DE2602975A1 - Diagnostic test reagent for body fluids - with highly absorbent carrier of inverse-phase polymeric material contg reagent-system - Google Patents

Abstract

Diagnostic reagent carrier for 'dip-read' testing of biological liq. is new comprising a synthetic, inverse phase polymeric carrier in which is incorporated a reagent or system of reagents sensitive to the substance to be detected in biological fluids. Arrangement provides relatively high absorptive power due to high vol. of interstitial pores; the carrier is of more constant reproducibility than a paper support; it will give a result after immersion in a liquid to be tested for one minute; it can be used in whole blood samples and interference by red blood cells is minimized.

Description

Description of the patent application relating to: "Test equipment and procedures for its production "The invention relates to test equipment for examining liquid samples, their manufacture and use.

It particularly relates to synthetic reagent carriers in which a Reagent is included, for use in qualitative and quantitative determinations chemical or biochemical substances in liquids, especially body fluids.

Test equipment for the detection of constituents of biological fluids, like blood and urine, are very widely used to aid medical diagnoses and treatments applied. The vast majority of such test equipment currently has those used for serial examinations in the clinical laboratory or in the doctor's office take the form of test strips in the form of elongated plastic strips on which one or several reagent carriers are attached. Such reagent carriers usually consist of pieces of absorbent paper tied with one or more Appropriate chemicals are impregnated, which when treated with an aqueous liquid Sample that contains the constituent to be detected come into contact with a Color change or colorometric reaction.

Test equipment suitable for urine tests are those of the "dip-and-read" type, which result in qualitative or semi-quantitative color reactions within 1 min, if immerse them in the urine sample for a moment.

Urine components that are usually determined in this way, include glucose, proteins, ketones, occult blood, bilirubin, urobilinogen, and nitrite.

Fast, convenient and reliable semi-quantitative definition of such Substances found in urine are extremely useful tools to assess the general condition of a patient with regard to many important ones clinical manifestations.

When analyzing blood samples the problem was the disruption of the color-forming test reaction due to the presence of colored blood cells in particular Erythrocytes overcome by surrounding the reagent matrix with a separating layer which is impermeable to red blood cells, but has a passage of the blood components to be detected in the matrix. Such erythrocyte barriers usually take the form of membrane coatings or foils over the exposed Part of the absorbent pad impregnated with reagent. It is also known that Test equipment for the examination of whole blood can be produced in such a way that the Reagent carrier matrix itself is designed so that it is a controlled Has limited porosity that prevents red blood cells from entering. Usually a drop of the blood sample is placed on the surface of the reagent impregnated pillow and after about 1 minute or more the surface will be rinsed, stripped or otherwise treated to remove the annoying staining Remove particles. The color reaction is then read off after a time of 1 minute or more for color development. Abnormal levels of glucose, urea and uric acid as well as other blood components are commonly used with such Test equipment proven. Test equipment of the "dip-and-read" type are also suitable for Examination of fluids other than body fluids. Usually be pH test papers, etc., extremely popular for various technical fields applied. It is well known that the presence or the amount is important certain components of a wide variety of industrial liquids, such as fermentation broths, To determine lubricating oils, electroplating baths, etc. While in the further description reference is made essentially to biological fluids, it should be noted that that the decisive advantages of the test equipment according to the invention also affect the investigation of industries and other liquids can be used.

Examples of "dip-and-read" test media, such as those used for urine testing are used are in U.S. Patents 3,048,475, 3,095,277, 3,112,420, 3,164,534, 3 447 905, 3 585 001, 3 814 586 and 3 814 688 are given. When making all This test equipment is an absorbent carrier, usually a cushion made of absorbent Paper, such as filter paper, with a solution or suspension containing the test reagent or the reagents, impregnated and then carefully dried. When bringing them together of Test equipment with the fluid sample that contains the substance to be detected, the paper reagent is rehydrated and it becomes possible that the color-forming Reaction is running.

In the manufacture and use of such paper-based test equipment certain disadvantages arise in the manufacture and in the product. One main reason for this are the differences in the structure and composition of paper products, which are available in stores.

In addition, some reagent systems cannot be used with good success in the Paper pillows are applied due to the presence of interfering substances in them the paper or the instability of the paper when it is strongly acidic, strongly alkaline or other aggressive reagent systems. The quality control, which is very critical when using the final product as an aid to medical diagnoses to be applied is complicated due to the variability of the current time for Test equipment applied commercially available paper.

Examples of test equipment that are particularly suitable for determination of components of whole blood due to the presence of a semipermeable Coating or membrane on the surface of the dry, impregnated with reagent Paper pillows are disclosed in U.S. Patents 3,092,465, 3,145,086, 3,298,789, 3,395,082, 3,427 225, 3,493,346 and 3,592,741.

US Pat. No. 3,607,093 describes a test device for testing of whole blood, including one permeable to liquid but erythrocytes impermeable membrane containing a test reagent. The test reagent is into the membrane, such as a cellulose acetate diffusion membrane, by impregnating the Membrane with a Solution of the test reagent and subsequent drying has been introduced. The resulting test equipment is relatively non-porous and not absorbent as it is specially designed to facilitate the diffusion of constituents of the sample to be examined, especially of erythrocytes, into the one containing the reagent Control membrane. When using the test agent as described, a drop Blood is applied to the membrane and left to stand for at least 2 min before the Sample is stripped off. Another 30 s are required before a visible color reaction occurs. So this test requires at least 2 1/2 minutes from the point in time, too which the sample is brought into contact with the test equipment until a readable Reaction occurs. Such a long test time is very inconvenient for urine tests. In addition, the sample has a residence time of 2 minutes, as is the case with the known test equipment is required, this is unsuitable for "dip-and-read" processes in which the Test equipment and the sample are only brought together briefly.

It is obvious that the type of reagent matrix like them in US-PS 3,607,093 is generally unsuitable for urine tests, because it is not absorbent and therefore a relatively long residence time of the Sample and time needed to read.

Another type of test equipment is described in US Pat. No. 3,630,957 in the form of a homogeneous, water-resistant film in which a color-forming test reagent is evenly distributed. The film is made by drying a mixture from an aqueous suspension or an organic non-aqueous solution of one organic film-forming polymer and the test reagent. The emerging density Slide in which the reagent is contained is not proportionate porous and non-absorbent as it consists of a single-phase polymer solution or suspension has been made. Films made in this way own Porosities less than 0.1 µm. Such a low porosity is well suited for Detection of constituents of whole blood as they effectively control the brythrocyteidaran prevents penetration into the reagent carrier matrix. However, such a test device is not suitable for use in urine tests because of the required test times go beyond 2 minutes and the required dwell time by far exceeds that that by an instant immersion in the test sample according to a "dip-alld-read" method is required.

A test medium for the detection of aldehydes in aqueous solutions is in U.S. Patent 3,784,358. This test equipment consists of a porous polymeric pad on which a chromogenic test reagent is adsorbed.

Examples of such porous polymeric substrates are porous polyethylene, polypropylene, nylon, and polystyrene, and they are made by processes requiring special techniques and apparatus. The test reagent adsorbed on the polymeric carrier is relatively unprotected against the harmful effects of the atmosphere, since the pores of the support are relatively large, on the order of 30 to 60 µm and the reagent is only adsorbed on the free surface of the support. Use various other known test media structural combinations of absorbent supports, foils and membranes, end hydrophobicity # agents etc., as disclosed in U.S. Patents 3,232,710, 3,418,083, 3,443,903, 3,798,004, and 3,811,840 Paper specified.

It is the object of the present invention to provide a new test device develop, which has a synthetic reagent matrix in which a chemical Test substance is built in and that is particularly suitable for "dip-and-read" analyzes of biological fluids, especially urine, and where you can get the test result Read off within 1 minute of contact with the liquid sample to be examined can. The test equipment should be able to be produced cheaply and it should be lower Quality control of the raw materials may be required than with the known Test equipment.

The reagent carrier matrix according to the invention should be relatively high absorbency, i.e. absorbency, in relation to the liquid to be examined have and an inner membrane-like structure with relatively large inner Cavities (pores), great uniformity in terms of structure and composition between batches of manufacture, stability in the presence of certain common liquid reagent test systems and uniformity as required for accurate and reliable instrumental studies of the reaction that occurs.

This object is achieved by a synthetic polymer Phase reversal obtained reagent carrier matrix in which a test substance or a test system is built in, which is to be detected in the liquid sample to be examined Substance or the condition to be detected. The membrane-like, Reagent matrices obtained by phase inversion are generally absorbent and wettable. lstur the usual paper matrices and can synthetically under regulated Conditions are established. These new reagent carrier matrices have proven to be Proven able to get an accurate test result within less than 1 min after the Immersion or contact with the liquid sample to be examined. The invention therefore relates to a very cheap and advantageous test equipment from "dip-and-read" type, which is particularly suitable for examining body fluids, like urine. The test equipment according to the invention can also be used for testing of liquids that require prior filtration, such as whole Blood.

The reagent carrier matrix according to the invention is produced by phase inversion precipitation of a dissolved polymer.

The phrase "matrix obtained by phase inversion" draws one Matrix obtained by a phase inversion precipitation method or the is characterized by the same essential features as one produced in this way Matrix. Such essential characteristics, the most important of which are discussed in detail later Can be broadly defined as those that directly relate to suitability of matrices made by reverse phase precipitation processes are like the test equipment of the type described here.

The reagent carrier matrix according to the invention is preferably produced using reverse phase membrane manufacturing processes. Phase reversal membranes can be made by the controlled precipitation of at least one polymer from a solvent system. The test +) or `` phasel reversal ilatrix '' reagent or test system is in the phase inversion carrier matrix in general on one of the in the following ways: (a) The phase inversion matrix is made and then the test reagent or test system and any additives that may be present, as they will be discussed in more detail later, by impregnation and drying, physical or chemical coupling or adsorbing, etc., or by a combination and / or a number of such steps1 incorporated. These manufacturing processes include the usual techniques, such as those used for installation in known paper backing polymeric film and fleece backing, etc. can be applied to the test reagents by a subsequent Impregnation or other treatment of the finished carrier matrix to be incorporated; (b) the phase inversion matrix is made from a solvent system containing the test reagent or test system and other additives that may be present, such as they are described in more detail later. This process, here referred to as the in siu phase inversion precipitation process is referred to, represents a means for producing a particularly advantageous Test means, as will be explained in more detail later, (c) if necessary, various Combinations of the two methods given above for the production of the test equipment can be applied. E.g.

one or more test reagents can first be added to the phase inversion matrix or components of the test system and one or more of the optional Existing additives can be incorporated by adding such substances to the solvent system from which the phase inversion matrix is precipitated. Then the phase inversion matrix with the remaining test reagents or components of the test system or, if applicable existing additional additives are provided, according to one or more Process steps as indicated under (a).

In order to produce a phase inversion support matrix, the polymer must or the polymers must be soluble in a solvent system that is at least a real one Solvent and at least one weak solvent and / or nonsolvent, that has a lower affinity for the polymer or polymers than one contains real solvent. The solvent system optionally contains one Solution, suspension or dispersion of the test reagent and / or any present further additions. The polymer or polymers must also have a certain minimum Have polarity and therefore a certain surface activity. The polymer solution is poured, sprayed on, rolled up or onto another on a suitable substrate Way brought up. When the resulting matrix is removed from the substrate this removal is facilitated by the use of a substrate made of is not wetted by the solvent or is inert to it, e.g. a glass substrate. If the resulting matrix and the substrate are to form an integral structure, a substrate is selected that is capable of chemically or physically interacting with the To couple polymer or is partially soluble in the polymer solvent system or this can absorb. Absorbent flat materials or a suitable plastic material that is in the polymer-solvent system is partially soluble are suitable for such substrates. The deposited polymer solution is freed from solvents (desolvated) using one of two basic processes: (aj Substantially by complete evaporation or (b) partial evaporation followed by immersion in a nonsolvent and subsequent removal the remaining solvents. Due to the presence of a weak and / or Non-solvent, the initially homogeneous solvent system separates as it progresses Evaporation into two mixed liquid phases, i.e. a coacervate. The coacervate comprises polymer-coated droplets of one phase which are converted into a Matrix from the second phase are embedded.

However, if the evaporation continues before it is complete is, the polymer goes into a gel phase. The fully formed phase inversion membrane or matrix is obtained after the solvent system is essentially complete evaporated or after immersion in a nonsolvent.

The phase inversion process involves the transition from the sol to the gel state gradually rather than suddenly.

Thus, during the sol-gel transition of the coacervate, the Coacervate droplets form polyhedra which ultimately form the lattice of the finished membrane represent. This gives a highly porous polymer structure. the one clearly has a larger cavity volume (porosity) than the known / Eolymertollen, obtained by precipitation from a single phase solution or suspension of the polymer have been. A void volume greater than 50% of the total volume of the matrix can can be easily achieved by a phase inversion precipitation process. Primarily due to the loss of solvent, the thickness of the resulting matrix is only one Fraction of the thickness of the deposited polymer solution. Because of the inclusion of cavities or pores, however, the phase inversion matrix has a thickness that is substantially larger than that of a dense film that has been produced is by depositing an equivalent amount and type of polymer from a single phase Medium.

Both the original polymer solution and the external factors during the evaporation can be varied within wide limits to the Formation of the gel structure created by the phase inversion and thus the structure of the finished matrix to regulate. Generally an increase in concentration results of weak solvent or nonsolvent or reduction of the real one Solvent in the polymer solution smaller and more numerous voids. While evaporation results in an increase in either temperature or relative humidity the surrounding area generally results in a larger pore size.

For a specific polymer or polymer mixture, the solvents be arranged in a solubility spectrum that of real solvents or Dispersants over weak solvents, sometimes called swelling agents are referred to, weak precipitants or gelling agents to nonsolvents or precipitants is enough. As a large number of known polymer solvents exists is a polymer solution that contributes to phase inversion precipitation Evaporation is appropriate, generally readily available for a given polymer or polymer mixture that is used with the test equipment or test equipment system, the to be incorporated into the finished matrix according to the in situ process, compatible To establish a rule of thumb for determining whether a solvent is considered genuine Solvent or nonsolvent is to be considered, with the latter mentioned The expression including weak solvents and true nonsolvents can have the relationship between the solubility parameters for the particular polymer and solvent. applied will. Solvent parameters are known useful tools in polymer technology and are in the Polymer Handbook, edited by Brandrup et al, Interscience Publishers (New York, 1967) defined, discussed and given. For the invention Purposes can be said to be a solvent with a solubility parameter within of 2 units of that of a specific polymer as a true solvent for the polymer can be considered. Such solvents, the solubility parameters outside this range can be considered nonsolvents, the true nonsolvents being those with more solubility parameters than 5 units from that of the selected polymer.

The reverse phase polymer precipitation process of the present invention yields a reagent carrier matrix that is highly porous and in relation to the one to be examined Liquid is absorbent. The liquid transport in the emerging, the reagent containing polymer matrix as well as within the matrix is greatly facilitated due to the inner open-cell structure. That by in situ reverse phase precipitation process The test equipment produced has various particularly advantageous properties.

Such a test means is characterized by the fact that while the test liquid easily gets to the inner surface of the matrix can and thus to the built-in reagent, this reagent proportionate evenly and is firmly immobilized in the highly porous structure of the membrane and not easily can be leached as it is both physically and chemically closely related to the Polymer material, which forms the structure of the matrix, is connected. The reverse phase reagent matrix generally has a high uniformity both on the surface and along a parallel plane, thereby reducing the smoothness of the test reaction is improved over the surface.

A more detailed discussion of the phase reversal process the relevant factors influencing membrane formation and methods for further Treatments of the resulting membrane are given in Kesting, R.E., Synthetic Polymer Membranes, McGraw-Hill, Inc. (New York, 1971). One of the most surprising features of the invention is that the versatile phase inversion method is used can be used to produce new reagent carrier matrices, which are very suitable for quick and easy examination of liquids.

The general macroscopic and microscopic character of the invention The reagent matrix corresponds in some respects to that of conventional paper matrices.

The reagent matrix is highly permeable for the sample to be examined and gives a strong and effective physicochemical interaction between the Components of the sample to be examined and the test reagent or reagent system in the matrix. The interaction between the components of the sample and the test reagent is enhanced by the general absorbent character of the matrix, i.e. by the ability of the matrix to absorb or adsorb the liquid sample and relatively to hold large volumes of the sample within the matrix.

The ability of the synthetic reagent matrix to be liquids to absorb and hold on to those in a manner and to an extent common paper matrices is based primarily on their hydrophilicity and membrane-like internal structure characterized by high porosity and high Pore volume is characterized. In contrast to the dense reagent foils that have substantially continuous coherent internal structures the matrix according to the invention interconnected open-cell and / or thin-walled Cavities which are present in great quantity and large and a strongly developed one Form membrane structure. The reagent carrier matrix according to the invention usually has a pore volume of more than about 30%, preferably more than about 70, based on the total volume of the matrix and an average pore size of more than about 1 / µm, preferably more than about 3.0 / µm.

The mean pore size of the carrier matrix is generally smaller than 30 / µm and preferably less than 20 / µm. Due to the general absorbent In nature, the reagent carrier matrix according to the invention is capable of a menpe of the liquid The sample to be investigated is to be absorbed around 8times1 which corresponds to its own weight and is preferably more than twice as large. In several ways, the reagent carrier matrix according to the invention as a synthetically produced paper-like one Material to be viewed. The matrices according to the invention therefore have the advantageous ones Absorption properties of conventional paper matrices with the essential advantage that the matrix material according to the invention can itself be produced synthetically. This eliminates the disadvantages of the Paper according to the invention replaces the advantages of well characterized synthetic polymers.

Synthetic polymers useful for the reagent matrix of the invention can be applied, include those against the liquid to be examined insoluble forms of such polymers, such as polyacrylates, such as polyacrylonitrile, polybutyl acrylate, Polyacrylate and polymethacrylate; the polyoxides, such as polyacetaldehyde, polyformaldehyde and polytetrahydrofuran; the polyesters, such as polyethylene terephthalate, the polyamides, the polyurethanes, the polyvinyls, such as polyvinyl acetate, polyvinyl alcohol, polyvinyl chloride and polyvinyl propionate and their derivatives and mixtures. Are particularly suitable the synthetic polysaccharides insoluble in the test liquids, especially cellulose products and their derivatives, such as cellulose ethers, such as methyl cellulose, Ethyl cellulose, propyl cellulose and butyl cellulose; the cellulose esters, such as cellulose acetate, Cellulose butyrate, cellulose acetate butyrate, cellulose acetate propionate and cellulose acetate hydrogen phosphate and others such as cellulose nitrate.

As used herein, the term reagent refers to a chemical compound or agent that is physically or chemically linked to a or several of the constituents present in the liquid sample to be examined or a state of the liquid sample producing a detectable reaction reacted.

Such a detectable reaction (response) can immediately after contact with the sample to be examined or a certain time afterwards. Such a reaction is said to be detectable if the physical or chemical effect through additional physical or chemical treatment or without one such with the help of the human senses or on can be demonstrated in another way, e.g. instrumentally.

The detectable response can therefore be a spectrophotometric, turbidometric (Turbidity measurement), radioactive, thermal or other reaction. Usually the reaction of a test reagent is a color change, preferably in the visible Area. The test equipment according to the invention is suitable for detecting a number of substances in liquid samples, e.g. urine or blood samples. The clinically important Substances that may be present in such biological fluids include Glucose, albumin or proteins, hydrogen ions, erythrocytes, hemoglobin, bilirubin, Urobilinogen, nitrite ions, ketones:, urea, uric acid, cholesterol, microorganism metabolites and cell components, etc.

The carrier matrix can also contain other customary ones that may be present Supplements included. Such additives include inert fillers, thickeners, Plasticizers, strength-increasing resins, inert background dyes and pigments etc.; when the polymeric material used to make the carrier matrix does not have a strong affinity for the liquid sample to be examined a surfactant or wetting agent can be added to the matrix, the is able to give it such an affinity for absorption or the penetration of the liquid to be examined into the carrier matrix after it has come into contact with the sample. E.g. standing. for the In the event that the sample to be examined is an aqueous sample and the polymeric material, which is used to produce the carrier matrix, hydrophobic or non-hydrophilic suitable surfactants or wetting agents are available to be incorporated into the Matrix either during their manufacture or through post-treatment can be introduced to give it a hydrophilic character; one A hydrophobic polymer matrix can be imparted with the absorbency by incorporating particles of a suitable absorbent material during the production.

The support matrix is usually included for easier handling connected to a base. Suitable test equipment can be manufactured by the carrier matrix with the help of adhesives or in some other way or through Formation of the ~ reagent matrix attached directly to the substrate to form an integral structure to obtain. The latter procedure can be performed by selects a solvent system from which the phase inversion matrix is formed and a pad that reacts chemically or physically with the solvent system. For example, a solvent can be selected that will clean the surface of the pad partially dissolves, so that the resulting phase inversion matrix when the solvent evaporates is integrally connected or fused to the base.

The pad or substrate generally has the shape of one Strips of plastic, paper, wood, metal foil or the like, as the carrier matrix can be prepared directly from a liquid with or without a test reagent, however, a large variation in the formats of the test equipment is possible.

Usually the carrier matrix is in the form of a thin film or a film or membrane in front of it in a laminate-like manner on an inert strip or substrate is attached. The strip serves as a base or handle for the matrix. A single reagent carrier matrix or different Such matrices can be attached to a strip. A test device for Various examinations can be made by placing a A variety of matrices are attached, each with a different test reagent or reagent system contain.

Another form of test equipment, which with the help of the inventive, carrier matrix impregnated with reagent can be produced is a test vessel, the inside of which is completely or partially covered with the carrier matrix. Such Vessels can be test tubes, dishes, capillary tubes, analytical columns, tight be sealable capsules or the like and when used, the one to be examined Sample placed in such a vessel and when the sample comes into contact with the carrier matrix the test reaction begins.

It is possible to use thin, film-like reagent carrier matrices as described Kind of laying them on top of each other like a laminate to form a layered structure or sandwich-like structure. With such a layered test medium, the individual matrix layers contain test reagents which refer to the same or different substances to be determined respond or the individually to different Address types or groups of connections. Optionally, semipermeable or selective separation layers or membranes between adjacent matrix layers can be inserted to control the transport of substances.

Some very advantageous features of the reagent matrix of the invention consist in the controlled porosity and high permeability and absorbency with respect to the liquid to be examined. One therefore obtains a structure in which a reagent is located, which is determined by its physiochemical Nature effectively facilitates fluid transport within the reagent matrix for a fast and thermodynamically favorable interaction between the test reagent and the liquid sample. The highly porous reagent matrix also facilitates penetration of atmospheric oxygen into the interior of the structure. That is special Importance if the test reaction used is aerobic. Liquids with Can be examined using the test means according to the invention, include of course occurring and artificially produced solutions, dispersions or suspensions. The test agent according to the invention is particularly suitable for examining biological Liquids, especially those that do not require filtration, such as Urine, serum, plasma, etc.; in addition, the porosity of the test means according to the invention be set so that interfering substances in the liquid to be examined can be kept away from the interior of the reagent matrix. Because the phase inversion method a precise regulation of the porosity for the production of the test equipment according to the invention the resulting reagent matrix allows the entry of substances from the liquid to be examined into the reagent matrix is also easily regulated will.

The fact that the test equipment according to the invention, when made so that the mean pore size is between about 0.1 and 5.0 to be adjusted, it is easy to detect substances in both urine as well can also be used in the blood.

Urine samples are examined in the usual dip-and-read manner. rehearse Whole blood can be examined by examining the reagent matrix beforehand wetted and then a certain time of less than 1 min with the blood sample brings in touch. The blood sample is then, for example, washed off or wiped off removed and the reaction observed immediately. Due to the static charge red blood cells remain on the surface of the polymer matrix Reagent matrix adsorbed even after washing if the matrix is directly in contact with the Blood sample is brought into contact without it being wetted beforehand.

However, this problem is overcome by having an anti-static Agent adds to the matrix. Such an antistatic agent is used to To balance charges on the surface of the matrix and a sticking of the red Prevent blood cells from attaching to the matrix so that they are easily washed off can.

Whole blood can also be made with a highly porous form of the invention Reagent carrier matrix to be examined, with a semipermeable film of the usual Kind of covered. If an antistatic agent is added to the reagent carrier matrix, which is impermeable to erythrocytes, according to the invention, a "dip-and-read test agent" is obtained for direct examination of whole blood.

This is not possible with the known test equipment.

Substances useful as antistatic agents are known. Such substances are generally ionic, such as quaternary ammonium compounds and amines or hydrophilic, such as polyglycols or ethylene oxide derivatives. Regarding more precise Information on antistatic agents, their composition and application will be provided see SPE Journal, Vol. 29 (1973), pp. 28-34 and 52-55.

The test means according to the invention has the advantages of flexibility the reagent matrix, uniformity of the matrix, resulting in a homogeneous test reaction which is particularly suitable for precise instrumental analysis, such as reflection spectrophotometry ' the possibility of relatively strongly alkaline or strongly acidic or aggressive Incorporate reagents into the matrix.

It is advantageous in the manufacture of the test equipment according to the invention possible the purity of the starting material and the quality of the structure and composition the reagent matrix can be easily monitored. All of these benefits can help with the known methods and conventional reagent carriers cannot be achieved.

The test equipment according to the invention can be used in a reproducible manner are manufactured within critical limits of quality control and can be used for dip-and-read analytical procedures. Such test equipment cannot be produced by known processes.

The invention also relates to a convenient method of manufacture of test media that are highly absorbent, with a phase inversion polymer matrix containing a test reagent. Since the resulting reagent carrier matrix Can be made from a single solvent system can be very different Test equipment, types and forms are obtained in which the reagent carrier matrix is absorbent.

The invention is illustrated by the following non-limiting examples explained in more detail.

Example 1 In this example, a test device was used to verify Glucose produced.

A first solution was prepared containing the following ingredients in the amounts given: glucose oxidase (4000 units / ml) 4.0 ml peroxidase (3000 units / mg) 0.289 g of 2,7-diamine fluorene. 2HCl 0.277 g ascorbic acid 0.020 g Gantrez AN * 20.0 ml tris-malonate buffer (pH 7.0) 10.0 ml ethanol 30.0 ml water 13.0 ml * An agent for solubilizing an equimolar copolymer of methyl vinyl ether and maleic anhydride (General Aniline and Film Corporation, New York).

6.0 ml of the first solution prepared as above and 3. Qnl of water 30.0 ml of a second solution were then mixed in, containing: ethyl cellulose ** 10.0 g acetone 114.0 ml ** G-50 from Hercules, Inc., Wilmington, Delaware.

The resulting solution was 1.27 mm (50 mil) thick with Applied using a Gardner's spatula and dried under standard room conditions.

The resulting phase inversion polymer matrix, which looked like paper, was peeled from the glass substrate and into approximately 12.7 mm blocks or pads cut. The pillows were then attached to plastic strips with the help of double-sided tape glued. The reagent pad area of six of the resulting test strips was currently immersed in one of six urine samples, each containing different ones known concentrations of glucose. The following results were obtained: Glucose concentration Color reaction g / 100 ml 0.0 yellowish-buff 0.1 yellowish-green 0.25 greenish-blue 0.5 pale blue 1.0 blue 2.0 deep blue In this way it could be shown that the test equipment according to the invention is suitable for the detection of glucose in a liquid Sample using the dip-and-read method.

Example 2 In this example, a test agent for the detection of Albumin produced.

A buffer solution containing the following ingredients was prepared in the specified quantities.

Sodium citrate 2 H20 8.4 g citric acid. H20 14.0 g water 195.0 ml of 66 ml of the resulting solution were then mixed with 30 ml of methanol containing 50 mg Tetrabromophenol blue mixed.

The resulting solution was brought to pH 3.3. 15.0 ml of the solution was mixed with 30.0 ml of a polymer solution containing 7.5 g Cellulose acetate (398-3 from Eastman Kodak Co., Rochester, New York) in 54.0 ml of acetone. The resulting clear solution was 1.0 mm thick on a glass plate poured and dried as in Example 1, cut into pieces and put on plastic strips attached.

The reagent pad portion of four of the resulting test strips was instantly immersed in each of four urine samples containing different ones known albumin concentrations. The following results were obtained: albumin concentration Color reaction mg / 100 ml 0 pale yellow 100 greenish yellow 300 yellowish green 1000 green It could thus be shown that the test equipment according to the invention is suitable Determination of albumin in a liquid sample ("dip and read").

EXAMPLE 3 In this example, a test device was used for the determination of pH of a liquid produced.

Two solutions were prepared with the following composition: Solution A methyl red 13 mg bromothymol blue 257 mg ethanol 270 ml water 400 ml solution B bromocresol red 2 mg ethanol 36 ml water 54 ml 20 ml of solution B were then mixed with 10 ml of solution A. 3.0 ml of the resulting solution were then with 4.0. ml of a 156 / own (w / w) solution of cellulose acetate (398-3 from Eastman Kodak Co .., Rochester, New York) mixed in acetone. The resulting clear solution was poured onto glass plates according to Example 1, dried and cut into pieces and attached to plastic strips.

The reagent pad portions of five of the resulting test strips were each instantly known in one of five urine specimens with the following pH values immersed. The following results were obtained: pH color reaction 5.0 yellow-orange 6.1 yellow 7.0 blue-green 7.9 green-blue 8.9 deep blue So could be shown that the test agent according to the invention is suitable for determining the pH value a liquid sample (d + r).

Example 4 According to this example, a test device was used for verification made from ketones.

Two solutions were prepared with the following composition: Solution A sodium nitroferricyanide H20 1.6 g methanol 50.0 ml water 50.0 ml solution B glycine 18 g sodium phosphate 20 g sodium biphosphate 9 g water 72 ml sodium nitroferricyanide and the buffer reagents are incompatible with this test system. Hence they became as specified later, incorporated in separate polymer layers, the the first-mentioned reagent was dispersed in a water-soluble film which was laminate-like with a phase inversion matrix containing the latter reagent had been.

20 ml of solution A were mixed with 0.5 g of sodium carboxymethyl cellulose (7-M from Hercules Inc., Wilmington, Delaware) mixed. The resulting solution was in a thickness of 0.25 mm on a glass plate using a Gardner spatula and dried in room conditions.

The resulting water-soluble film was peeled off the glass substrate.

7.5 ml of solution B were mixed with 7.5 ml of methanol and 20.0 ml of a 15% gene (W / w) acetone solution of ethyl cellulose (G-50 from Hercules Inc., Wilmington, Delaware) mixed.

The resulting clear solution was 1.27 mm thick with Applied to a glass plate with the help of a Gardner spatula and in room conditions dried.

After no more acetone could be smelled, that from the drying solution came, the previously prepared water-soluble film was applied to the damp surface applied to the resulting phase reversal matrix. The resulting laminate structure was completely dried, cut into squares approximately 12.7 mm and with Using a double-sided tape with the water-soluble side facing the strip glued to plastic strips.

The portions of four of the resulting test strips containing the laminate were each instantly immersed in one of four urine samples containing different ones known concentrations of the ketone acetoacetic acid. The following results were obtained: Concrete concentration color reaction mg / 100 ml 0 buff 15 purple buff 40 light purple 80 deep purple It could be shown that the inventive Test equipment is suitable for the detection of ketones in liquids. (d + r) example 5 In this example, a test device was produced for the detection of nitrite.

Two solutions of the following composition were prepared: Solution A p-arsanilic acid 0.2 g methanol 25 ml water 25 ml solution B N- (1-naphthyl) ethylenediamine HCl 0.1 g methanol 49.0 ml d-tartaric acid 0.5 g 10 ml of a mixture of the same Volumes of solutions A and B were mixed with 15 ml of a 10G / o (w / w) solution of ethyl cellulose (T-50 from Hercules, j: nc Wilmington, Delaware) mixed in acetone. The resulting clear solution was poured onto glass plates according to Example 1, dried, cut into pieces and attached to plastic strips. The part each of five test strips containing the reagent pad was briefly inserted into one dipped from five urine specimens containing various known concentrations of nitrite. The following results were obtained: Nitrite concentration color reaction mg / 100 ml 0.0 cream-colored 0.5 light pink 1.0 medium pink 5.0 medium purple 10.0 purple-violet So it could be shown that the test equipment according to the invention are suitable for Detection of nitrite-forming microorganisms in a liquid sample with the aid the dip-and-read process.

Example 6 In this example, a test device was produced for Determination of nitrite, the polymeric reagent matrix being a non-hydrophilic Polymer contained.

Solutions A and B were prepared according to Example 5.

1.5 ml of a mixture of equal volumes of solutions A and B were then mixed with 9.0 ml of the above (w / w) solution of polyvinyl chloride (No. 133-4 from Dow Chemical Company, Midland, Michigan) mixed in hydrofuran to the then 3.0 ml of a 21% (w / w) solution of an ethoxylated nonylphenol wetting agent (Renex 698 from Atlas Chemical Industries, Wilmington, Delaware) in chloroform became. The resulting clear solution was according to Example 1 on glass plates poured, dried, cut into pieces and attached to plastic strips. The reagent pad portion of three of the resulting test strips was each a time of less than 30 seconds in one of three aqueous solutions submerged, containing various known concentrations of nitrite. You got the following results: Nitrite concentration color reaction mg / 100 ml 0.0 cream-colored 2.5 light pink 5.0 pink So it could be shown that a test agent according to the invention, whose reagent carrier matrix contains a non-hydrophilic polymer, is suitable for Investigation of an aqueous sample by the dip-and-read method if the reagent matrix additionally contains a wetting agent.

Example 7 In this example, a test equipment was used for determination the pH of an aqueous solution produced, the reagent carrier matrix a non-hydrophilic polymer and made from a non-aqueous solution became.

A first solution was prepared with the following composition: Methyl red 13 mg bromothymol blue 250 mg ethanol 1000 ml to 3.6 ml of this first The solution was 3.0 ml of a 216-dose (w / w) solution of an ethoxylated nonylphenol wetting agent (Renex 698 from Atlas Chemical Industries, Wilmington, Delaware) in chloroform and 9.0 ml one 10Q / Oigen (w / w) polyvinyl chloride solution (No. 133-4 from Dow Chemical Company, Ivfidland, Michigan) in tetrahydrofuran. The resulting clear solution was poured onto glass plates according to Example 1, dried, cut into pieces and attached to plastic strips. The the Reagent-containing portions of four of the resulting test strips were each immersed in one of four aqueous solutions for a period of less than 30 s, the gave the various known pH values. The following results were obtained: pH color reaction 9.4 yellow 11.0 blue-green 12.0 blue So it could be shown that a suitable test equipment of the dip-and-read type for determining the pH value in aqueous media Liquids can be produced if the reagent matrix is non-hydrophilic Contains polymer and is made from a non-aqueous solution.

Example 1 8 In this example, a test device was used for the determination made of glucose in a sample of whole blood.

Test equipment according to Example 1 was produced.

That portion of six of the resulting rGesU strips containing the reagent pad was currently submerged in water. The test strips then became flat with the rehydrated Reagent pad put down. There were six blood samples with different known Glucose concentrations produced. One drop each Blood sample was applied to one of the rehydrated reagent pads. After 30 The blood samples were washed off the reagent pads with water. After washing up there were no more red blood cells on top of the reagent pads. The occurring Color reactions were identical to those given in Example 1.

It could be shown that the test equipment according to the invention is suitable are used to determine a component of whole blood.

Example 9 In this example, a test equipment was used for determination made of glucose in a sample of whole blood.

A first solution was prepared with the following composition: Glucose oxidase (4000 units / ml) 4.0 ml peroxidase (3000 units / mg) 0.289 g 2,7-diaminofluorene-2HCl 0.277 g ascorbic acid 0.020 g ris-malonate buffer (pH 7.0) 10.0 ml of ethanol 30.0 ml of water 13.0 ml of 2 ml of the specified first solution were then with 1.0 ml of water, 10.0 ml of a 106 / above (w / w) solution of ethyl cellulose (G-50 from Hercules, Inc., Wilmington, Delaware) in acetone and 1.0 g of an antistatic Mixed using (AL 15 from Argus Chemical Co., Brooklyn, New York). The emerging Solution was accordingly Example 1 cast on glass plates, dried, cut into pieces and attached to plastic strips. The that Each portion of six of the resulting test strips containing reagent pads was used instantly into one of six whole blood samples containing several known concentrations of glucose immersed. The color reaction that occurred was similar to those in Example 1.

It could be shown that if the reagent mixture for an inventive Test equipment contains an antistatic agent, the resulting test equipment as a whole Blood can be applied by the dip-and-read method.

B e i 5 p i e 1 10 In this example, a test device was used for verification made of nitrite by subsequent impregnation.

A first solution was prepared with the following composition: 10% cellulose acetate * in acetone 20 ml water 8.5 ml * Visc 45 from Eastman Kodak Co., Rochester, New York The resulting solution was 1.2 mm thick applied to a glass plate using a Gardner spatula and dried at 760C. The resulting phase inversion polymer matrix was detached from the glass plate and dipped successively in the following solutions A and B: solution A 2% Gantrez 15 ml arsanilic acid 75 mg malonic acid 1.8 g sodium lauryl sulfate 450 mg of methanol 45 ml of solution B polyvinylpyrrolidone / vinyl 3.7 ml of acetate copolymer * methanol 56.2 ml; N- (1-Naphthyl) -ethylene- 60 mg diamine 2HCl * E535 from the Antarac Chemical Division of GAFAC, New York The polymer matrix containing the reagent was then heated at 100C dried and cut into squares of about 5 mm. These reagent pads were then attached to plastic strips with the help of double-sided adhesive strips. The portion of the test strips obtained that contained the reagent pad was each Immediately immersed in one of five urine samples containing known concentrations of nitrite.

The following results were obtained: nitrite concentration 2'arbreaktion mg / 100 ml 0.0 white 0.2 light pink 0.5 medium pink 1.0 pink 2.0 dark pink So it could be shown that the test agents according to the invention for the determination of nitrite in a liquid are suitable if they are produced by subsequent impregnation have been.

Example 11 In this example, test equipment was used as evidence made of bilirubin through a subsequent impregnation process.

A first solution was prepared with the following composition: 15% cellulose acetate * in acetone 18 g 15 u, Ó tributoxyethyl phosphate in acetone 2 g Styrene maleic anhydride 1 ml toluene 3 ml methanol 4.5 ml water 4.5 ml * Visc 45 from Eastman Kodak Co., Rochester, New York. The resulting polymer solution was on Polystyrene plastic strips applied at a thickness of 1.27 mm and at approximately 70 ° C dried. The resulting structure, consisting of a phase inversion polymer matrix fused to a polystyrene sheet was added to the following reagent solution immersed: sulfosalicylic acid 9.8 g caffeine 14.0 g 1,5-naphthylene disulfonic acid 0.84 2,4-dichloroaniline 0.105 g 10% Gantrez 14M sodium nitrite 9.14 g water 126 ml the the polymer matrix-polystyrene film structure containing the reagent was dried at 70 ° C and then cut into reagent pad squares approximately 5 mm in size and the reagent pad with the polystyrene side facing the plastic strip attached to the plastic strip by double-sided adhesive strips. The reagent pad containing portion of two of the resulting test strips were each instant immersed in one of two urine samples containing certain concentrations of bilirubin.

The following results were obtained: bilirubin concentration on color reaction mg / 100 ml 0 white 1.6 pink It was thus possible to show that a test agent according to the invention, which has been obtained by subsequent impregnation of the matrix is suitable for the detection of bilirubin in a liquid.

B e i 5 D i e 1 12 In this example, a test device was used for verification made from ketones.

A mixture of the following composition was prepared: ethoxylated Fatty alcohol * 80 g sodium lauryl sulfate 14 g glycine 44 g sodium nitroferricyanide 14 g sodium phosphate 12 H20 17.5 g Sodium biphosphate 5.6 g amyl alcohol 20 ml * BIO-SOLF EA-10 from Stephan Chemical Co., Northfield, Illinois Mixture was tumbled (ground) in a ball mill for 15-18 hours and then 11 g of the mixture mixed with the following solution: 15% cellulose acetate * and 1.5 % Tributoxyethyl phosphate in 5.6% methylene chloride / 94.4% dioxane 20 g amyl alcohol 3 ml * Visc 45 from Eastman Kodak Co., Rochester, New York.

The resulting dispersion of reagents in the polymer solution was with the help of a Gardner spatula 0.64 mm thick on a polystyrene plastic strip applied and dried at room temperature. The resulting structure that consists of the phase inverse polymer matrix containing the reagent, bonded to the polystyrene film was fused, was made into square reagent pads of approximately 5 mm cut the pillow with the polystyrene side towards the plastic strip glued to plastic strips with the help of double-sided adhesive strips. The that Portion of two of the resulting test strips containing reagent pads were each Immediately immersed in one of two urine samples, one of which was no-ketone bodies and the other contained 2 o sodium acetoacetate.

The reagent pad that was in the urine sample containing acetoacetate was immersed, a clear change in color was observed.

It could be shown that the invention Test equipment for the detection of ketones in a liquid are suitable if they are with the help an in situ method in which the test reagent is in the Polymer 'solvent' system in the form of a dispersion or suspension is.

PATENT CLAIMS:

Claims (1)

PATENTANSPRt} CHE test equipment for examining a fluid sample, in that it is produced in a phase inversion polymer carrier matrix contains a test reagent. (2) Test means according to claim 1, characterized in that it is not possible to use it t that it also includes a pad for the matrix. (3) Test means according to claim 2, characterized in that there are no indications t that the matrix is fused to the substrate or glued to it. (4) Test means according to Claims 1 to 3, characterized in that they are e k e n n z e i c Note that the matrix has a mean particle size greater than about 0.1 µm owns. (5) Test means according to claims 1 to 4, characterized in that they are e k e n n z e i c It should be noted that the matrix has an average particle size of less than 30 μm. (6) Test means according to claims 1 to 5, characterized in that they are e k e n n z e i c that is, the void volume is greater than about 70 cju of the total volume of the Matrix matters. (7) Test means according to claim 1, characterized in that it is n -z e i c h n e t that the matrix consists of at least one polymer which is insoluble in the liquid sample, such as polyacrylate, polyacrylic acid, polyoxide, polyester, polyamide, polyurethane, Polyvinyl and / or polysaccharide consists. (8) Test means according to claims 1 to 6, characterized in that they are e k e n n z e i c n e t that the matrix consists of a synthetic which is insoluble in the liquid sample Cellulose derivative consists. (9) Test means according to Claims 1 to 8, characterized in that they are e k e n n z e i c n e t that the matrix consists of cellulose ether, cellulose ester or cellulose nitrate consists. (10) Test means according to claims 1 to 9, characterized in that they are e k e n n z e i c n e t that the matrix contains a hydrophilic substance. (11) Test means according to Claims 1 to 1D, characterized in that they are e k e n n z e i c h n e t that there is at least one selectively permeable layer over the matrix is located. (12) Test means according to claim 11, characterized in that it is a -k e n n z e i c h n e t that the selectively permeable layer retains erythrocytes. (13) Test means according to claims 1 to 10, characterized in that they are e k e n n z e i c h n e t that the matrix is impermeable to erythrocytes. (14) Test means according to claims 1 to 13, characterized in that they are e k e n n z e i c h n e t that the matrix contains an antistatic agent. (15) Test means according to claims 1 to 14, characterized in that they are e k e n n z e i c h n e t that the test reagent comprises a substance that interacts with glucose, albumin, Hydrogen ions, erythrocytes, hemoglobin, bilirubin, urobilinogen, nitrite ions, Ketones, uric acid, cholesterol and / or metabolites and / or cell components reacts to microorganisms. (16) A method for producing a test device according to claims 1 to 15, by the fact that one converts into a polymer by phase inversion The carrier matrix obtained introduces a test reagent. (17) The method according to claim 16, characterized in that g e k e n n -z e i c h n e t that a polymeric carrier matrix by phase inversion precipitation of at least a polymer from a solvent system for containing the test reagent. (18) Method according to claim 16 or 17, characterized in that c h n e t that the matrix is produced by evaporating the solvent from the solvent system, the solvent system at least one nonsolvent for the polymer contains. (19) The method according to claim 16 to 18, characterized in that it is possible to use it c h n e t that the solvent is allowed to evaporate essentially completely. (20) The method according to claims 16 to 19, characterized in that there is no e n n z e i c h n e t that evaporation can take place at normal room conditions or at a higher temperature performs. (21) The method according to claim 16 to 20, characterized in that it is e k e n n z e i c h n e t that one uses a solvent system that is a weak solvent or swelling agents for the polymeric substance. (22) The method according to claims 16 to 21, characterized in that there is no e n n z e i c h n e t that the solvent system is applied to an inert surface, from which the resulting matrix can be detached. (23) The method as claimed in claim 22, characterized in that it is e k e n n -z e i c h n e t that at least part of the matrix formed during evaporation from the Peeled off the surface and attached to a base. (24) The method according to claim 16 to 21, characterized in that it is possible to use it c h n e t that one applies the solution to a substrate with which it can react physically or chemically and the resulting matrix with the Substrate forms a uniform structure. (25) The method according to claim 24, characterized in that g e k e n n -z e i c h n e t that one uses a substrate that is at least partially in the solvent is soluble or the mixture is at least partially absorbed. (26) The method as claimed in claims 16 to 25, characterized in that it is e k e n n z e i c does not mean that the finished matrix is brought into contact with a liquid, which contains at least one (additional) test reagent and dries the matrix. (27) The method according to claim 26, characterized in that g e k e n n -z e It is noted that the first test reagent and the second test reagent together Form test system. (28) The method according to claim 16 to 27, characterized in that it is e k e n n z e i c h n e t that one starts from a solvent system that contains inert fillers, Thickeners, plasticizers, resins to increase strength, inert background dyes, Wetting agents, surface active agents, hydrophilic substances and / or absorbent Contains substances.