According to a special embodiment, it is provided that a nucleic acid micro-arrangement in the form of a matrix is formed from several security threads. The matrix can be produced by textile-technical processing of the security threads.

According to a further requirement of the invention, a tamper-proof marking is provided, in which one

Base body at least one security thread according to the invention is applied. The base body can be produced from a fabric, paper or non-woven material that enables liquid transport to the security thread. It can also have a suction pad. The aforementioned features enable targeted transport, for example of an identification liquid, to the security thread.

According to a particularly advantageous embodiment, several security threads can be arranged in parallel. The provision of several security threads, which are preferably modified with different nucleic acids, increases the security against forgery of the marking. A cover, preferably made of a plastic film, can be applied to the base body. The cover expediently has a first opening, preferably closed off from a plastic film, for the application of detection liquid. The cover can also have a second opening, preferably closed with a transparent film, for observing the security thread. After removing the cover, the detection liquid can be applied to the base body. There it is transported by capillary forces to the at least one security thread. Complementary nucleic acid molecules contained in the identification liquid are preferably designed here in such a way that they hybridize at room temperature with the nucleic acid molecules bound to the fiber surface. The hybridization expediently causes a fluorescence reaction. This can be identified optically by the cover by means of a reading device or, with a suitable design, even with the naked eye.

According to the procedural requirement of the invention, the following steps are provided for the forgery-proof marking of an object and for the identification of the marking:

a) providing at least one security thread according to the invention,

b) providing the object with the security thread,

c) contacting the security thread with a detection substance containing the complementary nucleic acid molecules and d) detection of the specific binding of the complementary nucleic acid molecules to the nucleic acid molecules on the object.

According to a particularly advantageous design feature, the steps lit. c and lit. d performed on the marked object. It is therefore not necessary to remove the security thread from the marked item. The proposed identification procedure can be carried out directly on site.

According to a further particularly advantageous embodiment, the steps lit. c and d performed in less than 5 min. This is achieved in particular by the fact that the nucleic acid molecules used for labeling and identification are already at room temperature, i.e. hybridize in a temperature range of 18 to 25 ° C. In particular, no heating is required. To carry out steps lit. c and d only one solution or suspension can be used. This also simplifies the process. Finally, the steps lit. c and d can be carried out without a washing step. Overall, the above-mentioned features indicate an extremely simple, quick and inexpensive method for identifying a tamper-proof marking, which is universally applicable and can be carried out on site.

The detection can be carried out by means of specific hybridization and a change in the optical properties brought about as a result of the hybridization, preferably by means of fluorescence or color reaction. For example, so-called “molecular beacons” can be used, each of which is specific for a sequence of the nucleic acid olec are cool and change their fluorescence after a specific binding, preferably at room temperature, with a complementary nucleic acid. According to a particularly advantageous embodiment, the marking is detected by applying a solution containing molecular beacons. The molecular beacons have nucleic acid molecules complementary to the nucleic acid molecules used for the labeling. In the case of hybridization, the bond between the nucleic acid molecules and the nucleic acid molecules complementary thereto can be carried out directly at the label

Fluorescence measurement take place. A particular advantage of using molecular beacons is that the marking can be detected without washing steps and directly on the • marked object. It is a one-step proof. It is also advantageous that detection is possible within a few minutes.

According to a further advantageous embodiment, the detection is carried out by means of laminar flow. A solution or suspension which contains labeled nucleic acid molecules complementary to the nucleic acid molecules used for the labeling is applied to an absorbent area of a carrier. The solution or suspension is transported to the security thread by capillary forces. The labeled complementary nucleic acid molecule is held on the security thread by hybridization. Excess complementary nucleic acid molecules are transported on by capillary forces. The marking is detected by binding the marked complementary nucleic acid molecules to the nucleic acid molecules immobilized on the security thread. The location of the proof and the location of the task of the identification means are different from each other. No washing steps are required in this process either. Also this identification can take place directly on site at the marked object.

Other methods known for the detection of the nucleic acid molecules N in the context of in situ hybridization and Southern or Northern blotting can also be used. These methods include methods that result in a color reaction. For example, the hybridization sample can be coupled directly or indirectly with an enzyme that converts a substrate to an insoluble dye. This dye can then be detected as a precipitate at the hybridization site. The specific hybridization can also be detected using a hybridization sample that is directly or indirectly bound to particles. The immobilization of the particles at the site of the hybridization is then used to detect the specific hybridization.

By binding different nucleic acid molecules N, Nl, N2 to one or more fibers, the unauthorized imitation or falsification of the label is difficult. For this purpose, non-specific nucleic acid sequences can also be bound to the fiber surface, so that non-specific nucleic acid detection does not lead to the disclosure of the specific label.

The sequence of the nucleic acid molecules attached to the security threads should only be known to authorized persons. Objects with such security threads can be marked at a specific position on or in the object. It can also have visible visual markings. The security threads thus make it possible to identify textiles, in particular items of clothing, in a forgery-proof manner. For this purpose, at least one security thread is expediently incorporated into the label, which is attached to the textile. It is essential in connection with the present invention that the nucleic acid molecules are bound to the fibers forming the security thread before the security threads are processed.

The security threads can also be used to generate security markings in the form of microarrays of nucleic acid molecules N. A suitable arrangement or matrix of the nucleic acid molecules N can be achieved with a textile fabric made from the security threads. These tissues can thus be used as nucleic acid microarrays. They have the advantage that they are comparatively simple and inexpensive to manufacture.

To form a matrix in the form of a textile fabric, the nucleic acid-modified security threads can be processed by textile technology processes such as weaving, knitting, crocheting, knotting, sewing or embroidery.

However, the security threads can also be applied to a fixed matrix at defined positions in a specific arrangement, for example in the form of a brush or tufts, without forming a fabric. For example, a plastic surface can be used as a matrix, through which security threads are drawn perpendicular to the surface at defined positions.

A nucleic acid microarray is thus created by producing nucleic acid-modified security threads by attaching the specific nucleic acid molecules N to defined areas of the fiber surfaces and forming a matrix using these nucleic acid-modified security threads. The fibers can be modified with different nucleic acids in different areas. In addition, fibers modified differently with nucleic acids can be used. The nucleic acid-modified security threads can comprise different nucleic acid-modified fibers and also fibers not modified with nucleic acids. The security threads have the properties mentioned above.

Exemplary embodiments of the invention are explained in more detail below with reference to the drawings. Show it

1 shows a directed binding of nucleic acid molecules N to fibers,

2 shows a specific detection of nucleic acid molecules N by complementary nucleic acid molecules N ',

3 shows a production of a fiber to which different nucleic acid molecules N are bound at defined sections,

4 shows a specific detection of different nucleic acid molecules N of a fiber by complementary nucleic acid molecules N ¹ ,

5 shows a synthesis of nucleic acid molecules N on a fiber, 6 shows a parallel synthesis of different nucleic acid molecules N on a fiber,

7a to c a parallel production of nucleic acid arrays on planar carrier materials,

8a to c schematically a process for the production of

Nucleic acid-modified threads made of fibers,

9a to c a first embodiment of a marking with nucleic acid-modified threads,

10a to d a proof of the marking shown in Fig. 9 and

11a to b show a second embodiment of a label with nucleic acid-modified threads.

In Fig. 1, the directional binding of nucleic acid molecules N to a fiber F is shown schematically. In a first

Step are generated on the surface of the fiber F by a suitable activation or reaction of linker groups L which are suitable for coupling to activated nucleic acid molecules N. This step is not necessary if the fiber surface already has suitable functional groups. In the case of wool or silk fibers, for example, free cysteine or amino groups are suitable for coupling activated nucleic acid molecules N. Alternatively, SH groups can be generated in the wool or silk proteins by reducing disulfide groups.

In a second step, nucleic acid molecules N are bound to the free linker groups L, so that the nucleic acid molecules acid-modified fiber FN is obtained. For this purpose, the nucleic acid molecules N are expediently modified with coupling groups K. Suitable coupling groups K are, for example, free SH or amino groups. Nucleic acid molecules N with such coupling groups K can be obtained by oligonucleotide synthesis. Preferably, there is the linking group K at ^λ 3 - or 5 'end of the nucleic acid molecule N. The terminal position of the coupling group K provides good accessibility of the nucleic acid N at a hybridization of a complementary strand. However, the nucleic acid N can also be bound to the fiber F via homofunctional or heterofunctional crosslinkers.

Binding to cellulose-containing fibers can take place by oxidation of sugars to aldehydes. The aldehydes can be linked covalently to amino-containing nucleic acid molecules N to form Schiff's bases and then reduced to amides.

Polycarbonate fibers can be linked to amino-containing nucleic acid molecules N by means of carbodiimide. Other plastics, such as polypropylene, can be covalently linked to nucleic acid molecules N after activation in the plasma. Gold threads can be bound to nucleic acid molecules N containing thiol groups. Glass or quartz fibers can be activated by means of silanization and then connected to the nucleic acid molecules N.

The linker group L can also be bound to the fiber F via a spacer. Polyglycol, polyimine, dextran, polyether can be used as spacers. With the help of the spacers, steric hindrance of the nucleic acids N during hybridization can be reduced, a certain surface layer fertilizer generated, an unspecific binding to the fiber F is reduced and the number of coupling groups K for the nucleic acid molecules N is increased.

2 shows the detection of the nucleic acid molecules N, which are bound to the fiber F, by hybridization. For this purpose, the nucleic acid-modified fiber FN is brought into contact with nucleic acid molecules N ^Λ which have a sequence complementary to the nucleic acid molecules N. The complementary nucleic acid molecules N ^x can have a signal group S. By means of the signal group S, for example, a modified electrical or optical signal can be generated after the hybridization. The signal group S can be a fluorophore, an antigen or an enzyme. By hybridizing the complementary nucleic acid molecules N with the nucleic acid molecules N, the signal group S is fixed at the site of the hybridization (FIG. 2 below) and can be detected on the basis of its properties.

3 shows the production of a fiber FN, to which different nucleic acids N1, N2, N3 are bound at defined sections. A fiber F, which has linker groups L, is divided into spatially separate reaction areas RB1, RB2, RB3. Each reaction area is implemented separately with different nucleic acids Nl, N2, N3. After coupling, a fiber FN1N2N3 is obtained in which different nucleic acids N1, N2, N3 are bound to defined sections.

4 shows the specific detection of different nucleic acid molecules N1, N2, N3, which are bound to different sections of a fiber FN1N2N3, by hybridization with complementary nucleic acid molecules N ^Λ 1, N ^x 2, N ^λ 3. For detection, the fiber FN1N2N3 is brought into contact with the complementary nucleic acid molecules N 1, N * 2, N 3. The specific hybridization can be detected on the basis of the signal groups S1, S2, S3 bound to the nucleic acid molecules N1, N2, N3. The signal groups S1, S2, S3 can be identical or different groups. By using different signal groups S1, S2, S3, for example fluorophores, patterns can be generated on the fiber.

5 shows a synthesis of nucleic acid molecules N on a fiber F. The nucleic acid molecules N are oligonucleotides synthesized from individual nucleotides. A fiber F is covalently provided with formation blocks Ba for the attachment of further nucleotides. In others

In each step an activated nucleotide (b, c, d, e, f, g) is added to the already bound nucleotides. A FBabcdefg fiber to which a defined sequence of nucleotides is fixed is obtained by the synthesis.

6 shows a parallel synthesis of different nucleic acids N on a fiber F. For this purpose, the fiber F is divided into reaction areas RB1, RB2, RB3. Each section is implemented separately with different activated education blocks Bai, Ba2, Ba3. In further steps, an activated nucleotide (b, c, d, e, f, g) is added to the previously immobilized nucleotide in each reaction area. The synthesis gives a fiber FN1N2N3 to which different oligonucleotides N1, N2, N3 are bound in defined sections.

7a to c show the parallel production of nucleic acid arrays on planar carrier materials M. For this purpose, a Number n of planar carrier materials M placed on top of one another (FIG. 7a). Different nucleic acid-modified threads FN1, FN2, FN3 are passed through the carrier materials M at defined positions. The security threads are then separated between the carrier materials M.

(Fig. 7b). In this way, a carrier is obtained on which oligonucleotides are fixed to security threads at defined positions.

8a to c schematically show a method for producing security threads Fd from fibers F. The fibers shown in FIG. 8a are modified with nucleic acid molecules N, whereby fibers FN are obtained (FIG. 8b). The fibers FN are spun into threads Fd in a spinning machine S (FIG. 8c).

9a to c show a first embodiment of a marking 1 with nucleic acid-modified security threads Fd. Four nucleic acid-modified security threads Fd are applied in parallel to a rectangular base body 2. A suction pad 2 is also located on the base body 2. The base body 2 is formed by a matrix which enables a lateral flow of a liquid. If the base body 2 is contacted with a liquid, it transports the liquid to the applied security threads Fd and the suction pad 3. The base body 2 can be formed from a fabric, absorbent paper or non-woven fabric, which are expediently applied to a liquid-impermeable plastic film. The plastic film prevents liquid from escaping into the environment and protects the marking. An adhesive plastic film can be used to apply the marking 1 to the object to be marked. The security threads Fd are in contact with the base body 2, so that a transfer of liquid that is applied to the base body 2 is possible. The absorbent pad 3 absorbs most of the liquid applied, so that only a minimal amount of liquid remains in the base body 1. For this purpose, the suction pad 3 is formed from a fabric with a high liquid binding capacity.

The marker 1 can comprise one or more nucleic acid-modified security threads Fd. Nucleic acid molecules N with different but known sequences can be bound to the security threads Fd. In addition, unknown substances such as randomly generated DNA can be bound in order to make the analysis of the security threads Fd more difficult. Such randomly generated substances do not bind any detection liquid, so that one is possible to check the detection liquid. In addition, security threads can be used that contain a substance such as DEAE cellulose that binds non-specifically nucleic acid molecules. Such a security thread, which binds any nucleic acid molecules N ^λ , is used to control the detection liquid. The use of several security threads Fd makes it possible to form complex markings. By using several, differently modified security threads Fd, a pattern can be formed which is only revealed if all nucleic acid molecules on the security threads Fd are identified. The security threads Fd can also form geometric patterns, for example numbers, letters, symbols, barcodes.

9b shows the marking 1 with a cover 4. The cover 4 expediently consists of an opaque material which covers the base body 2 and two Has recesses 4.1 and .2. The cover 4 serves to protect the marking 1 against mechanical stress, chemical stress or radiation. The first recess 4.1 shows the location on which a detection liquid for detecting the marking 1 is applied. The recess 4.1 can be closed, for example with a plastic film, and can only be opened if the marking 1 is to be identified. Such a closure serves to protect against contamination, for example by fat or similar substances, which could impair the flow behavior of the base body 1. The second recess 4.2 forms a viewing window which enables a view of the security threads Fd. The viewing window can be closed with a transparent film to protect the security threads Fd. The security threads Fd can then be seen through the viewing window 4.2, as shown in FIG. 9c. Before the identification of the marking 1, the security threads Fd can be invisible to the eye.

10a to d is a method for detecting the in

Fig. 9 shown marking 1, the marking 1 in Fig. 10a to c is shown for better illustration without a cover.

As shown in FIG. 10 a, a defined volume of a detection liquid is given through the recess 4.1 on the base body 2 to identify the marking 1. The detection liquid contains nucleic acid molecules N ¹ which are complementary to the nucleic acid molecules N bound to the security threads Fd. The nucleic acid molecules N ¹ used for the detection can be labeled by means of dye molecules, fluorogenic, gold or latex particles, so that the detection of a specific hybridization with nucleic acid acid molecules N is facilitated. The complementary single-stranded nucleic acid molecule N used for the detection preferably has a refolding. This increases the specificity of the hybridization.

10b, the lateral flow of the detection liquid in the direction of the suction pad 3 is identified by an arrow. During the flow, the detection liquid comes into contact with the security threads Fd. FIG. 10c shows the basic body 2 after the end of the lateral flow. The vast majority of the

Detection liquid has been absorbed by the suction pad 3. Security threads Fd, which have bound the nucleic acid molecules N *, are shown by the reinforced lines.

Fig. 10d shows the mark 1 with cover 4 after the identification of the mark 1. The thicker security threads Fd show the proof of the mark 1. The detection process is completed within a few seconds to minutes. When using colored particles such as gold particles, to which the nucleic acid molecules N ^{λ are} bound, only the eye and no further aids such as a photometric detector are necessary to detect the marking. This means that toxic or radioactive components can be dispensed with.

11a to b show a second embodiment of a marking 1 with nucleic acid-modified security threads Fd. In FIG. 11a, four nucleic acid-modified security threads Fd and a suction pad 3 are arranged on the base body 2. On the base body 2, a cover 4 is applied, which has two recesses 4.1, 4.2 (Fig. 11b). The recess 4.1 is used to apply the detection liquid on the security threads Fd. The recess 4.2 is used to observe the detection of the marking 1. In this embodiment, the security threads Fd themselves serve the lateral flow of the detection liquid.

The production of nucleic acid-modified fibers and their detection is explained below using an example.

1) Aldehyde activation of cellulose fibers

100 mg washed cotton (Machery and Nagel) is incubated in 10 ml 100 mM NaJ0 ₄ in PBS (pH 7.4) at 37 ° C overnight. The NaJ0 ₄ is removed by washing five times with 10 ml of PBS.

2) Binding of amino oligonucleotides to aldehyde-activated cellulose fibers

To the aldehyde-activated cotton are given 10 uM synthetic oligonucleotide N, the ^λ at the 5 -end carries a free amino group. The mixture is brought to 20 mM sodium cyanoborohydride in a final volume of 2 ml and over

Incubated overnight at room temperature with swirling. To saturate free aldehyde groups, 2 ml of 1 M TrisCl (pH 8) are added and incubated for a further 2 h at room temperature. To remove unbound oligonucleotides N, the cotton is washed five times with 10 ml of TBS and incubated for 1 h in an electrophoresis chamber at a voltage of 100 V in 10 mM trisacetate, 1 mM EDTA (pH 8). 3) Hybridization of oligonucleotides bound to cellulose threads

Cellulose threads with bound oligonucleotides N are incubated in 100 μl 10 mM TrisCl, 1 mM EDTA with 1 μM complementary oligonucleotides N ¹ at 37 ° C. for 30 min. The Oligonu- kleotide N have a sequence complementary to the oligonucleotides and N are labeled with a biotin group at the 5 -end ^λ. To remove unbound oligonucleotides, the cotton is washed five times with 10 ml of TBS and incubated for 1 h in an electrophoresis chamber at a voltage of 100 V in 10 mM trisacetate, 1 mM EDTA (pH 8).

4) Evidence of hybridization

Cellulose threads with hybridized oligonucleotides N, N 'are swirled with streptavidin-coated, superparamagnetic particles (Dynal) in 1 ml of 10 mM TrisCl, 150 mM NaCl, 1 mM EDTA (pH 8) (TBST) at room temperature for 1 hour incubated. Unbound particles are removed by washing five times with 1 ml TBST using a magnet. The binding of the particles to the fibers indicates the hybridization of oligonucleotide N ¹ to oligonucleotide N.

5) Silanization and aldehyde activation of a cotton thread

Approximately For the pretreatment, 2 m of cotton thread was incubated in 30 ml of 10% sodium dodecysulfate (SDS) at 55 ° C for one hour. The SDS was removed by rinsing five times in 30 ml of water. For silanization, the thread was divided and 50% of the thread in each case for 1 hour in 1% a) 3-aminopropyl-methyl-diethoxy silanes (Fluka) or b) 3-aminopropyl-triethoxysilanes, (ABCR) incubated in ethanol at 55 ° C. for one hour. The threads were rinsed in ethanol and dried at 85 ° C for one hour. To bind aldehyde groups, the threads were incubated in 20 ml of 5% glutaraldehyde for one hour at room temperature (RT) and rinsed intensively with water to remove excess glutaraldehyde.

6) Coupling amino oligonucleotides to silanized cotton threads

The threads were washed in 0.1 M Na ₂ CO ₃ solution pH 9.5 and 10 cm of the threads in 1 ml of 1 fM oligonucleotide N in 0.1 M Na ₂ CO ₃ solution were incubated at RT for four hours. To saturate any aldehyde groups still present, the suspension was brought to 1% ethanolamine and 2 mM EDTA and incubated at RT overnight. Unbound oligonucleotides were removed by intensive washing in 1% SDS solution in 10 mM TrisHCl, 1 mM EDTA, pH 8.8.

The following synthetic nucleic acids were used as oligonucleotides:

a) N-Tlk biotin:

Biotin-5> gca aca aga cca cca ctt cga aac 3> -C6 aminolink b) NH2-T9-Targetl-28

5'-C6-Aminolink -TTT TTT TTT CCA AGC CTG GAG GGA TGA TAC TTT

GCG C-3 '

7) Detection of the binding of the biotinylated oligonucleotides to the threads Approx. 5 mm of the threads were incubated with streptavidin peroxidase diluted 1: 5000 in phosphate-buffered saline (PBS) for 5 minutes. The threads were washed with 10 ml PBS and reacted with peroxidase substrate (Röche). Within five minutes a dark staining of the threads, which were labeled with the biotinylated oligonucleotide, was visible. Seams without a biotinylated oligonucleotide showed no staining.

8) Detection of the binding of the oligonucleotides by hybridization using molecular beacons

Approx. 20 mm of the threads were sewn into a cotton fabric with a needle. 5 μl of a 2 fM solution of a molecular beacon were dripped onto one end of each thread. The

Fluorescence of the threads was excited with a red laser light (wavelength = 650 nm) approx. 5 mm from the dropping point and determined using a fluorescence reader. Within two minutes an increase in fluorescence by more than 40% above the background fluorescence without molecular beacon addition was measurable for the thread with oligonucleotides of complementary sequence (NH ₂ -T9-Targetl-28). The threads with non-complementary sequence oligonucleotides (N-Tlk-biotin) only showed an increase in fluorescence of approximately 10% above the background signal.

Sequence of the molecular beacon

5'-Cy5-CCA AGC GCA AAG TAT CAT CCC TCC AGG CTT GG-BHQ2-3 SEQUENCE LISTING

<110> november AG Society for Molecular Medicine

<120> Security fiber, security thread, and methods of marking and identification

<130> 422132

<140> <141>

<160> 3

<170> Patentin Ver. 2.1

<210> 1 <211> 24 <212> DNA <213> Artificial sequence

<220>

<223> Description of the artificial sequence: oligonucleotide

<400> 1 gcaacaagac caccacttcg aaac

24

<210> 2 <211> 36 <212> DNA <213> Artificial sequence <220>

<223> Description of the artificial sequence oligonucleotide

<400> 2 tttttttttc caagcctgga gggatgatac tttgcg

36

<210> 3 <211> 32 <212> DNA <213> Artificial sequence

<220>

<223> Description of the artificial sequence: oligonucleotide

<400> 3 ccaagcgcaa agtatcatcc ctccaggctt gg 32

LIST OF REFERENCE NUMBERS

1 mark

2 basic bodies 3 suction pads

4 Cover 4.1, 4.2 Recess a, b, c, d, f, g nucleotides F fiber FB _a ... FB _abcdef g fiber to which nucleic acids N have been synthesized from nucleotides

Fd security thread

FN nucleic acid modified fiber

K coupling group L linker group

M carrier material

N nucleic acid molecule

N ^λ nucleic acid molecule which is complementary to the nucleic acid molecule N RB reaction area

5 signal group

Claims

claims

1. Security thread for counterfeit-proof marking of objects with at least one fiber (F), nucleic acid molecules (N) being bound at one end to a fiber surface of the fiber (F) and the other end of the nucleic acid molecules (N) being free, so that complementary nucleic acid molecules (N ^λ ) can be bound to the nucleic acid molecules (N).

2. Security thread according to claim 1, wherein the fiber (F) is formed from a natural polymer.

3. Security thread according to claim 2, wherein the natural polymer is selected from the following group: cellulose,

Chitin, silk, wool, cotton, hemp, flax or derivatives of these polymers.

4. Security thread according to claim 1, wherein the fiber (F) is formed from a synthetic polymer.

5. Security thread according to claim 4, characterized in that the synthetic polymer is selected from the following group: polyamide, polyacrylonitrile, nylon, polypropylene, polyvinylidene fluoride, polycarbonate, polystyrene or derivatives of these polymers.

6. Security thread according to one of the preceding claims, wherein the nucleic acid molecule (N) is bound to the fiber surface via a covalent or non-covalent bond.

7. Security thread according to claim 6, wherein the nucleic acid molecule (N) via a biotin / streptavidin bond, a Car- boxy, phosphate, amino, thiol, psoralen, cholesteryl or digoxigenin group is bound to the fiber surface.

8. Security thread according to one of the preceding claims, wherein the nucleic acid molecule (N) via one, preferably

Streptavidin-containing, intermediate layer is bound to the fiber surface.

9. Security thread according to claim 8, wherein the intermediate layer is a functionalized silane layer.

10. Security thread according to one of the preceding claims, wherein different nucleic acid molecules (N, Nl, N2,) are bound to the fiber surface.

11. Security thread according to claim 10, wherein the different nucleic acid molecules (N) are bound to defined regions of the fiber surface.

12. Security thread according to one of the preceding claims, wherein the nucleic acid molecule (N) is produced by chemical synthesis directly on the fiber.

13. Security thread according to one of the preceding claims, wherein the diameter of the fiber (F) is 100 nm to 100 μm.

14. Security thread according to one of the preceding claims comprising at least one further fiber.

15. Security thread according to claim 14, wherein the further fiber consists of another material and / or is a fiber (F) according to one of the preceding claims.

16. Security thread according to one of claims 12 to 15, wherein its diameter is 1 μm to 1 mm.

17. Textile with at least one security thread (Fd) according to one of the preceding claims.

18. Textile according to claim 17, - wherein it comprises security threads (Fd) modified with different nucleic acid molecules.

19. Textile according to one of claims 17 or 18, wherein nucleic acid-modified security threads (Fd) form a pattern which can be detected by means of the complementary nucleic acid molecules (N).

20. Label, in particular for textiles, the label containing at least one security thread (Fd) according to one of claims 1 to 16.

21. Label according to claim 20, wherein it contains security threads (Fd) modified with different nucleic acid molecules.

22. The label of claim 20 or 21, wherein a plurality of security threads is used to form a nucleic acid microarray in the form of a

Matrix is formed.

23. The label according to claim 22, wherein the matrix is produced by textile-technical processing of the security threads (Fd).

24. A counterfeit-proof marking in which at least one security thread (Fd) according to one of claims 1 to 16 is applied to a base body (2).

25. A counterfeit-proof marking according to claim 24, wherein the base body (2) is made from a tissue, paper or non-woven fabric that enables liquid transport to the security thread (Fd).

26. Counterfeit-proof marking according to claim 24 or 25, wherein the base body (2) has a suction pad (3).

27. Counterfeit-proof marking according to one of claims 24 to 26, wherein a plurality of security threads (Fd) are arranged in parallel.

28. Counterfeit-proof marking according to one of claims 24 to 27, wherein a cover (4), preferably made of a plastic film, is applied to the base body (2).

29. A counterfeit-proof marking according to claim 28, wherein the cover (4) has a first opening (4a), preferably sealed with a plastic film, for applying detection liquid.

30. A counterfeit-proof marking according to claim 28 or 29, wherein the cover (4) has a second opening (4b), preferably closed with a transparent film, for observing the security thread (Fd).

31. A method for counterfeit-proof marking of an object and for identifying the marking with the following steps:

a) providing at least one security thread (Fd) according to one of claims 1 to 16,

b) providing the object with the security thread (Fd),

c) contacting the security thread (Fd) with a detection substance containing the complementary nucleic acid molecules (N ^λ ) and

d) Detection of the specific binding of the complementary nucleic acid molecules (N to the nucleic acid molecules (N) on the object.

32. The method of claim 31, wherein the steps lit. c and d are carried out on the marked object.

33. The method of claim 31 or 32, wherein steps lit. c and d can be done in less than 5 minutes.

34. The method according to any one of claims 31 to 33, wherein lit. c and d only one solution or

Suspension is used.

35. The method according to any one of claims 31 to 34, wherein steps lit. c and d can be carried out without a washing step.

36. The method according to any one of claims 31 to 35, wherein the detection by means of specific hybridization and a consequent Ge the hybridization caused change in the optical properties, preferably by fluorescence or color reaction is carried out.

37. The method according to claim 36, wherein the detection is carried out by means of a molecular beacon.

38. The method according to any one of claims 31 to 37, wherein the detection is carried out by means of an enzymatic reaction and a change in the optical properties caused as a result of the enzymatic reaction, preferably by means of a fluorescence or color reaction.

39. The method according to any one of claims 31 to 38, wherein the detection is carried out by means of laminar flow.

40. The method according to any one of claims 31 to 39, wherein complementary nucleic acid molecules (NM are present bound to micro- or nanoparticles.

41. The method according to any one of claims 31 to 40, wherein during the detection a pattern formed by at least one security thread (Fd) is identified.

42. The method according to claim 41, wherein the pattern is formed from a plurality of security threads (Fd) which are provided with different nucleic acid molecules (N, Nl, N2).

43. The method of claim 41 or 42, wherein the pattern is formed by knitting, weaving, knitting, crocheting, knotting, sewing or embroidery.