DE2318044A1 - METHOD FOR THE ANALYTICAL DETERMINATION OF COMPONENTS IN A SAMPLE OF MATERIAL SUCH AS A BIOLOGICAL LIQUID - Google Patents

Description

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ο 2Ϊ, St *: i.Jdo.fitr. 11

65-20.516Ρ

April 10, 1973

DAMON Corporation
Needham Heights, Massachusetts,
United States

Method for the analytical determination of constituents in a material sample such as a biological one

liquid

The invention relates to a method for measuring or «determining a component or components in
a material like in a biological fluid.

More specifically, according to the invention, a method for measuring a component within a material is provided in which the sample material to be analyzed is in an optically thin layer of the reagents or reactants
subjected to a constituent-indicating chemical reaction
will. The layer is checked using electromagnetic radiation and receiving or scanning means,
which are linearly responsive to the concentration of the selected constituent indicating product of the reaction.

65- (DAH 196 ü ¹ ) NoHe

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The invention enables such measurements to be made repeatedly with considerable accuracy, even if the spatial Distribution of the material to be analyzed and / or the reactive agents over the thin layer both are not uniform and different from one another, provided that these distributions are in the case of successive Samples or tests are identical or analogous. In addition, according to the invention, compared with the prior art, highly sensitive measurements of constituent concentrations be performed.

The prior art relating to the invention can be found in the following US Patents: 2,129,754, 3,036,893, 3,216,804, 3,219,416, 3,260,413, 3,261,668, 3,331,665, 3,368,872, 3,502,438, 3,526,479, 3,526,480 and 3,552,925.

The aim of the invention is now an improved method for determining constituents in a sample, in particular a biological fluid within a "Faaer cuvette" or a fiber layer acting as a "reaction vessel". In particular, one or more soluble components should be used a fluid material contained in a fiber cuvette with higher sensitivity than before. Another goal is a method of the above type in which as a cuvette a fiber layer material or a fiber sheet free of material-restricting structures, such as a disc of limited size or locking rings can be used on the sheet material. In particular, the cuvette medium should be one or more may have unmounted or unlimited sample places or locations and an accurate and clean determination of components to be possible.

Another object of the invention is a method of the above Type in which only a very small amount of sample material

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- typically less than 30 μΐ - is required and can be carried out quickly and with minimal effort. Both reaction rate measurements (or the course of the reaction) and find point measurements should be possible.

Further objects of the invention emerge in part from FIG alone and will partly emerge from the following text.

The invention accordingly encompasses various steps and the relationship of one or more of those steps to the individual others, as evidenced by the procedures and examples described below.

Briefly, according to the invention there is provided an analytical method in which reagents and a sample solution are in one optically thin layer react chemically to form a reaction product, which is a known measure of a constituent of the sample solution. When the layer is irradiated with electromagnetic energy of a selected wavelength a radiation detector or other energy sensitive measuring device with linear response provides a linear one Keßwert or a linear measure for the concentration of the interest Constituent.

The reagents and the sample solution are spatially distributed in such a way that the amounts of the various reactants theoretically over the entire test area for an optimal chemical interaction is in equilibrium with each other (for the generation of the reaction product of interest). The preferred "reaction cuvette" or "container" is used a fibrous sheet or web material used to contain the reactants. With such a

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Arrangement, the equilibrium distribution of the reactants often requires the delivery of the reagents to the fiber cuvette in a specially chosen order with not necessarily uniform or identical distributions.

Also, on at least many occasions in practicing the invention, one is the reaction sites structure such as a Yagoda ring not required. Instead, the reaction site can be without a delimitation be.

In practicing the invention, apparatus such as that used in the present invention can be used related German patent application with priority dated April 11, 1972 (U.S. Serial No. 245 028) with the Title "Press for progressive pressing of an absorbent, liquid-soaked object" (AT April 10, 1973 DAMON Corporation). The press described in this application can (e.g.) be used to obtain a serum sample a sample of whole blood can be used for the analysis according to the invention described herein, furthermore the The iluorometer construction described in this parallel application is advantageous in the practical implementation of the one described here Invention can be used.

Am advantage of performing chemical analysis of Components according to the invention is that it takes little time, especially compared to previous methods will. Furthermore, the practice of the invention requires a minimum of equipment. For example, the lactate analysis of whole blood using a fiber cuvette already loaded with analytical reagents (as described below)

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is treated in such a way that the serum of interest of the blood sample by means of the in the above-mentioned parallel application The ultrafiltration press described is transferred to the "cuvette" and the lactate analysis is ended in about 5 minutes.

To better understand the nature and aim of the invention a more detailed description follows.

Practice of the invention typically involves the introduction of one of the things to be analyzed Liquid sample solution containing dissolved material at a selected point on an analysis cuvette Fiber layer or a fiber sheet. Several chemical reagents are used before or after the introduction of the Paser cuvette Sample solution or liquid sample supplied. Be there Sample solution and reagents are supplied to the fiber cuvette in such a way that there is always practically the same spatial concentration distribution for each performance of a given test or analysis. That is, in the inventive Analysis of multiple samples (of the same type) is the spatial distribution of the different samples (and Reagents) via the various fiber cuvettes (each) the same, i.e. repeatable. The same applies, as I said, to the spatial distribution of the reagents used in each (analog) test should be comparable.

The cuvette contains the sample solution and the reagents in an optically thin layer. This makes it possible determine the constituent of interest by examining the reaction mixture with electromagnetic energy and ensure a response linear to the concentration of the unknown component.

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In the context of the present description, a layer of sample solution and reagents is considered to be optically thin, if their transmission of the incident as well as the output energies to be detected at every point in the field of view of the measuring instrument essentially linear with the concentration of the constituent of interest up to the maximum concentration to be measured (or its reaction product) varies.

Bas in the reaction mixture under the above conditions The component-indicating reaction product of interest generated is preferably measured with a fluorometer or Another radiant energy-responsive instrument determined linearly to the concentration of the interest Substance appeals.

More specifically, in the examples given below, the reaction mixture according to the invention becomes fluorescent Reaction product is formed with a concentration that corresponds to the concentration of the unknown substance to be measured is equivalent to. When irradiated by the radiation source of a fluorometer, the fluorescent at every point on the cuvette im Field of view of the fluorometer the component-indicating reaction product in the reaction with an intensity, which is linearly proportional to the concentration as long as the layer is optically thin. The fluorometer detector speaks linearly to this radiation fluorescence from every point within its field of vision and thus generates an electrical one Signal that is a linear function of the product of fluorescence energy and the sensitivity profile of the fluorometer is integrated over the surface area of the cuvette surface within the fluorometer field of view. The sensitivity profile of the fluorometer corresponds to the spatial Distribution of the device's response over its field of vision

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for the fluorescence of his lamp. This profile is therefore made up of spatial characteristics of the fluorometer lamp and the i'luorometer detector together.

As has already been stated above, there is one in the aforementioned parallel application for the implementation of the present Invention suitable fluorometer construction described. The fluorometer according to this application has the same spectral Angle for the incident energy and for the measured fluorescence energy. That is desirable to use the device with a composite sensitivity profile (of lamp and detector) to provide that - despite a certain change radially to the axis - symmetrically and approximately uniformly around the axis around the viewing hole of the detector (for uniform ^ measurement or checking of all points in the fluorometer field of view). This means that in the case of the fluorometer with the same angle of incidence and angle for the received radiation, the entire optical The way from the fluorescence source to the reaction layer and then to the fluorometer detector is practically the same (at least initially Approximation) for all points within the fluorometer's field of view.

As an alternative to the fluorometer, an oscillometer can be used to practice the invention, which measures the interaction of reaction mixture components with an oscillating magnetic field. For example the leaf cuvette can form a plate capacitor be placed between two flat electrodes. The device is designed to measure the change in capacitance as a result of the reaction product of interest calibrated at a selected frequency.

The serving for the implementation of the invention as a reaction cuvette fibrous material upstream

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preferably made of fibers that are used for the sample solution and the analysis reagents inert and non-absorbent for the wavelengths of the electromagnetic radiation that are of interest in the context of the mixture and are transparent. However, these requirements are not mandatory but facilitate or support rather, measures or factors such as calibration and measurement accuracy and increase the measurement sensitivity. For example the invention was carried out successfully in practice with fibers of cellulose material or with glass fibers.

However, glass fibers are so susceptible to breakage that they can be compressed with the parallel application cited above break described press. So if such a press or other construction is used, with the The fiber materials are stressed, it should be advantageous if no fragile fibers or at least Fiber sheets made from a mixture of fibers that are less prone to breakage (e.g. cellulose fibers) can be used.

For measurements of a "Yerhältnisreaktion one under development reaction ¹¹ or (rate reaction) the sheet-like fiber material preferably has such a fiber structure that all liquid reagents stop within a time to propagate within the layer, which is short compared to the time, during which the reaction of interest progresses linearly. The reason for this is that the linear portion of the reaction should occur over a large (time) range after all reagents have stopped expanding due to the capillary action of the cuvette fibers. When measuring a typical ratio or In the course of the reaction, it is desirable that approximately three quarters of the time of the linear portion of the reaction elapse after any significant spreading is complete.

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With regard to the spread of the flowable materials within the fiber cuvette, it is also desirable that move all materials through the web material at the same speed. Otherwise, the distributions of the different reactants to become unbalanced at different locations within the surface area of the cuvette will. In view of the extremely different flow characteristics of materials that typically are included in component analyzes, this goal will often not be achieved to the desired extent. It was, however found that the dissimilar spread is different In many cases, materials can be limited by storing the materials of the i'aser cuvette 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 Propagation (or "expulsion") of the reagent applied first through the reagent applied last is essential is decreased. That is, it has been found that when solving constituents with small molecules, followed by a solution of constituents with large molecules, the heavy molecular material as it spreads tends to drive the lighter molecules in front of it, so that the small molecule size material is in a ring is concentrated outside the area of maximum concentration of the large molecular material. Such conditions are undesirable and lead to significantly lower measurement sensitivity than the reverse procedure, as in the following will be explained in more detail.

Examples of those useful in practicing the invention

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Sheet-like fiber materials are Schleicher & Schüll test papers Mr. 903, 903-0, 404, 410 and 25 and Whatman test papers GF / A, GP, B and GF / C, all of which have been used with success
became. In some cases, Yagoda-type rings delimiting (the analysis spot) have been found to be desirable. in the
in general, these rings appear to be more desirable on fiberglass-free papers and thinner papers. In particular, wax limiting rings were used to advantage with Schleicher & Schüll Papers 903-C and 410, while good results including high sensitivity were obtained with Schleicher & Schüll Paper 404 without limiting rings.

The above explanations regarding the order of application of the reagents also apply to the addition of the Sample solution.

The term sensitivity is intended in the context of the present. Invention the extent of by a certain concentration of the component to be measured given change in the received or detected energy such as the fluorescence intensity mean.

example 1

As a first example of practicing the invention, blood was considered in terms of glucose concentration
analyzed. The analysis was carried out using the known hexokinase reaction

Glucose + ATP - ^ * .G-6-P + ADP

G6PDH,

G-6-P + NADP> 6 PG + NADPH + H ⁺

their usual implementation, for example in that of Böhringer ,

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Mannheim published brochure "Diagnostische Test-Kombinen; Work instruction "is described.

According to the simplest procedure, one became all Solution containing necessary reagents by dissolving one "Smith Kline Eskolab Glucose" tablet in 0.5 ml Obtain distilled water. 10 Λ. of the to be analyzed Sample serums were placed on a fiber sheet such as "S & S inr. 903-G" brought. The sample was applied continuously rather than dropwise to the center of the reaction site - for example with a pipette. Then 10 λ of the "Eskolab" solution applied in the same continuous manner in the same location. The order of this addition is critical, since the reaction rate observed in the upper case is three times greater than when the "Eskolab" solution before the serum is applied.

The reaction site or the reaction spot was then monitored with a fluorometer through which the reaction site was irradiated at 340 mn wavelength; the stimulated thereby fluorescence was measured with a wavelength of 460 nm (the maximum NADPH-ilmission) responsive detector observed. That The output of the fluorometer was measured or recorded during the linear part of the reaction. Was measured preferably the speed of I \ IADPH formation, rather than the total amount of NADPH formation, reflecting the detection of fluorescence from NADPH in addition to from the background fluorescence radiation other materials at the test site as well as the cuvette itself. The latter radiation is essentially independent of time, while the NADPH fluorescence increases with WADPII formation increases. Care was also taken that contact of the fluorometer end window with the cuvette and the mixing of the reaction was avoided in order to prevent variations, which would otherwise be revealed in the detected fluorescence.

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The above glucose analysis was successfully performed on a variety of fibrous sheet materials as cuvette including S&S 404, 595 and 25 papers and Whatman Gi ¹ A, GF / B and GP / G papers.

Example 2

The lactate concentration of blood serum was measured with a • with pre-treated "fiber cuvette" such as S & S 903-C paper a wax ring around the test site. The cuvette was made by first separating out LDH in an ammonia suspension such as Sigma Type III bovine heart in the center prepared by the test site. This was followed at the same point by the deposition of 10 λ of an aqueous solution of 15 mg Pyridine nucleotide (NAD) per ml, along with a trace of a surfactant like "Brij".

When testing fresh serum, the pre-prepared fiber cuvette was first removed by depositing 10 λ glycine-hydrazine buffer treated at pH 9. This was followed by the separation of 10 Λ serum sample. About that with a fluorometer in the way The rate of NADH formation measured by Example 1 the lactate concentration is determined.

When preparing the fiber cuvette it turned out to be It is important that the LDH solution is given up before the NAD solution, since the lactate analysis is otherwise of significantly lower sensitivity. The reason for this is believed to be that the relatively heavy LDH molecules with the cuvette fibers a kind To enter into a binding or otherwise calibrate the following Adding the lighter NAD molecules to resist the spread, so that the two reagents are largely the same part

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occupy the fiber layer. If the reagents are applied in the reverse order, it is assumed that the lighter ones NAD molecules through the heavier LDH molecules from Application aorta moved or driven laterally outwards',,, with the result that the cuvette has a concentration of LDH molecules within a predominantly annular Has concentration of NAD molecules.

It was also observed that the reverse order of the addition of buffer and serum decreases the sensitivity, i.e., when the sample serum is added before the buffer.

(It should be noted that the final addition of sample serum in the lactate analysis according to Example 2 of the preferred Sequence in the oleose analysis of Example 1 is opposite).

Example 3

As a third example, a fiber cuvette was again prepared in the manner of Example 2, but the serum to be examined was available in the form of a dried whole blood spot on S&S 903-0 test paper. The paper containing the whole blood sample was - as described in the above-mentioned parallel application - arranged with the interposition of a filter sheet over a pre-prepared reagent sheet and placed in the press described in this application. Before the press was closed, the blood stain was "restored". With a fresh patch, this was done by addition of 20 X salt solution (0.9 "/ olge normal NAG1 solution). The press was then used to Überführung- of the serum through the intermediate filter layer to the fiber-cuvette toward closed. If

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the blood stain on the other hand was not fresh, it was vorzugaweise recovered surfactant by adding 20 X water with a trace "Brij" or the like., and then subjected to pressure for transfer to the reaction cuvette.

In each case the press was left closed for about 1 minute. A known proportion of the lactate in the sample was transferred to the reaction cuvette by the press, but typically not enough liquid was transferred to induce sufficient molecular mobility for the reaction to proceed Reagenzblatt withdrawn or removed and 20 X hydrazine glycine buffer (pH 9) was applied for moistening the lowermost reaction sheet. The rate of NADH formation was then again monitored with a fluorometer as indicated above.

In all of the above tests, the iluorometer

ο
a field of view of approximately 1 cm circular area centered on the point at which the reagents were applied to the reaction cuvette.

Example 4

To illustrate the application of the invention to a more complex chemical reaction in blood analysis, a fiber cuvette from S & S 903-0 paper was prepared as follows for checking blood serum with regard to the triglyceride concentration. The basic chemical reactions for the analysis are again known and are described, for example, in the above-mentioned brochure by Böhringer , Mannheim under "Neutral fat (triglycerides) and glycerine", although lipase was used for the hydrolysis of the neutral fat to glycerine.

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The first step in the preparation of the fiber cuvette was to apply 20 X Lipaee (Schwärtz-Mann No. 25) in a continuous stream or as single drops in the center of the reaction site on SAS 903-C paper and allow to dry. This was followed by the application of 20 "X enzyme solution of glycerokinase-glycerophosphate dehydrogenosis at the same point, whereupon the fiber layer was dried again. This was followed by 10 X of an Oo-factor solution with NAD and ATP as well as the Mg ⁺⁺ activator for G-lycerokinase and Oa ⁺⁺ activator for lipase in "Brij" water of at least 3 mg per ml.

It should be noted that the 3 reagents added so far in the order of decreasing molecular weight on the fiber layer were applied. The remaining preparation of the fiber layer consists in increasing the concentration of the combined enzyme solution by successive applications to prevent excessive spread of the material and rather it to a limited range of for example

ρ
Concentrate 1 cm of the fiber layer. Accordingly, prior to drying the Oo factor solution, an additional 10X enzyme solution was applied to the layer and then allowed to dry. Then 10 × glycine-hydrazine buffer of pH 9 was applied to the layer in the center of the reaction site and then a further 10 × of the enzyme solution and then dried again. The fibrous reaction layer was then ready for use and could be stored until use.

For carrying out a triglyceride analysis old dem Reagent paper prepared in the above manner were applied to the paper next 10 X-glycine-hydrazine buffer and then 10 X of the blood serum to be tested. The reaction spot was then measured with a fluorometer the rate of NADH formation is checked. A

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second test was performed with the same reaction pretreated paper in the same manner except that 10 X salt solution were applied instead of the sample serum and the rate of NADH formation in the same manner was measured. The difference between the two rates of NADH formation in the sample and in the saline solution provided the desired measure for the triglyceride concentration. Such a differential measurement ^ due to the contamination of the reagents and / or the küvettfoildenden fiber layer to achieve high accuracy deemed necessary ₀ This second blank can therefore be omitted in completely pure materials.

Although the analyzes in the preceding examples were carried out to produce NADH, the invention can equally applied when performing analyzes where a fluorescent substance is consumed and the rate of consumption is monitored.

Thus, it can be seen that the above objects are effective in addition to those apparent from the above description can be achieved.

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Claims (1)

Claims 1Λ Method for analyzing components of a liquid ? robe on or in a "üaser cuvette" through chemical reaction with appropriate reagents for generating a component-indicating reaction product, characterized , that a) a sheet-like i'aser cuvette with a test site and a strength that is optically thin for is the keßmaximum of the reaction product; b) the sample of the fiber cuvette in repeatable spatial Concentration distribution is taught across the test site; c) each reactant or each reagent with a repeatable spatial concentration distribution over .the test site is delivered away; d) the test site is exposed to incident electromagnetic radiation and e) electromagnetic radiation, which is excited by the incident radiation and which corresponds linearly to the concentration of the reaction product at the test spot, is recorded ₀ 2ο method according to claim 1, characterized in that at least one of the liquid materials and reagents of the test site with a non-uniform spatial concentration distribution is delivered " 309844/1049 3o method according to claim 1, characterized by the Generation of a measured value for the concentration of a constituent of the sample by integrating the recorded Radiation over the surface area of at least one zone of said test site. 4. The method according to claim 1, characterized in that the analysis with measurement of the changed by the Eeaktion Fluorescence radiation of the sample takes place. 5. The method according to claim 1, characterized in that the I'aser cuvette has an unmounted test site. 6. The method according to claim 1, characterized in that the measurement or recording of the radiation during formation of the reaction product takes place and the extent of the change in radiation is measured. 7. The method according to claim 6, characterized in that the speed or rate of the linear change of the radiation of a time after the capillary expansion of the liquids in the i'aser cuvette has ceased. 8. The method of claim 1 for measuring the amount of an ingredient a soluble material sample, characterized in that one a) a repeatable concentration distribution of the individual over a test site of a carrier element forms chemical reaction partners, which are able to produce a reaction product through chemical reaction with the sample to produce, which gives a measure of the concentration of said constituent, the distribution 309844/1049 of at least one reagent or reaction partner unevenly iat; b) that one optically thin layer of the sample over said test site with repeatable distribution the sample concentration is generated and the sample with the reagents at the test site to generate the reaction producta lets react; c) that at the test site electromagnetic radiation in excites to an extent linearly corresponding to the concentration of the reaction product; d) that one absorbs the electromagnetic radiation emitted by the test site and e) a measurement output corresponding to the integral of at least a selected zone of the test site forms received radiation emitted. 3098A4 / 1049