Note: Descriptions are shown in the official language in which they were submitted.
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The present invention relates to a method of making
a reagent test device, comprising a carrier and at least two
substances, supported by said carrier, to be activated upon
use of the reagent test device. The invention also comprises
a reagent test device, made according to this method.
Reagent test devices of the type mentioned above
have hitherto been maae in several different ways. In ac-
cordance with one method, one of said substances is encapsu-
lated in so-called microcapsules that are suspended in a
li~uid containing the other substance, whereupon the micro-
capsules and the liquid are applied to a carrier in one way
or other. The manufacture of such reagent test devices is
rather expensive.
According to another method, a reagent test device
of the type mentioned above is made by impregnating a carrier
with a porous structure in two ~ones, separate from each
other, with liquids containing the substances. The manufac-
ture of such reagent test devices is complicated due to the
difficulties to impregnate one and the same carrier with
two different liquids.
The present invention has for an object to provide
a method of making a reagent test device of the type men
tioned abo~e, which method is simple and cheap, and which
results in reagent test devices which make possible a quanti-
tative analysis of high accuracy.
According to the present invention, in a method ofmaking a reagent test device of the type mentioned by way of
introduction, at least two liquids, each of which containin~
one of said substances, are applied directly to one surface
of the carriex in such a way, that said substances will re-
main on the surface separated by a predetermined interspace
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along said surface, printing techniques known per se being
used for obtainingiu}-e~e~, predetermined interspace.
t
Preferably at least one of said substances is ap-
plied to the carrier in such a way, that it remains fixed to
it even upon use of the reagent test device.
In accordance with one preferred embodiment of the
present invention the substances in question are applied to
said surface of the carrier on a plurality of locations,
spaced apart with small interspaces, for example as dots
and/or stripes on the surface. The locations may be mixed
on the surface.
The invention is applicable for several reagent
test or indicator systems. Reference is made, by way of ex-
ample only, to U.S. Patents Nos. 3,092,463, 3,511,608,
3,549,328 and 3,926,732.
As mentioned above, according to one preferred em-
bodiment of the invention the substances, acting when the
indicator device is used, are applied to a plurality of loca-
tions on the surface of the carrier with very small inter-
spaces between them. This does not mean, however, that thescope of the invention is limited to cases where interspaces
of microscopic orders are necessary. Even in such cases,
where the distance between the substances shall be somewhat
larger, e.g., about l mm, the invention applies. Such an
interaction between different substances on the suxface of a
reagent test device carrier is also possible where the inter-
space between the substances is of this si~e, if a liquid
that is to be brought into contact with the reagent test de-
vice carrier has to be able to penetrate through the sub-
stances to provide a diffusion means, for example for partof one of the substances on its way to the other substance.
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In such a case it is obvious that utmost accuracy is needed
regarding the interspace between said substances, such as an
accuracy of one or a few hundredths of a mm.
The present invention arose as the inventor just
by chance happened to get acquainted with the details of
con~entional printing techniques. The inventor found that ~-
a printed text or picture might consist of a plurality of
minute little dots, situated in microscopic distances from
each other, which distances normally cannot be detected by
the naked eye. The inventor also got aware, that a printed
picture in color, which the eye perceives as just being of
one color, in fact can consist of a plurality of dots of
different colors. Thus a picture which the eye perceives as
green can consist of a plurality of blue and yellow dots.
The knowledge on conventional printing techni~ues
gained by the inventor led her to realize the unique poten-
tial of printing techniques to apply two or more reagent
substances to the surface of a reagent test carrier, the
interspace be~ween said substances being accurately predeter-
mined.
Thus the reagent test device in accordance with
the present invention is made according to printing tech-
niques, known per se, like photogravure printing, which
maans that the substances, dissol~ed in appropriate solvents,
are applied to the surface of the carrier by printing rollers,
provided with very small depressions or pores, with differ-
ent depths.
Silk screening is also part of well-known printing
techniques. This means that the substance in question is
dissolved in an appropriate solvent, which is pressed out
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through a screen provided with fine meshes. The screen is
placed around a rotatable roller.
There are several types of printing techniques,
known per se, which are not generally called "conventional".
There are, for example, different kinds of plateless printing
techniques, like direct electrostatic printing, indirect
electrostatic printing and ink-jet printing, which have
gained more and more importance lately.
Direct electrostatic printing means, that electro-
static charges are created direct and held on specially
coated paper which has a conducting layer covered by an in-
sulating layer. The electrostatic charge is developed into
visible image by a toner, which can be a liquid containing
the desired reagent substance.
Indirect electrostatic printing is an offset pro-
cess where the electrostatic charge is held on an intermedi-
ate surface (such as a drum) and only the toner, containing
the desired reagent substance, is transferred and fixed to
the paper. This method is used in the Xerox ~ copying sys-
tem.
Ink-jet printing has developed very fast lately.
There are many different systems, but they all depend on
continuous or discontinuous flows of very thin liquid jets,
that are directed with great accuracy in the desired direc-
tion toward the carrier in question.
An~ type of reproducing graphic techniques may be
used to perform the present invention.
Regarding the embodiment of the invention, accord-
ing to which the substances are applied to a plurality of
lGcations with very small interspaces, it should be noted
that there is an advantage that a color change, due to the
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activating of the substances as the reagent test device is
being used, will be perceived as a simultaneous color change
over a relatively large area. (The surrounding parts of the
reagent test device carrier should preferably have the same
color as said surface had before the color change.;) Such a
reagent test device, for example comprising two substances,
will give a more safe indication of an occurred reaction than
a reagent test device with one surface covered with one of
the substances only, which gradually changes its color from
one part to another part, as the second substance is diffus-
ing along the surface. Thus, i~ said substance is complete-
ly cons~med before it has diffused over the entire surface
area, whereby a color change will occur in just part of the
surface area, there may arise doubt as to the reliability
of the reagent test device. The possiblity of using a re-
agent test device for quantitative analysis is also improved,
if a color change occurs over a relatively large surface
area.
The present invention will now be described more
in detail, re~erence being made to the enclosed drawing.
Figs. 1 4 show schematically different embodiments of a re-
agent test device, made according to the invention. Fig. 5
discloses, by way of example only, schematically the prepa-
ration of an indicator device in accordance with Figs. 1-4.
Referring to Figs. 1-4, three different reagent
test devices are shown, for example intended for indication
of any occurrence of a certain enzyme in a liquid. The re-
agent test devices comprise carriers 1, 2, 3 and 4. Two
reagent substances A~ B have been printed with conventional
printing technique in various patterns on these carriers.
A reagent test device of this kind may be intended for
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dipping into a sample of said liquid, removing it from same
to let a thin liquid layer remain on the carrier. One of
the substances on the carrier, for example substance A, may
then be brought to diffuse through the thin liquid layer,
toward the other substance, i.e., substance B. The enzyme
to be indicated in the liquid may react with substance A, or
catalyze a chemical reaction caused by substance A. Thus
substance A is completely or partly consumed during its way
toward substance B dependent on the concentration of said
en~yme in the liquid. If substance A is completely consumed
by the enzyme in the liquid, no reaction can occur between
substance A and B. If part of substance A reached substance
B, these will react, substance A and B being of such a
nature that the color will change. Such a reaction will
cause the human eye to notice a color change over the total
surface area, onto which substances A and B are applied.
The intensity of this color change is dependent on the con-
centration of the enzyme to be quantitatively estimated in
the liquid. Substances A and B of course can interact in
any other way. For example, they may react with each other
in a first stage, giving rise to an intermediate substance
without any color change. Then in a second stage this
intermediate compound may react with any enzyme present to
bring about a color change, Alternatively, there may occur
a first color change as the intermediate substance is
formed, and a second color change when the intermediate sub-
stance reacts with the enzyme. Such a system would make it
possible to decide safely if a reagent test device has al-
ready been used, even if the enzyme reaction would not
occur due to the non-existence of the enzyme,
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In Fig. 4 a reagent test device for the indication
of any catalase enzyme present in a liquid is shown. The
reagent test device comprises a carrier 4 and substances A,
B and C applied to it with con~entional printing technique.
Substance ~ contains a peroxidase enzyme and a dye
like o-tolidine. Substance B contains the enzyme glucose
oxidase and substance C glucose. Enz~mes peroxidase and
glucose oxidase are both chemically fixed to cellulose par-
ticles, which are fixed to the carrier by means of any suit-
able binding agent using printing technique. After the
application of substances A and B the enzymes are immobile
in relationship to each other and to carrier 4.
The reagent test device just described will operate
in the following way when brought into contact with liquid
to be tested.
(1) The glucose in substance C is dissolved
by the liquid and is spread over carrier 4.
(2) In the vicinity of substance B enzyme
glucose oxidase catalyses the reaction between
glucose and oxygen, whereby hydrogen peroxide is
obtained as a reaction product,
(3) The hydrogen peroxide diffuses through the
liquid provided in the a~æas between substances A
and B, to the locations of substance A.
(4) At the locations of substance A the dye
o~tolidine is oxidized by hydrogen peroxide in the
presencenof enzyme peroxidase, and a blue color is
obtained.
~5) If there is any enzyme catalase present
in the liquid, the hydrogen peroxide is decomposed
completely or partly. CatalaSe in a high
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concentration in the liquid will decompose the hydrogen
peroxide completely before it has had time to diffuse to
the locations of substance A, and no blue color is obtain~d.¦
A small concentration of catalase will decompose the - '
hydrogen peroxide partly, and part of the hydrogen peroxide
will diffuse to the loaations of substance A to cause a
blue color. Thus, due to the concentration of enzyme
catalase in the liquid a more or less intensive color
change is obtained on the reagent test device. t
The reagent test devices disclosed in Figs. 1-4 are
prepared by printing techniques, as mentioned above. Reference
is made to Fig. 5, which shows the principle of conventional
printing. A sheet 5 is fed in the direction of the arrown With
two printing rollers 6 and 7 two different liquids, containing
substances A and B, are printed on the sheet. As already mentioned
there are many types of rollers, well known to those versed in
the art, so no detailed description is needed.
Of course, it is necessary to adjust the viscosity
of the liquids, containing the substances in question, dependent
20 on the printing technique chosen.
Example r
10 grams or particulate CMC (carboxi methyl cellulose)
were activa~d in the way well known to those skilled in the art. -
Glucose oxidase (Boehringe~) and peroxidase (Sigma Co)
were immobilized on different samples of the activated CMC, also
according to well known methods. The mixtures for printi~ were
prepared in the following way: ~
Glucose oxidase/CMC
2.5 grams of wet GO/CMC were stirred in 20 mls of -
30 distilled water with the air of a magnetic stirrer 0.085 grams of
colloidal CMC were added to adjust the viscosity of the mixture.
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.. .".,. ~
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Peroxidase/CMC
2.5 grams of wet PO/CMC wer stirred in 20 mls of distilled
water with the aid of a magnetic stirrer. 0.085 grams of
colloidal CMC were added to adjust the viscosity of the mixture.
0.033 grams of o-tolidine were added by stirring to the mixture.
The two mixtures thus obtained were printed by silk
screening as distinct parallel lines, according to the system
GO-PO-GO-PO etc, on a filter paper, that had been immersed in
a 10% gluclose solution in water and had been dried at 35C.
j:
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