JPS6361953A - Test piece for detecting peroxide active material - Google Patents

Abstract

PURPOSE:To obtain a test piece for detecting a peroxide active material which is improved in stability by completely isolating an org. hydroperoxide and chromogen. CONSTITUTION:An absorptive carrier is immersed in a soln. prepd. by dissolving or dispersing the org. hydroperoxide or org. hydroperoxide-cyclodextrin inclusion compd. buffer soln., wetting agent, and sensitizer in water or soln. mixture composed of water and org. solvent which can be mixed with water and is dried at 60-80 deg.C. A mesh is then immersed into the water or water/alcohol soln. mixture consisting of the chromogen, water soluble binder and buffer soln. and is dried at 40-60 deg.C. The resulted water absorptive carrier and mesh are stuck to a plastic sheet and are used after cutting. Such test paper when immersed into a sample such as urine is white to pale creamy if the peroxide active material does not exist in the sample. The paper shows the oxidized color of the chromogen if the peroxide active material exists therein. The content is, therefore, estimatable by comparing the color tone thereof with a preliminarily prepd. standard color tone.

Description

[Detailed Description of the Invention] [Field of Industrial Application] The present invention relates to a test piece for detecting peroxidation-active substances, and more particularly to a test piece used for detecting peroxidation-active substances such as blood in body fluids and excreta. It is.

[Conventional technology]

Detection of peroxide active substances, especially blood, in body fluids such as urine, vomit, gastrointestinal contents, and feces, and excreta is of great clinical importance from the viewpoint of early detection of vertebrae diseases.

Diseases in which blood is often detected in the above body fluids and excreta include gastritis tumors, hemorrhagic diathesis, cystitis, and scurvy.

Usually, the amount of hemoglobin in the excrement of these diseases is extremely small and often cannot be identified with the naked eye.For this reason, microscopic observation is performed, but this method has poor reproducibility and is complicated to operate. It has drawbacks such as difficulty in processing a large number of samples.

To compensate for this drawback, so-called test strips are widely used. The measurement principle for detecting peroxide-active substances using this test paper is based on the fact that hydroperoxide is decomposed using its peroxidase-like activity, and the oxygen generated during this process causes the chromogen to oxidize and become colored.

In this case, the intensity of color development is proportional to the amount of peroxide active substance present, so by comparing this with the standard density, the peroxide active substance in the sample can be quantitatively measured.

Conventionally, this type of analysis has been carried out by making the sample acidic with acetic acid, adding a chromogen such as pentidine and hydrogen peroxide, and observing the presence or absence of a single color, but since the reagent is unstable, It has the disadvantage that it requires adjustment when used.

Test strips containing organic hydroperoxides and chromogens were then developed (described in US Pat. No. 3,012,976 and US Pat. No. 5,092,464). In this test paper, the chromogen changes color not only due to the catalytic action of the peroxide active substance, but also through a redox reaction with the organic hydroperoxide itself, making it no longer suitable for analysis. [Problems to be Solved] Therefore, various methods have been considered to stabilize these problems. One method of stabilization is to add a substance intervening between the organic hydroperoxide and the chromogen as a stabilizer. For example, Published Patent Publication No. 49-54093 describes the use of phosphoric acid amide, Publication No. 115288-1983 describes the use of boric acid esters such as triethanolamine borate, Publication No. 1443396-1983 describes the use of acrylamide-based substances, In 1986-147066, ethylenediaminetetrakismethylenephosphonic acid, 4
p-prombenzoyl dimetheramides, 1980-1983
No. 656 discloses the use of polyvinylmethylacylamide.

However, these stabilizers have a stabilizing effect by reducing the reactivity of organic hydroperoxides, and therefore do not have the desired sensitivity. The stabilizing effect caused by this does not improve the stability due to layer build-up.

Another stabilization method is to completely separate the organic hydroperoxide and the chromogen. Japanese Patent Publication No. 39-14747 discloses a microencapsulation method in which organic hydroperoxides are physically encapsulated within a colloid material such as gelatin. Furthermore, Japanese Patent Laid-Open No. 51-65694 discloses a method of forming an amine or amino alcohol salt of an organic hydroperoxide and separating it from a chromogen using a film-forming polymer such as polyvinyl pyrrolidone.

However, these methods tend to cause non-uniform reaction on the test paper, and also cause uneven coloring. The microcapsule coating hardens over time and its reactivity decreases◇
Furthermore, if even a small amount of water is present, the reaction between the organic hydroperoxide and the chromogen will proceed, resulting in a disadvantage that the sensitivity will be significantly reduced.

Also, U.S. Pat. No. 5,511,608 shows a test strip in which separate pieces of paper are impregnated with a non-acceptable reagent and then pasted together, but this does not provide satisfactory sensitivity. Publication No. 58-2170 discloses a test piece consisting of two indicator layers, one of which is made of water-soluble paper, and in which the water-soluble paper contains a chromogen.
However, since the water-soluble paper dissolves relatively slowly, this method has drawbacks such as delayed reaction and non-uniform coloration.

Furthermore, as an example of separating unacceptable components using a mesh, there is a method of attaching an oxidizing agent to a mesh in order to remove reducing substances such as ascorbic acid, as shown in Japanese Patent Publication No. 60-224065. However, this is not intended to avoid the reaction between the organic hydroperoxide and the chromogen.

[Means for solving problems]

The present invention is intended to solve the above-mentioned problems in the prior art, and its purpose is to detect peroxide active substances with improved stability by completely separating organic hydroperoxides and chromogens. The purpose of the present invention is to provide a test piece for detecting peroxide-active substances, in which a component consisting of a chromogen, a water-soluble binder, and a buffer is attached to the surface of an absorbent carrier containing an organic hydroperoxide. It is characterized by being covered with a mesh.

The mesh used in the present invention is suitably made of nylon or Tetron, transparent or translucent, with a fiber thickness of 5 to 100 μm, and an opening area of 20 to 60 inches. ◇ The size and shape of the mesh are determined by absorbency. It may be sufficient to coat the surface of the carrier. To fix the mesh on the surface of the absorbent carrier.

Use conventional means such as fixing the four corners with double-sided tape or adhesive, or wrapping both ends of the absorbent carrier with mesh and fixing the mesh at both ends to the support base as shown in Figure 2. 〇The disadvantages that were thought to arise from attaching a chromogen to the mesh and separating it from the organic hydroperoxide, such as sensitivity to peroxide-active substances, delayed reaction time, and uneven coloration, etc. This point was improved using the measures shown below, and now it shows properties equivalent to those of the normally used single-layer test piece. The absorbent carrier used in the present invention includes: Examples include cotton, nonwoven fabric, and glass fiber, but nonwoven fabric is the most effective. This is because the nonwoven fabric has a high water retention capacity and water absorbency, so the movement of the chromogen in the mesh layer occurs very quickly. Due to the transparency of the fibers of the non-woven fabric, the color development inside the cine-woven fabric can be observed from the top, which is why the strong color development can be seen.

Furthermore, in order to increase the sensitivity of the test piece, it is necessary to attach a relatively large amount of chromogen to the mesh. Therefore, in order to uniformly adhere a certain amount or more of the chromogen to the mesh, a method is used in which the chromogen is mixed with a water-soluble binder having a good viscosity that is compatible with the mesh.

Examples of the water-soluble binder include polyvinylpyrrolidone, hydroxyglobil cellulose, carboxymethyl cellulose, polyvinyl alcohol, and water-soluble nylon, but polyvinylpyrrolidone is particularly preferred.◇The concentration of the water-soluble binder used is 1
．． 0 to 50 weight class.

Also, the transfer of chromogen from the mesh layer to the absorbent carrier layer,
In order to develop the mixture more smoothly, it is desirable to attach the same type of buffering agent as the carrier layer to the mesh layer. The pH of this buffer is adjusted to 4 to 7, particularly preferably 5 to O, for the carrier layer, but the pH of the mesh layer should be determined taking into account the solubility of the chromogen. Shi, 0 for chromogen
- When using an amine compound such as toridine, the pH
15 to 4.5 is appropriate.

The organic hydroperoxide used in the present invention may be either liquid or solid, but since hydroperoxides are generally unstable to light, heat, etc., it is desirable to select a more stable organic hydroperoxide. These are, for example, tert-butyl hydroperoxide, paramethane hydroperoxide, diisofurobylbenzene hydroperoxide, cumene hydroperoxide, 2,5-dimethylhexane-2,5-
It is a dihydroperoxide, which can be used in one type or in combination of several types, and contains 1 to 5 in the impregnating liquid.
Used in a concentration of % by weight.

The chromogen may be anything that produces color when oxidized.
Examples include orthotolidine, 3,3,5,5-tetramethylbenzidine, pentidine, para-anisidine, orthodianisidine, and 2,7-diamitfluorene.

These are normally used at a concentration of α1 to 1% by weight in the impregnating solution, but since the amount of adhesion is small in mesh, cL5 to 5% is used.
It is preferable to use it at 0% by weight.

In addition, in order to sensitively detect peroxide active substances, PH4-7
It is necessary to maintain this pH at 1% to 5-6 ◇ Therefore, this PH
Buffers are used to maintain citrate, phosphate, and succinate.

An example is a phthalate buffer. In the field of clinical chemistry, it is especially important to detect blood diluted 1,000,000 times, so the use of sensitizers is considered. ◇Cholera is known to contain quinoline derivatives, benzoquinoline conductors, pyridine derivatives, and Z-aminobenzothiazole. It is a derivative and α05 to 1.0 in the impregnating liquid.
It is used in a proportion of i% by weight.

Wetting agents can also be used, such as sodium lauryl sulfate,
Anionic wetting agents such as sodium dodecylbenzenesulfonate and sodium dioctylsulfosuccinate are preferred and are known to stabilize radical cations generated by oxidation of the chromogen.

In the present invention, an organic hydroperoxide is combined with a cyclodextrin to provide a secret, particularly stable test composition.

Further, it is practically convenient to apply the absorbent carrier to a suitable substrate such as a plastic sheet such as polyethylene, polypropylene, polystyrene, etc. and cut it into a desired size and shape. Although the size and shape of the plastic sheet substrate are not particularly limited, examples thereof include an elongated rectangular shape with a predetermined thickness.

An example of the method for manufacturing the test piece of the present invention will be described below.

An absorbent carrier is immersed in a solution in which an organic hydroperoxide, or an organic hydroperoxide-cyclodextrin clathrate buffer, a wetting agent, or a sensitizer is dissolved or dispersed in water or a mixed solution with an organic solvent that is miscible with water. 60~80℃
Dry with.

Next, the mesh is immersed in water or a water/alcohol mixed solution consisting of a chromogen, a water-soluble binder, and a buffer solution.
Dry at 0-60°C. The obtained water-absorbing carrier and mesh are pasted on a glass sheet, and the water-absorbing carrier portion is cut into squares (5W x 5 squares, for example) for convenient use.

[Effect]

When the test strip obtained in this way is immersed in a sample such as urine, it is white to pale cream in color if no peroxide-active substance is present, and when a peroxide-active substance is present, the chromogen is oxidized. Therefore, by comparing the resulting color with a pre-prepared standard color tone, the content can be estimated. Alternatively, the reflectance can be measured using an instrument and calibrated. It is also possible to determine the concentration from the line.

〔Example〕

The present invention will be explained in more detail in the following examples in comparison with comparative examples. However, the present invention is not limited to the following examples.

Examples 1 and 2: Solutions having the following compositions were prepared as the first solution and the second solution.

(1) First solution 1M citrate buffer PH5,2550 Sodium golauryl sulfate 2.0t 2-Aminobenzothiazole sulfate α3t Ethanol
30- Use distilled water to make all i 100ffi7! shall be.

(2) Second liquid orthotolidine 1.52
Polyvinylpyrrolidone-901,5t06M citrate buffer PH4,050ml ethanol
30-A nonwoven fabric (Palcross P-45, manufactured by Honshu Paper Industries) was impregnated with the first liquid component and dried at 60°C for 60 minutes.

Next, add mesh (NBC Kogyo 225T) as the second solution.

and dried at 60°C for 10 minutes. The obtained water-absorbing carrier and mesh were attached to a plastic sheet using double-sided tape, and the size of the water-absorbing carrier part was 5.
It was made into a stick of m×5M.

FIG. 1 shows a side view of the obtained test piece. In the figure, 1 is a stick, 2 is an absorbent carrier, 3 is a mesh, and 4 is an adhesion site.

Further, Fig. 2 shows another example in which the absorbent carrier was coated up to the end with Messy in the same manner as in Example 1.〇Comparative example: A solution having the same composition as in Example 1 was prepared as the first liquid. did. In addition, a solution having the following composition was prepared as a second solution.

Orthotolidine α52 Methylene chloride 10〇- Filter paper (Whatman 3MM) i Impregnated with the following cylinder 1 liquid component, dried at 60℃ for 60 minutes, impregnated with the 2nd liquid component, dried at 40℃ for 15 minutes, and wrapped with 1 double-sided adhesive tape. The size of the test paper portion was 5 m x 5 sticks.

Performance Comparison Test> The performance of the test pieces prepared in Example 1 and Comparative Example was compared.

Based on the present invention, the appearance of the test piece and comparative test piece is as follows:
Both were pure white and could detect blood diluted 1,000,000 times.

Further, both test pieces were heated at 60℃ for 72 hours and at 25℃ for 65%.
After leaving RH for 124 hours, hemoglobin (106■/dt
The color intensity for the aqueous solution was measured using a color difference meter (Murakami Color Research Institute C).
The results of reflection photometry using MS-1200 are expressed as /S values.

Here, the /S value is
Invention product α808 (1607 (75,1 inch)
0458 (56,7 卍 () is stored at room temperature f:, 10
As can be seen from the ratio of 0% to light, the test piece of the present invention exhibits much higher stability than the control test piece. Because it is clathrated, it has very good stability and is stable for one and a half years under normal storage conditions. The stability of the test piece of the present invention was even more improved than that of this comparative example, indicating that its stability was very high.

Example 5 In place of polyvinylpyrrolidone in Example 1.

Similar results were obtained using carboxymethyl cellulose and hydroxypropyl cellulose.

Example 4 In place of the nylon mesh in Example 1.

Almost the same results as in Example 1 were obtained even when Tetron Metsy (NBC Kogyo 225T) was used.

〔Effect of the invention〕

As mentioned above, the test piece for detecting peroxide active substances of the present invention has the following characteristics:
Since the surface of an absorbent carrier containing organic hydroperoxide is coated with a mesh to which components consisting of a chromogen, a water-soluble binder, and a buffer are attached, the organic hydroperoxide and chromogen are completely absorbed. Because it is isolated, it has the same detection sensitivity and greatly improved stability compared to conventional test pieces of the same type.

[Brief explanation of the drawing]

FIG. 1 is a side view of an embodiment of the test piece for detecting peroxide active substances of the present invention. FIG. 2 is a side view of another embodiment of the test piece of the present invention. In the figure, 1...stick 2...absorbent carrier 3...
Metsch 4... - Adhesive point Patent applicant Eiken Chemical Co., Ltd. Agent Patent attorney Yumi Kaede (and 2 others) Figure 1
Figure 2 1...Stick 2...Absorbent carrier 3...Mesh 4 -4 contact points

Claims (1)

[Claims] 1. A test piece for detecting peroxide active substances, characterized in that the surface of an absorbent carrier containing an organic hydroperoxide is covered with a mesh to which components consisting of a chromogen, a water-soluble binder, and a buffering agent are attached.