GB1601283A - Diagnostic test strips - Google Patents

Abstract

A flat substrate is printed with a network of microscopic points. After drying, a second network of points is printed using another ink. The two networks are superposed, but the points do not touch. When the device is wetted with a liquid sample to be analysed, a reaction takes place between the constituent to be detected and reagents contained in the two networks of points, producing a colour change. Each point furthermore contains a binder, a buffer and a wetting agent.

Description

(54) DIAGNOSTIC TEST STRIPS (71) We, MILES LABORATORIES INC., a Corporation organised and existing under the laws of the State of Indiana, U.S.A., of 1127 Myrtle Street, Elkhart, Indiana 46515, U.S.A., do hereby declare the invention for which we pray that a Patent may be granted to us, and the method by which it is to be performed, to be particularly described in and by the following statement: Background of the invention The present invention generally relates to diagnostic agents for testing biological liquids by providing rapid detection of given components therein, for example, for the detection of blood glucose, ketones and the like.

Known diagnostic test strips generally comprise an absorbent carrier which is impregnated with the reagents for carrying out a given coloured test reaction when wetted with the liquid to be tested.

The conventional carrier material in most test strips currently used consists of filter paper which provides distinct advantages, but also has certain drawbacks and limitations such as insufficient physical and chemical stability of the paper carrier when impregnated with certain test reagents having high concentrations to provide a satisfactory coloured test reaction.

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

In addition to these limitations with regard to the absorbent carrier material, the conventional production of test strips generally requires multiple impregnations which are quite difficult to achieve on a large scale in a highly reproducible manner and also considerably increase the production costs.

Summary of the invention A main object of the present invention is to obviate the abovementioned drawbacks as far as possible by providing a simplified method of producing diagnostic test strips on a large scale in a highly reproducible and economical manner.

The present invention thus provides a method of producing diagnostic test strips for detecting given components present in biological liquids such as urine, which comprises the steps of: (a) providing at least two reagent printing inks each @onsisting of a mixture mutually compatible components such that the inks taken together include all the reagents required for the desired test reaction while no single ink includes all the said required reagents, each said ink comprising (a) as solvent, water, an alcohol or a water-alcohol mixture, (b) a polymeric binding agent such as to confer on the ink rheological properties suitable for the printing technique used, (c) a wetting agent, and (d) in the case of at least one of the inks, a buffer such as to provide a specific predetermined pH upon wetting of the test strip by the biological liquid;; (b) successively printing the said reagent inks onto a sheet which is substantially insoluble in water and chemically inert to the reagent inks at least on the surface thereof, and has a surface suitable for printing with the said inks so as to form on the said surface at least two separate patterns of printed dots of the said reagent inks, the reagent ink dots of each said pattern being dried after printing and being arranged in closely spaced relationship with one another and with the dots of each different pattern, so that the printed dots of each reagent ink are separately arranged between the dots of each different reagent ink, in close proximity thereto but without contact between the dots of different inks, so that the reagents in the respective dry dots are thereby kept separate from one another, while allowing said reagents to diffuse along said surface and to thereby mutually interact thereon in the presence of said liquid analysate; and (c) cutting the said sheet having the said separate printing patterns printed on the surface thereof into a plurality of test strips of desired shape and size.

The use of printing to produce diagnostic test strips according to the present invention offers various practical advantages, and in particular the following: The possibility of readily separating different components of the reagent system which are ordinarily reactive, by including them in different reagent inks which are then used for printing separate dot patterns on the printing surface of the test strip.

The possibility of providing a readily reproducible, more rapid and relatively simple printing technique for applying exactly predetermined amounts of the reagents to the printing substrate, whereby the production of diagnostic test strips on a large scale may be greatly simplified and rendered more economical.

The possibility of eliminating the need for separate colour comparison charts, by also printing the respective test colours for comparison on given portions of the same test strip.

Various considerations which are of practical interest for providing such advantages in accordance with the principles underlying the present invention will now be discussed.

Reagent Ink Compositions It may be readily seen that the respective ink compositions will primarily depend on the particular colour reaction to be effected by means of the test strips, i.e. on the particular reagent system necessary to provide the desired test reaction in each case.

The constituents of each reagent ink should further be selected so as to be chemically and physically compatible with each other and with the printing substrate to which they are applied.

In order to ensure satisfactory printing, the physical properties of the reagent inks should moreover be adapted to the type of printing substrate as well as to the printing technique and printing means which are used to produce the test strips.

The term reagent ink as here used implies an intimate mixture of at least one main reagent necessary for the colour test reaction, with the above-specified auxiliary components and liquid vehicle or carrier medium, so as to provide an ink having suitable rheological properties adapted for printing by ensuring proper ink transfer from the printing means to the printing substrate used in each case.

The specific reagents which are included in the inks used for printing according to the invention will evidently depend in each case on the particular type of test reaction for which the test strips are to be produced.

However, the reagent system which is used to provide a given colour reaction on the test strips produced according to the present invention may generally comprise conventional reagents which are known from the prior art relating to diagnostic test reactions, known reagents being included in the inks used in the examples described further below to illustrate the invention.

On the other hand, as already mentioned above, all components of each reagent ink must be mutually compatible and should provide suitable rheological properties to allow proper printing. The choice of a suitable solvent, binding agent, wetting agent, and buffer, and of any auxiliary ink components other than the main reagent will thus depend on various considerations discussed below.

Ink Solvent The function of the solvent in the reagent ink is to provide a liquid medium which is necessary to retain the main reagent and the auxiliary components of the ink in solution or suspension, until the ink is used for printing on the substrate. After printing, the solvent has fulfilled its role and should be removed by drying in order to provide printed dots fixed on the substrate surface.

The solvent is preferably water or an aqueous mixture of water and ethanol. Higher alcohols may also be of interest as a solvent, e.g. glycol. The solvent used must be compatible with the other ink components in each case. However, certain inorganic salts, which may be included in the reagent inks as buffers or enzyme activators, have limited solubility in alcohols and this must be taken into account when selecting the ink components.

Ink Binder In order to impart to the inks the necessary rheological properties for ensuring satisfactory printing, the ink compositions generally include binding agents which are likewise included in the reagent inks used in the present invention.

Modern ink binding agents generally consist of polymer materials which dissolve or are suspended in the ink solvent in a sufficient amount to provide satisfactory rheological properties adapted to the printing method in which the inks are to be used.

The binders used in the reagent inks for the production of test strips for biological liquids, in accordance with this invention, must be compatible with the solvent and other ink components and should preferably be hydrophilic in order to be able to freely absorb biological test liquids, whereby to allow the desired test reactions to occur. Natural hydrophilic binders which are suitable for the reagent inks used in the invention are gelatin and gum arabic, both of which possess a high swelling power and are thus able to take up a considerable amount of water.

Moreover, during subsequent wetting of the printed test strip when it is used for the diagnostic colour test, the binder serves to prevent the primary reagents from going into the surrounding analysate solution.

The binder should be able to rapidly absorb water at room temperature in order to allow the reagents to diffuse along the printed surface whereby to effect the desired colour test reaction.

Various natural or synthetic binders may be contemplated for the reagent ink composition, such as the following, for example: cellulose derivatives gelatin collagen polyvinyl alcohol poly(N-vinylpyrrolidones) In the present invention, preferred binders for the reagent inks are the cellulosic binders which are used in the examples given further on.

Buffers At least one of the reagent inks contains a buffer in order to keep the pH of the printed reagents in a given small range to allow the desired colour reaction to take place in a suitable pH environment on the test strip.

The examples given below show various suitable buffers.

A major requirement for selecting the buffer in the reagent inks used to effect a given test reaction is that the buffer should be chemically compatible with the whole reaction system, and especially with the polymeric binder included in the reagent ink compositions.

In principle, the following four buffer types which are predominantly used to control the pH of biochemical test reactions between 5 and 7 may be contemplated: (i) Citric acid/sodium citrate (ii) Tris hydroxymethylamino-ethane (iii) a phosphate buffer (iv) a phthalate buffer.

Screening tests effected to determine the compatibility of various buffers with a number of polymers have shown that the following buffer systems may be envisaged for their buffering properties at the pH value (4-7) which is necessary for occult blood tests, 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) t3,P'-dimethylglutaric acid - NaOH (pH 5-6) 5) Na-citrate - citric acid buffer (pH 5-6) Moreover, sodium citrate/citric acid buffer (pH 5-6) may also be used in a glucose test reagent ink.

A phosphate buffer (pH 8-10) may be used in a ketone test reagent ink.

Table 1 below gives the results of a series of compatibility tests wherein the abovementioned buffer systems were respectively mixed with a number of polymers and the solubility of the polymer was observed both at normal room temperature and at 800C. TABLE 1 Compatibility of Buffer System Buffer System Dimethyl- Succinic Phthalate Maleate Citrate glutaric acid 0.2 M 0.1 M 0.2 M Polymer 0.8 M 0.1 M 25 C 80 C 25 C 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" - + + + + + + + + SODIUMCARBOXYMETHYL CELLULOSE "CMC 7 HF" + + + + + + + + + "CMC 4 LF" + + +(g) + +(g) + +(g) + +(g) POLYVINYLPYRROLIDONE "PVP K-90" - +partly + +partly + +partly + +partly + "PVP K-30" - +partly + +partly + partly + +partly + TABLE 1 (Cont.) Dimethyl- Succinic Phthalate Maleate Citrate glutaric acid 0.2 M 0.1 M 0.2 M 0.8 M 0.1 M Polymer 25 C 80 C 25 C 80 C 25 C 80 C 25 C 80 C HYDROXYETHYL CELLULOSE "Cellosize QP-4400H" - - + - + - + - + "Cellosize QP 100MH" - - + - + - + - + METHYLETHYL CELLULOSE "Edifas BX 100" (ICI) + + + + + + + + + "Edifas B 300" (ICI) + + + + + + + + + "Edifas B 1500" (ICI) + + + + + + + + + "Edifas B 10" (ICI) + + + + + + + + + "Edifas A" (ICI) - + - + - + - + POLY(METHYLVINYL ETHER ANHYDRIDE "Gantrez AN 169" - - - - - - - - +: dissolves; -: does not dissolve or swell; g: granular appearance "Natrosol", "Cellosize", and "Gantrez" are registered Trade Marks.

As may be seen from Table 1 above, the maleate, succinic, phthalate and glutaric acid buffers are compatible with most of the polymers tested whereas the citrate buffer is not.

Surface Active Agents The reagent inks may advantageously include surfactants as wetting agents.

Thus, for example, sodium dodecyl sulfate (SDS), gum arabic and gelatin are surface active agents which may be included as an emulsifier or emulsion stabilizer in a first reagent ink containing cumene hydroperoxide as a main reagent for detecting occult blood in urine, with test strips such as are produced according to the examples described further below.

Similarly, ethoxylated alkylphenol, such as is known by the Trademark "Renex 698" (Atlas Chemical Industries, Inc.) may be advantageously included as a surfactant in a second reagent ink containing o-tolidine as the second main reagent for detecting occult blood in urine.

Moreover, any other suitable surfactant may evidently be included in the reagent inks, such as polyoxyethylenesorbitan monolaurate known by the Trademark "Tween 20" (Atlas Chemical Industries, Inc.) or polyethylene glycol known by the Trademark "Carbowax" (ICI America. Inc.).

It is understood that the use of these terms: binder, surface active agent, emulsifier, stabilizer will largely depend on the primary function which one attributes to the respective ink components and that these functions may evidently overlap or have a relative importance of varying importance either in a given ink or in different reagent inks.

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

The various reagent ink components must evidently be selected so as to be mutually compatible in each case and relatively simple compatibility tests such as those providing the results in Table 1 above may be carried out for this purpose.

Thus, for example, it has been established that an occult blood test reagent ink may include cumene, water, cumene hydroperoxide (CHP), sodium-dodecyl sulfate (SDS), gum arabic and gelatin which are all mutally compatible with 0.5 M Tris Buffer and also with the following water soluble binders: Na - carboxymethyl cellulose (NaCMC) Hydroxyethyl-cellulose (HEC); and Methylethyl-cellulose (Trademark: "EDIFAS A", ICI) Printing Support Medium The reagent ink dots may be printed according to the invention on any suitable sheet which does not chemically react with the different reagent inks used for printing.

The printing sheet selected to produce the printed test strips will essentially depend on the reagent inks which are used in each case. This sheet should meet the following requirements as far as possible: (i) Substantial insolubility in water, and chemical inertness at the printing surface to the respective reagent inks used; fii) Ability to receive and fix the printed dots with good adherence of the reagent inks to the printed substrate surface; (iii) Wettability by the analysate liquid; (iv) Ability to diffusely reflect all wave lengths of the visible light spectrum, or full transparency thereto when mounted on an underlying white material; (v) Resistance to curling and heat.

A large number of synthetic resins may be used to provide a suitable printing support medium. Various thermoplastic resins which may be used to provide a suitable printing substrate for the test strips are given by way of example in Table 2 below: TABLE 2 Printing Support Materials polyethylene (low, medium or high density) polypropylene polyvinyl chloride (rigid) polyvinylidene chloride (rigid) polystyrene (e.g. high impart or A.B.S. resins) styrene - acrylonitrile copolymer acrylic resins (e.g. high impact a.r.) chlorinated polyether polychlorotrifluoroethylene fluorinated ethylene - propylene copolymer polytetrafluoroethylene Nylon (66 or 6) acetal resins polycarbonate resins polyurethane resins cellulose resins cellulose acetate butyrate cellulose propionate ethyl cellulose The printed sheet should provide a substantially white background in order to reflect as much visible light of all wave lengths as possible and thereby ensure proper visual (or photometric) detection of the colour change which occurs when the test strip is subsequently employed to effect the desired test reaction.

The printing substrate is preferably provided with a matt surface finish which diffusely reflects scattered light in all directions and provides much better detection than is the case with a high gloss finish which reflects light like a mirror.

In order to provide a high contrast, the substrate surface material may itself be made white in colour, either by incorporating a white pigment or else by providing a white surface coating. On the other hand, the printing substrate may also be made transparent and colourless while being supported on an underlying white sheet, e.g. of paper.

White sheets made entirely of plastics (or "synthetic papers") are commercially available and currently used in the graphic arts, particularly for photographic printing. One such "synthetic paper" known by the trademark name of "Polyart" is manufactured by Bakelite-Xylonite Ltd. U.K. and consists of modified polyethylene cast into a thin film. It has the feel and handling properties of a good quality art paper but its properties are otherwise those of a tough continuous plastic film. Another synthetic paper known by the trademark "Q' per" and manufactured by Nippon Art Paper Mfg. Co., Ltd., Japan, consists of a continuous polystyrene film.

Another material which may be used to provide a light-reflecting printing medium in the present invention is a plastic-paper composite material comprising a paper base coated with a thin transparent layer of plastics material. The optical properties of the paper stock are thus retained in this composite material, whereas its surface is polymeric. This type of material is produced by the firm Felix Schoeller, Osnabruck, West Germany and is commercially available in various grades with different surface finishes: silk grain, glossy and mat.

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

The use of a more or less porous printing medium provides two advantages which cannot be obtained with non-absorbent plastics materials. A first advantage is improved adherence between the reagent ink and the porous substrate, which has a much greater surface area than a non-porous material and thus presents a much larger area for adhesion and also for subsequently effecting the colour test reaction.

A second advantage of a porous substrate is related to subsequent mixing of the reagent inks in the presence of the analysate being tested. A thin porous membrane will allow the reagents to mix within the substrate itself. Moreover, as in the case of a non-absorbent substrate, mixing will also take place in the liquid phase between the reagent ink dots printed on the surface of the substrate.

It may thus be seen from the foregoing that the use of microporous plastics provides an absorbent substrate surface having the advantages of firstly presenting no ink adhesion problems and of secondly absorbing the printed reagents in a chemically inert matrix.

With reference to the resins listed in Table 2 above, it may be mentioned that polyethylene and polypropylene substrates may be pretreated to improve adhesion.

For the surface treatment of plastics film, there are several conventional techniques to improve adhesion by creating reactive chemical groups on the surface of the polymer: oxidation of the polymer can be effected by treatment with a sulphuric acid/dichromate solution or with similar solutions. The polymer is then washed before printing. The action of UV light particularly in the presence of ozone may also be contemplated to make certain polymer substrates more ink receptive. Electrical pre-treatment either by high-voltage corona discharge or by a glow discharge at reduced pressure can also be used to modify the polymer surface. An electron beam can likewise be used for this purpose.

In addition to "Polyart" sheets, other synthetic resin sheets such as a fibrous polyester sheet known by the Trademark "SILBOND" (Faserbrodukte GmbH, West Germany), a fibrous polyamide sheet known by the Trademark "SYNTOSIL" (SIHL, Papeteries Zürichoises sur Sihl, Ziirich, Switzerland), and a spunbonded polyethylene sheet known by the Trademark "TYVEK" (E.I. DuPont de Nemours and Co.) were successfully used as a printing support medium to provide printed test strips according to the present invention.

Another material which may be used to provide a light-reflecting printing medium is compounded glass fibre which is commercially available in thin sheets of density 70 g/m2 from Schleicher and Schuell, West Germany. Yet, another material which may be used to provide a light-reflecting printing medium is absorbent paper stock, which was successfully used as a print support to receive discrete dots of two reagent inks.

Printing Process In order to ensure a sufficient colour intensity to provide satisfactory visual or photometric detection by means of the test reaction occurring at the surface of the test strip, the thickness of the reagent ink film deposited in the form of printed dots should evidently be as great as possible.

It may be mentioned that each reagent ink should be sufficiently viscous for it to be able to form a thick dot-shaped printed layer on a given support medium, the thickness of the deposited ink evidently also being dependent on the printing process. This means that a high ink viscosity cannot by itself ensure the deposition of thick ink films by printing.

Thus, offset printing with a viscous ink allows layers to be printed which are only a few microns thick. A similar limitation applies to letterpress printing wherein a paste-like ink is used but provides only thin printed layers. Consequently, offset, letterpress, besides flexography and non-impact printing, all present certain inherent limitations with regard to the thickness of the printed ink layer (e.g. about 2-5F).

In rotogravure printing, an ink is generally used which is of low viscosity and contains a high proportion of an organic solvent (e.g. about 50%) that evaporates after printing so as to leave a relatively thicker dried ink layer, of about 121l thickness, for example.

In contrast, the technique to screen-printing may be considered particularly suitable for the purposes of the invention, inasmuch as it allows highly viscous inks to be printed with a thickness several times greater than with the above-cited processes, namely with a thickness of the dried printed ink layer which may range between 15 and 16,u in various conventional screen-printing processes.

It has been determined that conventional, commercially available reagent strips, which are impregnated to a thickness of about 300p can provide an occult blood test with colour changes which can be readily detected visually by light reflection from the reagents present in the upper 701l thick layer of the imbibed test strip.

It may thus be seen from the foregoing that the reagent ink dots 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 reagent inks used being in each case mutually adapted so as to provide printed dots of minimum size and maximum thickness.

The printed dots can have a diameter as small as 50cm, but reagent dots of a larger size lying in the range between about 30,u and 450 have been successfully obtained by screen-printing in the manner described further below.

The different reagent inks are deposited on the inert printing substrate in distinct, consecutive printing steps, i.e. a first array of printed dots of one reagent ink is deposited and dried before depositing a second array of printed dots of a second reagent ink, etc.

The dots of each array are preferably arranged between the dots of another array, the dots of different arrays being mutually spaced at a short distance, so that the main reagents in the respective inks are separately fixed to the printing substrate of the test strip, while providing only a short diffusion path for the reagents during subsequent wetting of the test strip with the analysate solution to be tested.

The printed dots are thus packed as closely as possible in order to provide a maximum concentration of the different reagents which are fixed by printing per unit area of the test strip surface, whereby to provide a rapid test reaction thereon with a resulting colour change of sufficient intensity to allow satisfactory visual detection.

The relative arrangement of the mutually spaced printed dots of different reagent inks, so as to provide a closely packed distribution thereof, may be explained in terms of arranging two types of dots say A and B, of radius R + E which exactly touch each other, R being the diameter of the printed dots and E the distance separating adjacent dots.

A very densely packed arrangement of dots A and B consisting of two different reagent inks can be printed in a particularly simple manner by using a highly simplified screen-printing technique using a metal sheet which is perforated with tiny holes spaced apart in parallel rows, with the holes of each row being staggered with respect to the holes of adjacent rows.

A basic triangular printing screen is thus formed in the said metal sheet by means of the perforations therein, whereby the imaging steps which are normally required for producing a desired hole pattern in a printing screen may be completely eliminated.

Thus, screen-printing with two different reagent inks, which will be designated by the letters A and B, may be simply effected by means of the said perforated metal sheet in the following manner: The perforated metal sheet is used as a printing screen stretched across a metal frame which is fixed to a screen printing machine.

The printing sheet, e.g. a sheet of Polyart synthetic paper, is placed on the printing table in a set position underlying said perforated sheet forming the printing screen.

A small array of register dots is first printed in one corner of the printing sheet by placing a small amount of a coloured (e.g. red) marking ink on a small edge portion of the perforated printing screen and passing a squeegee to spread this ink over a limited surface area at one corner of the screen.

A second small array of register dots can also be printed at the opposite corner of the printing sheet in order to more readily ensure accurate positioning of the screen on this sheet.

The first reagent ink is then applied to the edge of the screen furthest away from the printing operator in a quantity sufficient to print the whole sheet of the printing support medium.

A rubber squeegee with a hardness on the Shore scale lying between 80 and 90, and having a blade with a square profile, is now drawn slowly across the surface of the printing screen which is pressed down into contact with the printing sheet while the ink is forced through the holes of the screen. 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 squeegee has passed by, the ink remains on the printing sheet in the shape of an array of dots of diameter equal to or slightly larger than the diameter of the holes in the screen used. The overall pattern of the ink dots thus printed is then identical with the arrangement of the holes in the printing screen.

A first array of printed dots A is thus screen-printed on the sheet with the first reagent ink.

The frame bearing the printing screen is next removed from the machine, the screen washed in running water, rinsed with acetone and dried, the frame is placed once more on the machine in as near to the original position as possible, and each array of coloured register dots is then aligned with the corresponding perforations of the printing screen, by effecting any displacement of the printing table holding the printing sheet, which may be necessary to cause the register dots to exactly underlie the corresponding perforations, whereby all printed dots A of the first array are brought in to register with the perforations of the printing screen.

The printing sheet is next displaced parallel to the edges of the frame by shifting the printing table over predetermined distances X and Y in such a manner that the perforations of the printing screen are caused to overlie unprinted zones which are substantially centrally located between adjacent dots A already printed and which may each lie centrally either within a triangle of three adjacent printed dots A or within a rhombus of four adjacent dots A of the first array.

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

The second reagent ink is finally applied to the printing screen in the manner described above and used to print a second array of printed dots B on said zones of the printing surface which lie centrally between the already printed dots B of the first array.

Printing was carried out successfully in this manner with two reagent inks having the compositions which are given with reference to the Examples described further below.

For this purpose perforated nickel sheets manufactured by the firm Zeefplatenfabrick N.V. in Eerbeek/Holland were used as screens for printing in the manner described above.

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

TABLE 3 Printing Screens Veco sheet Thickness Hole Size Hole Spacing (mm) (mm) (mm) 25R 0.31 0.30 1 30R 0.25 0.25 0.83 40R 0.31 0.19 0.63 60P 0.09 0.17 0.42 80P 0.07 0.13 0.31 125P 0.05 0.08 0.20 Several series of printing tests with these sheets provided good results with the inks shown in Table 5 further below, the printed ink dot sizes being somewhat larger (5-20%) than the hole size of the Veco sheets used in each case, while the dot size evidently decreased as the hole size decreased.

Table 4 below shows the average sizes of the dots printed with four different Veco screens having the corresponding dimensions shown in Table 3 above, the inks used for printing having in this case the same formulations as the inks designated RI.A4 and RI.B4in Table 5 further below.

TABLE 4 Average Diameter of Printed Dots Veco Sheet Hole Diameter Average Dot Diameter (mm) 1st Reagent 2nd Reagent Ink RI.A4 RI.B4 25R 0.30 0.314 0.317 60P 0.17 0.187 0.180 80P 0.13 0.147 0.30 125P 0.08 0.083 0.078 The printed dots A and B of the two reagent inks do not touch each other and the proportion of printed area covered by the dots to the total printing surface of the sheet varied between 1.1 and 50%, and subsequently provided a clearly visible colour test reaction for detecting the presence of blood in urine.

It may also be noted that the dimensions of the printing screens should preferably be especially adapted to the reagent inks used so as to provide maximum coverage of the printing surface. Thus, for example, a Veco sheet of the type 125P has a hole size of 0-08 mm with an interaxial spacing of 0.20 mm and thus provides much more closely packed printed dot arrays than a Veco sheet of the type 25R having a hole size of 0.30 mm and interaxial spacing of 1 mm.

The increased density of the dots printed with screens of the 60P, 80P and 125P types (Tables 3 and 4) thus provides a more homogeneous and intense colour change during the test reaction.

Thus, the results of printing reagent ink dots, as herein described, consistently provided a visually detectable colour test reaction, although the packing density and film thickness of the printed reagent ink dots may certainly be considerably increased by printing through more closely spaced perforations and by using reagent inks with a substantially reduced solvent content to provide a correspondingly increased viscosity.

The invention may further be illustrated by means of the examples given hereinafter.

The compositions of the different reagent inks used in the following Examples 1 to 7 are summarized in Table 5 below, wherein abbreviations and Trademarks represent the following substances: Reagents: cumene hydroperoxide = CHP ortho-tolidine = (C6H3 (CH3) NH2)2 = o-tolidine Binders: hydroxyethyl-cellulose : "NATROSOL" (type 250 LR, Hercules) polyvinylpyrrolidone : "PVP" (type K90, BASF) sodium-carboxymethyl-cellulose : NaCMC (type 7HF, Hercules) methylethyl-cellulose : MEC : "EDIFAS A" (ICI) hydroxypropyl-cellulose : "KLUCEL" (type LF, Hercules) dextran (Pharmacia, Uppsala, Sweden) Filler: silica powder Surfactants: sodium-dodecyl sulfate : SDS ethyloxylated-alkylphenol : "RENEX 698" Buffer: sodium-hydrogen maleate buffer, pH 5.8:MALEATE TABLE 5 Occult Blood Test Reagent Inks Ink Solvent Reagent Binder, Surfactant Buffer wt% Filler Example 1 RI.A1 water CHP NaCMC SDS MALEATE 79% (3.14g) (0.5g) (0.5g) (30 ml) PVP (14 g) RI.B1 ethanol o-tolidine PVP Renex 698 82% (0.4g) (20 g) (0.5 g) Example 2 RI.A2 water CHP NaCMC SDS MALEATE 74.5% (3.14g) (0.6g) (0.5g) (30 ml) PVP (18 g) RI.B2 ethanol o-tolidine PVP Renex 698 83% (0.6g) (27 g) (0.6 g) Example 3 RI.A3 water CHP NaCMC SDS MALEATE 76% (3.14g) (0.6g) (0.5g) . (30 ml) PVP (16 g) RI.B3 ethanol o-tolidine PVP Renex 698 83% (0.6g) (27 g) (0.6 g) Examples 4-7 RI.A4 water CHP "NATROSOL" SDS MALEATE 68% (6.3g) (8 g) (1 g) (35 ml) "AEROSIL" (5 g) GELATIN (0.5g) RI.B4 ethanol o-tolidine "KLUCEL" Renex 698 (0.8g) (10 g) (1 g) Example 1 Diagnostic test strips for the detection of blood in urine, or so-called occult blood test strips are produced as follows: (a) Preparation of Reagent Inks Two reagent inks RI.A1 and RI.B1 (see Table 5) are prepared as a fluid carrier medium for the respective reagents cumene hydroperoxide (CHP) and o-tolidine which provide an oxidation colour test reaction in the presence of blood. They are obtained by admixing the aqueous buffer with an aqueous solution which contains the dissolved binder and emulsifier, (surface active agent) together with the respective reagent.The resulting mixture is energetically stirred so as to provide ink in the form of a viscous homogeneous, stable emulsion or colloidal suspension having the corresponding composition respectively given for RI.A1 and RI.B1 in Table 5 above.

The resulting reagent ink compositions have a relatively high viscosity which is particularly adapted to the screen-printing technique by which these inks are to be applied to the test strip support medium.

The inks given in Table 5 above respectively have a viscosity of AI = 1,000 to 20,000 CP and BI = 1,000 to 20,000 CP.

(b) Support Medium A sheet of white synthetic "paper" which is known by the Trademark "Polyart" (90 g/m2), has a matt surface finish and a thickness of = 80R, is used as printing support medium for producing test strips.

This type of Polyart paper consists of a modified polyethylene cast into thin film. It is commercially available for use in the graphic arts industry.

(c) Printing with Reagent Inks The two reagent inks RI.A1 and RI.B1 are consecutively applied by means af a special screen-printing technique onto the surface of the abovementioned white Polyart sheet used as the printing support.

Each reagent ink is printed in the form of tiny discrete dots which are mutually spaced apart at a short distance according to a triangular printing pattern.

A perforated nickel sheet of the type Veco 25R (see Table 4 above) was first used to screen-print a first triangular array of reagent ink dots A having a diameter between 300 and 3701l and an interaxial spacing of 1 mm, using the reagent ink RI.A1 (see Table 5 above) in the manner already described hereinbefore under the heading "Printing Process".

The same sheet Veco 25R was used to screen-print, as already described, a second triangular array of reagent ink dots B each centrally located with a rhombus formed by four adjacent dots A already printed. These dots B have a diameter lying between 300 and 350eel and the same interaxial spacing of 1 mm as the dots A and the holes of the perforated sheet used as a printing screen.

The proportion of the printed surface covered by both dots A and B amounted in this case to 17.7% of the total surface of the printing support sheet.

(d) Cutting up into Test Strips The printed sheet of Polyart is cut up into tests strips having a width of 4 mm.

A blue colour change may be distinctly observed when the test strips thus obtained are dipped in urine containing haemoglobin in an amount equal to or greater than 0.0014 mg/100 ml.

Example 2 Occult blood test strips are produced in substantially the same manner as described in Example 1, except that the reagent inks RI.A2 and RI.B2 (see Table 5 above) are here used to print through a perforated Veco sheet of the 30R type (see Table 3 above).

The interaxial spacing of the screen holes and hence of the printed dots is in this case 0.83 mm, while the printed reagent ink dots have respectively a diameter of 315 to 335u and 320 to 360F, the proportion of the printed area covered by all dots A and B being about 16.5% of the total printing surface.

The blue colour change which is observed when the printed test strips thus produced are utilized to detect blood in urine is substantially the same as with the test strips produced according to Example 1.

Example 3 Occult blood test strips are produced in substantially the same manner as described in Example 1 except that the reagent inks RI.A3 and RI.B3 (see Table 5 above) are here used to print through a perforated Veco sheet of the 40R type (see Table 3 above).

The interaxial spacing of the screen holes and hence of the printed dots is in this case 0.63 mm, while the printed reagent ink dots A and B respectively have a diameter of 235 to 255it and 255 to 260F, the proportion of the printed area covered by all dots A and B being about 15% of the total printing surface.

The blue colour change which is observed when the printed test strips thus produced are utilized to detect blood in urine is substantially the same as with the test strips produced according to Examples 1 and 2.

Example 4 Blood test strips are produced in substantially the same manner as described in Example 1, except that the reagent inks RI.A4 and RI.B4 (see Table 5 above) are used to print with a perforated Veco sheet of type 60P (see Table 3 above) on a sheet of absorbent white paper stock.

The interaxial spacing of the screen holes and hence of the printed dots of each array is 0.42 mm in this case, while the printed reagent ink dots A and B respectively have diameters of about 0.187 mm and 0.180 mm, the proportion of the printed area covered by all dots A and B being about 34% of the total printing surface.

The printed dots of inks RI.A4 and RI.B4 did not spread out on the absorbent paper stock but remained as small discrete dots on the surface of the paper. This non-flowing property of the inks is due to their high viscosities and paste-like rheology which nevertheless allows them to be correctly printed by the process described above.

A blue colour change is observed when the printed test strips thus produced are utilized to detect haemoglobin in solution in the same way as with the test strips produced according to Examples 1, 2 and 3, but with the added advantage of providing a more homogeneous and deeper colour response in the test strips produced in the present example.

Example 5 Occult blood test strips are produced in substantially the same manner as described in Example 1 with the same inks and Veco sheet (60P) as described in Example 4, except that the second reagent ink RI.B4 is printed in two successive impressions as two sets of discrete dots.

The ink RI.A4 is firstly printed on absorbent paper stock in the previously mentioned triangular dot array. When these printed dots are dry, the ink RI.B4 is printed with the same Veco screen (60P) within triangles of dots formed by the ink RI.A4, while there are adjacent triangles of dots formed by this RI.A4 ink which do not contain a dot of the RI.B4 ink. Therefore, a second impression of the RI.B4 ink is carried out to print dots in these vacant triangles.

The proportion of the total printed area covered by all dots A and B is then about 50% of the total printing surface.

A blue colour change is observed when the printed test strips thus produced are utilized to detect haemoglobin in solution in the same way as the strips described in Example 4, but with the added advantage of having a more homogeneous and deeper colour response than in the case of Examples 1 to 4.

Example 6 Occult blood test strips are produced in substantially the same manner as described in Example 4, except that the printing support medium used in the present case is a thin glass fiber-sheet special filter paper type 10,70 g/m2, from Schleicher and Schull GmbH, West Germany.

This printing surface is receptive to the ink and the ink dots adhere well thereon. The test strips printed on this material present a hydrophilic surface to the haemoglobin solutions and respond with a blue colour change to haemoglobin concentrations equal to or greater than 0.0154 mg/ml.

Example 7 Blood test strips are produced in substantially the same manner as described in Example 5, except that the printing support medium used in the present case is a microporous plastic sheet which is based on polyvinyl chloride and is produced by the Amerace Corporation, Microporous Division.

The test strips produced on this support medium were used for test reactions with haemoglobin in solution and provided a very satisfactory colour change, as in the case of Example 5.

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

The compositions of the glucose test reagent inks used in Examples 8 and 9 are given in Table 6 below wherein those abbreviations and Trademarks which have not already been explained with reference to the foregoing examples represent the following substances: Reagents: glucose oxidase = GOD peroxidase = POD potassium iodide = KI brilliant blue FCF dye = FD and C Blue No. 1 Binders: carboxymethyl-cellulose = CMC (type 7LF, Hercules) polyvinylpyrrolidone = PVP = 'PLASDONE" (type K29-32), GAF Corp.

Surfactants: polyoxyethylenesorbitan monolaurate = "Tween 20" (ICI Americas, Inc.) Buffer: 0.8 M citrate = 350 ml H2O + 257.3 g sodium citrate dihydrate + 25 g anhydrous citric acid TABLE 6 Glucose Test Reagent Inks Ink Solvent Reagent Binder Surfactant Buffer wt.% Example 8: water GOD it.06 g) CMC "Tween 20" Citrate RI.A8 85% POD (0.01 g) (6 g) (0.3 g) (50 ml) RI.B8 ethanol o-tolidine "Klucel" "Renex 698" 72% (0.8 g) (1û g) (1 g) - Example 9: water GOD (0.3 g) CMC "Tween 20" Citrate RI.A9 73% POD (0.04 g) (2 g) (0.06 g) (10 ml) RI.B9 water KI (0.08 g) PVP (0.2g) "Tween 20" Citrate 77% blue dye CMC (2.0g) (0.08 g) (15 ml) (0.06 g) Example 8 Diagnostic test strips for the detection of glucose in urine are produced as follows:: (a) Preparation of Reagent Inks Two reagent inks RI.A8 and RI.B8 (see Table 6 above) are prepared as a fluid carrier medium for the reagents glucose oxidase (GOD) and peroxidase (POD) on one hand and for ortho-tolidine on the other, which provide an oxidation colour test reaction in the presence of glucose.

The first reagent ink RI.A8 is obtained by admixing the aqueous CITRATE buffer (0.8 M) with two reagents (GOD and POD in powder form) and with the surfactant "Tween 20", and dissolving the binder CMC - 7LF therein. The resulting mixture is energetically stirred so as to provide the first reagent ink RI.A8 in the form of a viscous, homogeneous, stable solution with the composition given in Table 6.

The second reagent ink RI.B8 is the same as the second ink RI.B4 given in Table 5 above with reference to the foregoing Examples 4 to 7.

These reagent inks RI.A8 and RI.B8 both have a relatively high viscosity which is particularly adapted to use of a screen-printing technique for applying these inks to the test strip support medium.

(b) Support Medium A sheet of absorbent white paper is used as the printing support medium for producing the glucose test strips.

(c) Printing with Reagent Inks Glucose test strips are produced by printing in substantially the same manner as described in Example 1, except that the reagent inks RI.A8 and RI.B8 are used in the present case to print through a perforated Veco sheet of the 125P type (see Table 3 above).

The proportion of the printed surface covered by all the dots A and B amounts in this case to more than 30% of the total surface of the printing support sheet.

(d) Cutting up into Test Strips The resulting printed paper sheet is finally cut up into glucose test strips of 4 mm width.

A strong blue colour change may be distinctly observed when the test strips thus obtained are dipped in urine containing glucose in an amount equal to or greater than 0.1 g/100 ml.

Example 9 Diagnostic test strips for the detection of glucose in urine are produced in essentially the same manner as described in Example 8, except that the reagent inks RI.A9 and RI.B9 are used, which have the compositions given in Table 6 above.

These reagent inks RI.A9 and RI.B9 are used to print through a perforated Veco sheet of the 60P type (see foregoing Table 3) on an absorbent paper sheet, as in Example 8 above, and the resulting printed sheet is finally cut up into test strips of 4 mm width.

The unreacted printed glucose test strips thus obtained are blue in colour due to the presence of a small amount of an oxidizable blue dye (FD and C blue No. 1), in addition to the water soluble iodide salt (KI), in the second reagent ink RI.B9.

A colour change may be distinctly observed when the test strips thus obtained are dipped in urine containing glucose in an amount equal to or greater than 0.1 g/100 ml. The observed colour changes depend on the glucose concentration as follows: Glucose Colour Developed (g/100 ml) in 1 minute 0 blue 0.1 light green 0.5 brown-green 1 brown The following example illustrates the production of diagnostic test strips for the detection of ketone bodies (acetone, acetoacetic acid, betahydroxybutryric acid) in body fluids, especially acetoacetic acid in urine.

The compositions of the ketone test reagent inks used in the following Example 10 are given in Table 7 below, wherein the abbreviations and Trademarks used for the binder are the same as indicated above with reference to the foregoing examples.

"GAFAC RE-610" (see Table 7 below) is a Trademark for a surfactant manufactured by GAF Corp.

"PVP/VA" is a polyvinylpyrrolidone/vinyl acetate copolymer (50% in ethanol). TABLE 7 Ketone Test Reagent Inks Ink Solvent Reagent Binder Surfactant Buffer Example 10: RI.A10 water aminoacetic CMC-76F - Na3PO4.12H2O (36 ml) acid (9 g) (3 g) (10 g) Na2HPO4 anhydrous (4.3 g) RI.B10 EtOH Na-nitroferri- PVP/VA "GAFAC RE (54 ml) cyanide (2.9g) copolymer 610" (23 ml) (0.3 g) "KLUCEL JF" dioctyl (10 g) sodium- sulfoccinate Example 10 Diagnostic test strips for the detection of ketone bodies in body fluids are produced in essentially the same manner as described in Examples 8 and 9, except that the reagent inks RI.A10 and RI.B1û here used respectively comprise the aqueous phosphate compounds, the sodium nitroferricyanide and the ethanol-soluble binders (PVP/VA, "KLUCEL JF") in alcoholic phase which are given in Table 7 above.

These reagent inks RI.A10 and RI.B1û are used to print through a perforated Veco sheet of the 60P type (see foregoing Table 3) on an absorbent paper sheet, as in Examples 8 and 9 above, and the resulting printed sheet is finally cut up into test strips of a desired width.

A violet colour change may be distinctly observed when the printed ketone test strips thus obtained are dipped in liquids containing acetoacetic acid.

The colour varies from very light purple in the presence of 10-20 mg per 100 ml of acetoacetic acid to a very dark purple indicating over 100 mg per 100 ml.

It has been experimentally established, as appears from the foregoing examples, that the total printed area covered by all dots of the patterns printed with different reagent inks, to produce printed test strips in accordance with the present invention, should cover 30% and preferably 45% of the total area of the printing surface enclosing the different patterns, in order to provide the best colour changes and to thereby ensure the most satisfactory colour test reactions by means of the printed test strips in all cases.

Such coverage of at least 30% of the surface area of the printing support medium may be readily accomplished by suitably selecting perforated screens used for printing, namely screens having perforations whereof the size and spacing is as small as possible, while allowing satisfactory, successive printing of different discrete tiny dots which lie between each other and are very closely spaced together, without being in contact with each other.

Thus, for example, the Veco screens of the type 60P, 80P and 125P, having the dimensions shown in Table 3 above and used in the foregoing Examples 4 to 10, are particularly suitable for printing patterns of different dots covering about 30-50% of the corresponding surface area of the printing support sheet.

It has likewise been experimentally established that the total number of dots printed per unit area of the printing support sheet should further be equal to or greater than 1300 dots per square centimeter, in order to provide the best colour changes and to thereby ensure the most satisfactory colour test reactions by means of the printed test strips in all cases.

An overall density of discrete printed dots greater than 1300 dots per square centimeter may also be readily accomplished by suitably selecting perforated screens used for printing, such as for example, the said Veco screens of the types 60P, 80P and 125P used in the foregoing Examples 4 to 7.

In order to also ensure satisfactory colour test reactions in all cases, it has moreover been experimentally found that the test strips produced by printing in accordance with the present invention should be provided with printed dots having a thickness lying between 5 and 60 microns. It has further been found that, for similar reasons, the viscosity of the reagent inks used for printing test strips in accordance with the present invention should preferably lie in the range between 5000 and 300,000 centipoise in order to ensure satisfactory printing.

The reagent inks described above (Table 5) with reference to the foregoing examples are able to provide a sufficient ink viscosity and dot thickness, with a sufficient surface coverage and overall dot density, for ensuring most satisfactory colour test reactions by means of the printed strips in all cases.

It may be seen from the foregoing that the production of printed diagnostic test strips in accordance with the present invention may be likewise applied so as to provide similar advantages in a broad variety of colour test reactions using more or less conventional reagents and other ink components, whereof the chemically compatibility with each other and with more or less conventional printing support media may be experimentally established beforehand without great difficulty.

It is thus understood that the reagent ink compositions, the printing support medium and the printing means used may in each case be mutually adapted on the basis of prior art experience in the relevant fields of diagnostic agents and or printing methods.

The present invention is applicable to the presently known reactant compositions used in the preparation of diagnostic test devices. Typical test reactant compositions are set forth in U.S. Patents 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 will, however, be understood that the invention is especially applicable to diagnostic formulations in which it is important or desirable to keep reactants separated before use.

WHAT WE CLAIM IS: 1. A method of producing diagnostic test strips for detecting given components present

**WARNING** end of DESC field may overlap start of CLMS **.

Claims (21)

**WARNING** start of CLMS field may overlap end of DESC **. Example 10 Diagnostic test strips for the detection of ketone bodies in body fluids are produced in essentially the same manner as described in Examples 8 and 9, except that the reagent inks RI.A10 and RI.B1û here used respectively comprise the aqueous phosphate compounds, the sodium nitroferricyanide and the ethanol-soluble binders (PVP/VA, "KLUCEL JF") in alcoholic phase which are given in Table 7 above. These reagent inks RI.A10 and RI.B1û are used to print through a perforated Veco sheet of the 60P type (see foregoing Table 3) on an absorbent paper sheet, as in Examples 8 and 9 above, and the resulting printed sheet is finally cut up into test strips of a desired width. A violet colour change may be distinctly observed when the printed ketone test strips thus obtained are dipped in liquids containing acetoacetic acid. The colour varies from very light purple in the presence of 10-20 mg per 100 ml of acetoacetic acid to a very dark purple indicating over 100 mg per 100 ml. It has been experimentally established, as appears from the foregoing examples, that the total printed area covered by all dots of the patterns printed with different reagent inks, to produce printed test strips in accordance with the present invention, should cover 30% and preferably 45% of the total area of the printing surface enclosing the different patterns, in order to provide the best colour changes and to thereby ensure the most satisfactory colour test reactions by means of the printed test strips in all cases. Such coverage of at least 30% of the surface area of the printing support medium may be readily accomplished by suitably selecting perforated screens used for printing, namely screens having perforations whereof the size and spacing is as small as possible, while allowing satisfactory, successive printing of different discrete tiny dots which lie between each other and are very closely spaced together, without being in contact with each other. Thus, for example, the Veco screens of the type 60P, 80P and 125P, having the dimensions shown in Table 3 above and used in the foregoing Examples 4 to 10, are particularly suitable for printing patterns of different dots covering about 30-50% of the corresponding surface area of the printing support sheet. It has likewise been experimentally established that the total number of dots printed per unit area of the printing support sheet should further be equal to or greater than 1300 dots per square centimeter, in order to provide the best colour changes and to thereby ensure the most satisfactory colour test reactions by means of the printed test strips in all cases. An overall density of discrete printed dots greater than 1300 dots per square centimeter may also be readily accomplished by suitably selecting perforated screens used for printing, such as for example, the said Veco screens of the types 60P, 80P and 125P used in the foregoing Examples 4 to 7. In order to also ensure satisfactory colour test reactions in all cases, it has moreover been experimentally found that the test strips produced by printing in accordance with the present invention should be provided with printed dots having a thickness lying between 5 and 60 microns. It has further been found that, for similar reasons, the viscosity of the reagent inks used for printing test strips in accordance with the present invention should preferably lie in the range between 5000 and 300,000 centipoise in order to ensure satisfactory printing. The reagent inks described above (Table 5) with reference to the foregoing examples are able to provide a sufficient ink viscosity and dot thickness, with a sufficient surface coverage and overall dot density, for ensuring most satisfactory colour test reactions by means of the printed strips in all cases. It may be seen from the foregoing that the production of printed diagnostic test strips in accordance with the present invention may be likewise applied so as to provide similar advantages in a broad variety of colour test reactions using more or less conventional reagents and other ink components, whereof the chemically compatibility with each other and with more or less conventional printing support media may be experimentally established beforehand without great difficulty. It is thus understood that the reagent ink compositions, the printing support medium and the printing means used may in each case be mutually adapted on the basis of prior art experience in the relevant fields of diagnostic agents and or printing methods. The present invention is applicable to the presently known reactant compositions used in the preparation of diagnostic test devices. Typical test reactant compositions are set forth in U.S. Patents 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 will, however, be understood that the invention is especially applicable to diagnostic formulations in which it is important or desirable to keep reactants separated before use. WHAT WE CLAIM IS:

1. A method of producing diagnostic test strips for detecting given components present

in biological liquids such as urine, which comprises the steps of: (a) providing at least two reagent printing inks each consisting of a mixture of mutually compatible components such that the inks taken together include all the reagents required for the desired test reaction while no single ink includes all the said required reagents, each said ink comprising (a) as solvent, water, an alcohol or a water-alcohol mixture, (b) a polymeric binding agent such as to confer on the ink rheological properties suitable for the printing technique used, (c) a wetting agent, and (d) in the case of at least one of the inks, a buffer such as to provide a specific predetermined pH upon wetting of the test strip by the biological liquid;; (b) successively printing the said reagent inks onto a sheet which is substantially insoluble in water and chemically inert to the reagent inks at least on the surface thereof, and has a surface suitable for printing with the said inks so as to form on the said surface at least two separate patterns of printed dots of the said reagent inks, the reagent ink dots of each said pattern being dried after printing and being arranged in closely spaced relationship with one another and with the dots of each different pattern, so that the printed dots of each reagent ink are separately arranged between the dots of each different reagent ink, in close proximity thereto but without contact between the dots of different inks, so that the reagents in the respective dry dots are thereby kept separate from one another, while allowing said reagents to diffuse along said surface and to thereby mutually interact thereon in the presence of said liquid analysate; and (c) cutting up the said sheet having the said separate printing patterns printed on the surface thereof into a plurality of test strips of desired shape and size

2. A method as claimed in claim 1 wherein the said sheet comprises polyethylene, polypropylene, poly(vinylchloride), poly(vinylidene chloride), polystyrene, styrene acrylonitrile copolymer, an acrylic resin, chlorinated polyether, poly(chlorotrifluoroethylene), fluorinated ethylene/propylene copolymer, poly(tetrafluoroethylene), nylon-6,6 or nylon6, acetal resin, polycarbonate resin, polyurethane resin, cellulose acetate butyrate, cellulose propionate, or ethyl cellulose.

3. A method as claimed in claim 1 or 2 wherein said binding agent comprises gelatin, gum arabic, a cellulose derivative, collagen, poly(vinyl alcohol), poly(N-vinyl pyrrolidone), or mixtures thereof.

4. A method as claimed in claim 3 wherein the said binding agent comprises sodium carboxymethylcellulose, polyvinylpyrrolidone, hydroxyethyl cellulose, methylethylcellulose, hydroxypropylcellulose or dextran.

5. A method as claimed in any one of the claims 1 to 4, wherein the said buffer comprises citric acid and sodium citrate, tris(hydroxymethyl)aminoethane, a phosphate buffer, or a phthalate buffer.

6. A method as claimed in any one of claims 1 to 4 wherein the said buffer has a pH of 5 to 6 and comprises monosodium maleate and NaOH, succinic acid and NaOH, monopotassium phthalate and NaOH, P,P'-dimethylglutaric acid and NaOH, or sodium citrate and citric acid.

7. A method as claimed in any one of claims 1 to 6 wherein the said wetting agent comprises sodium dodecyl sulphate, ethoxylated alkyl phenol, polyoxyethylene-sorbitan monolaurate, or polyethylene glycol.

8. A method as claimed in any one of claims 1 to 6 wherein the said binder and the said wetting agent are the same substance, and the said substance comprises gum arabic, gelatin or a mixture thereof.

9. A method as claimed in any one of claims 1 to 8, wherein the said dots are printed by passing each reagent ink through a perforated screen having a regular array of holes spaced apart in accordance with the corresponding dot pattern.

10. A method as claimed in claim 9, wherein the holes of said screen are spaced apart in parallel rows with the holes of alternate rows being in a mutually staggered arrangement.

11. A method as claimed in claim 10, wherein two reagent inks are successively applied by passing a first reagent ink through said perforated screen onto the underlying surface of said sheet so as to thereby form thereon a first pattern of printed dots corresponding to the staggered hole arrangement of said screen, causing the printed dots of said first pattern to dry, changing the relative position of the said sheet surface having said first pattern printed thereon with respect to said screen, in such a mannr that the holes of the said screen come to overlie unprinted zones of said surface, and applying the second reagent ink through said screen in the same manner as the first ink so as to thereby form a similar second pattern of printed dots arranged between the dots of the first pattern.

12. A method as claimed in claim 11, wherein the said relative position is changed in such a manner that the said holes are caused to overlie unprinted zones which are each symmetrically located between adjacent dots of said first pattern.

13. A method as claimed in claim 12, wherein after changing said relative position the said holes are caused to overlie unprinted zones which are each substantially equidistant from diametrically opposite adjacent printed dots of said first pattern, so that the subsequently printed dots of said second pattern are thereby each centrally located within a rhombus formed by four adjacent dots of said first pattern.

14. A method as claimed in claim 12, wherein after changing said relative position said holes are caused to overlie unprinted zones which are each substantially equidistant from three adjacent dots of said first pattern, so that the subsequently printed dots of said second pattern are thereby centrally located within a triangle formed by three adjacent dots of said first pattern.

15. A method as claimed in any one of claims 12 to 14, wherein the printed area covered by the said patterns of different dots is at least 30% of the total surface area enclosing the said patterns.

16. A method as claimed in claim 15, wherein the total number of different dots printed per unit surface area of said support sheet is at least equal to 1300 dots per square centimetre.

17. A method as claimed in claim 16 wherein the thickness of the said different printed dots is between 5 and 60 microns.

18. A method as claimed in any one of claims 1 to 17 wherein each of the said reagent inks has a viscosity between 5,000 and 300,000 centipoises.

19. A method as claimed in any one of claims 1 to 18 for producing test strips for detecting blood in urine, wherein two reagent inks are provided, the first ink comprising the reagent cumene hydroperoxide, water as solvent, hydroxyethyl-cellulose as binder; a wetting agent, and a buffer, and the second ink comprising the reagent ortho-tolidine, ethanol as solvent, hydroxypropyl-cellulose as binder, and a wetting agent.

20. The method as defined in claim 1 substantially as described in any one of the foregoing Examples.

21. Diagnostic test strips when produced by the method of any of the preceding claims.