FI84625B - Process for detecting the threshold value of the analyte - Google Patents

Description

84625

METHOD FOR DETERMINING THE ANALYTICAL THRESHOLD

The invention relates to an enzymatic method and a reagent combination for detecting the concentration of an analyte above or below a certain threshold by means of PQQ dehydrogenases.

Quantitative colorimetric analysis is often used to measure the concentrations of organic or inorganic substances (analytes) in samples. The intensity of the color formed in the chemical reaction corresponds exactly to the concentration of the analyte to be determined in the sample. Such accurate quantitative assays are technically demanding to perform and require complex equipment as well as laboratory-trained personnel to succeed.

The most important area of analysis, which uses the measurement of the concentration of a substance based on changes in color intensity or hue, is the determination of concentration without visual measuring equipment, visual tests. Efforts have been made to expand the scope of visual test-based analytics by developing easy-to-use, one-man tests that do not require training. Implementations are often so-called. dry chemistry, in which a liquid sample dissolves dried or attached reagents in a test device and initiates reactions. The sample can also be diluted before analysis. In a visually interpretable test device, a color is generated or lost, the hue or intensity of which depends on the concentration of the analyte in the sample.

Thus, the analyte concentration corresponds to a change in the hue, intensity, or stained area of the test device.

The more accurately the color change can be detected, the more confident the concentration of analyte in the sample can be deduced.

However, in visual tests, there is rarely a need to accurately know the concentration of the analyte. In practice, 2 84625 decisions are often based on whether the concentration of the analyte exceeds a certain limit. These types of limits can be, for example, the maximum permissible concentration of air pollutants in the work or living environment (e.g. formaldehyde content) or the blood alcohol content in traffic, at the workplace or at the first aid station. A test performed without measuring equipment can act as an inexpensive screening test, depending on the result of which a decision can be made to perform an expensive laboratory assay.

The concentration or concentrations of the analyte determined in a suitable test device produce a clear color signal that guides the user's decision-making. The specified concentration, the threshold value, corresponds to a very sharp change in color, a kind of ΟΝ / NO signal. Such an analyte response is correctly interpreted and does not depend on the observer's subjective interpretations, such as in analysis tools based on differences in color intensity or hue.

Biochemical methods utilizing enzymes are generally best suited for colorimetric analyzes. Compared to methods based on conventional chemical reactions, biochemical analyzes are more specific, more convenient to perform, and are rapid at room temperature. Biological molecules are generally determined by two types of redox enzymes: 1. Dehydrogenases, which use NAD (P) H as their coenzyme. The change in the amount of NAD (P) H in the reaction can be measured directly in the UV range at 340 nm photometrically as a change in absorbance, or coupled to another reaction, often catalyzed by another enzyme, in which color is formed or lost.

2. Oxidases which catalyze the formation of hydrogen peroxide, the amount of which can generally be measured by a color reaction catalyzed by another enzyme.

3,84625

Abundant dehydrogenases are known which oxidize or reduce NAD (P) (H) in a stoichiometric reaction with a substrate. Patent application EP 154409 describes a test device and method for the determination of alcohols in which NAD (P) in an alcohol-dehydrogenase-catalyzed reaction is reduced by reaction with an alcohol. The resulting NAD (P) H reduces the dye in a diphorase-catalyzed reaction. Alcohol content is detected visually by observing a sliding change in color tone or by determining the time at which a certain degree of color change occurs. The weaknesses of such a system are obvious. The clarity of color change over the measurable analyte concentration range is an essential issue in assessing the reliability and usefulness of a test device. If a change in color intensity or hue is continuously sliding, this will result in a strongly dependent observation and performance-dependent interpretation. Perceiving differences in color intensity is very subjective. According to studies, the interpretation of different individuals for the same response varies greatly depending on vision, age, alertness, lighting conditions, and so on. The results of disposable test devices based on differences in color intensity or changes in color tone are not unambiguously interpretable. The problems are not mainly in the device structures, but in the reaction chemistry with which the analytical instrument is implemented.

A problem in determining the analyte directly from the sample matrix is usually also the high concentration of the analyte, in which case the color response obtained is too strong to be assessed visually or by measuring equipment. In this case, it is often necessary to dilute the sample before analysis. U.S. Pat. No. 4,490,465 describes a system in the presence of two analyte consuming enzymes, NAD (P) using dehydrogenase and oxidase. Thus, less than a stoichiometric amount of NAD (P) H is formed from the analyte, which can be determined by known methods. The analyte is derived into two different products in competing reactions catalyzed by two different enzymes.

84625

The method seeks to avoid sample dilution.

Efforts have been made to improve the clarity of the color change in the test device by adding a so-called a threshold component that prevents color changes before the analyte concentration exceeds a specified concentration. By changing the concentration of the threshold component, a certain cut-off concentration can be set for the analyte, which must be exceeded in the analyte sample in order to generate a color signal. Patent application WO 88/04694 describes an apparatus and method for measuring the content of NAD (P) H or indirectly the content of organic compounds which produce NAD (P) H by reaction with a specific dehydrogenase. In the process, the NAD (P) H formed in the dehydrogenase-catalyzed reaction primarily reacts with a competing threshold reagent that is consumed in the reaction. The amount of NAD (P) H that possibly exceeds the threshold concentration only causes a color change by reduction of the dye via an electron support or in a reaction catalyzed by a phosphorase. The aim of this method is to set a threshold value for the analyte, in which case the response would be an unambiguous ON / OFF color change. A weakness of the method is the use of poorly preserved coenzyme NAD (P). To improve its preservation, it is often necessary to use complex structural solutions, e.g. to dry it on a different layer than other chemicals. NAD (P) is expensive, so reducing its amount in a test device is an economic issue. On the other hand, the targeted substantial reduction in the amount of NAD (P) significantly reduces the shelf life of the test device. Diaphorase as another enzyme increases costs and impairs shelf life.

Often the problem in preparing enzymatic test solutions is to complete the reaction quickly enough. This problem clearly also applies to the solutions presented above. For example, the Michaels constant for NAD-dependent alcohol dehydrogenase (ethanol concentration at which a half-maximal reaction rate is reached) is quite high, about 13 5 84625 mmol / l, which corresponds to the order of magnitude of the ethanol concentration commonly found in blood or saliva. As a result, rapid and abrupt color change in the reactive zone is only feasible using large amounts of enzyme. In addition, the reaction of NAD (P) -dependent enzymes is often a reversible, bi-directional reaction with an equilibrium, e.g. in the ethanol assay, on the starting material side. To solve this problem, various auxiliary chemicals or enzymes can be used that react with the final product and help the reactions to complete. This, on the other hand, complicates the reaction chemistry and complicates the implementation of the analytical instrument, and may require the application of incompatible reagents to the apparatus design.

Impurities in enzyme preparations, instability of reagents and other substances and enzymes that cause side reactions are also often a problem when using enzymes using NAD (P). Enzymes using NAD (P) are very common in biological samples. In the light of the above, to date no suitable enzymatic method for the analysis of analytes containing oxo or hydroxy groups has been available to develop a fully reliable, unambiguous, easy-to-use and well-preserved visual test device for analyte concentrations above or below a certain threshold.

The method according to the invention and the combination of reagents provide a decisive improvement in the above-mentioned drawbacks. To accomplish this, the method according to the invention is characterized by what is set forth in the characterizing part of claim 1, and the reagent combination according to the invention is characterized by what is set forth in the characterizing part of claim 4.

An advantage of the method and reagent combination of the invention is that the concentration of analyte above or below a certain threshold can be detected enzymatically by a clear and sharp color reaction. Expensive and easily destroyable 6,84625 coenzymes such as NAD (P) are not required. At the same time, the likelihood of interfering side reactions is reduced. Furthermore, the enzyme reactions of the method of the invention are irreversible, and rapid even at low analyte concentrations. In addition, only one enzyme is required to determine the analyte.

The invention relates to a reaction system which can be used in a +/- type test to detect the concentration of an analyte above or below a preset limit value. The invention also relates to a reagent combination, kit or test device based on the described reaction system, by means of which the concentration of the analyte can be determined in the range of certain preset limits. The response given by the reaction system according to the invention takes place in the visible range of light and can be interpreted by a measuring device or visually.

The method and reagent combination of the invention utilize PQQ enzymes. PQQ enzymes is a recently described new group of enzymes that use pyrroloquinoline quinone (PQQ) as their prosthetic group. PQQ enzymes have been found in almost all groups of organisms. They have been described e.g. in the following overviews: Duine J A and Jongejan J A,

Ann. Rev. Biochem. 5, 403 (1989); Anthony C, Adv. Microbial. Physiol. 2T_, 113, (1986); Duine et al. Adv Enzymol. 59, 169, (1987) and in "PQQ and Quinoproteins", ed. Jongejan J A and Duine J A, Kluwer Academic Publishers, (1989). PQQ dehydrogenases have previously been used to determine various analytes photometrically in conventional solution assays (e.g., Ameyama M, Methods Enzymol. B_9, 20 (1982)). Dehydrogenases can oxidize the analyte with a variety of oxidants, including electron acceptors of PQQ dehydrogenases such as Fe (III) chelates such as ferricyanide or EDTA chelate, cytochrome c, and several 7,84625 quinoneimine dyes such as indurines. blue), indophenols (DCIP), azine (PMS, PES, 1-methoxy-PMS), oxazine (Meldolasin, Nilesin) and thiazine derivatives (thionine and methylene blue), which allows the use of PQQ enzymes in color change-based analysis directly, without further action. enzymes assisting in the indication phase, or added unstable k Enzymes can be used in kinetic or endpoint measurements. The reactions are irreversible and rapid, which are major advantages for analytics. Also advantageous when using PQQ enzymes are their relatively low Michaelis Constants, e.g. Acetobacter acetin MBADH (membrane bound alcohol dehydrogenase) has a Michaelis constant of 2 mmol / l for ethanol and an order of magnitude of 0.1 mmol / l for ferricyanide.

Surprisingly, it has now been found that PQQ dehydrogenases have a clear order of preference for some electron acceptors so that better oxidation is substantially consumed before the reaction begins to proceed with another electron acceptor, and this property can be used in limit chemistry-based indication chemistry. The method and reagent combination of the invention use PQQ dehydrogenases involved in the oxidation of oxo and hydroxy groups to determine analytes containing these groups in threshold chemistry. Such analytes typically include e.g. alcohols, carbohydrates and aldehydes.

According to the invention, dehydrogenase using PQQ as a coenzyme is used to detect the concentration of an analyte in excess of a certain amount by a color reaction. The method is based on the principle of competing oxidants, in which a first, substantially faster-reducing electron acceptor is consumed in the first step, and at the end, if there is any analyte to be analyzed, a second reaction begins with a noticeable color change as the chromogen is reduced. By varying the amount of the first electron acceptor β 84625, the method or reagent combination can be adjusted to indicate the amount of analyte in the sample above a certain limit. Likewise, the first electron acceptor can be used to reduce the color response of the analyte so that the rate of the color reaction or the amount of color at the end of the reaction can be measured by analytical equipment or evaluated visually.

To determine the analyte concentration, the method can also be used with PQQ dehydrogenases in a three-electron acceptor system in which the first electron acceptor essentially determines the cut-off, the second electron acceptor loses color, and the third electron acceptor (chromogenic) color is preferred in the third step. This provides a color change chain that can be used to estimate the analyte concentration.

Possible fields of application of the invention include industry (e.g. food industry), medicine, diagnostics, environmental hygiene, occupational health care, traffic control, etc., where it is often important to know whether the analyte concentration exceeds or falls below a certain value. An important area of application of the invention is disposable analysis devices suitable for "every man" use. It is clear to a person skilled in the art that the method according to the invention can also be used in a conventional laboratory analysis representing liquid chemistry.

In the method according to the invention, the PQQ enzyme is preferably PQQ alcohol dehydrogenase or PQQ aldehyde dehydrogenase, the isolation of which can be carried out according to procedures known from the literature, e.g. Ameyama, M., Methods Enzymol. 89, 20 (1982). The extent of the set of analytes to be determined depends on the substrate specificity of the PQQ enzyme selected in each case. When PQQ alcohol dehydrogenase is used, the analyte may be, for example, 9,84625 ethanol, n-propanol, n-butanol or 1,4-butyl glycol. When PQQ aldehyde dehydrogenase is used, the analyte may be, for example, formaldehyde or acetaldehyde. The only condition for the analyte to be determined by the method according to the invention is that it can serve as a substrate for the PQQ enzyme used in the method.

Figures 1-7 show the functionality of the limit value principle in some embodiments of the method according to the invention. Absorbances (A) with respect to time are expressed in relative units (optical distance <1 cm).

Various embodiments of the method of the invention will now be schematically described, in which PQQ dehydrogenases are used to detect the concentration of analyte above or below a certain threshold. The invention is not limited to the embodiments shown. Variations and modifications apparent to those skilled in the art may be made without departing from the spirit or scope of the invention.

A first embodiment of the method according to the invention can be described as follows:

E-PQQ

(1), d + Slox ► A0 x + Slred

E-PQQ

(3) Ar, d + S2 0 x * A0 x + S2 red where E-PQQ is the PQQ enzyme, Slox and Slred are the oxidized and reduced forms of the first electron acceptor S2 setting the threshold, S2ox and S2ced are the oxidized and reduced forms of chromogen S2, respectively , Ar # d and Aox are the reduced and oxidized form of the analyte, S2 is a better oxidant than S2 for E-PQQ. In this case, in the first step, the first electron acceptor S2 is consumed, and if there is any analyte left, the chromogen S2 only begins to run out.

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The amount of Sx in the reaction is critical. Sx is colorless or colored. The amount of S2 in the reaction is relatively small, and when it is reduced, there is an easily detectable color change, the detection of which is not hindered by the possible color of Sx. It is desirable that the molar absorbance of S2 be as high as possible. By changing the amount of substrate S3, the method can be adjusted to indicate the amount of analyte (Ared) in the sample exceeding a certain limit. Slred and S2ox can also react with each other:

E-PQQ

(!) Ar.d + Slox> Aox + Slred (2) slred + s2ox <slox + s2red

E-PQQ

(3) Ar # d + S2ox * A0 x + S2ted In this case, the reaction equilibrium must be very strongly to the left. The reaction initially consumes S3, and if Ar, d is present in excess, a color change occurs as S2 is reduced.

In the above reactions (1) to (3), S 1 may be a chelate of Fe (III), preferably ferricyanide, S 2 may be an indophenol dye, preferably DCIP (dichlorophenol-indophenol) or an oxazine dye, preferably Meldolasin.

Another embodiment of the method according to the invention can be described as follows:

E-PQQ

Ared + Slox Aox + Slred

E-PQQ

(5) Ared + S2ox * Aox + S2red (6) S2r, d + S3ox S2ox + s3red S3 is the first electron acceptor that runs out before S2 begins to act as an oxidant. Here, S2 acts as a redox catalyst between Ared and the chromogen S3. In this embodiment of 11,84625, St may be a chelate of Fe (III), preferably ferricyanide, or an indophenol dye, preferably DCIP. S2 may be a phenazine dye, preferably PMS (phenazine methosulfate), 1-methoxy-PMS or PES (phenazine ethosulfate), or an oxazine dye, preferably Meldolasin. S3 may be a tetrazolium dye, preferably MTT (thiazolyl blue) or INT (p-iodonitrotetrazolium violet), or indophenol dye (provided that S2 is not an indophenol dye), preferably DCIP.

In a third embodiment of the method according to the invention, the indication chemistry can also be used as a system of three competing substrates, whereby different colors and shades of color can be obtained in the test zone.

As a diagram, this embodiment can be represented as follows:

E-PQQ

("7) Ar · d + ®lox * ^ ox + ®lnd (E-PQQ) (8) Ar.d + S2ox" * Aox + S2r.d (this is slr.d + s2ox slox + s2r.d) ( 9) Ar · d + ®3ox * Ao x + 83red S2 and S2 are colorless or colored, the amount of S3 in the reaction is relatively small, and when it is reduced, an easily detectable color change occurs, the detection of which is not hindered by possible S2 and S2 By changing the amount of substrate S2 or S2, the method can be adjusted to indicate the amount of analyte in the sample above a certain limit, first the first electron acceptor S2 is consumed in the reaction, then the second electron acceptor S2 is consumed and finally the "worst" substrate S3 is chromogenic (or redox catalyst + chromogen), and changes color as it is reduced.A prerequisite for successful indication is that i2 84625 reduced St and S2 do not cause discoloration of S3.

S 1 here may be a chelate of Fe (m), preferably ferricyanide, S 2 may be an indophenol dye, preferably DCIP (dichlorophenol-indophenol), S 3 may be a combination of a phenazine dye (redox catalyst) + a tetrazolium dye (chromogen), preferably PMS + INT (p- iodonitrotetrazolium violet).

The necessary components or reagents of the process of the invention are brought together in a suitable medium, such as a solution, but most preferably they are impregnated on a solid support. Such solid carriers are known in the literature. Typically, the test device or reagent combination of the invention for analyte determination is disposable and includes all of the above reagents and enzymes necessary for the method of the invention, as well as a solid support. Typically, the test is performed by contacting the sample with a solid support containing the necessary reagents and enzymes. The sample material may be gaseous or liquid, e.g., a body fluid such as saliva, plasma, or urine, or exhaled air, workplace or residential air.

In the test, exceeding or falling below the limit value is shown as a sensitive ON / OFF type with an easy-to-interpret color change.

The color change can be detected visually, but also by suitable instrumentation, for example colorimetrically or fluorometrically.

The reagents or components of the process can be combined with a carrier by known techniques, e.g., dried on various surfaces or impregnated into porous materials prior to drying. In addition, well-known inert drying aids, reagent dissolution accelerators, and enzyme stabilizers and detergents can be used in the preparation of the dry reagent zone.

Suitable carrier materials for the test device include the i3 84625, all materials commonly used in this type of test. The most typical materials are glass, wood, metal, textile, gelatin, filter paper, porous cellulose products and plastic materials. The carrier material is not critical to the invention, but can in principle be any material capable of acting as a carrier for the reagents and components of the system.

For the purpose of the assay, the test device may also use well-known systems to provide a predetermined amount of liquid sample to the support. Such a system can be based on e.g. hydration or capillary action, and they have been shown e.g. in WO 88/04694. On the other hand, the method and test device according to the invention can also be applied in such a way that the amount of sample in contact with the carrier does not have to be constant.

In this case, the assay monitors whether a color change occurs in the test device over a period of time. Such an application can typically be used e.g. in the measurement of the formaldehyde content of the air.

The test device according to the invention may also have separate dried indicator areas, each of which indicates a separate defined limit value. One application of the method is to apply a limiting reagent at a continuously varying concentration (concentration gradient) to the indicator zone, thereby forming a color change point between the region where the analyte limit is exceeded and the region where it falls below. In this case, the exact numerical value of the analyte content of the sample can be obtained by comparing the color change point with a pre-calibrated scale.

The invention is illustrated by the following examples without, however, limiting the invention thereto.

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Example 1

Determination of ethanol by Acetobacter acetin PQQ alcohol dehydrogenase (MBADH).

Sx, i.e. the first electron acceptor acting as a threshold, is ferricyanide and S2 as a chromogen is DCIP (dichlorophenol-indophenol). Ferricyanide and DCIP are both substrates of MBADH and, in addition, are in redox equilibrium with each other being strongly left at pH 6. If unused ferricyanide (excess compared to ethanol) remains in the reaction, DCIP remains in the oxidized form S2ox, which is strongly blue. The reduced form S2red is colorless. A.acet's PQQ aldehyde dehydrogenase (MBALDH) behaves analogously in the experiment.

The cuvettes contained the following reagent concentrations: DCIP

0.16 mmol / l, ethanol 0.45 mmol / l, Mclainen buffer pH

6.0 40 mmol / l, Triton X-100 1.3%, MBADH 620 U / l and ferricyanide in cuvettes 1-6: 0, 0.31, 0.94, 1.09, 1.25 and 1.56 mmol / l. Figure 1 shows the results of the absorbance measurement in cuvettes 1-6 at a measurement wavelength of 570 nm. In cuvettes 1 and 2, the color of DCIP disappeared relatively rapidly, in cuvette 3 it decreased but remained strong, and in cuvettes 4, 5, and 6 it remained strongly blue. In cuvettes 2 and 3, a delay time is observed when the amount of DClP does not change, but ferricyanide is consumed.

Example 2

When 0.3 mmol / l PMS or 0.6 mmol / l 1-methoxy-PMS is added to the reaction mixture of Example 1, the rate of reduction of DCIP is approximately doubled. The rate of reduction of ferricyanide, on the other hand, is not affected by phenazine color additions.

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Example 3

The example illustrates another embodiment of the process according to the invention, in which the first electron acceptor S3 is DCIP, the redox catalyst S2 is PMS and the chromogen S3 is INT. The cuvettes contained the following reagent concentrations: PMS 0.3 mmol / l, INT 0.32 mmol / l, ethanol 0.5 mmol / l, Mclpain buffer (pH 7) 40 mmol / l, Triton X-100 1.3%, MBADH 700 U / l and DCIP in cuvettes 1 to 6: 0, 0.02, 0.04, 0.08, 0.12 and 0.16 mmol / l. Figure 2 shows the results of the absorbance measurement in cuvettes 1-6 at a measurement wavelength of 450 nm, where the blue color of DCIP does not significantly affect the absorbance. It can be seen from the figure that a red formazan dye absorbing at 450 nm is formed only after the blue DCIP has passed out of the reaction mixture. This could also be observed visually from the cuvettes.

In a further experiment, the amount of ethanol was halved and the amount of DCIP was tripled. When the molar DCIP / EtOH ratio was 1: 1, a light brown transition color was obtained which formed in less than 10 minutes and was stable for at least 30 minutes. When the DCIP concentration was below the ethanol content, the reaction mixture rapidly turned red, and with the proportions reversed, the reaction mixture remained blue. These colors were stable.

Example 4

Ethanol limit test using the MBADH / MBALDH combination.

The cuvettes had the following reagent concentrations: MTT 0.2 mmol / l, PMS 0.18 mmol, ferricyanide 43 mmol / l, 0.3 mol / l succinic acid buffer pH 4.5, Triton X-100 0.07% and ethanol in cuvettes 1-6 9.7, 9.7, 10.2, 10.77, 11.3 and 11.8 mmol / l. The enzyme activity in the cuvettes was: MBADH 35,000 U / l and MBALDH 5700 U / l. Cuvette 2 did not contain the 84625 enzyme. Figure 3 shows the results of the absorbance measurement in cuvettes 1-6 at a measurement wavelength of 570 nm. Upon completion of the reaction, cuvettes 1, 2, and 3 were colorless, cuvette 4 showed a fading blue, and cuvettes 5 and 6 were intense blue. The formation of a blue color began after four molecules of ferricyanide were consumed per molecule of ethanol. In the assay system, ethanol was converted to acetic acid. In this example, differences in ethanol content of less than 5% could be visually distinguished under the conditions used.

Example 5

Limit test for ethanol with almost pure MBADH preparation.

•

The cuvettes had the following reagent concentrations: MTT 0.2 mmol / l, PMS 0.18 mmol / l, ethanol 1.95 mmol / l, 0.28 mol / l succinate buffer pH 4.5, Triton X-100 1.1% and MBADH activity 2300 U / l. In cuvettes 1-6, the amount of ferricyanide was varied: 0, 1.11, 3.34, 3.89, 4.44 and 5.56 mmol / l. Figure 4 shows the results of the absorbance measurement in cuvettes 1-6 at a measurement wavelength of 570 nm. In cuvettes 1, 2 and 3, the more blue ferricyanide was formed the slower the blue color. In cuvette 4, color formation was very slow and in cuvettes 5 and 6 no blue color was formed.

A blue color began to form in the experiment when two molecules of ferricyanide had been consumed per molecule of ethanol and ethanol was still present in the reaction mixture. Ethanol was oxidized in the reaction to acetaldehyde.

Example 6 17 84625

Limit test for acetaldehyde with aldehyde dehydrogenase.

The cuvettes had the following reagent concentrations: MMT 0.19 mmol / l, PMS 0.17, acetaldehyde 0.42 mmol / 11, 0.27 mmol / l succinate buffer pH 4.5, Triton X-100 1.1% and aldehyde dehydrogenase activity was 2000 U / l. The amount of ferricyanide in the cuvettes was 1-6: 0, 0.22, 0.67, 0.78, 0.89 and 1.11 mmol / l. Figure 5 shows the results of the absorbance measurement in cuvettes 1-6 at a measurement wavelength of 570 nm. In cuvettes 1-4, a fairly rapid strong color formation was observed. After a long delay, a slowly pale blue color began to form in cuvette 5 and cuvette 6 remained yellow.

Example 7

Determination of formaldehyde by aldehyde dehydrogenase from a liquid sample.

The cuvettes had the following reagent concentrations: MTT 0.19 mmol / l, PMS 0.17 mmol / l, formaldehyde 0.42 mmol / l, 0.27 mmol / l succinate buffer pH 4.5, Triton X-100 1.1%, and cuvettes 1-6 aldehyde dehydrogenase activity (determined by acetaldehyde dehydrogenase hydrogenation) ) 2100 U / l. Cuvette 7 was free of formaldehyde. The amount of ferricyanide in the cuvettes was 1 to 7: 0, 0.21, 0.64, 0.72, 0.85, 1.06 and 0.21 mmol / l. Figure 6 shows the results of the absorbance measurement in cuvettes 1-7 at a measurement wavelength of 570 nm. In cuvettes 1-5, a blue color formed. The slow increase in absorbance, e.g. in cuvettes 6 and 7, which did not form a blue color, was due to the pale precipitate formed in the cuvettes.

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Example 8

Limit determination of ethanol: application of dry chemistry.

A 13 μΐ reaction mixture was applied to the polystyrene surface containing: succinate buffer 0.5 mol / l pH 4.5 760 μΐ MTT 8.7 mmol / l 30 μ PMS 7.8 mmol / l 30 μ ferricyanide 0.5 mol / l 58 μ

Triton X 100 10% 10 // MBADH 180000 300 μ Immediately the reaction mixture was air dried at 40 ° by blowing air.

13 μΐ ethanolic solutions of 8.3 and 6.2 mmol / l ethanol were pipetted into the dried reagents. The first formed a blue color in about two minutes and the second did not form a blue color. The concentration at which the color change occurs is adjustable by changing the concentration of ferricyanide in the example described above.

Example 9

Use of aldehyde dehydrogenase in the determination of air formaldehyde content. The rate of color change in standard toraldehyde dipality can be affected by changing the amount of ferricyanide or by the choice of carrier material.

Test 1

The formaldehyde concentration in the chamber air was about 130 mg / m3 (* 100 x HTP15 min for formaldehyde).

Solutions used: 19,84625

Succinate buffer 0.5 mol / l, pH 4.5 MTT 3.6 mg / ml PMS 2.5 mg / ml Triton X 100 10%

Ferrisyanide 10 mmol / l ALDH 16000 U / l

The reaction mixture of:

Succinate buffer 125 μΐ

To Triton X 100 25 "MTT 30" PMS 10 "38 μl of the mixture was added 20 μl of ALDH and the amount of ferricyanide used in each case (2 μΐ, 3 μΐ and 4μ1). From this mixture was taken into a well in PVC plastic (= TK ), UltraBind for MSV-450 paper strip (= UB) and fiberglass paper strip <»LK) 5 μΐ.

After pipetting the reaction mixture, the indicator zones were placed in a chamber containing formaldehyde in their atmosphere. Color change times were taken up. The results are presented. in Table 1.

Table 1 Color change times in the formaldehyde determination:

Ferrisyanide / Color change time / s

mmol x 10-6 tissue UB LK TK

1.7 20 25 45 2.5 40 35 70 3.4 55 45 80

In a similar experiment, no color formed on the enzyme-free strip. It can be seen from the table that the color change time depends on the amount of ferricyanide in the indicator zone at a constant formaldehyde content and on the support material used.

Test 2.

In this experiment, the rate of color change of the indicator zone was tested with a very low formaldehyde concentration in air. The formaldehyde concentration in the chamber air was about 1.3 mg / m3 (= HTP15 min for formaldehyde).

The solutions used were otherwise the same, except that the MTT concentration was 0.6 mg / ml and the PMS concentration was 1.20 mg / ml.

The reaction mixture of:

Succinate buffer 31.25 μΐ MTT 7.5 "PMS 2.5"

Triton X 100 6.25 "

Ferrisyanide 4.0 "ALDH 15.0"

A total of 66.5 μΐ

The reaction mixture may additionally contain stabilizers.

Of these mixtures, 10 μΐ was pipetted into a well (»TK) in PVC and 3 μΐ on the following strips of paper:

Whatman 3 mm chromatography paper (= W)

UltraBind paper (= UB)

Fiberglass paper (»LK)

Paper strips and PVC wells were placed in a chamber containing formaldehyde in the air space. Color change times were taken up. The results are shown in Table 2.

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Table 2 Color shrinkage times in the determination of formaldehyde using different support materials.

Carrier material LK UB W TK

min s min s min s min s Color change time 3 00 5 10 1 50 6 30

Example 10 This example illustrates a third embodiment of the process of the invention in a reaction system of three competing substrates in which the first electron acceptor Sx is ferricyanide, the second electron acceptor S2 is DCIP and the redox catalyst + chromogen combination is PMS + INT (S3). In this system, wear of ferricyanide first occurs and then the blue color of DCIP disappears (the color changes to yellow through blue and green). If there is more than a stoichiometric amount of ethanol, PMS / INT will begin to form a red color. The color then changed from pure yellow through orange to red. An example of such color changes is shown in Figure 7, in which the spectra (1-12) of a reaction mixture containing:

Mcllvainen 0.1 mol / l citrate / phosphate buffer pH 7 2 ml

Triton X 100 10% 0.4 ml

Ethanol 0.2% 0.15 ml DCIP 15 mmol / l 0.016 ml

Potassium ferricyanide 0.1 mol / l 0.030 ml PMS 7.8 mmol / l 0.1 ml INT 8 mmol / l 0.1 ml MBADH 120000 U / l 0.030 ml

Initially, at about 420 nm, a decrease in absorbance due to a decrease in ferricyanide 22,84625 is observed. When the ferricyanide is practically depleted, the blue color of the DCIP begins to disappear (maximum near 640 nm). When the DCIP is depleted (the yellow color of the PMS remains), a red color begins to form from the INT (in dashed curves, maximum near 440 nm).

Example 11

The color changes of the reaction of Example 10 were also monitored visually. In this experiment, the amount of DCIP was doubled and the amount of enzyme was 15-fold: t (s) color 0 green 10 blue 30 green 45 yellow 60 orange 75 red

The invention has been described above with reference to certain preferred embodiments and reagent combinations. However, it will be apparent to those skilled in the art that variations, modifications, and equivalent substitutions of reagents may be made without undue experimentation without departing from the spirit or scope of the invention. For example, it is well known in the literature that phenazine dyes act as electron carriers for a variety of dyes that can be applied to an invention such as that described (see Michal, G. et al., Methods of Enzymatic Analysis, Vol. I, 197, ed. Bergfmeyer HU, Verlag Chemie 1983).

Claims (18)

23 8 4 6 2 5 A method for determining an oxo- or hydroxy-containing analyte by means of a color signal generated when the amount of analyte exceeds a certain threshold value, characterized in that a chromogen is added to a measuring mixture containing a) a chromogen b) a first electron acceptor which is reduced by the analyte in the presence of PQQ dehydrogenase directly or via a possible redox catalyst substantially more rapidly than chromogen, thereby preventing chromogen reduction and color change reaction until the first electron acceptor is depleted of the first electron acceptor; and / or chromogen directly or via a possible redox catalyst, the analyte to be determined, wherein the substrate for PQQ dehydrogenase attina-acting analyte reacts by consuming the first electron acceptor or its depleted chromogen, and detecting whether a color change occurs. A method according to claim 1, characterized in that a second electron acceptor is further added to the measurement mixture, which the analyte in the presence of PQQ dehydrogenase directly or via a possible redox catalyst reduces substantially slower than the first electron acceptor but substantially faster than the chromogen. the second electron acceptor is depleted. A reagent combination for detecting an analyte containing oxo or hydroxy groups in a sample by means of a color signal which is generated when the amount of analyte exceeds a certain threshold value, characterized in that the following components are combined on a solid support; 24 84625 (a) a chromogen which is reduced by the analyte in the presence of PQQ dehydrogenase directly or via a possible redox catalyst, causing a color change reaction; b) a first electron acceptor which is reduced by the analyte in the presence of PQQ dehydrogenase directly or via a possible redox catalyst substantially faster than the chromogen; and c) PQQ dehydrogenase, which is capable of transferring electrons from the analyte to the electron acceptor and / or chromogen directly or via a possible redox catalyst. The reagent combination of claim 3, further comprising a second electron acceptor which, in the presence of PQQ dehydrogenase directly or via a possible redox catalyst, reduces the analyte substantially more slowly than the first electron receptor but substantially faster than the chromogen. Reagent combination according to any one of claims 3 to 4, characterized in that it further comprises said redox catalyst which, in the presence of PQQ dehydrogenase, is capable of receiving electrons from the analyte and transferring them to the chromogen and / or the first or second electron acceptor. Reagent combination according to Claim 5, characterized in that the redox catalyst is a phenazine dye or an oxazine dye. Reagent combination according to Claim 6, characterized in that the redox catalyst is PMS, 1-methoxy-PMS, PES, or Meldolasin. Reagent combination according to one of Claims 3 to 7, characterized in that the PQQ dehydrogenase is a membrane-bound alcohol dehydrogenase (MBADH). Il 25 84625 Reagent combination according to one of Claims 3 to 8, characterized in that the PQQ dehydrogenase is a membrane-bound aldehyde dehydrogenase (MBALDH). Reagent combination according to one of Claims 3 to 9, characterized in that the first electron acceptor is a complex, chelate or indophenol dye of Fe (III). Reagent combination according to Claim 10, characterized in that the first electron acceptor is ferricyanide or DCIP. Reagent combination according to one of Claims 3 to 11, characterized in that the chromogen is a tetrazolium dye, an oxazine dye or an indophenol dye. t Reagent combination according to Claim 12, characterized in that the chromogen is MTT, DCIP, INT or Meldolasin. Reagent combination according to one of Claims 5 to 13, characterized in that the first electron acceptor is ferricyanide, the chromogen is MTT and the redox catalyst is PMS or 1-methoxy-PMS. Reagent combination according to one of Claims 5 to 13, characterized in that the first electron acceptor is DCIP, the chromogen is INT and the redox catalyst is PMS or 1-methoxy-PMS. Reagent combination according to one of Claims 4 to 13, characterized in that the first electron acceptor is ferricyanide, the second electron acceptor is DCIP, the chromogen is INT and the redox catalyst is PMS or 1-methoxy-PMS. Use of a reagent combination according to any one of claims 3 to 16 for carrying out a method 26 8 4 6 2 5 according to any one of claims 1-2. Use according to Claim 17, characterized in that the analyte to be determined is a lower alcohol or a lower aldehyde. t li 27 84625