Diagnostic agent for the detection of component materials in liquid and process for producing same

Diagnostic agent for the detection of component materials, in liquids and method of production. The diagnostic agent includes a water resistant film composed of a film-former and contains a film opener in the form of fine, insoluble inorganic or organic particles.

The present invention is concerned with a diagnostic agent for the 
detection of components in liquids. 
The detection of component materials of liquids by means of test strips is 
of ever increasing importance. In many cases, they provide a simple, 
economical and rapid detection process. Test strips are widely used in 
urine diagnosis for qualitative and semiquantitative detection processes. 
Special test strips can also be used for the detection of component 
materials of blood and serum in medical diagnosis. Furthermore, test 
strips are widely used for investigating drinks, drinking water, waste 
water and other liquids occurring in industry. 
One difficulty for the quantitative evaluation of the detection methods, 
especially for high molecular weight and corpuscular components of 
liquids, on the basis of test strips has hitherto been the fact that paper 
has been used almost exclusively as absorbent carrier. By way of example, 
mention may be made of Tallqvist's method of determination of haemoglobin. 
The homogeneity with regard to layer thickness, structure and composition, 
which is necessary for quantitative tests, is difficult to achieve in the 
case of papers. Furthermore, when using papers for quantitative tests, it 
has proved to be disadvantageous that an exact measurement of the sample 
material is frequently necessary. 
A great advance in the quantitative determination of low molecular weight 
materials was provided by the use of films according to Federal Republic 
of Germany Patent Specification No. 1,598,153. The properties of these 
films can be adapted to the particular analytical process. They do not 
require a precise measurement of the material to be investigated and react 
uniformly with urine, plasma, serum and whole blood. After a short 
reaction period, excess sample material is simply wiped off. Because of 
the small pore size of these films, they permit a separation of dispersed 
or suspended components in the solution to be investigated, for example of 
erythrocytes in blood, from low molecular weight, dissolved component 
materials. 
For the improvement of their properties, these films can also contain small 
amounts of adjuvants, for example chalk or titanium dioxide, for 
increasing the remission. 
However, these films cannot be used when cellular components or large 
molecules, for example enzymes, are to be determined. Such substances do 
not penetrate or do not penetrate sufficiently into the film in order to 
bring about a measurable reaction. For this reason, with this film 
principle, it is not possible to produce tests which can be used, for 
example, for the detection of haemoglobin, cholesterol in lipoproteins or 
enzymes. 
Surprisingly, it has now been found that absorbent, "open" films are 
obtained when solid materials in the form of fine, insoluble organic or 
inorganic particles are added to aqueous dispersions or organic solutions 
of film-forming substances from which water-insoluble films are to be 
formed. 
Thus, according to the present invention, there is provided a diagnostic 
agent for the detection of component materials in liquids, comprising a 
water-resistant film containing a film opener in the form of fine, 
insoluble inorganic or organic particles and the reagents necessary for 
the detection. 
Since the solid material does not itself "react", its composition is not 
critical and use can be made, for example, of cellulose, kieselguhr, 
silica gel, precipitated gypsum, calcium carbonate, kaolin, a polyamide, 
glass or the like. 
The ratio of this solid material, which is herein called a "film opener", 
to the film former can be 20:1 to 0.5:1 and preferably 5:1 to 1:1. The 
ratio depends upon the nature of the film opener and the film former used, 
as well as upon the intended use. With increasing amounts of film opener 
and increasing specific surface area of the material employed, the film 
becomes more absorbent. 
When the diagnostic agent according to the present invention is to be used 
for the detection of high molecular weight and corpuscular materials, the 
ratio of film opener to film former is preferably 1:1 to 20:1 and more 
preferably 2:1 to 5:1 and when it is to be used for the detection of low 
to medium molecular weight substances, the ratio of film opener to film 
former is preferably 0.5:1 to 2:1. 
If the proportion of film opener exceeds a certain limit, then the film 
becomes mechanically unstable. If too little film opener is added to the 
film, then it is impermeable to high molecular weight and cellular 
components. Thus, for example, the ratio of film opener to film former is 
preferably 5:1 to 2:1 when polyamide or precipitated gypsum is used as 
film opener and an aqueous dispersion of polyvinyl propionate is used as 
film former. If, on the other hand, kieselguhr is used, then the ratio 
should preferably be 2:1 to 1:1. 
Preferred film formers include organic synthetic resins, such as polyvinyl 
esters, polyvinyl acetals, polyacrylic esters, polymethacrylic acid, 
polyacrylamides, polyamides, polystyrene and co-polymers of, for example, 
butadiene and styrene and of maleic acid esters and vinyl acetate. 
However, other film-forming, natural and synthetic organic polymers, as 
well as mixtures thereof, can be used, preferably in the form of aqueous 
dispersions. However, the film formers can also be dissolved in organic 
solvents, for example a co-polymer of vinyl chloride and vinyl propionate 
can be dissolved in ethyl acetate. 
The dispersions or solutions can be coated on a substrate to give a uniform 
layer which, after drying, gives a water-resistant film. 
A special advantage of the diagnostic agents according to the present 
invention in comparison with absorbent papers is that the films can be 
produced more simply, more uniformly and reproducably. The film can be 
used with the substrate as a carrier or, for carrying out the detection 
reaction, can be pulled off the substrate and/or applied to another 
carrier. Carriers for the coated films are preferably synthetic resin 
films. However, other films and foils, papers, synthetic resin plates, 
glass, metal and the like can also be used as carriers if it is 
appropriate for the purpose of use. 
The present invention also provides a process for the production of a 
diagnostic agent, wherein a solution or dispersion of a film former in an 
appropriate liquid is mixed with a film opener and optionally with the 
necessary reagents and adjuvants, the mixture is applied to a substrate to 
give a thin film and the liquid is evaporated, whereafter the resultant 
film is, if necessary, impregnated with reagents and adjuvants. 
The reagents necessary for the detection reaction are normally added 
directly to the dispersion. Insofar as it is advantageous to do so, the 
formed film can, however, also be impregnated with them. A preimpregnation 
of the film opener with the reagents is also possible. The processes can 
also be combined in such a manner that, for example, certain components 
are added to the dispersion and the others are subsequently impregnated 
into the film. In this way, a certain spatial separation of the components 
is achieved, which can result in more stable or more reactive tests. A 
further possibility of separating formulation components from one another 
is to distribute them among various coating masses which are then coated 
on in the optimum sequence one after the other so that a multi-layer 
system is obtained. 
If necessary, thickening agents, emulsifiers, dispersion agents, pigments, 
for example titanium dioxide, plasticisers, wetting agents and the like 
can also be added. 
Dispersion agents, emulsifiers and thickening agents serve for the 
production and stabilization of the dispersions. Pigments, for example 
titanium dioxide, which can also function as film openers, improve the 
remission properties of the films by providing for the smallest possible 
transparency and an increased remission of the films. This is of 
particular advantage when test agents so obtained are to be evaluated by 
remission photometry. The use of plasticizers makes it possible to obtain 
film coating masses, as well as films, with optimum properties. Thus, for 
example, their stability, their viscosity, their adhesion to the substrate 
to be coated and the like can be improved. Wetting agents are used in 
order to achieve a better wetting of the film by the sample material. At 
the same time, they can catalyze reactions or stabilize formulations or 
make the reaction colors more brilliant and with greater contrast. 
Although the above-described films were preferably developed for the 
detection of high molecular weight and corpuscular components, they can, 
of course, also be used for the detection and determination of low 
molecular weight compounds. In comparison with the films according to 
Federal Republic of Germany Patent Specification No. 1,598,153, they have 
the advantage of sucking up the solution to be investigated more strongly 
and quickly and thereby of showing more intensive reactions. With 
appropriate choice of the ratio of film opener to film former, which, in 
addition, also depends upon the nature of these materials. for example of 
0.5:1 to 2:1, it is, in some cases, possible to suppress the penetration 
of high molecular weight materials into the film, which otherwise could 
give rise to disturbances, and only to detect or determine low molecular 
weight materials. The diagnostic agents according to the present invention 
are preferably employed for the detection of component materials in body 
fluids, for example urine, blood, serum and saliva. However, in suitably 
modified form, they can also be used in other aqueous media, for example, 
drinking water, waste water and the like, and possibly also in organic 
solvents in which they are insoluble. 
A great advantage of the diagnostic agents according to the present 
invention, which contain the required reagents, is that they can be 
readily adapted to the intended purpose of use by selection of the most 
appropriate film formers, film openers and the like. A further great 
advantage is that a precise measurement of the sample can be omitted 
because the film itself measures the sample material, excess sample being 
simply wiped off after a definite reaction time.

The following Examples are given for the purpose of illustrating the 
present invention: 
EXAMPLE 1 
For the production of a reagent film for the detection of cholesterol in 
serum, a dispersion was prepared with the following composition: 
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cellulose 5 g. 
polyvinyl propionate dispersion 
3 g. 
(50% in water) 
methylhydroxypropyl-cellulose 
0.042 g. 
titanium dioxide powder 2 g. 
cholesterol esterase 1200 U 
cholesterol oxidase 800 U 
peroxidase 26,000 U 
gallic acid 0.0032 g. 
a solution of 0.2 g. 3,3',5,5'- 
tetramethylbenzidine and 0.17 g. 
dioctyl sodium sulphosuccinate 
1 ml. 
in 0.74 ml. acetone 
potassium dihydrogen phosphate 
0.049 g. 
disodium hydrogen phosphate dihydrate 
0.167 g. 
distilled water 19.5 ml. 
______________________________________ 
The mixture was applied to a polycarbonate film in a 300.mu. thick layer 
and subsequently dried with warm air. The reagent layer thus obtained 
gave, with cholesterol-containing sera, well graduated, blue colorations 
dependent upon the concentration of cholesterol. 
After adjustment of a remission photometer ("Reflomat") provided with a 
linear scale with an unused test strip to the 0 scale reading and with a 
black foil to the 100 scale reading, for the blue colorations 
corresponding to the cholesterol concentrations, there were obtained the 
following measurement values: 
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0 mg. 0 scale reading 
100 mg. 6 scale reading 
200 mg. 44 scale reading 
300 mg. 66 scale reading 
400 mg. 76 scale reading 
500 mg. 83 scale reading 
600 mg. 88 scale reading 
black 
control film 100 scale reading 
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EXAMPLE 2 
For a test for the detection of erythrocytes in urine, there was first 
prepared a film from the following mixture: 
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polyvinyl propionate dispersion 
50 g. 
(50% in water) 
kaolin 60 g. 
dioctyl sodium sulphosuccinate 
2 g. 
distilled water 140 ml. 
______________________________________ 
The mixture was applied in a 400.mu. thick layer on to a polycarbonate film 
and subsequently dried with warm air. The film so obtained was impregnated 
with the following Solutions I and II and, after each impregnation, dried 
with warm air. 
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Solution I 
______________________________________ 
ethylenediamine-tetraacetic acid 
1 g. 
disodium salt 
trisodium citrate dihydrate 
15.7 g. 
citric acid monohydrate 3.48 g. 
phosphoric acid trimorpholide 
30.5 g. 
benzolight yellow (C.I. direct yellow 
0.048 g. 
No. 12) 
distilled water 190 ml. 
______________________________________ 
3.2 g. 2,5-Dimethyl-2,5-dihydroperoxyhexane, dissolved in 60 ml. methanol, 
were added thereto. 
______________________________________ 
Solution II 
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3,3'5,5'-tetramethylbenzidine 
0.56 g. 
dioctyl sodium sulphosuccinate 
1.0 g. 
phenanthridine 1.4 g. 
phenyl semicarbazide 0.06 g. 
toluene 100 ml. 
methoxyethanol 10 ml. 
petroleum ether 90 ml. 
______________________________________ 
The film thus obtained gave green colorations with erythrocyte-containing 
urines. 
EXAMPLE 3 
A film for the detection of nitrite in urine was obtained in the following 
manner: 
Into the Solution (a) described below were stirred 10 g. cellulose, 
followed by filtering off and drying in warm air: 
______________________________________ 
Solution (a) 
______________________________________ 
3-hydroxy-1,2,3,4-tetrahydro- 
0.392 g. 
benzo-[h]-quinoline 
sulphanilamide 0.344 g. 
L-tartaric acid 5.00 g. 
methanol ad 200 ml. 
______________________________________ 
With the cellulose impregnated in this manner, there was produced the 
following coating mass: 
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impregnated cellulose 10.00 g. 
methylhydroxypropyl cellulose 
0.09 g. 
polyvinyl propionate dispersion 
5 g. 
(50% in water) 
10% solution of dioctyl sodium 
3 ml. 
sulphosuccinate in acetone 
distilled water 35 ml. 
______________________________________ 
A 400.mu. thick layer of the mass was applied to a polyester film and dried 
with warm air. The water-insoluble film gave red color reactions with 
nitrite-containing liquids, for example urine and industrial waste water. 
EXAMPLE 4 
For the detection of haemoglobin in blood, a reagent film was produced from 
the following mixture: 
______________________________________ 
cellulose 20 g. 
polyvinyl propionate dispersion 
15 g. 
(50% in water) 
dioctyl sodium sulphosuccinate 
0.3 g. 
0.5 mole phosphate buffer (pH 7) 
75 ml. 
______________________________________ 
The mixture was applied in a 200.mu. thick layer to a polyester film and 
dried with warm air. 
When, from a series of dilutions of blood with varying haemoglobin content, 
1 drop of each dilution was allowed to react for 1 minute on the test 
film, then, after wiping off the drop with wadding, graduated colorations 
were obtained. 
When the resultant colorations of the test strips were measured with a 
remission photometer (Zeiss PMQ 3) at 540 nm, then, in dependence upon the 
haemoglobin concentration, the following measurement values were obtained: 
______________________________________ 
g. haemoglobin/liter 
% remission 
______________________________________ 
53 46.1 
100 38.8 
150 31.7 
201 25.5 
______________________________________ 
EXAMPLE 5 
For the ascertainment of the limits of the process, the mixture of film 
opener and film former were, analogously to Example 4, varied and films 
obtained with the following properties: 
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properties in the case of 
reaction with erythrocytes 
film opener/film former 
in blood 
______________________________________ 
kieselguhr:polyvinyl propionate 
0.5:1 stable film, erythrocytes 
only penetrate at a few 
places 
1:1 stable film, uniform 
reaction of the whole film 
4:1 uniform reaction of the 
whole film; film material 
is partly removed by wiping 
off 
gypsum:polyvinyl propionate 
3:1 stable film, slightly 
colored 
4 to 10:1 stable film, uniformly 
deep coloration 
20:1 In the case of slight 
pressure, wipe-resistant 
film, uniform coloration, 
drops "chromatograph" on 
the edge 
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It will be understood that the specification and examples are illustrative, 
but not limitative of the present invention and that other embodiments 
within the spirit and scope of the invention will suggest themselves to 
those skilled in the art.