JP3236251B2 - How to measure specific component concentration - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method for measuring the concentration of a specific component contained in a liquid sample, in particular, the protein value (albumin concentration) and the sugar value (glucose concentration) of urine collected from humans and other animals. It relates to a measuring device.

[0002]

2. Description of the Related Art Urinary sugar levels and urine protein levels reflect a part of the state of health. Therefore, a method for easily and accurately measuring these is required. Conventionally, urinalysis
A test paper impregnated with a reagent corresponding to each test item, such as sugar or protein, is immersed in urine, and the color reaction of the test paper is observed by a spectrophotometer or the like. According to this method, different test papers are required for each inspection item. In addition, a new test paper is required for each inspection. Therefore, the running cost is high. Further, there is a limit in automation for labor saving. When used at home, it would require a layman to set up and replace test strips. This work was relatively hated, which hindered the spread of urine testing devices to homes.

On the other hand, International Publication No. WO97 /
No. 18470 proposes a urine test method that does not require consumables such as test papers. Glucose and albumin show optical rotation whereas other urine components show little optical rotation. Therefore, in this urine test method, the urinary sugar level and urine protein level are obtained by measuring the optical rotation of urine. When light propagates through a liquid containing an optical rotatory substance, the polarization direction of the light rotates in proportion to the concentration of the optical rotatory substance. That is, the following expression is satisfied. A = L × α (1) where L: measured optical path length A: optical rotation [degree] α: specific optical rotation of the optically rotating substance

For example, when light having a wavelength of 589 nm propagates 100 mm in an aqueous glucose solution having a concentration of 100 mg / dl, the polarization direction of the light rotates by 50 × 10 ⁻³ degrees. Therefore,
Utilizing such characteristics, a urine sugar value and a urine protein value are obtained from the above equation. Table 1 shows the specific rotations of the aqueous solutions of glucose and albumin at 20 ° C.

[0005]

[Table 1]

[0006] When N types of optically active substances are contained in the liquid, the following is obtained. A = L × (α ₁ × C ₁ + α ₂ × C ₂ +... + Α _N × C _N ) (2) where L: measured optical path length A: optical rotation [degree] α _n : substance n (however, n is a natural number of 1 to N) C _n : concentration of substance n [kg / l] α _n : specific rotation of substance n

As is apparent from equation (2), the optical rotation of the liquid obtained by the measurement includes information on a plurality of optically active substance concentrations. That is, in the case of urine, the obtained optical rotation includes the sum of the optical rotation by glucose and the optical rotation by albumin. Here, since the specific rotation differs depending on the wavelength of the light to be propagated, the optical rotation is measured using light of a plurality of wavelengths, and the urine sugar value and urine protein value are obtained from the simultaneous equations of equation (2). ing.

According to this method, when one type of light source is used, if one of the urine sugar value and the urine protein value is known, the other can be calculated. However, the urine sugar value and the urine protein value can be calculated. If the values are both unknown, multiple light sources are required. Furthermore, as shown in Table 1, since the rate of change of the specific rotation of glucose with respect to the change in the wavelength of light does not greatly differ from that of albumin, even when a plurality of light sources are used, the urinary glucose level and urine protein level are high. Judgment of accuracy cannot be expected. In particular, the accuracy is low because the urine protein value is one digit smaller than the urine sugar value.

[0009]

SUMMARY OF THE INVENTION The present invention solves the above-mentioned problems and provides an optimal method for urinalysis which can accurately determine the concentration of a specific component in a liquid sample, in particular, the protein concentration. The purpose is to:

[0010]

SUMMARY OF THE INVENTION In the present invention, a liquid sample containing protein such as urine is heated to make it cloudy, light is projected on the sample, and the intensity of light transmitted through the sample or scattered from the sample is increased. Is measured. Thereby, the protein concentration of the sample can be accurately determined. However, there is a sample that rarely becomes cloudy even when heated. For example, in urine having an abnormally high pH, turbidity of urine, that is, aggregation of albumin hardly occurs. As described above, for a sample that is hardly clouded by heating, a divalent metal ion or an acid is added to the sample before heating to promote clouding by heating.

[0011]

DESCRIPTION OF THE PREFERRED EMBODIMENTS In the present invention, light is projected onto a test sample which has been subjected to a heat treatment in advance and has become clouded, and the intensity of transmitted light or scattered light is measured. In addition, while heating a liquid sample containing protein, light is projected on the sample,
The intensity of the transmitted light or the scattered light may be measured, and the protein concentration may be determined from the amount of change in the intensity of the light relative to the sample temperature. The intensity of the transmitted or scattered light need not be measured continuously, but the intensity of the transmitted or scattered light is measured at two different temperatures, and the intensity of the transmitted or scattered light obtained at these two points is measured. A ratio may be used.
For example, the protein concentration can be measured at two points out of 60 to 80 ° C., and the protein concentration can be determined from the ratio of the transmitted light or scattered light intensity at these two points. The clouding of the sample containing the protein proceeds with increasing temperature. Thus, by simultaneously detecting the transmitted light or the scattered light while heating the sample, the protein concentration of the sample can be determined from the amount of change with respect to these temperatures. For example, the protein concentration is calculated from the ratio of the transmitted light intensity at 70 ° C. to the transmitted light intensity at 75 ° C. Thereby, the influence of the transmittance of the sample before heating can be eliminated. In addition, the influence of scattering and the like due to substances other than proteins can be eliminated.

When the sample contains an optically rotating substance other than the protein, the optical rotation of the sample is measured before heating. Thereby, in a urine test, a urine sugar value can be accurately determined together with a urine protein value. First, a urine protein value is calculated by comparing the amount of transmitted light or the amount of scattered light with a calibration curve, and the obtained urine protein value and the optical rotation of urine are substituted into equation (2) to obtain a urine sugar value. The method for measuring the concentration of a specific component according to the present invention is also useful for determining the protein concentration of, for example, a culture solution in addition to urine.

[0013]

EXAMPLES The method for measuring the concentration of a specific component according to the present invention is particularly useful for urinalysis because it can accurately determine the protein concentration of a liquid sample. That is,
ADVANTAGE OF THE INVENTION According to this invention, it becomes possible to measure a urine protein value accurately, without requiring a consumable.

Healthy adults usually have a daily average of 1000
Drain 1500 ml of urine. Among them, the total solid content is 5
0-70 g. Approximately 25 g of the solid content is an inorganic substance mainly composed of sodium chloride and potassium chloride, and most of these are dissolved in urine in an ionized state. The balance is organic, mainly urea and uric acid, but also phosphoric acid and, to a lesser extent, sugars, such as glucose and proteins. The protein in urine is basically albumin. Glucose is usually 0.1 mg per day.
13-0.5 g excreted in urine. When the glucose concentration in urine, that is, the urine sugar value is roughly estimated from this and the urine volume, the average is about 50 mg / dl or less. However, in the case of diabetics, this can be several hundred mg / dl and sometimes even several thousand mg / dl. That is, the value is increased by about one to two digits from the normal value. Albumin, on the other hand, is usually much less than glucose and is excreted in urine from 3 to 60 mg per day. When estimated from this and urine volume, the albumin concentration of urine, that is, the urine protein value is usually about 6 mg / dl or less. However, the urine protein level of patients with renal disease can be 100 mg / dl or more, that is, 10 times or more than the normal value.

Urine becomes cloudy when heated to 60-80 ° C. This is because albumin, a protein contained in urine, condenses into a giant colloid. All urine components other than albumin have small molecular weights, and the scattering of light by these can be ignored. Further, heating to such a temperature does not change other components such as glucose. Therefore, the intensity of the light transmitted through the urine or the light scattered from the urine among the light projected on the clouded urine depends on the urine protein value. In the albumin concentration range where the urine protein value can be taken, the intensity of the transmitted light is considered to be substantially proportional to the urine protein value. Therefore, the light is projected onto the clouded urine, the intensity of the scattered light or transmitted light is measured, and the intensity of the scattered light or transmitted light is measured.
By comparing with, the albumin concentration of urine, that is, the protein value can be determined.

[0016] However, depending on the composition ratio, even if a rare amount of protein is contained, it does not become cloudy even when heated. For example, urine having an abnormally high pH is unlikely to become cloudy.
In the case of such a test sample, albumin coagulation can be promoted by adding a divalent metal ion such as calcium ion or magnesium ion. These are added, for example, in a chloride state. When using calcium ions, it is desirable to add 0.2 mmol or more per 1 dl of the sample. When using magnesium ions, it is preferable to add 0.1 mmol or more per 1 dl of the test sample. Also, by adding an acid to the test sample in place of such a divalent metal ion to lower the pH of the test sample, preferably to 5.5 or less, it is also possible to promote the coagulation of albumin. Can be. As an acid to be added, for example, acetic acid or phosphoric acid is used.

When used for urinalysis, light having a wavelength of 500 nm or more is preferably projected on urine. The specific rotation of the optically rotating substance increases as the wavelength becomes shorter, as shown in Table 1, until the anomalous dispersion due to the optical rotation dispersion appears. Therefore, it is possible to perform determination with higher accuracy by using light having a shorter wavelength. However, in general, light having a wavelength of 500 nm or less is greatly absorbed by urine components such as urochrome, and therefore, when measurement is performed using light of such a wavelength, accuracy may be rather deteriorated. In addition, as described above, when the optical rotation of urine is measured, information on optical rotation substances contained in urine, that is, glucose and albumin can be obtained. Therefore, by measuring the optical rotation of urine in advance, and then heating the urine to make it turbid as described above and measuring the degree of turbidity, it is possible to accurately determine the urinary sugar level together. . This makes it possible to realize a urine test method that requires no consumables and is easy to maintain. Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings.

Embodiment 1 FIG. 1 shows the configuration of a measuring apparatus used in this embodiment. The projection module 1 includes an optical system using a semiconductor laser as a light source, and a drive circuit for the semiconductor laser. The projection module 1 projects substantially parallel light having a wavelength of 670 nm and an intensity of 5 mW to the sample cell 2.
The sample cell 2 is cylindrical and has both ends made of glass having a diameter of 10 mm, and transmits light projected from the projection module 1. The substantial optical path length of this sample cell 2 is 50 mm. The optical sensor 3 detects light projected from the projection module 1 and transmitted through the sample cell 2. The lock-in amplifier 4 performs phase-sensitive detection of the output signal of the optical sensor 3 with reference to the modulation signal emitted from the signal generator 5 to the projection module 1, and outputs an electric signal corresponding to the amount of transmitted light. here,
The signal generator 5 supplies a modulation signal to the projection module 1, and performs pulse modulation of the projection light in synchronization with the signal.

The concentrations are 100, 300 and 10 respectively.
An aqueous albumin solution of 00 mg / dl was prepared. Next, 22.2 mg (corresponding to 0.2 mmol) of calcium chloride was dissolved in these aqueous solutions and 1 dl of pure water. The resulting aqueous solutions were each heated at 80 ° C. for 5 minutes,
Until cooled. Each of the heat-treated aqueous solutions was injected into the sample cell 2, and transmitted light was observed. The obtained transmitted light intensity is plotted on the vertical axis, and the albumin concentration is plotted on the horizontal axis,
The calibration curve shown in FIG. 2 was created.

Next, a urine protein value of 10 was determined using a test paper.
Urine having a pH of 7.2 determined to be not less than 0 mg / dl and not more than 250 mg / dl is heated at 80 ° C. for 5 minutes, and is heated at 35 ° C.
Cooled to ° C. This is referred to as Comparative Example 1. Next, this urine was injected into the sample cell 2 of the above-mentioned measuring device, and transmitted light was observed. 22.2 mg of calcium chloride was dissolved in 1 dl of urine used in Comparative Example 1. Then, the solution was heated to 80 ° C.
For 5 minutes and cooled to 35 ° C. This is designated as Sample A. This urine was injected into the sample cell 2 and transmitted light was observed in the same manner.

Further, the urine protein value was 30 using a test paper.
Calcium chloride was added to 1 dl of urine having a pH of 7.0 determined to be 0 mg / dl or more and 500 mg / dl or less.
2.2 mg was dissolved. The urine was then heated at 80 ° C for 5 minutes and cooled to 35 ° C. This is referred to as Comparative Example 2. Further, the urine was injected into the sample cell 2, and transmitted light was observed using the above-described measuring device. 22.2 mg of calcium chloride was dissolved in 1 dl of urine used in Comparative Example 2.
The solution was then heated at 80 ° C for 5 minutes and cooled to 35 ° C. This is designated as Sample B. The urine was injected into the sample cell 2 and the transmitted light was observed.

FIG. 2 shows the transmitted light intensity of each urine described above. In addition, the range shown by the arrow in the figure shows the urine protein value range obtained using the test paper. From FIG. 2, it can be seen that in Comparative Examples 1 and 2, the degree of white turbidity due to heating was small, and the obtained transmitted light intensity deviated significantly from the calibration curve. The albumin concentrations of Sample A and Sample B were determined from a calibration curve to be 120 mg / dl and 400 mg, respectively.
/ Dl. These each correspond to the values obtained using test paper. Next, the degree of urinary cloudiness with respect to the amount of calcium chloride added was visually observed. As a result, it was confirmed that when the added amount of calcium chloride was increased, urine became more cloudy. At this time, when the amount of calcium chloride added per 1 dl of urine exceeded 0.2 mmol, the degree of cloudiness hardly changed. From this, it is considered that all albumin in urine can be coagulated by adding calcium chloride to 1 dl of urine in an amount of 0.2 mmol or more. As described above, albumin in urine can be coagulated by adding calcium chloride even in urine which is hardly clouded even by heating, and urine protein value can be accurately determined. In particular, in order to perform highly accurate measurement, it is preferable to add 0.2 mmol or more of calcium chloride to 1 dl of urine.

In addition, a similar study performed by adding magnesium chloride to urine in the same manner as above confirmed that the addition of magnesium chloride also promoted the aggregation of albumin in urine. At this time, when the amount of magnesium chloride added per 1 dl of urine exceeded 9.5 mg (corresponding to 0.1 mmol), the degree of cloudiness hardly changed. This indicates that the measurement can be performed with higher accuracy by adding 0.1 mmol or more of magnesium chloride to 1 dl of urine. Actually, 9.5 mg of magnesium chloride was dissolved in 1 dl of urine, and the transmitted light was observed in the same manner as described above using this. As a result, it was confirmed that accurate measurement was possible even when magnesium chloride was used. As described above, by adding a divalent metal ion, it is possible to accurately determine the urine protein value even in urine that is hardly clouded. However, when these divalent metal ions are added, the calibration curve fluctuates as compared with the case where they are not added. For example, when calcium chloride is added in an amount of 0.2 mmol or more to 1 dl of urine,
Although slightly, the calibration curve varies depending on the amount of calcium chloride added. Therefore, in any case, it is necessary to prepare a calibration curve using an aqueous albumin solution in which the same amount of divalent metal ions as the amount to be added to urine to be measured is dissolved.

Embodiment 2 In this embodiment, an acid is added to urine which is hardly clouded even when heated. Even if an acid such as acetic acid is added to urine to lower the pH of urine, albumin coagulation can be promoted. 0.1 ml of an aqueous solution of acetic acid having a concentration of 1% was added to 1 dl of urine similar to that used in Comparative Example 2 in the above example to make the urine cloudy. The pH of urine became 5.5 by the addition of the aqueous acetic acid solution. Then, change this to 8
Heat at 0 ° C. for 5 minutes and cool to 35 ° C. This is designated as Sample C. On the other hand, aqueous albumin solutions having concentrations of 100, 300 and 1000 mg / dl, respectively, were prepared. To these aqueous solutions and 1 dl of pure water, 0.1 ml of the same acetic acid aqueous solution as described above was added. Then, these aqueous solutions were each heated at 80 ° C. for 5 minutes and cooled to 35 ° C. The heat-treated urine and the aqueous solution were respectively injected into the sample cells, and the amount of transmitted light was measured as in Example 1. The result is shown in FIG. As shown in the figure, even when an acid is added instead of the divalent metal ion, the urine protein value can be accurately determined. It was also confirmed that the urine protein value could be similarly accurately determined even when the urine pH was further lowered by increasing the amount of acetic acid added. The same effect was obtained by adjusting the pH to 5.5 or less using phosphoric acid instead of acetic acid.

Embodiment 3 In this embodiment, a method for measuring the protein concentration in a sample by observing scattered light from a liquid sample will be described. FIG. 4 shows an outline of the measuring apparatus used in this example. As in the first embodiment, the projection light is projected from the projection module 11 to the sample cell 12. The signal generator 15 supplies a modulation signal to the projection module 11 and pulse-modulates the light projected by the projection module 11 in synchronization with the signal. A liquid sample containing a protein is injected into the sample cell 12. Optical sensor 1
Numeral 3 detects light scattered when propagating through the sample in the sample cell 12 and projected from the projection module 11, and outputs a signal corresponding to the intensity of the detected light. The lock-in amplifier 14 outputs the output voltage of the optical sensor 13 to the signal generator 1
5 refers to the modulation signal emitted to the projection module 11,
Perform phase sensitive detection.

Urine whose albumin concentration was previously determined to be 10 mg / dl or less by urine test paper was used as a solvent.
Albumin solutions at concentrations of 100, 300 and 1000 mg / dl were prepared. Then, magnesium chloride was added to 1 dl of urine used for the obtained albumin solution and solvent.
5 mg was added and dissolved. Each of these at 80 ° C
And then cooled to 35 ° C. Next, these solutions were injected into the sample cell 12, and the scattered light thereof was observed. The result, that is, the output voltage of the lock-in amplifier 14 is shown in FIG. Here, the output voltage is shown as a logarithm on the vertical axis, and the concentration of the albumin solution is shown on the horizontal axis. A concentration of 0 represents the solvent alone, ie urine. Thus, also in the measurement of the scattered light, the output voltage is approximated by a straight line with respect to the albumin concentration. Therefore, by using this straight line as a calibration curve, it is possible to determine the urinary albumin concentration, that is, the urine protein value.

Urine whose albumin concentration was previously determined to be 100 mg / dl or more and 250 mg / dl or less using urine test paper was examined using this apparatus. As a result, the output voltage of the lock-in amplifier 64 was 0.12V. The albumin concentration obtained from the calibration curve shown in FIG. 5 was 120 mg / dl. This is consistent with the test strip results. In addition, using this apparatus, albumin concentration was 300 mg / dl or more and 500 m
Urine determined to be less than g / dl was examined. As a result, the output signal intensity of the lock-in amplifier 14 was 0.35 V, and the albumin concentration determined from the calibration curve shown in FIG.
It became 0 mg / dl. This is consistent with the test strip results.

As described above, the urine protein value can be accurately determined by observing the scattered light as in the present embodiment. In addition, it was confirmed that the coagulation of albumin in urine was similarly promoted even when calcium chloride was added instead of magnesium chloride. As mentioned above,
The protein concentration in the sample can be similarly measured by adding a divalent metal ion to the sample and observing the scattered light. Even if an acid such as acetic acid is added to the sample to adjust the pH to 5.5 or less, the coagulation of the protein can be promoted, and the protein concentration in the sample can be measured in the same manner.

Embodiment 4 FIG. 6 shows the configuration of a measuring apparatus used in this embodiment. In this embodiment, the intensity of light transmitted through the sample is measured while heating the liquid sample containing protein such as urine and measuring the temperature. The projection module 21 projects light to the sample cell 22 similarly to the one used in the first embodiment. A sample is injected into the sample cell 22. After being projected from the projection module 21, the optical sensor 23 detects light that has propagated through the sample in the sample cell 22. Here, the signal generator 25 supplies a modulation signal to the projection module 21, and performs pulse modulation of the projection light in synchronization with the signal. The lock-in amplifier 24 outputs the output signal of the optical sensor 23 and the signal
The phase-sensitive detection is performed with reference to the modulated signal generated in step 1. The output voltage of the lock-in amplifier 24 corresponds to the transmitted light intensity. Around the sample cell 22, the sample cell 22
A heater 28 in the form of a solenoid coil for heating a sample in the inside is provided. The heater 28 is connected to the enameled wire 5
It is configured to be wound 00 times. The coil-shaped heater driver 29 supplies a current of 5 A at maximum to the heater 28 according to an instruction from the computer 27. The thermocouple 20 is installed in close contact with the sample cell 22 and detects the temperature of the sample in the sample cell 22 substantially. The temperature indicator 26
In addition to displaying the temperature of the sample detected by the thermocouple 20,
This value is sent to the computer 27. Lock-in amplifier 2
The output of 4, that is, a value indicating the transmitted light intensity, is also sent to the computer 27. Thereby, the computer 27 heats the sample according to a preset program and simultaneously measures the temperature and the intensity of the transmitted light.

Hereinafter, the measuring method of this embodiment will be described. Albumin concentration of 10 with urine test paper in advance
Urine determined to be not more than mg / dl was used as a solvent to prepare albumin solutions having concentrations of 100, 300 and 1000 mg / dl, respectively. 0.1 ml of an aqueous solution of acetic acid having a concentration of 1% by weight was added to each of the albumin solution and the urine used as the solvent. With the addition of acetic acid, the pH of each of these became about 5.5. Then, these were respectively injected into the sample cell 22, and the temperature was changed from 35 ° C. to 8
Heated to 0 ° C. at a heating rate of 2 ° C./min. The transmitted light was measured every 6 seconds while heating the sample. FIG. 7 shows the result. Here, the horizontal axis represents the temperature of the sample, and the vertical axis represents the output voltage of the lock-in amplifier corresponding to the transmitted light intensity of each sample. FIG. 8 shows the ratio R between the transmitted light intensity at 70 ° C. and the transmitted light intensity at 75 ° C. for each albumin concentration solution defined by the following formula. R = (transmitted light intensity at 75 ° C.) / (Transmitted light intensity at 70 ° C.) (3) Here, R is logarithmically indicated on the vertical axis. As is clear from FIG. 8, the relationship between R and albumin concentration is approximated by a straight line. Therefore, by using this straight line as a calibration curve, the protein concentration of the sample can be determined. According to the measuring method of the present embodiment, since the protein concentration is determined based on the ratio of the transmitted light intensity at two different temperatures while heating, it is possible to eliminate the interfering factors such as the transmittance of the sample before heating. The measurement can be performed with higher accuracy than in the first embodiment.

Urine whose albumin concentration was determined to be 100 mg / dl or more and 250 mg / dl or less in advance using a urine test paper was determined using the above-described measuring device. As a result, R was 0.89. When the albumin concentration was determined from this and the calibration curve in FIG. 8, it was 120 mg / dl. This is consistent with the test strip results. In addition, urine whose albumin concentration was determined to be 300 mg / dl or more and 500 mg / dl or less using a urine test paper was similarly examined. As a result, R was 0.67, and using this to determine the albumin concentration from the calibration curve of FIG.
mg / dl. This is consistent with the test strip results. As described above, the urine protein value can be accurately determined by using the ratio of the intensity of transmitted light at different temperatures as in the present embodiment.

Embodiment 5 FIG. 9 shows a configuration of a measuring apparatus used in this embodiment. In the present embodiment, as in the case of the fourth embodiment, the liquid sample is heated, and while measuring the temperature of the liquid sample, light is projected onto the liquid sample and the scattered light is observed. As in the first embodiment, the projection module 31 projects the projection light onto the sample cell 32. A liquid sample is injected into the sample cell 32. The optical sensor 33 detects light scattered when propagating the liquid sample in the sample cell 32 after being projected from the projection module 31. Lock-in amplifier 34
Performs phase-sensitive detection of the output signal of the optical sensor 33 with reference to the modulation signal emitted from the signal generator 35 to the projection module 31, and outputs a signal corresponding to the scattered light intensity. Here, the signal generator 35 supplies a modulation signal to the projection module 31, and performs pulse modulation of the projection light in synchronization with the signal. Around the sample cell 32, a heater 38 similar to that of the third embodiment is arranged. Coiled heater driver 3
9 is a heater 38 according to an instruction from the computer 37.
A current of 5 A at the maximum is passed through. The thermocouple 30 is installed in close contact with the sample cell 32 and detects the temperature of the liquid sample in the sample cell 32 substantially. Temperature indicator 36
Displays the temperature of the liquid sample detected by the thermocouple 30 and sends this value to the computer 37. The output of the lock-in amplifier 34, that is, a value indicating the intensity of the scattered light is also sent to the computer 37. Accordingly, the computer 37 heats the liquid sample according to a preset program, and simultaneously measures the temperature and the scattered light intensity of the liquid sample.

Hereinafter, a urine test method using the present measuring apparatus will be described. Urine whose albumin concentration was previously determined to be 10 mg / dl or less by urine test paper was used as a solvent to prepare albumin solutions having concentrations of 100, 300, and 1000 mg / dl. Next, 22.2 mg of calcium chloride was added to the urine used for the albumin solution and the solvent.
(Equivalent to 0.2 mmol) and dissolved. These are respectively injected into the sample cell 32, and are cooled from 35 ° C. to 8
Heated to 0 ° C at a heating rate of 2 ° C / min. At this time, the scattered light intensity was measured every 6 seconds. FIG. 10 shows the ratio r of the scattered light intensity at 70 ° C. and the scattered light intensity at 75 ° C. to the albumin concentration at this time. Here, r is defined as follows. r = (scattered light intensity at 75 ° C.) / (scattered light intensity at 70 ° C.) (3) Here, r is logarithmically indicated on the vertical axis. As is clear from FIG. 10, r is approximated by a straight line with respect to the albumin concentration. Therefore, by using this straight line as a calibration curve, the albumin concentration of the unknown urine, that is, the urine protein value can be measured.

Urine whose albumin concentration was previously determined to be 100 mg / dl or more and 250 mg / dl or less using urine test paper was examined using the above-mentioned measuring apparatus. As a result, r was 4.6. Using this, the albumin concentration was determined from the calibration curve shown in FIG.
/ Dl. This is consistent with the test strip results.
In addition, albumin concentration was 300 mg by urine test paper.
Urine judged to be not less than / dl and not more than 500 mg / dl was similarly examined. As a result, R was 13.3, and the albumin concentration was determined to be 400 mg / dl from this value and the calibration curve shown in FIG. This is consistent with the test strip results.

As described above, according to the urine test method, the urine protein value can be accurately determined. Also, no consumables such as test papers are used. Further, according to the inspection method of the present embodiment, since the influence of the urine transmittance before heating or the like is not exerted, the measurement can be performed with higher accuracy than in the fourth embodiment.

Embodiment 6 In this embodiment, before heating a liquid sample, its optical rotation is measured at room temperature. Then
While heating, measure the temperature of the liquid sample and the intensity of the light passing through it. By measuring the optical rotation, information about the optical rotation substance in the sample can be obtained. In particular, in a urine test, it is possible to calculate a urine sugar value together with a urine protein value. That is, as described above, since the optical rotation of urine is considered to be the sum of the one caused by glucose in urine and the one caused by albumin, the urine protein value and the optical rotation obtained by making the urine cloudy, The sugar level can be calculated.

FIG. 11 shows a measuring apparatus used in this embodiment. The basic principle of optical rotation measurement of this apparatus is an optical null method based on polarization plane vibration utilizing the Faraday effect. The projection module 41 projects substantially parallel light having a wavelength of 670 nm and an intensity of 5 mW in the same manner as that used in the first embodiment. The polarizer 53 transmits only a specific direction component of the projected light. The sample cell 42 is the same as that used in the first embodiment, and the urine injected therein modulates the deflection direction of the transmitted light to a minute width by the optical Faraday effect. The analyzer 54 is arranged in a crossed Nicols state with respect to the polarizer 53, and can be rotated with the transmission axis of the polarizer 53 as a rotation axis. Here, the polarizer 53 and the analyzer 5
If 4 is ideal, that is, if the extinction ratio is infinite, the transmitted light does not reach the optical sensor 43. But,
In practice, the extinction ratios of the polarizer and the analyzer do not become infinite. Polarizer 53 and analyzer 54 used in this embodiment
Has an extinction ratio of about 5000, so that transmitted light of about 1 μW reaches the optical sensor 43. This light intensity is sufficient to measure the transmitted light intensity.

The lock-in amplifier 44 performs phase-sensitive detection of the output signal of the optical sensor 43 with reference to the modulation signal emitted from the signal generator 45 to the projection module 41. The switch 55 is connected to the terminal “a” when measuring the optical rotation, and supplies the signal of the signal generator 45 to the coil-shaped heater driver 49 as a magnetic field modulation signal. Further, when measuring the transmitted light, it is connected to the terminal b and supplies a signal to the projection module 41 as a modulation signal of the projection light. The heater driver 49 supplies a maximum of 5 to the heater 48 in accordance with an instruction from the computer 47.
A current is applied. However, when the optical rotation is measured, the heater 48 and the heater driver 49 may also perform a function of applying a magnetic field to the liquid sample in the sample cell 42 under the instruction of the computer 47 and sweeping while modulating this magnetic field. it can. That is, in the present apparatus, the heater 48
Performs the function of applying a magnetic field to the sample at the time of optical rotation measurement, and performs the original function of the heater at the time of heating the sample. The thermocouple 40 is installed in close contact with the sample cell 42 and substantially detects the temperature of the sample in the sample cell 42. The temperature indicator 46 displays the temperature of the sample detected by the thermocouple 40 and sends this value to the computer 47. In the above configuration, the rotation of the sample is first measured by the instruction of the computer 47, and then the temperature and the intensity of the transmitted light are measured while heating the sample.

Hereinafter, a urine test method using the present measuring apparatus will be described. Urine which has been determined in advance by an urine test paper to have an albumin concentration of 10 mg / dl or less is used as a solvent, and the concentrations are 100, 300 and 1000 mg / d.
1 of albumin solution was prepared. Next, 22.2 mg of calcium chloride was added to the urine used for these solutions and the solvent. These were respectively injected into the sample cell 22 and heated from 35 ° C. to 80 ° C.
At this time, the heating rate was set to 2 ° C./min, and the transmitted light intensity was measured every 6 seconds. 70 ° C and 7 at each concentration of solution
The ratio R of the transmitted light intensity at 5 ° C. was calculated in the same manner as in Example 2.
This R is plotted for each concentration as shown in FIG. Here, the vertical axis is a logarithmic representation of R. By using the curve in FIG. 12 as a calibration curve, the urinary albumin concentration, that is, the urine protein value can be measured. The degree of polarization of linearly polarized light propagating in urine decreases as turbidity increases. That is, depolarization due to scattering occurs. Therefore, the calibration curve in FIG. 12 does not become a straight line like the calibration curve shown in FIG.

Next, using a urine test paper, the glucose concentration was 50 mg / dl or less and the albumin concentration was 100 mg / dl.
Urine judged to be not less than dl and not more than 250 mg / dl was examined by the same method as above using this apparatus. First, the optical rotation was measured, and A = −19.8 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the transmitted light intensity was measured.
From this, R = 0.85 was obtained. From this R and the calibration curve shown in FIG. 12, the albumin concentration is determined to be 120 mg / dl. By solving equation (2) using the albumin concentration and A, a glucose concentration = 30 mg / dl was obtained. This is consistent with the test strip results.

According to the urine test paper, the glucose concentration is 100 mg / dl or more and 250 mg / dl or less,
Albumin concentration 300mg / dl or more and 500m
Urine determined to be g / dl or less was examined using this apparatus. First, the optical rotation was measured. As a result, A = −56 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the transmitted light intensity was measured to obtain R = 0.62. From this R and the calibration curve shown in FIG. 12, the albumin concentration is determined to be 400 mg / dl. By solving equation (2) from this albumin concentration and A, a glucose concentration = 150 mg / dl was obtained. This is consistent with the test strip results.

As described above, according to the present embodiment, after measuring the optical rotation of urine, the urine is heated to make it turbid, and the degree of turbidity is measured. Can be inspected at the same time. Thereby, the urine protein value and urine sugar value can be inspected without using consumables such as test paper.

Embodiment 7 A measuring method according to the present embodiment will be described below in detail with reference to FIG. Also in this embodiment, the rotation of the liquid sample is measured first, and then the temperature and the transmitted light intensity are measured while heating the sample.
The basic principle of the optical rotation measurement of this apparatus is the same as that of the sixth embodiment. As in the sixth embodiment, the polarizer 73 transmits only a specific direction component of the substantially parallel light projected from the projection module 61. The sample cell 62 is the same as that used in the sixth embodiment. If the sample injected therein contains an optical rotatory substance, the deflection direction of the light passing through the sample is minutely modulated by the optical Faraday effect. . In addition, the polarizer 73
The analyzer 74 is arranged in a crossed Nicols state. The switch 75 is connected to the terminal a when measuring the optical rotation, and supplies a signal from the signal generator 65 to the coil-shaped heater driver 69 as a magnetic field modulation signal. Also, when measuring the transmitted light, it is connected to the terminal b and supplies the same signal to the projection module 61 as a modulation signal of the projection light. The beam sampler 78 extracts a part of the light transmitted through the sample. The optical sensor 76 detects the extracted light and emits a signal corresponding to the intensity of the light. The switch 77 is synchronized with the switch 75, and supplies the output signal of the optical sensor 63 to the lock-in amplifier 64 when measuring the optical rotation, and locks the output signal of the optical sensor 76 when measuring the transmitted light. Switch to supply to 64. When the optical rotation is measured, the heater 68 and the heater driver 69 can also perform a function of applying a magnetic field to the sample in accordance with an instruction from the computer 67 and sweeping while modulating the magnetic field. Thermocouple 60
Is installed in close contact with the sample cell 62, and substantially detects the temperature of the sample in the sample cell 62. The temperature indicator 66 displays the temperature of the sample detected by the thermocouple 60 and sends this value to the computer 67. In this embodiment, as in Embodiment 6, after measuring the optical rotation of a liquid sample, the temperature and the intensity of transmitted light are measured while heating the sample. However, in the present embodiment, unlike Embodiment 6, there is no influence of depolarization. Therefore, a linear calibration curve is obtained. Actually, as a result of inspection using the same albumin solution as in Example 6, a linear calibration curve was obtained.

The glucose concentration was 50 mg / dl or less and the albumin concentration was 100
Urine judged to be not less than mg / dl and not more than 250 mg / dl was examined using this device. First, the optical rotation was measured, and A = −19.8 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the transmitted light intensity was measured to obtain R = 0.89. From this R and the obtained calibration curve, the albumin concentration was determined to be 120 mg / dl. By solving the equation (2) from the albumin concentration and A, a glucose concentration = 30 mg / dl was obtained. This agrees with the judgment result using the test paper.

Further, the urine test paper shows that the glucose concentration is 100 mg / dl or more and 250 mg / dl or less, the albumin concentration is 300 mg / dl or more and 50 mg / dl or less.
Urine determined to be 0 mg / dl or less was similarly examined. First, the optical rotation was measured, and A = −56 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the transmitted light intensity was measured to obtain R = 0.67. From this R and the obtained calibration curve, the albumin concentration was determined to be 400 mg / dl. By solving equation (2) from this albumin concentration and A, a glucose concentration = 150 mg / dl was obtained. This is consistent with the test strip results.

As described above, according to the present embodiment, after measuring the optical rotation of the urine, the urine is heated, and the intensity of the transmitted light of the urine is measured to determine the degree of white turbidity. The sugar level can be checked at the same time. Thereby, the urine protein value and urine sugar value can be inspected without using consumables such as test paper. In particular, according to this example, since a linear calibration curve is obtained, the conversion from R to albumin concentration becomes easier.

Embodiment 8 In this embodiment, after measuring the optical rotation of urine, the temperature of urine and the amount of scattering of urine are measured while heating the urine. FIG. 14 shows a measuring apparatus according to the present embodiment.
The polarizer 93 transmits only a specific directional component of the substantially parallel light projected from the projection module 81 similar to that used in the first embodiment. The sample cell 82 is the same as that used in the first embodiment, and the urine injected therein modulates the deflection direction of the transmitted light in a minute width. The polarizer 93 and the analyzer 94 are arranged in a state of Nicols orthogonal to the optical axis direction of the projection module 81 with the sample cell 82 interposed therebetween. A coil-shaped heater 88 is provided around the sample cell 82.
Is arranged. The switch 95 is connected to the terminal a when measuring the optical rotation, and supplies a signal from the signal generator 85 to the coil-shaped heater driver 89 as a magnetic field modulation signal. Also, when measuring the scattered light, it is connected to the terminal b and supplies the same signal to the projection module 81 as a modulation signal of the projection light.
The optical sensor 96 detects light scattered when propagating through the liquid sample in the sample cell 82 after being projected from the projection module 81. The switch 97 switches so that the output signal of the optical sensor 83 is supplied to the lock-in amplifier 84 when the optical rotation is measured, and the output signal of the optical sensor 96 is supplied to the lock-in amplifier 84 when the scattered light is measured. The thermocouple 80 is installed in close contact with the sample cell 82,
The temperature of the sample in the sample cell 82 is substantially detected.
The temperature indicator 86 displays the temperature of the sample detected by the thermocouple 80 and sends this value to the computer 87.
Thus, in the urine test, after measuring the optical rotation of the urine in the same manner as in the sixth embodiment, it is possible to measure the temperature and the scattered light intensity of the urine while heating the urine.

Next, using a urine test paper, the glucose concentration was 50 mg / dl or less and the albumin concentration was 100 mg / dl.
Urine judged to be not less than / dl and not more than 250 mg / dl was examined using this apparatus. First, the optical rotation was measured, and A = −19.8 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the scattered light intensity was measured to obtain r = 4.6. From this r and the calibration curve obtained, the albumin concentration was determined to be 120 mg / dl. By solving the equation (2) from the albumin concentration and A, a glucose concentration = 30 mg / dl was obtained. This is consistent with the test strip results.

Urine having a glucose concentration of 100 mg / dl or more and 250 mg / dl or less and an albumin concentration of 300 mg / dl or more and 500 mg / dl or less was similarly examined using a urine test paper. First, the optical rotation was measured, and A = −56 × 10 ⁻³ [degree] was obtained. Next, the urine was heated and the scattered light intensity was measured to obtain r = 13.7. From this r and the calibration curve obtained, the albumin concentration was determined to be 400 mg / dl. By solving equation (2) from this albumin concentration and A, a glucose concentration = 150 mg / dl was obtained. This is consistent with the test strip results.

As described above, according to this embodiment, after measuring the optical rotation of urine, the urine is heated to make it turbid, and the degree of turbidity is measured. Can be inspected. Thereby, the urine protein value and urine sugar value can be inspected without using consumables such as test paper. In particular, according to the present embodiment, there is no need to use a plurality of optical sensors as in the measuring device of the seventh embodiment. As described above, the urine protein value can be obtained by making the urine cloudy and obtaining the degree of cloudiness from the intensity of transmitted light or scattered light. In addition, by measuring the optical rotation of urine before it becomes cloudy, the urinary sugar value can be determined together with the urine protein value.

[0051]

According to the present invention, it is possible to provide a urine test method without using consumables such as test paper.

[Brief description of the drawings]

FIG. 1 is a schematic diagram showing a configuration of a measuring device used in one embodiment of the present invention.

FIG. 2 is a characteristic diagram showing a relationship between an output signal of a lock-in amplifier and an albumin concentration of a sample obtained in the same example.

FIG. 3 is a characteristic diagram showing a relationship between an output signal of a lock-in amplifier and an albumin concentration of a sample obtained in another embodiment of the present invention.

FIG. 4 is a schematic diagram showing a configuration of a measuring device used in still another embodiment of the present invention.

FIG. 5 is a characteristic diagram showing a relationship between an output signal of a lock-in amplifier and an albumin concentration of a sample obtained in the same example.

FIG. 6 is a schematic diagram showing a configuration of a measuring device used in still another embodiment of the present invention.

FIG. 7 is a characteristic diagram showing a relationship between an output signal of a lock-in amplifier and a temperature of a sample obtained in the same embodiment.

FIG. 8 is a characteristic diagram showing a relationship between R (ratio of transmitted light intensity at 75 ° C. and transmitted light intensity at 70 ° C.) obtained in the example and albumin concentration of a sample.

FIG. 9 is a schematic diagram showing a configuration of a measuring device used in still another embodiment of the present invention.

FIG. 10 is a characteristic diagram showing a relationship between r (ratio of scattered light intensity at 75 ° C. and scattered light intensity at 70 ° C.) obtained in the example and albumin concentration of the sample.

FIG. 11 is a schematic diagram showing a configuration of a measuring apparatus used in still another embodiment of the present invention.

FIG. 12 is a characteristic diagram showing a relationship between R obtained in the same example and albumin concentration of a sample.

FIG. 13 is a schematic diagram showing a configuration of a measuring device used in still another embodiment of the present invention.

FIG. 14 is a schematic diagram showing a configuration of a measuring device used in still another embodiment of the present invention.

[Explanation of symbols]

1, 11, 21, 31, 41, 61, 81 Projection module 2, 12, 22, 32, 42, 62, 82 Sample cell 3, 13, 23, 33, 43, 63, 83 Optical sensor 4, 14, 24 , 34, 44, 64, 84 Lock-in amplifiers 5, 15, 25, 35, 45, 65, 85 Signal generators 26, 36, 46, 66, 86 Temperature indicators 27, 37, 47, 67, 87 Computers 28 , 38, 48, 68, 88 Heaters 29, 49, 69, 89 Heater drivers 30, 40, 60, 80 Thermocouples 53, 73, 93 Polarizers 54, 74, 94 Analyzers 55, 75, 95 Switches 76, 96 Optical sensor 77 Switch 78 Beam sampler

──────────────────────────────────────────────────続 き Continuation of the front page (56) References JP-A-3-170396 (JP, A) JP-A-4-214767 (JP, A) JP-A-8-113593 (JP, A) JP-A 9-1997 138231 (JP, A) (58) Field surveyed (Int. Cl. ⁷ , DB name) G01N 33/48-33/98

Claims (12)

(57) [Claims] 1. A step of adding a divalent metal ion to a liquid sample containing a protein, a step of heating the liquid sample to make it opaque, a step of projecting light onto the opaque liquid sample, and A method of detecting the light transmitted through the liquid sample or the light scattered from the liquid sample among the light, and a method of measuring the concentration of the specific component including the step of determining the protein concentration of the liquid sample based on the intensity of the detected light . 2. A step of adding divalent metal ions to a liquid sample containing a protein, a step of heating the liquid sample and projecting light on the liquid sample, and a step of heating the liquid sample. A method for measuring the concentration of a specific component, comprising: detecting light transmitted through a sample or light scattered from the liquid sample; and determining the protein concentration of the liquid sample based on the intensity of the detected light. 3. The method according to claim 1, further comprising the steps of: measuring the optical rotation of the liquid sample before heating; and determining the glucose concentration of the liquid sample based on the optical rotation and the protein concentration. Method for measuring the concentration of a specific component. 4. Light is projected onto the liquid sample at two different temperatures of 60 to 80 ° C., respectively, and the protein concentration of the liquid sample is determined from the ratio of the intensity of the transmitted light or scattered light obtained. The method for measuring the concentration of a specific component according to claim 2. 5. The method for measuring the concentration of a specific component according to claim 1, wherein the divalent metal ion is a calcium ion or a magnesium ion. 6. The liquid sample 1 according to claim 1, wherein
6. The method for measuring the concentration of a specific component according to claim 5, wherein the component is added at a ratio of 0.2 mmol or more to dl. 7. The method for measuring the concentration of a specific component according to claim 5, wherein said magnesium ion is added at a ratio of 0.1 mmol or more to 1 dl of said liquid sample. 8. The method according to claim 5, wherein said calcium ion or magnesium ion is added to said urine as chloride. 9. A step of adding an acid to a liquid sample containing a protein to adjust the pH of the liquid sample to 5.5 or less;
Heating the liquid sample to make it cloudy; projecting light on the cloudy liquid sample; and detecting light transmitted through the liquid sample or light scattered from the liquid sample among the projected light. And a step of determining the protein concentration of the liquid sample based on the detected light intensity. 10. The method according to claim 9, wherein the acid is acetic acid or phosphoric acid. 11. The liquid sample according to claim 1, wherein said liquid sample is urine.
Or the method for measuring the concentration of a specific component according to 9. 12. The method according to claim 11, wherein the wavelength of the light is 500 nm or more.