HK1081600B - Method and reagents system having a non-regenerative enzyme-coenzyme complex - Google Patents

Description

Methods and reagent systems containing non-regenerative enzyme-coenzyme complexes

The present invention relates to a method and a reagent system for detecting an analyte in a sample by an enzymatic reaction, comprising the use of an enzyme-coenzyme complex as a non-regenerative, in particular stoichiometric, amount of reactant for detecting the analyte present in the sample.

It is known to detect analytes, such as glucose in blood, by enzymatic methods. This requires contacting the analyte to be analyzed with a suitable catalytic amount of enzyme and coenzyme. The redox equivalents generated by the coenzyme during reduction or oxidation are transferred to the mediator, which is detected in a subsequent step by electrochemical or photometric methods. There is a calibration that provides a direct relationship between the measured value and the analyte concentration to be measured.

Sierra et al (anal. chem.69(1997), 1471-. In this method, a catalytic amount of enzyme and its coenzyme FAD is used, and the redox equivalent is transferred to the mediator oxygen.

Narayanswamy et al (Aanlytic letters21(7) (1988), 1165-1175) describe a glucose dehydrogenase and NAD fluorescence assay for glucose detection. The enzyme used in this case is catalytic, i.e.in non-stoichiometric amounts. Free NADH in the solution was detected by fluorometry.

In prior art detection systems, an electrochemically active substance (mediator) is required for the detection of the analyte to be detected, which is indirect, i.e. requires a series of chemical reactions. In this case, it is usually necessary to make complicated adjustments to the concentration of the substance used in order to increase the reaction rate as much as possible. Furthermore, the required electrochemically active substances are unstable during storage periods which are at risk for longer.

Furthermore, an excess of mediator has to be used in general with respect to the enzyme-coenzyme system. Coenzymes have a high reactivity and therefore decomposition of the mediator, even in small amounts, such as less than 1%, or exposure to foreign substances, such as evaporation of the substance from the packaging material, leads to a drastic reduction in the activity of the enzyme. This can lead to the generation of false signals in analyte detection. It is also a disadvantage that the detection time of the analyte is usually at least a few seconds, for example more than 4 seconds for glucose, and that the required sample amount is large, e.g. more than 0.5. mu.l.

The object of the present invention is to avoid the above-mentioned disadvantages of the prior art at least to a certain extent. In particular, a non-sensitive and rapid method for enzymatic detection of analytes is provided, which allows reliable assay results to be obtained even in the absence of a mediator or/and an indicator.

The object of the present invention is achieved by using the enzyme-coenzyme complex as a stoichiometric amount of the reactant, rather than as a catalyst as is usual. The detection of the analyte requires only a single reaction step and is therefore very rapid. The media and indicators and the complex reaction mixtures are no longer required here and therefore also no longer have low stability and high sensitivity to interference.

Accordingly, one aspect of the present invention provides a method for detecting an analyte in a sample by an enzymatic reaction, comprising the steps of; (a) contacting the sample with a detection reagent comprising an enzyme-coenzyme complex, wherein no regeneration of the coenzyme occurs, and (b) detecting a reaction of the analyte by a change in the enzyme-coenzyme complex.

Another aspect of the invention provides a reagent system for detecting an analyte in a sample, comprising: (a) a detection reagent comprising an enzyme-coenzyme complex wherein no coenzyme regeneration occurs, and (b) a carrier housing the detection reagent.

The invention makes possible a simple qualitative or quantitative detection of an analyte in a very short time, preferably < 5 seconds, particularly preferably < 1 second, most preferably < 0.1 second. The reaction is carried out under conditions such that no coenzyme regeneration occurs during the assay. In addition, the molecular enzyme-coenzyme complex can react with only one molecule of analyte. Advantageously, therefore, the reaction should be carried out in the absence of mediators or other substances capable of causing the regeneration of the coenzyme.

According to the described assay design, the detection reagent contains a sufficient amount of an enzyme-coenzyme complex for qualitative or/and quantitative detection of the analyte. In particular for the quantitative determination of an analyte, the amount of enzyme-coenzyme complex used should be such that the number of reactive molecules of the enzyme-coenzyme complex correlates with the concentration of the analyte present in the sample. It is particularly preferred that the amount of enzyme-coenzyme complex used should be at least a stoichiometric amount with respect to the analyte in the sample, preferably an excess of the stoichiometric amount with respect to the analyte. Herein, "at least a stoichiometric amount" means that the sample size needs to be adjusted relative to the number of enzyme-coenzyme complex molecules such that the number of molecules of the enzyme-coenzyme complex that react with the analyte correlates with the concentration of the analyte present in the sample, depending on the expected concentration of the analyte in the sample. The "stoichiometric amount" is preferably such that the number of molecules referring to the enzyme-coenzyme complex corresponds to the maximum number of analyte molecules expected in the sample under investigation.

The present method and detection system can use a minimum amount of sample, e.g., a sample amount of ≦ 1 μ l, and particularly ≦ 0.1 μ l. Optionally the sample may be diluted prior to contact with the detection reagent.

The method and test system of the invention are suitable for the detection of any analyte, for example a parameter of a body fluid, such as blood, serum, plasma or urine, but also waste water or food. The process may be carried out under wet conditions, such as in a test tube, or in the presence of a suitable reactant carrier in the dry state.

The analyte to be detected may be any biological or chemical substance capable of reacting with the enzyme-coenzyme complex, particularly a redox reaction, for example, glucose, lactic acid, malic acid, glycerol, alcohol, cholesterol, triglyceride, ascorbic acid, cysteine, glutathione, peptides, etc.

The enzymatic reaction is preferably a redox reaction in which the coenzyme in the enzyme-coenzyme complex is reduced or oxidized. In such reactions, the enzyme preferably used is an oxidoreductase. Particularly preferably used enzymes are dehydrogenases, for example selected from glucose dehydrogenases (e.c.1.1.1.47), lactate dehydrogenases (e.c.1.1.127, 1.1.1.28), malate dehydrogenases (e.c.1.1.1.37), glycerol dehydrogenases (e.c.1.1.1.6), alcohol dehydrogenases (e.c.1.1.1.1) or amino acid dehydrogenases, such as L-amino acid dehydrogenases (e.c. 1.4.1.5). Other suitable enzymes are oxidases, for example, glucose oxidase (e.c.1.1.3.4) or cholesterol oxidase (e.c. 1.1.3.6).

The coenzymes for the purposes of the present invention are preferably organic molecules which are linked covalently or non-covalently to the enzyme and which are altered, e.g.oxidized or reduced, by conversion of the analyte. Examples of preferred coenzymesRiboflavin, nicotine and benzoquinone derivatives, e.g. riboflavin derivatives, e.g. FAD, FADH₂、FMN、FMNH₂Etc., nicotinic nucleotide derivatives, such as NAD⁺、NADH/H⁺、NADP⁺、NADPH/H⁺Etc., or ubiquinones, such as coenzyme Q, PQQ, etc.

The change in the coenzyme upon reaction with the analyte can in principle be detected by any method. In principle, all methods known from the prior art for detecting enzymatic reactions can be used. However, it is preferred to use an optical method to detect the change in the coenzyme. Optical detection methods include, for example, measurement of absorption, fluorescence, Circular Dichroism (CD), Optical Rotatory Dispersion (ORD), refractometry, and the like. It is particularly preferred to detect the change in coenzyme by measuring fluorescence. Fluorescence assays are highly sensitive and can detect even at very low analyte concentrations in miniaturized systems.

The methods and detection systems of the invention may comprise liquid detection, in which case the reagents are, for example, in solution or suspended in an aqueous or non-aqueous liquid, or in the form of a powder or lyophilized powder. The methods and detection systems of the present invention preferably comprise dry detection, in which case the reagents are placed on a support. The carrier comprises, for example, a test strip containing an adsorbent or/and a swellable material, which can be wetted by the sample liquid to be tested.

In a particularly preferred embodiment, the detection reagent used is a gel matrix in which the enzyme-coenzyme complex is embedded. The gel matrix layer is preferably thicker than or equal to 50 μm, in particular thinner than or equal to 5 μm, and is placed on a support which is, for example, optically transparent at least to some extent. The gel matrix may be a matrix comprising one or more soluble polymers, as in known dry state detection systems (e.g.AccuChek Active), and may be cut with a knife and dried. The matrix is preferably based on a polymer of a substance having photopolymerisable structural properties, such as an acrylic monomer, such as acrylamide or/and an acrylate, such as polyethylene glycol diacrylate, or a vinyl aromatic monomer, such as 4-vinylbenzenesulfonic acid, or a combination thereof. Gel matrices of this type can be prepared by applying a liquid containing the enzyme, the monomers with photopolymerisable character and optionally the reagent for the coenzyme, the photoinitiator or/and the non-reactive components to an at least partially transparent support, for example a plastic plate, and irradiating the support, for example with UV light, so that the monomer or monomers polymerize on the support and reach a predetermined layer thickness. The layer thickness can be controlled by adding an adsorbent to the reagent or/and by adjusting the time and intensity of the irradiation. The excess liquid reagent can be removed after polymerization and reused (see, e.g., fig. 2).

Alternatively, the gel matrix may be prepared by a conventional coating procedure in which the liquid agent is coated onto the support, brought to the desired thickness using a suitable method, such as the use of a spatula, and then allowed to polymerize to completion.

After incorporation into the matrix by polymerization or intercalation, the enzyme is in a protected microenvironment. If the gel matrix of the polymer is sufficiently crosslinked, the enzyme molecules are in an immobile state. However, low molecular weight species or glucose or other analytes or coenzymes thereof can then diffuse freely in the polymer network.

The enzyme can either be included in the matrix together with its coenzyme by polymerization or the matrix can be contacted with a coenzyme solution after polymerization, so that a suitable enzyme-coenzyme complex is formed. The concentration of the enzyme in the gel matrix is preferably sufficient to allow a stoichiometric reaction with the analyte to be detected and to allow direct detection of the coenzyme altered by the reaction. The reaction consists only of a single catalytic reaction, such as a redox reaction, which can be completed in a time interval of milliseconds or microseconds. The coenzyme altered by the reaction is optimally protected from interfering influences by binding to the active center of the enzyme and can optionally also be embedded in the gel matrix.

The invention is described in detail below with reference to the figures and examples.

Drawings

FIG. 1 shows a first embodiment of the detection system of the present invention. A reagent layer (2), such as a gel matrix containing an enzyme-coenzyme complex, is applied to a transparent support (1). The enzyme-coenzyme complex is in a form such that regeneration of the coenzyme cannot occur during analyte detection. A sample (3), such as blood, is applied to the reagent layer. The enzymatic reaction between the analyte contained in the sample (3) and the enzyme-coenzyme complex contained in the reagent layer (2) is detected by an optical method. Light, such as a laser or LED, from a light source (4) is directed from behind (through the carrier) to the reagent layer (2). The absorbed light or fluorescence reflected back from the sample is detected by a detector (5). Optionally, especially for fluorescence detection, an optical filter element (6) is placed in front of the detector in order to prevent leakage of the excited fluorescence.

FIG. 2 shows the preparation of a detection system according to the invention. A liquid reagent (12), such as a first site (13), is applied to an optically transparent support (11), such as a plastic sheet. Light emitted from the light source (14) is irradiated from the rear through the carrier (11) to the liquid reagent (12) located at the second position. At the same time, the carrier is moved in the direction of the arrow shown in (15). Thus, the polymerized reagent layer (16) is directly formed on the carrier (11). There is an excess of liquid reagent on the polymeric layer (16). The thickness of the polymerized reactant layer (16) can be controlled by adjusting the composition of the reagent, the intensity and irradiation time of the light beam, and by the properties of the support (11).

Fig. 3 shows an embodiment of the fluorescence sensor from behind. The polymerized reagent layer, which is prepared, for example, by the sequential steps shown in FIG. 2, can be cut and placed on the support (21) using known techniques. The sample is placed on the side facing upward, and excitation light (23) such as UV emitted from a light source is incident from below. The analyte reacts with the enzyme-coenzyme complex of reagent layer (22) to produce fluorescence (24), such as blue light, which is detected by a detector.

It is also possible to place a plurality of reagents (identical or different) on the carrier. Figure 4 shows an embodiment in the form of one of the discs. A plurality of reagent dots (32) are arranged on a light-transparent carrier (31).

FIGS. 5A and 5B show fluorescence (glucose dehydrogenase and NAD) of the detection system of the present invention under a CCD camera⁺) Relationship to increased glucose concentration.

Examples

Example 1: glucose dehydrogenase (glucDH)/NAD in vitro⁺Detection of glucose stoichiometry under system

100mg/ml GlucDH was dissolved in buffer solution pH7 and mixed with appropriate amount of NAD⁺And (4) mixing. As the amount of glucose increased, an increase in fluorescence intensity was observed under an ultraviolet lamp (excitation wavelength of 366nm) (FIGS. 5A and 5B).

Solutions of the enzyme system without glucose do not fluoresce and only glucose and NAD⁺The solution of (a) does not fluoresce.

Example 2: on polymer films on GlucDH/NAD⁺Detection of glucose under system

Mixing a suspension of

Formulation 1

Substance(s)	Quantity (g)	Weight (%)
			Acrylamide	2.5	22.02
Methylene bisacrylamide	0.7	6.17
			2, 2-dimethoxy-2-phenylacetophenone	0.05	0.44
Glycerol	5	44.05
			Hydroxyethyl methacrylate	1.4	12.33
Methacrylic acid methyl ester	0.4	3.52
			Crodasinic O solution, pH8, 0.3g/1000ml	1	8.81
N, N' - (1, 2-dihydroxyethylene) bisacrylamide	0.3	2.64
			Total amount of	11.35	100

0.5ml of the suspension was mixed with 0.5ml of a solution of glucDH (100mg/ml) and the mixture was homogenized in an ultrasonic water bath to remove air bubbles.

The clear solution was poured onto corona-treated polycarbonate sheets having a thickness of 125mm and irradiated for 20 minutes with a customary irradiation apparatus (Isel UV irradiator 2). The plastic pieces were briefly rinsed with water and then dried in air.

The thickness of the layer obtained is < 2 μm. Preparation of fresh glucose/NAD⁺Solution (GKL-3 solution, 300mg/dl glucose, 1ml/6.4mg NAD⁺) And poured onto the film. Intense fluorescence was immediately visible under the uv lamp.

Example 3: effect of UV absorber addition on layer thickness

A polymer layer (formulation 2) containing a blue dye (absorption maximum wavelength ≈ 650nm) was prepared which is more attentive to the experimental results. In further experiments, a yellow dye was incorporated in the initial formulation as a UV absorber (formulation 3).

Formulation 2

Substance(s)	Measurement of	Weight (%)
			Acrylamide	37.5g(0.53mol)	25.78
Polyethylene glycolDiacrylate, Mw ≈ 575g/mol	52.5g (about 0.96mol)	36.10
			Crodasinic O (0.3g/l l) solution	50g	34.38
4-Vinylbenzenesulfonic acid	5g	3.44
			2, 2-dimethoxy-2-phenylacetophenone photoinitiator	350mg	0.24
New methylene blue N	100mg	0.06
			Total amount of	145.45g	100

The mixture was stirred and homogenized by ultrasonic bath treatment, distributed with a pipette onto 140 μm Pokalon plates (corona treatment, step 4) and irradiated for 1 minute with an ultraviolet irradiator (Actina U4, W.Lemmen GmbH).

The thickness of the obtained layer was measured with a gyrometer (Mikrometers chube), and it was 2405 μm.

Formulation 3

Substance(s)	Measurement of	Weight (%)
			Formulation 2	1ml	About 99.99
Yellow mordant 7(No.686) (ultraviolet absorber)	0.0001g	0.001
			Total amount of	About 1.0001g	100

The mixture was distributed onto the flakes as described above and subsequently polymerized. The thickness of the obtained layer was measured by a spiral meter, and it was 79.3. mu.m.

The test results show that the thickness of the layer can be influenced. The layer thickness was 240.5 μm without UV absorber under otherwise identical conditions; the layer thickness in the presence of the UV absorber (yellow mordant 7) was only 79.3. mu.m.

Claims (23)

1. A method for detecting an analyte in a sample by an enzymatic reaction, comprising the steps of: (a) contacting the sample with a detection reagent comprising an enzyme-coenzyme complex under conditions in which coenzyme regeneration does not occur, wherein the enzyme is a dehydrogenase, and (b) detecting the reaction of the analyte by a change in the enzyme-coenzyme complex.

2. The method according to claim 1, characterized in that the dehydrogenase is selected from glucose dehydrogenase (e.c.1.1.1.47), lactate dehydrogenase (e.c.1.1.1.27, 1.1.1.28), malate dehydrogenase (e.c.1.1.1.37), glycerol dehydrogenase (e.c.1.1.1.6), alcohol dehydrogenase (e.c.1.1.1.1) or amino acid dehydrogenase.

3. The method according to claim 1, characterized in that the coenzyme used is selected from the group consisting of flavins, nicotine and benzoquinone derivatives.

4. The method of claim 3, wherein the coenzyme is selected from the group consisting of FAD and FADH₂、FMN、FMNH₂Coenzyme Q, PQQ, NAD⁺、NADH/H⁺、NADP⁺And NADPH/H⁺。

5. The method according to any one of claims 1 to 4, characterized in that the change of coenzyme is detected by an optical method.

6. The method of claim 5, wherein the change in the coenzyme is detected by measuring absorption, fluorescence, CD, ORD, or refraction.

7. The method according to claim 6, wherein the change in coenzyme is detected by measuring fluorescence.

8. The method according to any one of claims 1 to 4, characterized in that a gel matrix having an enzyme-coenzyme complex embedded therein is used as a detection reagent.

9. The method of claim 8, wherein the gel matrix has a layer thickness of 50 μm or less.

10. The method of claim 9, wherein the gel matrix has a layer thickness of less than or equal to 5 μm.

11. A process according to claim 8, characterized in that a gel matrix based on photopolymerizable substances is used.

12. A method according to any one of claims 1 to 4, characterised in that the analyte is detected in a body fluid.

13. The method according to claim 12, characterized in that the detection of glucose in the blood is performed.

14. The method of any one of claims 1-4, wherein the analyte has a reaction time of 5 seconds or less.

15. The method according to any one of claims 1 to 4, characterized in that the reaction is carried out in the absence of a medium which can react with the coenzyme.

16. The method according to any one of claims 1 to 4, characterized in that the enzyme-coenzyme complex is used as a reactant in a stoichiometric amount with respect to the analyte.

17. The method according to claim 2, characterized in that the amino acid dehydrogenase is an L-amino acid dehydrogenase (E.C. 1.4.1.5).

18. A reagent system for detecting an analyte in a sample, comprising: (a) a detection reagent comprising an enzyme-coenzyme complex in a form in which coenzyme regeneration does not occur, wherein the enzyme used is a dehydrogenase, and (b) a carrier that holds the detection reagent.

19. The reagent system of claim 18, wherein the enzyme-coenzyme complex is embedded in a gel matrix.

20. Reagent system according to claim 18 or 19, characterized in that the carrier is at least partially optically transparent.

21. Reagent system according to claim 18 or 19, characterized in that the carrier comprises a flat structure.

22. A reagent system according to claim 18 or 19 characterised in that the carrier comprises a plurality of different detection reagents.

23. Use of a reagent system according to any one of claims 18 to 22 in a method according to any one of claims 1 to 17.