The way photochromic marking and/or ensure the authenticity of objects and composition for its implementation
The invention relates to a method for ensuring the authenticity of the subject by applying to a photochromic ink. To implement the method of the photochromic marking and/or ensure the authenticity of the items, and get the composition for its implementation used materials from bacteriorhodopsin and made from them a song. Such materials and compositions used for drawing objects, which use the characteristics of the low and high protection degree, preferably in combination. This provides optical detection protective trait, which under the action of light of the visible range of wavelengths may discolour or fade. 4 N. and 19 C.p. f-crystals, 2 Il.
The invention relates to a method for ensuring the authenticity of the subject by applying to a photochromic ink.
Protective technical measures to ensure the authenticity of documents or objects include the use of appropriate protective signs or markings of authenticity. The use of photochromic materials for protective purposes described, for example, in US 4927180. Photochromic identifying characteristic and Hiroshi sign is not recognized or badly recognized as such, consequently there is a risk that the user will not notice the absence of the identifying characteristic. Due to the use of ultraviolet light to the eyes of the Comptroller of authenticity requires appropriate protection. Therefore, the use of ultraviolet light to identify protective trait can be considered suboptimal. Such prior art is described in US 5807625. It also uses UV light to visualize the protective trait.
Organic photochromic materials disclosed in these documents are characterized by the typical number of cycles equal to 104-105. In the limited number of possible test operations on the identification of the protective trait. Therefore, the use of automatic methods of testing, as it is practiced in cash machines or at the entrance gate, for these security features is impossible or is possible only conditionally.
In addition, it is desirable to present an opportunity to identify specific portions of the marking composition and, for example, in case of loss or unauthorized use intended for protection circuits compositions can install their origin. Shchev.
Traditional photochromic materials have, in addition, the disadvantage that one of them both switching States are not characterized by marked its own color.
The objective of the invention is the creation of optically detectable protective trait, which under the action of light of the visible range of wavelengths may discolour or fade. In result, it becomes possible to cause the change of color with the use of cheap and commonly available light sources such as LEDs. In this case, the test would have been possible even using a conventional tube light and detect - to the naked eye. In addition, it is desirable to create a protective sign, characterized by the number of switching cycles in excess of 104- 105.
Another objective is the provision of a variety of structurally similar photochromic materials that differ from each other by their color and/or color change.
If known from the prior art security features can be identified due to the low technical effort to scan them as signs of a low degree of protection, hence the following problem: in addition, to ensure the signs of a high degree of protection,m
In WO 9806084 disclosed the use of nucleic acid molecules, particularly DNA molecules, as an invisible sign of a high degree of protection, which can be detected by appropriate amplification reactions, such as PCR reaction using specific seed.
According to the present invention was able to detect the material, such as bacteriorhodopsin, which can be combined in a sign of low degree of protection as, for example, photochromic, and the sign of a high degree of protection, allowing, for example, to identify the individual portions of the marking composition, and to use this material and to determine the authenticity of items.
The way to ensure the authenticity of items containing bacteriorhodopsin ink according to the invention set forth in paragraph 1 of the claims, and dependent claims listed preferred options for implementation.
The above problems are solved according to the invention by way of ensuring the authenticity of the subject by applying a photochromic ink on the subject, using photochromic ink containing at least one kind of bacteriorhodopsin as photochrom UEMOA, reversible change of state, used as a sign of low degree of protection while checking authenticity, in particular the color change, and which in addition to the characteristic low protection level has one or more are not visually detectable signs of a high degree of protection, which is (are) detected only by using instrumental analysis.
The object of the present invention is therefore the use of photochromic ink in a way to ensure the authenticity of the subject. Used ink according to the invention contain as a photochromic component at least one kind of bacteriorhodopsin. This option bacteriorhodopsin provides both visually detectable when checking on the authenticity of the sign of the low level of protection, and the inherent additional sign of a high degree of protection, detectable only by means of instrumental analysis.
The term "photochrome" means caused by light reversible change of state (in particular the change of color) of a substance which changes color (absorption spectrum) of the original substance. Then may be caused by the reverse reaction, naprorochila as photochromic component, which when exposed to light of the visible wavelength range changes its state, the need for irradiation with UV light according to the invention eliminates. The result can be eliminated associated with ultraviolet irradiation disadvantages, in particular fall associated requirements for equipment and protective measures.
Used according to the invention variants bacteriorhodopsin preferred are those in which the staining occurs when both, particularly preferably under all switching conditions.
Therefore, one aspect of the invention relates to a method of ensuring the authenticity of the items, and the photochromic composition in the ink containing bacteriorhodopsin and/or option bacteriorhodopsin as photochromic component, is applied to the subject, and the exposure of such photochromic composition with light of the visible wavelength range leads to a state change, in particular to a colour change that can be detected by checking authenticity. Find out the status, in particular the change of color, should preferably be reversible, and used according to the invention options bacteriorhodopsin sup>5more preferably >106and particularly preferably >107. In result, it becomes possible re-inspection protect the authenticity of the object by a low degree of protection in the framework of routine activities. If the status change becomes irreversible, for example, as a result of destruction photochrome-active bacteriorhodopsin, a sign of low degree of protection can be neutralized, i.e. destructed.
Check for authenticity is carried out preferably by irradiation of the photochromic ink visible light for discoloration bacteriorhodopsin, then photochromic ink is irradiated with light of a different wavelength range, so that a photochemical method to translate the bacteriorhodopsin in its original state, or is thermal relaxation for translation in neobeschannoe state. The change in optical properties during bleaching and/or damping can be observed with the naked eye or with optical measuring device.
Under the sign of low degree of protection refers to the characteristic, the presence or absence of which can be verified by the layman without the use of auxiliary means in a simple way, or p is AK, the presence or absence of which the layman cannot be determined and which can be verified only by specialists with a large technical expenditure.
Therefore the signs of low-grade protection are such signs, the analysis of which is spent small amount of funding (penny costs), which may be conducted by any person, at that time, as signs of a high degree of protection are the signs, the analysis of which can cost several hundred thousand DM and carried out by experts in laboratory conditions. Symptoms of low degree of protection provide protection from the methods of falsification "anyone and everyone", because fotochrome of novospassovka using known techniques. Indications of a high degree of protection include the individualization of individual protective paints intended for use or for user, up to the level of coding portions of the composition.
For example, photochrome options bacteriorhodopsin, i.e., change in color, easily traceable by an observer under irradiation of visible light, is a sign of low degree of protection. Other visually detectable signs of low-grade protection are naprosy properties.
In contrast to signs of a low degree of protection that simply perceived visually by any person, signs of a high degree of protection are tested only with the help of technically sophisticated analytical instruments, i.e., through instrumental analysis. Therefore to check for signs of a high degree of protection necessary technical AIDS. So, for example, replacement of amino acids in the sequence of bacteriorhodopsin leads to variants, the weight of which, deviating from the original type, can be identified via mass spectrometry. There is also an opportunity to form by coupling of the atoms and/or molecules options bacteriorhodopsin that can be detected, for example, on the basis of the difference between their masses, their model of fragmentation or other distinguishing characteristics, for example, by electron paramagnetic resonance, or NMR.
As a result of replacement of amino acids can be provided in particular by the following features high degree of protection. In the case of detection by liquid chromatography - mass spectrometry/mass spectrometry (e.g., ESI), you can measure the change in molecular weight and/or characteristic changed blondage or fluorescense detection can be detected by characteristic changes of the peptide splits, for example, dividing or joining the point of intersection, resulting in the number of fragments either increases or decreases. Using these methods it is also possible to detect the change in the number of aromatic amino acids. Connection-specific monoclonal antibodies to the sequence of bacteriorhodopsin or portions thereof can be detected by enzyme immunosorbent assay or similar methods.
Examples of indications of a high degree of protection, the resulting clutch, serve as spin labels that are detectable by electron paramagnetic resonance, and the modifications introduced by preteenmodelnude reagents, labeled stable isotopes (e.g.,13C,15N).
Used according to the invention the ink attached to the protected object as a sign of low degree of protection, for example, photochromic, and the sign of a high degree of protection, as, for example, the sequence information is used bacteriorhodopsin, allowing, for example, to identify a certain portion of the composition.
Consequently, using the method according to the invention is achieved double protection bulleted items. At that time, how to recognize the signs of a high degree of protection are hidden security features, which are only detected in the time-consuming analysis, and potential forger or forgers perhaps totally not detected. Potential forger not even know whether there should be a sign or not, as there are a number of indications of a high degree of protection, which may be combined with bacteriorhodopsin.
This additional protection provides a high degree of protection against forgery and simultaneously allows you to apply the coding of items, for example the encoding of the manufacturer or of the composition. As a result of application options bacteriorhodopsin symptoms of low and high degrees of protection, in addition, are inextricably linked, as are formed of the same molecule.
Check the bullet according to the invention objects can be implemented in different ways. In routine testing, as it is held at a Bank or credit institution at the beginning of every banknote can be applied, for example, a simple means to check only for the presence of characteristic low degree of protection. However, it is possible to check the presence of two or more signs of a low degree of protection. More thorough research is (options) bacteriorhodopsin. Proof of at least two signs of a high degree of protection can be obtained by applying two different options bacteriorhodopsin or twice a modified version of bacteriorhodopsin. In addition, the possible combination of checking for signs of low and high degrees of protection.
Bacteriorhodopsin is a membrane protein of halophilic bacteria. From microorganisms of the genus Halobacterium in large quantities to extract the protein bacteriorhodopsin. Bacteriorhodopsin original type and its main photochemical and physical properties specialist well known as a photochromic material, which is activated by light and passes a circular series of intermediate forms. In order modeling photochromic properties is applied greatly simplified photocell, consisting only of two States, denoted as state and In M After irradiation with light with a wavelength of 568 nm purple color status goes yellow state M, which, in turn, as a result of absorption of light with a wavelength of 412 nm again enters the state C. Therefore, the material bacteriorhodopsin can discolour yellow-green light, and the hemp is xali again will not happen coloring in yellow color or use the color blue for photochemical return material bacteriorhodopsin in state C. A list of named properties bacteriorhodopsin found in N. N. Vsovolodov, Biomolecular Electronics: An Introduction via Photosensitive Proteins, Birkhauser, Boston, 1998, and D. Oesterhelt, C. Brauchle, N. Hampp, Bacteriorhodopsin: A Biological Material for Information Processing, Quarterly Review of Biophysics, 24, 1991, page 425-278.
The expert knows that there are a number of options bacteriorhodopsin, which, although they have the same primary color as the original type, but sometimes significantly differ in the kinetics of photocycle. A preferred example is a variant of bacteriorhodopsin D96N. The properties of the latter are described in various publications, for example, in A. Miller, D. Oesterhelt, Kinetic Optimization of Bacteriorhodopsin by Aspartic Acid 96 as an Internal Proton Donor, Biochim. Biophys. Acta 1020, 1990, pp. 57-64.
According to the invention, it is preferable to apply the bacteriorhodopsin, which becomes colourless mainly visible light.
Turned out to be the optimal wavelength range from 500 to 600 nm. Bringing bacteriorhodopsin in its original state, you can provide thermal relaxation or irradiation of light of a different wavelength range. Such a different range of wavelengths, it is preferable to use a wavelength range from 400 to 450 nm.
Visible discoloration bacteriorhodopsin under the action of irradiation of the easier to detect than more produtivos power less than 100 mW/cm at wavelengths of 532 nm.
So far in the literature there was no description of the compositions, such as varnishes, inks or printing inks intended for application by means of printing equipment and/or for use in the field of protective equipment, in which bacteriorhodopsin contains as photochromic component. Compared with the aforementioned conventional photochromic materials bacteriorhodopsin provides the following benefits:
1. For change of color can be applied in the light of the visible wavelength range.
2. Both switching States have detectable own color.
3. The application of genetic techniques it is possible to obtain functional options bacteriorhodopsin by amino acid metabolism. Resulting variants differ from bacteriorhodopsin source type kinetics (D96N bacteriorhodopsin) and/or the initial absorption and motociclo (bacteriorhodopsin D85N).
4. The number of possible cycles is more than 105. In microorganisms of the genus lbterium bacteriorhodopsin is present in the so-called purple membrane form. Equipment acquisition and allocation of bacteriorhodopsin in the purple membrane form is well known (see, for example, EP 04068 Otsego exclusively from bacteriorhodopsin and lipids. This element is called the purple membrane. In this type bacteriorhodopsin particularly thermodynamically stable for protein even unusually stable. This is a prerequisite for many technical applications, also in the field of compositions according to the invention, where the bacteriorhodopsin is used as a pigment. So bacteriorhodopsin according to the invention or/and option bacteriorhodopsin particularly preferable to apply in the purple membrane form.
As bacteriorhodopsin can be applied to the original type according to the invention photochromic inks preferably contain at least one option bacteriorhodopsin as photochromic component. Option bacteriorhodopsin different from the original type at least one change. It is preferable to select bacteriorhodopsin of functional variants, variants of these sequences, variants branches of the chromophore options, isotopic variants and/or variants of the spin cell.
Variants of the sequences bacteriorhodopsin, which can also be functional variants of bacteriorhodopsin, can be subjected to expression in the environment of Halobacterium salinarum. This bacteriorhodopsin has no two-dimensional crystallization, but bacteriorhodopsin is associated with the membrane.
When receiving options bacteriorhodopsin target the exchange of individual amino acids as a result of this exchange in a minor for photochromic function ranges can be obtained variants of the sequences. Amino acid exchange can be carried out by locally based mutagenesis of the gene encoded on bacteriorhodopsin.
The result is the beginning for the formation of the characteristic high degree of protection, as the target amino acid metabolism in the molecule bacteriorhodopsin can be used to identify and therefore turns into a protective sign. When replacing, for example, only four amino acid positions 20 biogenic amino acids you can get the 4201012various materials bacteriorhodopsin.
In result, it becomes possible without great technical expenditure to get a huge number of molecules bacteriorhodopsin, including photochrome-functional identical materials bacteriorhodopsin, but clearly differ, in particular its amino acid sequence.
An additional sign of a high degree s can be reliably determine the composition of materials bacteriorhodopsin together with the mentioned modifications. First of all we should point out that mass-spectroscopy (see K. L. Schey, D. I. Papac, D. R. Knapp, R. K. Crouch, Matrix-Assisted Laser Desorption Mass Spectrometry of Rhodopsin and Bacteriorhodopsin, Biophys., J. 63, 1992, pp. 1240-1243; P. Hufnagel, U. Schweiger, C. Eckerskorn, D. Oesterhelt, Electrospray lonization Mass Spectrometry of Genetically modified Bacteriorhodopsin, Anal. Biochem. 243, 1996, No. 1, pp. 46-54; L/E/ Ball, J. E. Jr. Oatis, K. Dharmasiri, M. Busman, J. Wang, L. B. Cowden, Galijatovic, A., N. Chen, R. K. Crouch, D. R. Knapp, Mass Spectrometric Analisys of Integral Membrane Proteins: Application to Complete mapping of Bacterirhodopsins and Rhodopsin, Protein Sci.5, 1998, No. 3, pp. 758-764). Preferred materials bacteriorhodopsin provide a combination of sign of low degree of protection, namely optical easily find photochrome a sign of a high degree of protection, such as a variation in the sequence of the amino acid sequence of bacteriorhodopsin.
Signs of a low degree of protection provided by the invention include
1. Different initial coloring materials bacteriorhodopsin.
2. Different photo-series.
3. The change in kinetic properties.
A sign of low degree of protection according to the invention is preferably obtained by the use of functional options bacteriorhodopsin, but it can be obtained by using other options bacteriorhodopsin, such as, for example, variants of the branch bacteriana should be understood in particular ways, which differ in their absorption spectrum and/or its photocycle from the original type bacteriorhodopsin.
Known functional variants include, for example, D96N variant, in which aspartic acid substituted for asparagine at position 96. Such a functional variant of bacteriorhodopsin and some others are described in "N. Otto, T. Marti, M. Holz, T. Mogi, M. Lindau, H. G. Khorana, M. P. Heyn, Proc. Natl. Acad. Sci. USA 86, 1989, pages 9229-9232 and T. E. Thorgeirsson, S. J. Milder, L. J. Miercke, M. C. Betlach, R. F. Shand, R. M. Stroud, D. S. Kliger, Biochemistry 30, 1991, page 9133-9142".
Under options branch bacteriorhodopsin should be understood in particular ways to bacteriorhodopsin that are different from the original type covalent coupling of molecules. Before such molecules may be, for example, the goal: to increase the molecular weight of bacteriorhodopsin to mass spectroscopy, it was possible to identify such a molecule, or to serve a colored molecule to change the absorption spectrum of bacteriorhodopsin, or serve as a fluorescing molecule, so that it was possible to observe associated with bacteriorhodopsin fluorescence. Material bacteriorhodopsin can also be covalently bound to the polymer. Coupled reaction can be carried out, for example, according Chignell & Chignell, B the material bacteriorhodopsin or photochromic ink, and the color effect from the bleached state can greatly affect covalent bond of the respective dye molecules bacteriorhodopsin or/and easy adulteration of passive dyes or pigments. The visual effect from the resulting colorful mixtures can easily be seen by using the CIE chart. You can determine the color effects using known methods.
To bacteriorhodopsin can be attached to the linker molecules, which allow again to add other compounds. Molecules that attach in accordance with the objectives according to the invention, serve to increase the molecular weight of bacteriorhodopsin so that when the mass spektroskopii you can identify such a molecule.
Under the chromophore options bacteriorhodopsin should be understood in particular ways to bacteriorhodopsin that are different from the original type remove or replace the chromophore group retinylidene another molecule, in particular, the so-called retinaldehyde molecules. Radialnaya molecule can be covalently linked with bacteriorhodopsin via lysine-216.
Therefore, other options are getting fizicheskih properties preferably apply dehydroretinal or 4-katreina.
Under isotopic variants of bacteriorhodopsin should understand these options bacteriorhodopsin in which some or all of the amino acids partially or completely in the state of13With or15C. This is due to the fact that some of the labeled amino acids are added to the environment for growth, or that applies peptone, which is labeled as a whole. Through high-resolution NMR can be identified compounds, labeled thus.
Under variants of spin labels bacteriorhodopsin should be understood in particular such variants of bacteriorhodopsin that contain spin label covalently linked to a molecule of bacteriorhodopsin. This is achieved, for example, due to the fact that the derived TEMPO (2,2,6,6-tetramethylpiperidine-N-oxyl) or DOXYL (4,4-dimethyloxazolidine-N-oxyl) or PROXYL (of 188.8.131.52-tetramethylpyrrolidine-N-oxyl) covalently linked to material bacteriorhodopsin. Using electron paramagnetic resonance, you can check for the presence and type of spin labels.
Under variant sequence bacteriorhodopsin should in particular be understood such options bacteriorhodopsin, which differ in the e cause a significant impact on photocell. Variant sequences bacteriorhodopsin are, for example, D36C or variants, in which the terminator N or terminator With attached amino acids.
The combination of modifications of the above scenario leads to new versions, which makes it possible for a huge number of different compositions of bacteriorhodopsin. The result is the sign of a high degree of protection, as the cost analysis of very large and at the same time each portion of the composition can uniquely be identified due to the large diversity.
Options bacteriorhodopsin is preferable to choose from a number D36X, D96X and D85X, where X is one of the natural amino acids. Particularly, it is preferable to select bacteriorhodopsin from a number D36C, D96N and D85N.
Particularly preferable to apply the mutants with enhanced light sensitivity, and, in particular, the materials used in holography.
The composition of bacteriorhodopsin can contain along with bacteriorhodopsin as common photography pigment, or/and porogram and/or covalently associated with bacteriorhodopsin pigment, and/or additional photochromic pigment. By mixing with bacteriorhodopsin Nevestino may also be expedient to additionally apply along with visible light ultraviolet light.
Fluorescent and phosphorescent molecules can be bound with the molecules of bacteriorhodopsin, resulting in their emission is an additional sign. In the appropriate selection of the position of the fluorescence emission can be suppressed, provided that the material bacteriorhodopsin is not in the bleached state. This is due to the fact that the position of the initial absorption bacteriorhodopsin and position emission fluorescing or phosphorescent material strongly overlap each other. In this case, the material bacteriorhodopsin absorbs the emitted photons, and the naked eye fluorescence is not perceived. Fluorescence becomes noticeable only in the case when the material bacteriorhodopsin discolored photochemical method.
Check the composition of the supported bacteriorhodopsin may be performed, for example, a method in which using microanalytical sequencing determined wholly or partly amino acid sequence of material bacteriorhodopsin and by immunological methods used to measure reaction occurring with a specific antibody.
Materials bacteriorhodopsin containing indications of a high step is nochelatina sequences, moreover, changing the order does not affect the photophysical properties;
2. With covalently attached molecules;
3. With amino acids, which in the state of13And/or other isotopes. Particularly preferred combinations of two or more of the above variant types bacteriorhodopsin.
Particularly preferable to apply the method according to the invention at least one version of bacteriorhodopsin with the following characteristics:
a) necessary for the formation of purple membrane form of the protein band remains unchanged in relation to the original type bacteriorhodopsin;
b) in the loops, and/or terminator, and terminator N polypeptide is at least one amino acid exchange in comparison with the original type bacteriorhodopsin, accompanied by divisions, accessions, inclusions and/or replacements, and these amino acid exchanges do not cause changes photochromic properties bacteriorhodopsin, certain photochromic range.
Such bacteriorhodopsin remain unchanged in the range required for the formation of a purple membrane form of the protein, compared with the original type bacteriorhodopsin. Sufficient services the care, and further capable of forming Magenta membrane in this range.
Changes in amino acid sequence according to the invention include amino acid exchanges, such as deletion, inclusion, substitution and/or addition in any position within the entire polypeptide chain. Especially preferred attachment of amino acids to the terminators N, or/and, or/and to the loops of the polypeptide chain, based in particular outside of the membrane. Consequently carried out according to the invention changes when encoding the sign of a high degree of protection is preferable not to carry out a range of bacteriorhodopsin that affect the photochromic properties. Amino acid metabolism in the loops, and/or terminator, or the terminator N polypeptide chain when properly conducted, does not change the photochromic properties of the original bacteriorhodopsin. Here it must be emphasized that as the source bacteriorhodopsin can be used as bacteriorhodopsin original type, and modified, in particular the D96N bacteriorhodopsin.
Used in the analysis of amino acid exchanges cause changes in the mass of the molecule bacteriorhodopsin preferably 1 Yes, more preferably by mobs of ionization, known from the prior art, as, for example, FTMS (mass spectrometer with Fourier transform), and/or TOR (mass spectrometer time of flight), and/or MALDI (Matrix-assisted Laser Desorption lonisation (matrix laser desorption ionization)), and/or ESI (electrospray ionization).
When entering/joining of amino acids the number of additional amino acids can be achieved in particular to 1000, preferably up to 100, particularly preferably up to 50, and at least one of them, but most preferably up to 3-20 amino acids. You can attach at least 6 radicals histidine to the terminator With in order to detect the presence of variant bacteriorhodopsin through communication with metals by XRF or TXRF.
Deletion or substitution applies in particular 1-10, particularly preferably 1-4 amino acids.
As variants of substitution, it is preferable to apply a variant of bacteriorhodopsin, the residue is aspartic acid which is at position 36 is replaced by cysteine (option bacteriorhodopsin D36C).
Molecular weight variants of the amino acid sequence molecules bacteriorhodopsin can be determined by mass-spectrometry using electrospray ionization (ESI) or matrix-assisted laser desorption Iassy of the investigated substances with a resolution of up to mass number. When changing the amino acid sequence, for example, deletions or inclusions occur relatively large changes in mass number, which is analytically well detected. Even if only a few amino acids are substituted by other amino acids, the resulting mass change sufficient for analysis.
It is preferable to apply a variant of bacteriorhodopsin, in which the terminator is attached at least one amino acid. It is also preferable to apply options bacteriorhodopsin that contain at least one cysteine. In another preferred embodiment of the invention uses at least one version of bacteriorhodopsin, which is different from the original type photocell or/and different from the original type primary color.
It is most preferable to apply options bacteriorhodopsin, which are characterized by a reduced adaptation to light and darkness. Bacteriorhodopsin, which is characterized by a maximum absorption at 570 nm (state), in the dark undergoes relaxation slowly with time polyomino from 10 to 20 minutes with a partial transition to a state of maximum absorption at 548 sostoyaniye b and D, approximately 50:50. The state has the configuration of the "all-trans", D - 13-isconfiguration retinal. When exposed to light bacteriorhodopsin 100% goes to the state, which begins photocell, which is required intense color change (the shift of absorption to 412 nm).
If for a long time the ink containing bacteriorhodopsin, or labeled them the subject was kept in the dark, the bacteriorhodopsin goes in your so-called adapted to the dark state. If now controlled the first surface to irradiate light for discoloration bacteriorhodopsin, there is the impression that it has low light sensitivity or reduced rate of discoloration. This is for the reason that part used for the irradiation light is spent on translation bacteriorhodopsin from state D to state C. If now produced from discoloration to return to its original state, for example, by using a blue color, i.e., to restore lilac colouring initial state, when carried out immediately after the irradiation, the material will have a high sensitivity. One is A. Therefore, preferred materials for use according to the invention are variants of bacteriorhodopsin reduced or completely missing adaptation to light and darkness.
Options bacteriorhodopsin, which are characterized by a reduced adaptation to light and darkness, or its absence, can be obtained by applying analogs retinal or using options bacteriorhodopsin with a modified amino acid sequence. Particularly suitable examples include bacteriorhodopsin with chemically modified chromatogram, such as, for example, 13-dimethyl-11,14-epoxy-bacteriorhodopsin (M Muradin-Szweykowska et al., Rec.: J. R. Neth. Che.Soc. 102, 1983, pp. 42-46). Other preferred variants are bacteriorhodopsin, which contains 13-substituted retinal, in particular retinal, which in position 13 contains an atom of hydrogen, ethyl or sawn group (W. Gaertner et al., Biochemistry 27, 1988, page 3497-3502). Mutants Arg-82, Asp-85 and Asp-212 low light adaptation, which here is also preferable to use, as described, for example, in M. P. Krebs et al., Proc. Natl.Acad.Sci.USA 90, 1993, pp. 1987-1991. Other preferred mutants are Y185F (P. Rath et al., Biochemistry 1993, page 2272-2281), and the mutants described in S. pactically, generally, variants, which in conditions of darkness are able to and are not changed to D.
Molecules covalently associated with molecules of bacteriorhodopsin, provide additional choice of the signs of a high degree of protection. Attach molecules allows additional analytical dimension, for example, through the definition of the relevant properties of the attached molecules or through their mass. Attached molecules can be, for example, Biotin and/or avidin, and/or digoxigenin. In addition, you can attach molecules, which are separately detected by a mass spectrometer. Attaching molecules spin labels, such as TEMPO, DOXYL or PROXYL, can be detected by analysis of electron paramagnetic resonance, which can be limitations with the use of solids. Amino acids labeled with stable isotopes13C and15N, can be detected by the analysis of nuclear magnetic resonance (NMR) (M. Engelhard, B. Hess, G. Metz, W. Kreutz, F. Siebert, J. Soppa, D. Oesterhelt, High resolution carbon-13 solid state NMR of bacteriorhodospin: assignment of specific aspartic acids and structural implification of single site mutations, Eur. Biophys, J. I 8, 1990, pages 17-24).
Indications of a high degree of protection intended for checking autov which are known and which are optionally attached to the molecules of bacteriorhodopsin. As the polymer molecules can be used, for example, oligopeptides, polypeptides, proteins, nucleic acids, peptide nucleic acids or synthetic, is not a naturally occurring polymers. Polypeptides and proteins can contain deproteinize components, nucleic acid - denuclearisation components. Deproteinize and denuclearisation components may include, for example, glycosidic components, Biotin, or/and digoxigenin, or/and avidin.
It is also possible to mix to contain bacteriorhodopsin ink one or more additional polymers, which as the information-bearing components provide additional indications of a high degree of protection or, for example, serve only to set the viscosity or other mechanical properties of the label.
Attached to the bacteriorhodopsin polymer molecules, providing a sign of a high degree of protection, such as DNA or RNA, or/and NCP (PNA), or/and hybrids, consisting of these types of molecules can be detected, for example, as the result of these amplification reactions such as PCR reaction, using a special seed. When this detection may include SEB is coherence. Similarly, you can analyze and detect polypeptides using microanalytical determination of the sequence. Thanks to the capabilities provided by the organic synthesis of nucleic acids and polypeptides, can be used and not naturally occurring sequence.
The combination of the signs of low and high degree of protection provides interesting advantages. This combination can be obtained, for example, due to the fact that the photochromic properties bacteriorhodopsin as a sign of low degree of protection combined with one of the above signs of a high degree of protection. Particularly preferable to apply for this bacteriorhodopsin together with the polymer molecule. Bacteriorhodopsin or its variant can however be applied even as the analyzed polymer molecules. Despite the huge variability, security features are inextricably linked. Along with this it is possible to use in addition to the bacteriorhodopsin as a photochromic material of different polymers attached to bacteriorhodopsin or/and existing freely.
The method according to the invention to ensure the authenticity of items includes nanosize according to the invention contains, if necessary, along with bacteriorhodopsin original type or/and along at least one bacteriorhodopsin as photochromic component corresponding auxiliary substances and/or appropriate matrix materials.
During the coating process used excipients: to prevent pricing and improve the technological properties or/and to give stability to microcapsulation materials and composition for shielding ultraviolet rays or/and to increase the visual optical impressions for impact absorption spectrum, for example, in neobespecheno and bleached States of the material bacteriorhodopsin. Examples of such excipients are passive dyes and pigments, which are simply mixed into the ink to achieve the desired start or end color. Such a method can be created composite paint and/or can be shifted to the range.
The appropriate matrix materials are used for fixing composition as a result of the inclusion or/and covalent joining of the matrix material or/and the subsequent cross-linking of the composition, or matrix by chemical or photochemical methods. Cross connection can be formed by processing glutaraldehyde, transglutaminases, polymerization (radical) and/or photochemically.
The photochromic coating compositions according isoa, inkjet or pad printing, using a mechanical brush application and spraying, dipping or electrophoresis. Bacteriorhodopsin according to the invention can be fixed, followed by drying, and synthetic or biological matrix material is translated into an insoluble form additional processing. Be applied can the composition itself or the supporting substrate, which have a composition. The composition of bacteriorhodopsin can be applied in the form of microcapsules. The ink composition in the microcapsules are particularly suitable for use in the methods of printing.
In a particularly preferred embodiment of the method according to the invention when marking the subject with the use of photochromic ink last put on this subject, fix dried and fixed in a matrix material, and lighting it is possible to invoke change the color of the photochromic composition. The minimum light energy needed to change the color, it is preferable to install via the pH value of the photochromic composition.
In addition, it is preferable to apply them marking the subject of the two surfaces a and b, which, in particular, are adjacent to each other, and the first powergorilla, spectral brightness which in unexposed condition does not differ from the brightness of the first surface, but after exposure acquires a different brightness. In addition, it is also possible to apply to be marking the subject of two adjacent to each surface and the first surface And is marked with a photochromic composition containing the first bacteriorhodopsin, and the second surface In the second photochromic composition, sensitivity to light which is different from the light sensitivity of the first composition, resulting in the second surface in the unexposed state is not distinguished by its spectral brightness from the first, but after exposure detects different brightness. The second photochromic composition preferably contains the original type bacteriorhodopsin or/and option bacteriorhodopsin, which differs from the variant bacteriorhodopsin used in the first composition, and is, in particular, as the reference color.
The subject invention are also photochromic ink containing at least one, preferably at least two variants of bacteriorhodopsin. Preferred variants bacteriorhodopsin is predmetov to ensure their authenticity.
The ink according to the invention can be used in combination with traditional dyes known to the specialist, as, for example, porogram, pigments and/or other photochromic pigments. The ink according to the invention containing materials from bacteriorhodopsin, can be used as a regular ink, and can be used along with ensuring the authenticity of objects, for example, for decoration and other special effects.
Under the ink refers to any colored liquid for writing/liquid for printing and, if necessary, spray environment. The term "ink" is also subject to printing ink and other ink composition that can be used for printing on objects or which find wide application in obtaining prints, in the case of the use of materials from bacteriorhodopsin as ink solvents can serve as water or other solvents, such as solvents, alcohol-based. Preferably, the ink contained at least two variants of bacteriorhodopsin. When using at least two options bacteriorhodopsin or one variant of bacteriorhodopsin containing two modifications, anellini purple membrane form of bacteriorhodopsin in the ink according to the invention can optionally contain bacteriorhodopsin in solubilizing form.
Bacteriorhodopsin in solubilizing form can be obtained due to the fact that gene bacteriorhodopsin subjected to expression in the host, e.g. E. coli, and restore the addition of retinaldehyde. Another possibility is that bacteriorhodopsin is extracted from purple membrane by removing lipids. For example, a suspension of purple membrane (OD570<5) diluted in water or buffer solution 1% Teriton-X 100 and within hours continuously irradiated with ultrasound using the microprobe ultrasonic disintegrator. In the residue after centrifugation contains bacteriorhodopsin in solubilizing form.
Photochromic composition containing bacteriorhodopsin can be applied to any subject. The items of particular interest are, for example, documents, securities, Bank notes, artwork, certificates, clothing, cars, telltale signs, stamps quality, etc.
Another embodiment of the invention is the subject labeled, containing at least one kind of bacteriorhodopsin. This marking is applied mainly by the method according to the invention. The label contains pre-emption is a slight sensitivity, consequently when using light sources of normal intensity, such as sunlight, it is almost impossible to cause discoloration, well traceable to the naked eye. This low sensitivity is a consequence of the short duration condition M
However the duration of the state of the M source type bacteriorhodopsin can be increased through appropriate measures. Another object of the invention is a method of ensuring the authenticity of the subject by applying to him photochromic ink, and applied photochromic ink with the content source type bacteriorhodopsin as photochromic component, which after exposure to light of the visible wavelength range finds visually observed, a reversible change of state, used as a sign of low degree of protection when checking authenticity, and which includes the additional excipient, binding water or/and reduce the amount of protons. Thanks to the auxiliary substances that increase low density original type bacteriorhodopsin, in the ways to ensure authenticity can also be used source is longer state of the M source type bacteriorhodopsin. The duration of the state of the M source type bacteriorhodopsin increases almost complete absence of water. Therefore, the purpose of auxiliary substances is mainly to bind water or reduce the amount of protons. Suitable excipients are, for example, compounds which contain primary or secondary amino group. Particularly preferably be used as auxiliary substances arginine or guanidine x Hcl. Such excipients can also be used in embodiments of bacteriorhodopsin to increase sensitivity.
Thus in a way to ensure the authenticity of the object according to the invention applied photochromic inks contain preferably option bacteriorhodopsin or mutant bacteriorhodopsin, when this mutation is for use as information about the sign of a high degree of protection and/or to improve the sensitivity. Particularly preferable to apply options that contain at least two modifications, namely one modification that serves to increase the sensitivity of bacteriorhodopsin, and another modification, which mogadore type bacteriorhodopsin possible only conditionally because of low sensitivity (bacteriorhodopsin source type under normal light changes its color unnoticed), and because of its widespread availability, it is to protect the authenticity of relatively minor interest. When using combinations of the basic type of bacteriorhodopsin and one of the above described additives to improve the sensitivity applications can be interesting. In yet another preferred embodiment of the invention applies the above-described additives to improve the sensitivity or other additives, for example, to set the initial color, applying labels, etc. together with one or more options bacteriorhodopsin or mutants of bacteriorhodopsin.
Below the invention is explained more the following examples with reference to Fig.1 and 2. In this Fig.1 depicts the carrying out of the method of verifying the authenticity, Fig.2 depicts the copying process using the protective trait.
Check for any sign of a low degree of protection.
Easily check for the user are photochromic properties bacteriorhodopsin or its variants (Fig.1). The sign of 2 printed on the document 1, such as, for example, Bank tickets, securities, works of art or any other valuable item, and W it exposed to light 3 LEDs with an emission maximum in the green or yellow range, its color changes from purple to yellow. This is especially easy to see in Fig.1, position 4, when not irradiated the entire surface. Without additional measures, after a few seconds or minutes depending on the applied composition - purple color is returned and the original state is restored. Alternatively, the purple color can be restored by irradiation light 5 LEDs with an emission maximum in the blue range. Technical cost of the inspection is minimal. The user is able to observe the color change with the naked eye, the measurement can be carried out mechanically.
Document 1 with characteristic 2 according to the invention, which used a combination of a certain number of light-sensitive material is bacteriorhodopsin, for example, the source type bacteriorhodopsin, and material bacteriorhodopsin with high sensitivity, for example, a variant of bacteriorhodopsin D96N, in the unexposed state has a solid surface the same color (Fig.2). Instead of insensitive material bacteriorhodopsin can be applied to the corresponding dye of the same color. If such a document reproduced the capacity of the material bacteriorhodopsin discolored more than the surrounding material of lower density. So copy 4 will be the sign of the 5 low-grade protection, preserving for a long time its distinctive colouring. On this basis, the document is clearly identified as a copy.
Additional processing auxiliary substances and method of coating.
a) Additional crosslinking.
On a substrate together with dried photochromic layer comprising a matrix material and bacteriorhodopsin, was coated from a solution of 40% glutardialdehyde and stood for 15 minutes. Then a solution of glutardialdehyde was washed away. As a result of this processing photochromic layer was water-insoluble.
b) Photochemical processing.
10 mg of the purple membrane (D96N bacteriorhodopsin) was subtly dispersible in 4 ml of ink of the ultraviolet curing (IFS3000, F."Schmitt"). After applying the mixture using a doctor blade was held hardening during the night when exposed to ultraviolet light.
The principle of screen printing is similar to the technique of a solid patch is printed. Printed form consists of a screen fabric, provided impervious to paint predopredelennogo paint stencil using a squeegee. When this paint is applied on the button at the bottom of the base. To obtain ink for screen printing in the solution of 7.2% polyvinyl alcohol (Mowiol, type 56-98) during the night was mixed into 100 mg/ml pigment (source type bacteriorhodopsin). When the rheological properties of the mixture of the standard sample mixture could be used for printing on a conventional machine for screen printing.
In 5 ml of ink containing pigment (F. "Schmitt", IUF01) at 50° C was mixed into 1 g of the purple membrane (D96N bacteriorhodopsin). The resulting mixture was applied to conventional offset printing.
d) Mechanical protection.
Deposited on a substrate mixture containing bacteriorhodopsin sealed in the pocket of the film type GHQ-120TR using hot lamination device (GPM, Mylam 9) at a temperature of from 90 to 140° C.
e) Auxiliary substances.
Polyvinyl alcohol (type Moviol 56-98,68 mg/ml) dissolved in water at 50° C. To this mixture was introduced purple membrane after freeze-drying and received a concentration of 11 mg/ml of this mixture was mixed at room temperature for 1-octanol (1% in volume). The resulting mixture was characterized by improved is zlecenia.
To protect the photochromic pigment in the mixture was mixed into one of the following UV absorbers or derivative to a concentration of 1-30%, preferably 3-10% in weight ratio: benzophenone, hydroxynaphthoquinone, phenylbenzoxazole, ester of cinnamic acid, sulfonamide, ether aminobenzoic acid.
Document common use, such as a banknote, provided with a protective marking containing bacteriorhodopsin. Under the action of lighting the protective label changes its color from purple to yellow. After a short time (about 30-60 seconds) and/or when illuminated by the light of the blue wavelength range of the original purple color is restored. This document is for General use may be protected from prohibited reproduction or forgery.
Acted analogously to example 4, but the change of colour from yellow to purple conducted by irradiation with light of a blue wavelength range. After exposure to light environment, the original color was restored.
The document, for example, contract, provided with a strip of purple. This band creates a double printing with the use of two stencils, positive and negative. do a light. When the illumination light is white light-sensitive layer changes color from purple to yellow, while the second layer color either does not change or only very weakly. In this case there is color contrast. In the result, it is possible to distinguish at first a uniform surface word (for example, "original"), or any other series of characters and/or icons.
Acted as in example b, but the printing is performed on plain or photo paper, which was then eliminirovali. Being attached to a branded product (garment, for example, jeans etc) or transmitted together with a proprietary product in this paper can then ensure authenticity and thus prevent the production of pirated products.
To protect the document from prohibited reproduction, copying the received surface of homogeneous type as in example 6, with two stencils. Part of such a uniform surface during copying changed the color and as a result, copies of the formed heterogeneous surface.
1. The way to ensure the authenticity of the subject by applying to a photochromic ink, and apply the photochromic ink, measles irradiation with light of a visible wavelength range undergoes a visually detectable reversible change of state, employee sign a low degree of protection when checking authenticity, and which provide, in addition to the characteristic low-grade protection for one or more indications of a high degree of protection, which are not visually detected and confirmed only by means of instrumental analysis.
2. The method according to p. 1, characterized in that option bacteriorhodopsin choose from a number of functional variants, variants of these sequences, variants branches of the chromophore options, isotopic variants and/or variants of the spin label.
3. The method according to p. 1 or 2, characterized in that the apply option bacteriorhodopsin containing the following characteristics: a) the range required for the education purpurogallin form of the protein remain unchanged in relation to the original type bacteriorhodopsin, b) in the loops, and/or terminator, and terminator N polypeptide is at least one amino acid exchange in comparison with the original type bacteriorhodopsin, accompanied by divisions, accessions, inclusions, and/or substitutions, and these amino acid exchanges do not cause changes photochromic properties bacteriorhodopsin, certain photochromic range.
4. JV is at least one amino acid.
5. The method according to p. 3 or 4, characterized in that in a variant of bacteriorhodopsin contains at least one cysteine.
6. The method according to any of the preceding paragraphs, characterized in that the applied option bacteriorhodopsin is different from the original type photocell or/and different from the original type primary color.
7. The method according to any of the preceding paragraphs, characterized in that option bacteriorhodopsin choose from a number D36C, D96N and D85N.
8. The method according to any of the preceding paragraphs, characterized in that the photochromic ink additionally contains a polymer molecule.
9. The method according to any of the preceding paragraphs, characterized in that the apply option bacteriorhodopsin, in which the polymer molecule is attached to the bacteriorhodopsin.
10. The method according to p. 9, characterized in that the polymer molecule is a polypeptide containing, if necessary ones components.
11. The method according to p. 9, characterized in that the polymer molecule contains a nucleic acid or/and derivatives thereof, which are optionally present denuclearisation components.
12. The method according to p. 11, characterized in that the nucleic acids and/or derivatives thereof, attached to the bacteriorhodopsin, the selected amplification products.
14. The method according to any of the preceding paragraphs, characterized in that the photochromic composition contains at least two variants of bacteriorhodopsin or/and one version of it at least with two modifications.
15. The method according to any of the preceding paragraphs, characterized in that the optionally used auxiliary substances: (a) to prevent foaming and increase the wettability, or/and (b) for microcapsulation ink materials and shielding from ultraviolet rays to increase stability, or/and (C) for impact absorption spectrum of the material bacteriorhodopsin to improve visual optical effect.
16. The method according to any of the preceding paragraphs, characterized in that it further apply a matrix material for fixing the photochromic material as a result of: a) physical inclusion, or/and (b) covalent joining of the matrix material, or/and (C) cross-linking.
17. The method according to p. 16, characterized in that option bacteriorhodopsin finely dispersed in the paint, cured by UV light, and that the cross-links formed by irradiation with ultraviolet light.
18. The method according to any of the preceding paragraphs, different is it with ink.
19. The method according to any of the preceding paragraphs, characterized in that subject marking the subject used two adjacent to each surface and the first surface And mark the photochromic composition containing bacteriorhodopsin, and the second surface is applied photography dye, the spectral brightness is in the unexposed state is not different from the spectral brightness of the first surface, but which, after lighting acquires a brightness that is different from the brightness of the first surface.
20. The method according to any of the preceding paragraphs, and to be marking the subject used two adjacent to each surface and the first surface mark the first photochromic composition containing bacteriorhodopsin, and on the second surface, apply a second photochromic composition, the density of which differs from the first light, resulting in the second surface in the unexposed state is not distinguished by its spectral brightness from the first surface, but after lighting acquires a brightness that is different from the brightness of the first surface.
21. Photochromic ink, characterized in that they Soder-20.
23. The way to ensure the authenticity of the subject by applying to a photochromic ink, and applied photochromic inks that contain source type bacteriorhodopsin as photochromic component, which when exposed to light of the visible wavelength range undergoes a visually detectable reversible change of state, used as a sign of low degree of protection when checking authenticity, and which additionally contain excipients, binding water or/and reduce the amount of protons.