JPH071230B2 - How to measure ingredients - Google Patents

Description

The present invention relates to a method for measuring components in a solution using a test piece.

[Background of the Invention]

The target substance is colored by a chemical reaction or an enzymatic reaction,
Alternatively, the measuring method of the component in the solution which is changed into a measurable substance and measured is a solution method (hereinafter referred to as a wet method) in which a sample solution is added during a measurement test to cause a reaction, and a measuring reagent , A test paper method of impregnating a dried and prepared test paper piece with a sample solution to carry out a reaction, or a film prepared by dropping a test solution on a film prepared by laminating layers having various purposes The method can be roughly classified into a so-called dry method represented by a method.

The wet method has the merit of being able to perform more accurate measurement than the dry method, but the fact that the actual work such as preparation of measuring reagents, preparation of measuring instruments, and cleaning up is complicated, and the prepared measuring reagent solution is for a long time. There are drawbacks such as lack of stability over time.

On the other hand, the dry method is stable in the state of test strips and films for a long period of time and requires no preparation of a measurement reagent solution at the time of measurement. From the point of view of the above, there is a drawback that the reaction reach is low because the reaction phase is restricted. Especially in a measurement system using an oxidase, dissolved oxygen in the liquid phase is involved in the reaction, but in the dry method, the supply of oxygen to the liquid phase is insufficient, so the reaction stops halfway and It is inaccurate because the measured value of a high-concentration sample is lower than it actually is, and the oxygen supply rate is a rate-determining step, so the reaction rate of the target enzyme etc. can not be measured accurately was there. Further, the conventional optical measurement method of the dry method measures the concentration of the component in the sample solution by irradiating the surface or the back surface of the test paper piece or the film with the color exhibited based on the reaction and measuring the reflected light. However, because of this, there are restrictions such as the fact that the surface condition of the test strip or film must be dense, and in some cases, the measured value may have an error due to the influence of the surface condition.

[Object of the Invention]

INDUSTRIAL APPLICABILITY The present invention mainly has a problem in the method of measuring components in a sample solution which is a biological sample, that is, since the reaction is carried out under the condition that the reaction phase is restricted, the reaction reachability is low and the accuracy is high. The purpose of the present invention is to provide an optical measurement method of a so-called test strip method, which has been made to improve the point of lacking, and retains the advantages of the dry method as it is, and obtains accurate and reliable measurement values. .

[Structure of Invention]

 In order to achieve the above object, the present invention has the following constitution.

"A reaction liquid phase part generated by impregnating an absorbent carrier impregnated with a reagent composition for measurement and dried with a sample or a diluted sample to start a reaction and physically contracting the absorbent carrier The method for measuring a solution component is characterized in that the solution component is measured by optically measuring the progress of the reaction or the reaction completion state. What was impregnated with and dried (hereinafter abbreviated as reagent impregnated body) was put in a reaction vessel and diluted with water or an aqueous solution so that a sample solution or a liquid phase in an amount necessary for reaction and measurement existed. The sample solution is impregnated and reacted, and the reaction progress or the reaction completion state is optically measured by a liquid phase portion generated by physically contracting the reagent impregnated body portion without removing the reagent impregnated body from the reaction vessel. To measure And are characterized.

In the present invention, as a means for physically shrinking the reagent-impregnated body, when the reaction container is not limited, for example, as shown in JP-A-48-47877, more specifically, It is sufficient to use an insertion type optical measuring means as shown in FIG. 2 which is integrated with a means for contracting the reagent-impregnated body or which uses a device which operates in series. That is, for example, as shown in FIG. 2, it has an optical fiber probe composed of a reflecting mirror A, an optical connector B, an optical fiber C for irradiation light, and an optical fiber D for receiving light, and has a structure as shown in FIG. And a stabilized light source 5, a stabilized power source 6, a photodetector 7, an amplifier 8, an absorbance converter 9 and a display unit 10 are used as optical measuring means, and a light source unit composed of the stabilized power source 6 and the stabilized light source 5 is used. While the light having a specific spectral distribution emitted through the optical connector B and the irradiation optical fiber C reaches the light receiving optical fiber D reflected by the reflecting mirror A, the test drug 3
The amount of light absorbed by the photodetector 7, amplifier 8,
This can be done by measuring the absorbance as a value with a detection unit composed of the absorbance converter 9 and the display unit 10. First
The figure shows a cross-sectional view when this method is used. Further, when the measurement is performed using a quartz cell or the like used in a general spectrophotometer, for example, as shown in FIG. 5, the bottom surface shrinks the reagent impregnated body but allows the test solution to pass through. It is sufficient to use an insertion-type auxiliary device having a different structure, for example, a mesh structure.

Typical examples of the absorbent carrier forming the reagent-impregnated body include, but are not limited to, non-woven fabric, cotton, sponge-like foam, woven fabric, and glass wool. In addition, those that are generally elastic and are unlikely to cause fiber scraps are suitable.

As the measuring reagent solution impregnated in the absorbent carrier, an enzyme, a substrate, a color former, a buffering agent necessary for measuring an object to be measured,
A solution containing a coenzyme, an enzyme activator, a stabilizer for an enzyme or a color former, a film-forming agent, a solvent and the like as needed is used.

When the absorbent carrier is impregnated with the measuring reagent solution, it may be carried out by a single impregnation operation, but may be repeated several times if necessary, and depending on the purpose, the components of the measuring reagent solution may be mixed. It may be divided into several kinds of solutions and sequentially impregnated and dried.

Distilled water, ion-exchanged water or the like is usually suitable as a diluted solution of the sample solution used in the measurement by the method of the present invention, but if necessary, salts, buffers, surfactants, preservatives or It is also possible to prepare a solution that appropriately contains organic solvents and the like.

The reaction vessel used in the present invention does not have to be a light-transmissive cell as long as an insertion type optical means is used, and the material is not particularly limited as long as it does not affect the reaction.

As the measurement object that can be measured by the method of the present invention, for example, glucose, cholesterol, triglaceride, uric acid, urea nitrogen, bilirubin, creatinine, alkaline phosphatase, GOT, GPT, LDH, γ-GTP living body Examples thereof include components, but are not limited thereto.

Examples will be given below, but the present invention is not limited to these examples.

〔Example〕

Example 1. Determination of glucose in serum (tracking of reaction time) (Preparation of reagent impregnated body for glucose measurement) A rayon non-woven fabric (carrier) having a diameter of 1 cm and a thickness of 7 mm was impregnated with 80 μl of the following measuring reagent solution. It was then dried.

Test solution formulation for measurement glucose oxidase 50,000 units peroxidase 1,500 units mutarotase 100 units 4-aminoantipyrine 0.2 g phenol 0.5 g 1 potassium phosphate 0.5 g dipotassium phosphate 2.5 g bovine albumin 1.0 g purified water 100 ml in total.

(Sample) A serum sample diluted 100 times with water was used as a sample.

(Measurement Method) One piece of the reagent-impregnated body and 1.0 ml of the sample were added to a plastic container having a diameter of 1.2 cm and a depth of 1.5 cm and reacted at 37 ° C. On the way, the nonwoven fabric was expanded and contracted several times in order to accelerate the reaction between the reagent held on the absorbent carrier and the sample. As shown in FIG. 2, an optical fiber probe was inserted into the reaction vessel as shown in FIG. 1, and the absorbance at the wavelength of 505 nm (hereinafter abbreviated as E _S ) of the generated liquid phase portion was measured.

Instead of the serum sample, water or a glucose standard solution having a known concentration was used in the same manner, and the obtained absorbances were respectively blinded to the absorbance (hereinafter, abbreviated as E _Bl ) and the standard absorbance (hereinafter, E _B ). Abbreviated as _Std .).

FIG. 4 shows the change in absorbance with time.

As is clear from FIG. 4, the reaction of the reagent-impregnated body with glucose in the sample is completed within 5 minutes at 37 ° C.

Example 2. Determination of glucose in serum (preparation of reagent impregnated body for glucose measurement) The same as in Example 1.

(Sample) Ten samples of human serum diluted 100 times with water were used as samples.

(Measurement method) The measurement was performed in the same manner as in Example 1, and after 5 minutes, E _S , E _Std , and E _Bl were measured.

The glucose level in human serum was calculated according to the following formula.

[200 ^* : glucose level in glucose standard solution (mg / d
l)] Reference Example 1. Determination of glucose in serum Using the same sample as in Example 2, using a commercially available kit for glucose measurement [Glucose C-Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.] The glucose level was measured.

The measurement results of Example 2 and Reference Example 1 are also shown in Table 1.

As is clear from Table 1, the glucose measurement values obtained by the measurement method of Example 2 according to the method of the present invention show a good correlation with those of the conventional method using a commercially available kit.

Example 3. Determination of uric acid in serum (preparation of reagent-impregnated body for measuring uric acid) A urethane foam having a diameter of 10 mm and a thickness of 7 mm was used to measure 10
After impregnating with 0 μl, it was dried.

Test solution formulation Uricase 50 units Peroxidase 2,500 units Ascorbate oxidase 5,000 units Lipoprotein lipase 50,000 units 4-Aminoantipyrine 0.12 g N-ethyl-N- (2-hydroxy-3-sulfopropyl) -m-toluidine / Na salt 0.2 g 1 potassium phosphate 5 g 2 potassium phosphate 2 g Water-soluble gelatin 1 g Purified water Total amount 100 ml.

(Sample) A sample was prepared by diluting 10 human serum samples 60-fold with water.

(Measurement Method) One piece of the reagent-impregnated body and 1.0 ml of the sample were added to a plastic container having a diameter of 1.0 cm and a depth of 1.5 cm and reacted at 37 ° C. for 5 minutes. On the way, in order to accelerate the reaction between the reagent impregnated in the absorbent carrier and the sample, the urethane foam was expanded and contracted several times. As shown in Fig. 2, the optical fiber probe was inserted into the reaction vessel as shown in Fig. 1, and the wavelength of the generated liquid phase portion was 555n.
The absorbance E _S at m was measured.

E _Bl and E _Std were measured in the same manner by using water or a uric acid standard solution of known concentration instead of the serum sample.

The uric acid level in human serum was calculated according to the following formula.

[10 ^** : Uric acid value in uric acid standard solution (mg / dl)] Reference example 2. Determination of uric acid in serum Using the same sample as in Example 3, a commercial kit for measuring uric acid [Uric acid C-Test Wako , Wako Pure Chemical Industries, Ltd.] was used to measure the uric acid value.

The measurement results of Example 3 and Reference Example 2 are also shown in Table 2.

As is clear from Table 2, the measured value of uric acid obtained by the measuring method of Example 3 according to the method of the present invention shows a good correlation with that of the conventional method using a commercially available kit.

Example 4. Quantification of glucose in serum (Preparation of reagent impregnated body for glucose measurement) Same as in Example 1.

Ten human serum samples used in Example 2 were diluted 200 times with water and used as samples.

(Measurement Method) One piece of the above-mentioned reagent-impregnated body was placed in a quartz cell (10 × 10 mm, depth 4 cm), 2.0 ml of a sample was added, and the mixture was reacted at 37 ° C. for 5 minutes. On the way, in order to sufficiently react the reagent impregnated in the absorptive carrier with the sample, the nonwoven fabric was expanded and contracted several times with a device having a net at its tip as shown in FIG. Finally, the non-woven fabric was compressed using the above-mentioned instrument as shown in FIG. 6 so as not to cover the optical path of the spectrophotometer, and the absorbance E _S of the resulting liquid phase portion at a wavelength of 504 nm was measured.

Further, E _Bl and E _Std were measured in the same manner by using water or a glucose standard solution having a known concentration instead of the serum sample (using a Hitachi 556 spectrophotometer). The glucose level in human serum was calculated according to the following formula.

[200 ^* : glucose level in glucose standard solution (mg / d
l)] The measurement results of Example 4 and Reference Example 1 are also shown in Table 3.

As is clear from Table 3, the measured values of glucose obtained by the measuring method of Example 4 according to the method of the present invention show a good correlation with that of the conventional method using a commercially available kit.

〔The invention's effect〕

As described above, the present invention is an invention that exhibits remarkable effects in the following points, and is a great contribution to the art.

(1) In the measuring method of the present invention, the measuring reagent is stable and easy to handle because it is supplied as a dry type by impregnating and drying an absorbent carrier such as a nonwoven fabric.

(2) Since the reaction can be carried out in a liquid phase that is quantitatively sufficient, the reaction progresses faster and the degree of achievement is higher than that in the usual dry method reaction.

(3) Since it is not a method of measuring the reflected light from the surface of the test strip unlike the conventional optical measurement method of the dry method, there is no error due to the influence of the surface condition, and measurement with high accuracy and reproducibility is possible. .

(4) The operation is simple because it can be measured without removing the reagent-impregnated body in the optical measurement, and the operation is easier especially when an optical instrument that can be inserted into the reaction vessel is used. Become.

(5) In the measuring method using the insertion type optical instrument, it is possible to construct a disposable, convenient and inexpensive measuring method by selecting an appropriate combination of the reagent impregnated body and the reaction container. is there.

[Brief description of drawings]

FIG. 1 is a cross-sectional view when the measuring method of the present invention is carried out by using an insertion type photometer in which a means for physically contracting an absorptive carrier is integrated with an optical measuring means. FIG. 3 and FIG. 3 exemplify a schematic view of an insertion type photometer. FIG. 4 shows the time-dependent change in the absorbance during glucose measurement in serum obtained in Example 1, and the points obtained by plotting the absorbance obtained for each time (minute) on the horizontal axis along the vertical axis are connected. It is FIG. 5 shows an example of an insert-type auxiliary device used when the measuring method of the present invention is carried out using a general spectrophotometer, and FIG. 6 shows a case where it is used for actual measurement. It is a thing. The contents indicated by each symbol in the figure are listed below. 1: reaction vessel, 2: compressed carrier, 3: test solution, 4: optical fiber probe, 5: stabilized light source, 6: stabilized power supply, 7: photodetector, 8: amplifier, 9: absorbance converter, 10: Display unit, 11: Auxiliary equipment, 12: Quartz cell, 13: Compressed carrier, A: Reflector, B: Connector for connecting to light source unit and photodetector, C: Optical fiber for irradiation light, D: Light receiving Optical fiber for use, a: blinded, b: serum sample, c: standard.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (56) Reference JP-A-58-62561 (JP, A) JP-A-48-47877 (JP, A) JP-A-57-182638 (JP, A)

Claims (3)

[Claims] 1. A method in which an absorbent carrier impregnated with a reagent composition for measurement and dried is impregnated with a sample or a diluted sample to initiate a reaction and physically shrink the absorbent carrier. A method for measuring a solution component, which comprises measuring a solution component by optically measuring a reaction progress or a reaction completion state in a reaction liquid phase portion. 2. Means for physically shrinking the absorbent carrier comprises:
The measuring method according to claim 1, wherein the measuring method is integral with the optical measuring means or operates in series. 3. Means for physically shrinking the absorbent carrier comprises:
The measuring method according to claim 1, which is a method using an insertable instrument.