JP4674796B2 - Non-exposed surface coating composition and non-exposed surface coating - Google Patents

Description

  The present invention relates to a coating composition for a non-exposed surface such as an inner surface or back surface of a product and a coating film for a non-exposed surface, and more specifically, a coating composition for a non-exposed surface using an information nucleic acid that can be used for individual authentication. The present invention relates to an object and a non-exposed surface coating.

  Conventionally, for individual authentication, a license plate, watermark printing such as banknotes, an IC chip and a photo of a credit card are used.

  However, these individual authentication means have a drawback that they can be removed from the product by peeling, cutting, erasing or the like. For this reason, it has been expected to develop authentication information that cannot be removed from the product, that is, does not disappear.

On the other hand, DNA is an information biomolecule originally possessed by all living organisms and including all genetic information in each organism. Many of them correspond to the amino acid sequences of many proteins. That is, deoxyadenosine (dA), deoxyguanosine (dG), deoxycytosine (dC), and deoxythymine (dT) are bonded with a certain direction through a phosphate ester bond, and the number of bases is n. Thus, there are ⁴ⁿ types of DNA having that length. Thus, for example, there can be about 4.3 billion types of distinguishable DNAs with only 16 types of bases. At present, any DNA having a sequence of several tens of bases can be arbitrarily synthesized. In addition, if the amount of DNA exceeds a certain level, the sequence can be automatically determined by an automatic sequence reader (sequencer).

From such a background, it has been proposed to use a forgery-preventing label in which DNA is contained in a water-insoluble medium in a product to clarify the authenticity of the product based on the presence or absence of the DNA. (See Patent Document 1).
JP 2004-159502 A

  However, the technique described in Patent Document 1 basically relates to a method of mixing DNA and a water non-solvent, and the authenticity of a product is confirmed by the presence or absence of amplification of ribonucleic acid by PCR. This indicates that the target product containing ribonucleic acid is identified. In addition, authenticity verification data that uses the presence / absence of DNA as a test index is data on individual authentication that enables authentication for each product even if it is the same type of product, using the DNA sequence as a test index. Is not disclosed.

  On the other hand, when the perpetrator escapes in the case of theft or damage of an article such as a vehicle, there is a request to identify the target article at an early stage from the paint piece of the article left on the incident site.

  The present invention has been made in view of such problems of the prior art, and the object of the present invention is to individually identify the source / history product by containing information nucleic acid. It is providing the coating composition for non-exposed surfaces and the non-exposed surface coating film which can be specified specifically.

  As a result of intensive studies to solve the above-mentioned problems, the inventors of the present invention treat nucleic acid represented by DNA as one piece of authentication information, and detect information nucleic acid in the non-exposed surface coating later. The present inventors have found that the above problems can be solved and have completed the present invention.

  According to the present invention, even if it is a mass-produced product such as an industrial product, the target product can be individually authenticated by determining the sequence of the contained information nucleic acid.

  Hereinafter, the coating composition for unexposed surfaces of the present invention will be described in detail. In the present specification and claims, “%” indicates a mass percentage unless otherwise specified.

  The non-exposed surface coating composition of the present invention comprises an information nucleic acid provided with a site having an arbitrary and known base sequence in a non-exposed surface coating material. As a result, it can be easily applied as a non-exposed surface coating material to the product, and it is difficult to remove the non-exposed surface coating film from the product, so that it can be used as an excellent individual authentication means.

  Here, the above-mentioned information nucleic acid refers to DNA (deoxyribonucleic acid), RNA (ribonucleic acid) and derivatives thereof, which may be natural type or artificial type, but should be included in a primer coating composition which is used in a severe environment. In consideration, it is preferable to use an artificial mold that is structurally stable. In the artificial type, a sequence having a binding mode that does not exist in the natural type (for example, a linkage between nucleosides includes not only a phosphate ester bond but also a non-natural type such as a thiophosphate ester bond) can be formed.

  In addition, in the above-mentioned information nucleic acid, that the base sequence site is arbitrary means that it can be randomly selected as long as it is a detectable base sequence, and that the base sequence site is known is used for individual authentication. It shows that the base sequence to be grasped in advance.

  As for the size of the information nucleic acid, the number of bases in the whole nucleic acid is preferably 200 or less. When it exceeds 200, unreacted sites are generated little by little at the stage of synthesis, and the content of those lacking a base tends to increase. More preferably, it is about 100 bases.

  Furthermore, it is preferable that thymines are not adjacent to each other in the above base sequence. Thereby, it can suppress that thymine dimerizes.

  In addition, the information nucleic acid is preferably derivatized with a protecting group from the viewpoint of improving stability when used in combination with a compound that reacts with an OH group or when used in a severe environment. Specifically, a hydroxyl group at one or both of the 5′-position and the 3′-position is derivatized with a phosphate ester group, an acyl group, an alkoxycarbonyl group, a benzyl group, a substituted benzyl group, an allyl group, or the like. can do. FIG. 1A shows natural DNA, and FIG. 1B shows DNA derivatized at the 5 ′ position.

  Furthermore, from the viewpoint of enhancing the convenience of isolation and purification, it is preferable to derivatize the hydroxyl group at the 5 ′ position with biotin or a fluorescent molecule. Specifically, when vithion is used, it is easily adsorbed selectively on a column to which a protein called avidin is bound. On the other hand, when a fluorescent molecule such as fluorescein is used, the nucleic acid itself becomes fluorescent, so that it can be detected with high sensitivity and purification is facilitated. Thus, when the convenience of isolation and purification is increased, individual authentication becomes extremely easy.

  When RNA is used as the information nucleic acid, the 2′-position hydroxyl group can be derivatized with the protecting group from the viewpoint of improving stability.

  In addition, from the viewpoint of efficiently detecting the information nucleic acid even when the content in the non-exposed surface coating composition is low, the base sequence portion is a portion used for amplification of the information nucleic acid. Preferably there is. As a method for amplifying such information nucleic acid, polymerase chain reaction (PCR) that can be amplified synergistically can be appropriately employed.

  Typically, it is desirable to employ a PCR method that can be amplified extremely even if the information nucleic acid is extremely small. In this method, for example, when a heat-resistant DNA polymerase is allowed to act while temperature control is performed in the presence of a base (primer) complementary to several tens of bases of the DNA, the original DNA can be doubled. If this is repeated 30 times, for example, it can be amplified several hundred million times. This amplification makes it possible to obtain a sufficient amount for determining the sequence even from a very small amount of sample. As a result, the product containing the information nucleic acid from the information corresponding to the sequence (non-exposed surface coating The “identity” of the composition) will be revealed.

  In this case, it is preferable that the sites used for the amplification have primer-corresponding sites necessary for polymerase chain reaction (PCR) at both ends. This is because an information nucleic acid can be used without a primer, but it can be identified in a short time by providing a primer.

  For such a primer corresponding site, the lower limit of the number of bases is preferably 5 or more. More preferably, it is 10 or more. If the number of bases is less than 5, the number of nucleic acids that can be distinguished decreases, and particularly when many products (coating compositions for non-exposed surfaces) are mixed, it takes time for identification. On the other hand, the upper limit of the number of bases is preferably 100 or less. When the number of bases exceeds 100, the ratio of by-products lacking a base at any position increases, so that purification takes time or is difficult in some cases.

  In addition, when RNA is used as the information nucleic acid, DNA complementary to the sequence can be obtained using reverse transcriptase, and PCR can be performed using this DNA.

  The information nucleic acid preferably further has an authentication information site in addition to the base sequence site. In this case, individual authentication can be executed by more detailed information setting.

  For example, as shown in FIG. 2, in the case of information-oriented DNA provided with primer-corresponding sites at both ends, a sequence of m bases is placed in the center (B1 to Bm), and the sequence information of this part is used as authentication information. Make it correspond. At both ends, sequences (X1 to Xl, P1 to Pn) complementary to l (el) and n primers are ligated, respectively. The presence of this part makes it possible to adopt the PCR method for the first time. The information DNA can be used as an information element of this single-stranded DNA or a double-stranded DNA complexed with a complementary sequence of DNA. The sequence of the primer corresponding site can be devised so that the binding of complementary sequences is as stable as possible and amplification by the PCR method proceeds smoothly.

Further, the information nucleic acid is preferably contained in an amount of 0.5 to 2000 μg with respect to 100 g of the resin solid content from the viewpoint of improving the detection accuracy of the information nucleic acid and the dispersibility of the non-exposed surface coating material. It is. More preferably, it is 30-200 micrograms, Most preferably, it is 50-100 micrograms. When the amount added is less than 0.5 μg, it is difficult to distinguish the information nucleic acid from the unexposed surface coating. If it exceeds 2000 μg, the adhesion tends to be lowered. In addition, if a large amount of information DNA having a primer-corresponding site is contained, the cost increases.
In addition, the said resin solid content shows the non-exposed surface coating film which the coating composition for non-exposed surfaces solidified.

  As the raw material for the non-exposed surface, a general non-exposed surface paint can be used. Typically, a hydrophilic liquid such as an acrylic-based paint, a melamine-based paint or a urethane-based paint which is a lipophilic liquid. Examples thereof include hydrophilized acrylic paints, hydrophilized urethane paints, and hydrophilized melacin paints, such as polyester paints and acrylic paints that are powders. The lipophilic liquid may be a one-component type or a two-component type (for example, urethane resin paint).

  The non-exposed surface coating composition of the present invention may contain various additives in addition to the non-exposed surface coating material and the information nucleic acid. For example, various additives such as an organic pigment, an inorganic pigment, a dispersant, and a curing accelerator can be appropriately contained.

  The information nucleic acid is preferably carried on fine particles. As a result, it is possible to prevent the information nucleic acid contained in the non-exposed surface coating material from flowing out of the non-exposed surface coating material, thereby extending the life of the non-exposed surface coating composition.

  Furthermore, the average particle diameter of the fine particles is preferably 0.01 to 60 μm from the viewpoint of improving the detection accuracy of the information nucleic acid and the dispersibility of the non-exposed surface coating material. More preferably, it is 0.02 to 10 μm, and particularly preferably 0.02 to 5 μm. If the average flow diameter is smaller than 0.01 μm, the detection accuracy tends to be lowered, and if it is larger than 60 μm, the adhesion tends to be lowered.

  Furthermore, the content of the fine particles is in a ratio of 0.5 to 70% with respect to the solid content of the resin, so that the detection accuracy of the information nucleic acid and the dispersibility of the coating material for the non-exposed surface are good. From the viewpoint of: More preferably, it is 0.5 to 30%, and particularly preferably 0.5 to 10%. When the content is less than 0.5%, since the amount of fine particles in the sampled undercoat film is too small, the detection accuracy may be lowered. If it exceeds 70%, the adhesion tends to decrease.

  As the fine particles, for example, titanium oxide, molybdenum oxide, tungsten oxide, barium titanate and the like are preferably used in addition to silica and zinc oxide.

  The fine particles are supported on a dispersion by dispersing in sterile distilled water to form a suspension, and the information nucleic acid solution is prepared by dissolving the information nucleic acid in the suspension as it is or dissolving the information nucleic acid in sterile distilled water. And can be produced by drying. At this time, a part of the information nucleic acid may be added as it is without forming an aqueous solution, and the remaining part may be added as an aqueous solution.

  At this time, the suspension includes alcohol (eg, methanol, ethanol, propanol, butanol, pentanol, hexanol, heptanol, octanol, nonanol, etc.), ester (eg, ethyl acetate, butyl acetate, propyl acetate, etc.) ), Ketones (eg, acetone, dimethyl ketone, methyl ethyl ketone, diethyl ketone, etc.) and aromatic solvents (toluene, hexane, cyclohexane, xylene, etc.) are preferably added, thereby As well as improving dispersibility, the volatilization of water and solvent after the addition of the information nucleic acid is promoted.

  In addition, these solvents are not limited to only 1 type, It is also possible to use 2 or more types of solvents together. These solvents may be added at the same time as the information nucleic acid or after the information nucleic acid is added.

  As the addition amount of the solvent, in the case of alcohol, the volume ratio of sterilized distilled water / alcohol is preferably in the range of 1 to 99. In the case of a solvent other than alcohol, that is, the ester, ketone, aromatic solvent In some cases, the volume ratio of sterile distilled water / solvent is preferably in the range of 1 to 75.

  That is, if the addition amount of the solvent is too small, the above-mentioned effect due to the addition of the solvent cannot be obtained sufficiently, and conversely, even if it is too much, the water tends to remain without volatilization due to a decrease in compatibility with water. There is a tendency to become insufficient.

Next, the non-exposed surface coating film of the present invention will be described in detail.
Such a non-exposed surface coating film is formed by disposing and solidifying the above-described non-exposed surface coating composition on an arbitrary substrate. For example, as shown in FIG. 3, a non-exposed surface coating film 2 formed on the back surface (upper surface in the drawing) corresponding to the non-exposed surface of the substrate 1 can be mentioned. The provided information DNA can be contained.

  The film thickness of the non-exposed surface coating is not particularly limited, but is preferably about 20 to 30 μm from the viewpoint of the detection accuracy of the information nucleic acid.

  In addition, as said arbitrary base materials, typically various metal materials, such as iron, aluminum, copper, various organic materials, such as a polypropylene and a polycarbonate, various inorganic materials, such as quartz and ceramics (calcium carbide etc.) Is mentioned. Moreover, as a method of coating these base materials with the non-exposed surface coating composition, a known and commonly used method can be employed. For example, a brush coating method, a spraying method, an electrostatic coating method, an electrodeposition coating method, and a sputtering method can be used. Furthermore, the non-exposed surface coating film only needs to be coated in a range where the information nucleic acid can be detected, and can be coated on the whole or a part of the substrate.

  In the non-exposed surface coating composition and the non-exposed surface coating film described above, individual authentication can be performed by detecting information nucleic acid.

  At this time, in determining the base sequence, the information nucleic acid data extracted from the non-exposed surface coating composition or the non-exposed surface coating, and the information nucleic acid database including at least the information nucleic acid data, It is desirable to contrast. The time required for product authentication can be significantly reduced by comparing with a database of information nucleic acids that has been grasped in advance.

  Examples of data stored in such a database include electrophoresis time and movement distance when gel filtration is performed (this is sufficient if the information nucleic acid itself is allowed to flow in the control lane).

  In the individual authentication method, when the information nucleic acid is amplified by the PCR method, the extracted information nucleic acid solution, PCR buffer solution, sterile distilled water, at least one primer, 2,3-dideoxynucleoside triphosphate Acid (dNTP) and polymerase are mixed, (1) heated at 92-95 ° C. for 2-5 minutes, then (2a) at 92-95 ° C. for 30-60 seconds, (2b) at 20-50 ° C. for 30- It is preferable that the heating cycle of (2c) at 70 to 80 ° C. for 30 to 120 seconds is repeated 20 to 50 times for 60 seconds, and then (3) heat treatment is performed at 70 to 80 ° C. for 1 to 10 minutes. In addition, it is preferable to use two types of primers from the viewpoint of increasing the arbitraryness of the base sequence of the information nucleic acid.

  In (1), 5 minutes at 94 ° C. is particularly preferable. If it is shorter than 2 minutes at 92 ° C., it will be difficult to separate the DNA into two differences, and if it is longer than 5 minutes at 95 ° C., the enzyme will be inactivated. In addition, it is unnecessary when the information nucleic acid contained in the coating composition for non-exposed surface or the non-exposed surface coating film is a single strand.

  Further, in (2a), 30 seconds at 94 ° C. is particularly preferable. When the temperature is shorter than 30 seconds at 92 ° C., the amplification factor decreases, and when it is longer than 60 seconds at 95 ° C., the enzyme is inactivated.

  Further, in (2b), 30 seconds at 40 ° C. is particularly preferable. When it is shorter than 30 seconds at 20 ° C., it becomes difficult to bind the primer and DNA, and when it is longer than 60 seconds at 50 ° C., the enzyme is inactivated.

  Further, in (2c), 30 seconds at 72 ° C. is particularly preferable. If it is shorter than 30 seconds at 70 ° C., the elongation becomes insufficient, and if it is longer than 120 seconds at 80 ° C., the enzyme is deactivated.

  Further, in (3), 7 minutes at 72 ° C. is particularly preferable. If it is shorter than 1 minute at 70 ° C., the elongation becomes insufficient, and if it is longer than 10 minutes at 80 ° C., time is wasted.

  Furthermore, the repetition of the heating cycles (2a) to (2c) is particularly preferably 30 times. When the heating cycle is less than 20, the amplification factor decreases, and when it is more than 50, time is wasted.

FIG. 4 shows a flowchart of an embodiment of the individual authentication method.
As shown in the figure, in S1, information DNA is extracted from the non-exposed surface coating composition or the non-exposed surface coating. In S2, it is concentrated by lyophilization. In S3, two kinds of primers and a polymerase are added. In S4, DNA is amplified by repeating PCR. In S5, the remaining excess primer is degraded by a single-stranded DNA cleavage enzyme. In S6, the double-stranded information DNA is purified by gel filtration. In S7, sequencing is performed by a sequencer.

  Here, in S1, for example, the non-exposed surface coating film may be powdered and mixed with a small amount of water. For example, when the information DNA is chemically bonded to the fine particles, it is hydrolyzed. Thus, it can be extracted efficiently. Moreover, in S2, you may concentrate using a centrifugal evaporator, for example. Furthermore, in S5, for example, Taq DNA polymerase, Tth DNA polymerase, Tfl DNA polymerase, Vent polymerase, Pfu polymerase, Bca BEST polymerase, KOD DNA polymerase, etc. can be used as the single-stranded DNA cleaving enzyme. Further, the target DNA may be amplified between S6 and S7 by repeating the same operations as in S3 and S4. In S7, sequencing may be performed by a mass spectrometer, or may be combined with sequencing by a sequencer.

  From the viewpoint of enhancing the convenience of isolation and purification of the information nucleic acid, it is preferable to derivatize the 5′-position hydroxyl group with biotin or a fluorescent molecule. Specifically, when vithion is used, it is easily adsorbed selectively on a column to which a protein called avidin is bound. On the other hand, when a fluorescent molecule such as fluorescein is used, the nucleic acid itself becomes fluorescent, so that it can be detected with high sensitivity and purification is facilitated. Thus, when the convenience of isolation and purification is increased, individual authentication becomes extremely easy.

  Furthermore, for example, when the 5′-position is substituted with sulfur, it can be easily separated by passing the column extracted with water through a carrier column coated with gold (Au).

  Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited to these examples.

1. Preparation of primer coating composition containing informational DNA with primer-corresponding sites Add a specified amount of fine particles with immobilized informational DNA of different particle sizes to Planet PP-7 made by Origin Electric Stir for 1 hour.

2. Fine particles used (1) Zinc oxide having an average particle size of 0.02 μm: ZS-032 manufactured by Showa Denko KK
(2) Aluminum oxide having an average particle diameter of 60 μm: AW40-74 manufactured by Micron Corporation
(3) Aluminum oxide having an average particle size of 70 μm: AW70-125 manufactured by Micron Corporation

3. Formation of Laminated Coating Film On a polypropylene plate with a thickness of 2 mm and 70 mm × 150 mm, Planet PP-7 manufactured by Origin Electric Co., Ltd. was coated so that the dry film thickness was 20 μm, and then baked at 80 ° C. for 30 minutes.

With respect to the obtained coating films of Examples 1 to 18, Reference Example 19, Reference Example 20, Example 21 and Example 22, whether DNA was detected or not was evaluated as follows.

<Detection of information DNA>
(1) The test piece of the information nucleic acid composition was cut into pieces using a cutter.
(2) 5 mL of sterilized distilled water was added to the test strip and stirred with a magnetic stirrer to extract DNA into the aqueous layer.
(3) The test strip and the aqueous layer were separated using a centrifuge, and the aqueous layer was concentrated using a centrifugal evaporator.
(4) DNA solution (5 μL) eluted and collected, PCR buffer (5 μL), Taq polymerase (0.25 μL), sterile distilled water (24.75 μL), 5 μM primer 1 (5 μL), 5 μM primer 2 (5 μL) , And 2 mM dNTP (5 μL).
・ Primer 1 ... 5'-TGCACGCACCGTGTACTC-3 '
・ Primer 2 ... 5'-CCGACCAACGTGTCCACT-3 '
(5) After heating at 94 ° C. for 5 minutes, [94 ° C. for 30 seconds → 40 ° C. for 30 seconds → 72 ° C. for 30 seconds] was repeated 30 times.
(6) After being treated at 72 ° C for 7 minutes, it was stored at 4 ° C.
(7) Using a single-stranded DNA cleaving enzyme (S1 nuclease), the excess primer was decomposed and the target double-stranded information DNA was purified by gel filtration.
(8) One kind of primer (primer 1: 5′-TGCACGCACCGTGTACTC-3 ′) and fluorescently labeled 2,3-dideoxynucleoside triphosphate were mixed with the purified information DNA.
(9) The same operations as in the above steps (4) to (6) were performed.
(10) After gel filtration purification, it was subjected to sequencing by using an automatic sequencer.
As shown in Tables 1 and 2, when these steps were performed and detection of informational DNA was attempted, the case where the informational DNA was easily identified was ○, and the case where further PCR treatment was necessary was Δ It was.

<Evaluation of adhesion>
After leaving it in an atmosphere of 50 ° C. and a relative humidity of 95% for 500 hours, it was taken out and checked for adhesion. The adhesion force is determined by drawing the parallel lines of 11 vertical and horizontal lines that reach the coating substrate with a cutter knife (specified in 7.2 (2) (e) of JIS K 5400) at intervals of 2 mm. A cross-cut was formed. A cellophane tape (specified in JIS Z 1522) was brought into close contact with the grid-like coating film and peeled upward at a stretch, and the number of grids remaining in the 100 grids was measured. The results are shown in Tables 1 and 2.

As is apparent from Tables 1 and 2, the non-exposed surface coatings of Examples 1 to 12 can identify the information DNA well and show good adhesion. In other words, the desired adhesion can be obtained with the same workability as normal coating, and the unexposed surface coating can be identified.
In contrast, the non-exposed surface coatings of Examples 13 to 18, Reference Example 19, Reference Example 20, Example 21 and Example 22 have the information DNA content, the particle size and the amount of fine particles used. Since any of these deviates from the preferred range of the present invention, it can be seen that either the discriminability or adhesion of the information DNA is reduced.

This is a structural formula showing natural DNA and DNA derived from this 5 'position. It is the schematic which shows the information nucleic acid which has a primer in both ends in an authentication information site | part. It is a cross-sectional schematic diagram which shows an example of a non-exposed surface coating film. It is a flowchart which shows an example of an individual authentication method.

Explanation of symbols

1 Polypropylene plate (base material)
2 Informatized DNA-containing non-exposed surface coating layer with primer

Claims (5)

A non -exposed surface coating composition comprising an information nucleic acid having a site having an arbitrary and known base sequence in a non-exposed surface coating material,
A non-exposed surface coating composition, wherein the information nucleic acid is carried on fine particles having an average particle diameter of 0.01 to 60 µm .   The non-exposed surface coating composition according to claim 1, wherein the information nucleic acid is contained in an amount of 0.5 to 2000 μg per 100 g of resin solid content. The non-exposed surface coating composition according to claim 1 or 2 , wherein the content of the fine particles is 0.5 to 70% of the resin solid content. The non-exposed surface coating material is any one of a lipophilic liquid, a hydrophilic liquid, and a powder. The non-exposed surface according to any one of claims 1 to 3, Paint composition for exposed surface. A non-exposed surface coating film obtained by solidifying the non-exposed surface coating composition according to any one of claims 1 to 4 .

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