TWI225097B - Anti-fake biolabeling system - Google Patents

Abstract

A receptor-ligand recognition biolabeling system comprising one or a plurality of receptors; a basal layer, which one part is immobilized by the receptors and the other part is blocked by an inert material; one or a plurality of bio-active ligands; and signal-producing means which is manipulated to bind to the bio-active ligand is recognizable to the receptor and the signal is produced on the surface of the basal layer.

Description

1225097 V. Description of the invention (l) Field of the invention The present invention relates to a biological anti-counterfeiting barcode, and in particular to the use of identification and binding power between biological molecules and / or small molecule compounds to generate specific signals or patterns. , Which can be used in the field of anti-counterfeiting, anti-counterfeiting or anti-piracy, and get results quickly and accurately. Background of the invention <dot; == product barcode ′ is the use of stripes with different thicknesses as a permutation combination, printed on the surface of the product.

Y leather is counterfeit and has no anti-counterfeiting function at all. In recent years, full photos (ograra) have been introduced to the surface of products, providing another: using a laser pattern as an anti-counterfeiting function: :: to be trained professionals to ^ ^ break and require space for application. In addition, this year has: Judgment 'so restrict the knowledge system, A uses a specific method, and the industry has introduced DNA anti-counterfeiting discrimination ~ ~ Fu Fu DNA fragments are coated on the primer of Rui σ μ ^ Guan as a cumulative test; but / On the quotient, and the application of phase (PCR) technology, and / or the use of polymerase chain reaction preparation, therefore, not only time consuming = requires m :; the operation of the operator to obtain results. Therefore, in the process of studying timeliness, & L _ π editions, there is a need for Luju to prevent the detection of hidden evidence, counterfeiting, and high-cost and low-cost prevention. Li can quickly know the results and accuracy. First of all, the purpose of property rights is to effectively detect illegal and to ensure the use of antibodies or receptors. Α ά has been widely used in immune diagnostic tests = special compounds in urine ^ fluid, such as ovulation test, pregnancy test 0643-5754TWF .ptd _ Page 4 1225097 V. Description of the Invention (2), Amphetamine Test Reagent, etc. This reagent usually contains a pair of antibodies, and the knife does not recognize different positions of an antigen. One of the antibodies is fixed in the test, and the other carcass is bound to enzymes, liposomes, or colored beads. At one end, when the test object is bound to the adsorbed antibody, it is moved on the test 4 membrane by Mao Erli, until the complex of the test object and the adsorbed antibody 蟪 = the first antibody fixed on the membrane can be generated. Obvious anti-21β 1fit ^ irnbaUm, An ^ ytical Biochemistry 206: 168-171, 1992. Immunity :: 2Γ '° 46, ° 58 discloses a test piece device, which uses the ϊ liquid specimen to contain a certain special substance, and continues to move to the c ”body knot containing the standard special substance to generate a color band, which indicates the specimen. It does not contain the special shell to be tested, on the contrary, the positive or negative result is determined by the generation of color bands. Quality, this device US Patent No. 5,260, 1 94 discloses a substance that measures% absorption fixed on two This kind of receptor is placed in the first piece, this -HRP, the second receptor can bind the antibody to the test object, = the higher the concentration of the test object, the test object_HRP moves to the first receptor :: The larger the test substance in the body, the stronger the signal produced by the first receptor, the more the quantity of the test substance is due to the amount of dioxin. 'Analysable in the sample to be tested US Patent No. 5,770,460 discloses a non- Test device, this non-absorbent material is divided into three-fold single-step specimen entry zone, the second zone is the zone containing the test substance, the first zone is-colored beads, the third page 5

1225097 V. Description of the invention (3) The zone fixation can be combined with the test object-anti-test object-when the sample contains the substance to be tested, u should be in line. $ 1 can produce anti-US patent No. 4,703,01 7 which can be observed with the naked eye-Lutian ^ specific receptors on the nitrocellulose material, the method of oral phase / knife analysis, fixing lipids When the body forms a complex, the total energy of the object and the object to be tested-wired to determine the existence of the object to be tested. 〃, limb, ，, mouth 5, and a colored reaction appears. These previous techniques and principles listed above use specific antibodies to detect specific 5 applications.

Easy for everyone = Designed to enable epidemiological principles to be applied in the field of anti-counterfeiting. There are teaching techniques to avoid this. In addition, the US patent No. 5776-dimensional material surface, and then with denaturation; single-stranded open-light reaction, and by electrochemical or gene gene sample US patent No. 5 on detection Party detection. Method, which produces a aptamer that can be bound to the blood number master / a kind of oligo sequence. Appropriate factors and all surface molecules are suitable for the above-mentioned prior techniques using nucleic acids, and the ancient and basal layers of the face have not been neutralized, and probes have been fixed in the field of anti-counterfeiting. The surface is connected to a coloring agent, which is combined with the identification of the application. Table ::: Soil molecules and / or small molecule compounds, ^ generation tool, on the surface of the product Λ: 0643.5754TTVF.ptd Page 6 1225097 5 2. Description of the invention (4) Arrangement and combination of certain signals or patterns, and ^^ And 'this kind of anti-counterfeiting label is in appearance; = Du Ersi's identification system. Otherwise, it is impossible to know the behavior of the anti-counterfeit through the test solution. 'The style of wearing, so that counterfeiting can be eliminated. Summary of the invention = Ming Er for a biological anti-counterfeiting test = multiple receptors; ⑻-basal layer, where the receptor is solid. 二-said surface _ surface, the other of the basal layer Some are based on ―㈣ ^ on the substrate =-one or more biologically active ligands;

ΐ :; produced by artificially binding with the biologically active ligand; ΐ: ，, the biologically active ligand can identify the characteristic;-wherein a signal is generated on the surface of the basal layer. Another aspect of the present invention that is fixed in the party body is to provide: (a) a thief overnight, a biological security label, including: (a) # or multiple carcasses; and基底 A base layer is fixed on the surface of the base layer with a specific pattern. ^ == The body is blocked with -inert material. On the other side of the base layer, another aspect of the present invention imitates the present invention is to provide-a method for detecting whether the product is a 2 or profitable version, including: (1) combining the biological anti-counterfeit label provided by the present invention with a Solution contact, wherein the solution contains one or more active ligands, and the biologically active ligand is operatively combined with a signal generating tool, and (2) monitoring the biological anti-counterfeiting label for a period sufficient to detect a The time at which the signal is generated visually to confirm whether the biological anti-counterfeiting label has a specific pattern designed. In another preferred embodiment, the present invention provides a biological protection

0643-5754TWF.ptd Page 7 1225097 V. Description of the invention (6) 22 ~ A solution containing a biologically active ligand. DETAILED DESCRIPTION OF THE INVENTION The present invention provides a biological anti-counterfeit test sleeve, which is used on a base = (for example, filter paper, nitrocellulose fiber membrane, fortune membrane, poly-smoke membrane or glass, · ', quasi membrane), and will The receptors are fixed in different positions, for example, they can be configured into different graphics; when reacting with various biologically active ligands = test solution, the same receptors will bind their specific ligands to form a complex and present The authenticity of the specific color "graphics" and the location of the source of the product. Π Yu Ji's !! ΐ is to provide a unique receptor arrangement pattern surface of a variety of products. Now its ^ = wide bottom, can be joined by different The agent is fixed on the merchandise table arrangement chart. Fortunately, the surface of the product can be directly pressed, that is, the receiver sounds on the human body = ^, or the base capillary phenomenon of different shapes is made and the material of the liquid is absorbed. ： The base layer of Jiadi is composed of porous carrier material that can provide Shangjietian. Graphical receptors, including π, nucleic acid, oligobiotics specifically designed antibodies, enzymes, and small molecule compounds. Good, can be used to dilute with j = Or fix the graphics on the base layer to reveal more A and stylistics to increase the PP / 丨 / V type. The fixing method is not limited to nan. Any biomolecules can be fixed, especially and these The technique is familiar with this technique: technique = can be used, and the salt solution or organic solvent, the receptor solution point :: two :! For example, the conventional method using carbonic acid is coated on the substrate layer. Then two on, or In any case, when the solution is removed, the base 1225097 V. Description of the invention (7) The surface of the bottom layer is blocked with an inert substance (b 1 0ck i ng) to avoid unwanted immune adsorption reactions. Can be used Inert substances include bovine serum albumin (BSA), gelatin (geiatin), or the like, and this blocking reaction is well known to those familiar with enzyme-linked immunosorbent analysis (elisa).

Another feature of the kit of the present invention is that it can recognize the biologically active ligands of the above-mentioned specific receptors. Among them, the specific ligands and the receptors have an identification binding power, such as the binding power between antigens and antibodies, nucleic acids and The forces of the complementary sequence include non-covalent forces, hydrogen bonds, van der Waals forces, hydrophobic forces, ionic forces, etc. The source of the biologically active ligands that can be used in the present invention is not particularly limited, as long as it can produce a binding force sufficient for identification with the selected receptor, including antibodies, single-stranded nucleic acid probes, lecithin ( lecithin), lipids, carbohydrates or proteins. In addition, 'on the biologically active ligand of the present invention, a signal generating tool can be operatively linked according to design requirements, whereby when the biologically active ligand is bound to a receptor fixed on the surface of the basal layer, visual observation can be generated Detectable signal or pattern 0 The above-mentioned signal generating tools are well known to those skilled in the art, and can be used as required, including 'indicators, fluorescent immunoassay agents (for example, steel fluorescent materials) , Cold light markers (for example, biological cold light markers or chemical cold light markers), enzymes or secondary antibodies; where the secondary antibody system refers to antibodies (including single or multiple strains) that can be used against the biologically active ligands used And is conjugated to an enzyme (eg, alkaline phosphatase, peroxidase, or callase)

1225097 V. Description of the invention (8) ^^ (rr; rridase) #) Including — = ί ”Health tools, the kit of the present invention can immunologically analyze shore reactions to generate signals. The relevant methods and reaction conditions can be See Ant i body: A τ A ^ M ai, —Ed. Hari〇w & Dayid Lane, Laboratory beads, in the example, the indicator can choose its own color micro

C; U granules include polystyrene ... Dilute toluene, polyethylenimide Peptides: polymers of dilute acid; metal colloidal solutions that can be used include:-inventions can be made in advance in large quantities to produce biological security labels, including Attached to :: Two and one or more types of receptors fixed on it, used to attach ... This method is only used as an example. See Figure 1 and Figure Not Sound (10 ;: the scope of various applications. Refer to Section Figure 1: Base f 2 条 Strip-shaped porous membrane, this porous membrane is activated: amine fiber, quasi-membrane (Nylon) or nitrocellulose membrane, the receptor BSA-biotin is immobilized on the membrane (12) and BSA_TNP (14), after drying and blocking, that is, I stick to the back of various stickers or trademarks (16), the back of stickers or trademarks ^ 3 There are sticky substances such as polyester, where the front of the stickers or trademarks is like (20) All colored beads, Oi-Og micrometer (" m), bean surface with carboxyl group (-C00H), using the coupling reagent EDC (Ethyl_3_; [, 3_ Di 曱Aminopropyl] carbodiimide) can react with carboxyl and amine groups simultaneously

1225097 V. Characteristics of the invention (9) 'After reacting the carboxyl group of the colored microbeads with EDC, then reacting with streptavidin or the amine group on the structure of the anti-TNP antibody' A streptavidin or anti-TNP antibody is bonded to the colored microbeads, and BSA is added to block the unbonded portion of the microbeads. The microbeads can be replaced with emulsion particles, metal colloidal solutions or dye colloidal solutions. The final microbead concentration is 0.2% (w / v).

The anti-counterfeiting label attached to the surface or hidden place of the product can be removed separately during testing or the trademark sticker (18) with the anti-counterfeiting label can be directly immersed in the solution containing the bioactive ligand ( 22), or the solution (22) containing the bioactive ligand is dropped on the anti-counterfeit label (10), and the capillary solution of the porous membrane gradually moves the microbead solution above the membrane, and the red microbeads ( Streptavidin) and blue microbeads (anti-TNp antibodies) ', agglutinated at the positions where BSA-biotin and BSA-TNP were fixed, respectively, forming red lines (see Figure 2). Because the position of the line or the figure is /, 'two paired with the test solution' cannot be counterfeited. The intensity of the signal can be determined by the concentration of the fixed receptor. After the fixed BSA-Biotin comparison signal of 0-5_1 millimeters is generated, the surname must be produced, and the striker can be found: the best results can be obtained if the ancient itchy, ancient, and fruit are found; the degree of love is 1 G / ml MA-biotin.

^ Tested the stability of BSA-biotin or BSA-TNP immobilized on the filter 'for other preparation-batch 0.5 × 3 cm filter membrane, and in .tnp' It sticks to the surface of the sticker, and then pastes this sticker on the surface of the item. They were placed at 4t (2-0-, 30t :), and color test was performed at different time points-to determine the stability of the filter. According to the results, this diaphragm can be at least 6,

1225097 V. Description of the invention (ίο) Normal appearance within months. Examples The present invention will be described in further detail by the following examples. However, these examples are merely examples, and are not intended to limit the scope of the present invention. Example 1 · Bonding of small molecules with bovine serum albumin (A) Biotinylation of BSA (biotinyiation)

20 mg of BSA was dissolved in 50 μm sodium decomposing buffer (pH 8.0) at a concentration of 2 mg / ml and placed in a 15 ml centrifuge tube. Another 1 mg of lucyl-NHS-biotin (Pierce; v. 21 335) was dissolved in 1 ml of water (1 mg / μl), and 0.5 ml of the sulfur-NHS-biotin solution was directly Add a centrifuge tube containing the BSA solution, and react at 4 ° C for 2 hours. After the reaction, put the solution in a dialysis bag to completely remove unreacted sulfur-NHS-biotin, and finally save the BSA-biotin Reserve in 4 arts. (B) Combined co-consumption of 2,4,6-dinitrobenzene was combined to prepare 2 mg / ml of BSA solution dissolved in 0 μm borate buffer (pH 9.2), 10 ml was added per 4 mg of BSA. 〇μg of TNBS (2,4,6-trinitrobenzene sulfonate; S i gma), after mixing, protected from light, and allowed to stand at room temperature for 20 hours for reaction. After the reaction, the unreacted iTNBS was removed by dialysis against phosphate buffered saline (pBS), and TNP-BSA was replaced in the PBS solution, and finally stored at 4 t for future use. Example 2: Preparation of biological anti-counterfeit label BSA-biotin or BSA-TNP at a concentration of 10 mg / ml was added to a solution of BSA-biotin at a concentration of 1 μ NaCl

0643-5754TWF.ptd Page 13

Claims (1)

1 · A biological anti-counterfeiting test kit, which includes ·· one or more receptors, which hurts people and people. θ π //, selected from the group consisting of antigens, antibodies, enzymes, small molecules and white matter, and combinations thereof; wherein the receptor is fixed on the surface of the basal layer, and the trauma of the basal layer is An inert substance blocking; one or more biologically active ligands; and a flood generation tool operable to generate signals with the biologically active ligand; wherein the biologically active ligand can recognize the specific receptor, This generates a signal on the surface of the substrate to which the receptor is fixed. 2. The biological anti-counterfeiting test kit according to item 1 of the scope of patent application, wherein the base layer is composed of a porous carrier material that can provide capillary phenomenon and absorb liquid. '3 · The biological anti-counterfeiting test kit according to item 2 of the scope of the patent application, wherein the base layer is selected from the group consisting of filter paper, nitrocellulose membrane, nylon film, polyolefin film and glass fiber membrane. 4. The biological anti-counterfeiting test kit according to item 1 of the patent application, wherein the receptor is fixed on the surface of the base layer with a specific pattern. 5. The biological anti-counterfeiting test kit according to item 1 of the scope of patent application, wherein the biologically active matching system is selected from the group consisting of antibodies, single-stranded nucleic acid probes, lecithin, lipids, carbohydrates and proteins. 6. The biological anti-counterfeiting test kit according to item 1 of the scope of patent application, wherein the signal generating tool is selected from the group consisting of an indicator, a fluorescent immune test agent, a cold light marker, an enzyme and a secondary antibody. . 7. The biological anti-counterfeiting test kit as described in item 6 of the scope of patent application 0643 -5754 twf3; ch i umeow.ptc Page 16 ^ No. 8912585 ^ No. 6, patent application scope, group, where the indicator is selected from the solution and dye colloidal solution colored beads, milk particles, metal colloids 8 · As in the ethnic group of patent application scope f. Group, in which the biological micro-counterfeit test kits described in item I of the colored micro-specials, and the ethylenic-acrylic milk ear granules include polystyrene and polyethylenimine 9. As the scope of patent application Polymer of aldehyde. Group, in which the metal colloid is soluble, and the biological anti-counterfeiting test kit described in item 7 includes the silicone solution as # in the scope of the patent application. Group, wherein the cold-light marker is the biological anti-counterfeit test kit marker described in item 6. Select from biological cold light markers or chemical cold light II. As in the patent application scope group, the biological anti-counterfeiting test kit described in the secondary antibody system b /, the antibody is more conjugated with an enzyme antibody against the biologically active ligand. And 12. · As claimed in the scope of the patent application group, the enzyme is selected from the family consisting of the biological anti-counterfeiting test kit lactosidase enzymes described in the item ^ = phosphatase, catalase and / 5 -half 13 · If the scope of patent application is °, it also includes a substrate, the biological anti-counterfeit test cover 1 4 described in item w 1. If the towel asks for patent 4 enzyme reaction to generate a signal. Group, wherein the inert substance includes the biological anti-counterfeit test kit described in the item *. Bovine serum albumin, gelatin or similar 1 5 · a kind of biological wound prevention ^ tags include: ",, π iron, which is characterized by the biological anti-counterfeit one or more receptors, natsuko compounds, proteins and their ... Self-antigens, antibodies, enzymes, small fractions --- --- ~ &group; and III nu 鼸 JJtlWJIUU-VU Ji. · IU sinks, · 1 I · ^ 0643-5754twf3; chiumeow.ptc Chapter 1225097 Modified a base layer, in which the receptor is fixed on the surface of the base layer with a specific pattern, and the other parts of the base layer are blocked with a / inert substance. 16 · The biological anti-counterfeiting label as described in item 15 of the scope of the patent application, wherein the base layer is composed of a porous carrier material that can provide a capillary phenomenon and a material that adsorbs liquid. 17 · According to the biological anti-counterfeiting label described in Item 16 of the scope of the patent application, wherein the base layer is selected from the group i of filter paper, confirmation fiber film, nylon film, polyolefin film and glass fiber film in. 1 8. The biological anti-counterfeiting label as described in item 15 of the scope of patent application, wherein the inert substance includes bovine serum albumin, gelatin or the like. 1 9 · A method for detecting whether a product is counterfeit or pirated, comprising: (1) contacting a biological anti-counterfeit label as described in any one of item 15 and item 18 of the scope of patent application with a solution, wherein The solution contains one or more biologically active ligands, and the biologically active ligands are operatively combined with a signal generating tool; and (2) monitoring the segment of the biological security label is sufficient to observe a visually generated signal. Time to confirm whether the biological anti-counterfeiting label has a specific pattern designed. 2. The method as described in item 1g of the scope of Shen Qing's patent, wherein the biologically active ligand is selected from the group consisting of antibodies, single-stranded nucleic acid probes, lecithin, lipids, carbohydrates, and proteins. ′ Wherein the signal is a cold light marker, 2 1 · The method as described in item 19 of the scope of patent application The production tool is selected from the group consisting of fingerless agent, glorious immune test agent, enzyme and secondary antibody. Wherein this instruction 2 2 · The method described in item 21 of the scope of patent application --- page 18 L 厶 厶 I VI. Patent application ^ 5855 The correction agent is selected from its own solution Beads, milk particles, colloidal solutions, and dye colloids. , 23 Such as the microbeads colored in item 22 of the scope of patent application. .1 The method described in item τ π π and π π. Brewery: Porous particles include polymers of polystyrene, polyvinyl toluene 24, and polyacryl.腴 所 & · According to the method described in item 22 of the scope of application for patent, the gumbello solution includes a stone gum solution. 2 ζ »• As the method described in the scope of patent application No. 21, ... Λ Do not choose the biological cold light marker or chemical cold cursor 3 ^ 2 6 · The method described in the scope of patent application No. 21 Among them, the secondary antibody system is against the antibody of the biologically active ligand, and the antibody is linked to an enzyme. 27. The method according to item 26 of the scope of the application for patent application f's enzyme is selected from the group consisting of self-testing discontase, catalase and galactosidase. The method of 0, 0, 10, and more includes the addition of Z8. As in the patent application No. 26, the pigmented polystyrene-wherein the metal in which the cold light is a substrate, which can react with the enzyme and Generate signal § 0643-5754twf3; chi umeow.ptc Page 19 1225097 Figure 3