SE444085B - TESTING DEVICE FOR PAVISANING CARBAMIDE INCLUDING BERARMATRIS IMPREGNATED WITH SURGERED CATION EXCHANGER AND REAGENT SOURCE FROM EACH OTHER WITH A POLYMER MATERIAL, AND PROCEDURE FOR USING THESE TEST ORDERS - Google Patents

Description

"7905266-8 2 value. In the past, arthritic wet chemical methods have been used to perform such analysis. Other methods have also been used, such as conductivity methods, spectrophotometric and colorimetric methods. Although these methods are usually accurate, they are time consuming. and may require careful interpretation.

In perhaps the most common method for determining urea in biological fluids, the urea is hydrolyzed to ammonium carbonate using the enzyme urease in the presence of a buffer solution. The amount of ammonia released is determined colorimetrically. This method is very specific and sensitive, but it has the disadvantage that the urease can be inhibited and inactivated. Furthermore, there is a problem with the stability of the reagent, and a further disadvantage is that endogenous ammonia is measured in this method. According to another method that uses hydrolysis with urease, special equipment is required, which is not always available in laboratories intended for routine analyzes. According to another method, a direct colorimetric reaction is obtained between urea in a protein-free filter and an organic reagent, such as diacetyl monoxime. This diacetyl reaction has the disadvantages that the color formed is unstable, that the color develops at a temperature of about QSOC, and that the volatile diacetyl compound has an odor which is unpleasant to at least some people.

U.S. Patent No. H OYU 912 discloses an assay method for determining urea, wherein a sample of a biological fluid is reacted with an acidic reaction solution of o-phthaldehyde and 8- (U-amino-1-methylbutylamino) -6- methoxyquinoline. It is not stated or suggested that the reagent solution may be incorporated into a test device, such as a carrier matrix, not to mention a carrier matrix prepared with a strong cation exchanger in acid form.

This patent also does not disclose any way of overcoming the stability problem which arises when the reagents in the acidic reagent solution are combined. Another problem with this analysis method is that the determinations must be made within the wavelength range H70-5UO nanometer, which is close to the wavelength at which a blank without carhamide gives maximum color absorption. This increases the risk of measurement errors during spectroscopic measurement of the reflection.

In industry, urea is an important additive to, such as plating baths, and a rapid method for determining the urea concentration therein is of great value. Urea was also used as a fertilizer, and a rapid method of analysis of this substance is important as a control measure in the production and use of the urea.

In both the medical and industrial fields, there is thus a need for an improved method for determining the urea content in fluids, which method is fast and does not require any special equipment or trained personnel to handle such equipment.

It is an object of the present invention to provide a test device for determining the urea content in fluids, in which test device the reagents used are stable.

Another object of the invention is to minimize disturbances due to serum turbidity and the presence of bilirubin, when the urea content of blood serum is to be determined. Another object is to provide a test device with high specificity and sensitivity for determining urea in fluids within a wide concentration range. A further object of the invention is to provide a colometric test device with which urea can be detected when the amount of liquid to be analyzed is limited. A further object of the invention is to provide a method for determining urea nitrogen in blood, which method is fast and does not require any special equipment.

The new test device according to the invention for detecting urea in a test fluid comprises a carrier matrix prepared with a strong cation exchanger in acid form, and that two reagents which are not in contact with each other are incorporated in the carrier matrix, one reagent being of o-phthalaldehyde and the second reagent consists of 3-hydroxy-1,2,3,4-tetrahydrobenzophenquinoline, 3-acetoxy-1,2,3,4-tetrahydrobenzofquinoline or the dihydrochloride salt of any of these compounds. The reagents are suitably separated from each other by a polymeric material.

When using the test device, it is immersed for a moment in a sample, or a sample is otherwise applied to the support matrix, after which any detectable color change is observed by measuring the change in reflection at a wavelength above 550 nm.

The term "fluid" in the present specification refers to both body fluids, such as blood serum, blood plasma, urine and spinal fluid, and other aqueous solutions containing urea. Although the most preferred application of the invention is the determination of urea in body fluids, such as blood serum, it should be understood that the invention can also be applied to the analysis of industrial fluids.

In accordance with the present invention, a unique reagent strip is prepared by incorporating with a carrier matrix two different reagents which are separated from each other on the carrier matrix, for example by means of a polymer barrier. The term "carrier matrix" includes both moisture-absorbing and non-moisture-absorbing matrices. which are insoluble in and maintain their structural integrity upon exposure to water and other physiological fluids. Examples of suitable moisture-absorbing matrices are paper, cellulose, wood, synthetic resin webs, woven and nonwoven fabrics, and the like. Examples of non-moisture-absorbing matrices are matrices made of glass fibers and plastics, such as polypropylene and the like. In order for the carrier matrix to be easier to use, it can suitably be attached to an insoluble carrier member, such as a plastic strip, for example of polystyrene. The fastening is done in some suitable way, such as with the help of a double-sided tape.

The reagents incorporated into the carrier matrix react with urea under acidic conditions to form a chromogenic compound which absorbs light at wavelengths above 550 nm, usually between 55 ° C and 700 nm. A quantitative analysis can be achieved by measuring the change in reflection at about 620 nm after the test device has been brought into contact with the sample.

In order to achieve the acidity required for the reaction, it has been found necessary to modify or impregnate the support matrix with strong cation exchangers in acid form. Ion exchange prepared carrier matrices containing Amberlite IR 120 (Rohm & Haas, Philadelphia, Pennsylvania) or Dowex®) 50 (Dow Chemical Co., Midland, Michigan) are particularly preferred sulfonic acid modified carrier materials, especially when analyzing serum. The ion exchangers mentioned above are sulfonic acid modified polystyrene crosslinked with different amounts of divinylbenzene. Each carrier matrix is prepared with between 25 and 50% by weight of ion exchanger, preferably 35-50% by weight.

Two reagents were used to prepare the test device.

One reagent is o-phthalaldehyde, and the other reagent is 3-hydroxy-1,2,3,4-tetrahydrobenzoylquinoline, 3-acetoxy-1,2,3,4-tetrahydrobenzo [§7quinoline or the dihydrochloride salt of any of these compounds. dan prefers to use 3-hydroxy-1,2, j, U-tetrahydrobenzoic quinoline. Although according to the invention the two reagents must be applied separately to the carrier matrix, it is irrelevant in which order the two reagents are applied. Thus, any reagent can be applied first. The cation exchanger can be applied to the support matrix at the same time as the first reagent, or a support matrix prepared with an ion exchanger can be used.

According to one, in a first step, one reagent is applied to the carrier matrix.

In a second step, a polymer is usually applied to the support matrix, the polymer being present in a solvent which does not dissolve either the first reagent or the sulfonic acid material. Examples of useful polymers include methylcellulose, cellulose acetate, polyethylene oxide, styrene-butadiene copolymer, styrene-isoprene copolymer, polystyrene and styrene-acrylonitrile copolymer. In a third, final step, the second reagent is then applied to the support matrix. , this reagent being present in a solvent which does not dissolve the polymer which has been applied in the second step.

The reagent strip is usually allowed to dry between each step.

Suitable solvents for o-phthalaldehyde are water, methanol, acetone and hexane. Water, methanol or any mixture thereof is the preferred solvent if the reagent o-phthalaldehyde is applied in the first step. Acetone or hexane is the preferred solvent if o-phthalaldehyde is applied in the third step. Suitable solvents for the polymers are chloroform, acetone, benzene, toluene and the like. Suitable solvents for the second reagent are water, methanol and mixtures of methanol and acetone. Water is the preferred solvent if the second reagent is applied in the first step, and either methanol or a mixture of methanol and acetone is the preferred solvent if the second reagent is applied in the third step.

The reagents can be used in different concentrations. The reagent o-phthalaldehyde is usually present in an amount of 300-800 mg%, preferably 500-600 mg%. The second reagent is usually present in an amount of 200-500 mg%, when a dibydrochloride salt is used, and in an amount of 600-1200 mg%, when the free base is used. The concentration of the polymeric material is not critical provided that an amount of between 0.5 and 5 g% is used. The polymeric material is preferably present in an amount between 1 and 3 g fl. Although the preferred method of making the test device of the invention comprises three impregnation steps and subsequent drying, the test device may also be made by any other suitable method, such as printing on one or both sides of the matrix or applying different reagents to opposite sides. of a substrate or a matrix.

When using the test device according to the invention, it is suitably immersed briefly in a sample, or a sample is applied in some other way to the proximity matrix, after which any detectable color change is observed.

The invention is illustrated by the following examples.

Erempelml. A test breath was prepared as follows.

Nan was based on a SA ~ filter paper from Reeve Angel, Clifton, New Jersey. This filter paper was prepared with a strong cation sheath, and it had been pre-washed with 10-2OZig hydrochloric acid. The ion exchange capacity was about U mill equivalents per g of dry yen exchange. After rinsing with deionized water and drying, the paper was impregnated in three steps with the solutions given below. The filter paper SA-2 is a paper containing the sulphating cation exchanger Amberlite® IR 120. The ion exchanger is supplied in sodium form, and the paper contains between H5 and 50% by weight of ion exchanger.

PsLföf S ß ß- .i media; 11 2 taken - The filter paper was impregnated_by dipping in a solution containing the following substances in the specified amounts. water 100 ml maleic acid ~ methyl vinyl ether- Copolymer (stabilizer) 100 mg boric acid (color stabilizer) 2.5 g o-phthalaldehyde 200 mg 7 - (?,} - dihydroxypropyl) theophylline (stabilizer) 10 g After impregnation, the paper was dried at SOOC under 30 minutes. The second impregnation step.

The dried, impregnated paper from the first step was impregnated by dipping it in a solution of 2 g of polystyrene (5tyFOH, Dow Chemical Co., Midland, Michigan) in 100 ml of toluene.

The third impregnation step. The dried, impregnated paper from the second stage was impregnated by dipping it in a solution containing the following substances in the indicated amounts. methanol 30 ml acetone YO ml hydroxypropylcellulose (stabilizer) 0.5 g 3-hydrogly-1,?,}, N-tetrehydro-b <, »r1s«> _ / _ l_1 / quinoline-hydrochloride '100 mg He first dissolved the kinnlin derivative in the methanol, after which the hydroxypropylcellulose was separately dissolved in the acetone. The latter solution was then added to the former solution.

After the impregnation, the paper was dried at 6000 for 10 minutes, after which the paper was attached to a polystyrene carrier by means of a double-sided tape. The polystyrene carrier is a suitable handle, by means of which the reagent-impregnated paper can be dipped in a solution in and for determining the urea content of the solution.

The table below shows the color intensity obtained after immersion of the prepared strip strips in samples with different carmide concentrations.

A portion of a sample containing the specified amount of curbamide amide nitrogen; is diluted with two parts of water, after which 0.050 ml of the resulting mixture is applied to the surface of a test device prepared as described above. After incubation at IYOC for 1 minute, the reflectance of the reagent strip was measured at 620 nm, and the reflectance was expressed as a percentage of the reflection from a white reflective surface of barium sulfate. f ¥ ëP§ll-l Carbamide nitrogen content, Reflection, Carhamid nitrogen content, Reflection, mg% 72 mg% Û 75 50 '50 5 62 60 27 10 5H 70 25 20 uu 90 21 50 58 12o H8 Ho 53 150 15 Example 2. This example describes the manufacture of another test device according to the invention. A filter paper was used. ®. . . content HO weight% Dowex 50W 1 acid form; The ion exchanger was the 8% divinylbenzene cross dog (Dow Chemical Company., Midland, Michigan). 7905266-8 8 This filter was impregnated in three steps with the solutions given below.

The filter paper was impregnated by dipping it in a solution containing the following substances in the indicated amounts of water 100 ml of polyethylene oxide 50 mg of o-ft 300 mg of 300 mg of o-ft................................. mg Y ~ (2, §-dihydroxypropyl) -tenphylline (stabilizer) 10 g boric acid 9.5 g After impregnation, the paper was dried at GOOC for 50 minutes. _'2 <_f_ß__¿1! = fâf: ê_ _ il fl ßgf-ïßti fi æfln The dried, impregnated paper from the first step was then impregnated by dipping it in a solution of 2 g of styrene-acrylonitrile copolymer in 100 ml of toluene.After the impregnation with the styrene-copolymer, the paper was dried at 6000 for 15 minutes. 42.93 - The dried, impregnated paper from the second stage was finally impregnated by dipping it in a solution containing the following substances in the indicated amounts: methanol 30 ml of acetone 70 ml of hydroxypropylcellulose 0.5 g of 5-acetyl 1,2,}, U-tetrahydro-binosyn / quinoline-hydro chloride 500 ng The quinoline derivative was first dissolved in the methanol, after which the hydroxypropylcellulose was separately dissolved in the acetone. The two solutions were then mixed with each other.

The impregnated paper was then dried at 6000 for 10 minutes, after which it was attached to a polystyrene support using double-sided tape. The polystyrene carrier is a suitable handle with which the test reagent impregnated test reagent can be dipped in a solution to determine the urea content of the solution.

Other test devices were prepared in the same manner as above, using Dowex 50w® crosslinked with 2% divinylbenzene and Dowef) SOW crosslinked with 16% divinylbenzene. It was found that the intensity of the dye obtained after dipping the test device in a PPOV containing carhamide could be controlled by changing the degree of crosslinking. The color intensity varied inversely proportional to the degree of crosslinking.

The table below indicates the color intensity of reagent strips prepared with ion exchangers with different degrees of crosslinking, ie. different levels of divinylbenzene (DVB).

Reagent strips were prepared from paper prepared down the Dowex® 50 cation exchange with 2%, 8% and 16% divinylbenzene crosslinking grades, respectively. Urea samples were diluted in the same manner as in Example 1 and applied to the test devices, after which they were incubated at 3700 for 1 minute and the reflectance was measured.

Tê? 9} loš Reflection,%, at different urea concentrations and different degrees of crosslinking.

(Reflection increases as color intensity decreases) Urea content of urea, "__Ev§ïÉindninßs§r§d¿ Å DYÉ_ mg% 2. 8 16 20 57 nu 66 HO 27}} S6 60 21 27 50 Table 3 below shows the energy intensity of reagent strips. prepared with carrier matrices containing different amounts of ion exchangers.

In the same manner as in Example 1, reagent strips were prepared from filter paper containing 25, 55 and 50% by weight of cation exchangers crosslinked with 8% divinylbenzene, respectively. In the same manner as in Example 1, urea samples were diluted, the samples were placed on the test devices, the test devices were incubated for one minute at 5700 and the reflectance was measured by a reflectometer.

Table ä Reflection,%, at different urea levels and ion exchange amounts Content urea nitrogen content, Ion exchange amount, weight% mgí 25 55 50 ___ PO 58 S1 Uh NO Ü8 U0 ÉÉ '60 U2 53 27 Example_â. Using the ion exchange paper described in Example 2, a test device was prepared by impregnation in three steps. ï * 7905266-8 10 The first impregnation step.

The paper was impregnated by dipping it in a solution containing the following substances in the amounts indicated. water 25 ml polyethylene oxide 100 mg Y - (?, j-dihydroxypropyl) - theophylline 10 ß ethylenedinitrile tetraacetic acid or sodium salt 1.5 g sodium lauryl sulphate 100 mg methanol 15 ml glycerol 0,? ml boric acid H, 5 g o-phthalaldehyde 600 mg After impregnation, the paper was dried at 5000 for 20 minutes.

Rek The paper was impregnated with a solution of 5 g of polystyrene in 100 ml of talc. After impregnation with the styrene polymer, the napkin was dried at 50 DEG C. for 10 minutes. ÉÉÉaEÉÉÉÉÉÉMÄÉBÄ9QÉÉEÄP§? To B92 'The dried, impregnated paper from the second stage was finally impregnated ¿except that it was soaked in a solution containing the following substances in the specified amounts. methanol 25 ml of acetone 75 ml of hydroxypropylcellulose 0.75 g of hydrogen sulfide, 2,5, of tetrahydrobenzo [Q / quinoline 900 mg He first mixed the acetone, the hydroxypropylene cellulose and the quinoline derivative, after which the methanol was added so that a solution was obtained. - held.

The impregnated paper was dried at 5000 for 5 minutes.

The resulting test device could be used for analysis of euthanasia blood serum or plasma (dilution was required using the test devices of Examples 1 and 2). With this test device, urea nitrogen contents of up to 70 mg% could be determined.

The use of hydroxypropylcellulose as a stabilizer is not necessary according to the present invention. One can further use such a stabilizing agent as caffeine instead of dyphylline used in the following example. It has been found that dyphylline or the like should be used in an amount greater than 5% by weight.

Example Q. For comparison, a test device was prepared as follows.

P3? í <è01š§i; §1_¿1_fe1_i <ïu_nf: fluaäëgeeàí- Eilter paper Whatman EMM was acid-anodized by being immersed in a solution containing the following substances in the specified wñngderna. water 100 ml benzenedisulfonic acid JO Q N-naphthylethylenedine-minidine hydrochloride H00 mg dyphylline 10 g maleic anhydride-methyl-vinyl ether-copolymer (s * abilizer) 0.1 g _. _ ._ .o, _. hfter the impregnation the paper was dried at G0 t for 20 minu- Uxk The second i; The dried, impregnated apparatus from the first step was dissolved by dipping it in a solution of 1 g of polyethylene oxide in 70 ml of chloroform and 50 ml of the acetate. After the drying tor- 20 minutes. "i L; Û, C; U) 'CT i)' U 'UO * 1 Q rr <p.. fl O \ CO Ö C» _ C v C' 1 Det torkr-nie, i1: zpn = tz¿ ¿R1 «2r¿1 <ï <:; ..;: p'peret from» let .z »ha stmreï; ïrpregnerndes finally by dipping it in a solution of 500 ng o phthalaldehyde in 100 ml hexane. at 6000 for 20 minutes The resulting two impregnated paper was attached to a polystyrene support using double-sided eJp The resulting reagent strip could be used for quantitative CI 'C' determination of urea in protein free samples.Unwanted precipitation occurred when tried to analyze samples containing protein.

This fact underlines the importance of the present invention.

From the above it appears that according to the present invention several advantages are obtained. Three specific problems are solved by the reagent strips of the present invention. ~ First, serum proteins are usually precipitated under the usual acidic assay conditions, with the precipitated proteins interfering with their development. According to the invention, precipitation of iron proteins is eliminated by using an acidic tube matrix prepared with a strong cation exchanger. Secondly, the purifiers used are usually unstable. According to the invention, the purifiers are applied to the carrier nuts in such a way that the reagents are stabilized.

Third, the reflection error in spectroscopic measurement is reduced to a minimum when determining the urea content of blood serum. According to the invention, a coupling agent is used, which gives a mature compound which can be detected at wave lands above 5§0 Gm,, _ \. of a blit * fl thereby overcoming the yiro problem as a pre-emission which gives the test strip a yellow color and overcoming the problem of disturbing color ïrsmmallad by hilirubin and / or turbidity in the serum, when the color intensity is determined by Cbsorptionsspeztrosko or reflection spectroscopy. The use of ~-hydruxy-1,2-h, tetr @ nydrobenzo157quinoline or its dihydrochloride salt minimizes the blind reaction which takes place in the absence of carbonium. When, on the other hand, primaquine diphosphate uses few- (Ü-amine l-methylbutylamino) -6-methoxyquinoline7 or N-naphthylethylenedianine-di-, the formed crown compound develops a fewer rums must be measured at wavelengths close to the nbsorptine nuxium for the color formed at The use of 5-hydroxy-1,?,}, H-tetrahydrobenzo [§7quinoline or its dihydrochloride salt makes it difficult to increase the measurements at such wavelengths where the color as f: kt, ioriezi Lip;> vi :: n: “miiiis il ¿zo :: @ 1¶> ti nildes vid blindrt According to n pïinningen, all reindeer are incorporated into a single carrier netris without one but need to use corrosive floating redgens. Although the present invention is of great importance for the early detection of physiological diseases by determining the content of carbamide in body fluids, the invention can also be used for precise harbunid determination in industrial process fluids, where the carbamide concentration must be regulated within certain limits. Test devices for determining the urea content of industrial or physiological fluids are of the utmost value if the test devices give a rapid exposure result, are reliable and easy to use. In addition, in the case of a medical diagnosis, the test devices must be sufficiently accurate to reflect variations in an individual's condition. These objects are achieved according to the present invention.

Many different modifications and variations are, of course, possible within the scope of the present invention.

Claims (7)

Claim 7905266-8 Test device for the detection of urea, characterized in that it comprises a carrier matrix prepared with a strong cation exchanger in acid form, and that two reagents, which are not in contact with each other, are incorporated in the carrier matrix, one reagent being o-Phthalaldehyde and the second reagent are 3-hydroxy-1,2,3,4-tetrahydrobenzo [h] quinoline, 3-acetoxy--1,2,3,4-tetrahydrobenzo [h] quinoline or the dinhydrochloride salt of any of these compounds . Test device according to claim 1, characterized in that the reagent o-phthalaldehyde is present in an amount of 300-800 mg%, and that the second reagent is present in an amount of 200-500 mg%, when a dihydrochloride salt is used, and in an amount of 600-1200 mg%, when a free base is used. Test device according to Claim 1, characterized in that the reagents are separated from one another by means of a polymeric material. 4. Test device according to claim 1, characterized in that the carrier matrix is a moisture-absorbing paper. Test device according to claim 4, characterized in that the support matrix contains 25-50% by weight of ion exchanger. Test device according to claim 5, characterized in that the ion exchanger is crosslinked with 2-16% divinylbenzene. A method for detecting urea in a liquid sample, characterized in that a test device according to any one of claims 1 ~ 6 is briefly immersed in the sample, after which any detectable color change is observed by measuring the change in reflection at a wavelength above 550 nm. .