DE3701833A1 - Method for the optical determination of a catalytic enzyme activity of a sample, and an arrangement for carrying out the method - Google Patents

Abstract

Known methods for the optical determination of the catalytic enzyme activity of a sample, which use enzyme substrates which are cleaved under the influence of the enzyme to be measured and decompose to coloured or fluorescent products, where the increase in colour or fluorescence intensity per unit time is regarded as a measure of the enzymatic activity, have the disadvantage that they are suitable only for optically transparent sample solutions. Measurements on whole blood, in particular in vivo determinations of enzyme activity, are therefore impossible. The method according to the invention avoids these disadvantages by an enzyme substrate being assigned to the end of a light conductor and being contacted with the sample to be measured, and by the change, owing to the enzymatic reaction, in spectral properties of the enzyme substrate or its reaction products per unit time being measured and used for determination of the enzyme activity.

Description

The invention relates to a method for optical Determination of the catalytic enzyme activity of a sample, and an arrangement for performing the method, wherein the sample to be measured in contact with an enzyme substrate stands.

Determination of the catalytic activity of enzymes plays in biochemical and clinical analysis a very big role. Deviations from normal activity are often characteristic of the occurrence of diseases. For example, the activity of the enzyme is "acid Phosphatase "when bone or prostate tumors appear significantly increased, so that their determination is considerable diagnostic importance.

A variety of methods are available for the determination enzymatic activities become known. They are summarized in the books by G.G. GUILBAULT: Enzymatic Methods of Analysis, Pergamon Press, Oxford, 1970; such as H. BERGMEYER: Fundamentals of Enzymatic Analysis, Verlag Chemistry, Weinheim - New York, 1977. Also identification bacterial pathogens with the help of synthetic Protease substrates have recently become of interest [published by COBURN, LYTLE and HUBER, in Anal. Chem. 57, 1669 (1985)].

It is known to help the activity of enzymes for example synthetic, colored or fluorescent To determine substrates. Enzyme substrates are used for this, which are split under the influence of the enzymes and disintegrate into colored products. The increase of Color or fluorescence intensity over time serves as Measure of the enzymatic activity. Among the hydrolases, which is particularly good with the help of this kinetic method can be determined, mention should be made of carboxylesterases, Phosphatases, sulfatases, dealkylases, glycosidases and Proteases.

A typical example is the method known from Analytical Biochemistry, 143, 146 (1984) by KOLLER and WOLFBEIS for determining the phosphatases, which is based on the fact that a phosphoric acid ester of the following structure A is split into two fragments by the enzyme "Acid Phosphatase". An ionic hydrolysis product B and a free phosphate ion are formed. In contrast to the starting substrate A, the phenolate ion B is characterized by an intense yellow color and a blue-green fluorescence. The increase in color or fluorescence with time can be used as a direct measure of the activity of the "acid phosphatase".

This method of determination is very sensitive and allowed the photometric detection of approximately 0.001 activity units Phosphatase per milliliter sample, as well as from approx. 60 micro units per milliliter with the fluorimetric Determination.

Corresponding methods for the determination of carboxylesterases, Sulfatases, or amylases are also known.

Proteases can, as in the work cited above described by COBURN, LYTLE and HUBER, in an analogous manner can be determined with the help of synthetic peptides or amides. All these processes have in common that the enzymatic Reaction a different colored or fluorescent Product is created.

Thereby the determination of enzymatic activities until now only in vitro, since none the known methods for use on the living organism suitable is. However, this appears from various Desirable because, for example, an in vitro Determination can be done much faster and in real time  can. In contrast, with the help of previously known Methods first a sampling is necessary, whereby falsifications, as in sampling and sample preparation occur, cannot be prevented.

In addition, the above and all others Methods of determination with the help of synthetic enzyme substrates practically only suitable if optically transparent solutions or samples are available. For whole blood as well as for they are unsuitable for in vivo determinations.

The object of the present invention is a method or an arrangement for determining the catalytic To introduce enzyme activity, which the above Does not have disadvantages and in particular an in vivo determination the enzyme activity allows.

According to the invention, this object is achieved by that an enzyme substrate associated with the end of an optical fiber and brought into contact with the sample to be measured and that the change caused by the enzymatic reaction spectral properties of the enzyme substrate or whose reaction products per unit of time recorded and Determination of the enzyme activity is used. The present Invention solves the problem of in vivo determination thus in that an enzyme substrate for example hydrolytically active enzymes assigned to the end of a light guide becomes. This end then comes into contact with the sample brought, by the enzymatic activity of Sample the enzyme substrate in its spectral properties is changed. The change is made via the light guide tracked and used as a measure of current enzyme activity. For the first time, this is also an activity determination in whole blood and other optically non-transparent liquids possible without prior sample preparation.

In one embodiment of the method according to the invention it is provided that the enzymatic reaction conditional change in the absorption of the preferably synthetic Enzyme substrates detected via the light guide and is used to determine the enzyme activity.  

In another embodiment of the invention the spectral change caused by the enzymatic reaction the fluorescence radiation of the preferably synthetic enzyme substrate, for example the change in fluorescence intensity or the shift of the fluorescence maximum detected via the light guide and for determining the enzyme activity be used. The first Method that of enzyme substrate or its reaction products backscattered excitation light measured, which in its Intensity depends on that due to the catalytic enzyme activity influenced absorption capacity of the substrate. The other method uses, for example a filter or an energy dispersive detector system caused by the catalytic activity of the enzyme Change in the fluorescence intensity of the enzyme substrate measured via the light guide and the measured values of an evaluation fed.

An embodiment of the method provides that in addition for enzyme activity in a manner known per se the pH of the sample is measured, and that this reading to correct the predetermined enzyme activity is used.

It is known that the speed of many enzymatic Reactions strongly depend on the pH of the solution. In addition, the degree of dissociation of many is also the hydrolysis of released dyes depends on pH. Since the pH values of physiological samples vary within certain ranges can, it is necessary to measure the actually measured Correct enzyme activity with regard to pH. This is best done empirically. The pH dependency the enzymatic activity is very many Enzymes already known, but must be used in the event of one immobilized enzyme substrates can be redetermined. To So it is measuring the individual pH of the sample sensible, at the same time as measuring the enzyme activity also to record the pH. The determination of the true Enzyme activity is best done by:  an empirically determined relationship between activity and relative signal change as a function of the current pH value for the determination.

An arrangement of the type mentioned for implementation of the method according to the invention, the one to be measured Sample is in contact with an enzyme substrate given that the enzyme substrate the end of an optical fiber is assigned, with a photodetector with subsequent Signal evaluation is provided, which of the Enzyme substrate or its reaction products emitted Measures fluorescent light or reflected excitation light. It is advantageous if using a beam splitter part of the excitation light a reference photodetector is fed, causing fluctuations in the excitation light source can be eliminated. Depending on the type of The question and the nature of the enzyme are different metrological arrangements possible. All orders is common, however, that a synthetic, for example Enzyme substrate assigned to the end of an optical fiber is present and that the change in spectral properties is tracked with the help of an optical fiber.

Photomultipliers are expediently used as photodetectors, Phototransistors or photodiodes. Because the latter cheap, but only sensitive in the visible spectral range are preferably used such enzyme substrates, their cleavage at wavelengths above 450 nm, ideally over 500 nm, can be tracked. The advantages an analytical wavelength of over 460 nm the possibility of using inexpensive light-emitting Diodes as light sources.

Incandescent lamps, gas discharge lamps, light emitting diodes (LEDs) or lasers. The beam splitter can also be dichroic Mirrors can be replaced so that the scattered or reflected Better to separate excitation light from fluorescent light. The light guide is preferably a single fiber, can also be a fiber bundle.  

The fluorescent light can be from the reflected excitation light can of course also be separated with the help of filters.

One of the arrangements according to the invention provides that the enzyme substrate at the end of the light guide chemically or physically immobilized. The enzyme substrate directly on the polished exit surface of the light guide immobilized. However, it is also possible that Enzyme substrate after removal of the jacket in the end area of the light guide on the peripheral surface of the core of the light guide to immobilize. The light weaknesses or the In this case, fluorescence is caused by the so-called evanescent Light wave causes because of the total reflection the interface of the light guide the electromagnetic Wave a few nanometers deep into the enzyme substrate, which here is the optically thinner medium. The excitation light can be absorbed there or fluorescence induce when within the reach of the evanescent Wave there is a dye. Reflected or emitted light is returned through the light guide and measured.

The enzyme substrates can also be immobilized in various ways. So z. B. the coumarin-derived substrate of the following structure, which is suitable for a determination for butyryl cholinesterase, is immobilized at the end of a light guide in the following way: The bare end of a 200 micron thick quartz light guide was finely polished and activated with a 20% aqueous solution of sulfuric acid. This removes surface ionic impurities and makes the surface more chemically reactive. The glass surface was then reacted in a known manner with the glass derivatization reagent aminopropyltriethoxysilane. This reaction essentially consists in heating the active surface of the glass in a 10% solution of the silane in toluene at 100 ° for 2 hours, then washing with acetone and drying at 120 °. This gives a light guide, the end of which carries free amino groups.

These amino groups can now be used with help known chemical methods enzyme substrates can be linked. The aforementioned substrate I was made using the peptide coupling Reagent ethyl N ′ - (3-dimethylaminopropyl) carbodiimide- immobilized hydrochloride.

Alternatively, the substrate can also be placed in the corresponding one Transfer carboxylic acid chloride and then with the amino groups cause the glass surface to react.

Bring the optical end pieces thus obtained Fibers in contact with a solution made from butyryl enzyme Cholinesterase contains, one observes in a measuring arrangement, a clear in a short time according to claim 5 Increase in light absorption at an analytical wavelength of 410-430 nm, as well as an increase in fluorescence intensity at 460-470 nm.

In an analogous manner to this example, the typical substrates for sulfatases, amylases or proteases of the following structures II, III, IV derived from the same basic molecule can be immobilized: R = -OSO ^- ₃ Na⁺ (II)
R = -O-amylose (III)
R = amino acid (s) -NH- (IV)

In all cases the enzymatic activity causes the sample an increase in the absorption at 420 nm or Fluorescence at 460 nm.

The substrate for the enzyme lipase of the following structure V, which is derived from 1-hydroxypyrene-3,6,8-trisulfonate, designated VI, can serve as an example of a synthetic enzyme substrate which can be immobilized electrostatically. The immobilization is based on the interaction between the negatively charged sulfonato groups of the substrate and the positively charged surface ammonium groups of the anion exchanger, which is used as the carrier material.

In a further embodiment of the invention also be provided that the enzyme substrate on a thin Carrier film immobilized, which is on the end of the light guide is spanned. The following is in typically the manufacture of a membrane with a described electrostatically immobilized enzyme substrate:

An anion exchange membrane is immersed in a solution 1 g of 1-hydroxypyrene-3,6,8-trisulfonate VI in 50 ml Methanol. After about 2 minutes you pull the yellow colored and green fluorescent membrane and washes it first with approx. 0.06 molar phosphate buffer of pH 7, then with water. After drying, it is placed in a solution of 100 mg Lauric anhydride placed in dioxane, which makes the surface bound VI changes to V. The solution of lauric anhydride was prepared by dissolving 0.1 g lauric acid in 1 ml of dioxane, addition of 0.1 g of N, N'-dicyclohexylcarbidiimide and filtering off the resulting precipitate. After two hours the membrane can be removed from the solution and washed with dry acetone. She now points a blue-violet fluorescence, its yellow color is  disappeared.

This membrane is stretched at the end of an optical fiber and in contact with a solution of the enzyme lipase the substrate is turned into a yellow, transferred to a strongly green fluorescent hydrolysis product. The increase in the absorption at 460 nm or the green fluorescence at 520-535 nm is followed with the help of the light guide. It is a direct measure of the prevailing sample Enzyme activity. This substrate is cleaved but not only through the enzyme lipase, but also by serum albumin. Because the activity of the latter is often considerably higher, the sensor can also be used for determination human serum albumin, e.g. B. in the blood will.

The last method described is also suitable for the production of other immobilized enzyme substrates for Hydrolases. It is not just an alternative method to Understanding the production of enzyme-sensitive layers but can also be used for regeneration Serve sensors.

The cleavages caused by different hydrolases of the immobilized enzyme substrates take place on a solid surface, namely the substrate for the substrate. Such cleavages occur with chemically immobilized Substrates often much slower than in solutions, where the substrates attack the enzymes from everyone Sides are exposed. The slower the hydrolysis gets, the stronger the disturbing influences are the non-enzymatic, which always runs to a small extent Noticeable hydrolysis of the substrates.

It is now found that the hydrolysis rate immobilized synthetic substrates by enzymes can accelerate significantly if according to the invention between the surface of the light guide or that of the carrier film and the enzyme substrate long chain spacer groups are located.

Z serve as spacer groups. B. long chain diamines,  like the hexamethylenediamine. A spacer group can can also be introduced directly with the silylation reagent, with the help of so-called "long chain amines".

For disturbing influences of strongly colored sample materials, e.g. B. blood to prevent the optical system can the end of the light guide with a protective cover are covered, which prevents the penetration of colored large particles, e.g. B. erythrocytes. Enzymes can however, penetrate this protective cover. An ideal material for protein-permeable protective covers is cellulose.

Instead of the enzyme substrates directly at the end of an optical fiber According to the invention, it is also possible to immobilize that the end of the light pipe from a reaction space forming enzyme-permeable membrane which contains the enzyme substrate and for cellular Components of the sample are impermeable. Such a reaction space must be accessible for the enzyme to be determined but at the same time the enzyme substrate can be made from it cannot diffuse out.

The enzyme substrate can be diffused out according to the invention thereby preventing the enzyme substrate bound to a water-soluble or water-swellable polymer is present, e.g. B. by chemical immobilization. As water-soluble or swellable polymers which are used as carriers serve for the enzyme substrates located in the reaction space can come z. B. in question: dextran, agarose, polyethyleneimine, Polyacrylamide or polyalcohols. Crucial in this case the choice of polymeric carrier is The fact that the molecules of the carrier polymer are large enough to avoid leakage through the protective cover. The immobilized substrate becomes by enzymatic hydrolysis split and thereby undergoes a change in its spectral properties. This change will be made via the Optical fiber tracks and serves as a measure of enzyme activity. Because the released dye to a certain extent diffuse through the cellulose membrane, it is imperative required that the enzymatic reaction be essential  takes place faster than the diffusion from the reaction bubble.

In the last mentioned arrangement only such can Enzyme substrates are used, their spectral Properties become clear through the enzymatic reaction to change. This is because the light guide both the enzyme substrate and its cleavage products are optically detected. Therefore, in a further development of the invention suggested that the enzyme substrate on the inner surface one at the end of the light guide, preferably cylindrical reaction space is immobilized, the reaction space on its side facing the sample Side has an enzyme permeable membrane, which for cellular components of the sample are impermeable and that the reaction products formed by enzymatic reaction diffuse into the reaction space. Thereby, that now the substrate of its fission products spatially is present separately, you can specifically the formation of Track fission products.

The optical separation is made possible in a simple manner in that a direct excitation of the enzyme substrate can be avoided by the given exit angle α of the excitation light in connection with the coordinated diameter and the length of the cylindrical reaction space. The cylinder wall together with the superficially applied substrate is now outside the numerical aperture of the light guide given by the exit angle α , so that the substrate is not covered by the light guide as long as no enzymatic reaction takes place.

Now the enzyme to be determined penetrates into the reaction space an enzymatic reaction takes place under Release of a dye instead. The dye spreads itself in the reaction space and its color or fluorescence is further captured by the light. The increase in color or fluorescence intensity is used as a measure of the catalytic Enzyme activity used.

The latter arrangement has some basic ones  Advantages on.

• a) Enzyme substrate and cleavage product can because of the spatial Spectral separation no longer overlap, so that high blank values can be avoided.
• b) Not only synthetic enzyme substrates can be used but also natural substrates, which are marked with dye.

The following examples illustrate the wide range of applications the arrangement according to the invention, being almost always the immobilization by chemical linkage of polymeric carrier, spacer group, and the actual dyes he follows:

• 1.) The immobilization of 7-hydroxycoumarin-3-carboxylic acid via a spacer group (adipic acid) on a cellulose membrane or on the surface of another OH group-bearing polymer leads to a surface of the following structure: The enzymatic cleavage of the CO-O bond by an enzyme from a group of carboxylesterases leads to the release of the dye, which is then diffused into the field of view of the light guide and determined.
• 2.) The immobilization of 3-cyano-7-hydroxycoumarin on OH-containing polymers via a CH ₂ -CH ₂ -CH ₂ -CH ₂ spacer group leads to a structural element of the following chemical formula: The resulting immobilized dye is a synthetic substrate for dealkylases. These cleave the ether groups and release the fluorescent dye 7-hydroxycoumarin-3-carboxylic acid, which can be detected again by the light guide.
• 3.) Below is the structural element of one of a glass surface immobilized protease substrate:

A can also be on the surface of the reaction space immobilized with a natural substrate labeled with a dye be, e.g. B. a protein. For this, e.g. B. Chicken egg white Lysozyme immobilized on the surface of the wall and then fluorescently labeled with fluorescein isothiocyanate. This substrate is cleaved in contact with the enzyme trypsin. Fluorescein and fluorescein-labeled fragments of the lysozyme diffuse optically into that of the light guide recorded space and can be measured fluorimetrically there will.

Immobilized proteins are easy to produce, can also be purchased. Saccaride too, So sugar-like compounds are in immobilized form commercially available, e.g. B. immobilized N-acetylglucosamine, Lactose, melibiosis, N-acetylgalactosamine, fucose, Mannose, cellobiose, galactose and glucose. These materials can be attached to the wall of the reaction space and be marked with a dye. As such dyes are suitable fluorescein isothiocyanate, dansyl chloride, Fluorescamin, naphthyl isocyanate and europium chelates. By enzymatic reaction the labeled molecules are released, whereupon they diffuse into the reaction space and can be detected by the light guide.  

The material that surrounds the reaction space can be the substrate itself. It can be z. B. the amylose, which belongs to the polyglucosides, which fluorescence marks on the inner surface must become. Polypeptides or fatty acid esters also come as materials that are both substrates and wall material are in question. Of course, this can only be about trade water-insoluble materials.

The benefit of using stained results in natural enzyme substrates, is that one in this way can take advantage of the high specificity which Have enzymes for natural substrates. Combined with fluorimetry has the goal of being both sensitive as well as specific method of determination.

Many enzymes do not only appear in one form, but also form further subgroups. A typical example of this are the phosphatases. Among these there are those which have an activity maximum in alkaline solution, So-called alkaline phosphatases, and those which in acidic solution have an activity maximum, namely acidic Phosphatases. Determining the clinically important Acid phosphatase can be used in e.g. B. pH 7.4 no longer take place without simultaneously the activity of the alkaline phosphatases to be included at least in part.

To get around this problem, it will be necessary to create a Arrangement with two enzyme-sensitive fiber-optic catheters to use. These must differ in that the two enzyme substrates are different at each end Possess affinity for the enzymes and also in distinguish the maximum implementation speed. The Affinity of an enzyme for the substrate is determined by the Michaelis-Menten constant indicated the maximum speed by a size representing the maximum implementation speed indicates.

The invention is explained in more detail below with reference to the exemplary embodiments shown in FIGS. 1 to 5. In the drawings Fig. 1 is a schematic representation of an arrangement according to the invention and Figs. 2 to 5 respectively different embodiments of a detail of Fig. 1.

In FIG. 1, the light falling from the light source 1 is collected with a collimator lens 2 and, after passing through a filter 3, is coupled through a focusing lens 4 into the entry-side end 5 of a light guide 6 . The wavelength of the light is adjusted with the help of the filter 3 to the absorption maximum of the dye released by the enzymatic hydrolysis. Part of the light is directed by a beam splitter 7 onto a reference photodetector 8 . This reference detector is used to eliminate fluctuations in intensity of the light source 1 .

The light is guided via the light guide 6 to the synthetic enzyme substrate 9 , for example, which is located in immobilized form at the exit end 10 of the light guide 6 . If the enzyme substrate 9 is now cleaved by an appropriate enzyme, the incident light is more and more absorbed due to the formation of colored reaction products 9 ' and thus weakened. If the corresponding dye is capable of fluorescence, on the other hand an ever increasing fluorescence intensity will be observed.

Both reflected light and fluorescent light are returned through the same light guide 6 and directed through the semi-mirrored surface 11 of the beam splitter 7 with the aid of the lenses 12, 13 onto a photodetector 14 . If the fluorescence is measured, a second optical filter 15 is connected between the beam splitter 7 or lens 12 and the detector 14 , which is opaque to reflected excitation light, so that only the fluorescent light can be detected very selectively. The signal S measured at the photodetector 14 or the reference signal R measured at the reference photodetector 8 is amplified and fed to a display or evaluation device, not shown here.

Fig. 2 shows magnified the end 10 of the light guide 6, which has a core 16 and a cladding 17 having different refractive indices. The substrate 9 is on a thin carrier film 18 , for. B. immobilized from cellulose, ion exchange membrane or polyacrylamide, which is then clamped onto the outlet end 10 of the light guide 6 .

It is also possible, as shown in FIG. 3, to apply the enzyme substrate 9 to the outer surface 19 of the cylindrical core 16 of a light guide 6 , the jacket 17 being removed in the end region of the light guide. The light guide 6 can carry a light-reflecting cap 21 on its circular exit surface 20 .

Fig. 4 shows again schematically the end 10 of a light guide 6 , on which there is a bubble-shaped reaction space 22 which is separated by a protein-permeable membrane 23 , for. B. from cellulose is limited. In the reaction space 22 there is the enzyme substrate 9 , which, however, since it is made up of relatively small molecules, would diffuse out through the wall of the membrane 23 . To prevent this, the enzyme substrate 9 is immobilized on a water-soluble polymer with larger molecules. Water-soluble or swellable polymers are particularly suitable as carriers for the enzyme substrates present in the reaction chamber 22 .

With the arrangement shown in Fig. 5 it is possible to spatially separate the reaction products 9 ' from the originally present enzyme substrate 9 . At the end 10 of a light guide 6 there is a cylindrical reaction chamber 24 , the side facing the sample with an enzyme-permeable membrane 23 , for. B. made of cellulose or porous polycarbonate. Is located on the inner wall 25 of the reaction chamber 24 forming cylinder 26, the colored or fluorescent enzyme substrate. 9 In contrast to the arrangements described above, it is not distributed homogeneously in the reaction space, but is only chemically, electrostatically or physically immobilized on the inner wall 25 of the cylinder 26 .

As soon as the enzyme to be measured enters the reaction space 24 along the pillar 29 through the membrane 23 , the enzymatic reaction takes place with the release of the reaction products 9 ' . The reaction products 9 ' can move freely in the reaction space 24 and also diffuse in that area which is defined by the exit angle α of the excitation light. Care should be taken to ensure that the diameter 27 and the length 28 of the cylinder 26 delimiting the reaction space are selected so that the emerging excitation light does not strike the wall of the cylinder or the enzyme substrate 9 immobilized thereon.

Claims (12)

1. A method for the optical determination of the catalytic enzyme activity of a sample, characterized in that an enzyme substrate is assigned to the end of an optical fiber and brought into contact with the sample to be measured and that the change in spectral properties of the enzyme substrate or its reaction products caused by the enzymatic reaction per Unit of time recorded and used to determine the enzyme activity. 2. The method according to claim 1, characterized in that that the change due to enzymatic reaction the absorption of the preferably synthetic enzyme substrate detected via the light guide and for determination the enzyme activity is used. 3. The method according to claim 1, characterized in that the spectral caused by enzymatic reaction Change in the fluorescence radiation of the preferably synthetic enzyme substrate, for example the Change in fluorescence intensity or shift of the fluorescence maximum, via the light guide recorded and used to determine the enzyme activity becomes. 4. The method according to any one of claims 1 to 3, characterized characterized in that in addition to the enzyme activity in the pH of the sample is measured in a manner known per se and that this measured value for correcting the  predetermined enzyme activity is used. 5. Arrangement for performing the method according to one of claims 1 to 4, wherein the sample to be measured is in contact with an enzyme substrate, characterized in that the enzyme substrate ( 9 ) is associated with the end ( 10 ) of a light guide ( 6 ), wherein A photodetector ( 14 ) with subsequent signal evaluation is provided, which measures the fluorescent light or reflected excitation light emitted by the enzyme substrate ( 9 ) or its reaction products ( 9 ' ) ( Fig. 1). 6. Arrangement according to claim 5, characterized in that the enzyme substrate ( 9 ) at the end ( 10 ) of the light guide ( 6 ) is chemically or physically immobilized. 7. Arrangement according to claim 5, characterized in that the enzyme substrate ( 9 ) is immobilized on a thin carrier film ( 18 ) which is stretched onto the end ( 10 ) of the light guide ( 6 ) ( Fig. 2). 8. Arrangement according to claim 6 or 7, characterized in that there are long-chain spacer groups between the surface of the light guide ( 6 ) or that of the carrier film ( 18 ) and the enzyme substrate ( 9 ). 9. Arrangement according to claim 5, characterized in that the end ( 10 ) of the light guide ( 6 ) is enclosed by a reaction space ( 22 ) forming enzyme-permeable membrane ( 23 ) which contains the enzyme substrate ( 9 ) and for cellular components of the sample is impermeable ( Fig. 4). 10. The arrangement according to claim 9, characterized in that the enzyme substrate ( 9 ) is present bound to a water-soluble or water-swellable polymer. 11. The arrangement according to claim 5, characterized in that the enzyme substrate ( 9 ) is immobilized on the inner surface ( 25 ) of a preferably cylindrical reaction space ( 24 ) arranged at the end ( 10 ) of the light guide ( 6 ), the reaction space ( 24 ) on its side facing the sample has an enzyme-permeable membrane ( 23 ) which is impermeable to cellular components of the sample and that the reaction products ( 9 ' ) formed by the enzymatic reaction diffuse into the reaction space ( 24 ) ( Fig. 5). 12. The arrangement according to claim 11, characterized in that a direct excitation of the enzyme substrate ( 9 ) can be avoided by the given exit angle α of the excitation light in connection with the coordinated diameter ( 27 ) and the length ( 28 ) of the cylindrical reaction space ( 24 ) ( Fig. 5).