JPH0979982A - Method for measuring constituent in humor optically and simultaneously - Google Patents

Abstract

PROBLEM TO BE SOLVED: To simultaneously perform the qualitative and quantitative analysis of multiple constituents by applying Raman excitation light to a humor sample and measuring and analyzing the Raman spectrum intensity at each measurement wavenumber of each constituent of measurement. SOLUTION: A flux of light 24s for sample from a light source part 1 is reflected by a half mirror 12 arranged at a measurement target optical adjustment part 3 and is applied to a sample 11 of a sample part 2. Scattered light from the sample 11 is cleared of the wavelength constituent which is the same as that of excitation light via the adjustment part 3 and enters a spectroscope 21 from an entrance slit 25 via a half mirror 16. On the other hand, with a flux of light 24r for correction which is divided by a half mirror 9 at the excitation light source part 1, the quantity of light is adjusted via a correction optical adjustment part 4 and the flux of light 24r enters the spectroscope 21 from the slit 25 via the mirror 16. The fluctuation of spectrum light intensity caused by the fluctuation of the excitation light intensity is corrected by the flux of light 24r, thus detecting the Raman spectrum intensity of each constituent.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to components in body fluids (urine, blood, serum, plasma, saliva, sweat, etc.) such as albumin, globulin, hemoglobin, glucose and acetoline by utilizing Raman light in the field of clinical examination. The present invention relates to a method for qualitatively and quantitatively measuring lithium acetate, β-hydroxybutyric acid, acetone, ditaurobilirubin, urobilin, sodium nitrite, urea and the like simultaneously in a short time.

[0002]

2. Description of the Related Art As methods for measuring components in body fluids in clinical tests, there are a reagent method, a test strip method, a chemiluminescence method, an immunoassay method, an enzyme method, a chromatography method and the like.

The test paper used in the test paper method is often one in which a reaction part of cellulose containing a reaction reagent is fixed to a plastic support with an adhesive or the like and dried. If the reaction part contains moisture, a reaction will occur between the reagents, and it will also be transformed by high temperature and light, and the sensitivity will often decrease.Therefore, the container for the test paper should be sealed and stored at high temperature. , Must be used within the expiration date. For example, urine test strips include pH, protein, glucose, ketone bodies, bilirubin, occult blood, urobilinogen, bacterial infection,
It is possible to simultaneously measure multiple items such as specific gravity within 1 minute, but the reaction of the reagent part is affected by the reaction temperature and humidity as well as the endogenous promoters and inhibitors, and Can only quantify. The judgment method by the test paper method is mainly a colorimetric method, and the reaction mechanism is mainly an enzymatic reaction or a chemical reaction such as a redox reaction. The reagent method is mainly a colorimetric method via an indicator, an enzyme / chemical reaction, or a turbidimetric method used for protein measurement.

The enzyme method has a high specificity to the substance to be measured and is simple, but has a risk of masking the positive reaction due to the color tone of the body fluid itself, and furthermore, it is endogenous when the redox reaction is involved. The reaction may be suppressed by various exogenous oxidation and reduction reaction substances, and there is a risk of false negatives and false positives.

However, since each of these methods is an indirect measurement method in which the measurement is carried out via a mediated reaction, there is a potential error.
Generally, when a colorimetric method is used for determination, an error is likely to occur in the determination of the result, and the one that is mediated by chemical reaction has low specificity.
In addition, since many substances interfering with these tests are present in the body fluid, the reaction is inhibited, a false positive reaction is caused, a color tone different from that of the positive reaction is exhibited, and the positive reaction is masked. The system that originally uses an enzyme is unstable, and the system mediated by a chemical reaction is easily affected by coexisting substances. Also,
When the clinical significance of the test items are correlated (bilirubin and urobilinogen, protein and occult blood, glucose and ketone bodies), it is useful to conduct multi-item tests at the same time and make a comprehensive judgment. It is cumbersome to perform each of the steps individually, and moreover, it is easily affected by an interfering substance. Multi-item test papers are often avoided because they are expensive and difficult to judge.

The reagent method, test strip method and enzyme method require reagents, test strips or enzymes which are consumables, and there is a problem of storage stability of reagents before use and disposal after use. Further, there is a possibility that an error such as an added amount of a reagent or a sample may occur due to a mistake during the operation, the operation is complicated, and there is a disadvantage that it is interfered by other components such as ascorbic acid which are not intended for measurement. Although the reagent method and the enzyme method can measure quantitatively with a single component, it is not possible to measure multiple components at the same time, and the test strip method can simultaneously measure multiple components, but only semi-quantitative. There are drawbacks.

The chemiluminescence method requires a luminescent substance,
An immunoassay method via an antigen-antibody reaction generally requires washing, requires a lot of man-hours, and has a risk of influence of interfering substances and nonspecific adsorption.

The chromatographic method requires expensive equipment, and the measurement is generally time-consuming. Further, the column, which is a consumable item, is expensive and there is a problem that the cost becomes high.

[0009]

The present invention eliminates the need for consumable reagents, test strips, enzymes and the like, and further eliminates the problems of storage safety before use and disposal after use of such consumables. It is an object of the present invention to provide a method for quantitatively measuring multiple components at the same time, while eliminating problems such as complicated operations that cause the occurrence and interference effects of other components.

[0010]

Means for Solving the Problems The present invention obtains a unique Raman spectrum from each component in a body fluid in a substance to be measured, and utilizes the spectrum to obtain a spectrum intensity or spectrum integrated intensity or a plurality of wave numbers in an arbitrary wave number range. Each component is qualitatively and quantitatively measured from the Raman spectrum intensity at the wave number. In the first aspect of the present invention, for each component in the body fluid to be measured, a wave number having a good correlation between the concentration of the component and the Raman spectrum intensity is selected in advance as a measurement wave number specific to the component, and a body fluid sample is prepared. On the other hand, the Raman excitation light is irradiated to measure the Raman spectrum intensity at each measurement wave number for each component to be measured, and the calibration prepared in advance for the Raman spectrum intensity and the concentration of that component at each measurement wave number for each component Qualitative / quantitative analysis of each component in the body fluid at the same time using lines. The Raman spectrum intensity at the measured wave number means the peak height of the peak of the Raman spectrum. Instead of the peak height, the peak area of the Raman spectrum peak in the measurement wave number range including the measurement wave number may be measured for qualitative and quantitative determination.
The Raman spectrum integrated intensity in the measurement wave number range means the peak area of the Raman spectrum peak.

In the second aspect of the present invention, a sample of body fluid is irradiated with Raman excitation light, and the wave number at which the correlation between the concentration of each of the plurality of components in the body fluid to be measured and the Raman spectrum intensity is good is determined for each component. The Raman spectrum intensity at those measurement wavenumbers is measured as a unique measurement wavenumber, or the Raman spectrum intensity at a plurality of wavenumbers in an arbitrary wavenumber range is measured, and multiple components in a body fluid sample are simultaneously qualitatively determined by multivariate regression analysis. Quantitative analysis.

The term "Raman" used in the present invention means not only Raman scattering in a narrow sense but also fluorescence generated at the same time when fluorescence is generated, so that it includes Raman scattering and fluorescence. Used in.

Regarding the components in each body fluid to be measured,
The correlation coefficient R ² between the concentration of the single-component aqueous solution and this specific Raman spectrum intensity is 0.8 or more, preferably 0.0.
A wave number of 9 or more is selected as the measured wave number specific to the component. The correlation coefficient R ² is given by the following equation.

[Equation 2] xi: concentration of each point of component in body fluid yi: Raman spectrum intensity with respect to xi X: average value of concentration of each point of component in body fluid Y: average value of Raman spectrum intensity

The preferred measurement wave number or measurement wave number range of each component is about 590 to 650 cm ⁻¹ for albumin and 800
~ 860 cm- ^1, near 2342 ~ 2402 cm- ¹ (see Figure 6), for globulin 158 ~ 218 cm- ^1, near 40
2 ~ 462 cm- ¹ vicinity, 623 ~ 683 cm- ¹ vicinity, 807 ~ 867 cm
^-1 area, 822-862 cm- ¹ area, 1353-1413 cm- ¹ area,
1562 ~ 1622 cm- ¹ vicinity, 1847 ~ 1907 cm- ¹ vicinity, 2095 ~
2155 cm- ¹ vicinity, 2340-2400 cm- ¹ vicinity, 2403-2463c
Select from around m- ¹ (see Fig. 36). For hemoglobin, around 807-867 cm- ¹ , 893-953 cm- ¹ , 13
53-1413 cm- ¹ vicinity, 1562-1622 cm- ¹ vicinity, 1840-19
Near 00cm- ¹ , 2095-2155cm- ^1, Near 2340-2400cm
^-1 near, 2403-2463cm- ¹ near (see Figure 37), for glucose, 200-554cm- ¹ near, 810
~ 944 cm- ¹ and 2590 ~ 2940 cm- ¹ (see Fig. 8)
799-859 for lithium acetoacetate
cm- ¹ vicinity, 1353 ~ 1413cm- ¹ vicinity, 1840 ~ 1900cm- ¹
Near, 2347 to 2407 cm- ¹ Near, 2907 to 2967 cm- ¹ (Fig. 3
4 reference) chosen from the vicinity of, for β- hydroxybutyrate, 1420～1480Cm- around ^1, 1557～1617Cm- around ^1, 16
Select from around 00 to 1660 cm- ^{1 and} around 2870 to 2971 cm- ¹ (see Fig. 35). For acetone, 775 to 845 cm- ¹
Neighborhood, 1050-1110cm- ¹ neighborhood, 1207-1267cm- ¹ neighborhood,
1399 ~ 1459 cm- ¹ vicinity, 1680 ~ 1740 cm- ¹ vicinity, 2911 ~
Select from around 2971 cm- ¹ (see Fig. 14), and for ditaurobilirubin, near 927-987 cm- ¹ , 1233-1293
cm- near ^1, selected from around 1586～1616Cm- ¹ (see FIG. 38), for the urobilin, select an arbitrary position from 332～2900Cm- ^1, for the sodium nitrite, 78
3~843cm- near ^1, selected from around 1318～1378Cm- ¹ (see FIG. 39), with respect to urea, 501～561Cm- around ^1, 567～627Cm- around ^1, 978～1048Cm- near ^1, 1033
~ 1093 cm- ¹ near, 1138 ~ 1198 cm- ¹ near, 1583 ~ 1643
cm- ¹ (see Fig. 10), uric acid from 640 to 700 cm- ¹ (see Fig. 40), and folic acid from 631 to 691 cm- ¹ and 816 to 876 cm. - around ^{1, 901~961cm-} near ^{1, 1355~1415cm-} near ^1, 1562
~ 1622 cm- ¹ near, 1847 ~ 1907 cm- ¹ near, 2093 ~ 2153
cm- ¹ vicinity, 2339-2399cm- ¹ vicinity, 2400-2460cm- ¹
Select from the vicinity (see FIG. 41), for ascorbic acid, 800 to 860 cm ⁻¹ , 1673 to 1733 cm ⁻¹ , 29
19～2979Cm- ¹ selected from around (see Figure 42), with respect to vitamin B _2, 404~464cm- around ^1, 626～686Cm-
¹ area, 798-858 cm- ¹ area, 860-920 cm- ¹ area, 131
7 ~ 1377 cm- ¹ vicinity, 1546 ~ 1606 cm- ¹ vicinity, 1834 ~ 189
4 cm- ¹ vicinity, 2106-2166 cm- ¹ vicinity, 2360-2420 cm-
¹ near, 2419 ~ 2479 cm ^-1 (see Figure 43), for creatine monohydrate, select from 800 ~ 860 cm ^-1, near 1563 ~ 1623 cm ^-1 (see Figure 44),
For creatinine, near 538-598 cm- ¹ , 580-6
40 cm- ¹ vicinity, 665-725 cm- ¹ vicinity, 817-877 cm- ¹ vicinity, 868-928 cm- ¹ vicinity, 1021-1081 cm- ¹ vicinity, 1550
~ 1610cm- ¹ vicinity, 2868-2900cm- ¹ vicinity, 2900-2950
cm- near ^1, selected from around 2950～2996Cm- ¹ (see FIG. 45), for the α- amylase, 393～453Cm- around ^1, 631～691Cm- around ^1, 792～852Cm- around ^1, 868 ~ 9
28 cm- ¹ vicinity, 1333-1393 cm- ¹ vicinity, 1546-1606 cm
- around ^1, 1834～1894Cm- ¹ around, selected from around 2334～2394Cm- ¹ (see FIG. 46), for the β- amylase, 376～436Cm- around ^1, 622～682Cm- around ^1, 783～ 8
43 cm- ¹ vicinity, 868-928 cm- ¹ vicinity, 1334-1394 cm- ¹
Neighborhood, near 1546 to 1606 cm- ^1, near 1834 to 1894 cm- ¹ ,
Select from around 2665 to 2725 cm- ¹ (see Fig. 47), and for ammonia, around 3190 to 3250 cm- ¹ and 3292 to 3350
cm- near ^1, selected from around 3377～3437Cm- ¹ (see FIG. 48), for the inositol, 404～464Cm- around ^1, 805～865Cm- around ^1, 1014～1074Cm- near ^1, 1438
~ 1498cm- ¹ near, 2966 ~ 3026cm- ¹ near (see Figure 49), for galactose, 466 ~ 526c
m- ¹ neighborhood, 835-895 cm- ¹ neighborhood, 1032-1092 cm- ¹ neighborhood, 1237-1297 cm- ¹ neighborhood, 1332-1392 cm- ¹ neighborhood, 14
Select from 38 to 1498 cm- ¹ and 2946 to 3006 cm- ¹ (see Fig. 50). For fructose, 569 to 629c
m- ¹ neighborhood, 772-832 cm- ¹ neighborhood, 1044-1106 cm- ¹ neighborhood, 1237-1297 cm- ¹ neighborhood, 1438-1498 cm- ¹ neighborhood, 29
Select from around 66 to 2996 cm- ¹ (see Fig. 51).

When the body fluid sample contains two or more of the above components, the Raman spectrum obtained by irradiating the body fluid sample with excitation light of a single wavelength and receiving the scattered light from the body fluid sample. Of these, it is desirable to calculate the concentration of each component based on the spectral intensities in the wave number regions set as described above for each component and in the wave number regions that do not overlap each other.

In the multivariate regression analysis calculation, data analysis is performed using a multivariate regression analysis method such as a principal component regression analysis method (PCR method) or a partial least squares method (PLS method). In the multivariate regression analysis method, regression analysis can be performed using many spectral intensities at a time, so that quantitative analysis with higher precision than in simple regression analysis is possible. The multiple regression analysis is most frequently used, but requires a large number of samples, and when the correlation between the spectral intensities at each wave number is high, the accuracy of the quantitative analysis is low. On the other hand, the PCR method, which is a multivariate regression analysis method, can aggregate spectral intensities in a plurality of wavenumber regions into principal components independent of each other, and can eliminate unnecessary noise data. Accuracy is obtained. In addition, since the PLS method can also use the data of the sample concentration when extracting the main component, high quantitative analysis accuracy can be obtained as in the case of the PCR method. For multivariate regression analysis, "Multivariate Analysis" (Kazuo Nakatani, Shinyosha) can be referred to.

Computer data processing is very useful for extracting necessary information from a spectrum that is complicatedly changed by various fluctuation factors. A typical processing method is also stored in processing software installed in a commercially available near infrared device or the like. As commercial software, C
There is an unscrambler from OMO. Typical processing methods are the multiple regression analysis, the PLS method, the principal component regression analysis, etc. mentioned above.

A major flow of data processing applied to quantitative analysis is creation of a calibration model, evaluation of the calibration model, and quantification of an unknown sample. In order to perform the calibration, it is necessary to measure an appropriate number of samples for preparing a calibration curve with sufficient accuracy. If necessary, the obtained spectrum is pretreated. Typical pre-processing includes spectrum smoothing, differentiation, and normalization, all of which are general processing.

Next, the calibration is a process of constructing a mathematical relational expression between the spectral data and the analysis value of the target characteristic, that is, a model. The model is created by a statistical method using the analysis values and the spectrum data of the sample for preparing the calibration curve.

In order to accurately evaluate the accuracy of the prepared calibration curve for an unknown sample, the evaluation sample requires a measurement error for the unknown sample. When it is determined that the accuracy of the calibration curve is insufficient, the type of processing method and parameters are changed as necessary to correct the calibration curve. The calibration curve, which has been confirmed to have sufficient accuracy, is used as a relational expression for predicting the value of the target characteristic from the spectral data when analyzing an unknown sample, and is used for quantifying the concentration of the unknown sample.

FIG. 54 shows a general procedure of multivariate regression analysis as a flow chart. The Raman spectrum of the sample for making the calibration curve (analysis value is known) is measured, and if necessary, pretreatment such as smoothing and normalization is performed, and then the obtained Raman spectrum data (Raman spectrum intensity at each wave number) is used. , Calibrate using multivariate regression analysis to create a calibration model.

Next, a Raman spectrum of an evaluation sample (analyzed value is known) for evaluating this calibration model is measured, and if necessary, pretreatment such as smoothing or normalization is performed, and then the obtained sample is obtained. Substituted Raman spectrum data into the calibration model, the measured value of the evaluation sample is compared with the calculated value from the calibration model, and the accuracy of the calibration model is evaluated. If the accuracy is insufficient, return to the stage of spectrum measurement and pretreatment of the sample for calibration curve preparation, preparation of the calibration model, etc., change the processing as necessary, modify the calibration model and evaluate. The evaluation is repeated according to the Raman spectrum data of the sample for use. If it is determined that sufficient accuracy has been obtained, Raman spectrum data obtained by Raman spectrum measurement of an unknown sample (analysis value unknown) is substituted into this calibration model, and the concentration is calculated.

The present invention is used as a method for a clinical test using a body fluid such as urine or blood as a sample. Albumin and globulin are nephrotic syndrome and nephritis, hemoglobin is inflammation and tumor of urinary system, glucose is diabetes, lithium acetoacetate and β-hydroxybutyric acid and acetone are ketoacidoses, bilirubin is liver and biliary disease, and urobilinogen is liver and biliary tract. Diseases, hemolytic diseases, and sodium nitrite are indicators of bacterial infections of the urinary system. Urine in the urine is useful for knowing protein metabolism in the body, liver / renal function, etc., and if it increases, it may be due to increased body protein catabolism or taking drugs such as quinine. Be done. Furthermore, it has been found that the amount of urea in urine serves as an index of specific gravity of urine ("New Edition Laboratory Handbook 3").
Nozomu Kozai, Yakuji Nippo), and in general, osmotic pressure, refractive index, and urine specific gravity have a good correlation, and the determination of the three is substituted by specific gravity ("Clinical Examination Method Recommendation" Masamitsu Kanai, Kanehara Publishing Co., Ltd.).
In addition, uric acid is urinary tract stones and hyperuricemia, folic acid is malabsorption syndrome and malignant tumor associated with gastric and intestinal incision, creatine is steroid myopathy, creatinine is muscle disease and renal disease, acute pancreatitis and pancreatic duct obstruction, and ammonia is It is an index of metabolic disorders and severe liver diseases.

Since bilirubin is insoluble in water,
Zitaurobilirubin was used in the experiment. Ditaurobilirubin is a taurine-conjugated bilirubin, which has a common basic skeleton and two carbonyl groups dehydrated and bound to taurine. You-Zing Hsieh et al. (Langmuir 198
7,3,1141-1146) compared with the SERS (surface-enhanced Raman spectroscopy) spectrum of bilirubin, the Raman spectrum obtained this time has almost the same spectrum except for the peak near 690.065 cm ⁻¹ . It can be said that the other peaks are derived from bilirubin. Also, the peak near 957.5 cm- ¹ which seems to be CH ₂ lateral vibration is considered to be -CH ₂ -longitudinal vibration, but since CH _{2 of} taurine part is also included in the spectrum, the relative intensity of bilirubin is It can be expected to be slightly lower.

With respect to urobilin, urobilinogen is unstable and is easily converted into urobilin by air oxidation. Therefore, urobilin was used for the measurement. Urobilin is one in which one of -NH- of urobilinogen is oxidized to become -N =. The peak position of this --NH-- is 3530 to 3480.
cm- ¹ and OH stretching vibration of water (3650-3400)
Since it overlaps with the position of cm- ¹ ) and is hidden, the spectra of both can be expected to be almost the same. 2372.8 of albumin
The peak near cm- ¹ is considered to be due to NH + stretching vibration.

The peak near 188.232 cm- ¹ and the peak near 432.919 cm- ^{1 of} globulin are vibrations from CCC, 653.
The peak around 937 cm- ¹ is CH ₂ , vibration from CH, 837 cm- ¹
The peak in the vicinity is vibration from CH ₂ , the peak in the vicinity of 852 cm ⁻¹ is vibration from CH ₂ CH, the peak in the vicinity of 1383.49 cm ⁻¹ is vibration of amide III, the peak near 1594.41 cm ⁻¹ is vibration of amide II, The peak near 1877.64cm- ¹ is vibration from CH ₂ , 21
The peak near 25.39 cm- ¹ is vibration from CC, 2370 cm- ¹
It is considered that the peak in the vicinity is due to vibration from NH + and the peak near 2433 cm- ¹ is due to vibration from SH.

The peak near 837 cm- ^{1 of} hemoglobin is C
Vibration from H _2, 923.373cm- ¹ vibration from the vicinity of the peak CH3, 1383.49cm- ¹ near the peak vibration of amide Bu III, 1592.41cm- ¹ peak around the vibration of amide II, 187
0.82Cm- ¹ near peak vibration from CH _2, 2125.39cm
- ¹ peak around the vibration from CC, 2370cm- ¹ near the peak vibration from NH +, peak near 2433Cm- ¹ is believed to be due to vibration from SH. In addition, the spectrum of oxyhemoglobin by resonance Raman spectroscopy is described in "Raman spectroscopy" (written by PRCAREY), which was referred to.

The peak near 405 cm ^{-1 of} D-glucose is C
Vibration from CO, peak near 524 cm- ¹ is basic skeletal vibration, peak near 648 cm- ¹ is vibration from CC, 779 cm- ¹
The near peak and the peak near 840 cm- ¹ are vibrations from CH, the peak near 914 cm- ¹ is the vibration from CH and COH, 1054
cm- ¹ near peak vibration from CH, 1076Cm- ¹ near the peak vibration from CH and COH, the peak of the peak and 1346cm- around ¹ near 1276Cm- ¹ vibration from COH, 1426c
^m-1 near the peak vibration from CH _2, 1640cm- ¹ peak around the vibration from HOH, 2900cm- ¹ near the peak CH
It is thought that this is due to vibrations from CH ₂ and CH ₂ .

[0029] 829.379Cm- ¹ near the peak of acetoacetate lithium, 1383.49cm- ¹ peak around and 1870.82cm
- ¹ peak around the vibration from CH _2, peak around 2937Cm- ¹ is believed to be due to vibration from CH3 and CH _2.

The peak of β-hydroxybutyric acid near 1450.98 cm ^-1 is vibration from CH ₂ and CO ₂ −, 1587.69 cm.
- ¹ near arsenide ° over click is CO ₂ - vibrations from, 1630Cm- ¹ near the peak vibration from CO, vibration from the peak around 2900cm- ¹ CH _2, vibration from 2959Cm- ¹ peak around the CH ₃ It is thought to be due to.

Peak of 805 cm- ^{1 of} acetone, 1080
cm- ¹ near the peak, 1429cm- ¹ peak in the vicinity of and 2940
cm- ¹ near peak vibration from CH _3, 1237cm- ¹ peak around the vibration from CH ₃ C, 1710cm- ¹ peak near the
Probably due to vibration from CO.

The peak near 690.065Cm- ¹ of ditaurobilirubin vibration from CC, the peak of the peak and 1458.88cm- around ¹ near 957.5Cm- ¹ vibration from CH _2, 1,263.9
The peak around 6 cm- ¹ is the vibration from CH ₂ and the vibration from CO,
The peak around 1616.29 cm- ¹ is thought to be due to the vibrations from CC and CO.

Urobilin itself has a strong fluorescence,
Raman peak was not obtained. 81 of sodium nitrite
3Cm- ¹ near the peak and 1339.5Cm- ¹ peak near the N
Probably due to vibration from O.

The peak of urea near 531.939 cm- ¹ , 1008
cm- ¹ peak and 1168.35Cm- ¹ near peak near the C
Vibration from N, vibration from 597.248Cm- ¹ peak near the CO, peak near 1613.67Cm- ¹ is considered to vibration from CO and NH _2.

When measuring urine as a sample, the concentration of other urinary components is standardized based on the concentration of creatinine whose excretion amount per hour is constant, so that the concentration of urinary components changes with the amount of urine. Can be corrected.

Hereinafter, the present invention will be described in more detail by way of examples, but the present invention is not limited thereto.

EXAMPLES An example of a measuring apparatus for carrying out the measuring method of the present invention is shown in FIGS. 1 and 2. FIG. 1 is a block diagram of the device configuration, which comprises a light source unit 1, a sample unit 2, a measurement target optical adjustment unit 3, a correction optical adjustment unit 4, a spectral detection unit 5, and a data processing unit 6. FIG. 2 shows a specific example showing the block diagram of FIG. 1 in detail. The light source unit 1 transmits an excitation light source 7 that generates a single wavelength light and a bandpass filter 27 that transmits only the excitation wavelength from the excitation light source 7 and reflects the other light.
And a half mirror 9 for splitting the light beam from the excitation light source 7 into a sample light beam 24s and a correction light beam 24r, and a light source for converging the sample light beam 24s on the sample 11 of the sample portion 2 with the half mirror 9 interposed therebetween. Condensing lens 8 and condensing lens 10
It has.

As the light source 7, for example, a laser device is used. As a laser device, a continuous oscillation Ar ion laser, Kr ion laser, He-Ne laser, He-
A Cd laser, an Nd: YAG laser, a pulse laser, or the like can be used, and a laser having a wide wavelength range from the near ultraviolet region to the near infrared region can be used. As a light source other than the laser device, a light source that generates multi-wavelength light such as a halogen lamp can be used in combination with the spectroscope.

A body fluid sample 11 is placed in a cell 26 in the sample section 2 and installed therein.
s is irradiated.

The measurement target optical adjusting unit 3 removes the same wavelength component as the excitation light from the scattered light generated by irradiating the sample 11 stored in the sample cell 26 with the sample light beam 24 s, and the fluorescence and Raman scattered light are obtained. It is provided with a filter means 14 for taking out the light to be measured including the above, and optical systems 13 and 15 for adjusting the beam so that the scattered light is converged on the entrance slit 25 of the spectroscope. At the exit position of the measurement target optical adjustment unit 3, the sample light beam 24s from the measurement target optical adjustment unit 3 and the correction light beam 24r emitted from the correction optical adjustment unit 4 serve as a combining unit that places them on the same optical path. A half mirror 16 is provided.

The filter means 14 in the optical adjustment section 3 to be measured may be a holographic notch filter containing the excitation light wavelength in the notch region, or a cut filter containing the excitation light wavelength and blocking the shorter wavelength side. desirable. The holographic notch filter blocks only a desired wavelength region and transmits light in other wavelength regions. By using the shielded region (notch region) containing the excitation light wavelength, the sample light beam 24s emitted from the measurement target optical adjustment unit 3 contains only the measurement target light component.

The holographic notch filter can be obtained from, for example, KAISER OPTICAL SYSTEMS. INC. (USA). The holographic notch filter 14 has a characteristic of completely blocking, for example, wavelength light included in the notch region, and transmitting 80% or more of light in a wavelength region other than the notch region.

The correction optical adjustment unit 4 includes an attenuation filter 17 that attenuates the light amount of the correction light beam 24r split by the half mirror 9 in the excitation light source unit 1, and a mirror 18 that bends the optical path. The correction light beam 24r is for correcting the fluctuation of the spectral light intensity due to the fluctuation of the excitation light intensity from the light source, and when such correction is not required,
The half mirror 9 in the light source unit 1, the correction optical adjustment unit 4, and the half mirror 16 serving as a combining unit are not necessary. Since the correction light beam 24r includes only the excitation light from the light source 7 and does not pass through the sample, it does not depend on the sample and faithfully represents the intensity fluctuation from the light source.

The spectroscopic detection unit 5 takes in the sample light beam 24s from the measurement target optical adjustment unit 3 and the correction light beam 24r emitted from the correction optical adjustment unit 4 from the half mirror 16 through the entrance slit 25 and spectroscopes the light beam. 21 and a detector 2 for detecting the spectral light dispersed by the spectroscope 21
It has 0 and.

The spectroscopic detector 5 is preferably a polychromator which includes a multi-channel photodetector as the detector 20 and simultaneously detects the wavelength region to be measured.
When the spectral detection unit 5 is a polychromator, the wavelength region to be measured can be detected at the same time, and the measurement target light spectrum and the excitation light in the predetermined region can be simultaneously detected. As a result, there is no difference between the detection time of each wavelength of the measurement target light and the detection time of the excitation light. However, when a difference may occur between the detection time of each wavelength of the light to be measured and the detection time of the excitation light, the spectral detection unit 5 includes a wavelength scanning type spectroscope as the spectroscope 21 and the detector 20 as the spectroscope 21. It may have a single-channel photodetector and sequentially detect a wavelength region to be measured.

The data processing section 6 is provided with a processing calculation control section 22 and an output device 23, and the detector 2 of the spectroscopic detection section 5 is included.
It has a function of correcting the intensity of the light to be measured based on the detected intensity of the excitation light component in the spectrum detected by 0.

The processing calculation control section 22 controls the operation of each section and performs processing such as spectrum analysis and multivariate analysis of the signal detected by the spectroscopic detection section 5 to obtain the spectral spectrum detected by the spectroscopic detection section 5. Data processing is also performed to correct the detection intensity of the measurement target light based on the detection intensity of the excitation light component, and the Raman scattering spectrum in which the fluctuation of the light source is corrected is calculated, and the qualitative and quantitative determination of the sample is also performed from the measurement target light intensity. To do. The output device 23 is a processing operation control unit 22
Printers and displays that output the data processed in.

The operation of this embodiment will be described below.
The sample light flux 24 s is reflected by the half mirror 12 arranged in the measurement target optical adjustment unit 3 and is applied to the sample 11 of the sample unit 2. The scattered light from the sample 11 has the same wavelength component as the excitation light removed through the measurement target optical adjustment unit 3, and passes through the half mirror 16 and the entrance slit 25 to the spectroscope 21.
Incident on. On the other hand, the correction light beam 24 r split by the half mirror 9 in the excitation light source unit 1 is adjusted in the amount of light through the correction optical adjustment unit 4, and enters the spectroscope 21 through the entrance slit 25 through the half mirror 16. Correction light beam 24r
By this, the fluctuation of the spectrum light intensity due to the fluctuation of the excitation light intensity is corrected, and the Raman spectrum intensity of each component is detected.

When the angle formed by the scattered light from the substance to be measured 11 with respect to the incident light is θ, in the embodiment shown in FIG.
Although θ = 180 °, it is not limited to this and 0
It suffices if θ ≦ θ <360 °.

FIG. 3 shows the device for θ = 90 °,
The same parts as those of the apparatus shown in FIG. 2 are designated by the same reference numerals and the description thereof will be omitted.

FIG. 4 shows the substance to be measured 11 when using the flow cell.
It is the figure which showed the example of the path | route of the inflow and discharge. The substance 11 to be measured flows into the flow cell in the cell holder 30 from the sample liquid inflow line 32, and the excitation light incident from the excitation light incident window 34 opened on the side surface of the cell holder 30 is irradiated to the flow cell, and in the flow cell. The excited Raman light exits from the Raman light exit window 36 and enters the measurement target optical adjustment unit 3. After the measurement, the substance 11 to be measured passes through the sample liquid discharge line 38 and is discarded in the waste liquid bottle 40. Further, the sample liquid discharge line 38 may directly communicate with a water channel such as sewage.

In the flow cell of FIG. 4, θ = 90 °, but the present invention is not limited to this, and the Raman light emitting window 36 may be located at any position as long as 0 ° ≦ θ <360 °.

(Example 1) Raman measurement of normal urine FIG. 5 is a Raman spectrum of normal urine. The peak around 1000 cm- ¹ is derived from urea, and is around 1650 cm- ¹ and 3000
The peak around -3700 cm- ¹ originates from water as the solvent.

(Example 2) Quantification of albumin in urine using Raman spectroscopy FIGS. 6 and 7 show the results of an increase amount test for albumin in normal urine. Fig. 6 shows about 15 mg / m2 albumin in normal urine
1 shows the spectrum of the sample with increased amount. Since multiple components present in urine are present at different concentrations, it has a complex spectrum. 2370 of this spectrum
The result of examining the correlation between the cm- ¹ intensity and the concentration is shown in FIG.
It is.

Example 3 Quantification of glucose in urine using Raman spectroscopy FIGS. 8 and 9 show the results of a glucose increase test on normal urine. FIG. 8 shows a spectrum of a sample obtained by increasing glucose saturation in normal urine. FIG. 9 shows the result of examining the correlation between the Raman spectrum intensity and the concentration in the wave number 0 to 4000 cm ⁻¹ of this spectrum.

Example 4 Quantification of Urea in Urine Using Raman Spectroscopy FIGS. 10 and 11 show the results of a test for increasing the amount of urea in normal urine. FIG. 10 shows the spectrum of a sample obtained by increasing the amount of urea in normal urine by 300 mg / ml. FIG. 11 shows the result of examining the correlation between the intensity at 1008 cm ⁻¹ of this spectrum and the concentration.

Example 5 Quantification of Acetone in Urine Using Raman Spectroscopy FIGS. 12 and 13 show the results of a test for increasing the amount of acetone in normal urine. Figure 12 shows 60% acetone in normal urine
(V / V) shows the spectrum of the increased sample. FIG. 13 shows the result of examining the correlation between the intensity at 1080 cm ⁻¹ of this spectrum and the concentration.

Example 6 Simultaneous quantification of acetone, urea and glucose in urine using Raman spectroscopy FIGS. 14 to 16 show results obtained by adding acetone, urea and glucose to normal urine at different concentrations. Is. FIG. 14 is a spectrum of a sample in which acetone was added to normal urine so as to be 37.5% (V / V). FIG. 15 is a spectrum of a sample obtained by adding 17% (V / V) of acetone to normal urine, 25 mg / ml of urea and 416 μg / ml of glucose, and FIG. 16 shows the spectrum of acetone at 17% (V / V). ) Is a spectrum of a sample to which urea was added at 50 mg / ml and glucose was added at 1.17 mg / ml.

17 to 22 show 1008 cm for urea.
^{- 1, 1063cm- 1, 1168cm- 1} , 531cm- 1, 597cm- 1 and 1613Cm- ¹ in wave number position, in which took the correlation between Raman spectral intensity and concentration. 23 to 26, regarding acetone, 805 cm- ¹ , 1237 cm- ¹ , 1429 cm-
^{The same} correlations were obtained at the wave number positions of ¹ and 2941 cm ⁻¹ , and FIG. 27 is the same correlation at the wave number positions of 2924 cm ⁻¹ for glucose, and the correlation coefficient R ² is Good results were obtained at 0.9 or higher.

Example 7 Simultaneous quantification of protein and lithium acetoacetate in urine using Raman spectroscopy FIGS. 28 to 30 show results obtained by adding albumin and lithium acetoacetate to normal urine at different concentrations. Is. FIG. 28 is a spectrum of a sample to which albumin was added at 0.66 mg / ml and lithium acetoacetate was added at 1.4 mg / ml.

FIG. 29 shows the results of examining the correlation between the intensity of 620 cm ⁻¹ and the concentration of albumin. Further, FIG. 30 shows the result of examining the correlation between the intensity and the concentration of 829 cm ⁻¹ for lithium acetoacetate. In all cases, the correlation coefficient R ² was 0.9 or more, and good results were obtained.

Example 8 Simultaneous quantification of urobilin and ditaurobilirubin in urine using Raman spectroscopy FIGS. 31 to 33 show results obtained by adding urobilin and ditaurobilirubin to normal urine at different concentrations. Is. FIG. 31 is a spectrum of a sample to which urobilin was added at 0.13 mg / ml and ditaurobilirubin was added at 0.286 mg / ml.

FIG. 32 shows the results of examining the correlation between the intensity and the concentration of urobilin at 2370 cm ^-1 . Further, FIG. 33 shows the results of examining the correlation between the intensity of 1263 cm ⁻¹ and the concentration of ditaurobilirubin. In all cases, the correlation coefficient R ² was 0.9 or more, and good results were obtained.

Example 9 Detection of Components in Urine Using Raman Spectroscopy FIGS. 34 to 39 show lithium acetoacetate (1
3.3 mg / ml), β-hydroxybutyric acid (16%, V /
V), globulin (30 mg / ml), hemoglobin (30 mg / ml), ditaurobilirubin (0.3 mg)
/ Ml), sodium nitrite (55.28 mg / ml)
Is the result of measurement by adding each.

By confirming the change in intensity of an arbitrary peak, the presence or absence of the peak can be confirmed. For example, in the case of lithium acetoacetate shown in FIG. 34, a Raman peak which cannot be obtained in the spectrum of normal urine appears at 829 cm ⁻¹ . This confirms the presence of lithium acetoacetate. Similarly, the β-hydroxybutyric acid in FIG. 35 has a peak at 1630 cm ⁻¹ , the globulin in FIG. 36 has a peak at 852 cm ⁻¹ , and the hemoglobin in FIG. 37 has a peak at 1592 cm ⁻¹ .
Peak of Sita In uronium bilirubin 1616Cm- ¹ in FIG. 38, it is confirmed that the peak at the sodium nitrite 813Cm- ¹ in FIG. 39 appears, respectively, the presence or absence can be confirmed.

Example 10 Detection of Components in Body Fluid Using Raman Spectroscopy FIGS. 40 to 51 show uric acid, folic acid, L-ascorbic acid, vitamin B ₂ , creatine monohydrate, creatinine in distilled water. Saturated amounts of α-amylase and β-amylase, respectively, 25% (V / V) of ammonia, inositol,
It is the result of measuring by adding galactose and fructose so as to be saturated amounts respectively.

As in Example 9, the presence or absence of the peak can be confirmed by confirming the intensity change of an arbitrary peak. Peak of 670Cm- ¹ against uric acid in Figure 40, the peak of 661Cm- ¹ relative folate FIG 41, the peak of 1703Cm- ¹ against ascorbic acid Figure 42, vitamin 43 peak of 890Cm- ¹ for B ₂ is the peak of creatine 1 for the monohydrate 830Cm- ¹ in FIG. 44, the peak of 695Cm- ¹ for creatinine of Fig. 45,
Figure peak of 1864Cm- ¹ for α- amylase 46, the peak of 898Cm- ¹ for β- amylase FIG 47, the peak of the relative ammonia 3407Cm- ¹ of FIG. 48, FIG. peak of 443Cm- ¹ for 49 inositol, relative to the galactose of FIG 50 495cm- ¹
The peak of is 599 cm-for fructose in FIG.
By confirming that each of the ¹ peaks appears,
You can check its existence.

FIG. 52 is a chart showing the measured wave numbers of the respective components. Duplicate peaks by selecting wavenumber positions enclosed with broken lines, mixed Ya can be avoided, also near 2370Cm- ¹ is in measuring the total amount of protein, when measuring the total ketone bodies is chosen to be close to 2940Cm- ¹ And measure it. In addition, the correlation coefficients obtained for several kinds of substances to be measured are shown in a list so that the accuracy of the present invention can be confirmed. Figure 53 is that. Further accurate measurement can be performed by using an analysis method such as multivariate regression analysis.

Example 11 Simultaneous measurement of multiple components in urine using multivariate regression analysis Urea was added to normal urine at 150 mg / dl, 300 mg / dl,
450 mg / dl, sodium nitrite 600 mg / d
1, 1.2 g / dl, 1.8 mg / dl, 30 acetone
0 mg / dl, 600 mg / dl, 900 mg / dl were added in arbitrary combinations, spectral measurements were performed, and concentrations were quantified using multivariate regression analysis.

The concentration C of any substance present in urine can be approximated by the following equation.

(Equation 3) k (λi): Proportional constant at wave number λi A (λi): Raman light intensity at wave number λi k (λi) is set so that the correlation coefficient between the concentration of the analytical value known sample and the estimated concentration is the highest. It is determined in the procedure of random regression analysis. This calculation is pre-installed in commercially available processing software and is automatically performed, and this formula is created for each urinary substance whose concentration is to be determined.

The processing from the spectrum measurement to the concentration quantification was performed according to the flowchart of FIG. In this example,
No pretreatment was performed, and QUANT + manufactured by Perkin Elmer was used as processing software. The PCR method was adopted as the processing method. Also, when performing full cross validation, evaluation of the accuracy of the calibration model can be omitted.

FIG. 55 is a correlation diagram between the calculated value and the actually measured value (reference value) obtained in this example. Correlation coefficients R ² and SEP are 0.986 and 18.13 for urea and sodium nitrite, respectively. Is 0.992 and 57.17, and acetone is 0.956 and 6.
It was 9.74. Here, SEP is a predicted standard deviation, and is calculated by the following calculation formula.

(Equation 4) di: difference between calculated value and actual measured value by calibration model D: average value of di n: number of samples for evaluation Smaller SEP means higher accuracy of the calibration model.

[0072]

[Effects of the Invention] A plurality of components can be qualitatively / quantitatively determined simultaneously by the method described above. In the present invention, the body fluid sample, for a plurality of components in the body fluid to be measured from the spectrum obtained by irradiating with Raman excitation light, to obtain the intensity of the appropriate wave number position, so that the components in the body fluid can be quantitatively analyzed. Becomes
Consumables such as test papers and reagents are unnecessary, and there is no problem of disposal after use. Further, even when there is interference with other components in the body fluid, it becomes possible to simultaneously perform a qualitative / quantitative analysis of a plurality of components in the body fluid by using a technique such as a multivariate regression analysis method.

[Brief description of drawings]

FIG. 1 is a block diagram showing an outline of a measuring apparatus to which the method of the present invention is applied.

FIG. 2 is a device configuration diagram showing a specific example of the block diagram of FIG. 1 in detail, which is an example of extracting scattered light from a substance to be measured in a 180 ° direction with respect to incident light.

FIG. 3 is a device configuration diagram showing a specific example of the block diagram of FIG. 1 in detail, which is an example of extracting scattered light from a substance to be measured in a 90 ° direction with respect to incident light.

FIG. 4 is a perspective view showing the inflow and outflow paths of a substance to be measured for use in a flow cell.

FIG. 5 is a diagram showing a Raman spectrum of normal urine.

FIG. 6 is a diagram showing a Raman spectrum of a sample obtained by increasing albumin in normal urine by about 15 mg / ml.

FIG. 7 is a diagram showing a correlation between albumin concentration in urine and Raman scattered light intensity at a wave number of 2370.75 cm ⁻¹ .

FIG. 8 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of glucose in normal urine.

FIG. 9 is a diagram showing a correlation between glucose concentration in urine and Raman scattered light intensity at a wave number of 0 to 4000 cm ⁻¹ .

FIG. 10 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of urea in normal urine by about 300 mg / ml.

FIG. 11 is a diagram showing the correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 1008 cm ⁻¹ .

FIG. 12 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of acetone in normal urine to 60% (V / V).

FIG. 13 is a diagram showing the correlation between the acetone concentration in urine and the Raman scattered light intensity at a wave number of 1080 cm ⁻¹ .

FIG. 14 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of acetone in normal urine to 37.5% (V / V).

FIG. 15 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of acetone in normal urine to 17% (V / V), urea to 25 mg / ml, and glucose to 416 μg / ml.

FIG. 16: 17% acetone (V / V), 50 mg / ml urea and 1.17 mg / ml glucose in normal urine
It is a figure which shows the Raman spectrum of the sample increased so that it might become.

FIG. 17 is a diagram showing a correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 1008 cm ⁻¹ .

FIG. 18 is a diagram showing a correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 1063 cm ⁻¹ .

FIG. 19 is a diagram showing the correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 1168 cm ⁻¹ .

FIG. 20 is a diagram showing a correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 531 cm ⁻¹ .

FIG. 21 is a diagram showing the correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 597 cm ⁻¹ .

FIG. 22 is a diagram showing a correlation between the urea concentration in urine and the Raman scattered light intensity at a wave number of 1613 cm ⁻¹ .

FIG. 23 is a diagram showing a correlation between acetone concentration in urine and Raman scattered light intensity at a wave number of 805 cm ⁻¹ .

FIG. 24 is a diagram showing a correlation between acetone concentration in urine and Raman scattered light intensity at a wave number of 1237 cm ⁻¹ .

FIG. 25 is a diagram showing the correlation between the acetone concentration in urine and the Raman scattered light intensity at a wave number of 1429 cm ⁻¹ .

FIG. 26 is a diagram showing the correlation between the acetone concentration in urine and the Raman scattered light intensity at a wave number of 2941 cm ⁻¹ .

FIG. 27 is a diagram showing a correlation between glucose concentration in urine and Raman scattered light intensity at a wave number of 2924 cm ⁻¹ .

FIG. 28: About 0.66 mg / ml of albumin in normal urine
FIG. 3 is a diagram showing a Raman spectrum of a sample in which lithium acetoacetate was increased by about 1.4 mg / ml.

FIG. 29 is a diagram showing a correlation between albumin concentration in urine and Raman scattered light intensity at a wave number of 620 cm ⁻¹ .

FIG. 30: Urinary lithium acetoacetate concentration and wave number 829c
It is a figure which shows the correlation with the Raman scattered light intensity in m- ¹ .

FIG. 31: Urobilin in normal urine of about 0.13 mg / ml
FIG. 3 is a graph showing a Raman spectrum of a sample containing 0.228 mg / ml of ditaurobilirubin increased.

FIG. 32 is a diagram showing a correlation between urobilin concentration in urine and Raman scattered light intensity at a wave number of 2370 cm ⁻¹ .

FIG. 33: Urine ditaurobilirubin concentration and wave number 1263c
It is a figure which shows the correlation with the Raman scattered light intensity in m- ¹ .

FIG. 34: Normal urine with lithium acetoacetate 13.3 mg
It is a figure which shows the Raman spectrum of the sample which increased / ml.

FIG. 35 shows 16% (V / V) of β-hydroxybutyric acid in normal urine.
It is a figure which shows the Raman spectrum of the sample increased so that it might become V).

FIG. 36 is a diagram showing a Raman spectrum of a sample obtained by increasing globulin by 30 mg / ml in normal urine.

FIG. 37 is a diagram showing a Raman spectrum of a sample obtained by increasing hemoglobin by 30 mg / ml in normal urine.

FIG. 38: Normal urine containing 0.3 mg / day of ditaurobilirubin
It is a figure which shows the Raman spectrum of the sample which increased ml.

FIG. 39: Normal urine 55.28 mg sodium nitrite
It is a figure which shows the Raman spectrum of the sample which increased / ml.

FIG. 40 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of uric acid saturated with distilled water.

FIG. 41 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturated amount of folic acid in distilled water.

FIG. 42 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of L-ascorbic acid in distilled water.

FIG. 43 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of saturation of vitamin B _{2 in} distilled water.

FIG. 44 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of creatine monohydrate saturated with distilled water.

FIG. 45 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of creatinine saturated in distilled water.

FIG. 46 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of α-amylase in distilled water.

FIG. 47 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of β-amylase in distilled water.

FIG. 48 is a diagram showing a Raman spectrum of a sample obtained by increasing the amount of ammonia in distilled water to 25% (V / V).

FIG. 49 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of inositol in distilled water.

FIG. 50 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturated amount of galactose in distilled water.

FIG. 51 is a diagram showing a Raman spectrum of a sample obtained by increasing the saturation amount of fructose in distilled water.

FIG. 52 is a chart showing measured wave numbers of components in each body fluid.

FIG. 53 is a chart showing the correlation coefficient between each concentration and Raman scattered light intensity for the extracted components in the body fluid.

FIG. 54 is a flow chart diagram showing the procedure from spectrum measurement to concentration quantification when the multivariate regression analysis method is applied.

FIG. 55 is a correlation diagram between calculated values and measured values calculated by a multivariate regression analysis method for a plurality of components in urine.

[Explanation of symbols]

 1 light source part 2 sample part 3 measurement target optical adjustment part 4 correction optical adjustment part 5 spectroscopic detection part 6 data processing part

Claims (4)

[Claims] 1. For each component in a body fluid to be measured, a wave number having a good correlation between the concentration of the component and the Raman spectrum intensity is selected in advance as a measurement wave number specific to the component, and Raman excitation light is applied to the body fluid sample. The Raman spectrum intensity at each measurement wave number for each component to be measured or the Raman spectrum integrated intensity at each measurement wave number range is measured, and the Raman spectrum intensity at each measurement wave number for each component or A measurement method characterized in that each component in a body fluid is simultaneously qualitatively and quantitatively analyzed using a calibration curve prepared in advance for the Raman spectrum integrated intensity and the concentration of the component in each measurement wave number range. 2. A body fluid sample is irradiated with Raman excitation light,
The Raman spectrum intensity at those measurement wave numbers is measured as the wave number with a good correlation between the concentration of each component in the body fluid to be measured and the Raman spectrum intensity, and the Raman spectrum intensity at those measurement wave numbers is measured, or in any wave number range. The measurement method is characterized by measuring Raman spectrum intensities at a plurality of wave numbers and simultaneously performing a qualitative / quantitative analysis of a plurality of components in a body fluid sample by multivariate regression analysis. 3. The wave number having a good correlation between the concentration of each component and the Raman spectrum intensity is a wave number having a correlation coefficient R ² of 0.8 or more, preferably 0.9 or more. Measuring method. However, the correlation coefficient R ² is a value calculated by the following equation. [Equation 1] xi: concentration of each point of component in body fluid yi: Raman spectrum intensity with respect to xi X: average value of concentration of each point of component in body fluid Y: average value of Raman spectrum intensity 4. A body fluid component to be measured is albumin, globulin, hemoglobin, glucose, lithium acetoacetate, β-hydroxybutyric acid, acetone, ditaurobilirubin, urobilin, sodium nitrite, urea, uric acid, folic acid, ascorbic acid. , Vitamin B ₂ , creatine monohydrate, creatinine, α-amylase, β-amylase, and ammonia, at least one component is included, and the measurement wave number or the measurement wave number range of each component is 590 to 650 cm for albumin. -Near ¹ , 800-860c
m- ¹ near, 2342-2402cm- ¹ selected, for globulin near 158-218cm- ¹ , 402-462c
m- ¹ vicinity, 623-683cm- ¹ vicinity, 807-867cm- ¹ vicinity,
822 ~ 862 cm- ¹ vicinity, 1353 ~ 1413 cm- ¹ vicinity, 1562 ~ 16
22 cm- ¹ vicinity, 1847-1907 cm- ¹ vicinity, 2095-2155 cm
^-1 near, 2340-2400cm- ¹ near, 2403-2463cm- ¹ near, for hemoglobin, 807-867cm- ¹ near, 893-9
53 cm- ¹ vicinity, 1353-1413 cm- ¹ vicinity, 1562-1622 cm
- around ^1, 1840～1900Cm- around ^1, 2095～2155Cm- around ^1, 2340～2400Cm- around ^1, selected from around 2403～2463Cm- ^1, with respect to glucose, 200～554Cm- around ^1, 810～ 944
cm- near ^1, selected from around 2590～2940Cm- ^1, for the acetoacetate lithium, 799～859Cm- around ^1,
1353 ~ 1413 cm- ¹ vicinity, 1840 ~ 1900 cm- ¹ vicinity, 2347 ~
2407Cm- ¹ near, chosen from the vicinity of 2907～2967Cm- ^1, for the β- hydroxybutyrate, 1420～1480Cm- around ^1, 1557～1617Cm- around ^1, 1600～1660Cm- around ^1, 28
Select from around 70 to 2971 cm- ¹ , and for acetone, near 775 to 845 cm- ¹ , 1050 to 1110
cm- near ^{1, 1207~1267cm-} near ^{1, 1399~1459cm- 1}
Near, near 1680～1740Cm- ^1, selected from around 2911～2971Cm- ^1, for the ditaurobilirubin, 927～987Cm- around ^1,
1233～1293Cm- ¹ near, chosen from the vicinity of 1586～1616Cm- ^1, for the urobilin, select an arbitrary position from 332～2900Cm- ^1, with respect to sodium nitrite, 783～843Cm- ¹ near ,13
18～1378Cm- ¹ selected from around, for urea, 501～561Cm- around ^{1, 567~627cm- 1}
Near, 978-1048cm- ¹ Near, 1033-1093cm- ¹ Near, 1
138～1198Cm- ¹ near, chosen from the vicinity of 1583～1643Cm- ^1, for the uric acid, selected from around 640～700Cm- ^1, for the folate, 631～691Cm- around ^1, 816～876Cm- ¹
Neighborhood, 901-1961 cm- ¹ neighborhood, 1355-1415 cm- ¹ neighborhood, 15
62 to 1622 cm- ¹ near, 1847 to 1907 cm- ¹ near, 2093 to 21
53 cm- ¹ vicinity, 2339-2399 cm- ¹ vicinity, 2400-2460 cm
- ¹ selected from around, for ascorbic acid, 800～860Cm- near ^1, 1673
～1733Cm- ¹ near, chosen from the vicinity of 2919～2979Cm- ^1, with respect to vitamin B _2, 404~464cm- around ^1, 626 to 68
6 cm- ¹ neighborhood, 798-858 cm- ¹ neighborhood, 860-920 cm- ¹ neighborhood, 1317-1377 cm- ¹ neighborhood, 1546-1606 cm- ¹ neighborhood, 18
34-1894 cm- ¹ vicinity, 2106-2166 cm- ¹ vicinity, 2360-24
Select from around 20 cm- ^{1 and} around 2419-2479 cm- ¹ , and for creatine monohydrate around 800-860 cm- ¹ ,
Select from around 1563 to 1623 cm- ¹ , and for creatinine, around 538 to 598 cm- ¹ and 580 to 6
40 cm- ¹ vicinity, 665-725 cm- ¹ vicinity, 817-877 cm- ¹ vicinity, 868-928 cm- ¹ vicinity, 1021-1081 cm- ¹ vicinity, 1550
~ 1610cm- ¹ vicinity, 2868-2900cm- ¹ vicinity, 2900-2950
cm- ¹ near, 2950-2996cm- ¹ near, for α-amylase, 393-453cm- ¹ near, 631
~ 691 cm- ¹ vicinity, 792-852 cm- ¹ vicinity, 868-928 cm-
^1, around 1333 to 1393 cm- ^1, around 1546 to 1606 cm- ^1, around 1834 to 1894 cm- ^1, around 2334 to 2394 cm- ¹ , and for β-amylase, around 376 to 436 cm- ^1, around 622
~ 682 cm- ¹ near, 783 ~ 843 cm- ¹ near, 868 ~ 928 cm-
^1, near 1334 to 1394 cm- ^1, near 1546 to 1606 cm- ^1, near 1834 to 1894 cm- ^1, near 2665 to 2725 cm- ¹ , and for ammonia, near 3190 to 3250 cm- ¹ and 3292 to
Select from around 3350 cm- ^{1 and} around 3377 to 3437 cm- ¹ . For inositol, around 404 to 464 cm- ¹ and 805 to 8
65 cm- ¹ vicinity, 1014-1074 cm- ¹ vicinity, 1438-1498 cm
^-1 near, 2966 ~ 3026cm- ¹ near, for galactose, 466 ~ 526cm- ¹ near, 835 ~ 8
95 cm- ¹ vicinity, 1032-1092 cm- ¹ vicinity, 1237-1297 cm
- around ^1, 1332～1392Cm- around ^1, 1438～1498Cm- around ^1, selected from around 2946～3006Cm- ^1, for the fructose, 569～629Cm- around ^1, 772-8
32 cm- ¹ vicinity, 1044-1106 cm- ¹ vicinity, 1237-1297 cm
4. The measuring method according to claim 1, wherein the measuring method is selected from around ^−1, around 1438 to 1498 cm ^{−1, and} around 2966 to 2996 cm ⁻¹ .