DD224951A1 - ANALYTICAL MATERIAL FOR DETERMINING H LOW 2 O LOW 2, ORGANIC PEROXIDES AND H LOW 2 O LOW 2-FABRIC MIXTURES - Google Patents

Abstract

The invention relates to an analytical material for the determination of H2O2, organic peroxides and H2O2-forming mixtures, which is preferably used in medical diagnostics for the determination of glucose in body fluids. The material should be easy and reproducible to handle and suitable for screening and patient self-monitoring. On a transparent carrier layer there is only one swellable indicator layer, which contains the indicator system in a diffusion-resistant manner and has good physical-mechanical properties. The indicator layer contains as film former gelatin and a polycation of the general formula I, as indicator system a p-phenylenediamine derivative and a coupler with a CH-acid group and as gelatin crosslinking agent Cr (III) salts. The polycation forms salts with the indicator system.

Description

Analytical material for the determination of HpO ₂ , organic peroxides and H ₂ O-forming substances

Field of application of the invention

The invention relates to a material for the determination of H ₂ O ₂ , organic peroxides and H-Op-forming substance mixtures, which finds application in industry, in agriculture, in environmental protection, but preferably for glucose determination in medical diagnostics.

Characteristic of the known technical solutions

Materials for the determination of organic peroxides and H 0 are well known in practice. Increasingly, these analytical methods of determination are subject to rapid or strip tests which allow a quantitative or samiquantitative determination by means of color reactions in the swollen solid indicator layers. Here, two principal methods have prevailed, which differ by the nature of the layer formers for the indicator layers. Often, paper is used as fibrillar layer former (DE-OS 2546252, US-PS 3092465,

DD-PS 143379).

Increasingly, due to the requirement of quantitative evaluation, however, thin polymer layers settle as carrier material, which are produced from film-forming natural or synthetic polymers (DE-OS 2910134, US-PS 3992158, DD-PS 140175). These conforming polymers make it possible to reproducibly produce layer thickness-defined indicator layers, a prerequisite for quantitative test strips. As support for the indicator layer, for example, transparent support materials in the form of films are used. The desired reflection is achieved by applying a pigmented layer, which usually still acts as a filter and distributor layer (US Pat. No. 4,050,898). Common to these systems is that the H O or organic peroxide to be detected, in a reaction catalyzed by a peroxidatively active compound, forms colorants whose color density is proportional to the HO ₂ or peroxide concentration, thereby permitting photometric or visual quantification. The detection can be carried out by a one- or two-stage reaction. In the single-stage process, a colorless indicator is usually present in the reaction layer, which is converted via the intermediate stage of the indicator oxidation product into a more or less stable seraichinoid form. In Zweistufsn- prozaß first the indicator oxidation product is also formed, in which case, however, the dye formation is carried out by reaction with corresponding couplers to form kova-Isnter bonds between indicator oxidation product and coupler, lsi the first case as an indicator bisphenyl derivatives, such as o-tolidine and o-dianisidine and in the second case substituted p-phenylandiamines and as coupler z, B. substituted naphthols or pyrazolones (DO-PS 135243) are used.

For the system of bisphenyl derivatives, it has proved to be advantageous to stabilize the dye system by adding so-called fixing agents (VVP C Ol N / 236 500, WP C 01 N / 244.218).

As a laminating agent for these indicator systems, gelatin has become increasingly prevalent because, in addition to its good protective colloid effect, it offers the advantage of thermoreversibility of its gels, which substantially facilitates the production of the materials and their handling. (DD-PS 135 843, 140 175, 145 428).

Critical in these systems is the use of Q-tolidine or o-oianisidine as indicators because they are highly toxic and known as carcinogens. With insufficient diffusion resistance in the layer, these compounds can deposit on the surface of the materials, creating a hazard in their application. "AuSerdsra has a clear diffusion of these indicators into the liquids during the application of the liquids to be examined, which leads to a loss of indicator in the detection layer" and the accuracy of the matrial.

Similar disadvantages have become known from materials in which a water-soluble dye is formed as a result of the detection reaction. Here, large portions of the dye are bound by the pigmented topmost distribution layer present in these materials and are not detected by reflected light measurement. Ss has been attempted to solve the problem by adding Beizraltteln (US-PS 3585 112 3917 _r 453). By this procedure, the problem could not be solved satisfactorily, so that a practical application is out of the question.

An additional layer as a diffusion barrier for the water-soluble dye (DE-PS 2834 713) to prevent losses. In addition to the necessary application of another layer, these materials require very exact evaluation conditions due to the dye diffusion. which can only be guaranteed by machines.

Fast patient self-monitoring is not possible here. When using p-phenylenedianiinderivaten and couplers with OH-acidic groups and long-chain alkyl radicals as indicator system diffusion processes are also not completely excluded and the color formation process is reduced »Further disadvantages are caused by the hydrophilic couplers, which cause significant viscosity increases in the presence of gelatin (Ewa E *, Bertyp.v Colloid «Z. 149 (1956)). As a result, the required reproducible production of thin indicator layers is impossible. This effect is particularly pronounced with increasing pH. The coupler also significantly embrittles the detection layer, which, for example, results in an undesired tendency of the materials to move, making their handling difficult or impossible. Gelatine itself exhibits different brittleness at different humidities, which also results in a constant variation in the quality of the physico-mechanical properties. By applying extremely strong gelatin coatings, it is possible to counteract the deposition of the indicators on the surface, the uncontrolled diffusion of the indicator in the coating remains however. In addition, the problems of breakage become even more prominent. "These materials are usually unsuitable for a whole blood determination, which is essential in the care of diabetis mellitus.

Object of the invention

The aim of the invention is an analytical material for the determination of H ^ O _2, organic peroxides and H_0 ₂ -forming Stoffgeraischen, in particular for Glucosebestiaraung in body fluids, ÖAS material is simple and reproducible to handle in the form of test strips and be suitable for screening and patient self-testing,

Explanation of the essence of the invention

The invention is based on the object ,, by using suitable components to develop an analytical material for the determination of HO ₂ , organic peroxides and H ₂ 0 ₂ -forming mixtures whose Indikaorsystera is anchored diffusion-proof in the indicator layer · The material is good physical-meehanische Have properties »be easy to handle and show a stable reproducible color reaction during use ·

According to the invention the object is achieved in that the indicator layer as a layer former gelatin tnd a PoIykation the general

Formula I

CHR - CR - CR "- CHR ₀ 2 j 3 j 4 2

CH, CH,

wherein R - * R ₂ , R ₃ , R ₄

^R 5 ' ^R 6 η

nCl "η

= H, alkyl of 1-6 carbon atoms a alkyl of 1-6 carbon atoms i 50-1000

mean,

as an indicator system for an interstage process, a p-

Phenylenediamine derivative of the general

Formula II

X-R

, _t

R a is alkyl of 1-8 carbon atoms

X = H, Y

Y = ^-SO ₃ H, · -COOH

mean and a coupler of the general

OH formula III-si ** ^ - ^^ - Z-

wherein Z = -CONH-, - ₂

R = alkyl radical having 12-20 carbon atoms, if appropriate substituted phenyl R- = alkyl radical having 1-20 carbon atoms U = H, -SO ₃ H, -COOH

or a coupler of the general formula IV CH -CZR _±

wherein Z = -CH ₂ -, -NH-CO-

R ₁ = alkyl radical having 10 to 20 carbon atoms R ₂ a substituted phenyl radical containing as substituent -SO ₃ H or -COOH

mean and

gelatin crosslinking agent containing a chromium (III) compound.

An essential advantage of the material according to the invention is that only one layer is applied to the substrate, while other known materials consist of several layers.

Preferably, the material is used in the form of test strips. As a transparent substrate is suitable only hydrophilized Filoanterlags, other transparent films are also suitable.

In the indicator layer, the gelatin can be replaced within wide limits by a polycation according to the invention. Polycations of general formula I having a degree of polymerization of η => 300-600, which have good compatibility with gelatin in aqueous solution, are preferably suitable, so that neither turbidity phenomena nor viscosity changes occur. Gelatin / polycation layers have better physical-mechanical properties than pure gelatin layers. Instead of the polycation of the formula I, it is also possible to use copolymers of vinyl monomers, such as acrylic acid, acrylamide, acrylonitrile, diacetic acrylamide or vinylpyrrolidone, and the monomer of the formula I. use.

The inventive polycation is capable of salting with the indicator system. This stain effect prevents diffusion of the p-phenylenediamine derivative and the coupler from the layer during application of the sample solution, so that a quantitative detection of the dye formed is ensured in the application

 By addition of chromium (III) salts of

Formula V a)

NH

^{Cr | 0 = c} C '/ I ^C1 3 ·

b) K ₃ [cr (C ₂ O ₄ ) ₃ ]. 3 H ₂ OC) JCr Cl ₂ (H ₂ O) 1 Cl ₂ H ₂ O

e) JCr ₃ (OH) ₂ (CH ₃ COO) ~ | CH ₃ -COO.6H ₂ O

f) ^ r ₃ (OHJ ₂ (CH ₃ COO) 5 Cl. 8 H ₂ O

g) [(NH ₂ CH ₂ COO) ₂ Cr (OH) ₂ Cr (00CCH ₂ NH ₂ J ₂ ] .H ₃ O

but preferably Cr (CH ₃ -COO) ₃ , 6 HO

As a gelatin crosslinking agent, a defined liquid absorption rate of the indicator layer is achieved so that the HO ₂ or the content of the sample solution can be quantitatively determined on the basis of the density of the dye formed.

Transparent indicator layers are obtained which, after application of the sample, enable a visual evaluation of the resulting dye against a comparison scale or a measurement in a commercially available photometer. The azomethine or indophenol dyes formed in this indicator system are stable. Test and evaluation can be separated in time, which allows a wide application of the test strips.

In the presence of glucose oxidase and peroxidase in the indicator layer, the material is suitable for rapid determination of glucose in body fluids. Broad application of this test material is possible in medical diagnostics in screening and especially in the self-control of diabetis mellitus.

embodiments Beispiell

BegieSlösung A contains 50 g / kg gelatin BegieSlösung B-O = gelatin is gradually replaced by the polycation of

Formula Ia

CH-CH-CH-CH ₁ -

² Ii ²

CH CH-

CH.

CH

3 ^y

η = 300 (Table 1)

Begießlösung K contains 45 g / kg gelatin and 5 g / kg polycation according to. DE-PS 2,834,713 of

Formula Vi 4CH-CH "-CH-CH-1

CpH *)

Z ¹ Z

CH-

n Cl "

η = 250

The viscosity measurements of the Begießlösungen AK were carried out at 4O ⁰ C, the values obtained are shown in Table 1. To determine the permeability of the Begießlösungen AK were cast on hydrophilized Callulosetriacetatunterlage and measured the resulting layers on the meter Spekol VEB C.Zeiss Jena

 The layer thickness is 6, USi

 The results obtained are shown in Table 1.

Table 1

Begieß gelatin Ia / g / KGj polycation fresh ^ In the Pas] after 24 h Durchlässig- "" " solution fg / kg] Vl ^ g / KGj 7 ^ after 4 h 7.5 of the layer [% l A 50 5 - 7.7 7.7 8.0 100 B. 45 10 - 8.4 8.2 8.9 100 C 40 15 - 8.9 8.9 9.5 100 D 35 20 9.0 9.6 10.0 100 e 30 25  * * 9.2 9.9 10.0 100 F 25 30 mm 9.0 9.2 9.1 100 G 20 35 - 9.1 9.1 9.2 100 H 15 40 -. 9.0 9.3 9.0 100 I 10 45 ml 9.0 9.1 9.0 100 α 5 - "Μ 20,6 _; j 8.9 36.8 100 K 45 ", .., .., J 5 ,, 33.0 96

- 11 -

~ 12 -

In the pouring solutions, the polycation Ia has a good compatibility with gelatin and causes no turbidity and no increase in viscosity, so that a problem-free replacement of the gelatin by the polycation is possible within wide limits . The polycation V | (DE-PS 2,834,713) does not show this advantage. It causes an increase in viscosity, which increases with prolongation of Stehzait.

Example 2

To characterize the physico-mechanical properties, a pure gelatin layer and 3 other gelatin layers with increasing content of polycation Ia are prepared and measured, the values obtained are shown in Table 2

- 12 -

Table 2

Γ%] Gelatin [% ~ jpolykation Ia WRV |% Η ₂ θ7 Fp ( ⁰ C? D / X) Red/ 100 , 790 31 6 13ο ¹³ 95 5 460 45 16 10 ⁸ 90 10 490 45 24 10 ⁷ 80 20 510 46 30 10 ⁶

WRV a water retention capacity

D a Elongation (measurement at 30 % relative humidity) Ro a Surface resistance

The water retention capacity of the layer is not impaired by addition of the polycation Ia, the gel sol-Urawahdlungspunkt advantageously increases slightly and the brittleness is improved. In addition, an antistatic finish of the material results, which is always advantageous for polymer films.

Example 3

Production of the 3-layer solutions for indicator layers BegieSolutions L-S

Stratifier (gelatin and polycation of Forl © a) see Table 3

pimp

4- (NN-cf-sulfobutyl-buty! Ammonium J-aniline sulfate glucose oxidase peroxidase

Na-hexadecyl sulfohat chromium (III) acetate

3.7 g / kg

2.4 g / kg

100 000 U / kg 4 000 U / kg 0.03 g / kg 4.5 g / kg

Adjust to pH - 7.4 with sodium hydroxide solution. After 1 h of standing time, the viscosity is measured at 40 ° C

Table 3

film formers Polycation / g / kgj 6 pimp ^ / ra Pas / ' BegieSlösung Gei tine fg / kgj 12 1 46.2 L 60 5 IS 1 11.6 M 54 12 1 N 48 18 1 9.2 O 42 - 2 66.8 P 60 2 12.6 O. 54 2 9.1 R 48 2 8.9 S 42

- 14 -

Coupler 1 a 1- (3-sulfo-4-phenoxyphenyl) -3-carboxyhepta-

Decylamino-pyrazolone (5) Coupler 2 = 1-Hyciroxy-4-sulfo-2-naphthoic acid octa

decylatnid

The usual increase in viscosity of the BegieSiösungsn containing only gelatin as a layer former with the addition of the coupler is almost in the presence of the polycation of the formula Ia

inhibit * Compare the viscosities

the pouring solution A (Table 1) of the pouring solution L, .P (Table 3) and the pouring solution N, N ₁ O, Q, R, S (Table 3)

Example 4

Casting solution M according to example 3 is poured onto hydrophilised Ceilulasetriacetat JJterlag * is obtained

Material A,

As a comparison pouring solution L according to Example 3 ,: but without chromium (Ili) acetate shed on the same surface,

Haterial B.

Both materials determine the physico-mechanical properties, such as water retention, gel solubility and surface resistance. The dimensional stability, which is a measure of the required wiping resistance of the indicator layer, is assessed by the vulnerability of a layer through a measuring pin on which different masses are applied »The test for diffusion resistance of the Indikatorsystsms by first watering the material, then applied Glucoselösung and then the color density in the absorption maximum of the resulting dye was measured. '

Table 4

material WRV fei Mp [° C] D I «] tear] wet strength Loss of color density [%] another 10 '' 20 '' 3C water treatment A 220 70 20 10 ⁷ 500 0 0 2 B t *> ~~ - "- ¹ -" ** - · - 875 28 4 ".ν ..-. _ "I 10 ¹³ 50 4 14 20

Layer thickness of the indicator layer 6, Um

- 16 -

- OK -

Table 4 clearly shows the advantages of the material A according to the invention. The indicator system remains non-diffusible in the layer even during prolonged washing and allows quantitative glucose determinations. The good physical-mechanical properties allow easy handling of the Testraaterials in the application. Material B has much poorer physico-mechanical properties and does not allow quantitative glucose feed due to the diffusion of the indicator system.

- 17 -

Claims (1)

invention claim 1. Analytical material for the determination of HpO ₂ * organic peroxides and H ₅ O ₂ forming substance mixtures, consisting of a transparent substrate and an indicator layer, characterized in that the indicator layer as a layer former gelatin and a polycation of the general Formula I CHR. -CR ₃ -CR ₄ -CHR ₂ J- n Cl " ^R 5 " ^R 6 wherein R _± , R ₂ , R ₃ , ₄ = H, alkyl. With 1-5 carbon atoms h = 50-1000 R ₅ , R ₆ = alkyl with 1-δ carbon atoms mean, as Indikatorsystera for a two-stage process, a p-Phenylendiarainderivat the general Formula II X-R - 18 - wherein R, Rp = alkyl having 1-8 carbon atoms X = H, Y Y = - SO_H, -COOH mean and a coupler of the general OH Formula III «s =» -> ^ _r - ^ 5s, -ZR _H CO. * wherein Z = -CONH-, -CONR ₂ - Ra alkyl radical having 12-20 carbon atoms, optionally substituted phenyl radical R ₂ a alkyl radical having 1-20 carbon atoms U = H -, - SO ₃ H * -COOH mean or a coupler of the general Formula IV CH ₀ -CTR ' I ² P ¹ wherein Z = -CH ₂ -, -nh-CO- R a is an alkyl radical of IO-20 carbon atoms R is a substituted phenyl radical which, as a substituent, must contain --SO.sub.H or -COOH and, as gelatin crosslinking agent, contains a chromium (III) compound.