JPH0380262B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Field of Application] The present invention provides an improved test strip for detecting bilirubin in body fluids, particularly urine. (Prior Art) In clinical medicine, bilirubin excreted in body fluids, especially urine, is regarded as an important indicator for diagnosing liver and biliary diseases and jaundice. Methods for testing urinary bilirubin are well known, including methods for observing the green color of biliverdin produced by oxidizing bilirubin (rosin method, gmelin method, etc.);
It is broadly divided into a method (diazo method) that observes an azo dye produced by a coupling reaction between bilirubin and a diazonium salt. Nowadays, when testing for urinary bilirubin requires simplicity and speed, test strips based on the diazo coupling reaction are playing an important role in clinical medicine. Diazonium salts that undergo an azo coupling reaction with urinary bilirubin are already known, such as sulfanilic acid, 2,4-dichloroaniline, 2,
Examples include diazonium salts derived from compounds such as 5-dichloroaniline. However, this method has a relatively slow reaction rate and is not sensitive enough to detect low concentrations of bilirubin (about 0.5 mg/dl). Various reaction accelerators have been studied to solve this problem. According to Japanese Patent Application Laid-open No. 52-43493,
Addition compounds of ureido compounds and aromatic sulfonic acids are disclosed. According to this method, the reaction between bilirubin and diazonium salt is promoted to some extent, but the reaction with inhibitory substances such as 5-hydroxyindoleacetic acid and indican present in urine is also promoted, making it difficult to determine the concentration of bilirubin. Have difficulty. Moreover, so-called acid capter derivatives are disclosed in JP-A-58-18168. However, the results of the re-run test show that the sensitivity is clearly low and it is not possible to detect bilirubin concentrations as low as 0.5 mg/dl. (Problems to be Solved by the Invention) In view of the above circumstances, an object of the present invention is to provide a highly sensitive test piece for detecting bilirubin that suppresses reaction with inhibitory substances such as 5-hydroxyindoleacetic acid. It's about doing. (Means for Solving the Problem) As a result of extensive research, the present inventor has found that in the reaction between bilirubin and diazonium salt, when a compound represented by the general formula () is contained as a reaction accelerator, 5-hydroxy It has been found that the color reaction with inhibitory substances such as indole acetic acid is suppressed, and the reaction with bilirubin is significantly enhanced. [In the formula, R ₁ , R ₂ and R ₃ may be the same or different, and have a linear or branched chain and have 1 to 1 carbon atoms.
18 alkyl group; cycloalkyl group having 5 or 6 carbon atoms; substituted or unsubstituted aryl group. That is, the present invention provides bilirubin in body fluids, which contains as essential components a diazonium salt capable of coupling with bilirubin in body fluids, an acid in an amount sufficient for the coupling reaction, and a compound represented by the general formula (). This is a test piece for detecting. (Function) Although it is not clear for what reason the compound of the general formula () in the present invention suppresses the reaction with inhibitory substances such as 5-hydroxyindoleacetic acid and increases the reactivity with bilirubin, it is In particular, it is necessary to reduce bilirubin in urine at low concentrations (0.5mg/
It has become possible to specifically detect even dl level). Residues of compounds of general formula () in the present invention
R ₁ , R ₂ and R ₃ are preferably the same, and in the case of linear or branched alkyl groups having 1 to 18 carbon atoms, the reaction promoting effect increases as the number of carbon atoms increases. , the effect varies depending on the hydrophobicity of the group. Among them, phosphine oxide in which R ₁ , R ₂ and R ₃ are n-octyl groups has a great effect. The compound of general formula () is 0.1-20% in the impregnating solution
, preferably in amounts of 0.5 to 10%. The diazonium salt contained in the test piece may be any known one, particularly aryl diazonium salts having a halogen or nitro group in the molecule. Examples include 2,4-dichlorobenzenediazonium, 2,4-dibromobenzenediazonium, 2,4,5-trichlorobenzenediazonium, and 4-nitrobenzenediazonium. In order to stabilize the diazonium group, it is advantageous to use a known salt, such as sulfate, tetrafluoroborate, arylsulfonate, etc., and tetrafluoroborate is particularly preferred because it has excellent thermal stability. It is. The diazonium salt is preferably from 0.02 to about 1% in the impregnating solution.
Use in amounts of 0.05-0.5%. Furthermore, it is necessary to use a sufficient amount of acid for the bilirubin to react with the diazonium salt;
It is advantageous to maintain a strongly acidic PH range, in particular a PH of 1 to 3. Preferred acids that can maintain this pH and are solid at room temperature include oxalic acid, citric acid, sulfosalicylic acid, and metaphosphoric acid. The amounts used are from 3 to 30%, preferably from 5 to 20%, of the impregnating solution alone or in mixtures. In addition, a surfactant may be contained in order to impart wettability to the test piece. For this purpose, any nonionic, anionic or cationic surfactant may be used, which has a surface-active effect even in strongly acidic media, but anionic surfactants are preferably used. Some examples include sodium lauryl sulfate, sodium dodecylbenzenesulfonate, and sodium dioctyl succinate. In terms of quantity, amounts of 0.1 to 2%, preferably 0.2 to 1%, are used in the impregnating solution. Furthermore, it is advantageous to include a diazonium salt stabilizer known from Diazo Chemistry in that retention is increased. As such a stabilizer, for example, sodium fluoroborate, sodium arylsulfonate, sodium metaphosphate, etc. are used alone or in combination. In terms of quantity, it is used in an amount of 1 to 15%, preferably 5 to 10%, in the impregnating solution. Next, the solvent or solvent mixture for impregnation may be one that does not react with the diazonium salt and dissolves all its components, or one that has a low boiling point so that it is not necessary to expose it to high temperatures in the drying process.
It is also advantageous to impregnate each component in separate working steps, for example first impregnating and drying a mixture of diazonium salt and acid in water and then impregnating and drying acetate ester or phosphine oxide in chloroform. Examples of the absorbent carrier used in the present invention include filter paper, polyester fleece, porous plastic, etc., and filter paper is particularly preferred. The test piece according to the present invention is manufactured, for example, as follows. The acid is dissolved in purified water, optionally a surfactant and stabilizer solution dissolved in water or a water-miscible solvent is added, and then the diazonium salt is dissolved to form the impregnating solution. A carrier such as a filter paper is impregnated with the first impregnating solution obtained in this way, and
Dry at 60℃. Next, the compound represented by the general formula () is dissolved in acetate or chloroform to prepare a second impregnating solution, and the first impregnating solution is again added to this impregnating solution.
Impregnate the dried carrier with the impregnating solution for 40~
Dry at 60℃. The test piece thus obtained can be attached to a plastic film using double-sided adhesive tape or the like for convenient use. When this test piece is used to detect bilirubin in body fluids, especially urine, it is carried out, for example, as follows. Bilirubin concentration can be determined by immersing a test piece in the sample urine and immediately pulling it out, and comparing the color produced after a certain period of time with a standard color table created in advance, or by determining the spectral reflectance from the color obtained within a certain period of time. It is also possible to measure the reflection function using a meter and determine the bilirubin concentration from a calibration curve. (Examples) Next, the present invention will be explained in more detail with reference to the following examples, but the scope of the present invention is not limited thereby. Example 1 Filter paper (Toyo Roshi No. 525) was impregnated with the following solutions in sequence and dried under ventilation at 50°C. Solution 2,4-dichlorobenzenediazonium tetrafluoroborate 0.08g Oxalic acid 10g Sodium lauryl sulfate 0.3g Purified water Total volume 100ml Solution Triphenylphosphine oxide 1.1g Ethyl acetate Total volume 100ml The test paper thus obtained was cut into a 5 mm square. Cut it into strips and attach it to one end of a 5 mm x 8 cm plastic sheet using double-sided adhesive tape. When this test strip was immersed in test urine, urine that did not contain bilirubin showed a pale yellow color, but urine that contained bilirubin showed a reddish-pink to red color after 20 to 30 seconds depending on the concentration, and the sensitivity limit was approximately 0.3 mg. /dl was hot. Test strips containing no triphenylphosphine oxide but of the same composition took about 2-3 minutes to react and had a sensitivity limit of about 1.0 mg/dl. Example 2 Using the test piece obtained in Example 1, the reactivity with 5-hydroxyindoleacetic acid was investigated. The results are shown in Table 1. The numbers in the table are 0 if no color development is observed.
When color development is observed, it is assigned according to the intensity of the color development.

[Table] Example 3 A test paper is prepared by replacing triphenylphosphine oxide in the solution of Example 1 with tri(n-octyl)phosphine oxide. When the test strip obtained in this way was immersed in the urine to be tested, urine that did not contain bilirubin showed a light yellow color, and urine that contained bilirubin showed a reddish-pink to red color after about 20 to 30 seconds depending on the concentration. The sensitivity limit was approximately 0.2-0.3 mg/dl. Similarly, sensitivity limits when other phosphine oxides are used are shown in Table 2.

[Table] Example 4 A test paper is prepared by replacing triphenylphosphine oxide in the solution of Example 1 with triphenylphosphine. The reactivity of the obtained test paper decreased and bilirubin could not be detected. Example 5 2,4-dichlorobenzenediazonium tetrafluoroborate in the solution of Example 1 was converted to 2,4-dichlorobenzenediazonium tetrafluoroborate.
A test paper is prepared in place of dibromobenzenediazonium tetrafluoroborate. The obtained test paper has the same performance as Example 1. Similarly, prepare test paper using the following diazonium salt. ●2,4,5-trichlorobenzenediazonium tetrafluoroborate ●4-nitrobenzenediazonium tetrafluoroborate The obtained test paper has almost the same performance as Example 1. (Effects of the Invention) According to the present invention, it is possible to provide a test piece for detecting bilirubin with extremely high sensitivity, and therefore the present invention has extremely great industrial benefits.

Claims (1)

[Claims] 1. A diazonium salt capable of coupling with bilirubin in body fluids, an acid in an amount sufficient for the coupling reaction, and a general formula [In the formula, R ₁ , R ₂ and R ₃ may be the same or different and are linear or branched and have 1 to 18 carbon atoms.
represents an alkyl group; a cycloalkyl group having 5 or 6 carbon atoms; a substituted or unsubstituted aryl group. ] A test piece for detecting bilirubin in body fluids, which is characterized by containing a compound represented by the following as an essential component.