CH620300A5 - Reactive strip for analysis - Google Patents

Description

The subject of the invention is a test strip for analysis, in particular in diagnostics for the analysis of biological liquids, for the detection of a given constituent in a sample to be analyzed.

Test papers are well known. It is also in principle known to print a two-dimensional support, such as a strip of paper, of inks in discrete areas, see US Patent No. 3,549,328 where other literature is cited.

Known test strips intended for biological analyzes generally comprise an absorbent support impregnated with reagents which, when wetting with the liquid to be analyzed, produce a colored reaction, allowing the detection of a given constituent.

The conventional support material in most commonly used test strips is filter paper which provides advantages, but also has certain drawbacks and limitations such as insufficient physical and chemical stability of the paper support during its impregnation. with certain reagents having high concentrations in order to obtain a satisfactory color analysis reaction.

It has been proposed to make the filter papers more stable by laminating them with a synthetic resin film, or by using synthetic resin fibers in order either to reinforce the paper or to completely replace cellulose fibers. However, it has been found that cellulose fibers swell during impregnation and thus absorb the reagents within the fibers, while impregnation only allows a relatively small amount of the reactants to be deposited on the surface of synthetic fibers.

In addition to these limitations relating to the absorbent support material, the production of test strips by conventional methods generally requires multiple impregnation operations which are difficult to perform on a large scale in a highly reproducible manner and which also increase the cost of comes back in a big way.

The main object of the present invention is to provide a test strip, in particular for biological analyzes, and this on a large scale, in a highly reproducible and economical manner, so as to be able to avoid as much as possible the abovementioned drawbacks.

Thus, the reactive strip according to the invention is characterized in that it comprises a support material printed with two reactive inks, the printing by each of the inks being in the form of a network of points, the points of the first network being neighbors of those of the second network but not touching each other, and the two networks being at least partially superimposed, in that the two inks each contain

(a) water or a water-alcohol mixture as a solvent;

(b) a binder to give the ink rheological properties suitable for printing,

(c) a buffer capable of giving the ink, when it is wetted with the said sample, a specific pH, and

(d) a surfactant, and

(e) a different reagent for each ink,

these reagents being capable of reacting with each other in the presence of said constituent to be detected by producing a detectable change in color.

The production of these test strips, by means of printing, makes it possible to obtain various practical advantages, of which the following may be mentioned in particular:

- Possibility of easily separating different constituents of the reactive system, which can normally react with each other, by their inclusion in different reactive inks which are then used to print separate networks of dots on the surface of the sheet constituting the support of the strips reactive.

- Possibility of using a very simple and well reproducible printing technique to quickly apply exactly predetermined quantities of reagents to said support, thus making it possible to considerably simplify the production of the reactive strips and to make it more economical.

- Ability to eliminate the need to provide comparative colorimetric scales separately by also printing, on the same tape, the respective colors allowing the comparative analysis.

Various considerations, which are of practical interest in obtaining such advantages in accordance with the basic principles of the present invention, are discussed below.

Composition of reactive inks

It is understood that the composition of the respective reactive inks will depend first of all on the color analysis reaction which is to be carried out by the reactive bands, or on the system

60

3

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particular reagent which is necessary in order to obtain the desired analytical reaction in each case.

The constituents of each reactive ink must also be chosen so that they are chemically and physically compatible with each other and with the substrate on which printing is carried out with the reactive inks.

In order to ensure satisfactory printing, the physical properties of the reactive inks should however be adapted to the type of substrate as well as to the printing technique and means used to produce the reactive strips.

The term reactive ink as used herein implies an intimate mixture of at least one main reagent necessary for the color analysis reaction, with auxiliary constituents and an appropriate liquid vehicle or carrier, so that the 'an ink having rheological properties is obtained,. suitable for printing so as to ensure correct ink transfer to the substrate used for printing in each case.

The main reagents which will be included in the respective inks used for printing will obviously depend in each case on the particular type of analysis reaction for which the reactive strips will be manufactured.

The reagent system which will be used to obtain a color analysis reaction given on the strips manufactured in accordance with the invention may however comprise conventional reagents, which are known from the state of the art relating to diagnostic analysis reactions , known reagents being contained in the inks used in the examples described below to illustrate the invention.

All the constituents of each reactive ink must therefore be mutually compatible and they must also give the ink appropriate rheological properties to allow correct printing. The choice of an appropriate solvent and auxiliary constituents of the ink, other than the main reagent, will thus depend on various considerations discussed below.

Solvents

The function of the solvent in the reactive ink is to provide a liquid agent which is necessary to maintain the main reagent and the auxiliary constituents of the ink in solution or in suspension, until the ink is used for the printing on the substrate. After printing, the solvent has served its purpose and should be removed by drying to provide dry printed dots on the surface of the substrate.

When enzymes or other bioactive substances are contained in the composition of the reactive ink, the solvent will be water or an aqueous mixture of the water-ethanol type.

Binders

In order to give the inks the 5 „rheological properties required to ensure satisfactory printing, inks include binders, and such binders will therefore also be contained in the reactive inks used

Modern binders are generally formed from polymeric materials which dissolve or are suspended in the solvent of the ink in sufficient quantity to be able to give it satisfactory rheological properties adapted to the method used for printing with ink.

The binders used in reactive inks for the production of test strips must be compatible with the solvent and the other constituents of the ink, and should preferably be hydrophilic so as to be able to freely absorb the biological liquids to be analyzed. and thus promote the desired analytical reaction. Natural hydrophilic binders which lend themselves to the reactive inks used in the present invention, are gelatin and gum arabic, which have a high swelling power and can therefore absorb a considerable amount of water.

In addition, upon subsequent wetting of the test strip when used for the diagnostic test color reaction, the binder is used to prevent reagents from leaving the surrounding test solution.

The binder should be capable of rapidly absorbing water at room temperature to allow the reagents to diffuse along the printed surface and to carry out the color reaction desired for analysis.

Various natural and synthetic binders can be envisaged for the composition of the reactive ink, such as the following for example:

- cellulose derivatives

- gelatin

- collagen

- polyvinyl alcohol

- poly (N-vinylpyrrolidone)

Preferred binders for the reactive inks used in the present invention are the cellulose binders which are used in the examples given below.

Stamps

Reactive inks contain a buffer to maintain the pH of the printed reagents in a small given range to allow the color reaction desired for analysis to take place in an appropriate pH environment on the test strip.

The examples given below show various suitable buffers.

A major requirement for the selection of the buffer in the reactive anchors used to carry out a given analysis reaction is that the buffer should be chemically compatible with the entire reaction system, and in particular with the polymeric binder contained in the compositions of the reactive inks. .

In principle, the following four types of buffer can be envisaged which are used above all to obtain a pH between 5 and 7 of biochemical analysis reactions:

(i) Citric acid / sodium citrate

(ii) Tris (hydroxymethylamino) -ethane

(iii) Phosphate

(iv) Phthalate

Systematic tests carried out to determine the compatibility of various buffers with several polymers have demonstrated that the following systems can be envisaged for their buffering powers at the pH value (4-7) which is necessary for occult blood analyzes, and also for their relatively low cost:

1) Na-H-maleate-NaOH (pH 5-6)

2) Succinic acid -NaOH (pH 5-6)

3) K-H-phthalate-NaOH (pH 5-6)

4) ß-ß'-dimethylglutaric acid-NaOH (pH 5-6)

5) Na-citrate-citric acid buffer (pH 5-6)

In addition, a sodium citrate-citric acid buffer (pH 5-6) can be used in a reactive glucose analysis ink.

A phosphate buffer (pH 8-10) can be used in a reactive ketone analysis ink.

Table 1 below gives the result of a series of compatibility tests in which the above-mentioned buffer systems were mixed respectively with several polymers and the solubility of the polymer was observed both at normal room temperature and '' at 80 ° C.

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Table l:

Compatibility of Buffer Systems with Polymers

Polymer

Buffer system Citrate Dimethyl acid- Succinic 0.8 M glutaric 0.2 M

0.1M

25 ° C 80 ° C 25 ° C

0.1 M Phthalate

0.2 M maleate

80 ° C 25 ° C 80 ° C 25 ° C 80 ° C

Hydroxyethyl cellulose

"Natrosol 250 HXR" -

"Natrosol 250 HHR" -

"Natrosol 250 MH" -

"Natrosol 250 M" -

"Natrosol 250 HR" -

"Natrosol 250 MX" - Sodium carboxymethyl cellulose

"CMC 5 HF" +

"CMC 4 LF" + Polyvinylpyrrolidone "PVP K-90"

"PVPK-30"

Hydroxy-ethyl cellulose "Cellosize QP-4400H"

"Cellosize QP-100MH"

Methyl ethyl cellulose "Edifas BX 100" (HERE) + "Edifas B 300" (HERE) +

"Edifas B 1500" (HERE) + "Edifas BIO" (HERE) +

"Edifas A" (HERE)

Poly (methyl vinyl ether anhydride) "GantrezAN 169" -

+

+

+

+

+

+

+ in part

+ in part

+ + + + +

+

+

+

+

+

+

+

+ (g)

+

+

+ + + + + +

+

+

+

+

+

+ in part

+ in part

+ + + + +

+

+

+

+

+

+

+

+ (g)

+

+

+ + + + + +

+

+

+

+

+

+ in part

+ in part

+ + + + +

+

+

+

-f- +

+

+

+ (g)

+

+

+ + + + + +

+

+

+

+

+

+ in part

+ in part

+ + + + +

+

+

+

+

+

+

+

+ (g)

+

+

+ + + + + +

+: dissolves

-: does not dissolve and does not swell g: granular appearance

As shown in Table 1 above, the maleate, succinic, phthalate and glutaric acid buffers are compatible with most of the polymers tested while the citrate buffer is not.

Surfactants

Reactive inks contain surfactants as a wetting agent.

Thus, for example, sodium dodecyl sulfate (SDS), gum arabic and gelatin are surface-active agents which can be included as emulsifier or emulsion stabilizer in a first reactive ink containing eumene hydro-peroxide in as the main reagent for the detection of occult blood in the urine, with analysis strips which are produced according to the examples described below.

Likewise, ethyloxylated alkylphenol, as it is known under the registered trademark “Renex 698” (Atlas Chemical Industries Inc.) can advantageously be included as surfactant in a second reactive ink containing o-tolidine as a second main reagent for the detection of occult blood in the urine.

In addition, any other suitable surfactant can obviously be included in the reactive inks, such as the polyoxyethylene sorbitan mono-laurate known under the registered trademark "Tween 20" (Atlas Chemical Industries Inc.) or the polyethylene glycol known under the registered trademark "Carbowax" (ICI America Inc.).

It is understood that the use of these terms: binder, surfactant, emulsifier, stabilizer will largely depend on the primary function which is attributed to the respective constituents of the ink and that these functions can obviously be overlap or have relative importance of varying importance in a given ink or in different reactive inks.

Thus, for example, gum arabic and gelatin are natural polymers which can absorb a large amount of solvation water, thus acting as an emulsion stabilizer, while at the same time increasing the viscosity of the ink. reactive and thus simultaneously serving as an ink binder.

S (The various constituents of the ink such as those described above must obviously be chosen so that they are mutually compatible in each case and, for this purpose, relatively simple compatibility tests can be carried out such as those giving the results of Table 1 above.

55

So, for example, it has been established that a reactive ink for the detection of occult blood can contain eumene hydroperoxide (CHP), sodium dodecyl sulfate (SDS), gum arabic and gelatin which are all mutually compatible 60 with the 0.5 m Tris buffer and also with the following water-soluble binders:

- Na-carboxymethyl cellulose (NaCMC);

- hydroxyethyl cellulose (HEC); and

65 - methyl-ethyl cellulose (registered trademark "Edifas A", HERE).

Print media

Reactive inks can be printed on any sheet

5

suitable which does not react chemically with the different inks used for printing.

The printing sheet chosen to produce the printed tapes will depend mainly on the reactive inks used in each case. This sheet should further fulfill the following requirements as much as possible:

i) be essentially insoluble in water and chemically inert on the printing surface with respect to the respective reactive inks used;

ii) be capable of receiving and fixing the printed dots i n uvee good adhesion of the reactive inks to the surface of the substrate;

iii) be wettable by the liquid analyte;

iv) be capable of diffusely reflecting all the wavelengths of the visible light spectrum, or else be completely transparent when it is mounted on an underlying white matter;

v) be resistant to winding and heat.

A large number of synthetic resins can be used to obtain a suitable support material.

Various thermoplastic resins which can be used to obtain a suitable printing medium for the test reagent strips are given by way of example in Table 2 below.

Table 2:

Print media materials

Polyethylene (low, medium or high density)

Polypropylene

Polyvinyl chloride (rigid)

Polyvinylidene chloride (rigid)

Polystyrene (e.g. impact resistant resins or ABS) Arylonitrile styrene copolymer

Acrylic resins (eg impact resistant acrylic resins)

Polyether lorinated

Polychlorinated trifluoroethylene

Fluorinated ethylene-propylene copolymer

Polytetrafluoroethylene

Nylon (66 or 6)

Acetal resins

Polycarbonate resins

Cellulose resins

Cellulose acetate butyrate

Cellulose propionate

Ethyl cellulose

The printed sheet should provide an almost white background in order to reflect as much visible light as possible of all wavelengths and thus ensure good visual (or photometric) detection of the color change that takes place when the test strip is subsequently used to carry out the desired analytical reaction.

The printing substrate is preferably given a mat surface finish which diffusely reflects light scattered in all directions and allows much better detection than in the case of an icy finish which reflects light like a mirror.

In order to provide good contrast, the surface material of the substrate may itself be white in color, by incorporating a white pigment therein, or else by providing it with a white surface coating. The printing substrate can however also be transparent and colorless while it will be supported in this case on a white sheet, of paper for example.

White sheets made entirely of plastic (so-called “synthetic papers”) are obtained commercially and commonly used in the graphic arts, in particular for photographic printing. Such “synthetic paper” known under the registered trademark “Polyart” is manufactured

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by Bakelite-Xylonite Ltd., U.K. and consists of a modified polyethylene, cast in the form of a thin film. It exhibits the properties of a good quality fine art paper when touched and handled, but its properties are in other respects those of a continuous resistant plastic film. Another synthetic paper known under the trademark “Q 'per” and manufactured by Nippon Art Paper Mfg. Co, Ltd., Japan consists of a continuous film of polystyrene.

Another material which can be used to provide a light reflective printing medium in the present invention is a plastic-paper composite comprising a paper base coated with a transparent thin layer of plastic. The optical properties of paper are thus retained in this composite material, while its surface is polymeric. The company Felix Schoeller, Osna-brueck, R.F.A., manufactures this type of product which is obtained commercially in different qualities with different surface finishes: evening grain, shiny and matt.

In order to promote the adhesion of the printed dots, the printing medium can be given a more or less rough or porous surface, or both.

The use of a more or less porous printing medium gives two advantages which cannot be obtained with non-absorbent plastics. A first advantage is the improved adhesion between the reactive ink and the porous substrate, which has a much larger surface area than a non-porous material and thus has a much larger surface area for adhesion and also for the colored reaction d analysis done later.

A second advantage of a porous substrate concerns the subsequent mixing of the reactive inks in the presence of the analyzed analyte. A thin porous membrane will absorb the test solution and allow the reagents to mix inside the substrate itself.

However, as in the case of a non-absorbent substrate, the mixture. will take place in the liquid phase between the dots of reactive ink printed on the surface of the substrate.

It therefore follows from the above that the use of microporous plastics gives an absorbent substrate surface having the advantages of firstly exhibiting no adhesion problem and secondly absorbing the reagents printed in a chemically inert matrix.

Regarding the resins listed in Table 2 above, it can be noted that polyethylene and polypropylene substrates can be pretreated to improve adhesion.

For the surface treatment of plastic films, there are several conventional techniques to improve adhesion by creating reactive chemical groups on the surface of the polymer: the polymer can be oxidized by treatment with a sulfuric acid solution / dichromate or similar solutions. The polymer is then washed before printing. One can also consider the action of UV light in particular in the presence of ozone in order to make certain polymeric substrates more receptive to ink. Similarly, an electrical pretreatment can be used to modify the surface of the polymer, either by a high-voltage Corona discharge or by an incandescent discharge at reduced pressure. One can also use an electron beam for the same purpose.

In addition to the “POLYART” sheets, other synthetic resin sheets have been used with success such as a polyester fibrous sheet known under the brand name “SILBOND” (Faserprodukte GmbH, RF from Germany), a known polyamide fibrous sheet under the brand "SYNTOSIL" (SIHL, Papeteries Zurichoises on Shil Zürich) and a woven polyethylene sheet known under the brand "TYVEK" (EI DuPont

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de Nemours) as a support material for producing test strips according to the present invention.

Another material that can be used to provide a light-reflecting printing medium is a glass fiber-based medium from the company Schleicher und Schuell (RFA) which is obtained commercially in the form of thin sheets of grammage 70 g / m2. Yet another material that can be used to provide a light reflecting medium is absorbent paper, which has been used successfully as a printing medium to receive separate dots of two reactive inks.

Printing process

In order to ensure sufficient color intensity to allow sufficient visual or photometric detection by the reaction taking place on the surface of the test strip, the thickness of the film of reactive ink deposited in the form of printed dots should obviously be as large as possible.

It should be noted that each reactive ink should be sufficiently viscous for it to form a specific layer: printed on a given support, the thickness of the ink deposited being obviously also a function of the printing process. This means that a high viscosity of the ink alone does not allow the deposition of thick films by printing.

Thus, offset printing with a viscous ink can only print layers with a thickness of only a few microns. A similar limitation applies to typography where pasty ink is used but only provides thin printed layers. As a result, offset printing, letterpress, as well as flexography and no-touch printing all have certain inherent limitations on the thickness of the printed ink layer (eg 2-5 | i).

When printing by rotogravure, an ink is generally used which is of low viscosity and contains a high proportion of an organic solvent (for example about 5 (1 '/) which evaporates after printing so as to leave a relatively thicker dried ink layer, about 12 µm thick for example.

On the other hand, it can be considered that the screen printing technique is particularly suitable for the purposes of the present invention, insofar as it makes it possible to print with highly viscous inks and with a thickness several times greater than with the others. above-mentioned methods, namely with a thickness of the layer of printed and dried ink which may be between 15 and 65 μm in various conventional screen printing methods.

It has been determined that conventional test strips such? as obtained commercially, which are impregnated to a thickness of 300 u, can provide an occult blood detection reaction with color changes which can be easily detected visually by light reflection from the reagents present in an outer layer 70 | t thick of the impregnated reactive strip.

As appears from the above, the dots of reactive ink will preferably be printed by screen printing or a related process using viscous inks, the dimensions of,

the printing screen and the rheological properties of the reactive inks used being in each case mutually adapted so as to be able as far as possible to obtain printed dots of a minimum size and a maximum thickness.

The printed dots can have a diameter as small as 50 | t, but reactive ink dots of larger size have been obtained in a range between 80 [i and 450 [t, and this by printing by screen printing as described below.

The various reactive inks are deposited on the inert printing substrate by separate consecutive printing operations, that is to say a first network of printed dots is deposited and dried, formed of a reactive ink before depositing a second network of printed dots, formed of a second reactive ink, etc. . .

The points of each network are preferably arranged between the points of another network, the points of different networks being spaced a short distance from each other, so that the main reagents contained in the respective inks are separately from the substrate d printing of the test strip, while presenting only a short diffusion path for the reagents during the subsequent wetting of the test strip with the analysis solution to be examined.

The printed dots will thus be tightened as closely as possible between them in order to obtain a maximum concentration of the various reagents which are fixed by printing per unit of the surface of the reactive strip, and thus to produce therein a rapid reaction for the analysis. which leads to a color change of sufficient intensity to allow sufficient visual detection.

The relative arrangement of the mutually spaced printed dots, formed of different reactive inks, so as to obtain their tight clamping, can be explained in terms of two types of dots, which will be called A and B, of a diameter R + E touching exactly one another, R being the diameter of the printed dots and E 1 the gap separating the adjacent dots.

A very dense, tight-fitting arrangement of dots A and B formed of two different reactive inks can be printed in a particularly simple manner using a very simplified screen printing technique using a metal sheet which is perforated by tiny holes spaced apart between them. parallel rows, the holes in each row being staggered and staggered relative to the holes in the neighboring rows.

A triangular base network is thus formed in said metal sheet by means of these staggered perforations, which makes it possible to completely eliminate the imaging operations which are normally required to produce a desired arrangement of holes in a screen. printing as used in conventional screen printing.

Thus, the screen printing with two inks, which will be designated by the letters A and B, can be carried out simply by means of said metal sheet by proceeding as follows:

The perforated metal sheet is used as a printing screen stretched through a metal frame which is fixed to a screen printing machine.

The sheet serving as a printing medium, for example a sheet of synthetic paper of the Poiyart type, is placed on the printing table in a predetermined fixed position below the perforated sheet forming the printing screen.

We first print a small network of landmarks in a corner of the printing sheet by applying a small amount of a colored marking ink (red for example) and, by passing a doctor blade so as to spread this ink on a small area in a corner of the screen.

The second array of landmarks can also be printed in the opposite corner of the print sheet to make it easier to ensure exact positioning; the screen on this sheet.

The first reactive ink is then applied to the edge of the screen which is furthest from the operator of the printer, and this in sufficient quantity to print on practically

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the entire surface of the sheet constituting the printing medium.

A rubber doctor blade with a hardness on the Shore scale between 80 and 90, having a square profile blade, is now moved slowly along the surface of the printing screen which is pressed down and put in contact with the print sheet while the ink is forced through the screen holes. The ink is thus brought into contact with the printing sheet and adheres sufficiently to it, so that when the printing screen returns to its initial position after the passage of the doctor blade, the ink remains on the printing sheet in the form of a network of dots whose diameter is equal to or slightly larger than the diameter of the holes of the screen used.

The network of all the dots thus printed is then identical to the arrangement of the holes in the printing screen.

A first network of dots A printed on the sheet is thus obtained by screen printing with the first reactive ink.

The frame carrying the printing screen is then removed from the machine, the screen is washed in running water, rinsed with acetone and dried, the frame is again placed on the machine in a position which is as close as possible to its initial position, and each network of colored reference points is then aligned with the corresponding perforations of the printing screen, and this by effecting any displacement of the printing table which is necessary so that the reference points are positioned exactly below the corresponding perforations; all the printed dots A of the first network are thus identified with the perforations of the printing screen. i

The printing sheet is then moved parallel to the edges of the frame by displacements of the printing table over predetermined distances X and Y chosen so that the perforations of the printing screen are now positioned above unprinted areas which <are practically located in the middle between neighboring points A already printed and which can be centrally located, either inside a triangle made up of three neighboring points A, or inside a rhombus made up by four neighboring points A of the first network. -j

The physical displacements X and Y of the printing table are precisely determined by means of microganges associated with this table.

The second reactive ink is finally applied to the printing screen in the manner described above and is thus used i to print a second network of printed dots B on said areas of the printing surface which are located centrally between the points already printed B of the first network.

Satisfactory printing is carried out in this way with two reactive inks having the compositions used in the examples described below.

To perform the printing as described above, perforated nickel sheets were used as the printing screen which are manufactured by the company Zeefplatenfabriek N.V. in Merbeck, Netherlands. "

Six types of sheets known as Veco, obtained commercially, which are designated by the references 25R, 30R, 60P, 80P and 125P, and which have the dimensions shown in the Table respectively, were used as the printing screen. 3 which follows.

Board

Print screens

Sheet type Veco No

25 R 30 R

Thickness mm

0.31 0.25

Hole diameter mm

0.3 0.25

Hole gap mm

1

0.83

40 R

0.31

0.19

0.63

60 P

0.09

0.17

0.42

80 P

0.07

0.13

0.31

25 P

0.05

0.08

0.20

Several series of tests of printing with these sheets have given good results, are the object of the

Table 5 below, the dots printed being iege. ^ :: -; larger (from 5 to 20 tonnes) than the size of the Veco sheet holes used in each case, it being understood that the size of the points decreases when the size of the holes is reduced.

Table 4 below shows the average sizes of the printed dots obtained with four Veco screens having the corresponding dimensions given in Table 3 above, the inks used for printing having the same compositions in this case as the inks designated by RI .A4 and R1 .B4 in Table 5 given below.

Table 4:

Average diameter of printed dots

Typical sheet

Hole diameter

Average point size (mm)

Veco No

I. Ink

2. Ink

reactive reactive

RI. A4

R1.B4.

25 R

0.30

0.324

0.317

60 P

0.17

0.187

0.180

80 P

0.13

0.147

0.30

125 P

0.08

0.083

0.078

The printed dots A and B of the two reactive inks do not touch each other and the ratio of the printed surface covered by the dots to the total printing surface of the sheet varied between 11 and 50rr and we obtained later a clearly visible colored analysis reaction for detecting the presence of blood in the urine.

It may also be noted that the dimensions of the printing screens should preferably be adapted to the reactive inks used, so that maximum coverage of the printing surface is obtained. Thus, for example, a Veco sheet of type No 125 P has a hole size of 0.08 mm with an interaxial distance of 0.2 mm and thus provides networks of tight points much more tightly than a sheet of type 25 R having a hole size of 0.3 mm and an interaxial gap of 1 mm.

The higher density of the dots printed with the screens of the types 60 P, 80 P and 125 P (Tables 3 and 4) thus turned a more homogeneous and intense color change during the analysis reaction.

The results of the printing of reactive ink dots, as described here, have always given a colored analysis reaction which is visually detectable, it being understood that the density and the film thickness of the printed dots of reactive ink could be increased considerably by printing through more closely spaced perforations and using reactive inks having a greatly reduced solvent content to give them increased viscosity.

The invention can be further illustrated by means of the examples given below.

The compositions of the various reactive inks used in Examples 1 to 7 which follow are summarized in Table 5 below, in which abbreviations and registered trademarks represent the following substances;

Reagents

Hydroperoide of eumene Ortho-tolidine- [C ,, H3 (CU,) NH ;;] 2

= CHP

O-tolidine

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8

ones

Hydroxyethyl cellulose: "NATROSOL" (Type 250 LR, Hercules)

Polyvinylpyrrolidone: "PVP" (Type K90, BASF) Sodium-carboxymethyl cellulose: Na CMC (Type 7 HF, Hercules)

Methyl-ethyl cellulose: MEC: "EDIFAS A" (ICI) Hydroxypropyl cellulose: "KLUCEL" (Type LF, Hercules) Dextran (Pharmacia, UPPSALA, Sweden)

Filling material Silica powder

Surfactants s Sodium dodecyl sulfate: SDS Oxyethylated alkylphenol: "RENEX 698"

Buffer

Sodium-hydrogen maleate buffer, pH 5.8: MALEATE

Table 5:

Reactive ink for occult blood analysis

Reagent Solvent Binding Agent Buffer

% by weight Surfactant charge

Example 1 RI. Al ear 79%

R1.B1 ethanol 82%

CHP NaCMC

(3.14g) (0.5g) PVP 14g o-tolidine PVP 0.4g (20g)

SDS 0.5g

Renex 698 (0.5g)

Maleate (30 ml)

Example 2 R1.A2 water

74.5%

R1.B2 ethanol 83%

CHP NaCMC

(3.14g) (0.6g) PVP (18g)

o-tolidine PVP 0.6g (27g)

SDS

0.5g

Renex 698 0.6g

Maleate (30 ml)

Example 3 RI. A3

R1.B3

water 76%

ethanol 83%

CHP NaCMC

3.14g 0.6g PVP 16g o-tolidine PVP (0.6g) (27g)

SDS 0.5g

Renex 698 (0.6g)

Maleate 30 ml

Example 4-R1.A4

R1.B4

water 68%

ethanol

CHP (6.3g)

o-tolidine (0.8g)

"Natrosol" (8g)

"Aerosil" (5g)

gelatin 0.5g

"Klucel" (10g)

SDS (lg)

Renex 698 (lg)

Maleate (35 ml)

Free 1

Diagnostic test strips for detecting blood in the urine, called occult blood test strips, are produced as follows:

a) Preparation of reactive inks

Two reactive inks RI .Al and RI, B1 (see Table 5) are prepared as fluid carrier medium for the respective reagents eumene hydroperoxide (CHP) and o-tolidine which produce a colored oxidation reaction in the presence of blood. They are obtained by mixing the aqueous buffer with an aqueous solution which contains the dissolved binder and emulsifier (surfactant), as well as the respective reagent. The resulting mixture is stirred vigorously so as to obtain the ink in the form of a homogeneous, viscous, stable emulsion or of a colloidal suspension having the corresponding composition given above for RI.Al andRl.Bl in Table 5 .

The reactive inks obtained with these compositions have a reactively high viscosity which is particularly suitable for the screen printing technique by which these inks are to be applied to the support of the reactive strip. The inks ('' shown in Table 5 above have a viscosity between 1,000 and 20,000 cp.

b) Support

„5 A white synthetic“ paper ”sheet which is known under the trademark“ Polyart ”(90 g / m2) and which has a matt surface finish and thickness is used as the printing medium for the production of reactive strips. about 80 jt.

9

620,300

This type of Polyart paper is formed from a polyethylene The blue coloration that is observed when the modified strips flowed in the form of a thin film. Obtained printed reagents are used to detect blood in the trade for use in the graphic arts industry - the urine is essentially the same as in the case of the bands. reagents produced according to Examples 1 and 2.

c) Printing with reactive inks ^ E l 4

The two reactive inks RI .Al and RI.B1 are applied _, xempe

- t i-- j - • u- Test strips for blood detection are produced-

consecutively by a particular screen printing technique. „,," ,,. 6, *

i f a i -ii „ui u n„ i ”„ ”-„ „„ To, are essentially in the same manner as in Example 1, except on the surface of the aforementioned white sheet of Polyart that ,,, ■

,, „„ „. that 1 we use RI.A4 and R1.B4 reactive inks (see

I use as a print medium. 2, ,, _. ,. „„. v. . „

Each reactive ink is printed in the form of dots (ablet 5t ^ above) to carry out Printing with a separate tiny sheet which are spaced at a short distance from each other Veco Pe [forf du ^ Pe 60 P (see Table 3) on a sheet of others following a triangular printed network. white absorbent paper.

A perforated nickel sheet of the Veco 25 R type is used. The deviation, ntuerax, has holes of and d ° nC of P01 "1 *.

(see Table 4 above) as screen printing for ^ pnmcs of each network is 0.42 mm in this case, while

,,, j. . . ,. J - 4. a that the points printed with reactive ink A and B have respectively-

First print a first triangular network of points A 1 ^. .

reactive ink with a diameter between 300 and 370 n and a dlamfre ° '187 mm and °' 180 mm approximately1 'la. an interaxial deviation of 1 mm, using reactive ink R1.A1 Proportion of the printed surface covered by all points A.

(see Table 5 above), and this in the manner already described and B being approx. 34% of the area d -,

above under the title "Printing process". The dots printed with RI .A4 and RI .B4 inks do not spread

We use the same Veco 25 R sheet to print, as 2 (1 Not on PaPier ^ orbiting but remain as small already described a second triangular network of ink dots B distinct dots on the surface of the paper. This property of ab- reactive, each centrally arranged in a diamond formed so that this ink flow is due to their v.scositesleves by four neighboring points A already printed. These points B have and their rheoloS, Pateuse 1UI eur P6 ™ * nûannï ° mus d être a diameter between 300 and 350 p and the same deviation correctly impnmees by the process described above, interaxial of 1 mm than the points A and that the holes of the sheet 25 ° n to observe, ve a blue coloration when the bands reactiv

perforated used as a printing screen. you're ai, nsi. Products are useful, see P0l! R hemoglobin detector

The proportion of the printed surface covered by the dots "olut.on in the same way as with the reactivated strips

A and B in this case amount to 17.7% of the total area ob, held ^ sel ° "the examples \ -2 and 3 'with, 1 additional advantage-

available on the sheet forming the print medium. neIue 1ue 1 on, obl'entt ^ more homogeneous and intense color reaction on the test strips produced according to the present d) Cutting into test test reaction strips example.

The sheet printed in Polyart is cut into reaction strips- Example 5

analysis tives with a width of 4 mm. Blood test strips are produced

A blue coloration can be observed distinctly when essentially in the manner described in example 1 with the strips thus obtained being immersed in urine with the same inks and the same Veco sheet (60 P) as described containing 1 hemoglobin in an amount equal to or greater in Example 4, except that the second reagent is printed at 0.0014 mg / 100 ml. R1.B4 in two successive prints in the form of two distinct sets of points.

Example 2 ^ RI .A4 ink is first printed on a sheet of

Occult blood test strips are absorbent paper in the triangular arrangement of dots produced essentially as described in the example mentioned above. When these printed dots are dry, 1, except that the reactive inks RI .A2 and RI .B2 are used here (see] RI ink, B4 is printed with the same Veco screen (60 P) in Table 5 above) to print with a sheet the interior of triangles of dots formed by RI .A4 ink,

Veco perforated type 30 R (see Table 3 above). ^ while there are other neighboring triangles formed of this

The interaxial gap of the holes of 1 screen and therefore of the RI .A4 ink dots which do not contain a dot of RI .B4 ink.

printed in this case is 0.83 mm, while the dots ^ consequently, a second printing is carried out with the reactive ink in-printed have respectively a diameter ri b4; (I try to print points in these triangles tre of 315-335 | A and 320-360 | i, the proportion of the vacant printed surface covered by all points A and B being 16.5% jo The proportion of the total printed surface covered by approximately the total printing surface. all jes a and B is then approximately 50% of the surface

The blue coloration which is observed when the total reactive printing bands thus obtained are used for the detection of blood A blue coloration is observed when the reactive bands

in 1 urine is essentially the same as that obtained with your produced ajnsj are used to detect hemoglobin the reactive bands produced according to 1 example 1. ^ in seution, in the same way as the reactive bands described in example 4, but with the additional advantage that Example 3 one obtains in this case a more homogeneous color reaction

Reactive strips for detecting blood are produced and intense only in the case of Examples 1 to 4. You are essentially as described in Example 1 except that the reactive inks RI .A3 and RI are used here .B3 (see 60 Example 6

Table 5 above) for printing with a sheet Reactive strips for detecting blood are produced with Veco perforated type 40 R (see Table 3 above). essentially as described in Example 4, except

The interaxial gap of the screen holes and therefore of the points that the printing medium used in this case is a fire-

in this case is 0.63 mm, while the thin white dots of glass fiber paper (printed filter paper A and B have a diameter of 235-255 | i ft5 Special Type 10.70 g / m2 respectively , de Schleicher and Schuell, GmbH, RF and 255-260 [i, the proportion of the printed surface covered by from Germany).

all the points A and B being approximately 15% of the printing surface. The printing surface of this sheet is receptive to total sion. ink and ink dots adhere well. The reactive bands

620,300

10

ves imprinted on this material have a hydrophilic surface to hemoglobin solutions and react with a blue color to hemoglobin concentrations equal to or greater than 0.0154 mg / ml.

Example 7

Test strips for detecting blood are produced essentially as described in Example 5, except that the support used in this case is a white sheet of a microporous plastic material based on polyvinyl chloride, manufactured by Amerace Corporation, Microporous Division.

The reactive strips on this support are used to carry out analysis reactions with hemoglobin in solution and then provide a very satisfactory coloration, as in the case of Example 5.

The following examples 8 and 9 further illustrate the production of diagnostic test strips for the detection of glucose in urine.

The compositions of the reactive glucose detection inks used in Examples 8 and 9 are given in Table 6 below, in which the abbreviations and trademarks which have not yet been specified above represent the following substances:

Reagents

Glucose oxidase Peroxidase potassium lodide Bright blue dye FCF

GOD POD Kl

FD and C Blue No. 1

: CMC (Type 7LF, Hercules): PVP

Binders

Carboxymethyl cellulose Polyvinylpyrrolidone "PLASDONE" (Type K29-32, GAF Corp.)

Surfactants

Polyoxyethylene sorbitan monolaurate:

"Tween 20" (ICI America Inc.)

Buffer: n 0.8 M citrate

= 350 ml H20 + 257.3 g sodium citrate dihydrate

+ 25 g anhydrous citric acid

Table 6:

Reactive glucose detection inks

Reactive Solvent Ink% by weight

Binder

A. Buffer surfactant

Example 8 R1.A8

R1.B8

water 85% ethanol 72%

GOD (0.06g) POD (0.01g) o-tolidine (0.8g)

CMC (6g)

"Tween 20" (0.3g)

(10g)

(lg)

Citrate (50 ml)

"KLUCEL" "Renex 698"

Example 9 R1.A9

R1.B9

water 73% water 77%

GOD (0.3g) POD (0.04g) Kl (0.08g) blue dye (0.06g)

CMC (2g)

PVP (0.2g) CMC (2g)

"Tween 20" (0.06g) "Tween 20" (0.08g)

Citrate (10 ml) Citrate (15 ml)

Example 8 45

Test strips for detecting glucose in urine are produced as follows:

a) Preparation of reactive inks

Two reactive inks RI.A8 and RI .B8 are prepared (see S () Table 6 above) as a fluid carrier for the reagents glucose oxidase (GOD) and peroxidase (POD) on the one hand and ortho-tolidine d on the other hand, which provide a colored oxidation reaction in the presence of glucose.

The first RI, A8 reactive ink is obtained by mixing 55 aqueous citrate buffer (0.8 M) with the two reagents (GOD and POD powder) and with the "Tween 20" surfactant, and dissolving the CMC-7LF binder there. The resulting mixture is stirred vigorously so as to obtain the first reactive ink R1.A8 in the form of a viscous, homogeneous, 60 solution stable with the composition given in Table 6.

The second reactive ink RI, B8 is the same as the second ink R1.B4 given with respect to the previous examples 4 to 7 in Table 5.

These two inks RI.A8 and RI .B8 have a relatively high viscosity fi5 which is particularly suitable for the use of a screen printing technique for the application of these inks on the support of the reactive strips.

Support

A sheet of white absorbent paper is used as the printing medium to produce the test strips for detecting glucose.

c) Printing with reactive inks

Glucose detection test strips are produced essentially as described in Example 1, except that the reactive inks RI.A8 and RI .B8 are used in this case for printing using a perforated sheet of the Veco 125 type. P (see Table 3 above).

The proportion of the printed surface covered by all the points A and B is in this case greater than 30% of the surface of the sheet of printing support.

d) Cutting into reactive strips

The printed sheet of paper is finally cut into reactive glucose detection strips with a width of 4 mm.

A strong blue color can be observed distinctly when the test strips thus obtained are immersed in urine containing a quantity of glucose equal to or greater than 0.1 g / 100 ml.

11

620,300

Example Q

Diagnostic test strips for detecting glucose in urine are produced essentially as described in Example 8, except that RI, A9 and RI .B9 inks having the compositions given above are used in the Table 6.

These reactive inks RI, A9 and RI, B9 are used to print by means of a perforated sheet of the Veco 60 P type (see Table 3) on a sheet of absorbent paper, as in Example 8, and cut out. finally the sheet printed in reactive strips 4 mm wide.

Before serving the reaction, the reactive bands thus obtained are blue in color due to the presence of a small amount of an oxidizable blue dye (FD & C Blue No. 1), in addition to iodide salt (Kl) soluble in water, in the second reactive ink RI, B9.

A color change can be clearly observed when the reaction strips thus obtained are immersed in urine containing a quantity of glucose equal to or greater than 0.1 g / 100 ml. The color changes observed depend on the glucose concentration as follows:

Glucose (g / 100 ml)

0

; 0.1 0.5

1

Color developed in 1 minute blue light green brown-green brown

The following example illustrates the production of test strips for the detection of ketones (acetone, acetoacetic acid, betahydroxybutryric acid) in body fluids, in particular acetoacetic acid in urine.

The compositions of the ketone analysis test strips used in the following example 10 are given below in Table 7, in which the abbreviations and marks used for the binder are the same as those indicated with respect to the previous examples.

In Table 7 below:

- "GAFAC RE 610" is a registered trademark for a surfactant manufactured by GAF Corp.

2 (1 - "PVP / VA" is a polyvinyl pyrrolidone / vinyl acetate copolymer (50% in ethanol)

Table 7:

Reactive Ketone Inks

Ink

Reactive solvent

Binder

A. Buffer surfactant

Example 10 RI .AIO water

(36 ml)

aminoacetic acid

(9g)

R1.B10 EtOH nitroferri-cyanide (54 ml) of sodlium (2.9 g)

CMC-76F (3g)

PVP / VA «GAFAC

copolymer (23 ml) "KLUCEL JF"

(10g)

Na3 P04 • 12H20

(10g)

Na2HP04

anhydrous

(4.3g)

RE 610 "(0.3g) sodium dioctylsul-foccinate

Example 10

Test strips for detecting ketones in body fluids are produced essentially as already described in Examples 8 and 9, except that the RI reactive inks. AIO and RI. BIO used in the present case respectively include the phosphate salts in the aqueous phase, the sodium nitro ferricyanide in the alcoholic phase and the binders soluble in ethanol (PVP / VA, "KLUCEL JF") which are given above in Table 7.

These reactive inks R1.A10 and R1.B10 are used for printing by means of a perforated sheet of the Veco 60 P type (see Table 3) on a sheet of white absorbent paper, as in Examples 8 and 9 above, and the printed sheet obtained is cut into reactive strips of a desired width.

A purple coloration is observed distinctly when the reactive strips thus obtained are immersed in a liquid containing acetoacetic acid.

The coloring varies from very light purple, in the presence of 10-20 mg of acetoacetic acid per 100 ml of liquid, to very dark purple indicating the presence of more than 100 mg per 100 ml.

As is clear from the above, it has been established experimentally that the total area covered by all the points

4S networks printed with the different reactive inks, to produce reactive bands printed in accordance with the present invention, should cover 30% and preferably 45% of the available printing surface containing the different networks, and this in order to be able to obtain the better coloration 50 and thus ensuring the most satisfactory analysis color reactions in all cases by means of the printed test strips.

Such a covering of at least 30% of the surface area of the support can be easily achieved by an appropriate choice of the 55 perforated screens used for printing, namely screens having perforations whose size and distance are as small as possible, while allowing a satisfactory, successive impression of different distinct tiny dots which are located one between the other and are spaced very narrowly-60 ment, without they being in contact with each other .

Thus, for example, said screens of the Veco 60 P, 80 P and 125 P types, which have the dimensions indicated in Table 3 and f) 5 are used in Examples 4 to 10 above, are particularly suitable for printing of networks of different dots covering approximately 30 to 50% of the corresponding surface of the sheet forming the printing medium.

620,300

12

It has also been established experimentally that the total number of dots printed per unit area of the sheet forming the support should also be equal to or greater than 1300 dots per square centimeter, in order to be able to provide the best colorings and thus ensure reactions. colored them? more satisfactory in all cases for carrying out the analysis using the test strips.

One can also easily achieve an overall density greater than 1300 dots printed per square centimeter, and this by an appropriate choice of screens used for printing, such as for example said screens of the Veco 60 P, 80 P and 125 P used in Examples 4 to 7.

In order to also ensure a satisfactory color reaction for analysis in all cases, it has also been found experimentally that the test strips which are produced by printing in accordance with the present invention should be provided with printed dots having a thickness between 5 and 60 microns. Likewise, it has been found that, for similar reasons, the viscosity of the reactive inks used for printing test strips according to the invention should preferably be in a range between 5,000 and 300,000 centipoise in order to ensure a satisfactory impression.

The reactive inks described above (Table 5) with respect to the preceding examples are capable of providing sufficient viscosity and point thickness, with a covering of:> surface area and a sufficient overall point density,

to be able to ensure very satisfactory color reactions in all cases for the analysis carried out by means of the printed test strips.

As can be seen from the above, the production of test strips can also be applied so as to obtain similar advantages in a wide range of color analysis reactions, using more or less conventional reagents and other constituents of inks whose chemical compatibility with each other and with more or less conventional printing supports can be established experimentally beforehand without great difficulty.

It is therefore understood that the compositions of the reactive inks, the printing medium and the means used for printing can be adapted to each other in each case based on the experience available in the relevant technical fields relating to diagnostic agents on the one hand and printing processes on the other.

The present invention can therefore advantageously be applied to the compositions of the reagents known and used for the preparation of diagnostic analysis devices. Types of reagent compositions which can be cited by way of example are described in US Pat. Nos. 3,438,737, 3,095,277.3 212,855, 3,164,534, 3,050,373.2 981,606, 3,123,443.3 252,762, 3,290 117, 3,092,463.3 012,976, 3,122,420.3 453 180, 3,585,001, 3,585,004, and 3,447,905.

It is understood, however, that the invention applies especially to diagnostic compositions in which it is important or desirable to keep the reagents separate before use.

VS

Claims (6)

620,300 CLAIMS 1. Reactive strip intended for analysis for the detection of a given constituent in a sample to be analyzed, characterized in that it comprises a support material printed with two reactive inks, the printing by each of the inks being under - form of a network of points, the points of the first network being close to those of the second network but not touching, and the two networks being at least partially superimposed, in that the two inks each contain. (a) water or a water-alcohol mixture as a solvent; i „ (h) a binder to give the ink rheological properties suitable for printing, (c) a buffer capable of giving the ink, when it is wetted with the said sample, a specific pH, and (d) a surfactant, and i - (e) a reagent, different for each ink, these reagents being capable of reacting with each other in the presence of said constituent to be detected, producing a detectable change in color. 2. Reactive strip according to claim 1, characterized in that the support is made of polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, a copolymer of styrene and acrylonitrile, polyacrylate, chlorinated polyether, polyehlorotrifluoroethylene, a copolymer of fluorinated ethylene and propylene, polytetrafluoroethylene, polyamide, an acetal resin, a polycarbonate resin, or a cellulose derivative such as acetate butyrate, propionate or ethylcelulose. 3. Reactive strip according to claims 1 or 2, characterized in that the ink binder is chosen from the following: gelatin, gum arabic, cellulose derivatives, collagen, polyvinyl alcohol, poly-N-vinyIpyrrolidone, and their mixtures. 4. Reactive strip according to one of claims 1 to 3, characterized in that the buffer is chosen from the following substances: tris- (hydroxymethylamino) -ethane, a phosphate, a ^ phthalate, sodium acid maleate / NaOH giving a pH 5 at 6, succinic acid / NaOH for a pH of 5 to 6, potassium acid phthalate / NaOH for a pH of 5 to 6, ß acid, ß'-dirnethylglutaric NaOH for a pH of 5 to 6, and sodium citrate / citric acid for a pH of 5 to 6. ■> " 5. Reactive strip according to one of claims 1 to 4, characterized in that the surfactant is chosen from sodium dodecyl sulfate, ethoxylated alkylphenols, polyethylene glycols and polyoxyethylensorbitan monolaurate. 6. Reactive strip according to claim 1, characterized in that the binder and the surfactant are the same substance, namely gum arabic and / or gelatin.