U.S. patent application number 10/477158 was filed with the patent office on 2005-09-29 for secueity thread for the forgery-proof making of objects.
Invention is credited to Josten, Andre, Kosak, Hans.
Application Number | 20050214532 10/477158 |
Document ID | / |
Family ID | 7684359 |
Filed Date | 2005-09-29 |
United States Patent
Application |
20050214532 |
Kind Code |
A1 |
Kosak, Hans ; et
al. |
September 29, 2005 |
Secueity thread for the forgery-proof making of objects
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
suface and 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).
Inventors: |
Kosak, Hans; (Bonn, DE)
; Josten, Andre; (Nurnberg, DE) |
Correspondence
Address: |
FISH & RICHARDSON P.C.
PO BOX 1022
MINNEAPOLIS
MN
55440-1022
US
|
Family ID: |
7684359 |
Appl. No.: |
10/477158 |
Filed: |
July 16, 2004 |
PCT Filed: |
May 8, 2002 |
PCT NO: |
PCT/EP02/05079 |
Current U.S.
Class: |
428/364 |
Current CPC
Class: |
D06M 15/01 20130101;
C12Q 1/6813 20130101; Y10T 428/2913 20150115; G07D 7/14 20130101;
C12Q 2563/185 20130101; C12Q 2527/125 20130101; C12Q 2563/155
20130101; C12Q 1/6813 20130101 |
Class at
Publication: |
428/364 |
International
Class: |
D02G 003/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 11, 2001 |
DE |
101228368 |
Claims
1. A security thread for the forgery-proof marking of objects
having at least one fiber (F), where one end of one or more nucleic
acid molecules (N) are linked to a fiber surface of the fibers (F),
and where the other end of the nucleic acid molecules (N) is free,
so that complementary nucleic acid molecules (N') are able to
undergo linkage to the nucleic acid molecules (N).
2. The security thread as claimed in claim 1, where the fiber (F)
is formed from a natural polymer.
3. The security thread as claimed in claim 2, where the natural
polymer is selected from the following group: cellulose, chitin,
silk, wool, cotton, hemp, flax or derivatives of these
polymers.
4. The security thread as claimed in claim 1, where the fiber (F)
is formed from a synthetic polymer.
5. The security thread as claimed in claim 4, where the synthetic
polymer is selected from the following group: polyamide,
polyacrylonitrile, nylon, polypropylene, polyvinylidene fluoride,
polycarbonate, polystyrene or derivatives of these polymers.
6. The security thread as claimed in claim 1, where the nucleic
acid molecule (N) is linked via a covalent or noncovalent linkage
to the fiber surface.
7. The security thread as claimed in claim 6, where the nucleic
acid molecule (N) is linked via a biotin/streptavidin linkage, a
carboxy, phosphate, amino, thiol, psoralen, cholesteryl or
digoxigenine group to the fiber surface.
8. The security thread as claimed in claim 1, where the nucleic
acid molecule (N) is linked via an intermediate layer to the fiber
surface.
9. The security thread as claimed in claim 8, where the
intermediate layer is a functionalized silane layer.
10. The security thread as claimed in claim 1, where different
nucleic acid molecules (N, N1, N2) are linked to the fiber
surface.
11. The security thread as claimed in claim 10, where the different
nucleic acid molecules (N) are linked on defined zones of the fiber
surface.
12. The security thread as claimed in claim 1, where the nucleic
acid molecule (N) is produced by chemical synthesis directly on the
fiber.
13. The security thread as claimed in claim 1, where the diameter
of the fiber (F) is from 100 nm to 100 .mu.m.
14. The security thread as claimed in claim 1, further comprising
at least one second fiber.
15. The security thread as claimed in claim 14, where the second
fiber consists of another material.
16. The security thread as claimed in claim 1, where the diameter
of the security thread is from 1 .mu.m to 1 mm.
17. A textile having at least one security thread (Fd) as claimed
in claim 1.
18. A textile as claimed in claim 17, which comprises security
threads (Fd) modified with different nucleic acid molecules.
19. The textile as claimed in claim 17, where nucleic acid-modified
security threads (Fd) form a pattern which can be detected by means
of the complementary nucleic acid molecules (N').
20. A label where the label comprises at least one security thread
(Fd) as claimed in claim 1.
21. The label as claimed in claim 20, where the security threads
(Fd) are modified with different nucleic acid molecules.
22. The label as claimed in claim 21, where a nucleic acid
microarrangement in the form of a matrix is formed from a plurality
of security threads.
23. The label as claimed in claim 22, where the matrix is produced
by techniques of textile processing of the security threads
(Fd).
24. A forgery-proof marking, where at least one security thread
(Fd) as claimed in claim 1 is applied to a basic article (2).
25. The forgery-proof marking as claimed in claim 24, where the
basic article (2) is produced from a fabric, paper or flow agent
which makes transport of liquid to the security thread (Fd)
possible.
26. The forgery-proof marking as claimed in claim 25, where the
basic article (2) has an absorbent pad (3).
27. The forgery-proof marking as claimed in claim 24, where a
plurality of security threads (Fd) are arranged in parallel.
28. The forgery-proof marking as claimed in claim 24, where the
covering (4) is applied to the basic article (2).
29. The forgery-proof marking as claimed in claim 28, where the
covering (4) has a first orifice (4a) for application of detection
liquid.
30. The forgery-proof marking as claimed in claim 29, where the
covering (4) has a second orifice (4b) for observing the security
thread (Fd).
31. A method for identifying a forgery proof marking on an object
having the following steps: a) providing an object comprising at
least one security thread (Fd) as claimed in claim 1, b) bringing
the security thread (Fd) into contact with an indicator comprising
the complementary nucleic acid molecules (N') and c) detecting the
specific linkage of the complementary nucleic acid molecules (N')
to the nucleic acid molecules (N) on the object.
32. (canceled)
33. The method as claimed in claim 31, where steps c and d are
carried out in less than 5 minutes.
34. The method as claimed in claim 31, where only a solution or
suspension is used for carrying out steps c and d.
35. The method as claimed in claim 31, where steps c and d are
carried out without a washing step.
36. The method as claimed in claim 31, where detection is carried
out by means of specific hybridization and by a change in optical
properties brought about as a result of the hybridization.
37. (canceled)
38. The method as claimed in claim 31, where the detection is
carried out by means of an enzymatic reaction and by a change in
optical properties brought about as a result of the enzymatic
reaction.
39. The method as claimed in claim 31, where the detection is
carried out by means of laminar flow.
40. The method as claimed in claim 31, where the complementary
nucleic acid molecules (N') are linked to micro- or
nanoparticles.
41. The method as claimed in claim 31, where a pattern formed by at
least one security thread (Fd) is identified in the detection.
42. The method as claimed in claim 41, where the pattern is formed
by a plurality of security threads (Fd) which are provided with
different nucleic acid molecules (N, N1, N2).
43. The method as claimed in claim 41, where the pattern is formed
by formed-loop knitting, weaving, drawn-loop knitting, crocheting,
knotting, sewing or embroidery.
44. The security thread as claimed in claim 8, where the
intermediate layer contains streptavidin.
45. The forgery proof marking as claimed in claim 28, where the
covering (4) is plastic.
46. The method as claimed in claim 36, where the optical properties
are fluorescence or a color reaction.
47. The method as claimed in claim 46, wherein the detection is
carried out by means of a molecular beacon.
48. A method for the forgery proof marking of an object having the
following steps: a) providing the object, and b) contacting the
object with the security thread as claimed in claim 1.
Description
[0001] 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.
[0002] 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.
[0003] 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.
[0004] 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.
[0005] 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.
[0006] 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.
[0007] 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.
[0008] 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.
[0009] 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.
[0010] 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'.
[0011] 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.
[0012] Besides these, the fiber may also consist of inorganic
materials such as, for example, glass, quartz or a metal,
especially gold or platinum.
[0013] 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.
[0014] 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.
[0015] 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.
[0016] 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.
[0017] 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.
[0018] 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.
[0019] The diameter of the fibers can be from 100 nm to 100 .mu.m.
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.
[0020] 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 .mu.m to 1 mm.
[0021] 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.
[0022] 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.
[0023] 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.
[0024] 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.
[0025] 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.
[0026] 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.
[0027] The method provided by the invention for the forgery-proof
marking of an object and for identifying the marking has the
following steps:
[0028] a) providing at least one security thread of the
invention,
[0029] b) providing the object with the security thread,
[0030] c) bringing the security thread into contact with an
indicator comprising the complementary nucleic acid molecules
and
[0031] d) detection of the specific linkage of the complementary
nucleic acid molecules to the nucleic acid molecules on the
object.
[0032] 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.
[0033] 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.degree. 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.
[0034] 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.
[0035] 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.
[0036] 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.
[0037] 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.
[0038] 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.
[0039] 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.
[0040] 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.
[0041] 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.
[0042] 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.
[0043] 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.
[0044] Examplary embodiments of the invention are explained in more
detail below by means of the drawings. These show
[0045] FIG. 1 a directed linkage of nucleic acid molecules N to
fibers,
[0046] FIG. 2 a specific detection of nucleic acid molecules N by
complementary nucleic acid molecules N',
[0047] FIG. 3 a production of a fiber to which different nucleic
acid molecules N are linked at defined sections,
[0048] FIG. 4 a specific detection of different nucleic acid
molecules N in a fiber by complementary nucleic acid molecules
N',
[0049] FIG. 5 a synthesis of nucleic acid molecules N on a
fiber,
[0050] FIG. 6 a parallel synthesis of different nucleic acid
molecules N on a fiber,
[0051] FIG. 7a to c a parallel production of nucleic acid arrays on
planar support materials,
[0052] FIG. 8a to c diagrammatically a method for producing nucleic
acid-modified threads from fibers,
[0053] FIG. 9a to c a first embodiment of a marking with nucleic
acid-modified threads,
[0054] FIG. 10a to d a detection of the marking shown in FIG. 9
and
[0055] FIG. 11a to b a second embodiment of a marking with nucleic
acid-modified threads.
[0056] FIG. 1 depicts diagrammatically the directed linkage of
nucleic acid molecules N on a fiber F. In a first step, linker
groups L which are suitable for coupling to activated nucleic acid
molecules N are produced on the surface of the fiber F by a
suitable activation or reaction. This step is unnecessary if the
fiber surface already has suitable functional groups. In the case
of wool or silk fibers, for example, free cystein or amino groups
are suitable for coupling activated nucleic acid molecules N.
Alternatively, SH groups in the wool or silk proteins can be
generated by reducing disulfide groups.
[0057] 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.
[0058] 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.
[0059] 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.
[0060] 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.
[0061] FIG. 2 depicts the detection of the nucleic acid molecules
N, which are linked to the fiber F, by hybridization. For this
purpose, the nucleic acid-modified fiber FN is brought into contact
with nucleic acid molecules N' which have a sequence complementary
to the nucleic acid molecules N. The complementary nucleic acid
molecules N' may have a signal group S. It is possible by means of
the signal group S for example to generate an altered electrical or
optical signal after the hybridization. This signal group S may be
a fluorophor, an antigen or an enzyme. The signal group S is
immobilized at the hybridization site (bottom of FIG. 2) through
the hybridization of the complementary nucleic acid molecules N'
with the nucleic acid molecules N, and can be detected on the basis
of its properties.
[0062] FIG. 3 shows the production of a fiber FN to which different
nucleic acids N1, N2, N3 are linked on defined sections. A fiber F
having linker groups L is divided into spatially separate reaction
zones RB1, RB2, RB3. Each reaction zone is reacted separately with
different nucleic acids N1, N2, N3. The coupling results in a fiber
FN1N2N3 with different nucleic acids N1, N2, N3 being linked on
defined sections.
[0063] FIG. 4 shows the specific detection of different nucleic
acid molecules N1, N2, N3, which are linked on different sections
of a fiber FN1N2N3, by hybridization with nucleic acid molecules
N'1, N'2, N'3 complementary thereto. For the 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 depicted
by means of the signal groups S1, S2, S3 linked to the nucleic acid
molecules N1, N2, N3. The signal groups S1, S2, S3 may be identical
or different groups. Patterns can be generated on the fiber through
the use of different signal groups S1, S2, S3, for example
fluorophors.
[0064] FIG. 5 depicts 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 construction blocks Ba for attaching further
nucleotides. In further steps, in each case an activated nucleotide
(b, c, d, e, f, g) is attached to the nucleotides already linked.
The synthesis results in a fiber FBabcdefg to a defined sequence of
nucleotides is fixed.
[0065] FIG. 6 shows a parallel synthesis of different nucleic acids
N on a fiber F. For this purpose, the fiber F is divided into
reaction zones RB1, RB2, RB3. Each section is separately reacted
with different activated construction blocks Ba1, Ba2, Ba3. In
further steps, in each case one activated nucleotide (b, c, d, e,
f, g) is attached to the previously immobilized nucleotide in each
reaction zone. The synthesis results in a fiber FN1N2N3 to which
different oligonucleotides N1, N2, N3 are linked in defined
sections.
[0066] FIG. 7a to c show the parallel production of nucleic acid
arrays on planar support materials M. For this purpose, a number n
of planar support materials M is placed one on top of the other
(FIG. 7a). Different nucleic acid-modified fibers FN1, FN2, FN3.
are passed through the support materials M at defined positions.
Subsequently, the security threads between the support materials M
are severed (FIG. 7b). This results in a support on which
oligonucleotides are immobilized on security threads at defined
positions.
[0067] FIG. 8a to c show diagrammatically a method for producing
security threads Fd from fibers F. The fibers shown in FIG. 8a are
modified with nucleic acid molecules N, resulting in fibers FN
(FIG. 8b). The fibers FN are spun to threads Fd in a spinning
machine S (FIG. 8c).
[0068] FIG. 9a to c depicts 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 basic article 2. An absorbent pad 2 is also located on
the basic article 2. The basic article 2 is formed from a matrix
which enables lateral flow of a liquid. If the basic article 2 is
contacted with a liquid, it transports the liquid to the applied
security threads Fd and the absorbent pad 3. The basic article 2
may be formed of a fabric, absorbent paper or flow agent, which are
expediently applied to a liquid-impermeable sheet of plastic. The
sheet of plastic prevents liquid escaping into the surroundings and
protects the marking. An adhesive sheet of plastic can be used to
apply marking 1 to the object to be marked.
[0069] 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.
[0070] 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.
[0071] FIG. 9b shows the marking 1 with a covering 4. The covering
4 expediently consists of an opaque material which covers the basic
article 2 and has two cutouts 4.1 and 4.2. The covering 4 serves to
protect the marking 1 from mechanical stress, chemical stress or
radiation. The first cutout 4.1 indicates the place where a
detection liquid is supplied to detect the marking 1. Cutout 4.1
can be closed for example with a film of plastic and be opened only
when an identification of the marking 1 is to take place. Such a
closure serves to protect from soiling, for example by grease or
similar substances which might impair flow characteristics of the
basic article 1. The second cutout 4.2 forms a viewing window which
makes it possible to look at the security threads Fd. The viewing
window can be closed with a transparent sheet to protect the
security threads Fd. The security threads Fd are then evidenced
through the viewing window 4.2, as depicted in FIG. 9c. Before
identification of the marking 1, the security threads Fd may be
invisible to the eye.
[0072] FIG. 10a to d depicts a method for detecting the marking 1
shown in FIG. 9, with the marking 1 being depicted in FIG. 10a to c
without covering for better visualization.
[0073] As depicted in FIG. 10a, a defined volume of a detection
liquid is added to the basic article 2 through the cutout 4.1 to
identify the marking 1. The detection liquid comprises nucleic acid
molecules N' which are complementary to the nucleic acid molecules
N linked to the security threads Fd. The nucleic acid molecules N'
used for the detection may be marked with dye molecules,
fluorogens, gold or latex particles, so that detection of a
specific hybridization with nucleic acid molecules N is
facilitated. The complementary single-stranded nucleic acid
molecule N' used for the detection preferably has a back-folding.
This increases the specificity of the hybridization.
[0074] In FIG. 10b, the lateral flow of the detection liquid in the
direction of the absorbent pad 3 is indicated by an arrow. During
the flow, the detection liquid comes into contact with security
threads Fd. FIG. 10c shows the basic article 2 after the end of the
lateral flow. Most of the detection liquid has been absorbed by the
absorbent pad 3. Security threads Fd to which the nucleic acid
molecules N' are linked are depicted by the thicker lines.
[0075] FIG. 10d shows the marking 1 with covering 4 after the
identification of the marking 1. The security threads Fd which are
shown thicker indicate the detection of the marking 1. The
detection process is complete within a few seconds to minutes. On
use of colored particles, such as gold particles, to which the
nucleic acid molecules N' are linked, only the eye, and no further
aid such as, for example, a photometric detector, is necessary for
detecting the marking. It is thus possible to dispense with toxic
or radioactive components.
[0076] FIG. 11a to b shows 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 one absorbent pad 3
are disposed on the basic article 2. A covering 4 having two
cutouts 4.1, 4.2 is disposed on the basic article 2 (FIG. 11b).
Cutout 4.1 is used for applying the detection liquid to the
security threads Fd. Cutout 4.2 is used for observing the detection
of the marking 1. In this embodiment, the security threads Fd
themselves serve for lateral flow of detection liquid.
[0077] The production of nucleic acid-modified fibers and their
detection is explained below by means of an example.
[0078] 1) Aldehyde Activation of Cellulose Fibers
[0079] 100 mg of washed cotton (Machery and Nagel) is incubated in
10 ml of 100 mM NaIO.sub.4 in PBS (pH 7.4) at 37.degree. C.
overnight. The NaIO.sub.4 is removed by washing with 10 ml of PBS
five times.
[0080] 2) Linkage of Amino-oligonucleotides to Aldehyde-activated
Cellulose Fibers
[0081] 10 .mu.m 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.
[0082] 3) Hybridization of Oligonucleotides Linked to Cellulose
Threads
[0083] Cellulose threads with oligonucleotides N linked thereto are
incubated in 100 .mu.l of 10 mM TrisCl, 1 mM EDTA with 1 .mu.M
complementary oligonucleotides N' at 37.degree. 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.
[0084] 4) Detection of the Hybridization
[0085] 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.
[0086] 5) Silanization and Aldehyde Activation of a Cotton
Thread
[0087] About 2 m of cotton thread was pretreated by incubating in
30 ml of 10% sodium dodecysulfate (SDS) at 55.degree. 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.degree. C.
for one hour. The threads were washed in ethanol and dried at
85.degree. 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.
[0088] 6) Coupling of Amino Oligonucleotides to Silanized Cotton
Threads
[0089] The threads were washed in 0.1 M Na.sub.2CO.sub.3 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 Na.sub.2CO.sub.3 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.
[0090] The oligonucleotides used were the following synthetic
nucleic acids:
[0091] a) N-Tlk-Biotin:
[0092] Biotin-5> gca aca aga cca cca ctt cga aac 3>-C6-amino
link
[0093] b) NH2-T9-Target1-28
[0094] 5'-C6-amino link-TTT TTT TTT CCA AGC CTG GAG GGA TGA TAC TTT
GCG C-3'
[0095] 7) Detection of the Linkage of the Biotinylated
Oligonucleotides to the Threads
[0096] 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.
[0097] 8) Detection of the Linkage of the Oligonucleotides by
Hybridization Using Molecular Beacons
[0098] In each case about 20 mm of the threads were sewn using a
needle into a cotton fabric. 5 .mu.l 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
(NH.sub.2-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.
[0099] Sequence of the molecular beacon
[0100] 5'-Cy5-CCA AGC GCA AAT TAT CAT CCC TCC AGG CTT GG-BHQ2-3'
Sequence CWU 1
1
3 1 24 DNA Artificial Sequence Oligonucleotide 1 gcaacaagac
caccacttcg aaac 24 2 36 DNA Artificial Sequence Oligonucleotide 2
tttttttttc caagcctgga gggatgatac tttgcg 36 3 32 DNA Artificial
Sequence Oligonucleotide 3 ccaagcgcaa agtatcatcc ctccaggctt gg
32
* * * * *