CS260663B1 - Analytical means for the determination of diagnostically important body fluid components - Google Patents

Abstract

The analytical device for determining the components of body fluids, especially blood, consists of an absorbent carrier containing all the reagents necessary for performing the analysis, is provided with a semipermeable membrane formed from a mixture of a hydrophilic, water-soluble hydroxyalkyl ether of cellulose and a hydrophobic, water-insoluble, polymeric substance from the group of ethers or esters or acetals of polyvinyl alcohol. This analytical device enables rapid and efficient determination of individual components of blood serum without the need for laborious and lengthy separation of serum from blood elements.

Description

The subject of the invention is an analytical device for determining diagnostically significant components of body fluids, especially blood, consisting of an absorbent carrier containing all the reagents necessary for performing the analysis and provided with a semipermeable membrane for separating blood elements, formed from a mixture of a hydrophilic, water-soluble hydroxy alkyl ether of cellulose and a hydrophobic, water-insoluble polymeric substance from the group of ether or ester of cellulose and/or ester or acetates of polyvinyl alcohol. This analytical device enables rapid and efficient determination of individual components of blood serum without the need for laborious and lengthy separation of serum from blood elements.

For decades, complex laboratory methods have been used to examine body fluids, such as blood, urine or cerebrospinal fluid, based on the addition of a precise amount of the sample of the examined fluid to a reagent solution with subsequent spectrophotometric evaluation of the reaction that has occurred - usually color. When examining blood, this already lengthy process is further complicated by the need to separate blood elements from serum, either by centrifugation or so-called de-bleaching. These methods are therefore only feasible in a perfectly equipped clinical biochemistry laboratory.

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To eliminate this deficiency, special reagent strips have recently been developed, in which all the reagents required for the specific analytical reaction to take place are contained in a solid state in a suitable absorbent carrier, such as filter paper, synthetic fiber fleece or even porous polymer film. With the use of these strips, the entire examination procedure has been simplified to applying the examined liquid to the strip or dipping the strip into the examined liquid and visually or reflectometrically evaluating the specific color reaction that has occurred. However, these strips have so far only found general use in urine examination. Their wider application for blood examination is still hindered by, among other things, the fact that only serum can be examined with them, so that the actual examination must be preceded by separation of blood elements by centrifugation. These strips can therefore only be used in the laboratory and not, for example, directly at the patient's bedside or even by the patient himself.

However, strips have also been developed that allow direct examination of whole blood. In these strips, the absorbent carrier with reagents is layered with a semipermeable membrane, which, after a drop of blood is applied, retains blood elements on its surface and allows only serum or plasma to pass to the reagents contained in the absorbent carrier. The blood elements retained on the thus treated surface of the strip are removed by rinsing or wiping, and after a certain time, a visual or reflectometric evaluation of the color of the indicator area of the strip is performed, the intensity of which is proportional to the content of the determined component of the blood serum.

The common shortcoming of all these blood test strips, however, is again the unsatisfactory properties of the semipermeable membranes used so far. These membranes, formed by applying water-insoluble and strongly hydrophobic cellulose esters or ethers, such as ethyl cellulose, nitrocellulose or cellulose acetate, or simply by hydrophobicizing the surface of the carrier with fats, oils, paraffin, silicone or waxes, prevent, thanks to their strongly hydrophobic properties, the rapid and uniform diffusion of serum into the absorbent carrier, which is the result of their 260 063

- 3 consequences not only of the relatively long time of the entire determination, but especially the inhomogeneity of the resulting discoloration of the strip area leading to significant inaccuracy of the results, ranging up to + 40% relative. A certain improvement in this direction was brought about by the invention which describes the use of a semipermeable membrane composed of relatively hydrophilic methyl polymethacrylate and hydrophobic polyvinyl formal, but even this solution does not eliminate all the aforementioned shortcomings.

It has now been found that a semipermeable membrane made of a mixture of hydrophilic and hydrophobic substances allows for the complete separation of blood elements from serum and its rapid and completely uniform penetration into the reaction system in the absorbent carrier, whereby the hydrophilic component must also be soluble in water. The subject of the invention is therefore an analytical device for determining diagnostically significant components of body fluids, in particular blood, consisting of an absorbent carrier containing all the reagents necessary for performing the analysis, characterized in that it is provided with a semipermeable membrane formed from a mixture of a hydrophilic, water-soluble hydroxyalkyl ether of cellulose, such as hydroxymethyl-, hydroxypropyl- or methylhydroxypropylcellulose, and a hydrophobic, water-insoluble polymeric substance from the group of ethers or esters of cellulose, such as methyl-, ethyl- or nitrocellulose, and/or esters or acetals of polyvinyl alcohol, such as polyvinyl acetate, polyvinyl propionate, polyvinyl formal or polyvinyl butyral. The semipermeable membrane of this composition stands out against all previously known solutions with excellent filtration properties, which is manifested not only by the unusually rapid and completely uniform penetration of serum into. carrier, but also the ease with which blood elements can be removed from its surface by rinsing or simply wiping. In addition, this membrane does not have to be applied only to the surface of the carrier, but the carrier can be saturated with it throughout its mass; Thanks to this, the carrier is protected against the penetration of blood elements even on the surfaces created by cutting a larger surface of the carrier, so that even on these surfaces only serum or plasma penetrates the carrier, which prevents the unevenness of the resulting coloration at the edges of the indicator area, which is typical of all previously known devices and which is also generally described in the literature as an undesirable so-called “edge effect”. The semipermeable membrane forming an integral part of the device according to the present invention and consisting of a mixture of hydrophilic water-soluble cellulose derivatives and hydrophobic, water-insoluble polymeric substances, has, in addition to these excellent filtration properties, a number of other advantages over the membranes used so far, created exclusively from water-insoluble components. This is primarily its significant stabilizing effect on the reaction system of the device. The mechanism of this effect is not entirely clear, but the fact remains that this membrane not only significantly prolongs the stability of the product during its storage under normal conditions, i.e. in a hermetically sealed package with a desiccant, but significantly reduces its sensitivity to the decomposing effect of atmospheric moisture, which in the case of existing products is manifested, for example, when the package is not perfectly closed or when it is reopened during analysis.

In addition, the membrane also has a significant positive effect on the quality of the coloration of the indicator area of the analytical agent after the reaction with the determined component of the body fluid.

This effect is manifested in the formation of a cleaner and more brilliant color, which allows a more unambiguous and accurate quantitative evaluation of the examination result both in objective reflectometric determination and especially in visual comparison of the color of the device with a colored, experimentally made comparison scale*. The analytical device according to the present invention can be used for the examination and determination of all clinically important components of blood, such as the determination of glucose, urea, uric acid or bilirubin. However, due to its generally stabilizing effect on the reaction systems of analytical devices from the field of dg.strips, the membrane according to the invention can be advantageously used also on devices for the examination of such body fluids as urine or liquor, for which it is not necessary to remove interfering components, such as blood elements in the examination of whole blood, using a membrane. In all these devices, the reaction system, i.e. a set of reagents and chemicals, required for the actual analytical determination of the relevant diagnostically significant component of the body fluid

- 5,260,663 known per se and has been generally used for this purpose for a long time. The most significant and, in its composition and properties, completely new element of the analytical device according to the invention is therefore a semipermeable membrane, intended for the separation of blood elements, or for the stabilization of the reaction system of the analytical device. The desirable properties of this semipermeable membrane are then given both by its basic composition defined above, and by the mutual ratio of its individual components and the method of application to the absorbent carrier.

As follows from the above description of the semipermeable membrane ₉ , its excellent functional and stabilizing properties are given by the combination of a hydrophilic, water-soluble cellulose derivative with a hydrophobic, water-insoluble polymeric substance, i.e. a mixture of two or more of these substances, from the group of cellulose esters or ethers and/or polyvinyl alcohol esters or acetals. The mass ratio of the hydrophilic, water-soluble and hydrophobic, water-insoluble components of the membrane can vary within relatively wide limits from 5:1 to 1:5, depending on the desired properties. In general, however, the best properties of the membrane with respect to its filtration and stabilizing capabilities are achieved with a ratio of both components of about 1:1 to 1:1.5. It is further preferred, although not absolutely necessary, that the hydrophobic, water-insoluble component consists of two different substances, i.e. a suitable cellulose ether or ester and an ester or acetal. polyvinyl alcohol* The mass ratio of these two hydrophobic, water-insoluble components can again lie within relatively wide limits from 10:1 to 1:10, but it is preferable to choose a ratio of 1:1.5*. In summary, in most cases, a semipermeable membrane made of 2 parts by mass of hydrophilic, water-soluble modified cellulose, 1 part by mass of hydrophobic, water-insoluble cellulose ether or ester and 1.5 parts by mass of polyvinyl alcohol ester or acetal* can be considered the most advantageous.

As mentioned above, the properties of the membrane are determined - in addition to the already mentioned ratio of their individual components - also by the method of application to the absorbent substrate. In principle, the membrane can be applied to the carrier from a suitable non-aqueous volatile solvent, such as methanol, ethanol, propanol, dichloromethane, chloroform, trichloroethane, benzene or toluene. However, the most preferred

260,663 showed the coating of a membrane from a mixture of non-aqueous solvents consisting of a lower aliphatic alcohol such as methanol and/or ethanol, a lower halogenated alkane such as dichloromethane, chloroform or trichloroethane and benzene or toluene.

The ratio of the individual components of these solvents in the said mixture is not critical and can be varied more or less arbitrarily.

Usually the membrane is applied to the support as the last impregnation, i.e. after the application of all reagents and substances necessary for the course of the actual analytical reaction. However, it is possible - and in some cases advantageous - to add some of these analytical reagents to the solution for forming the membrane. These possibilities, together with the general method of preparing the composition according to the invention, are illustrated in more detail by some typical examples given below.

Blood glucose test strips

1) Impregnation solution 1 Ethanol 24.0 ml Acetone 42.0 ml Methanol 16.0 ml rain water* 16.0 ml 3,315,5-Tetramethylbenzidine 0.72 grams Sodium dioctyl sulfo succinate o,ia g Polyvinylpyrrolidone K 90 1.20 grams Impregnation solution II Glucose oxidase, 28 U/mg 0.24 grams Peroxidase, 100 U/mg 0.10g Citrate buffer, pH 5.3 20 .0 ml Polyvinylpyrrolidone K 90 t»0 g sodium lauryl sulfate 0.04 grams Gelatin, 1.5% aqueous solution 20.0 ml Water dist. 26.0 ml Methanol 12.0 ml

Impregnation solution III

Hydroxypropyl cellulose,

5.3% solution in a 2:1 mixture of trichloroethane and methanol Nitrocellulose, 6.8% solution in toluene Polyvinyl acetate, 2.9% solution in ethanol

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43.0ml 28.0ml 28.0ml

The individual impregnation solutions are prepared by dissolving or, if necessary, mixing the individual components in the order listed and applied so that the paper is just saturated with the impregnation solution.

After each impregnation, the paper is thoroughly dried with a stream of hot air. The dried, creamy white paper is then cut into 6x6 mm squares and glued - for example using double-sided self-adhesive tape - to the end of a white plastic strip measuring 6x80 mm. If a drop of capillary blood is applied to the indicator area prepared in this way, the blood elements can be removed from the surface of the strip after 3θ β, for example by rinsing with water or wiping with moistened cellulose cotton, and the pure blue color of the area, the intensity of which is proportional to the glucose content in the blood being examined, can be evaluated, for example by visually comparing it with a color comparison scale made on an experimental basis. These strips indicate the concentration of glucose in the blood in the range from 1 to 8 mmol/l and are therefore suitable for determining severe hypoglycemia through normal values up to slightly elevated hyperglycemia.

2) Blood glucose test strips

Impregnation solution I

Sodium dihydrogen phosphate 0.08 g

Sodium hydrogen phosphate (12 HgO) 1.33 g

Ethylenediaminetetraacetic acid, tetrasodium salt 2.28 g

Water distilled· 57.0 ml

Gelatin, 1.5% aqueous solution 38.0 ml

Glucose oxidase, 28 U/mg 3.04 g

Peroxidase, 100 U/mg, 1.8 aqueous solution 5.7 ml

- 8 Methanol 68.0 ml

Distilled water 24.0 ml

1-(4-sulfophenyl)-3-methyl-5-pyrazo Ion 2.16 g

4-aminoantipyrine 0.63 g

Sodium dioctyl sulfosuccinate,

2% solution in ethanol 50.0 ml Tartrazine, 1% aqueous solution 4.8 ml

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Impregnation solution II

Impregnation solution III

Methylhydroxymethyl cellulose,

3% solution in a mixture of methanol-dichloromethane 1:1 20.0 ml

Ethylcellulose, 4% solution in toluene 15.0 ml

Polyvinylbutyral B 20 H, 5% solution 17.5 ml in ethanol

All impregnation solutions are applied to paper with a surface weight of 180 g/m in the manner described in Example 1, according to which further processing of the impregnated paper on the strip is also carried out. After applying a drop of capillary blood to their indicator area, it is possible to remove the blood elements from the surface of the indicator area after 30 s and evaluate after another 30 - 60 s - for example by comparing with a trial-made color comparison scale - the intensity of the resulting, pure red color, which is directly proportional to the concentration of glucose in the blood in the range of approximately 4 - 45 mmol/l. The strips are therefore suitable for determining glucose in individuals with slightly reduced blood glucose levels up to highly pathological hyperglycemia, which occurs, for example, in severe, uncompensated diabetes.

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- 9 3) Strips for determining total bilirubin in blood

Impregnation solution I

2,4 - Dichlorobenzenediazonium- 0.6g tetrafluoroborate Dyphylline 10.8 grams Metaphosphoric acid 3.7 grams Slajelic acid 4.2 grams sodium lauryl sulfate 0.3g Polyvinylpyrrolidone K 90, 4.7 grams Eoda dest· 100.0 ml Impregnation solution II Hydroxyethylcellulose,4 * 4% 35.0 ml solution in ethanol-chloroform 1.3»1 Ethylcellulose, 5.7% solution solution 35.0 ml

in benzene

The analytical agent is prepared by successively applying both impregnation solutions to 180 g/m2 paper and further processing the impregnated and dried paper according to the procedure given in Example 1). The strips obtained, used for the determination of bilirubin in blood, are used by applying 20 µl of blood to the indicator area and after 30 s removing the blood elements from the surface of the indicator area by wiping with moistened cellulose cotton wool. After another 90 s, the resulting reddish-purple color of the indicator area is evaluated again either visually by comparison with a color scale, experimentally made, or by measuring the reflectance using a suitable reflection photometer. The dependence between the measured values of the reflection density and the concentration of bilirubin in blood is given in the attached graph. No. 1.

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4) Blood urea nitrogen test strips

Impregnation solution I

Methylhydroxymethylcellulose,

4% aqueous solution 30.6 ml

Citrate buffer 0.3 mol, pH 5.2 20.4 ml

Cetylpyridinium bromide 0.255 g

Glycine 0.082 g

2,3 - Dithioerythritol 0.086 g

Urease, 2800 U/g 0.50 g

Impregnation solution II Methylhydroxymethylcellulose,

3% solution in a mixture of methanol and dichloromethane 1:1 20 ml

Ethyl cellulose, 2% solution in toluene 15 ml

Polyvinyl butyral B 20 H, 2.5% solution in ethanol 17.5 ml Bromothymol blue 0.026 g

Impregnation solutions are prepared by gradual mixing or

by dissolving the individual components in the above order and then applying them to 180 g/m paper in the manner described in example no. 1. However, impregnation solution II is applied twice.

After applying a drop of capillary or venous blood to the strip and removing the erythrocytes 30 seconds after application, it is possible to visually evaluate the resulting blue coloration with relative accuracy, the shade and intensity of which is proportional to the amount of urea in the examined blood in the concentration range of 1.5 - 40 mmol/l.

5) Urine glucose test strips

Impregnation solution I

Citrate buffer, pH5.23 34.0 ml

Gelatin, 1.5% aqueous solution 18.0 ml

Glucose oxidase (22 U/g) 0.99 g

Peroxidase (95 U/mg), 1.18% aqueous solution 2.545 ml

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Impregnation solution II

Methanol 40.0 ml

1-phenyl-3-methyl-5-pyrazolone 2.14 g

4-aminoantipyrine 0.94 g

Water distilled 30.0 ml

Polyvinylpyrrolidone K 90, 74.0 ml

5% aqueous solution

Sodium lauryl sulfate 0.6 g o-Dianisidine 0.92 g

Tartrazine, 1% aqueous solution 3.6 ml

Impregnation solution III

Composition according to example 2), but diluted with ethanol in a ratio of 1:9.

These strips are used to determine glucose in urine, which is detected by the formation of a red to dark brown color in the concentration range of 0.35 - 30 g/l. Compared to strips of the same composition, but without III. impregnation, i.e. without a semipermeable membrane, they show significantly increased stability to air humidity with the same reactivity to glucose.

Claims (1)

OBJECT OF THE INVENTION Analytical means for the diagnosis of significant components of body fluids, in particular blood, consisting of a water-borne carrier containing all the reagents necessary for carrying out the analysis, characterized by a semipermeable membrane formed of a mixture of hydrophilic, water-soluble cellulose hydro-alkyl ether, such as hydroxymethyl- hydroxypropyl or methylhydroxypropylcellulose and hydrophobic, water-insoluble polymeric substances from the group of cellulose ethers or esters, such as methyl, ethyl or nitrocellulose and / or polyvinyl alcohol esters or acetals, such as polyvinyl acetate, polyvinylpropralionate or polyvinylbutyralpropionate, polyvinylbutyralpropionate or polyvinylbutyralpropionate. Price: 2,40 Kčs Printed by Moravian printing works, center. 11 100, the class of People's Militia 3, Olomouc