DE2536886A1 - METHOD AND DEVICE FOR ANALYTICAL DETERMINATION OF SOLUBLE COMPONENTS IN FIBER MATERIALS AS CARRIER MEDIA - Google Patents

Description

Damon Corporation, Needham Heights, Massachusetts, V.St.A.

Method and device for the analytical determination of soluble components in fiber materials as

Carrier media

The invention relates to a method and a device for the analytical determination of soluble components of materials such as biological fluids
Main patent ... (patent application P 2j5l8o44.1), in particular an analytical method in which the sample material to be analyzed is subjected to a chemical reaction in an optically thin layer or another form of distribution of the reactants, with which the to
determining constituents can be determined analytically, the reactants being in a fibrous carrier medium such as paper. The layer is exposed to electromagnetic radiation which reaches a detection device, the signal of which in a known manner

646) SPF

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and preferably linearly with the concentration of the chosen analytical determination of the constituent serving reaction product depends.

The invention enables the reproducible implementation such measurements with high accuracy even if the apparent background absorption or -emission of the fibrous medium (hereinafter referred to as the fibrous medium for short), which is used as the carrier for the Reaction mixture is used, is relatively high and varies significantly from one test site to another, as well as when the local distribution of the material to be analyzed and the reagents used for the reaction are not uniform and are different from each other so that the reaction product is not evenly distributed.

The method provides repeatable, reproducible measurement results provided that the relative distribution in successive samples is always the same is. The method according to the invention and the device according to the invention also allow In contrast to methods and devices according to the prior art, the analytical determination of components with high sensitivity.

The invention is based on the object of an improved method and a device for determining Specify constituents of biological fluids in a fiber medium, with which it is particularly possible to have a trace chemical reaction and / or a reaction product determine which is contained in a Fais medium, the dependence of the result on the uniformity of the distribution of the reactants to only a minor extent

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is dependent. One or more soluble constituents of a fluid material that is present in a fiber medium is contained, can be determined with greater sensitivity than before; as an analysis vessel should be a Fiber layer material are used (fiber cuvette), which has no material-delimiting structure such as a disc of a defined size or locking rings on the layer material. The fiber material should be have one or more non-limited test sites, with an accurate and clean determination of the ingredients should be possible. The procedure also seeks to implement the determinations with unlimited test sites as well as with minimal loss of accuracy due to changes in the volumes of the test areas enable applied reactants. The method should also only use a small sample volume, often below 1 / ul require, be quick to implement and require the least possible effort. The method as well as the invention Device should finally both measurements of speeds (kinetic measurements) and Allow endpoint determinations.

The invention provides a method and an apparatus in which the reagents and the solution of the sample are chemically interconnected in an optically thin layer Formation of a reaction product react which is known as the product to determine the corresponding constituent the sample solution is used. When the layer is irradiated with electromagnetic radiation, the chosen wavelength is determined provides a radiation detector or other energy sensitive device of known and preferably linear type Response behavior is a known or linear measure of the Concentration of the component under consideration. That

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The reaction mixture is adjusted so that the concentration of the reaction product at the closest point is so low that only a small part of the through radiation passing through the fiber medium is absorbed.

The reagents and the sample solution thus become three-dimensional or locally distributed that the amounts of the various reactants ideally for an optimal chemical Interaction to generate the reaction product of interest over the entire test area with one another are balanced. Preferred chemical systems are kinetic systems; it is accordingly desirable, optimal Maintain balanced conditions across the field of view of the device. The fiber medium contains the Reactants for incubation as well as 1 hour tube stirring of the measurements. With such an arrangement, the balanced Distribution of the reactants often involves adding the reagents to the reaction site on the fiber medium in a specially designed manner chosen order with not necessarily even or identical distribution that with regard to the chromatographic migration of reactants must be carried out with the addition of subsequent solutions.

With numerous types of implementation of the invention Procedure, it is also ■ not necessary that Reaction sites through delimiting structures such as Limit Yagoda Ranks. Instead, the reaction parts to be without boundary. The device according to the invention is used for the preferred implementation of the device according to the invention Procedure. The device generally comprises a self-adjusting and reproducibly operating optical system Arrangement of radiation source, the reactants

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holding fiber medium and radiation detector. This arrangement remains over the course of the chemical reaction away the same. The device therefore carries the fiber material in an essentially fixed manner Geometry in a fixed position. The device also delays the evaporation of liquids the layered fiber medium at least during the measuring time and prevents undesired compression or compressing the site containing the reactants.

For the practical implementation of the method according to the invention, a device can be used which is a press for progressively compressing an absorbent, liquid-beverage article. These Press can be used, for example, to produce a protein-free ultrafiltrate of a sample solution such as blood serum or whole blood can be used for analysis by the method according to the invention. A fluorometer assembly can be used advantageously in the practical implementation of the method according to the invention.

There is an advantage in carrying out chemical component analyzes according to the method according to the invention in that only a short time is required, especially compared to previous methods. Carrying out the invention also requires a minimum of equipment. For example, the lactate analysis of whole blood under Use of a fiber medium (fiber cuvette) that has already been used with the analytical reagents (described below) is treated, take place in such a way that the corresponding serum ultrafiltrate of the blood sample with the said ultrafiltrate

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transfer press to the fiber material and the entire lactate analysis from finger puncture to the final analytical result is completed within three to five minutes.

The method according to the invention enables accordingly a quick, simple and low-cost chemical analysis or "spot tests", d. H. the Determination of the product amount of a chemical reaction that takes place in liquids contained in a fiber medium. The determination of the reaction product can be carried out both at the end of the reaction, i. H. as end point determination, or during the reaction, d. H. as a kinetic measurement. The invention is primarily there, however not exclusively, on measurement and determination methods based on the measurement of an electromagnetic Based on radiation, d. H. to photometric methods based on absorption or fluorescence.

The photometric measurement of chemical reactions of materials in a fiber medium such as paper or a similar fibrous sheet material has long been used because of the ease of handling such Media and its low cost intensively pursued, especially as a medium suitable for analytical purposes. In connection with the said press, the method according to the invention allows samples to be collected on filter paper and subsequent analysis without the need for centrifugation as is the case with conventional methods.

However, there are some problems that make it difficult to make such measurements with the required

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To carry out accuracy and reproducibility. One problem is that the paper or a similar Medium delivers a high photometric background signal that differs from one reaction site to another and changes significantly even within a reaction site. For example, it was particularly based on investigations in the field of paper chromatography marked changes in optical transmittance, optical reflection, and native fluorescence, occurring from one point to another on sheets of analysis paper. This location-dependent underground signal sets the accuracy of measurements of a reaction signal of interest in chemical S pot-Test-A na Iy se decisively down. In addition were Attempts to solve this problem in paper chromatography do not rely on the slightly different ratios Applicable in chemical spot test analysis.

In spite of these difficulties, US Patents 3,036,893, 3,502,438, corresponding to the prior art and 3,526,480 indicate the unknown concentration of a chemical constituent in a sample Compare the optical reading on a reagent-containing site on the fiber sheet with that of interest .Probe- was added, with a corresponding optical measured value of the same, known with calibration solutions Concentration offset point can be determined. To achieve reproducible and more accurate quantitative Results in this way, however, require that the measured difference in optical properties at different points caused by the chemical reaction depending on the concentration of the to be determined Component is caused is large compared to the difference in the optical background that is at these points

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Was already present in the fiber material.

With the mentioned, the state of the art Method Accordingly, an uneven distribution of the reactants in the test area is the main source of error. According to this prior art, a large number of methods have been specified in order to achieve a uniform Distribution within the measuring field of the device to achieve a higher accuracy. So is for example stated in US Pat. No. 3,06,893 that one to be analyzed Solution to produce an even distribution over a reagent strip through a porous sieve is applied through. In said US-PS as well as US-PS 3,526,480 the advantages of a highlighted even distribution of reagents. However, these procedures are also subject to changes in the carrier medium self dependent.

According to the state of the art, this is the so-called ring formation phenomenon known as the major limitation on the accuracy of quantitative determinations on fiber materials. This Phenomenon occurs in the prior art method due to different concentrations of the reaction product at different points of the optical measuring field that do not contribute proportionally to the total measured variable, namely in processes that work in transmission, reflection or fluorescence.

The invention is based on the assumption that said ring pattern ungs phenomenon does not interfere with such measurements if the ring itself emitted the incident or re-emitted Light is only slightly absorbed. The method according to the invention works accordingly in such a way that all absorbent molecules, which form the ring, as do the molecules within the

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of the ring are exposed to essentially the same incident light intensity and that of the neighboring molecules does not weaken emitted light. The reduction of the concentration in the ring to such a low level however leads to a chemical change in the optical measured value which is small compared to the difference in the Underground from one place to another. At such low absorptions, methods work according to the state of the art the technology is not satisfactory.

The method according to the invention avoids the two difficulties mentioned by measuring the chemically caused ones Change of the optical properties of each point in relation to the subsurface at the same point the reaction causing the change. The resulting relative measured variable of the corresponding test areas is then compared with the corresponding calibration areas. The method according to the invention thus uses any Reaction area as a separate, associated reference blank sample The resulting measured variable is essentially the same as the original optical conditions at the relevant point of the fiber medium and speaks accordingly only to the maximum effect that is actually of interest. The independence from the described disadvantageous The effects of ring formation associated with this process allow the concentration of reaction products to be determined, which result from the direct application of a sample solution to a reagent-provided fiber material, quite in contrast to the state of the art mentioned.

In detail, the method according to the invention is based on the finding that an accurate determination of a Product of a reaction that takes place with non-uniformly distributed reactants in a fiber medium is then possible is when the optical absorption of the product is low,

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especially well below the minimum value of 0.2. that of conventional in vitro absorption spectrometry is usually regarded as indispensable for reliable measurements (see, for example, Spectro-Chemical Methods of Analysis, ed. by J.D. Winefordner, Wiley-Interscience, page 344). It also stands the measured variable with the corresponding concentration of the reaction products in an essentially linear relationship.

The mentioned condition of a low optical absorption is equated in the following with the condition

it t '

that the system is optically thin. When the reactants, out which the reaction product of interest is formed, are present in an amount such that a correspondingly low concentration occurs, the sensitivity of the Measurement in relation to non-uniform distribution over the optical measuring field and in particular in relation to ring formation negligibly small.

In addition, the invention is based on the further finding that the of a reaction product in an optical measurement result produced by a fiber medium significantly depends on the degree of moisture of the medium and the degree of compression depends on the reaction site. The moisture problem occurs in the method according to the invention only when kinetic measurements are carried out, which require the presence of a liquid phase to ensure molecular mobility. This Problem is according to US-PS 3,0 ^ 6,893 for example by Drying the reaction surface after the reaction and before the optical measurement avoided.

The invention thus provides a method as well as a Device for determining the concentration of certain components of a fluid system by a controlled one

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chemical reaction that has a small change to the optical Properties of the subsoil of a very variable fiber medium. The respective measurement requires no uniform distribution of the reaction product over the measuring field of the device, but only a distribution that is reproducible for different measurements is.

The method according to the invention and the method according to the invention Devices use a memory measurement of the high or apparently optically thick background of the fiber medium at the reaction site as a reference for the subsequent one at the same site Measurement of the 'optically thin' change, that of the actual Measured variable corresponds. According to a preferred development of the invention, the moisture is also the compression of the fiber material at the reaction site controlled. With this method, reliable measurements are possible even if the apparent absorption of the substrate medium ranges from 1.0 when completely wet to 1.7 when dry.

The invention also provides preferred methods for producing reproducible distributions of the constituents within the reagent disk or other fibrous media, as well as the creation, combinations or equalization of concentrations Components resulting in a continuously high reaction rate, at least as much as desired Measurement interval leads. The consistently high reaction speed leads to a correspondingly high measurement sensitivity. The preferred modes of implementation of the method according to the invention described below permit

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furthermore, the implementation of reactions with a relatively uniform reaction rate over a relatively long time Times, which enables kinetic measurements with high sensitivity.

The reproducible distribution is achieved in that the successive application is more liquid Reagents are applied to the fiber medium in a selected order in order to wash out a reagent through the to control and compensate for subsequently applied further reagents. Alternatively, the Concentrations of different reagents in a single solution adjusted so that differences in their spread or affinity for the fiber medium can be compensated for when the solution is applied to the reaction site and the process is carried out further. The quantities agree reagents v / are also selected so that there is a relatively optimal balance of all reacting ingredients over the Test area results. To this end, it was found that the necessary to achieve a balance relative volumes are often different from those normally used be used in liquid mixtures to achieve optimal reaction conditions, d. H. in liquids, which are in a vessel like a test tube in contrast to a fiber medium, the chromatographic one Separation is subject to how it is used according to the invention.

Be used in a preferred embodiment of the invention in which not _v .begrenzte test sites whose area is larger than the corresponding measurement field, the capillary properties of the pretreated fiber medium create a buffer zone, the excess reagent or sample solution were brought to the reaction site, subtracting or sucks. Under certain conditions that is

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The resulting measurement result is primarily proportional to the concentration of the unknown substance in the solution and only relatively slightly dependent on the exact volume of the sample or the added reagent solutions.

The invention accordingly comprises both the individual process steps in their mutual dependence as well as the device according to the invention in all constructive Details and arrangements of each item necessary to carry out the appropriate steps to serve.

The invention is illustrated below by means of examples as well as the drawing explained in more detail; show it:

1: a graphic representation of the usual dependence of the optical transmittance of a Liquid from optical absorption;

2: a simplified representation of the implementation of the method according to the invention or of the Operation of the device according to the invention;

Fig. 3: various graphic representations of the Relationship between the concentration of ilaterials and the corresponding optical permeability, each depending on the local or spatial distribution;

Fig. ¹ I: a perspective view, partly in front elevation view of an apparatus for carrying out the Invention erfindungsgemäl3en ~ err.äJen method;

FIG. 5: a partial view of the device from FIG Compression division;

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-U-

Fig. Β: a shown partially open and for Partial perspective view of a fourth device according to the invention;

7: a cross-sectional representation of a further structural design of the analysis device j

Fig. 8: Schematic circuit diagrams of "" according to the invention u.8a: measuring devices as well

9 and 10: graphic representations for carrying out the method according to the invention.

That of conventional methods for chemical spot test analysis The range of between about 0.2 and 0.8 used is indicated in FIG. 1 as section 12 on curve 10. The curve itself corresponds to a graphic representation of the Lambert-Beer's law, where the percentage optical permeability of a liquid (transmission) as a function of the concentration of the absorbent Materials depicted therein 1st '. The optical absorption of the liquid is proportional to this concentration and represents in detail the product of the total number of molecules along the optical measuring path and the molar absorption coefficient of the molecules. Fig.l thus represents the relationship between the percent optical transmittance and the concentration of a liquid. The transmission in curve section 12 is less linear on the absorption urü thus the concentration than at the upper end of the curve, i.e. i.e., with absorptions below 0.2.

In contrast to all of the methods specified so far, the method according to the invention works through in the range of one the reaction of interest caused change the optical absorption below the curve section and in particular in the lower absorption range 14.

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This section corresponds to differential absorption values of 0.2 and less, preferably below 0.15 This absorption range and the correspondingly high percentage transmission of the beactant mixture of the fiber matrix define an optically thin preparation or distribution of the material components. Larger optical Densities corresponding to section 12 and sections to the right of it on curve 10 are referred to here as' optical thick "considered.

In Fig. 2 is the operation of the invention Method shown with an absorption photometer 20. A sheet 16 made of a material with continuous A fibrous structure such as paper contains a liquid solution 18 of the chemical reactants. The reactants are selected so that a reaction product is formed from them, which is used to determine a selected constituent the original reactants can be used. An illumination unit 20a of the absorption photometer 20 illuminates the solution on sheet 16 with intensity I. An optical detector as well as a Output unit 20b of the photometer, which are located in the optical axis of unit 20a and sheet 16, register the resulting light intensity I transmitted through the sheet containing the solution. The measuring field 22 of the Unit 20b on sheet 16 lies within the spreading zone of solution 18 on sheet, such as can be seen in the figure. The wavelength of the incident radiation with the intensity I and the wavelength of the resulting radiation of intensity I to which the unit 20b is responsive are selected so that in the unit 20b a measurement signal is generated, which primarily

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if not exclusively responsive to the concentration of the selected reaction product of solution 18.

As shown in broken lines in FIG. 2, the photometer can also be arranged in such a way that the light intensity reflected by the solution 18 is measured. This means that the illumination unit 20a ¹ and the output unit 20b 'can be optically arranged in such a way that they are at the same angle of incidence or reflection to the sample; however, other angles can also be set depending on the light scattering of the sample.

Such as 2 further apparent from Fig., Can by means of the photometer 20 simultaneously using a single light source with an arrangement of the illumination unit according 20a ', of the measuring unit of the reflection 20b ^f, and the measuring unit for transmission 20c' respectively aligned optically for Light source 20a 'are located, are measured in reflection and in transmission. With the same arrangement, fluorescence measurements can also be carried out ^{with the unit 20b 1} and absorption measurements with the unit 20c ^f. The method of operation of the method according to the invention and of the device according to the invention shown in FIG. 2 corresponds to generally customary measuring methods. First, however, the term "optical thickness" of a solution contained in a fiber medium will be explained in more detail with reference to FIG.

The fiber sheet 16 itself lets through only a small part of the incident radiation, which is for illustration purposes for example a wavelength of 3 ^ 0 nm having. The transmission is low, primarily due to the scattering of the paper fibers, but also /, albeit in little, owing to their absorption. Anged. sees this circumstance the expression "weakening" is ~ chooses about the influence of the sheet material on the passing through Describing light and distinguishing it from normal absorption. The term attenuation refers to it

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So it refers to the sum of absorption by the material plus the loss of intensity of the light beam due to scattering.

The attenuation as well as the reflection and emitted fluorescence of the sheet 16 against the incident radiation proved to be highly dependent on the moisture content and the compression of the leaf. This becomes understandable if one looks at the influence of a liquid and the influence of pressure on the fibers and their spaces illustrated. Paper scatters and reflects light most when it is dry because of the spaces in between Fibers are filled with air, resulting in a refractive index that is significantly different from that of paper fibers differs. Water, on the other hand, has a refractive index closer to that of the fibers, so that, when water is applied to the leaf, lower scattering, lower reflectance and higher apparent Transmission (i.e., reduced "attenuation") and reduced fluorescence emitted occur. This phenomenon lies based on the use of translucent agents in paper chromatography, with liquids used for To match the refractive index of the paper fibers, a dried chromatogram can be added to it for subsequent use To make densitometry as transparent as possible.

According to this theoretical model, it can be predicted that a beam of light falling on the sheet 16 is scattered several times in the sheet before it is finally absorbed or as reflected or continuous light emerges again. It is accordingly to be expected that the effective path length of the light through the fiber sheet is larger than the actual thickness of the sheet itself.

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This increased effective path length became the method according to the invention in analytical investigations established. In detail, such an increase in the path length was found in a device which is described below with reference to Fig. 7, the fluorescence and transmission observed of paper discs that are wetted with known volumes of NADH solutions at various concentrations with the corresponding fluorescence and transmission values of the same volumes of the solutions in vitro, d. H. instead of the paper discs. The apparent increase in the effective path length was found between three and five in this type of implementation.

On this basis it can be understood that an absorbing solution, which is located on a fiber medium such as paper , the apparent "weakening" of the light by

Ability to increase absorption at any point along the increased length. If this absorption is a Reemission of fluorescent radiation follows, the fluorescent light must pass through in the same way as the excitation radiation the scattering fibers penetrate until it emerges from the leaf or is absorbed inside.

From IB-PS 255 ^ 321 it is also known that

Fluorescence measurements not linear · v / ground when the concentration the fluorescent substance becomes so high that the incident Light beam is greatly weakened when passing through the solution to be measured. The reason for such Non-linearity lies in the fact that the fluorescence causing absorption by the external liquid, which first interacts with the incident radiation occurs, the contribution of the internal fluid to the re-emitted Light is reduced by the fact that the effective excitation intensity in the internal liquid decreases.

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As used herein, the term "optical thickness" of a solution is based on the increased effective path length, such as described above.

The optical absorption of the in the solution 18 im Sheet 16 existing reaction product is according to the invention in the spectral region of interest and for the effective path length defined by the scatter within the leaf at the respective humidity to less than 0.2 and generally kept below 0.15. This means ob.fi the solution on the sheet 16 behaves optically thin in the spectral region of interest, at least within of the measuring field 32 of the photometer unit 20b.

FIG. 3 shows the mode of operation of the device shown schematically in FIG. 2 with different distributions of the measured material, ie the material components, to which the photometer responds. As mentioned above, it is necessary according to the prior art that these materials are evenly distributed over the entire measuring field of the photometer. The graphical representation of the spatial or local distribution of the measured material in FIG. 3 & shows such a uniform distribution. FIG. 3 shows the associated transmission, ie the ratio of the resulting light intensity I to the initial intensity I ₀ for this distribution of the measured material. As expected, the profile of the latter representation is identical to that of FIG. JA .

In Fig. 3C is an uneven spatial or local distribution of the measured material shown on the sheet 16, as it is ideally created when the materials are applied in the center of the measuring field and expand radially. The distribution shows a greater consistency

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centering in the periphery of the resulting "spot" than in its center, which leads to the typical "ring formation", d. H. for washing out liquid or dissolved Material from the center of a spot towards its outer zone.

If the concentration of the materials distributed in Fig. ^ C is significantly "optically thick", ie the absorption is well above 0.2 (see. Fig. 1), the optical transmittance of this distribution has a profile that corresponds to the curve 24 in Fig . 3D corresponds. This profile therefore does not represent a linear correspondence to the distribution profile in FIG. 3C. Rather, the profile is extremely uneven with less than the proportional permeability of the highly concentrated portions of the profile in FIG. ^ C. Because of these non-linearities, it is necessary in accordance with the prior art to apply the materials to be measured in a uniform spatial distribution, as in FIG. JA.

With a lower concentration of the measured materials in a distribution as in FIG. JG, which, however, is still above an "optically thin" absorption, the resulting optical transmission has a profile as in curve 26 of FIG. JD. This profile is not linear with respect to the concentration profile of Fig JC, but corresponding less than the "optically-thick" ratios curve 24. If the ratio of "mountain" to "valley" of the concentration curve in Fig J example 2:.. 1 is , the optically thin proportions should be appropriate. Curve 28 will have the same "mountain" to "valley" ratio, but curve 26 will have a lower ratio

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the ratio for curve 24 being even smaller, for example close to 1.

The device shown in Fig. 1 consequently provides no linear measure for the concentration of the reaction product, if the materials to which the corresponding measuring method responds, under optically thick conditions are unevenly distributed. The importance of a possibility for linear concentration determination is shown in Fig. ^ E showing a concentration distribution specific to spot test analysis in the absence Procedural measures of a conventional kind is typical and the case according to the invention concerns.

A linear measurement is possible, however, if the uneven distribution is optically thin. So if the local distribution profile of the reaction product in Fig. ^ C is optically thin, d. H. shows an absorption below 0.2 at the densest points, the corresponding shows Transmission profile almost linear behavior. The curve in Fig. 3D corresponds to this condition.

The device shown in Fig. 2 accordingly provides an essentially linear concentration measurement variable, if the materials that have the wavelength of interest absorb, exist under "optically thin" conditions or comparable conditions. This finding remains valid even in the presence of another, strongly weakening background, for example through the sheet of paper 16 itself or the original reactant solution is conditional, provided that the subsurface weakening is constant during the blank and signal measurement or varies in a known manner that is reproduced from sample to sample changes.

In addition, consistent accurate measurements can be made

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of different samples of the solution 18 can be carried out at different locations on the sheet 16 when the local distribution is the same for different samples, i.e. it is reproducible. In contrast, according to the state technology has always assumed that reproducible measurements can only be obtained if they are uniform Material distribution is present.

In Figs. 4 and 5, a fluorometer 30 is shown, that for carrying out the method according to the invention can be used and is more suitable for this purpose than the absorption device described above. The statements made in FIGS. 1 and 3 regarding the condition "Optically thin" and for reproducibility also apply to a device that responds to fluorescence. A fluorometric measurement also provides greater sensitivity than absorption measurements.

The fluorometer 30 shown in Fig. 4 is with a Press 32 connected that only the fraction of small molecules a liquid sample is applied to a fiber sheet, where the chemical reaction intended for the measurement with it Proportion is carried out. The compression of the fiber sheet by the press is usually done during the measurement canceled again. In particular, a dry, fibrous reagent sheet J54, which was previously used to hold the corresponding Reactant has been treated under a white dry "fibrous sample sheet 36 is provided, with sick in between an ultrafiltration membrane or a similar one another filter 38 is located. The liquid to be analyzed is applied to the test sheet, usually with a pipette. It becomes "when moving the press head 5I in arrow shape from the in'Fig. 4 * position shown

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in the in Pig. 5, a significant portion of the small molecule fraction of interest of the sample is pressed from sheet 36 through filter 38 onto reagent sheet 34, whereupon the desired chemical reaction of the liquid fraction with the reagents in sheet 34 begins. The -S ¹ IuOr ometer measures the reaction product of interest, either continuously, for example to carry out kinetic measurements, or after suitably preselected times. The lower part 42 of the press forms the housing of the fluorometer 30. The fluorometer illuminates the reagent sheet 34 of the radiation from a lamp 44 through the plate 46 of the press; the plate represents an optical window which is transparent to the incident excitation radiation of the lamp 44 and the fluorescence generated by this radiation in the reaction product of interest in the reagent sheet 34.

In particular, the lower part 42 has a first passage 48 and a second passage 50 on the passages are coplanar, v / whether their central axes are at an angle to each other form and coincide at sheet 34. The lamp 44 is mounted in the first passage 48 and is optical aligned through the optical window of platen 46 to the portion of the reagent sheet that is below the center of the press is located, d. H. which is located under an inner foot part of the press. A first filter 54 is also in the first passage is provided between the lamp 44 and the plate 46 to avoid unwanted radiation during illumination away from the reaction surface. A reference detector 56, which is typically an electrical one corresponding to the intensity and modulation of the illumination from lamp 44 Provides signal, is also located in the first passage, preferably outside the light path between the lamp and of the plate as shown in FIG.

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Although not essential, the second is Passage 50 of the pluorometer shown below Oriented angle at which the incident radiation from the lamp 44 is reflected by the reagent sheet 34. The second Passage 50 of the pluorometer 30 shown is therefore arranged at the angle at which the incident energy from the lamp from the surface of the blade 34 is reflected, which is adjacent to the optically transparent plate. Such a geometry is preferred because it leads to substantially equal optical path lengths from lamp 44 to detector 58 in second passage 50, the illumination of all points of the reagent sheet 34 detected, which are within the pluorometer measuring field. the The same optical path lengths in turn lead to high measurement accuracy.

The second passage 50 carries a fluorescence detector 58 and a lens 60 which is optically aligned between the detector 58 and the reagent sheet and which focuses the desired fluorescence radiation onto the detector 58. The second filter 62, 64 and 66 are also located in the second passage. These filters together keep reflected primary radiation from the lamp 44 as well as the background fluorescence and other radiation above and below the passage band of the fluorescence to be measured.

The second filters are further selected so that they - at least in the wavelength range dpr measurements - are non-fluorescent when they are excited with reflected radiation from the lamp 44 and any fluorescence. As filters 63, 64 and 66 with the desired level of non-fluorescence, dielectric and metal film filter assemblies are preferred. The non-fluorine-decorating η second filters provided according to the invention lead to a

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In contrast, increased sensitivity and improved separation of the basic signal of the fluorometer j50 to pluorometers of similar construction with glass or other secondary filters that are known to produce broadband fluorescence which includes the wavelength range of the measurements.

As an alternative to the joint arrangement of press and photometer in a single device as in FIG these parts can also be used independently of one another. For example, a liquid sample ready for analysis applied directly to a fibrous sheet of paper and reacted with the reagents to form a reaction product, which is measured with a fluorometer built as in FIG.

In Fig. Β is a preferred development 70 of the inventive fluorometer for carrying out the inventive Procedure shown in detail. Although the arrangement below is in terms of a fluorometer is described, the arrangement can also be used with other types of photometer. The optical components the device, d. H. Lamp, filter, signal detector and reference detector can in the arrangement described in FIG. 4 in the optical passages 70a and 70b to be ordered; The lamp and signal detector are only indicated schematically in FIG.

The carrier of the fiber medium as well as the exposure or optical connection of the medium with the lamp and the Signal detector provide further characteristic details of the invention. In detail, the device 70 carries a fiber medium shown as a paper strip 72 on a flat support surface 74a. The reaction site 72a on the strip that used to generate the reactant mixture

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of the selected reaction product is located between an optical window 78, which is set off with a spacing space 8o from the underside of the support surface, and an overlying counter plate 82 which extends laterally over the reaction surface and rests on the paper strip above the support surface. The strip 72 is wrapped or coated with an optically transparent film 66. The film is contiguous with the top and bottom surfaces of the strip and encloses the one bearing the reactants. Location 72a to avoid evaporation of liquid from this location. The film j6 also prevents contamination of parts of the device by reactants on the strip, thereby eliminating the need to clean the optical window 78, support surface 74a and backplate 22 between measurements at different reaction sites. The film 76 can consist of an optically transparent, thermoplastic resin which is produced, for example, by polymerizing vinylidene chloride, for example from "Saran Wrap" (trade name).

The housing 74, which forms the support surface 74a, is for opaque the radiation at which the device operates and the window 78 for the wavelengths of interest permeable. The window 78 is attached in an opening of the housing 74 and to the two optical Aligned passages 70a and 70b, which are for example at an angle of 45 ° to each other.

The counter plate 82 rests on the paper strip 72 above the housing 84; the center is above the window The illustrated counter plate 82 is hingedly mounted on the housing of the device in order to easily fold away

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to enable the carrier plate in such a way that that the counter plate, if access to the paper strip or to the support surface and the window is required, can be easily removed or replaced. The counter plate 82 is in the form of a disc with a flat Floor area shown. A holder 86 goes up from the rear upper side of the disc into a horizontal one Arm 88 over that on a vertical bracket 90 on the housing is hinged. The counterplate serves to flatten the strip 72 because of its light weight to hold against the support surface 74a, with a saran or similar film is pressed against the strip. In this way, the distance of the reaction site is relative to the Source and the signal detector fixed. The plate, along with the applied film 76, also prevents one Deformation of the strip at the reaction site due to the there liquid present. Although the strip can easily mold into spacer 80, one is such deformation due to the support effect of the applied film 76 and the shallow depth of the space only small, which is preferably less than the strip thickness. For a paper strip of for example 0.45mm (0.017 inch) thick, the spacer space 80 is approximately 0.25mm (0.01 inch). The distance space 80 serves, among other things in addition, a defor nation of the fiber material of the strip at the reaction point in this space and thereby a significant compression of the fiber material to prevent under the counter plate 82. However, it should be emphasized that the counter plate on the Stripe exerted weight is only small.

The counter-plate 82 is sufficiently large to be able to rest on the dry paper of the strip 72, which outside of the liquid-bearing and consequently

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wet spot 72a is located. The thickness of the dry paper that the plate 82 due to its limited Weight not compressed, so it automatically determines the distance between the lower surface of the plate and the support surface 7 4a. The plate 82 presses the surface accordingly of the wet spot 72a is flat and therefore coplanar with the top surface of the dry portion of the strip As already mentioned, the wet spot can be easily deformed into the spacer 80 as a result, what is desirable because in this way the fiber material remains relatively uncompressed at the reaction site.

The counter plate 82 is fixed in the b: very flat manner the reaction site relative to the support surface 74a and consequently relative to lamp and signal detector in a desirable manner. The counter plate also performs automatically and with high and reproducible accuracy and automatically Compensating for changing thickness at different locations on the strip 72 and even from one strip to another for such a positioning, since the dry part of the strip surrounding the reaction site the distance of the Counter plate determined over the support surface.

Another function of the counterplate 82 is based on the fact that it represents an optically uniform surface behind the reaction point 72a. This surface can be highly reflective or be highly absorbent, albeit a reflective one Surface is generally aimed at generating a higher fluorescence output signal. Of importance is that the surface is optically uniform over the measuring field of the device. Such a one optical uniformity also avoids air

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inclusions between the strip 72 and the counterplate.

The photometer device 70 of FIG. 6 also has elements for temperature control or regulation, to fogging of the photometer measuring field by the Reaction site 72a to avoid any moisture present. Fogging occurs if the temperature is not controlled the upper surface of the window 78 and the underside of the counter plate 82 in an otherwise identical device as in Fig. 6. The fogging occurs obviously then on when the underside of the counter plate 82 is at a different temperature than the support surface 74a or the window 78 so that liquid on the Reaction site 72a evaporates and condenses on the colder surface of the window or the counterplate. Such a thing Command & st is generally undesirable as it is a Represents a source of error and at the same time impairs the accuracy of the measurements. It was found that the Fogging can be avoided in that the counter plate 82 and the window 78 together with the adjacent Cash box of the housing 74 kept at the same temperature will. As can be seen from the drawing, a thermocouple is located in the counterplate 82 for this purpose 91 or another temperature sensor and heating elements 92. The temperature sensor 9I shown is located exactly above the window 78 and thus above the reaction point 72a. The heating elements 92 are electrically connected to a controllable heating source. The heating source can after conventional method for heating the counter-plate to a selected, specific temperature be built. the However, if necessary, the device can also have a Thermistor or another temperature sensor 93

that is in good thermal contact with the Window 78 is located and is typically mounted next to it in housing? 4. The feeler 93 is also included connected to the controllable source supplying the heating elements 92, thereby placing the counter-plate on the same Temperature as the window 78 is maintained.

The device 70 can also be thermally selectively comprised of a layer 84 of a thermal insulating material can be provided on the underside of the counterplate 82. This layer is particularly desirable if it is aimed at. that the reaction site 72a Is in thermal equilibrium with the window 78, if the counter plate is made of metal or another thermally conductive material and / or no external temperature control as provided by heater 92. The layer 84 can easily be given the desired property Establish optical uniformity as described above. In the case of the further training presented, there is the layer 84 is, for example, made of polytetrafluoroethylene (Teflon, trade name).

In Fig. 7 is a further embodiment of the invention a photometer for the analysis of reaction products on paper disks 94 is shown. The device has in detail a housing 96 'with an optical Window 89 is provided. The window sits in a chemically inert sealing ring 100 that extends over the housing surface 96a protrudes and supportingly receives a carrier sheet 102 having an opening into which the therein seated disc 94 is added. The opening in the carrier sheet fits so exactly to the size of the paper disk that one

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sufficient fit results that the disc can be guided with the carrier sheet. The depicted Carrier sheet 102 is preferably slightly thinner than the dry paper from which the disc is cut, for example for a dry disk of 0.43 * nm (0.017 inch) thickness 0.38 mm (0.015 inch) thick. That Carrier sheet consists of optically opaque material and can be made of metal for washers or spacers be made.

The apparatus of Figure 7 provides essentially one Vapor-tight space 106 between the pane 94 and the window 98. In detail, this is the upper surface of the window is preferably deposited below surface 96a, as described for window 78 in FIG. As already mentioned, the carrier sheet 102 sits on the upper edge of the seal 100, which extends over the surface 96a protrudes and creates a tight connection between them.

The disk 94 can be covered with a film such as that in FIG. described film 76 must be coated to avoid evaporation therefrom. Alternatively, it can also be used without Such an envelope can be used, the evaporation from it on the small enclosed space ΙΟβ between the carrier sheet and the disc 98 is restricted.

The photometer device shown in FIG. 7 has an optical window 104 which extends over the pane 94 and the carrier sheet 102 lies. The window 1O4 can be placed on the carrier sheet, attached to it or in one Spacing above it. With each of the specified fastening methods, an optical passage of the disk 94 carrying the reactants to the rest (not

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- 38 -

shown) optical device, which is located in the passages 108a and 108b of a further housing 108. The rest of the optical device can comprise, for example, a fluorescence or an absorption detector, if the transmission properties of the pane 9k are to be measured. The arrangement of FIG. 7 was used, for example, with a light source in passage 96b: a fluorescence detector in passage 96c and an absorption detector opposite the light source in passage 108a. The device carrying the pane shown in FIG. 7 can of course alternatively also be operated with a measuring device described in FIG. 6, the second window 104 being replaced by an opaque counterplate.

As mentioned above, the inventive method resolves point-to-point changes in the optical Properties of the fiber medium carrying the reactants in that each reaction site is its own Blank is used for reference purposes. This means that the same optical properties that are used for Measurement of the reaction product of interest can be used for the fiber medium on which the reactants are attached supporting point before the production of the reaction product be measured. This measurement determines the desired optical property of the fiber medium at the point at which the reaction product of interest will be generated, this site being wetted with any reactants that were used prior to the generation of the constituent being measured are present. During this measurement, the optical background parameter is determined accordingly,

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.33- - 253688S

which is present at the reaction site when the reaction of interest occurs. A comparison of this reading with the same meiwert that subsequently, i. H. while of the reaction of interest, provides a resultant signal that is essentially

only '' corresponds to the reaction product of interest. The resulting comparison signal is only slightly dependent on the optical background, in the presence of which the reaction product of interest is generated.

Referring to Figure 8, there is a preferred circuit for implementation specified blind measurement. A modulated power supply 110 powers the photometer source with alternating voltage for illuminating the sample 114, that is for example the constituent of interest at a reaction site on the fiber medium, and illuminates one Reference detector 116. The lamp is powered by the power supply 110 modulated at a frequency many times over 60 Hz as well as multiples or harmonics thereof away in order to exclude interference from the network and similar sources of this frequency. A signal detector 118 responds to the resulting intensity emanating from this point and generates an intensity corresponding to the sample Measurement signal S, which is fed to a ratio circuit 120. The ratio circuit also receives the reference signal R from detector 116, which corresponds to the lamp intensity. A synchronous demodulator 122 receives the resulting Ratio signal proportional to the amplitude ratio S / R and from fluctuations or other components is unaffected, which are caused by undesirable changes in the output intensity of the source.

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The demodulator that synchronizes with the reference signal is demodulates the changing ratio signal to separate the original AC voltage modulation, that of the lamp lighting by the power supply unit 110 had been impressed. The output of the demodulator is accordingly a direct current signal, dss is fed to an input of a differential amplifier 126 will. The circuit shown has the modulated power supply and the demodulator 122 to interference by avoiding βΟ-Ηζ frequencies, and uses a Ratio circuit 120 for the elimination of lamp-related fluctuations.

The circuit shown in FIG. 8 also has an adjustable voltage divider 124, which supplies a direct voltage of magnitude k, which corresponds to the S / N value at time t = 0, ie before the development of the reaction product of interest, to the second input of the Difference:. Amplifier 126 delivers. The output signal of the differential amplifier based on these two signals is the desired difference signal, which is proportional to the value (S / R - k), which is finally fed to a ratemeter IJO for further processing, which consists, for example, in a display or registration. can.

The voltage divider 124 and the differential amplifier

value

enable the aforementioned blink determination of the high ground at the sample-carrying point 114. For this purpose, the voltage divider is either manual or with known automatic reset method so that the output of the amplifier 126 before the point of interest Measurement is zero. The voltage divider is preferably set so that the amplifier output the Signal zero during the incubation period of the interest

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Indicating reaction., D. H. immediately after development of the reactant solution ■ at reaction site 114, but prior to the creation of the component to be displayed. The so The set voltage divider is used to store the information that the optical background at the point and is measured at the same optical wavelength as the reaction product of interest.

The circuit shown in FIG. 8 is only an example; also others, the principle according to the invention corresponding circuits are included in the general idea of the invention. In Fig. 3a, for example, is a Circuit diagram given, which has a GIeinspannungs ratio circle contains.

From the description it appears that the inventive Method typically relies on a liquid sample solution containing the material to be analyzed contains dissolved, at a selected point of a fibrous Sheet is brought, which serves as an analysis vessel, d. H., contains the reaction mixture in the measuring field of the measuring device. Various chemical reagents are thereby in the fibrous reaction vessel or fiber medium before or introduced after application of the sample solution. The sample solution as well as the respective reagents are applied to the Fiber medium applied so that they each have essentially the same local distribution of concentration in each implementation of a given test or analysis. When performing an analysis on several Samples according to the method according to the invention are therefore the local distribution of different samples over the different ones fibrous reaction parts each have the same, d. H. reproducible. The local distribution of the

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respective reagents are the same when examining each sample. The fiber medium contains the sample solution and the reagents in an optically thin layer. In this way it is possible to identify the components of interest to be determined by measuring the reaction mixture with electromagnetic radiation, a measurand which is essentially linearly related to the concentration of the unknown component.

According to the invention, an optically thin preparation of a sample solution and chemical reagents is provided which result in a reaction product for the detection of a constituent is used; the product is based on a linear relationship between measurand and Determined concentration of the corresponding reaction product. Sample and reagents are for the reaction and the measurement applied to or contained in a fibrous sheet. The same place on the fiber sheet that the contains reactive materials, is used to generate a blank value as a reference signal for the measurement.

As already mentioned, an optically thin distribution of the sample solution and the reagents in the fiber medium leads to the fact that the transmission of the intensity of the incident radiation and the exiting intensity measured by the detector at each point in the measurement of the measuring device essentially linearly from the concentration of the interest Component up to the maximum concentration to be measured depends.

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The reaction product of interest generated in the reaction mixture under the conditions mentioned, which for Evidence of an ingredient being used is preferably based on a fluorometer or another Radiant energy responsive device measured linearly to the concentration of the substance of interest appeals to.

In detail, in the examples given below, according to the invention, a fluorescent reaction product of a concentration which corresponds to the concentration of the unknown substance to be determined is formed in the reaction mixture. When irradiated by the fluorometer source, the reaction product used to detect a constituent fluoresces in the reaction mixture at every point of the fluorometer measuring field with an intensity that is linear with the concentration. is proportional as long as the layer is optically thin over the effective length through the fiber medium, ie the paper. The fluorometer detector responds linearly to the fluorescence radiation from every point within the measuring field and accordingly generates an electrical signal that is a linear function of the product of fluorescence energy and the sensitivity profile of the fluorometer integrated over the area of the Γε-surface in the pluorometer field Sensitive profile of the fluorometer corresponds to the local distribution of the response of the device over its measuring field to the lamp fluorine essence. The profile is therefore from the spatial hzvr. local characteristics of the fluorometer lamp and the fluorometer detector.

The fluorometer shown in Fig. 4 also has like the devices shown in Fig.β and 7 <as already

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given equal spectral angles for the incident energy and the measured fluorescence energy. This is desirable in order to give the device a composite, ie lamp and detector-dependent sensitivity profile that - despite a certain change radially to the axis - is symmetrical and approximately uniform around the viewing axis of the detector, whereby all points in the fluorometer measuring field are uniform can be measured. This means that in a fluorometer with the same angle of incidence and angle for the registered radiation, the entire optical path length from the fluorescence source of the reaction layer and the fluorometer detector is essentially, at least in a first approximation _} within the fluorometer measuring field for all points uniform.

Alternatively to the use of a fluorometer or absorption photometer can be used for the practical implementation According to the invention, an oscillometer is also used to show the interaction of the constituents of the reaction mixture measures with an alternating electric field. The fiber sheet can, for example, between two flat electrodes are arranged, which result in a plate capacitor. The device will operate at a selected frequency to measure the change in capacity by the interesting Reaction product calibrated.

According to the invention for taking up the reaction mixture The fiber material provided preferably consists of fibers are inert and non-absorbing with respect to the wavelengths of the electromagnetic radiation used in the measurement and are permeable. These demands are

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although not mandatory, they make the calibration easier, increase the measuring accuracy and increase the measuring sensitivity .

The invention has been successful with, for example, cellulosic fibers as well as glass fibers carried out.

However, glass fibers are so fragile that when compressed with the mentioned, in the US application 243 028 break the press described. So if a such a press or another corresponding device is used with which the fiber materials are stressed it is considered cheaper if there are no fragile fibers or at least fiber sheets. from a mixture with fibers that are less prone to breakage, such as for example cellulose fibers can be used.

For kinetic measurements, the fiber sheet preferably has such a fiber structure that all applied liquid reagents cease to spread within the layer within a time which is short compared to the time during which the reaction of interest takes place linearly. This is because the linear part of the reaction should take place over a large area after all reagents have stopped spreading due to the capillary action of the fibers. In typical kinetic measurements, it is desirable that about J> / K of the linear portion of the reaction occur after the significant spreading is complete.

With respect to the spread of fluid materials in the fiber sheet, it is also desirable that all materials

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move through the sheet material at the same speed. Otherwise, the distributions of the various reactants at different points in the material imbalanced in response and analysis. In view of the very different pleating properties of Materials that are commonly used in such analyzes cannot achieve this goal in many cases can be achieved to the desired extent. However, it has been found that the different spreads are different Materials in many cases can be limited by the fact that the materials are the fiber medium in a special selected order.

In particular, it has been found that when applying Large molecule reagents with or without drying prior to application of smaller molecule reagents the spread of the reagent applied first is substantially reduced by the reagent applied thereafter. This finding means that in the task of solving components with small molecules on the Fiber medium that follows the task of solving constituents with large molecules, the material with the larger molecules tend to spread and propel the material with the smaller molecules in front of it, so that the material with the smaller molecule size in a circular ring outside of the flecken area is more maximal Concentration of the material with the large molecules concentrated will. Such conditions are undesirable and lead to a significant reduction in the measurement sensitivity compared to the reverse procedure, which is explained in more detail below.

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2B36886

Examples of fiber sheets suitable for practicing the invention are Schleiche; ·· & Schüll test papers No. 90.5 * 903-C, 404, 410 and 25, and Whatman test papers GF / A, GP, B and GF / C, all of which have been used with success. In some cases, so-called Yagoda rings have proven to be desirable for delimiting the analysis spot. In general, these rings appear to be cheaper on fiberglass-free papers and thinner papers. In particular, wax limiting rings were advantageously used together with Schleicher & Schüll papers 903-C and 410, while good results and high sensitivity were obtained with Schleicher 3: Schüll paper 404 without limiting rings.

The above explanations regarding the order in which the reagents are applied also apply to the Adding the sample solution.

The term sensitivity here means the rate of change the registered intensity, for example the fluorescence intensity, for a given Concentration of the measured component.

Example I.

The determination of the glucose concentration serves as a first example for the practical implementation of the invention in blood serum ^ the analysis is based on the well-known hexokinase reaction

HK
Glucose + ATP ■ } G-6-P + ADP

G6PDH
G-6-P + IHDP> 6 PG + NADPH + H ⁺

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their usual implementation, for example, in the Boehringer Mannheim Corp. published brochure "Diagnostic test combinations, instructions for use" is described.

Example Ia

In a simplest implementation, an all necessary reagents containing solution was obtained by dissolving a "Smith Kline Eskalab glucose tablet in 0.5 ml of distilled water. 10A of the sample to be analyzed serum was appropriately diluted to a dry sheet of fibers such as Schleicher Sz sound No. 903-C applied. the sample was continuously applied (better than dropwise) in the center of Ree'ctionsstelle with a pipette. then, 10 of the diluted { '' Eslcalab "solution were else applied in the same continuous V / drying without at the same location . The order of this addition is important - since the observed initial rate of the reaction in the above case is three times greater than when the solution was added before the serum was applied.

The reaction site is then checked with a fluorometer such as the fluorometer 30 in FIG. 5 measured that the reaction site with a wavelength irradiated from j5 ^ 0 nm, the fluorescence with a Detector is measured, which is to the length of 400 nm of the maximum of the NRD PH emission. The fluorine ornet er-A output signal is generated during the linear part of the reaction process measured. The speed of NADPH generation is preferably measured, which is cheaper than the determination of the total amount of NADPH produced, in order to achieve an easy separation of the measured fluorescence of the NKDPK from the background fluorescent radiation of others Materials at the test site as well as from the fiber sheet itself

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su enable. The latter radiation is essentially time independently, while the K ^ DPH fluorescence with the Increase in MDPH produced increases. It is further on Ensure that there is a compression contact of the fluorometer end window with the fiber material and the reaction mixture was avoided in order to prevent changes that would otherwise have shown in the measured fluorescence.

FIG. 9a shows the time dependency of the measured fluorescence signal over the background fluorescence of 903-C paper after application of the reagent solution. Various standard reagent solutions of glucose were used as samples. These solutions were prepared by diluting a starting solution of 150 mg / dl, which were diluted 1:50 with deionized water to give samples with concentrations of 5 mg / dl, 2.5 mg / dl and 1.25 mg / dl, as can be seen from FIG. 9 ^a . The sensitivity of the resulting measurements is sufficiently high that human sera which are in the normal range of 65-10 mg / dl require a dilution of 1:30 in order to fall within the scope of this detection method.

From the curves it can be seen that the fluorescence increases over an approximately linear part sohneil that at ends about 45 seconds. The initial slopes are there proportional to the concentration. The observed increase for water as a sample, i.e. H. with a concentration of 0 mg / dl, shows a significant change in the first minute from the change in the optical connection between the counterplate, the fiber medium and the fluorometer window.

At the four concentration values shown in Fig. 9a 17 experiments were carried out. The observed initial slopes showed a linear concentration dependence with a correlation coefficient of 0.962. Four of the

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Measurements were taken at 5 mg / dl and found a standard deviation of 0.5 mg / dl. The standard deviation of the background fluorescence of the dry Paper before application of the sample and reagents was over after conversion into clinical units the fitted linear regression compares to 1.0 mg / dl or about twice that for kinetic measurements observed value.

Example Ib

The reagent system of Example I was designed to suppress differential spreading and the above mentioned washout reformulated to a minimum optimize the linear part, d. H. to make it as big as possible. The production was done using the the following starting ingredients in deionized water according to the following formulation:

component

concentration
(Amount / ml)

Trade name

Buffer pH 7.5

Tris (hydroxymethyl) -

a minome tha n-hy dr ο ch 1 or id succinic acid Magnesium chloride

Glucose-6-phosphate dehydrogenase (G-6-EDH)

0.18 mmol
0.12 mmol
0.16 mmol

Sigma Trizma T4003 JT Baker ^ r 0 ^ 46 Sigma M-0250

20 IU (Inter-Sigma Bakers Yeast, national Units) sulfate-free

Type XU
MR. G6378

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component

Concentration (amount / ml)

Trade name

Hexokinase

Adenosine triphosphate (ATP)

Nicotinamide adenine dinucleotide phosphate (MVDF)

25 IU (International Units)

0.02 mmol

Signa Bakers Yeast sulfate-free type F -300
Mr. H-4502

PL Biochemicals No. 900

To prepare the reaction sites, 20 ml of this reagent solution were applied to dry, unbound paper of type 902-C and the paper was then desiccated in a vacuum. The reagent solution and the dried strips were stable for at least one month in the frozen state.

An unused reaction site is used to carry out the measurement placed at room temperature over the window of the fluorometer from Fig. 6, whereupon 20 / U1 of the solution as above be applied in the center of the treated area. A layer of "saran" film or some other thin, optically transparent plastic film can be used on both sides of the paper to allow direct contact of the liquid with the fluorometer window and the counterplate

to avoid. In this way, the entrainment of sample material from one test to the next is avoided, but the optical measurement is not influenced. The film also keeps the humidity ^{constant at the A} ev reaction site because evaporation is prevented. The resulting kinetic curves are shown in FIG. 9b for three concentrations as well as pure water. Ik: area

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from 1-2 min the curves are practically linear and can be evaluated for at least 3 min. The measurement results with pure Water as a sample show a behavior similar to that in Example Ia, but the measurement can now be carried out according to the Stabilization of the physical situation can be carried out at one minute.

Example Ic

A slightly different result is achieved by pretreatment of 903-C type paper with a low polymer molecular weight obtained to suppress the absorption of the solution in the paper fibers as much as possible himself serves. Paper strips 2.5 cm wide and For example, a length of 20 cm is soaked in an aqueous solution of 6 mg / nl polyox resin; this product is a crystalline, high molecular weight polyethylene oxide with a molecular weight of 6000,000 (grade WSR-205, manufacturer Union Carbide Corp.).

According to the manufacturer, this is resin or its production is described in the following US-PS:

2,866,761, 2,897, 178, 2,991,229, 3 030, 315, 3,062,755, 3,085, 071, 3,214,387 and 3,274,129.

The soaked strips are then dried in a vacuum desiccator. The dry polyox paper is slightly thicker than uri-treated 903-C paper and v / e is 0.48 mm (0.019 inch) versus 0.43 mm (0.017 inch) thick.

The treated paper shows significantly reduced spread of liquids applied to it, what is called is considered desirable. As a reason for this advantageous behavior, it is assumed that the treated

1Q / üG8a

Pasern does not absorb liquid and move outside let get. When 10, 15 and 20 / μl of fluid are on applied to different spots · are the diameters of the resulting translucent spots (Spots) 9.5 mm, 11.43 mm and 12.07 mm (0.375 »0.450 or 0.475 iJich) for untreated paper, but only 8.26 mm, 9.5 mm and 10.16 mm (0.325, 0.375 and 0.400 inches) for the treated paper.

The polymer resin reduces the capillary expansion of the fluid substance accordingly. In addition, the liquid absorption capacity of a punched disc made of treated paper seems to be around 10-15% higher than that of untreated paper. A 9.5 mm (0.375 inch) diameter disk of treated paper contains about 38-40 µl of liquid without overflow, while treated paper contains 42-45 µl.

The reaction sites on the polyotraded paper 903-C v / ground as produced in Example Ib. However, as the paper contains more liquid, 30 / Ul des optimized reagent applied to the appropriate areas and then vacuum-dried.

The reaction is then carried out again in the fluorometer of FIG. 6, with the dry stain over the optical window is centered and again a thin plastic film to prevent overflow as well as the Evaporation is used; as before it will be 20 / ul Glue solution applied.

The individual results are very similar to the results shown in FIG. 9b, with the difference that the time scale is doubled, d. H. up to 6 minutes. The curve for V / water flattens out at 2 minutes, and the rise The upper curve is compared to FIG. 9t> approximately twice, from which the approximately double chemical sensitivity

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is evident. The observed increase in water in the first 2 minutes resulted from fogging of the saran film (or the counterplate itself, if no saran film was used), as a result of which the optical connection with the counterplate was interrupted. The fogging was caused by a temperature difference of about 5 ^° C. between the counterplate and the window during the measurement. The slope of the blank measurement was greatly reduced when the counterplate was heated to a temperature closer to the temperature of the window. The standard curve is linear up to about 60 mg / dl (dilution 1:50), so that patient sera must be diluted by a factor of 50-100 so that the normal range of 65-100 mg / dl is in the linear measuring range.

It should also be emphasized that such measurements show an amount of 0.2-0.4 / ul of the original patient serum require, with the end result being obtained in 2-4 min.

When the volume of glucose solution that is dry Feagensfleck is added, varied from 15-30 / ul it is found that the measured speeds in the linear part of the curves compared to an expected value vary from 2 by a factor of only about 1.5. (It is believed that this change in the Slope can be further reduced if the blank value is mixed with pure water by means of suitable temperature control is reduced). This decrease in sensitivity versus volume of liquid also facilitates the practice of the invention with accurate results without corresponding accuracy when applying or distributing the reactant solutions. .

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Example Id

Polyox treated paper and the optimized reagent can be used in conjunction with a press constructed in accordance with the aforementioned U.S. application 243,028 is; in this way glucose can be determined in whole blood samples which have been applied to 90J-C paper became. For this purpose, the reaction points are created by creating depressions in the treated paper, by folding the press onto the paper. A ring about 1 mm deep and 1 cm in diameter is made very strongly compressed in the process, with the center only being subjected to moderate compression. 11 / ul of the optimized Glucose saws are then placed in the center added to this well and vacuum dried. This volume of reagent fills the center of the well straight out and does not penetrate the highly compressed ring.

To carry out the measurement, a depression containing the reagent is placed in the dry paper under the press centered and initially with an ultrafiltration membrane and then covered with the sheet of paper containing the dried blood stain. The blood stain will subsequently with 20 μl saline solution or an aqueous solution of a surfactant such as "Brij" and the press closed for 20-40 seconds.

During this time, a small volume of ultrafiltrate, which contains 1-10 <fo of the glucose contained in the blood stain, penetrates through the membrane into the well. The volume of the liquid is sufficiently small that the molecular mobilities are low and the reaction takes place slowly, if at all. When the press is opened and the sheets are removed

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the stain dries. The reaction site will then given via the window of the fluorometer, which is constructed, for example, as in Fig. 6, taking 15 / ul water to reconstitute the dry Flecks and initiation of the reaction are added. This volume is in turn selected so that the Liquid not in the highly compressed annulus. entry. ,

The internal pressure exerted by the press and the time it remains closed become like this adjusted that the transferred glucose concentration lies in the process area of the reaction.

Example Ie

To further investigate the optical phenomena that take place in the fiber medium, simultaneous measurements of UV transmission and fluorescence in reflection were carried out, using the aforementioned polyox-treated paper and optimized glucose reagent. Two optical attachments in the arrangement shown in FIG. 7 with lamp and reference detector in the upper unit (ie in passages lo8a and l03b) were used to generate and measure the incident light of 1/8 nm. The detector in the lower unit (ie in passage 96b) was placed behind a 340 nm filter, while the upper detector was placed behind a non-fluorescent filter set centered at 46o nm. To facilitate the measurement, disks of 9 * 5 mm (5/8 inch) diameter made of polyox-treated paper were wetted with 11 μl of the reagent solution and vacuum dried. The slices were in a recess

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9.5 mm (J./8 inch) diameter in a 0.33 mm (0.015 inch) thick stainless steel disk (see backing sheet 102 in Fig. 7) that is placed against the Window (104) of the upper device through the rubber seal (100) of the lower device was pressed. The distance was such that the piece of paper to be analyzed was in contact with the upper window and extended down through the pane, however, the lower window (48) did not touch. The left out Space (106) was closed with the seal, so that the volume in which the liquid evaporate could, was limited. This airtight seal also served as a temperature buffer against the colder one lower window.

To carry out the analysis, 25 / ul were diluted Commercially available serum standard (Ortho Diagnostics Instruments low AcuChem calibration fluid, lot no. T) with a Glycose concentration of 50 mg / dl applied to several different discs and the corresponding Curves recorded. There were a total of 24 different Measurements made at 6 different dilutions. 8 of them were done at a 1:20 dilution, 5 of them contained water as a sample. The test results are shown in Fig. 10 together with a recorder of the dry or wet Paper to compare chemically induced changes in native fluorescence and with lower transmission of the Paper.

With the mean slopes obtained in each determination as a function of the dilution, a

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linear regression analysis performed. The results show that the evaluation "of water up to a dilution of 1:10 is linear. The correlation coefficient is 0.998 for the fluorescence measurement and 0.995 for the absorption measurement. The 8 results for the 1:20 dilution provide a standard deviation of the observe speed corresponding to 2.4 mg / dl or 4.9 % of the 50 ml / dl contained in the original fluid. The transmission measurements were almost as accurate and showed a standard deviation (in clinical units) of 5 * 5 mg / dl or 11 % of the 50 mg / dl contained in the original solution.

The results show that, provided that the fluctuations in the weakening of the fiber medium are eliminated from spot to spot, transmission measurements can be carried out just as well as fluorescence measurements. These fluctuations show up in the differences between the starting fluorescences at t = 0. The standard deviation of the 8 initial values of the data obtained with the 1:20 dilution is 17.9 mg / dl after conversion into clinical units. The corresponding value for the 5 water samples is 16.8 mg / dl. The fluctuations at the starting point thus far exceed the change caused by the chemical reaction in one minute. These initial fluctuations are typical, as can be seen in the right third of FIG. 10, in which the observed fluorescence and transmission signals are shown with a saran-wrapped strip of paper 2.5 20 cm (1 8 inches) being pulled through the space between the upper and lower windows of the device, 98 and 104 in Figure 7, and the carrier sheet 102 removed was. The 50 % moist

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Strip was made by fully immersing a strip prepared in water and then brought together with a dry strip, if possible was given to spread evenly between the two strips. It should also be noted that the upper housing 108 of the device of FIG. 7 for ease of access in light of the hinged attachment the counter plate 82 of Fig. 6 was hingedly mounted. Between the housing loS and the lower part however, there is preferably a small gap in the device so that the housing does not close completely tightly, as can be seen from Fig. 7 '.

Example If

Fresh whole blood was concentrated on the Prepare galactose using previously prepared reagent on paper of the type 9OJ5-C, which is examined with a solution of 6 mg / ml Polyox was treated as above.

The reaction used is the enzymatic conversion of galactose to galactonic acid by galactose dehydrogenase with simultaneous conversion of nicotinamide adenine dinucleotide (NAD) to the reduced form NADH.

Galactose dehydrogenase (GDH) was developed by Bοehringer-Männheim Corp. as a suspension of 5 rag / ml in ammonium sulfate solution. The enzyme must first from Ammonium sulfate is separated, which is in an ultrafiltration funnel happened (Minicon 25; manufacturer Amocon Corporation). 120 yul of the GDH-S us pens ion were in the Ultrafiltration device introduced and saline solution up to 750 / Ul mark added.

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The solution was then up to the 150 / UI mark concentrated five times. This washing process was still going on ZV / ein.I repeated, whereupon one concentrated tenfold to 75 / Ul Final solution was obtained. In this process, the GDH remains in the funnel, with the ammonium sulfate is removed with the wastewater.

The GDH is then by adding 0.1 M-Tris ^ PH 6.2 buffer. ■■ · up to the 750 / UI mark and pour the solution removed into a container. The GDH concentration of the solution is about 5 IU / ml. The reagent will completed by adding .6 mg of NRD.

The reagent strips are by adding 15 / ul this solution to preformed wells in polyox treated Paper produced as in Example Id and subsequent vacuum drying.

To this end, it should be noted that the buffer with the low pH value of 6.2 serves to keep the reaction outside to carry out their maximum speed, which is pH 8. This is done to suppress the reaction

during or after the ultrafiltration in the press and before the start of the reaction in the fluorometer

To carry out the galactose test, the pretreated wells are centered under the press and with the ultrafiltration membrane and a leaf untreated 903-C paper covered. A whole blood sample of 20 / Ul is then applied to this top sheet and the press closed for 75-100 seconds to let through as much galactose as possible. This procedure can also include a saran polie to avoid one Sample contact with the press itself and its plates can be used.

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When the press is opened, the upper layers are removed and the reaction zone over the window of the Fluorometer placed as in Fig. 6. The reactants will by applying 11 µl of a 0.2 M Tr is buffer of pH 8.6 reprocessed or reconstituted and the reaction followed for a few minutes. The investigation will by repeating the procedure with 20 / Ul different Standard solutions calibrated instead of whole blood. The examination can also be performed directly using centrifuged and separated serum can be performed by adding 10 μl of diluted serum and 5 μl of pH 8.6 buffer applied to an unused reagent well.

A calibration curve was obtained by examining various standard galactose solutions instead of whole blood. A total of 11 examinations were carried out with 9 different solutions in the concentration range from 0.0-90 mg / dl carried out. The linear regression analysis gave a correlation coefficient of 0.999 and also showed that the chemical process is linear up to 90 mg / dl. The accuracy of the examination can be seen from the deviations can be estimated from the regression line for each of the measuring points. The standard deviation of these differences is 1.48 mg / dl.

A standard detection experiment was also performed by adding galactose to four patient sera at different incremental values. The mean coverage of 10 such additions to the 4 sera was 96 % with a standard deviation of 8 ^.

The above glucose analysis has been carried out with numerous

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Pas erb latter η successfully carried out as a reaction vessel, including the Schleicher & "Schüll-Papiere 4o4, 595 and 25 and the Whatman papers GP / A, GP / B and GP / C.

Example II

It was Sehüll-paper examined 905 Pas -C than he reaction vessel, the lactate concentration of blood serum on system before dissecting Schleicher &, wherein a guard ring was used to the test site. The paper was initially produced by applying LDH in an ammoniacal suspension (for example Sigma Type III from bovine hearts) in the center of the test site. Then, without drying, 10 / L of an aqueous " ¹ solution of pyridine nucleotide (HID) at a concentration of 15 mg / ml with a trace of a surfactant (for example" Brij ") was applied to the same place. The leaf was then placed in In a similar way other fiber materials can be used for this purpose.

When fresh serum to be examined, which is pre-prepared, but dried fibrous "reaction vessel" at first (hereinafter referred to as Paserblatt) by applying, treating a glycine hydrazine buffer at pH 9 of 10 A. This is followed by the application of 10 λ, the serum sample, which is diluted 10: 1, without drying. By measuring the rate of NkDH formation with a fluorometer in the manner described in Example I, the lactate concentration can be determined within 5 % of the amount determined by a comparative method.

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When preparing the paser sheet, it was found to be important that the IDH solution was applied before the NAD solution otherwise the lactate analysis will be of significantly lower sensitivity. As a reason for this it is believed that the relatively heavy IDH molecules form some kind of bond with the fibers of the sheet material or in another way resist the spreading of the subsequent addition of the lighter NAD molecules, so that the two reagents cover substantially the same part of the fiber sheet. When the reagents are in reverse If the sequence is applied, it can be assumed that the lighter MD molecules are replaced by the heavier ones IDH molecules laterally outwards from the application site be driven so that the stain on the fiber sheet has a concentration of IDH molecules within a predominantly ring-shaped concentration of NAD molecules having.

It was also found that the sensitivity decreases when buffer and serum are added in the opposite direction, i.e. when a serum sample is added before the buffer. Here is to note that the last addition of the sample serum in the present lactate analysis is the reverse of the preferred order in glucose analysis of example I corresponds to.

Example III

As a third example, a fiber sheet was first again produced according to Example II, the one to be examined Serum in the form of a Schleicher & Sqhüll test paper 903-C present dried whole blood stain. The paper containing the whole blood sample was as described in the above-mentioned parallel application,

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arranged a filter sheet to an ultrafiltration membrane on a pre-prepared dry Reajensblatt under Zviischenschaltung (see. Fig. ¹ I) and in the press described in this application (see. Fig. k, 32) placed. The blood stain was reconstituted before closing the press. In the case of a fresh stain, this was done by adding 20% Brij water or 0.9% normal IiaCl solution. The press was then brought to the reagent sheet to transfer the Serurn ultrafiltrate through the filter sheet placed in between.

On the other hand, if the blood stain is not fresh, it is preferably reconstituted by adding 20 / L '..'& sser with a trace of "Brij" or similar borderline-aictive [littein] and then subjected to pressure for transfer to the reagent sheet. If the stain is old, it proved to be advantageous first to 25-30 L · Diäthylather for penetration of the stain immediately before the "Prij" apply.

In each. ^1st The trap would be left closed for about 1 min. A known part of the lactate of the sample was transferred to the reagent sheet by the press,

however, typically not enough fluid for Bringing about sufficient molecular mobility for the reaction to proceed. The leaf, that originally carried the blood sample, as well as the intermediate filter sheet, were removed from the reagent sheet accordingly peeled off and 20 · ^ glycine-hydrazine buffer (pH 9) for humidification of the lowest reverse leaf applied. then was again the speed of HVDH formation with followed on a fluorometer as described above.

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In the above-mentioned investigations, the fluorometer had a circular measuring field of about 1 square centimeter, centered on the point where the reagents were applied to the fiber sheet.

A reaction site with this area is through the Specified sample and reactant volumes are not saturated.

Example IV

To illustrate the applicability of the invention A fiber sheet from Schleicher & Schüll. paper 9OJ5-C was used for more complex chemical reactions in blood analysis Examination of blood serum for triglyceride concentration prepared as follows. This analysis underlying chemical reactions are known and, for example, in the cited brochure of Boehringer-Mannheim Corp. under "neutral fat (triclycerides) and glycerine ", although lipase is used to hydrolyze the neutral fat to glycerine became.

The first step in the production of the fiber sheet consisted of applying 20 λ_ Lipase (Schwartz-Mann No. 25) in a continuous stream or as single drops to Schleicher & Schüll paper 903-C and allowing it to dry. Then 20 Å enzyme solution of glycerokinase-glycerophosphate dehydrogenase was applied to the same place, whereupon the fiber sheet was dried again. Then 10 Λ, a C o factor solution with NAD and ADP as well as Mg ⁺⁺ activator for glycerokinase and Ca ⁺⁺ activator for lipase in "Bri j water" of at least

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3 mg / ml applied.

It should be noted that the three are on the paser sheet applied reagents were applied in the series of decreasing molecular weight, so the solution with glycerophosphate dehydrogenase added last as the lowest molecular weight. The rest Preparation of the fiber sheet consists in increasing the concentration of the combined enzyme solution by successive Deposits to prevent excessive spreading of the material and to keep it in a limited area of, for example, 1 square centimeter of the paser sheet. Accordingly, before drying the Co-pact or solution add another 10 / L enzyme solution to the leaf applied and then allowed to dry. Then 10Λ glycine-hydrazine buffer of pH 9 on the leaf in the Abandoned in the middle of the reaction site and then added 10Λ of the enzyme solution, whereupon it was again dried. The dry reaction paser sheet was then ready for use and could be stored until use.

To carry out the triglyceride analysis with the reagent sheet produced in this way, 10 μg of glycerine-.hydrazine buffer applied to the sheet and then 10 of the blood serum to be examined. The reaction point was then measured with a pluorometer with respect to the rate of NRDH formation. A second The test was carried out with the same pre-prepared reaction sheet,

but carried out at a different spot or at a different location,

instead of the sample serum, 10 / L saline solution was applied and the rate of N & DH formation was measured in the same way. The difference between the two rates of NftDH formation in the sample

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or the salt solution corresponds to the desired measured value for the triglyceride concentration; any speed of response is related to the subsurface at the same point as the actual reaction speed is eaten. Such a differential measurement is used to achieve high accuracy in the presence of impurities in the reagents and / or the the fiber sheet representing the reaction or analysis vessel deemed necessary; Such a blank sample can consequently be dispensed with in the case of pure materials.

The triglyceride concentration determined in the manner mentioned is within 5% of the concentration which determined according to standard laboratory procedures.

Although the examples mentioned refer to analytical methods in which MDH is formed, the Invention can also be used in the same way to carry out analyzes in which a fluorescent Substance consumed and accordingly the speed of the Consumption is tracked.

The given description of embodiments according to the invention is purely exemplary; also other den Features of the invention are procedural or apparatus-related embodiments or developments included in the general inventive concept.

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Claims (1)

Patent claims 1 .//"Experience for the analysis of components of a liquid PTo ¹ De on a fiber medium by chemical reaction with appropriate reagents to generate a reaction product used to detect the respective component, the sample material and the reagents in the liquid state with reproducible local concentration distribution on a selected analysis point of the fiber medium applied and distributed over it, the analysis site with a conspicuous electromagnetic gene Radiation Illuminated and that from the incident radiation resulting and from the concentration of the reaction product affected electromagnetic radiation is eaten, according to Ilauptpatent. (patent application P 2j5lSc44), characterized that the reaction product at the analysis point only v.'ird is produced in an optically thin concentration and the measurement field lies within the analysis parts or coincides with it. 2. The method according to claim 1, characterized in that the speed of change of the resulting radiation is measured. J5. Method according to claim!, Characterized in that the sample material and the reagents are applied and distributed in a concentration that the medium at the B09810 / 0689 Analysis site not saturated. 4. The method according to claim 1, characterized in that the moisture content of the reactants at least ° ür held essentially constant for a certain period of time during the formation of the reaction product 5. The method according to claim 1, characterized / leave a reproducible on the fiber medium at the analysis point Compression state is maintained. 6. The method according to claim 1, characterized in that the fiber medium at the point of analysis shows essentially no capillary suction. 7. The method according to claim 1, characterized in that the measurement is carried out at a specific wavelength will. 8. The method according to claim!, Characterized in that the difference function from the resulting radiation to generate a measurement signal corresponding to the sample, that of the background of the corresponding medium, the sample material and the reagents as well as the reaction product originates, and from the pure background radiation of the medium with the sample material and the reagents before the reaction leading to the reaction product is formed in the measuring field. 9. The method according to any one of the preceding claims for spot test analysis on a test spot on a fiber medium, characterized in that the concentration of the 609810/0689 The measurement signal corresponding to the reaction product is generated by ' Measurement on the same test spot before the formation of the reaction product and Difference formation from the measurement signal obtained before the formation of the reaction product and the measurement signal during or after the reaction. 10. The method according to claim 9 »characterized in that that the measurement of the concentration of the reaction product corresponding signal made over a certain time during the image of the corresponding product will and the moisture content of the test patch during this Time is kept essentially the same. 11. The method according to claim 10, characterized in that the compression state of the fiber medium at least on Test spot and in the neighborhood is kept the same during the measurement time. 12. The method according to any one of the preceding claims, characterized characterized in that the ^ tightness of the sample material and the Reagents at the analysis site to generate an im substantially uniform reaction speed for at least part of the implementation of the measurement during application is balanced at the analysis point. 13. The method according to any one of the preceding claims, characterized in that at least some of the reagents in one of the molecular size of an effective ingredient of the corresponding reagent can be applied in the appropriate order, starting with the reagent that corresponds to the Contains component with the highest molecular weight. 609810/0689 14. The method according to any one of the preceding claims, characterized in that at least the analysis site or the test spot on the fiber medium is treated with an aqueous solution of a crystalline high molecular weight polyethylene oxide to reduce the liquid absorption by the fibers of the fiber medium before the application of the reagents forming the reaction product 15 · The method according to claim 14, characterized in that the polymer solution on the fiber medium before application the reagents is dried. 16. Device for carrying out the method according to the main patent (Patent application P 2 ^ lSo44) and possibly a of the preceding claims for the determination of a certain chemical reaction product at an analysis point on a fiber medium with a device for irradiation with electromagnetic radiation as well as for the selective measurement of the electromagnetic energy parameters caused by the presence of the corresponding reaction product, geke marked by a measuring device for generating a measuring signal the registered radiation energy that the reaction product corresponds, based on the radiation energy, from the corresponding analysis point due to the incident electromagnetic radiation energy is registered before the formation of the reaction product. 17. The device according to claim 16, characterized in that that the measuring device generates the measuring signal with monochromatic radiation. 609810 / U689 13. Apparatus according to claim 16, characterized by (1) a facility for measurement and storage a first measurement of the energy of the electromagnetic radiation of the selected wavelength emitted by the analysis site is registered before the formation of the reaction product, (2) a device for measuring a second measured value of the energy of the electromagnetic radiation of the selected one Wavelength registered by the analysis site during the formation of the reaction product, as (3) a device for generating a measured value for the reaction product by comparing the measured values (1) and (2). 19. Device according to one of claims 16-18, characterized in that the fiber medium at the reaction site and in its vicinity has a substantially uniform fiber structure and consists mainly of a material without capillary absorbency in which the reactants spread in the liquid solution therein comes to a standstill essentially before the end of the radiation measurement directed at the product. 20. The device according to claim I9 »characterized by a measuring device for registering the Product influenced radiant energy in the selected time interval in which the reactants the corresponding Reaction product at a substantially constant rate form, as a material with a fiber structure in which the spread of the reactants in the liquid solution is significant BO 9.8 10/0689 comes to a standstill before the selected measurement interval ends. 21. Device according to one of claims 16-20, characterized in that the fiber medium consists essentially of Paper fiber is made up of minimal liquid absorption and are made by treating with a relatively high molecular weight polymer. 22. Device according to one of claims 16-21, characterized characterized in that the paper fibers with an aqueous solution of a high molecular weight, crystalline polyethylene oxide treated and dried prior to application of the chemical reactants. 23. Photometer device for illuminating one of the reactants having test site on a fiber material with electromagnetic radiation and for measuring the of the Test site due to the radiation emitted and the electromagnetic energy resulting from the presence of the corresponding reaction product, marked by a first and a second housing part, between which a sheet-like carrier material for the test site is inserted can be, wherein the first housing part on one side of the carrier material and the second housing part on the other, opposite side of the carrier material lies, with the incident radiation illuminating one side of the test site and the resulting energy on at least one side of the test site is measured, and a housing part in close contact with the carrier material and the test site is 609810/0683 the other housing part is in contact with the other side of the substrate and from the other Side of the test site usually has a gap and the substrate sheet by contact with the housing parts with respect to the light path of the Specimen striking and the resulting energy is fixed locally and the close concern of the one Housing part at the test point this centered relative to it. ÖÜ.9 810 / U683 Le first page