JPS61202142A - Analyzing method and apparatus using absorbance - Google Patents

Abstract

PURPOSE:To enable accurate and stable analysis, by altering the specified wavelength of a transmission light corresponding to the concentration of a specified material in a liquid to be inspected and/or altering the transmission light path length in the liquid being inspected before the measurement of the absorbance. CONSTITUTION:A test tube having a colored liquid is put into a test tube insertion port 5 and them, a switch 1 is depressed. Light with the wavelength of 660nm hits the liquid in the test tube from a light source 3 and the transmission light hits a detector 7, where the light is converted into an electrical signal and then, into concentration in the liquid being inspected according to a calibration curve memorized into an arithmetic unit 9, the results obtained are shown on a concentration display section 9. When the display value of the display section 9 exceeds a specified value, the test tube is taken out of the insertion port 5 to put it into a test tube insertion port 6 and then, a switch 2 is depressed. Light with the wavelength of 560nm hits the liquid in the test tube from the light source 4, the transmission light hits a detector 8 and the concentration obtained with the arithmetic unit 9 is shown on a concentration display section 10. Thus, accurate and stable analysis can be done.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Field of Application] The present invention relates to a quantitative analysis method and a quantitative analysis apparatus using absorbance. More specifically, the present invention provides a quantitative analysis method and an apparatus for the same, which allows accurate and stable analysis even in a region where the concentration of an analyte substance in a test liquid is high. In particular, various components contained in body fluids such as blood and bile, and excretions such as urine,
For example, hormones, vitamins, enzymes, and other biological components.

The present invention provides a simple routine analysis method and device suitable for analyzing drugs and the like.

[Prior Art] In conventional quantitative analysis using light, it has been usual to measure the transmitted light of a test liquid containing a target substance to determine the absorbance. In this case, the intensity of the incident light on the test liquid is Io, the intensity of the transmitted light is 1, the concentration is C9, the optical path length is
When ε is a constant, the absorbance E can be expressed by the following equation, and is known to be proportional to the concentration C.

For E-LiO(+(10/T)-εC) However, this proportional relationship holds because the concentration of the target substance in the test liquid is relatively low and the intensity I of the transmitted light is equal to the intensity IO of the incident light. It is limited to a range that does not become too small in comparison.If the concentration of the target substance becomes higher than this range, not only will the above proportional relationship not hold;
Since the difference in absorbance due to the difference in apox becomes extremely small, accurate quantitative analysis cannot be performed. As a strategy for this purpose, dilution of the test solution is used, but
This method has a major drawback in that the operation is complicated.

[Object of the Invention] The object of the present invention is to solve the above-mentioned drawbacks in analytical methods using absorbance.

That is, the object of the present invention is to provide an analysis method and an analysis apparatus that can accurately, stably, and extremely easily analyze a substance contained in a test liquid at a high concentration.

[Structure of the Invention] As a result of intensive research to achieve the above object, the present inventor found that it is very effective to prevent an extreme decrease in the intensity of transmitted light in a high concentration region of the target substance, We have arrived at the following invention as a measure to accomplish this with an extremely simple operation.

That is, the present invention provides an analytical method for measuring the concentration of a specific substance in a test liquid by irradiating the test liquid with a light beam and measuring the absorbance of the transmitted light at a specific wavelength. An analysis method using optical intensity, which is characterized by changing the specific wavelength and/or changing the transmitted optical path length in the test liquid in accordance with the concentration range of the specific substance; and In an analyzer for a specific substance in the test liquid, the analyzer has a means for irradiating light, and a means for receiving the transmitted light and measuring the absorbance at a specific wavelength. The present invention provides an analyzer using absorbance, characterized in that it is equipped with a means for changing a specific wavelength and/or a means for changing a transmitted optical path length in the specimen in accordance with the concentration. .

A more detailed explanation of the invention follows.

The measurement principle of the quantitative analysis method using transmitted light according to the present invention is based on the fact that the absorption spectrum of the test liquid changes depending on the concentration of a specific substance in the test liquid. A device that selects a specific wavelength, photoelectrically measures the change in absorbance of the transmitted light at that wavelength, and then converts it into a concentration unit and displays it based on the correlation between concentration and absorbance obtained from the absorption characteristics of the specific substance, etc. It is.

In quantitative analysis using this absorbance, the present invention involves changing the specific wavelength and/or changing the length of the transmitted optical path in the test liquid depending on the concentration of the specific substance to be analyzed in the test liquid. It is characterized by:

The method of changing this specific wavelength is not particularly limited, but the following method is one that can accurately and stably measure the absorbance of the specific substance over a wide range of 'a degrees, and the measurement operation and measuring device are simple. The format shown is preferred.

That is, it is desirable to divide the concentration range of the specific substance in the test liquid into a plurality of regions and use different specific wavelengths corresponding to each region. As the specific wavelength, it is preferable to select a wavelength at which the absorbance can change as much as possible depending on the concentration change based on the absorbance characteristics of the specific substance in each concentration range. In particular, in the high concentration region of the specific substance, if the same specific wavelength as in the low concentration region is used, the absorbance will be too large and it will be impossible to measure. A specific wavelength should be selected. Normally, it is preferable to divide the concentration range into two regions and use two corresponding wavelengths because the operation is simple.

Furthermore, the method of changing the length of the transmitted optical path in the test liquid of the present invention is not particularly limited, but the length of the transmitted optical path can be changed by providing a spacer in a part of the transmitted optical path in the test liquid. A method that reduces the amount of water is preferred. It is preferable that such a spacer has a low absorbance so that inserting it will have little effect on analysis accuracy, and preferable materials include inorganic materials such as glass and quartz, and organic polymers such as polystyrene and methyl methacrylate. Examples include substances, and may have a space inside. Although two or more spacers may be used as a means for changing the length of the transmitted light path, from the viewpoint of operability and simplicity of the device, the effect of Part 7 can be sufficiently obtained by using one spacer.

Another method of changing the transmitted optical path length is to change the optical path length by changing the shape of the cell to be measured. For example, a method can be considered in which the inner diameter or depth of the upper part of the same cell containing the test liquid is increased, and the inner diameter or depth of the lower part thereof is decreased. That is, using a measuring cell as illustrated in FIG. 2, first irradiate the upper part 20 with the thicker inner diameter with light, and if the transmitted light obtained is stronger than a predetermined intensity, the lower part with the narrower inner diameter Shine a light on 21.

Further, in the present invention, both the above-mentioned change of the specific wavelength and change of the transmitted optical path length may be used together, but it is usually preferable to use either one of them. In particular, the method of changing the specific wavelength is a preferable embodiment since it has good operability.

The test liquid referred to in the present invention may contain a small amount of solids such as emulsions and suspensions depending on the case, but it is preferably in a uniform solution state that does not contain solids. The test liquid is placed in a measurement cell made of an inorganic material such as glass or an organic polymer material for analysis. The solvent for such a test liquid is not particularly limited, although a solvent suitable for dissolving the substance to be analyzed can be used.

The specific substance to be analyzed in the present invention is not particularly limited, but the specific substance particularly suitable for the present invention is a peptide hormone.

Non-peptide hormones, other hormones, enzymes.

Viruses, specific antigens, serum protein components, vitamins.

Drugs can be mentioned.

That is, examples of peptide hormones include insulin, C-peptide, glucagon, parathyroid hormone, calcitonin, ``squirrel poetin,'' secretin, cholecystokinin, gastrin, angiotensin, ``pappressin,'' and oxytocin.

Melanocyte stimulating hormone, adrenocorticotropic hormone, thyroid stimulating hormone (TS)-1), TSH secretagogue hormone, growth hormone, prolactin, luteinizing hormone (LH), LH secretagogue hormone (LHRH), chorionic gonadotropin (HcG) ).

Examples include follicle stimulating hormone. Examples of non-peptide hormones include steroid hormones such as glucocorticoids, aldosterone, testosterone, estriol, and estradiol.

Examples include progesterone. Other hormones include, for example, thyroid hormone (thyroxine, triiodothyronine, reverse triiodothyronine).
, cortisol, adrenaline, noradrenaline, melatonin, acetylcholine, etc. Furthermore, examples of enzymes include C1 esterase and alkaline phosphatase.

Pepsinogen, trypsin, kinase, etc.; specific antigens include, for example, α-fetoprotein.

Carcinoembryonic antigen (CEA), etc.; Serum protein components include, for example, thyroxine-binding globulin, β2 microglobulin, I (IG, rgv, It)A.

10D, I (IE, etc.; drugs include, for example, diphenylhydantoin, phenobarbital, mephobarbital, carbamazepine, and palproic acid.

Primidone, ethosuximide, methosuximide.

mefenytoin, fensuximide, glutethimide, clonazepam, chlordiazepoxide, nitrazepam,
Amphetamine, imipramine, amitributyline, protributyline, phenothiazine, mebrobamate, cocaine, etc.; vitamins such as vitamin A (retinol), vitamin B+ (thiamine), vitamin B2 (riboflavin), vitamin B6 (pyridoxal phosphate), vitamin B12 ．． Vitamin C (ascorbic acid), vitamin D, vitamin E (α-tocopherol), folic acid, di]tinic acid, pantothenic acid, biotin, etc., such as rheumatoid factor, IBs antigen, H
Examples include Bs antibody and myosin.

In the conventional analysis method using absorbance, when it is desired to measure a specific substance contained in the test liquid in a concentration range of 10 to 1000 expressed by a certain concentration standard, for example,
In this concentration range, the absorbance at a certain wavelength, that is, O, D,
(Optical density) may exhibit a value of about 0.1 to 5.0. In this case, because of the nature of the photometer, absorbances with O, D, greater than about 2.0 have too large an error, so absorbances in this range are not used for measurements. Therefore, in the analysis method of the present invention, by changing the specific wavelength to B, for example, Q, l'
), 5.0 can be lowered to O20,1,0, thereby making it possible to measure in the high concentration range, for example 200-1000, which could not be measured at wavelength A.

However, in this case, it is not preferable to measure the low concentration range (10 to 200) at wavelength B because both sensitivity and accuracy deteriorate.

In this way, by measuring the low concentration region with wavelength A and the high concentration region with another wavelength B, it is possible to accurately measure a wide range of 10 to 1000 with the same test liquid preparation.

Furthermore, the quantitative analyzer using absorbance according to the present invention performs the quantitative analysis as described above, and selects a specific wavelength for measuring absorbance corresponding to the concentration of the target substance in the test liquid. It is characterized by comprising a means for changing and/or a means for changing the transmitted optical path length in the test liquid.

The specific wavelength changing means is preferably of a type that can be selected from two or more, preferably two, specified wavelengths and can be easily switched and used, for example, with a changeover switch or the like. Further, when a spacer is used as a means for changing the length of the passage, the operation of inserting and removing the spacer may be performed manually or automatically using a motor or the like.

Furthermore, the analyzer is equipped with means for converting the measured absorbance into the concentration of a specific substance in the test liquid. Such a concentration conversion means has a function of storing a calculation method that uses mathematical conversion such as an approximation formula or graphical conversion such as pattern recognition to express a correlation between absorbance and concentration, that is, a calibration curve. This is very effective in practice. It is desirable that this calculation method can be updated and stored.

Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples at all.

Example 1 (1) Preparation of sample WHO2nd International, an international standard product of human chorionic gonadotropin (hereinafter abbreviated as 11CG)
onal 5 standard for ) IgG (
5300 international units (IU/ampule) to 0.01 M
Phosphate buffered saline (abbreviated as PBS) [Na C
98.5g, KH2PO40,266g, K2H
Dissolve 41,401g of PO in distilled water to make 1Jl! ] Dissolved in 5.3m of pH'7.2 to 1000IU
/d HCG stock solution was prepared. This was appropriately diluted with the above PBS and 10.25.50. 100. 200°40
0, 600. A solution of 900 Tl'L IU/d was prepared and used as a sample.

(21m immunoassay method (EnzyIIle l 1m
Enzyme immunoassay was carried out using a HOG quantitative kit consisting of the following components.

■ Antibody-conjugated stick: (Plastic stick bound with antibody against GCG-beta subunit ■ Enzyme-antibody conjugate: Horseradish peroxidase bound with antibody against HCG-beta subunit
Raddish Peroxidase) ■ Substrate solution; 0.03% hydrogen peroxide solution ■ Coloring solution: Tetramethylabendicine solution, i.e. 12X 75. Sample 0.5mm in a glass test tube! and enzyme-antibody conjugate■ 0.5 liters were added and mixed well. An antibody-bound stick was placed therein and incubated in a 25°C water bath for 15 minutes. During this 15 minute incubation, 0.7 d of substrate solution (1) and 0.3 d of color developer (2) were added to another 12X 75 m glass test tube and mixed well. After 15 minutes had elapsed, the stick was taken out and thoroughly washed with tap water, placed in the previously prepared mixture of substrate solution and coloring solution, and incubated in a 25°C water bath for 10 minutes. After 10 minutes, the stick was discarded and the absorbance of the liquid in the test tube was measured. (3) Measurement of absorbance Measure the absorbance of the colored liquid in the test tube with an optical path length of 10IllI1
Table 1 shows the results of measurement using cuvette No. 1 at a wavelength of 660 tv. The spectrophotometer 1 used was a spectrophotometer UVIDEC505 manufactured by JASCO Corporation.

The same liquid was measured in the following manner using the apparatus shown in FIG. 1, which is an embodiment of the analyzer of the present invention.

First, put a test tube containing a colored liquid into the test tube insertion port 5 and press the switch 1. Then, from the light m3, the wavelength is 660n
The n+ light hits the liquid in the test tube by hand, and the transmitted light hits the detector 7. Here, it is converted into an electrical signal, which is converted into a concentration in the test liquid according to a calibration curve stored in the arithmetic unit 9, and the obtained result is displayed on the concentration display section 9. However, if the obtained concentration is lower than 5 m 1 U/d, it will be displayed as "5 squid", and if it exceeds 150 m I Ll /-, it will be displayed as "150 m U/d".

When "150 yen" is displayed, take out the test tube from the test tube insertion port 5 and insert it into the test tube insertion port 6. By pressing the switch 2, light with a wavelength of 560 nm is emitted from the light 8!4 and hits the test tube, and the transmitted light hits the detector 8.

Here, it is converted into an electrical signal, which is converted into a concentration in the test liquid according to another calibration curve stored in the arithmetic unit 9. The density thus obtained is displayed on the density display section 10. The results obtained are shown in Table 2.

(Leaving space below) Table 2 As is clear from the above results, at a wavelength of 660 nm, l-10G in the range of 5 to 1507+'L I U /d
can be measured, but it is impossible to measure values of 1507+'L I U /d or higher because the absorbance is too high. But wavelength 5
By adopting eoiv ioo~1oooom
The concentration of Iu/Id can be measured with high accuracy.

Example 2 (1) W1-10I st Inte, an international standard product of human response hormone (hereinafter abbreviated as LH)
rnationalReference Prep
arrangement of LH (77 international units (IU)
)/ampule) was dissolved in 7.7 m PBS as described in Example 1 to make a 10 IU/ml HCG stock solution. Dilute this appropriately with PBS to give 5, 10.25°50, 1
00. 200. A solution of 400 mIU/udl was prepared and used as a sample.

(2) Enzyme immunoassay
o A 5say.

Implementation of E IA) Enzyme immunoassay was carried out using LH quantitative determination fluids 1~ consisting of the following components.

■ Antibody-binding stick; plastic stick bound to an antibody against LH ■ Enzyme-antibody conjugate; horseradish peroxidase bound to an antibody against LH
Peroxidase (abbreviated as IRP) Substrate solution: 0.03% hydrogen peroxide solution Color development solution: Tetramethylbenzidine solution Quantification was carried out in exactly the same manner as described in Example 1.

(3) Measurement of absorbance The absorbance of the colored liquid in the test tube was measured at a wavelength of 660 nm using a cuvette with an optical path length of 10 mm. Table 3 shows the results. The spectrophotometer used was spectrophotometer U manufactured by JASCO Corporation.
It is VIDEC505 type.

Table 4 shows the results of measuring the same liquid using the device shown in Figure 1 (however, in this case, the calculation device used was one made for LH).
Shown below.

(Leaving space below) Table 4 As is clear from the above results, the wavelength of eeonm is 5~150m1U/#li! It is possible to measure LH in the range of 150 m I LJ / rd or more, but it is impossible to measure it because the absorbance is too high. However, by adopting a wavelength of 560 nm, 100 to 500 m I Ll /
The concentration of 1uft can be measured with high accuracy.

Example 3 (1) Sample preparation The same procedure as in Example 1 was carried out.

(2) Enzyme immunoassay assay The procedure was the same as in Example 1, except that the test tubes shown in FIG. 2 were used to hold the substrate solution and coloring solution.

(3) Measurement of absorbance The colored liquid in the test tube shown in FIG. 2 was measured using the measuring device shown in FIG. 3, which is an embodiment of the analyzer of the present invention.

In other words, insert the test tube containing the colored liquid into test tube insertion port 1.
4 and press switch 11. Then, the light from the light source 13 hits the thick inner diameter part of the test tube, and the transmitted light is transmitted to the detector 15.
is converted into an electrical signal. The arithmetic unit 16 converts this into ill-pox and the resulting concentration is displayed on the 8I display section 17. The concentration is 5TILIU/mi! If it is lower than 15071iL IU/d, it will be displayed as "5 IU/d", and if it is higher than 15071 iL IU/d, it will be displayed as "150 IJ". "
150" is displayed, press switch 12. Then, the part of the test tube insertion port 4 that supports the test tube rises a little, and the part with the narrower inner diameter of the test tube enters the optical path. Next, light is emitted from the light source 13 and the density is displayed on the density display section 17 in accordance with the above-described order.

The results thus obtained are shown in Table 5.

(Margins below) Table 5 [Effects of the Invention] The present invention provides quantitative analysis using absorbance, which enables accurate and stable analysis even in areas where the concentration of the target substance in the test liquid is high, and with an extremely simple operation. A method and quantitative analysis device are provided.

In particular, by using wavelengths with high and low absorption coefficients for the analyte, by inserting a spacer into the measurement test tube, or by devising the shape of the test tube to easily convert the optical path length. , provides a method and apparatus for accurately measuring substances in a wide range of concentrations.

[Brief explanation of the drawing]

FIG. 1 shows a block diagram of one embodiment of a transmission type analyzer according to the present invention. FIG. 2 is a cross-sectional view of a test tube that is one embodiment of a measuring cell suitable for the present invention.The upper part has a thinner wall and therefore has a larger inner diameter, and the lower part has a thicker wall and has a smaller inner diameter. FIG. 3 shows one embodiment of the measuring device according to the present invention when measuring using the test tube shown in FIG. 2. Figure 1 Figure 2 Figure 3

Claims (7)

[Claims] (1) In an analysis method that measures the concentration of a specific substance in a test liquid by irradiating the test liquid with a light beam and measuring the absorbance at a specific wavelength of the transmitted light, the specific substance in the test liquid is An analysis method using absorbance, characterized in that the specific wavelength and/or the length of the transmitted optical path in the test liquid are changed in accordance with the concentration of the sample. (2) The length of the transmitted optical path is changed by inserting a spacer into a part of the transmitted optical path in the test liquid and removing the spacer from the part. Method described. (3) The method according to claim 1, wherein the concentration range of the specific substance in the test liquid is divided into a plurality of regions, and the change is made in accordance with the regions. (4) The method according to claim 3, in which the region is divided into two parts and two types of specific wavelengths are used. (5) In an analyzer for analyzing a specific substance in the test liquid, which has a means for irradiating the test liquid with a light beam and a means for receiving the transmitted light and measuring the absorbance at a specific wavelength, Analysis using absorbance, characterized by comprising means for changing the specific wavelength in accordance with the concentration of a substance and/or means for changing the length of the transmitted optical path in the specimen in accordance with the concentration. Device. (6) Claim 5, comprising means for converting the measured absorbance into the concentration of a specific substance in the test liquid.
Apparatus described in section. (7) The device according to claim 6, wherein the conversion means has a function of storing a calculation method for conversion.