WO2001053516A2

WO2001053516A2 - Detection system for verifying the originality of an object and a test device for carrying out said verification

Info

Publication number: WO2001053516A2
Application number: PCT/EP2001/000764
Authority: WO
Inventors: Peter Schneider; Armin Nass
Original assignee: Sension, Biologische Detektions- Und Schnelltestsysteme Gmbh
Priority date: 2000-01-24
Filing date: 2001-01-24
Publication date: 2001-07-26
Also published as: DE10002819B4; AU2001242348A1; WO2001053516A3; DE10002819A1; EP1257660A2

Abstract

The invention relates to a detection system for verifying the originality of an object. At least one component of the detection system is connected to the object or contained therein, whereby said component acts as the marker. The invention is characteterised in that the detection system contains a) at least one substrate for at least one enzyme, b) a catalyst comprising b1) at least one enzyme for at least one substrate and/or b2) at least two components which, together, form at least one enzyme for at least one substrate and c) optionally at least one indicator system that is in tune with the enzyme-catalysed substrate conversion and is used for generating signals. A signal is generated by means of the combination of the substrate and enzyme only or the combination of the substrate, enzyme and indicator system. The invention also relates to a test device, especially for carrying out such a verification. The inventive detection system and the inventive test device are suitable for verifying or identifying products, product packings or labels for instance.

Description

Detection system for checking the originality of an object and test device for performing the test

The invention relates to a detection system for checking the originality of an object, at least one component of the detection system being connected to or contained in the object as a marker.

The invention further relates to a test device, in particular for carrying out such a test.

The detection system according to the invention and the test device according to the invention are suitable for testing or identification, e.g. of products, product packaging or labels.

Both non-selective and selective methods for checking the authenticity of objects are already known, the authenticity check preferably being carried out using a selective method.

WO 98/33162 describes a selective method for checking the authenticity of objects which is based on the selective interaction between a "print molecule" or an analog and a "molecularly imprinted molecule". In this method, the component to be detected ("print molecule") is impressed on a polymer which then contains three-dimensional impressions ("molecularly imprinted molecule") of the molecule. The proof of originality is based on the (more or less) specific recognition of the imprinted molecule by the polymer.

From EP-A-0327163, US-A-5,429,952 and WO 95/06249 the use of specific antibodies in an immunoassay for authenticity testing is known. WO 87/06383 uses macromolecular test components, such as proteins and nucleic acids, for labeling products, the specific detection being carried out in the case of proteins with labeled antibodies and in the case of nucleic acids with correspondingly labeled complementary nucleic acid probes. A disadvantage of this method is that the formation of a complex by two complementary binding partners does not lead directly to an evaluable signal, and consequently steps connected in series, for example to separate non-specifically bound reagent, are necessary for the originality check in order to obtain a specific signal.

In the described methods, several steps connected in series for the authenticity check are always necessary in order to receive a signal, with each of these steps being able to be carried out incorrectly. Due to the number of necessary process steps, the analyzes are extended and the costs are increased. In addition, a quick, simple and non-error-prone procedure is particularly desirable for an originality check that should be carried out quickly, regardless of instrument, and also by untrained people.

Due to the increasingly widespread counterfeiting of branded products or the manipulation of information that is directly related to the product, e.g. Expiry dates of drugs, there is a strong need for a selective, difficult to counterfeit, difficult to circumvent or manipulate, simple and very quickly feasible procedure for checking the originality of objects or information related to the object.

The object of the present invention is therefore to provide a detection system which enables simple and quick checking of the originality of objects and / or information or data related to the object.

Another object of the invention is to provide a test device, in particular for simple and quick checking of the originality of objects.

In a first aspect, the invention provides a detection system for checking the originality of an object, at least one component of the detection system as Marker associated with or contained in the object, characterized in that the detection system

a) at least one substrate for at least one enzyme,

b) comprising a catalyst

bi) at least one enzyme for at least one substrate and / or

b ₂ ) at least two components which together produce at least one enzyme for at least one substrate, and

c) optionally contains at least one indicator system for signal generation which is matched to the enzyme-catalyzed substrate conversion,

whereby a signal is generated by the combination of substrate and enzyme b- or substrate and at least the two components b ₂ ) alone or by their combination with an indicator system.

The two components b ₂ are preferably at least one apoenzyme and at least one coenzyme.

Substrates which can be used according to the invention are chemical compounds whose catalytic conversion by enzymes leads directly to an identifiable product; or their implementation leads to a product that leads to an identifiable product through a downstream indicator system. The identifiable product can be detected, for example, by adding chromogens or fluorogenic substances. Substrates that can be used in connection with the detection system are basically all known substrates of oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases, the enzymatic conversion of which leads directly or indirectly to an identifiable product. The substrates can preferably be converted by galactosidases, glucosidases or phosphataseπ. For example, the galactosidase treatment of a galactose chromogen can release the chromogen. sets and then be verified directly. Also preferred as substrates are p-nitrophenol derivatives, the catalytic conversion of which causes the release of p-nitrophenol.

The detection system according to the invention can also contain an indicator system for signal generation that is matched to the enzyme-catalyzed substrate conversion. If the enzyme-catalyzed conversion of the substrate does not lead directly to an identifiable, in particular colored, product, the detection system contains the indicator system. The indicator system can contain the components of a downstream enzyme reaction in which the product of the first reaction is reacted with a second enzyme, so that a directly identifiable, in particular colored, product is produced. The indicator system can also or alternatively contain chromogenic or fluorogenic substances which react with the product to form an identifiable, in particular colored, product.

The chromogenic or fluorogenic substances can, for example, be antibodies specific for the product, which are linked to known chromophores or fluorophores, e.g. FITC, can be coupled.

The detection system according to the invention enables the originality of objects, such as products, packaging and labels, to be checked simply, quickly and without errors.

In addition to solid surfaces, the object to which the marker is connected can also include, for example, pulverized objects or other states, such as liquids.

The detection system is difficult to forge if its composition is unknown to the counterfeiter. It can also be used by untrained people without any problems.

The detection system according to the invention can contain its components which lead to signal generation in various possible arrangements, with the proviso that that at least one component must be linked to or contained in the object as a marker. The marker is preferably invisible on or in the object. The other components can be contained individually or as a mixture of components in the respective embodiment. In a preferred embodiment, these components are contained on the reaction field of the test device described later.

According to a preferred embodiment, the substrate can be connected to the object as a marker or contained therein. Advantages of this embodiment are:

Greater flexibility in use

There are a variety of enzyme substrates with different chemical properties (hydrophilic-hydrophobic, light-absorbing or not, inert or reactive e.g. against oxidizing agents, liquid-solid). A selection can thus be made according to the application of the method for marking an object. In contrast, the properties of enzymes and binders are not so variable.

Greater stability of the marking

Enzyme substrates can be selected according to their stability. Enzymes and enzyme fragments as well as binders, on the other hand, require defined conditions for good long-term stability with regard to biological activity. These conditions are often not compatible with the circumstances of an application process.

In addition to the important long-term stability of the marker (eg on a label), the stability of the marker also plays an important role in the production of a label, for example. There is an extremely wide range of different manufacturing processes, such as labels. External factors in the manufacturing process such as temperature, chemicals and solvents or conditions in drying processes play a role. Chemically relatively resistant organic molecules have a significantly higher resistance than complex enzymes, which can easily be subject to denaturation, for example. Different components, such as varnishes or paints, are often used on an object. Each of these components has its own chemical characteristics. Organic markers are much easier to adapt to these different chemical components than the biomolecules (enzymes), which are generally sensitive to external influences.

When testing new markers, e.g. on labels, their stability must always be guaranteed and must therefore be checked accordingly. This important factor is much easier to ensure with a system that uses a less sensitive substrate as a fixed component instead of a sensitive biomolecule.

Less susceptibility to errors

This embodiment also has a low susceptibility to errors compared to the connection of the enzyme to the object, since even small amounts of entrained enzymes, e.g. the body's own enzymes on skin surfaces or unspecific catalysis can lead to false positive results.

For detection that is not based on enzymatic activity, the detection procedure is more complicated and involves several steps to separate unbound material from the binding complex and then to detect it. This limits the practicability and increases the susceptibility to errors in the test.

Greater economy

The marking must be on every object. Especially for products with large quantities and low intrinsic value, attention should be paid to low additional costs through the marking. Enzymes and enzyme fragments as well as binders are complex to produce and often expensive, while enzyme substrates are often inexpensive.

Furthermore, either an enzyme, an apoenzyme, a coenzyme, a mixture of apoenzyme and coenzyme, a mixture of substrate and coenzyme or a mixture of substrate and apoenzyme can be linked to the object as a marker or contained therein. In a preferred embodiment, a coenzyme is linked to the object

The amount of coenzyme required can ideally be chosen to be so small that it is practically impossible to clarify the structure due to the wrong person. Great advantages are therefore achieved under the safety aspect which is generally extremely valued by the user

A mixture of substrate and coenzyme associated with the object is particularly preferred. Advantages of this embodiment are

a) low amounts of labeling substances (eg by increasing the sensitivity of detection), b) the establishment of systems which only produce the desired signal when several marker substances are present, and c) the deactivation of the analyzability of the test device explained below by the wrong person in any case to force on the costly and time-consuming way of structure elucidation, since the test device does not contain any active enzyme in the absence of the coenzyme

The small amount of coenzyme is possible because the coenzyme is later part of the catalytic system and is therefore not required stoichiometrically for signal generation. The test device does not contain the complete enzyme, but at least one apoenzyme. The wrong person had to identify the coenzyme in addition to the labeling substance to generate a signal with the test device. In this case, the coenzyme acts as a molecular switch for the enzyme system on the test device

The detection can, for example, be carried out in such a way that, for example, a textile fabric impregnated with one or more components of the detection system or a pen impregnated therewith is brought into contact with the object to be checked and a check is carried out to determine whether a signal is obtained. The components required next to the marker of the detection system can, for example, also be in the form of a solution or in another suitable form, such as a suspension, which is applied to the object The enzyme in the detection system according to the invention can be, for example, an oxidase which reacts for the reaction of the corresponding substrate or cosubstrate, an enzyme which is specific for the reaction of mono-, di- or ogosaccharides or for other polyhydroxy compounds, a diaphorase, phosphatase, hydrolase, Urease, esterase or polymerase or a mixture of such enzymes may be included

Many enzymes belong to the proteids, which consist of a protein portion (apoenzyme) and a prosthetic group (coenzyme). The enzyme shows its full enzymatic activity only after the coenzyme has bound to the apoenzyme. According to the invention, the expression "coenzyme" also includes cofactors The terms “coenzyme” and “prosthetic group” are used synonymously with one another. Examples of suitable coenzymes are NAD, NADP, FMN, FAD, ubiquinone, lipoic acid, ATP, pyoxydoxalphosphate, adenosylmethionine, tetrahydrofolate, biotin and coenzyme A. Examples of apoenzymes are NAD- or NADP-dependent dehydrogenases, flavoprotems, ammosau reoxidase, xanthine oxidase, aldehyde oxidase and ferredoxins

According to the invention, a signal can be generated, for example, directly by an enzyme-catalyzed conversion of the substrate, which leads, for example, to a visible change in color

The indicator system leads directly through one or more known subsequent reactions after the enzyme-catalyzed conversion to a signal, for example a color change

With the detection system according to the invention, it is possible to generate sufficient signals, for example color signals, even through the conversion of relatively small amounts of substrate

In the detection system according to the invention, any compound which can be reacted with an enzyme and which leads to an evaluable signal - optionally with the additional use of an indicator system - can be contained as substrate

For example, the substrate can also be present in a form bound to a polymer on the object, as in a label According to the invention, the test results are preferably evaluated with the naked eye alone, without measuring devices or other aids.

The invention further relates to a test device, in particular for carrying out the testing of an object for originality using the detection system according to the invention. A preferred embodiment of the test device according to the invention is shown in FIGS. 1 and 2.

1 shows a top view of a preferred test device, and

2 shows a cross section of this test device.

In its simplest form, the test device according to the invention comprises a carrier with a reaction field 1, which in particular contains the components required for carrying out the detection. The use of the test device according to the invention enables quick and uncomplicated testing, which can also be carried out by persons who have not been specially trained. It also has the advantage that after checking, no signal remains on the marked object.

The test device is not restricted to the method according to the invention. The test device is also e.g. usable for performing diagnostic assays.

If the test device is used for checking the authenticity, it can be used, for example, with detection systems based on immunoassays (EP-A-327 163, US 5,429,952, WO 95/06249) or on macromolecular test components such as proteins and nucleic acids (WO 87/06383) , The immunoassay is preferably a homogeneous immunoassay. The detection system suitable for implementation with the test device can also be based on complexometric detection.

In a preferred embodiment, the carrier of the test device further comprises a transmission field which can be brought into contact with the reaction field. Preferably the transfer field is brought into contact with the reaction field by kinking the support.

Advantageous properties of the preferred test device are:

1. The possibility of collecting, tapping, binding, etc. a substance that completes the detection system.

2. The availability of a detection system minus the substance to be detected.

3. The setting of an optimal reaction environment (solvent, pH etc.).

4. Optimal storage conditions (e.g. reactive zone dry for higher enzyme stability).

Another advantage is the possibility of spatially separating the components of the detection system. In some cases this is necessary in order to achieve the desired stabilities or to avoid premature, undesirable reactions of the components with one another.

Homogeneous immunoassays can also be carried out with this system.

A preferred embodiment of the test device according to the invention is explained in more detail with reference to the drawings.

The test device consists of a customary carrier 3, which preferably has a predetermined kink 4 in the middle to facilitate folding. It contains a reaction field 1 at one end and a transmission field 2 at its other end. The two fields can be brought into contact by folding over. In order to facilitate contact with the object and / or the reaction field 1, the transmission field 2 is preferably designed to be raised above a surface of the carrier 3. The reaction field 1 is preferably also raised above a surface of the carrier 3. Furthermore, the test device according to the invention can contain an airtight and moistureproof outer packaging (not shown in the drawings), for example a conventional plastic or aluminum foil.

In a preferred embodiment, the transmission field 2 contains at least one solvent for the marker located on the object. Such solvents can e.g. Water, aqueous buffer solutions, alcohols, etc. If the transfer field contains a solvent for the marker, it is preferred that the test device has an airtight and moistureproof outer packaging.

The reaction field 1 containing the at least one component and the transmission field 2 can e.g. made of paper, cardboard, a plastic film, glass fiber or a membrane such as nitrocellulose. The carrier 3 can consist of any suitable material, for example of polyethylene, polyester, polyvinyl chloride or cardboard.

In the test device according to the invention, the components of the detection system can be contained in any arrangement, both on the transmission field 2 and on the reaction field 1, as long as it is ensured that there is still no signaling reaction by contact of the transmission field 2 with the marker itself.

The test device according to the invention is particularly suitable for transferring a subset of the marker from an object provided with a marker to the transfer field 2 and then bringing the transfer field 2 into contact with the reaction field 1. The solvent contained in the transmission field 2 also serves as a reaction medium for the components involved. The use of the transmission field 2 prevents all components necessary for the reaction from coming into contact with one another on the object. After the subsequent contact of the transmission field 2 with the reaction field 1, the detection system is completed and a signal is generated. The generation of a signal directly on the object to be tested may be undesirable, for example, in the case of expensive objects, since this would impair the external impression of the object. In addition, it is not apparent to third parties that the object has been subjected to an authenticity check. Conversely, the test device can also be designed so that the signal is generated on the object itself. This is desirable, for example, for permanent test documentation. The signal can also be designed in such a way that, for example, a pattern, a text or an image is generated.

The examples illustrate the invention.

example 1

P-Nitrophenyl phosphate in micromolar concentration is used as the object marker in its function as an enzyme substrate. For this purpose, p-nitrophenyl phosphate is taken up in diethanolamine buffer (pH 10.3) and applied to the surface of a label. The label coated in this way is then dried.

The transfer field 2 of the test device consists of gel blotting paper from Schleicher & Schuell (GB004) and contains diethanolamine buffer (pH 10.3) in aqueous solution. By contacting the transfer field with the coated label, a partial amount of the marker is released, and the marker is thereby simultaneously brought to an alkaline pH value necessary for the enzymatic conversion.

The reaction field 1 of the test device consists of gel blotting paper from Schleicher & Schuell (GB002), on which alkaline phosphatase is contained as an enzyme in immobilized form in dried form.

The arrangement of the transmission field 2 and the reaction field 1 on the test device is shown in FIGS. 1 and 2.

To check the authenticity of the label, the transfer field 2 impregnated with buffer solution is pressed onto the label by finger pressure for a few seconds, a subset of the marker being picked up by the transfer field 2. The transmission field 2 is brought into contact with the reaction field 1 of the test device under pressure for a few seconds by kinking along the desired kink point 4. After separating the two fields, a yellow color is immediately visible on the reaction field 1 due to the enzyme-catalyzed formation of nitrophenolate anions. If not or On the other hand, incorrectly marked labels leave reaction field 1 colorless during the test process.

Example 2

Cholesterol is used as the object marker and cholesterol oxidase as the enzyme. Cholesterol is converted into cholest-4-en-3-one and hydrogen peroxide by cholesterol oxidase.

To prepare a cholesterol solution for object marking, the cholesterol is added to Triton X-100 ^® in a water bath with heating (about 100 mg cholesterol

® sterol / ml Triton X-100). This solution is mixed with water and boiled briefly.

After cooling, it is applied to a label and the label is dried.

The transfer field 2 of the test device contains only aqueous potassium phosphate buffer (pH 7.0), mixed with Triton X-100 ^® , in order to transfer a portion of the cholesterol from the marked label to the transfer field 2 during the test process. The indicator system, consisting of 4-aminoantipyrine, phenol (in a molar ratio of about 1:20) and peroxidase, as well as cholesterol oxidase for converting the cholesterol, is immobilized on reaction field 1.

When the transmission field 2 is brought into contact with the reaction field 1, the hydrogen peroxide formed reacts with 4-aminoantipyrine and phenol in a subsequent reaction under the influence of peroxidase (horse radical peroxidase) to give a purple dye.

In an analogous manner, other oxidases can also be converted to dyes with other mono-, di-olido or poly-hydroxy compounds.

Example 3

Example of an Apoenzyme Reactivation Immunoassay (ARIS) A FAD hapten derivative is bound via an antibody on the reaction field. At the same time there is an apoenzyme (apo-glucose oxidase) in this field that shows no activity. HRP (horseradish peroxidase - horseradish peroxidase) and a chromogen are required for signal generation. These components could be immobilized on the reaction field. The transmission field could contain glucose. The object is marked with a hapten, which is transferred to the test device by pressing on the moist transmission field. The FAD hapten derivative is released from the antibody by squeezing the transfer field and the reaction field. The apoenzyme can thus be supplemented to form a functional enzyme. The active glucose oxidase converts glucose to produce hydrogen peroxide, which is used to oxidize the chromogen under the influence of the HRP. This generates a signal in the presence of the hapten.

Claims

claims

1. Detection system for checking the originality of an object, at least one component of the detection system being connected to or contained in the object as a marker, characterized in that it

a) at least one substrate for at least one enzyme,

b) comprising a catalyst

bi) at least one enzyme for at least one substrate and / or

whereby a signal is generated by the combination of substrate and enzyme bi), or substrate and at least the two components b ₂ ) alone or by combining them with an indicator system.

2. Detection system according to claim 1, characterized in that the two components b _{2 are} at least one apoenzyme and at least one coenzyme.

3. Detection system according to claim 1, characterized in that the substrate is connected to or contained in the object as a marker.

4. Detection system according to claim 1, characterized in that the enzyme is linked to or contained in the object as a marker.

5. Detection system according to claim 1, characterized in that the apoenzyme is connected to or contained in the object as a marker. Detection system according to claim 1, characterized in that the coenzyme is connected to or contained in the object as a marker

Detection system according to claim 1, characterized in that a mixture of apoenzyme and coenzyme is connected to the object or contained in it as a marker

Detection system according to Claim 1, characterized in that the substrate and the coenzyme, or the substrate and the apoenzyme, are connected to the object or contained in it as a marker

Test device, in particular for carrying out the test of an object for originality using the detection system according to one of claims 1 to 8, wherein the test device has a carrier with, in particular for performing the test of an object for originality using the detection system according to one of claims 1 to 18 suitable reaction field (1)

Test device according to claim 9, characterized in that the carrier further comprises a transmission field (2) and a reaction field (1) which can be brought into contact with it

Test device according to claim 10, characterized in that it has an air- and moisture-tight outer packaging both for the transmission field (2) and for the reaction field (1)

Test device according to claim 11, characterized in that the transmission field (2) contains at least one solvent for the marker

Use of the detection system according to one of claims 1 to 8 for checking the originality of an object

4. Use of the test device according to one of claims 9 to 12 for checking the originality of an object.