Technical Field

The present invention concerns test strips for evalua- ting substances dissolved in aqueous Solutions, for instan- ce biological fluids. Strips of this kind, are generally made from a thin porous rigid or flexible laminated mate- rial in which reagents are incorporated, said reagents being responsible for the development of typical color reac- tion when the strip is contacted with the substances to be analyzed, for instance by dipping the strip into an aqueous solution of said substances. Such strips are particularly useful for making rapid qualitative or semi-quantitative determinations in medical diagnosis. A typical example of deterrαination is the checking of blood or glucose in urine. There exists already many modeis of coπimercial strips for achieving the above purpose, among which the following can be mentioned.

Back Ground of the Art

US Patent No 3,092,463 discloses the use of strips for analyzing so e blood constituents. These strips are made of filter paper impregnated with dtifferent reagents such as encapsulated hydroperoxides, an indicator (o-tolidine) and a buffer (citrate) . Under normal storage conditions, no reaction occurs between the reagents and no discoloration of the indicator is observed but when the strips are dipped into a solution containing a substance to be analyzed, e.g. the prosthetic groups of blood, the capsules break hydroly- tically with consecutive liberation of the hydroperoxide

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and a color reaction with the indicator is allowed to deve- lop. However, these strips have the disadvantages that the filter paper (bibulous material) which Supports the reagents often contains impurities which may interfere with the co- lor development and, further, its sampling capacity for the solution to be analyzed is not well constant fro test to test because of unavoidable differences in the fibrous mate¬ rial used for aking the strips; thus the method is only qualitative. Another disadvantage is that the encapsulation is performed by means of a colloid substance such as gelatin, gum arabic or carboxy-vinyl polymers that produces very fra¬ gile thin walled capsules of questionable mechanical and storage stability. Another disadvantage is that the color will develop within the bibulous material itself which is often opaque and not homogeneous, thus introducing further sources of errors in the color evaluation.

USP 3,926,732 discloses the use of test strips for the color determination of catalase in milk. This method is b.ased on the catalase dependent inhibition of the color reac- tion involved upon oxidation of a leuko-dye (o-tolidine) by hydrogen peroxide in the presence of peroxidase. In one em- bodi ent of said reference, the strips comprise an inert so¬ lid absorption and diffusion medium in which are embedded physically separated individual microcapsules. The microcap- sules are of the "Semi-permeable aqueous" type (see Can.J. Physiol.Pharmacol. 4_4 (1966) , 115) and the medium is made of cellulose fibers. ^" When the strip is dipped into the solution to be analyzed, the hydrogen peroxide generated by the rea- gents of a first kind of microcapsules .will migrate through the medium whereby it will be partially inhibited in propor- tion to the a ount of catalase to be analyzed and, finally, it will react with the dye having diffused from microcapsule of a second kind. The color will then be released within the diffusion medium with consecutive disadvantages as men- tioned heretofore. Also in this method the sampling capacity of the strip per unit area is very difficult to keep constan

and the method is not accurate. Another disadvantage of the strips involving layers of fibrous absorbing media is the fact that such media have a largely opened structure which does not adequately filter out unwanted colored species which may be present in the liquid to be analyzed and which may interfere with the desired color reaction. This has been partly remedied by using strips with a low porosity media layer in which the reagents are dispersed or dissol- -ved. Such kind of strips are disclosed for instance in TJSP 3,630,957, but they have the drawback that they possess no instant sampling capacity for the liquid to be analyzed. Indeed, when such strips are immersed in said liquid, the latter will slowly penetrate into the pores of the medium (in the ränge of 0,1-1 m) and the substances to be analyzed will diffuse toward the reagents leaving out the possibly interfering materials. However, this process will take seve¬ ral minutes or more and it is not possible to exactly say at which time the process is really effective and when the color is sufficiently developped. Therefore, the test is slow and qualitative only. Some of the above drawbacks can be obviated by depositing a drop of the liquid to be analy¬ zed on the strip. However, this is also a slow process and there is no way to teil that the drop will always be absor- bed by a known given area of the strip; therefore the test is also qualitative only.

The above disadvantages have been partly cured by using the strips disclosed in French Patent No 2.303.290. These "strips comprise as a diffusing medium a synthetic polymer obtained by the phase Inversion precipitation technique. According to this technique, a solution of a polymer is pre- pared in a mixture of two solvents, one of which being a poorer solvent for this polymer and less volatile than the other solvent. When the solution is allowed to dry, there becomes a moment when the good solvent has sufficiently eva- porated for causing the polymer to slowly precipitate which results, after complete drying, in an opened porous gell«

bibulous structure resembling cellulose fibers and having favorable sampling properties for the liquid to be analyzed The reagents necessary to develop the analytical color reac tions of the strips can be either incorporated by impregna- tion or by dissolving into the original polymer solution. This is advantageous because, in case of mutually incompa- tible reagents, one of which can be put within the body of the polymer itself whereas the other can be imbibed in the opened porous structure whereby contact between said rea- gents in the dry State under storage is minimized. These strips have any advantages, however the intimate structure and the porosity of the matrix is strongly dependent on the preparative conditions and reproducibility of the specifica tions is difficult to maintain from baten to batch. Also, because of its rather fibrous nature, this material can in- troduce differential diffusion effects on the solution to b analyzed which can be a source of inconsistency in color de velopment. Further, the color develops within the f ll body of the absorptive medium which, because of light diffractio problems related to the structure thereof, may limit the se sitivity in some cases.

USP 3,993,451 discloses test strips an embodiment of which comprises a strip of hydrophilic paper coated, on'one side, with a uniform layer of a homogeneous mixture of two kind of particles. These particles are formed of agglomera- tes of hydrophilic absorbent materials such as powdered cel lulose, alumina, silicagel, ete ... impregnated by the rea¬ gents Solutions and dried thereafter. One kind of the parti cles contain a first reagent and the second kind of parti- cles contain a second reagent, said two reagents being not compatible and being thus kept apart during storage. ^" When dipped into the solution to be analyzed, a portion of said solution is sampled by the absorbent materials and the rea¬ gents are allowed to contact the substance to be tested wit consecutive development of the color reaction. This color test is therefore very fast but the sampling action is rath

erratic because of the powdery nature of the absorbent mate¬ rial and the uncontrolled size of the agglomerates containing the reagents.

The present invention has for object to correct as uch as possible the above discussed deficiencies. Briefly summarized an ideal test strip would fullfil the following needs and properties : a) Accurately sample a given amount of solution to be analyzed per unit area of the strip. b) Achieve such sampling instantaneously in the course of one Single move : dip and withdraw. c) Allow intimate contact between the substance to be analyzed and the reagents and filter out undesirable component of the solution which may interfere with the desired analyti- cal reactions. d) Allow rapid and reproducible color development with good sensitivity and good measurability (visual or spectro- photometer) . e) Allow good Separation under storage of reagents when mutually incompatibles,. f) Accomodate reagents of different compatibilities , e.g. water-soluble reagents and liposoluble reagents. g) Avoid as much as possible so called "inert" media which may mask or interfere with the color reaction. h) Offer simple and economic fabrication routes. i) Be strong enough mechanically to withstand acciden- tal abuse (rupture by abrasion) . j) Have a long shelf-life, i.e. have reagents well shielded and preserved from evaporation or decomposition. Disclosure of the Invention

The test strip of the invention is a very valuable approach to the above requirements. This strip essentially encompasses a supporting non porous, non absorbant base Sub¬ strate, e.g. a plastic sheet or strip coated (at least on one side) with a continuous layer of polymeric hydrophilic microspheres or beads, the reagents necessary for achieving

the desired analytical color reactions being incorporated in said beads. Alternatively, the base material could be a etal sheet, e.g. an aluminum sheet or a glass sheet. The beads are simply attached by adhesion to the supporting str without any dispersive and absorbent matrix for embedding t beads like in the strips of the prior-art in which said ma¬ trix serves usually as a sampling medium for the Solutions to be tested and also as a development medium for the desi¬ red colored analytical reaction. Therefore, in the strip of the invention, the color will develop within the beads them selves which is much advantageous with respect to color de¬ velopment reproducibility because an erratic spreading of the reagents is thus strongly minimized. Further, despite the lack of an absorbent medium, the present strip displays a very accurate sampling capacity. In this connection, it should be noted that the absorbent power of the beads the - selves (or rather of the hydrophilic polymer which constitu tes the beads) has no particular influence, because it is the space between the beads which is adapted for achieving the sampling action on the solution to be analyzed. This is possible because the beads are made of a .hydrophilic polyme which gets wetted by said solution when the strip is im er- sed therein and which will therefore instantaneously retain a constant portion of liquid per unit area due to capillary effects. Therefore, depending on the wetting properties of the polymer used and on the beads average diameter, the amount of liquid sampled per unit area of the strip can be determined and kept under control. For this, the beads will preferably have a uniform average diameter and they will be applied on the strip in a uniform fashion, e.g. as a mon layer coating. In such case, the space between the beads will result from placing the beads on the strip in substan- tially close relationship to each other so as to provide between the beads on said strip voids of regulär distribu- tion. The sum of these voids will furnish a constant volu- me per unit area to be filled by the solution to be analy-

zed when the strip is dipped therein. The beads will be adhered to the strip preferably by means of an adhesive but other means such as, for instance, heat softtening the surfa- ce of the plastic support to the point that the beads will directly adhere thereto is also possible. It should be re- marked at this stage that the principle of putting beads side by side on a strip to achieve a sampling action on a liquid is not novel per se. It has been disclosed for instance in Prench Patent No 2.191.734 which teaches a strip for detec- ting and evaluating substances by applying a drop of a solu¬ tion of said substances on the strip and spectrophotometri- cally determining the color developped by some reaction bet¬ ween reagents of the strip and said substances to be analy¬ zed. In the strip of this patent, there is at least three layers having independent functions : one of the layers ser- ves as a reagent holding medium, a second layer serves as a filter element and a third external layer function as a dis- tributing medium for equally spreading the liquid to be ana¬ lyzed on the surface of the strip. This effect is obtained by using spherical beads of glass or polymer resin placed side by side as a spreading layer and coated onto the surfa¬ ce by means of an adhesive. However, it is said nowhere in the reference that such beads can also contain the analyti¬ cal reagents of the strip like in the invention. Preferably, in the invention, the size of the beads and the che ical nature of the polymer are selected for ha¬ ving the sampled liquid penetrate the beads at a rate suffi- cient to have an observable color develop within a inute. or so. The polymer therefore must be- adequately structured semi-per eability) and, in these conditions, the unwanted materials possibly present in the solution will be filtered out because the polymer is adapted for allowing the substan¬ ces to be analyzed to diffuse into the beads and for blo- cking other substances of larger molecular weights (proteins or red cells which are constituents of blood for example) . Some data relative to the positionning of the reagents

within the beads will be provided hereinafter.

The beads can all be of the same kind and contain the reagents all ixed together if they are mutually compatibl or separated from each other if necessary; the method for achieving this Separation will be described hereinafter. Alternatively, the beads can be of different kinds, i.e. one kind will contain one of the reagents and another kind will contain another reagent, said reagents being possibly not compatible with each other. In such case, the color will develop in one kind of the beads only but this is of no i portance because the beads can be made small enough ( r > 20-200 -Λ ) for having the colored area to appear unifo

Normally, for good color observation, the polymer use for the beads is preferably transparent or translucent and in such case, the plastic sheet supporting the beads can b made opaque for better reflectivity, e.g. with titanium di xide or any other opaque filier (BaCO-., CaSO,, paper coati pigments ete ...). Surprisingly enough, it has been found that the TiO_ can also be incorporated within the beads, i desired, which is possible because the site of the beads which is the most important for color development is near the periphery thereof i.e. the presence of the white pigme in the beads has no detrimental effect on the observation the color but rather enhances the contrast. The polymer of the beads of the present strips can be selected from most water-insoluble hydrophilic polymers provided they can be dissolved in non-miscible organic sol vents for reasons which will be discussed hereinafter. As such, produets derived from cellulose, hydroxyacrylic poly mers, polyalkylene-oxy polymers and polyamides can be used In the cellulose group, cellulose esters and ethers such a methylcellulose, ethylcellulose, butylcellulose, cellulose acetate, propionate and butyrate can be used. The amount o the reagents which can be incorporated in the beads is ex- tremely variable and, of course, depends on the particular reagents solubility, the sensitivity to be given to the

tests and other needs which will appear to people skilled in the Art from case to case. This will be illustrated in the Examples hereinafter.

Best Modes of Carrying out the Invention The method for preparing the beads is derived from known procedures and, more particularly, from the following reference: T.M.S. CHANG : "Microencapsulation of Enzymes and Biologicals", from "METHODS IN ENZYMOLOGY", Vol.44 (1976) , p.210. The method used depends on the particular product desired. Thus, in one particular embodirαent of the invention, a test strip was designed for ascertaining gluco- se in urine. The chemical reactions involved were the clas- sical oxidation of glucose in the presence of a glucose-oxi- dase and the detection of the hydrogen peroxide liberated by means of o-tolidine in the presence of peroxidase. For a- king the beads, an ester of cellulose was selected and dis- solved in a water-immiscible organic solvent containing o-tolidine. Then, a buffered water phase containing the en¬ zymes was emulsified within this organic solution, thus for- ming droplets of the water phase within the organic solution. Thereafter this emulsion was again dispersed in a second wa¬ ter phase which resulted in the formation of polymer solu¬ tion drops in said second water phase, each of said drops still containing, suspended therein, the droplets of the first water phase. Then, the dispersion was subjected to eva- poration whereby the organic solvent was removed, thus pro- viding hard cellulose ester beads containing srαall vacuoles filled with the water solution of the enzymes. The o-toli¬ dine stayed dissolved in the polymer resin itself and thus was kept apart from mixing with the enzymes. The beads were filtered, dried and subjected to calibration with proper wire-mesh sizes, the beads in the 100 Λ region being selec¬ ted. Then the beads were applied as a continuous monolayer coating on a plastic support strip using an adhesive. As such, any usual adhesive in liquid form can be used, e.g.

Solutions of carboxymethylcellulose (CMC) , polyvinylalcohol

(PVA) , gum arabic, butadiene-styrene rubber (3M) , ete ... In order to bond the beads to the plastic strip, a very thin layer of the adhesive solution can be sprayed or otherwise deposited on the latter and the beads are regularly adhered thereto using moderate pressure and an equalizing flat sur- face. In this case the beads were applied as a monolayer, the beads substantially touching each other in a continuous fashion. Alternatively, the beads can be wetted with a dilu- ted solution of the adhesive and applied on the plastic stri with a doctor-knife or any similar tool. In this case thicke layers öf beads (double or triple layers) can be contemplate

When under use, the .strip is dipped into the solution containing the glucose and immediately withdrawn. Then it is allowed to stand for a given ti e sufficient to enable the solution trapped in the space between the beads to penetrate the beads the selves and reach the vacuoles containing the enzymes. There, the reactions oeeur and the oxygen liberated by the peroxidase acting on the H_0_ generated by the oxida¬ tion of glucose will convert the o-tolidine located in the near vicinity of the vacuoles to the desired blue dye. On a practical ^• stand point, in order to have the color develop within a reasonable time, say one minute, the depth at which the substance -to be analyzed must penetrate into the polymer to substantially reach the reagents of the beads should be about a fraction of a icron to one micron. Therefore, the thickness of the semi-permeable membrane protecting the va¬ cuoles situated within easy reach from the outside should preferably be comprised betwen 0,1 Ain and 1 um.

In another embodiment, a strip was fabricated involving the use of two mutually uncompatible liposoluble reagents. In this case a first kind of beads were prepared by dissol- ving a polymer and the first reagent in a suitable non water-soluble solvent. This solution was dispersed in a water phase and thereafter subjected to evaporation which produced an aqueous dispersion of polymer beads of essen- tially uniform size (about 60 Λ<.m) containing the first rea-

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gent in the dissolved State. These beads of the first kind were then filtered, dried and calibrated. Beads of a second kind were made identically but with incorporation of the se¬ cond reagent. After drying, both kinds of beads were mixed in proportion such that suitable equivalent amounts of the reagents were present in the mixture, then the latter was used to prepare the test strips as described hereinabove in the case of the first embodiment. The field use for the strips of both these embodiments will be described herein- after in ore detail.

In the preparation of the beads discussed above, many solvents insoluble in water but having good dissolving power for the hydrophilic polymers selected can be used; as such chlorinated solvents such as methylene Chloride, Chloroform and trichlor-ethylene can be used, as well as esters and ethers such as ethyl acetate, butylacetate, diethylether, ^' ete ... In general such solvents must be volatile enough to be removed by evaporation under gentle heating and air blo- wing or reduced pressure. For carrying out the emulsion and dispersion operations mentioned above the use of emulsifying agents is strongly recommended. As such most usual commercial emulsifying agents with neutral properties are convenient, e.g. sodium-lauryl sulfate, "Tween 20" (made by ICI) , ete It can be seen from the above description that the objeets of the present invention are substantially fulfil- led by the present strip components. Thus, due to the pre¬ sence of hydrophilic beads arranged regularly on a non po- ξous water impervious supporting plastic film, an aecurate and fast sampling of the liquid to be analyzed is achieved. Then, because of the polymer intimate structure resulting from the method of preparation, the reagent will be effi- ciently separated and shielded under storage (fundamentally important when storing together a hydroperoxide and an oxi- dizable indicator) although the beads will allow intimate contact of the substances to be analyzed with the reagents

contained in the beads and simultaneously exclude unwanted materials. Further, the concentration of the reagents near the surface of the beads will be sufficient for obtaining useful color developments with a penetration of the solu- tion to be analyzed involving only a small fraction of the total thickness of the beads. In this respect, it should be pointed out that also with regard to contact surface consi- derations, there is a significant differehce between the prior-art and the invention. In the prior-art, the observa- ble reagent containing surface is a flat surface the absor- gent power and the reflectivi ^' ty of which is related to its planar ^* dimensions; In the invention, the surface is not fla but constituted by a succession of spherical beads the tota

2 surface of which is equal to 4 If r (r being the radius of the beads) . It can be easily calculated that the ratio of the area of said beads to the area of the surface supportin said beads is IT , i.e. the available area is now more than 3 times the area available in the strips of the prior-art. Also from the color observation stand point, it can be cal- culated that the observable area from an array of beads on a flat surface visible from the outside is about 1.4 times that of the flat surface itself; therefore the observable color density and sensitivity will be increased as compared with the strips of the prior-art. i can also be pointed out that in most test-strip em- bodiments of the prior-art, the bibulous materials used for sampling the solution to be analyzed will have to simulta¬ neously accomodate the compound to be detected, the dye and the other reagents. Further such materials have a great in- ternal contact surface and generally lack homogeneity becau they cannot well reconcile two opposing properties : a high absorption capacity for the liquid to be analyzed and an equalizing action on the migration rates of the various Chemicals involved (no chromatography effects) . All such drawback have been eliminated in the invention. Also, in th invention the reproducibility in color development will be

good because of the relatively narrow space concentration of the colored sites and the avoiding of the diffusion of the color outside the beads. Another favorable feature is the complete suppression of the fibrous embedding matrix of the strips of the prior-art, thus avoiding the differential diffusion effects related to the Chromatographie properties of such fibrous media.

Finally, the strips used in the invention can be fabri- -cated very simply and economically since they involve only two construetive elements i.e. the beads and the supporting strips, said beads being of a strong and resistant structu¬ re as compared with the frangible microcapsules of the prior- art. In such beads, the reagents are well shielded from the outside and the present strips have a very long shelf-life without modification of their analytical properties. The following Examples illustrate the invention. Industrial Applications Example 1

Two g of cellulose triacetate and 0,25 g of o-tolidine were dissolved in 50 ml of CH„C1_ with 0,1 g of a surfac- tant ("Tween 20" from ICI = polyoxyethylene-sorbitan mono- laurate) to form a polymer solution (solution A) .

A water solution was prepared by mixing together 3.5 ml of glucose-oxidase solution at lOOO I.U./ml, 60 mg of peroxidase (60 I.U./mg) and 185 mg of a citrate buffer (pH 4.7). This gave solution B.

Solution B was then emulsified in solution A under the following conditions : temperature : room temperature, stir- rer : 2000 rpm, time : 20 min..The emulsion consisted in droplets of the B solution (l-5 u ) suspended in the polymer organic solution. Then this emulsion was dispersed in a se¬ cond aqueous phase consisting of 460 mg of sodiu -lauryl Sul¬ fate in 800 ml of 0.1 M aqueous acetate buffer, care being taken not to stir too fast in order to avoid obtaining too small particles. This dispersion consisted thus in drops of the organic solution A suspended in the second phase, each

drops containing, evenly distributed, the droplets of solu tion B.

The dispersion was heated under agitation to 35 C for 30 min while air was blown at the surface. The CH-Cl^ was progressively driven off which resulted in the formation o solid polymer beads containing the o-tolidine and solution B of the enzymes as tiny vacuoles trapped within the micro porous body of the beads. When the CH^Cl- was completely r moved (which can be ascertained by the smell of the solven the beads were filtered and dried. They were examined unde the microscope and were shown to have dia eters ranging from about 40 to lOO j with the vacuoles situated near the periphery quite visible, the membrane separating said exte nal vacuoles from the outside being only a fraction of a z _* . Such configuration is illustrated by the photographs taken with a scanning electron microscope and given in annexe :

Photograph 1 pictures the beads at 200x magnification and shows the size distribution thereof.

Photograph 2 is an enlargement of one of the beads of photograph 1.

Photograph 3 is an enlargement of a portion of one of the beads of photograph 1 and shows the presence of the va cuoles normally filled with solution B. Of course, most of the vacuoles shown on the photographs have broken due to the pressure developped in the inside thereof when subjec¬ ted to the vacuum of the electron-microscope.

The beads were subjected to calibration with a wire mesh for selecting the beads in the ränge of 100 - diamete Then, the selected beads were uniformly deposited as a con nuous monolayer (each bead substantially touching its neig bors) on an auto-adhesive plastic strip (type "EGAFIX" mad by R. BURKHART, Lucerne, Switzerland) so as to obtain a strip in accordance with the invention.

The strips obtained as described were dipped for one second into Solutions of glucose of various concentrations (shown below) and the color was allowed to develop for one

minute after which a color readiήg was made. The color was due to the oxidation of o—tolidine by the peroxidase cata- lyzed action of the H ₂ 0 ₂ liberated by the glucose-oxidase catalyzed oxidation of glucose, thus producing a blue indi- cator dye. The results are shown below :

Glucose solutions Colors of the strips (concentration g/1)

1 very light blue

2,5 light blue

5 medium blue

10 deep blue

It is important to note that for reproducible results, the color readings must be taken at given intervals after dipping the strips because at the time of reading the reac- tion is not yet terminated and some of the glucose is not consumed yet.

The above described strips were very stable when stored under normal dry conditions. The membranous wall of the va- euoles being semi-permeable, the enzymes were efficiently trapped therein because of their molecular weight which was too high for the enzymes to filter out through said semi- permeable membrane. Example 2 Beads were prepared as described in Example 1 but o i- ting the o-tolidine in the polymer solution. This indicator was added afterwards to the dried beads by impregnating the beads with a solution of o-tolidine in toluene and subsequent drying. Then, test strips were prepared with the beads as described in Example 1 and behaved identically under field uses.

Example 3

The procedure of Example 1 was repeated but with the following Variation : solution A of the polymer was made from 4 g cellulose triacetate, 0.5 g of "Tween 20", 1 g of TiO _? and 50 ml of CH„C1 . The beads were prepared and

used for making test strips exactly as in Example 1. When the strips were tested with glucose Solutions they gave equally good results but with enhanced color contrast. Example 4 A first organic solution was prepared by mixing toge- ther : CH ₂ C1 ₂ 25 ml; sodium-lauryl sulfate 0.5 g; cellulos triacetate 2 g; cumene hydroperoxide 0.5 g; 6-methoxyquino line 0.16 g; ethylene glycol (stabilizer) 0.5 g.

This solution was emulsified in 400 ml of 0.1 M aceta te buffer (pH 5.8) containing 0.5 g of sodium-lauryl sulfa te; then it was evaporated as described in Example 1 to gi beads of a first type (C) which were filtered and dried.

A second type of beads (D) were prepared the same way but using as the organic solution the following mixture : CH-Cl^ 25 ml; cellulose triacetate l g; sodium-lauryl sulfate 0.5 g; o-tolidine 0.25 g. The dispersion aqueous phase was the same as above.

Thereafter•strips were prepared as described in Examp 1 using a 1/1 by weight mixture of beads C and D. The test strips thus prepared were tested to determine traces of blood in aqueous solutions. The test mechanis is the same as that discussed in connection with USP 3,092,463 and is based on the ability of some components of blood (hemoglo- bi e constituents) to catalyze the transfer of oxygen from the peroxide to the o-tolidine with consecutive formation the blue dye. The following results were obtained :

Hemoglobine conc. (mg/1) Color of the strip (After one min)

0.923 light blue

6.16 blue

The above Examples shall not be considered as limitin as many other strip types can be prepared for measuring other biologically produced substances such as galactose, cholesterol, ketone compounds and bilirubine. Further, the strips can be applied to many other domains where the rapi

checking of water solution is important, e.g. chemical syn- thesis, plating, surface finishing, ete ...

Also strips can easily be made with one kind of bead system on one side and another kind on the other side for measuring selectively two different components in a solu¬ tion.

One factor is very important and should always be care- fully considered when manufacturing beads suitable for the present invention in the case said beads contain a water solution of a water soluble component. In order to be within rapid reach of the solution to be analyzed, the vacuoles containing said water solution must be rather near the sur¬ face of the ^' beads but, of course, not too much otherwise the vacuoles will not resist accidental bursting. In other words, the wall separating the vacuoles from the outside will have to be strong enough to conform with the mechanical strength speeifications required for the present beads but thin enough to allow the diffusion of the substances to be analyzed within a reasonable time. Now, such wall thickness can actually be controlled by properly adjusting the osmotic factors of a) the solution to be incorporated in the beads and b) the water phase serving as a final dispersion phase in the manufacture of the beads. Such osmotic factors will be kept within acceptable limits when the ratio of the molar concentration of the ingredients of the first water phase to that of the ingredients of the second waterphase is from about 1/5 : 5/1.

Beyond the lower limit, the entrapping yield may be low and the vacuoles may be distributed too far from the periphery of the beads which may result in having the color reaction to develop too slowly to be practical. On the other- hand when the upper limit of this ränge is exceeded, there is a risk that the external wall of the vacuoles becomes too thin (the external water will tend to penetrate into the beads to dilute the trapped solution) and the vacuoles might partially break during preparation or under storage with

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consequent loss of part of the encapsulated products.

Example 5

An organic solution (O) of cellulose triacetate was prepared by dissolving under agitation 2 g of the granula- ted polymer in 25 ml of CH ₂ C1_ containing 0.1 g of "Tween 20" and 0.25 g of o-tolidine. Then a first water phase Wl was prepared by measuring and mixing successively 3.5 ml of glucose oxidase solution (1000 International ünits/ml) , 60 mg of peroxidase, 133.8 mg of trisodium citrate and 51.4 mg of citric acid, the two latter ingredient being first mixed together in the dry State before dissolving. Then the first waterphase Wl was emulsified in the organic solution at room temperature and high speed until a regulär emulsion Wl/0 is obtained (drops of the waterphase about 1-5 c-m dispersed in the organic solution) . During this Ope ration, cooling may be necessary in order to avoid heating and possible denaturation of the enzymes.

In the meantime, the three following second waterphase

Solutions (W2a, W2b and W2c) were prepared by dissolving the following ingredients. in 350 ml of distilled H ₂ 0.

Ingredients W2a W2b W2c

Trisodium citrate 6.69 g 13.38 g (0.13M) 26.76 g

Citric acid 2.8 g 5.4 g (0.07M) 10.8 g

(NH ₄ ) ₂ S0 ₄ 2.31 g ^• 4.62 g (0.13M) 9.24 g Sodium lauryl-sul- 202 mg 202 mg 202 mg fate

Water to make 350 ml 350 ml 350 ml

Then the emulsion Wl/0 was dispersed in W2b by adding Wl/0 slowly and with rapid stirring into W2b. After the addition was complete, stirring was continue and the temperature was gradually raised to 40 C while a stream of nitrogen was swept over the surface of the agita- ted dispersion until the ethylene Chloride was removed by evaporation. The resulting Suspension of polymer beads was filtered and dried and the filtered waterphase was ana¬ lyzed for residual untrapped enzy e (glucose oxidase) by

OMPI

usual means. It was found that only 3% of the originally used glucose oxidase was not trapped within the beads which called for high trapping efficiency.

The above dispersion step was repeated with new lots of Wl/0 emulsions using the above water phases W2a and W2c. In such cases the encapsulation yields for the enzymes were 72% and 82% respectively. This indicates that the best re¬ sults are obtained when the buffer concentrations in the _^ first and the second water phase are about equivalent. Example 6

Preparation of a test-strip for ascertaining the presence of catalase in cows milk

The presence of catalase in the milk of ruminant is the sign of diseases and a simple method for detecting the enzyme may help detecting such deseases at an early easily curable stage. The reactions involved in the present strip are the following : Splitting the milk's lactose into galactose with galactosidase. Oxidizing the galactose in the catalysis pre¬ sence of galactose oxidase, thus producing hydrogen peroxide. Decomposing the H ₂ 0 ₂ into water and 0 ₂ by the catalase possi- bly present. Ascertaining the residual H ₂ 0_ present by its action on o-tolidine in the presence of peroxidase (same co¬ lor reaction as in the previous Examples) .

Four gram of cellulose triacetate and 0.02 g of o-toli- dine were dissolved in 50 ml of CH ₂ C1 ₂ with 0.4 g of "Tween 20". The solution was divided into two equal 25 ml portions called El and E2.

Then a first water phase (Fl) was prepared by dissol¬ ving 10 I.ü. of catalase free ß -galactosidase and 150 I.U. of catalase free ß -galactose oxidase into 3.5 ml of 0.1 M Phosphate buffer (pH 7.5) containing some magnesium Chlori¬ de (0.003 M MgCl ₂ ) .

Another water phase (F2) was prepared identically with 100 I.U. of peroxidase in 3.5 ml of the same phosphate-MgCl ₂ buffer. Then, Fl was e ulsified with El in the same condi- tions described at Example 1 in the case of emulsifying B

in A; and F2 was emulsified with E2 identically.

Afterwards, Fl/El and F2/E2 were gently mixed together (to avoid the microdrops of El collapsing with that of E2) and the mixture was dispersed in a third water phase G which was made by dissolving the following ingredients :

Sodium lauryl sulfate 0.2 g; ammonium sulfate 4.6 g; 0.1 M phosphate buffer (pH 7.5) 350 ml. The obtained disper¬ sion was subjected to evaporation under nitrogen as descri¬ bed in Example 4, after which beads of polymer were obtained (50-120.u) which contained tiny vacuoles (1-5 ) of two kinds. The first kind contained the first ^" water phase Fl and the second kind contained the second water phase F2. The vacuoles of the two kinds were homogeneously and statisti- cally distributed within the periphery of the beads. Then the beads were calibrated and applied on the surfa ce of an adhesive strip as in the previous Examples.

When the strips were used to test the milk of healthy or infected animals, the lactose dissolved in the milk was allowed to react with the enzymes of Fl whereby H-,0 ₂ was li- berated. Then the solution containing the catalase and the H ₂ 0 ₂ did migrate from the first type of vacuoles to the se¬ cond type in which the H ₂ 0 ₂ having not yεt been destroyed by the catalase did oxidize the tolidine because of the pre¬ sence of the catalase. The color development after one minu- te was therefore inversely proportional to the original con- ^• centration of the catalase in the infected milk. In the case of healthy cows, the color was very deep blue. In the case of cows with heavy astitis. The strip kept colorless or very pale blue.