|Publication number||US20050214532 A1|
|Application number||US 10/477,158|
|Publication date||Sep 29, 2005|
|Filing date||May 8, 2002|
|Priority date||May 11, 2001|
|Also published as||DE10122836A1, DE10292112D2, WO2002093504A2, WO2002093504A3|
|Publication number||10477158, 477158, PCT/2002/5079, PCT/EP/2/005079, PCT/EP/2/05079, PCT/EP/2002/005079, PCT/EP/2002/05079, PCT/EP2/005079, PCT/EP2/05079, PCT/EP2002/005079, PCT/EP2002/05079, PCT/EP2002005079, PCT/EP200205079, PCT/EP2005079, PCT/EP205079, US 2005/0214532 A1, US 2005/214532 A1, US 20050214532 A1, US 20050214532A1, US 2005214532 A1, US 2005214532A1, US-A1-20050214532, US-A1-2005214532, US2005/0214532A1, US2005/214532A1, US20050214532 A1, US20050214532A1, US2005214532 A1, US2005214532A1|
|Inventors||Hans Kosak, Andre Josten|
|Original Assignee||Hans Kosak, Andre Josten|
|Export Citation||BiBTeX, EndNote, RefMan|
|Patent Citations (19), Referenced by (11), Classifications (9), Legal Events (2)|
|External Links: USPTO, USPTO Assignment, Espacenet|
The invention relates to a security thread for the forgery-proof marking of objects and to a method for the forgery-proof marking and identification of objects.
It is known to secure objects with markings which can be detected only on use of a particular indicator. The purpose of these markings is to establish the genuineness of an object in an indisputable manner. The precondition for this is that the marking cannot be altered, decrypted, copied or removed by third parties.
EP 0 408 424 B1 discloses a method for the concealed security marking of objects in which a chemical compound is applied to the objects. A proposed chemical compound is a nucleic acid with selected sequence, which is applied in solution to the object. The nucleic acid can then be detected with a suitable detection means, whereby the object is identified. This method has the disadvantage, however, that the applied nucleic acid is incorporated into the object, which is intended to be accomplished, in particular, by impregnating the object with the nucleic acid-containing solution. However, the precondition for this is that the object is able to absorb the nucleic acid. An alternative proposal is to apply the nucleic acid to a support made of a suitable material and then to incorporate this support in the object. However, this has the disadvantage that it is possible comparatively simply to separate the impregnated support and the object from one another, thus preventing identification of the object. The fundamental disadvantage of both variants is that a nucleic acid applied by impregnation can be removed for example by solvents. A further disadvantage of the method disclosed in EP 0 408 424 B1 is based on the fact that the linkage of the nucleic acids to the support therein is undefined. Such an undefined linkage is relatively weak. Because the linkage is undefined, nucleic acids complementary to the nucleic acid are capable of linkage to only a small extent. In addition, nonspecific linkages may occur. In this case, therefore, only a very small amount of nucleic acid is available for sequence-specific linkage. This reduces the specificity and sensitivity of the known method.
WO 01/09607 A1 describes a microarray in which a plurality of fibers are taken up on a support in a preset fixed position relative to one another in order to detect substances possibly present in a solution. Each of the fibers is provided on its surface with a particular chemical detection reagent. For the detection, a solution possibly containing the substances which are sought is brought into contact with the microarray and examined for whether and, where appropriate, on which fibers a reaction takes place. It is possible to conclude from this whether and, where appropriate, which of the substances which are sought are present in the solution. Such microarrays are unsuitable for the forgery-proof marking of objects. Identification of a detection reaction taking place on the fibers requires highly complex apparatus. It cannot be carried out on the spot.
DE 197 38 816 A1 describes a method for marking solid, liquid and gaseous substances. In this case, a synthetically prepared nucleic acid sequence formed from a plurality of sequence sections is releaseably connected to the object to be marked. For identification, the nucleic acid sequence is removed from the object and depicted by means of PCR using predetermined primers. The known method is complicated because of the need to separate the nucleic acids and to carry out a PCR necessary for the identification. The identification cannot be carried out on the spot.
It is an object of the invention to eliminate the prior art disadvantages. It is particularly intended to indicate a simple possibility for the forgery-proof marking of objects. It is to be possible to produce a marking as simply and at as low a cost as possible and in a way amenable to a conventional textile-processing method. According to a further aim of the invention, it is intended to indicate an identification method which can be carried out simply and with which the forgery-proof marking can be identified on the spot.
This object is achieved by the features of claims 1 and 31. Expedient embodiments of the inventions are evident from the features of claims 2 to 30 and 32 to 43.
The invention provides a security thread for the forgery-proof marking of objects having at least one fiber, where the nucleic acid molecules are linked with in each case in their one end to a fiber surface of the fibers, and where the other end of the nucleic acid molecules is free in each case, so that complementary nucleic acid molecules are able to undergo linkage to the nucleic acid molecules.
Nucleic acid molecules means for the purposes of the present invention organic molecules which have a specific affinity for organic molecules complementary thereto. The specific affinity brings about a specific linkage of such molecules. A possible example is one strand of a DNA which hybridizes with a complementary strand. Examples of further suitable nucleic acid molecules are RNA, PNA, proteins, peptides, synthetic oligonucleotides and the like.
The proposed security thread is suitable for secure marking and identification because of the nucleic acid molecules N linked thereto. The nucleic acid molecules N can be linked in defined positions on the fiber surface. A linkage preferably takes place where the fiber surface has a corresponding functional group. The nucleic acid molecules N bound to the fiber surface can be specifically detected using complementary nucleic acid molecules N′.
The fiber can in principle be formed from any filamentary 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.
Besides these, the fiber may also consist of inorganic materials such as, for example, glass, quartz or a metal, especially gold or platinum.
The nature of the linkage of the nucleic acid molecules N to the fiber surface depends on the chemical nature of the fiber material and the purpose for which the fiber is used. The nucleic acid molecules N are preferably connected via a defined linkage to the fiber. A defined linkage means in this connection a known chemical linkage. Undefined linkages as, for example, in UV-crosslinked DNA on nylon are, by contrast, linkages for which it is not possible to indicate the atoms on the nucleic acid molecules which is the origin of the linkage to the fiber. In addition, the number of linkages with which a nucleic acid molecule N is linked to the fiber is unknown. The linkage of the nucleic acid molecules N to the fiber via defined linkages has the advantage that the nature of the linkage of all the nucleic acid molecules N on the fiber is substantially identical. The nucleic acid molecules N can be coupled to the fiber at defined positions, so that the change caused by the linkage in the activity and the accessability of the nucleic acid molecules N is identical and known.
The nucleic acid molecule N can be linked via a covalent linkage to the fiber surface. The high linkage coefficient of a covalent linkage prevents simple removal of the nucleic acid molecules N from the fiber surface, e.g. by using a solvent. The nucleic acid molecule N is preferably linked via a carboxyl, phosphate, amino, thiol, psoralen, cholesteryl or digoxigenine group to the fiber surface.
The nucleic acid molecule is expediently linked to the fiber surface via a preferably streptavidin-containing intermediate layer. Such a linkage is particularly preferred because of its high affinity constants. This linkage cannot be broken even on use of a strong base such as sodium hydroxide solution. The intermediate layer may, however, also be a functionalized silane layer. For example, the nucleic acid molecules N can be linked to quartz/glass fibers via derivatized silanes. The fiber surface is silylated for this purpose. Nucleic acid molecules containing SH groups are capable of linkage to gold fibers.
Not all surface groups of the fiber which are suitable for linkage to a nucleic acid molecule need be saturated with a nucleic acid molecule. The free functional groups remaining after attachment of the nucleic acid molecules to the fiber surface can remain in this state or be saturated by suitable reactions. Free thiol groups can, for example, be oxidized to disulfides or be reacted with low molecular weight substances such as iodoacetamide.
Nucleic acid molecules N with in each case the same specific sequence or different nucleic acid molecules N1, N2, that is to say nucleic acid molecules with different sequence, can be linked to the fiber surface.
In addition, further nucleic acid molecules with nonspecific sequence can be linked to the fiber. If there is use of fibers to the surface of which nucleic acid molecules with different sequence are linked, the nucleic acid molecules N are preferably linked to defined regions of the fiber surface.
The diameter of the fibers can be from 100 nm to 100 μm. It is possible to use such fibers to produce security threads by employing known methods. However, it is also possible for the nucleic acid molecules N to be linked to the fibers only after production of the thread. This can expediently take place by chemical synthesis.
The security thread may 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. More than one of the physical properties of a security thread, for example length and tensile strength, are greater than those of one fiber. It is possible through suitable choice of particular parameters, for example number of fibers per thread, nature of the twisting and/or the use of different fibers, to adjust the properties of the security thread in a targeted manner. The security threads of the invention may be formed from fibers of different materials. In addition, besides fibers not modified with nucleic acid molecules it is also possible to employ fibers modified with different nucleic acid molecules N, N1, N2. The diameter of the security thread is expediently from 1 μm to 1 mm.
The security thread of the invention is expediently incorporated into textiles. The textiles may have safety threads modified with different nucleic acid molecules. The textiles are produced using known methods such as spinning, weaving, drawn-loop knitting, crocheting, knotting, macrame, sewing or embroidery. The nucleic acid-modified security threads may moreover form a pattern in the textile which can be detected by means of the complementary nucleic acid molecules N′. This pattern may be designed for example as geometric pattern in the form of a symbol or of a bar code.
The invention further provides a label, in particular for textiles, which comprises at least one security thread of the invention. The label may, of course, also comprise security threads modified with different nucleic acids. Such a label can be sewn for example into textiles, shoes or head coverings.
A particular embodiment provides for a nucleic acid microarray in the form of a matrix to be formed from a plurality of security threads. The matrix can be produced by techniques of textile processing of the security threads.
The invention further provides a forgery-proof marking where at least one security thread of the invention is applied to a basic article. The basic article may be produced from a fabric, paper or smooth agent which enables liquid to be transported to the security thread. It may furthermore have an absorbent pad. The aforementioned features enable for example an identification liquid to be transported directly to the security thread.
In a particularly advantageous embodiment, a plurality of security threads can be disposed in parallel. The provision of a plurality of security threads which are preferably modified with different nucleic acids increases the security of the marking against forgery.
A covering, which is preferably produced from a sheet of plastic, can be applied to the basic article. The covering expediently has a first orifice, preferably from a film of plastic, for applying detection liquid. The covering may further have a second orifice, preferably closed with a transparent sheet, for observing the security thread. After removal of the covering, the detection liquid can be applied to the basic article. There, it is transported by capillary forces to the at least one security thread. Complementary nucleic acid molecules present in the identification liquid are in this case preferably designed so that they hybridize with the nucleic acid molecules linked to the fiber surface at room temperature. The hybridization expediently brings about a fluorescence reaction. This can be identified optically through the covering by means of a reader or, if suitably designed, even with the naked eye.
The method provided by the invention for the forgery-proof marking of an object and for identifying the marking has the following steps:
According to a particularly advantageous feature of the embodiment, steps c and d are carried out on the marked object. It is therefore unnecessary to remove the security thread from the marked object. The proposed identification method can be carried out directly on the spot.
According to a further particularly advantageous embodiment, steps c and d are carried out in less than 5 min. This is achieved in particular through the nucleic acid molecules used for marking and identification hybridizing even at room temperature, i.e. in a temperature range from 18 to 25° C. In particular, no heating is necessary. Steps c and d can be carried out by using only a solution of suspension. This also simplifies the method. Finally, steps c and d can be carried out without a washing step. Overall, a method which can be carried out extremely simply, rapidly and cost-effectively, and which can be applied universally and can be carried out on the spot, for identifying a forgery-proof marking is indicated with the aforementioned features.
The detection can take place by means of specific hybridization and be carried out by means of a change, brought about as a result of hybridization, in the optical properties, preferably by fluorescence or color reactions. It is possible, for example, to use so-called molecular beacons which are in each case specific for a used sequence of the nucleic acid molecules and which change their fluorescence after a specific linkage, preferably at room temperature, with a complementary nucleic acid. Detection of the marking takes place in a particularly advantageous embodiment by applying a solution comprising molecular beacons. The molecular beacons have in this case nucleic acid molecules which are complementary to the nucleic acid molecules used for the marking. In the event of a hybridization, the linkage between the nucleic acid molecules and the nucleic acid molecules complementary thereto can take place directly on the marking by fluorescence measurement. A particular advantage of the use of molecular beacons is that the detection of the marking can take place without washing steps and directly on the marked object. It is a one-stage detection. A further advantage is that the detection is possible within only a few minutes.
In a further advantageous embodiment, the detection takes place by means of laminar flow. In this case, a solution or suspension which comprises marked nucleic acid molecules which are complementary to the nucleic acid molecules used for the marking is applied to an absorbent film of a support. The solution or suspension is transported by capillary forces to the security thread. The marked complementary nucleic acid molecule is firmly held on the security thread through hybridization. Excess complementary nucleic acid molecules are transported further by capillary forces. Detection of the marking takes place by linking the marked complementary nucleic acid molecules to the nucleic acid molecules immobilized on the security thread. In this case, the detection site and the site of application of the identifying means is different from one another. No washing steps are necessary in this method either. This identification can also take place on the marked object directly on the spot.
It is also possible to use other methods known for detecting the nucleic acid molecule N within the framework of in situ hybridization and of Southern or Northern blotting. These methods include methods resulting in a color reaction. For example, the hybridization probe can be coupled directly or indirectly to an enzyme which converts a substrate into an insoluble dye. This dye can then be detected as precipitate at the hybridization site. The specific hybridization can moreover also be detected by means of a hybridization probe which is linked directly or indirectly to particles. Immobilization of the particles at the hybridization site is then utilized for detecting the specific hybridization.
The linkage of different nucleic acid molecules N, N1, N2 to one or more fibers makes unauthorized copying or counterfeiting of the marking difficult. For this purpose it is also possible to link nonspecific nucleic acid sequences to the fiber surface so that a nonspecific nucleic acid detection does not lead to the specific marking being revealed.
The sequence of the nucleic acid molecules immobilized on the security threads should be known only to authorized persons. Marking of objects with such security threads can take place at a particular position or in the object. It may additionally have optically visible markings.
The security threads thus make it possible for textiles, especially items of clothing, to be identified in a forgery-proof way. For this purpose it is expedient to incorporate at least one security thread into the label fastened to the textile. It is essential in connection with the present invention that the nucleic acid molecules are linked to the fibers forming the security thread before the processing of the security threads.
The security threads can also be used to produce 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 of the security threads. These fabrics can thus be employed as nucleic acid microarrays. They have the advantage that they can be produced relatively easily and cost-effectively.
A matrix in the form of a textile fabric can be formed by processing the nucleic acid-modified security threads by textile-processing methods such as weaving, knitting, crocheting, knotting, sewing or embroidery.
The security threads may, however, also be applied at different positions to a solid matrix in a particular arrangement, for example brush-like or cluster-like, without forming a fabric. It is possible to use as matrix for example a plastic surface through which the security threads are drawn in defined positions perpendicular to the surface.
A nucleic acid microarray is thus produced by producing nucleic acid-modified security threads by attaching particular nucleic acid molecules N at defined zones 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 zones. It is moreover possible to use fibers modified differently with nucleic acids. The nucleic acid-modified security threads may comprise different nucleic acid-modified fibers and also fibers not modified with nucleic acids. The security threads have the abovementioned properties.
Examplary embodiments of the invention are explained in more detail below by means of the drawings. These show
In a second step, nucleic acid molecules N are linked to the free linker groups L, resulting in the nucleic acid-modified fiber FN. For this purpose, the nucleic acid molecules N are expediently modified with coupling groups K. Examples of suitable coupling groups K are free SH or amino groups. Nucleic acid molecules N with such coupling groups K can be obtained by oligonucleotide synthesis. The coupling group K is preferably located in the 3′ or 5′ end of the nucleic acid molecule N. The terminal position of the coupling group K makes it possible for the accessibility of the nucleic acid N to be good on hybridization with a complementary strand. The linkage of the nucleic acid N to the fiber F can, however, also take place via homo- or heterofunctional crosslinkers.
Linkage to cellulose-containing fibers is possible through oxidation of sugars to aldehydes. The aldehydes can be covalently linked to amino-containing nucleic acid molecules to give Schiff's bases and subsequently reduced to amides.
Polycarbonate fibers can be linked by means of carbodiimide to amino-containing nucleic acid molecules N. Other plastics such as polypropylene can be covalently linked to nucleic acid molecules N after plasma activation. Gold threads can be linked to thiol group-containing nucleic acid molecules N. Glass or quartz fibers can be activated by silanization and subsequently connected to the nucleic acid molecules N.
The linker group L can also be linked to the fiber F via a spacer. Spacers which can be used are polyglycol, polyimine, dextran, polyether. It is possible with the aid of the spacers to minimize steric hindrance of the nucleic acids N on hybridization, to generate a particular surface charge, to reduce a nonspecific linkage to the fiber F and to increase the number of coupling groups K for the nucleic acid molecules N.
The security threads Fd are in contact with the basic article 2 so that liquid applied to the basic article 2 is able to be transferred. The absorbent pad 3 absorbs most of the applied liquid, so that only a minimal amount of liquid remains in the basic article 1. For this purpose, the absorbent pad 3 is formed from a fabric with a high liquid-binding capacity.
The marking 1 may comprise one or more nucleic acid-modified security threads Fd. Nucleic acid molecules N with different, but known, sequences can be linked to the security threads Fd. In addition, unknown substances such as, for example, randomly generated DNA may be linked in order to make analysis of the security threads Fd difficult. Such randomly generated substances do not bind any detection liquid so that one for control of the detection liquid is possible. In addition, security threads which comprises a substance, such as, for example, DEAE-cellulose, which links nucleic acid molecules nonspecifically can be used. A security thread of this type, which links any nucleic acid molecules N′, is used for controlling the detection liquid. The use of a plurality of security threads Fd makes it possible to form complex markings. It is possible by using a plurality of differently modified security threads Fd to form a pattern which is revealed only when all nucleic acid molecules on the security threads Fd are identified. The security threads Fd may also form geometric patterns, for example numbers, letters, symbols, bar codes.
As depicted in
The production of nucleic acid-modified fibers and their detection is explained below by means of an example.
1) Aldehyde Activation of Cellulose Fibers
100 mg of washed cotton (Machery and Nagel) is incubated in 10 ml of 100 mM NaIO4 in PBS (pH 7.4) at 37° C. overnight. The NaIO4 is removed by washing with 10 ml of PBS five times.
2) Linkage of Amino-oligonucleotides to Aldehyde-activated Cellulose Fibers
10 μm of synthetic oligonucleotide N which has a free amino group at the 5′ end are added to the aldehyde-activated cotton. The mixture is brought to 20 mM sodium cyanoborohydride in a final volume of 2 ml and incubated at room temperature with agitation overnight. To saturate free aldehyde groups, 2 ml of 1 M trisCl (pH 8) are added and incubated at room temperature for a further 2 h. Unbound oligonucleotides N are removed by washing the cotton five times with 10 ml of TBS and incubating in 10 mM tris acetate, 1 mM EDTA (pH 8) in an electrophoresis chamber at a voltage of 100 V for 1 h.
3) Hybridization of Oligonucleotides Linked to Cellulose Threads
Cellulose threads with oligonucleotides N linked thereto are incubated in 100 μl of 10 mM TrisCl, 1 mM EDTA with 1 μM complementary oligonucleotides N′ at 37° C. for 30 min. The oligonucleotides N′ have a sequence complementary to the oligonucleotides N and are marked with a biotin group at the 5′ end. Unbound oligonucleotides are removed by washing the cotton five times with 10 ml of TBS and incubating in 10 mM tris acetate, 1 mM EDTA (pH 8) in an electrophoresis chamber at a voltage of 100 V for 1 h.
4) Detection of the Hybridization
Cellulose threads with hybridizated oligonucleotides N, N′ are incubated with streptavidin-coated, superpara-magnetic particles (Dynal) in 1 ml of 10 mM trisCl, 150 mM NaCl, 1 mM EDTA (pH 8) (TBST) at room temperature with agitation for 1 h. Unlinked particles are removed by washing five times with 1 ml of TBST with the assistance of a magnet. The linkage of the particles to the fibers shows the hybridization of the oligonucleotide N′ to oligonucleotide N.
5) Silanization and Aldehyde Activation of a Cotton Thread
About 2 m of cotton thread was pretreated by incubating in 30 ml of 10% sodium dodecysulfate (SDS) at 55° C. for one hour. The SDS was removed by washing in 30 ml of water five times. For the silanization, the thread was divided and in each case 50% of the thread was incubated for one hour in 1% each of a) 3-aminopropyl-methyl-diethoxysilane (Fluka) or b) 3-amino-propyl-triethoxysilane (ABCR) in ethanol at 55° C. for one hour. The threads were washed in ethanol and dried at 85° C. for one hour. For linkage of aldehyde groups, the threads were incubated in 20 ml of 5% glutaraldehyde at room temperature (RT) for one hour and thoroughly washed with water to remove excess glutaraldehyde.
6) Coupling of Amino Oligonucleotides to Silanized Cotton Threads
The threads were washed in 0.1 M Na2CO3 solution of pH 9.5 and in each case 10 cm of the threads were incubated in 1 ml of 1 fM oligonucleotide N in in 0.1 M Na2CO3 solution at RT for four hours. Aldehyde groups still present were saturated by bringing the suspension to 1% ethanolamine and 2 mM EDTA and incubating at RT overnight. Nonlinked oligonucleotides were removed by thorough washing in 1% SDS solution in 10 mM trisHCl, 1 mM EDTA, pH 8.8.
The oligonucleotides used were the following synthetic nucleic acids:
In each case about 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 of PBS and reacted with peroxidase substrate (Roche). Within five minutes, the threads marked with the biotinylated oligonucleotide were visibly dark-colored. Threads without biotinylated oligonucleotide showed no color.
8) Detection of the Linkage of the Oligonucleotides by Hybridization Using Molecular Beacons
In each case about 20 mm of the threads were sewn using a needle into a cotton fabric. 5 μl of a 2 fM solution of a molecular beacon were added dropwise to one end of each thread. The fluorescence of the threads was excited about 5 mm from the addition using a red laser light (wavelength=650 nm) and determined using a fluorescence reader. Within two minutes there was a measurable increase in the fluorescence, by more than 40% above the background fluorescence without addition of molecular beacon, for the threads with oligonucleotides of complementary sequence (NH2-T9-Target1-28). The threads with oligonucleotides not of complementary sequence (N-T1k-Biotin) showed an increase in fluorescence of only about 10% above the background signal.
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|Cooperative Classification||Y10T428/2913, G07D7/14, D06M15/01, C12Q1/6813|
|European Classification||D06M15/01, C12Q1/68B, G07D7/14|
|Feb 3, 2004||AS||Assignment|
Owner name: NOVEMBER AKTIENGESELLSCHAFT GESELLSCHAFT FUR MOLEK
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:KOSAK, HANS;JOSTEN, ANDRE;REEL/FRAME:014303/0260
Effective date: 20040114
|May 5, 2010||AS||Assignment|
Owner name: SECUTECH INTERNATIONAL PTE. LTD.,SINGAPORE
Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNOR:NOVEMBER AKTIENGESELLSCHAFT GESELLSCHAFT FUR MOLEKULARE MEDIZIN;REEL/FRAME:024335/0331
Effective date: 20100406