RU2266543C2 - Reagent systems for detecting for reduced cofactor - Google Patents

Abstract

FIELD: bioassay methods.

SUBSTANCE: invention, in particular, relates to a reagent system for detecting substances. Signal generation system for detecting substances in a sample contains at least one first and second electron transfer agent and redox indicator. Electron transfer agents utilized are those of protein and nonprotein nature, for example phenazine compound and diaphorase. System further contains at least one of following compounds: enzyme cofactor, enzyme manifesting oxidative activity relative to tested substance, e.g. corresponding dehydrogenase. Proposed systems, reagent compositions, test strips, and kits find use in detection of a large number of substances in a sample such as a biological sample, e.g. blood or blood fraction.

EFFECT: increased reaction rate of signal generation system and reduced cost.

8 cl, 1 dwg, 3 tbl

Description

Technical field

The present invention relates to the detection of analytes, in particular to a reagent system for use in the detection of analytes.

State of the art

Detection of analytes in physiological fluids, such as blood or in products derived from blood, is becoming increasingly important in modern society. Often it is necessary to conduct analyzes with the detection of the analyzed substances in clinical laboratory studies, studies at home, etc., where the results of the study play a decisive role in the diagnosis and treatment of a variety of painful conditions. Analyzers of interest include alcohol, formaldehyde, glucose, glutamic acid, glycerin, beta-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvic acid, steroids. Numerous methods and devices have been developed for detecting analytes, intended for use both in clinical and at home. Some of the methods and devices that have been developed to date use signal generation systems to identify the analyte of interest in a physiological sample, such as blood.

One of the signal generation systems that finds application in the detection of many different analytes is a system in which dehydrogenase oxidizes the analyte and at the same time reduces the cofactor of the enzyme, such as NAD (P) +. Then, the reduced form of the cofactor, for example NAD (P) H, is detected by subsequent reaction with a cofactor oxidizing agent, for example phenazine with metosulfate or diaphorase, which transfers the electron to an oxidation-reduction indicator such as a tetrazolium salt to obtain a detectable product.

Although a variety of signal generation systems have been developed to date for use in detecting a large variety of different analytes, there is a need for further development of such systems.

US patents of interest include 4,629,697; 5126247 and 5902731, as well as Raap et al., Histochem. J., 1983.

US Pat. No. 4,629,697 discloses a testing system having extended measurement limits and an appropriate method for determining NAD (P) H or substrates or enzymes that interact with the formation or consumption of NAD (P) H in liquids. The testing system contains at the same time several substances acting independently as electron acceptors for NAD (P) H and having different electrochemical potentials. Adding a testing system to the sample solution leads to the formation of various end products that can be analytically differentiated and which can be evaluated visually or with different instruments.

US Pat. No. 5,126,247 discloses oxygen-free methods, systems, and devices for enzymatic colorimetric evaluation and determination of biochemical analytes. The patent discloses two systems that make it possible to obtain less than one dye equivalent per equivalent of a substrate, while dye concentrations are maintained within a range where, according to Beer, there is a linear color-concentration relationship. One system allows you to get an analog color signal from an analog analyte input signal. Another system allows you to obtain a digital signal from an analog analyte input signal.

As the closest analogue, the applicant indicated US patent 5902731, which claims a reagent for measuring the concentration of analyte in hemoglobin containing biological fluid, such as blood. The reagent contains a dehydrogenase enzyme that is specific for an analyte, NAD (nicotinamide adenine dinucleotide) or NAD derivative, a tetrazole dye precursor, a diaphorase enzyme or its analogue, and a nitrous acid salt. The reagent causes the formation of dye, which is a measure of the concentration of the analyte. The nitrous acid salt suppresses the dye formation caused by hemoglobin (non-enzymatic). Preferably, the reagent is used for colored strips for measuring ketone bodies, such as betahydroxybutyrate.

Summary of the invention

The basis of the present invention is the task of creating a signal generation system that provides increased reaction speed and lower cost.

The problem is solved by creating a signal generation system, reagent composition, test strips and their sets, as well as a method for their use in the detection of analytes in the sample.

The signal generation system is characterized in that it contains at least first and second electron transfer agents and an oxidation-reduction indicator. In a preferred embodiment, the system comprises protein and non-protein electron transfer agents, for example a phenazine compound and a diaphorase. In many preferred embodiments, the systems and kits further include an enzyme cofactor and an enzyme having oxidative activity with the analyte, for example, analyte dehydrogenase. Consider systems, reagent compositions, test strips and kits are used to detect a large number of analytes in a sample, such as a physiological sample, such as blood or its fraction.

Brief Description of the Drawings

The invention is illustrated by the description of preferred embodiments with reference to the drawing, which shows a diagram of the reaction rate in the test strip depending on the theoretical expected reaction rate for the test strip, which uses both PMS and diaphorase, from the diagram it follows that the use of both non-protein and a protein electron transfer agent, for example PMS and diaphoresis, provides an unexpected increase in the reaction rate according to the invention.

DESCRIPTION OF PREFERRED EMBODIMENTS

A signal generation system, reagent compositions, test strips and their kits, as well as methods for their use in detecting analytes in a sample are proposed. The signal generation system comprises at least first and second electron transfer agents and an oxidation-reduction indicator. The system also includes protein and non-protein electron transfer agents, for example, a combination of phenazine and diaphorase. In many preferred embodiments, the system and kits further include an enzyme cofactor and an enzyme having oxidative activity with the analyte, for example, analyte dehydrogenase. The system, reagent compositions, test strips and kits find use in the detection of a large number of analytes in a sample, such as a physiological sample, for example, blood or its fraction.

The signal generation system according to the invention is designed to detect the presence of a reduced enzyme cofactor in a sample. A signal generation system is understood to mean a combination of two or more compounds or molecules that, when combined, generate a detectable signal indicating the presence and quantity of a particular analyte in a given sample. The term signal generation system is used in a broad sense to indicate both a mixture of all the reagents that make up the signal generation system, and a system in which one or more of the reagents that make up it are separated from the rest of the reagents, for example, a system presented as a set.

An essential feature of a signal generation system is the presence of two different electron transfer agents. An electron transfer agent is a compound or molecule that can transfer an electron in the form of a hydride ion from a reduced cofactor of the enzyme to an oxidation-reduction indicator. In the considered signal generation systems, the first of the electron transfer agents is a low molecular weight molecule, and the second electron transfer agent is a high molecular weight molecule. Low molecular weight means a molecular weight that does not exceed about 2000 daltons, typically about 1000 daltons, and in many cases, about 500 daltons. High molecular weight means a molecular weight of approximately 5,000 Daltons, and in many cases 10,000 or 20,000 Daltons or higher. The high molecular weight of the electron transfer agent does not exceed about 100,000 daltons. The low molecular weight electron transfer agent is a non-protein compound, while the high molecular weight electron transfer agent is a protein compound. By protein compound is meant a polypeptide or a polymer mimetic thereof.

Of interest are a variety of non-protein low molecular weight electron transfer agents selected from the group consisting of flavins such as riboflavin (RBF), alloxazine (ALL) and lumichrome (LC); phenazines such as phenazine, phenazine methosulfate (PMS), phenazine ethosulfate, methoxyphenazine metosulfate and safranin; methyl-1,4-naphthol (menadione), phenothiazines such as PT and its cationic radical, PT +, thionine (TH), azur A (AA), azur B (AB), azur C (AC), methylene blue ( MB), methylene green (MG) and toluidine blue O (TOL); phenoxazines such as phenoxazine (POA), basic blue 3 (BB3) and cresyl bright blue ALD (BCBA), benzo-α-phenazoxonium chloride (Medola blue); indophenols such as 2,6-dichlorophenol indophenol (DCIP); and indamines such as Bindschedler green and phenylene blue. Of particular interest are phenazine compounds, for example PMS, phenazine ethosulfate, methoxyphenazine metosulfate and safranin, where PMS is a non-protein low molecular weight electron transfer agent.

A high molecular weight electron transfer agent is an enzyme that is capable of oxidizing a reduced cofactor, such as NAD (P) H, and at the same time restores the oxidation-reduction indicator. This electron transfer enzyme is a diaphorase such as lipoic dehydrogenase, ferredoxin-NADP reductase, lipoamide dehydrogenase, NADPH dehydrogenase. A variety of diaphorases are available and can be used in the signal generation systems under consideration, they include bacillus diaphoresis, clostridium diaphoresis, vibrio diaphoresis, pig diaphoresis.

In the considered signal generation systems, the ratio of the first electron transfer agent to the second agent is selected to accelerate the reaction rate compared to a control, for example, a signal generation system, with one electron transfer agent with only PMS or diaphoresis. Typically, the ratio of the first electron transfer agent to the second agent in the systems in question varies from about 0.001 to 10, preferably from about 0.01 to 1.0, and more often from about 0.05 to 0.5 (nmol / unit) .

Additionally, signal generation systems include an oxidation-reduction indicator. By an oxidation-reduction indicator is meant a compound that can reduce electron transfer agents to produce a detectable chromogenic product. When the oxidation-reduction indicator is a chromogen, suitable chromogens are any compound capable of changing color when one or more electrons are reduced, i.e. chromogens accept electrons from electron transfer agents.

Redox indicators are selected from the group consisting of oxazines, thiazines and tetrazolium salts. Of particular interest are tetrazolium salts, which are capable of accepting the captured hydride from electron transfer agents to form a colored formazan product. In many cases, these salts have a peculiarity - they are pale yellow in the oxidized form, but take clearly visible colors upon electron reduction and conversion to formazan dyes. Tetrazolium compounds or salts of particular interest include 2- (2'-bezothiazolyl) -5-styryl-3- (4'-phthalhydraedyl) tetrazolium (BSPT); 2-bethiazolyl- (2) -3,5-diphenyl tetrazolium (BTDP); 2,3-di (4-nitrophenyl) tetrazolium (DNP); 2,5-diphenyl-3- (4-styrylphenyl) tetrazolium (DPSP); distyryl tetrazolium nitro-blue (DS-NBT); 3,3 '- [3,3'-dimethoxy- (1,1'-biphenyl) -4,4'-diyl] bis [2- (4-nitrophenyl) -5-phenyl] -2H tetrazolium (NBT) ; 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H tetrazolium (MTT); 2-phenyl-3- (4-carboxyphenyl) -5-methyl tetrazolium (PCPM); tetrazolium blue (TV); thiocarbamyl tetrazolium nitro-blue (TCNBT); blue tetrazolium tetranitrogol (TNBT); tetrazolium violet (TV); 2-benzothiazothiazolyl-3- (4-carboxy-2-methoxyphenyl) -5- [4- (2-sulfoethylcarbamoyl) phenyl] -2H-tetrazolium (WST-4); 2,2'-dibenzothiazolyl-5,5'-bis [4-di (2-sulfoethyl) carbamoylphenyl] -3,3 '- (3,3'-dimethoxy-4,4'-biphenylene) ditetrazolium, disodium salt ( WST-5); 2- (p-nitrophenyl) -3- (p-iodophenyl) -5-phenyltetrazolium chloride (INT); etc. WST-5 is preferred in many embodiments because it is readily soluble in an aqueous medium that is most compatible with biological samples. In addition, the resulting formazan compound exhibits strong spectral absorption in the purple-blue region, which reduces the need for correction of the background signal from hemoglobin. Other suitable tetrazolium salts are described in US Pat. Nos. 4,490,465; 4,491,631; 4,598,042; 4,351,899; 4,271,265; 4,247,633; 4,230,090; 4,215,917; 4,142,938; 40,24021; 3,867,259; 3,867,257; 3,791,931; and 4254222.

The signal generation systems described above are capable of detecting the reduced cofactor of the enzyme in a sample, in particular in an aqueous sample, and more particularly in a physiological sample, for example in whole blood, or in a fraction thereof or product. Many different reduced enzyme cofactors can be detected using signal generation systems where representative reduced enzyme cofactors include reduced forms of the following cofactors: beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate (beta-NADD), , thionicotinamide adenine dinucleotide phosphate, nicotinamide 1, N6-ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate and pyrroloquinoline quinone (PQQ). These signal generation systems are preferred for detecting NADH or NAD (P) H.

The reduced enzyme cofactor is that obtained after oxidation of the analyte of interest in the sample. Therefore, the signal generation systems of interest also include an enzyme cofactor and an enzyme oxidizing an analyte that is capable of oxidizing an analyte and simultaneously restoring an enzyme cofactor. Enzyme cofactors of interest include those described above, i.e., beta-nicotinamide adenine dinucleotide (beta-NAD), beta-nicotinamide adenine dinucleotide phosphate (beta-NADP), thionicotinamide adenine dinucleotide, thionicotinamide adenine dinucleotide 1, dinucleotide phosphate ethenoadenine dinucleotide, nicotinamide 1, N6-ethenoadenine dinucleotide phosphate and pyrroloquinoline quinone (PQQ). Cofactors of enzymes that may be included in the signal generation system under consideration include NADH or NAD (P) H.

The enzyme that oxidizes the analyte present in the signal generation system necessarily depends on the nature of the analyte to be detected using the system. Enzymes that oxidize the analytes include alcohol dehydrogenase for alcohol, formaldehyde dehydrogenase for formaldehyde, glucose dehydrogenase for glucose, glucose 6-phosphate dehydrogenase for glucose-6-phosphate, glutamate dehydrogenase for glutamic acid, glycerol hydroxyhydrogen dehydrogenase for beta-hydroxybutyrate, hydroxysteroid dehydrogenase for steroid, L-lactate dehydrogenase for L-lactate, leucine dehydrogenase for leucine, malate dehydrogenase for malic acid and pyruv t dehydrogenase for pyruvic acid. Thus, the enzyme that oxidizes the analyte, as a rule, is a dehydrogenase.

The invention also provides a reagent composition for detecting at least the reduced cofactor of the enzyme, and in many cases the analyte in the sample. The reagent compositions may be a fluid, for example aqueous, or dry compositions, more often the reagent compositions are dry compositions.

Reagent compositions are those compositions that include first and second electron transfer agents and an oxidation-reduction indicator, these components are described above. These reagent compositions are suitable for detecting reduced cofactors of enzymes, for example NAD (P) H in a sample. However, the reagent compositions further include an enzyme cofactor and an analyte oxidizing enzyme.

Reagent test strips are also provided according to the invention for detecting the presence of analytes in a sample. In particular, dry strips for an analyte in whole blood, for example beta-hydroxybutyrate, glucose and the like, are provided.

The reagent test strip contains a solid support and a dry reagent composition placed on it, which is made of all reagent compounds necessary to generate a detectable signal in the presence of the analyte. The dry reagent composition on the test strip includes the following elements: an enzyme that oxidizes the analyte, an enzyme cofactor, first and second electron transfer agents, and an oxidation-reduction indicator.

Typically, test strips contain a membrane layer for analysis, which is fixed on a solid substrate. The substrate may be made of polystyrene, nylon or polyester, or a metal sheet, or any other suitable material. The analysis layer preferably contains moisture-absorbing material, for example filter paper or a polymer membrane.

The REAGENT COMPOSITION is associated with a layer for analysis, for example, it can be applied to a layer for analysis, included in the layer for analysis. The strip may also be a more complex system, for example, when the analysis layer is placed between the substrate and the surface layer, while one or more reagents used to process the sample may be present in the surface layer. Additionally, channels for streams may be provided on the test strip.

Test strips can be made by any known method. One method is to immerse at least a portion of the strip in an aqueous composition that includes all of the elements of the reagent composition that must be present in the test strip analysis layer. It is convenient to immerse part of the strip in an aqueous composition, hold there for a sufficient period of time, and then dry. In this case, a test strip with a layer for analysis with reagents is obtained.

The aqueous composition includes various elements of the reagent composition to be introduced into the test strip analysis layer, the various elements being present in sufficient quantities to provide the desired amount of reagents in the composition that is created on the analysis layer.

The concentration of the non-protein electron transfer agent present in the aqueous composition ranges from about 1 μM to 1000 μM, usually from about 10 μM to 500 μM.

The concentration of the protein transfer agent for the electron transfer in the aqueous composition ranges from about 50 units. up to 3000 units, usually from about 100 units. up to 1000 units The concentration of the redox reagent present in the aqueous composition ranges from about 3 mm to 36 mm, usually from about 6 mm to 24 mm. The concentration of the cofactor of the enzyme ranges from about 1.5 mm to 28 mm, usually from about 3.5 mm to 14 mm. The concentration of the enzyme that oxidizes the analyte varies from about 100 units. up to 2000 units, and usually from about 200 units. up to 1000 units Other components that may be present in the aqueous composition used to prepare the reagent test strip include sodium chloride, magnesium chloride, Tris, PSSA, Tetronic 1307, Crotein-SPA, sucrose, oxamic acid sodium salt, and the like.

Many different analytes can be detected using the claimed method, and the analytes include those described above, for example, alcohol, formaldehyde, glucose, glutamic acid, glycerin, beta-hydroxybutyrate, L-lactate, leucine, malic acid, pyruvic acid, steroids. Considered methods can be used to determine the presence, and often concentration, of analytes in many different physiological samples, such as urine, tears, saliva, they can be used to determine the concentration of analytes in the blood or in blood fractions, for example, in samples derived from blood, and more specifically from whole blood.

An essential feature of the claimed method is that the signal generation systems, which include both the first and second agents for electron transfer, provide an acceleration of the reaction time compared to a control system that includes one electron transfer agent. As a rule, the reaction rate increases by 1-3 times, depending on the ratio of PMS and diaphorase. Moreover, the reaction rate is 1-3 times higher than the theoretical or predicted rate, which would be expected by adding up the speeds provided by individual agents for electron transfer. When the reaction rate is measured in K / S per second (see examples below), this value for the reaction using the signal generation systems in question ranges from about 0.01 to 10, preferably from about 0.05 to 5, and more preferably from about 0.1 to 2.

The sample and the signal generation system are combined in the reaction mixture, the reaction proceeds for a time sufficient to generate a signal indicating the presence (and often the quantity) of the analyte in the sample. The resulting signal is detected and aligned with the presence (and often the quantity) of the analyte in the sample. In the broadest sense, the reaction mixture can be prepared in a cuvette or other convenient means for containing the fluid. In many embodiments, the above steps take place on a reagent test strip as described above.

When implementing the method according to the invention in the first stage, the application of a certain amount of a physiological sample to the test strip is carried out. The amount of a physiological sample, such as blood, that is applied to the test strip may vary, but typically ranges from about 2 μl to 40 μl, usually from about 5 μl to 20 μl. The amount of blood sample that is applied to the test strip can be relatively small and ranges from about 2 μl to 40 μl, preferably from about 5 μl to 20 μl. When the physiological sample is blood, samples with many different hematocrit can be tested, and the hematocrit can range from about 20% to 65%, preferably from about 25% to 60%.

After applying the sample to the test strip, it interacts with the elements of the signal generation system to obtain a detectable product, which is present in an amount proportional to the initial amount of the analyte of interest present in the sample. The amount of the detected product is determined, that is, the signal obtained using the signal generation system, and this amount is brought into correspondence with the amount of the analyte in the original sample. In some cases, automatic tools are used. At this stage, the obtained concentration of the analyte takes into account the component from parallel reactions, for example, by appropriate calibration of the instrument.

Kits for implementing the claimed method are also provided according to the invention. The kits contain the signal generation system described above, wherein the components of the signal generation system can be combined into a single reagent composition or separated, that is, located in separate containers. In some embodiments of the invention, the signal generation system is in a kit in the form of a test strip with reagents, as described above. Consider kits may further include means for obtaining a physiological sample. For example, when the physiological sample is blood, the kit may further include means for obtaining a blood sample - a device for perforating a finger, means for actuating a device for perforating a finger. In addition, the kits may include a control solution or a standard solution, for example, a control solution of the analyte, which contains a standardized concentration of the analyte. In certain embodiments of the invention, the kits comprise an automated tool for determining the amount of product obtained on the strip after application of the sample and determining the correspondence between the detected product and the amount of analyte in the sample. Finally, kits include instructions for using the components in determining the concentration of an analyte in a physiological sample. Instructions may be presented on one or more items, including packaging, liner, containers present in kits.

A. Preparation of the ketone test strip

A 0.8 μm Cuno nylon membrane (Meridien, CT) was immersed in the reagent disclosed in Table 1 until saturation.

Table 1 Component number Water 100 ml Tris (hydroxymethyl) aminomethane 1.2 grams Sodium Chloride (MW 56.44, Sigma St. Louis. MO) 560 mg Magnesium Chloride (MW 203, Sigma, St. Louis, MO) 2.5 grams PSSA Polystyrenesulfonic acid sodium salt, (MW 70,000) 3 grams Crotem-SPA (Croda Inc., Parsippany, NJ) 3 grams Oxamic acid sodium salt 250 mg Tetronic 1307 (BASF Corporation, Mount Olive, NJ) 2 grams Sucrose (MW 342.30, Aldrich Chemicals, Milwaukee WI) 5 grams NAD (MW 663.4, N7004, Sigma. St. Louis, MO) 450 mg D-3-hydroxybutyrate dehydrogenase 50,000 units WST-5 (MW 1331.37, Dojmdo, Japan) 1.8 gram Diaphorase 0-15000 units Phenazine Metosulfate (PMS) 0-3 mg

Excess reagent was carefully removed (scraped) with a glass rod. The resulting membrane was hung for drying at 56 ° C in an oven for 10 minutes. Porex (0.6 mm thick) was impregnated in a 5% nitrite solution and then hung for drying at 100 ° C in an oven for ten hours. Finally, the membrane was laminated between a polyester backing (0.4 mm Melenex® polyester from ICI America, Wilmington DE) and Porex impregnated with nitrite.

B. Assays

The analysis was carried out as follows.

10 μl of aqueous samples containing 40 mg / dl of (D) β-hydroxybutyrate were examined in strips as described above. Strips varied in terms of the amount of PMS and / or diaphorase present on the strip. A 10 ml aqueous sample was applied to a freshly prepared test strip. A strip was inserted into the OTDR and data was collected. The reflection coefficient of the test strip was monitored at 660 nm at one second intervals for two minutes. Then the data was transferred from the OTDR memory buffer to a personal computer using a modified serial cable. The reaction rate was calculated based on the initial speed in K / S, in the range where the reaction profile is linear. The results in Table 2 are the average of five repetitions. For each individual analysis, a reaction rate of K / S / s is observed, where K / S is the magnitude of the reflection coefficient.

Table 2 presents the results.

table 2 The drug DAD and PMS, separately DAD and PMS cooked together DADed / ml Speed (K / S / sec) PMS (μm) Speed (K / S / sec) Separately ⁺ (K / S / sec) DAD units / ml PMS (μm) Together (K / s / sec) 150 0.0166 10 0.013 0.0296 150 10 0.0385 300 0.0392 twenty 0.0225 0.0617 300 twenty 0.1054 600 0.0943 40 0.0484 0.1427 600 40 0.3095 900 0.1666 60 0.078 0.2446 900 60 0.5861 1200 0.1787 80 0.1085 0.2872 1200 80 0.9107 1500 0.2866 100 0.13 0.4166 1500 100 Too fast* ⁺ Theoretical speed, that is, the speed predicted on the basis of the sum of the reaction rates carried out in the presence of diaphorase and PMS separately.
* The reaction is too fast, and it is difficult to determine the speed accurately.

C. Dependence of reaction rate on theoretical rate

Table 3 compares the actual reaction rate and the expected or theoretical reaction rate for the analyzes described above.

Table 3 Theoretical reaction rate (K / S per second) Actual reaction rate (K / S per second) 0.0296 0.0385 0.0617 0.1054 0.1427 0.3095 0.2446 0.5861 0.2872 0.9107

The drawing shows a diagram of the dependence of the observed speed on the theoretical speed.

From the diagram it follows that the reaction rate of the test strip signal generation system is accelerated in the presence of both PMS and diaphoresis, the magnitude of the observed acceleration is unexpectedly greater than the predicted acceleration based on the sum of the reaction rates of the systems including PMS or diaphoresis separately.

From the above results, it is clear that the claimed invention provides a significant and unexpected increase in the reaction rate when detecting an analyte based on the oxidation of the analyte and the concomitant reduction of the oxidation-reduction indicator. In addition, according to the invention, a more economical method for detecting an analyte is obtained in comparison with known methods based on diaphorase as an electron transfer agent.

Claims (10)

1. A signal generating system for detecting the presence of a reduced cofactor in a sample, comprising a dry reagent composition comprising a first low molecular weight electron transfer agent and a second high molecular weight electron transfer agent capable of oxidizing the reduced cofactor, the ratio of the first agent to the second agent is in the range of 0.001 to 10, an indicator of oxidation-reduction. 2. The signal generation system according to claim 1, characterized in that the first electron transfer agent is a low molecular weight electron transfer compound. 3. The signal generation system according to claim 2, characterized in that the low molecular weight electron transfer compound is a phenazine compound. 4. The signal generation system according to any one of claims 1 to 3, characterized in that the second electron transfer agent is a high molecular weight electron transfer compound. 5. The signal generation system according to claim 4, wherein the high molecular weight electron transfer compound is a protein compound. 6. The signal generation system according to claim 5, characterized in that the protein compound is an enzyme. 7. The signal generation system according to any one of claims 1 to 6, characterized in that the oxidation-reduction indicator is a tetrazolium compound. 8. The signal generation system according to any one of claims 1 to 7, characterized in that the dry reagent composition is present on the test strip. 9. A method for detecting the presence of analytes in a sample, which consists in using a signal generation system containing a dry reagent composition according to any one of the preceding claims 1 to 8. 10. A kit for detecting the presence of analytes in a sample containing a signal generation system according to any one of claims 1 to 8.