Patent Application
20130010293
This application claims the benefit of Japanese Patent Application No. 2011-150436, filed on Jul. 6, 2011, the entire disclosure of which is incorporated by reference herein.
The present invention relates to a spectrophotometer, a spectrophotometer tool, a spectrophotometric method, and a recording medium, which are used to analyze the concentration of an analyte in a test object by absorption spectrophotometry.
The wavelength of light absorbed by substances varies among the substances. The absorbance changes according to the concentration of a substance in solution and optical path length. It is known that when sample concentration is low, the absorbance of light passing through the same distance in solution is proportional to the sample concentration (Lambert-Beer law). The Lambert-Beer law is used for quantitative sample analysis by absorption spectrophotometry. Absorption spectrophotometry is a technique for quantitatively analyzing the concentration of an analyte in a sample solution by measuring the magnitude of light absorption by the analyte, namely the absorbance of the analyte when light applied to the sample solution passes therethrough.
Generally, in absorption spectrophotometry, a container holding a sample solution is called a cell. In the measurement of absorbance of transmitted light, a length in which light passes through a sample can be called a cell length. To accurately measure the concentration of a substance by absorption spectrophotometry, it is necessary to accurately determine the length (cell length) in which light passes through the sample.
For example, Unexamined Japanese Patent Application KOKAI Publication No. H05-018823 has disclosed a technique for performing quantitative analysis by spectrophotometry in which cell length correction is conducted for quantitative analysis using a plurality of cells having different cell lengths. In the spectrophotometric technique described in the Patent Literature, for spectrophotometric analysis using an apparatus performing spectrophotometric measurement at a plurality of predetermined wavelengths, various output variations are measured in advance in each wavelength and regarded as vectors in the dimensional space of the number of wavelengths measured to obtain a subspace orthogonal to all the vectors. Then, regarding a reference cell and a measurement cell, various error variations are removed in advance by measuring a plurality of measurement samples and projecting the data on the subspace. Using the projected data, a correlation between measured values of the reference cell and those of the measurement cell is obtained to correct changes due to cell length. The corrected values and a calibration curve equation are used to obtain output values. This allows the use of a plurality of cells having different cell lengths.
In the technique of the Unexamined Japanese Patent Application KOKAI Publication No. H05-018823, upon the cell length correction, many wavelengths (six in the Examples) are used and complicated calculations are performed for the cell length correction.
However, when performing sample component analysis by colorimetry, it is necessary to determine a wavelength in which signal change occurs by reaction. In addition, a wavelength region with the signal change is not so wide. For example, to simultaneously measure the six wavelengths, it might be possible to use a large apparatus such as a spectrophotometer with a halogen lamp or a D2 lamp (deuterium lamp) as a light source. However, a small analytical instrument with LED as a light source would require the incorporation of six or more LEDs having different wavelength characteristics. Thereby, since the number of controllers increases, instrument miniaturization becomes difficult and the system of the instrument becomes complicated.
The present invention has been accomplished in view of the circumstances and is directed to provide a spectrophotometer, a spectrophotometer tool, a spectrophotometric method, and a recording medium, which allows absorbance correction using an optical path length in which light passes through a test object in measurement in each spectrophotometer tool.
A spectrophotometer according to a first aspect of the present invention includes:
Preferably, the corrector calculates the corrected wavelength component absorbance by subtracting the wavelength component absorbance of the light absorbed by the pigment from the detected wavelength component absorbance.
Preferably, the analyte and the pigment are any one of the following combinations (a) to (f):
A spectrophotometer tool according to a second aspect of the present invention is a spectrophotometer tool holding a test object and includes a pigment holding portion, a hollow cavity provided between two planes inside the spectrophotometer tool, and a pathway leading to an inside of the hollow cavity from an outside of the tool, in which a substance forming at least one of the two planes having the hollow cavity therebetween is transparent,
Preferably, the analyte and the pigment are any one of the following combinations (a) to (f):
A spectrophotometric method according to a third aspect of the present invention includes:
Preferably, the correcting step calculates the corrected wavelength component absorbance by subtracting the wavelength component absorbance of the light absorbed by the pigment from the wavelength component absorbance detected at the spectrometric step.
Preferably, the analyte and the pigment are any one of the following combinations (a) to (f):
A computer-readable non-transitory tangible recording medium having recorded thereon a program according to a fourth aspect of the present invention causes a computer controlling a spectrophotometer performing spectrophotometric measurement of a test object to execute:
According to the present invention, when performing measurement in each analysis tool, absorbance can be corrected by the optical path length in which light passes through a test object.
A more complete understanding of this application can be obtained when the following detailed description is considered in conjunction with the following drawings, in which:
Hereinafter, embodiments of the present invention will be described in detail with reference to the drawings. The same or equivalent parts in the drawings are given the same reference numerals.
In
In the spectrophotometer 1, light with a plurality of wavelengths is applied to the test object 3 to measure a concentration of an analyte in the test object 3 from a absorbance of each wavelength component of light passing through the test object 3.
The light emitter 11 emits the light with the plurality of wavelengths towards the test object 3 held in the spectrophotometer tool 2. To produce the light with the plurality of wavelengths, for example, light from a light source having a continuous spectrum is separated to be consecutively emitted. To separate the light, for example, an interference filter or prism can be used. Alternatively, by consecutively switching on/off a plurality of light sources with different wavelength characteristics, light with a plurality of wavelengths may be applied.
The light receiver 13 is composed of an optoelectronic device or the like and receives the light with the plurality of wavelengths passing through the test object 3 after being emitted from the light emitter 11 in order to convert the light into an electric signal corresponding to an intensity of the light. In the consecutive emission of light with a plurality of wavelengths, the intensity of light of each wavelength component can be determined according to the emission timing. The light emitter 11 may simultaneously emit light with a plurality of wavelengths, and the light passing through the test object 3 may be separated to be simultaneously received by a plurality of light receiving elements.
The solution of the test object 3 injected in the recess 21 is held in the hollow cavity 23 through the pathway 22. The air pathway 24 is disposed to introduce the test object 3 injected in the recess 21 into the hollow cavity 23 and also to release air from in the hollow cavity 23. The spectrophotometer tool 2 is supported by the sample chamber 12 such that the hollow cavity 23 is located in a position to which light from the light emitter 11 is applied in the state of holding the test object 3 in the hollow cavity 23. The light applied from the light emitter 11 enters one surface of the spectrophotometer tool 2, then passes through the test object 3 held in the hollow cavity 23, and is output from the other surface thereof.
The spectrophotometer 1 may be of transmission type as shown in
In the present embodiment, a pigment that absorbs light with a wavelength other than a wavelength of light absorbed by the analyte in the test object 3 is dissolved in the test object 3 in advance at a predetermined concentration. Then, in wavelength components of light having a plurality of wavelengths passing through the test object 3 containing the dissolved pigment, an absorbance of a wavelength component of light absorbed by the added pigment is used to calculate the optical path length.
To do that, the absorbance of a solution of the added pigment with a predetermined concentration is measured in advance using a plurality of optical path lengths to predetermine an absorbance of a wavelength absorbed by the pigment with the predetermined concentration in a reference optical path length. Next, for light with a plurality of wavelengths passing through the test object 3 containing the dissolved pigment, a wavelength component absorbance of a wavelength absorbed by the pigment is compared with a predetermined value of the wavelength component absorbance to calculate an optical path length.
More specifically, in the spectrophotometer 1 of the present embodiment, the light emitter 11 of
In the case of the reflected type shown in
The detector 14 of
Next, the test object 3 is held in the spectrophotometer tool 2 to measure the wavelength component (intensity) of light transmitted through the test object 3. A difference obtained by subtracting the wavelength component (intensity) of the light transmitted through the test object 3 from the wavelength component of the reference spectrum for each wavelength is equivalent to the wavelength component absorbance.
The optical path length calculator 16 of the calculator 15 compares, in the wavelength component absorbances detected by the detector 14, the wavelength component absorbance of light absorbed by the pigment that absorbs light with a wavelength other than a wavelength of light absorbed by the analyte in the test object 3 and a predetermined value of the wavelength component absorbance to calculate an optical path length. When the concentration of the added pigment is constant, the absorbance of the pigment is proportional to optical path length, so that the optical path length can be calculated from the absorbance of the pigment. Accordingly, the optical path length can be determined by precision of the concentration of an added pigment.
The corrector 17 corrects the absorbance of a wavelength component other than the wavelength of light absorbed by the added pigment in the wavelength component absorbances detected by the detector 14 using the optical path length calculated by the optical path length calculator 16 to calculate a corrected wavelength component absorbance in the reference optical path length. When the concentration of a light-absorbing substance is constant, the absorbance thereof is proportional to the optical path length. An actual optical path length of the test object 3 is the optical path length calculated by the optical path length calculator 16. Thus, by calculating a proportion between the actual optical path length (the optical path length calculated by the optical path length calculator 16) and the wavelength component absorbance detected by the detector 14, the corrected wavelength component absorbance in the reference optical path length can be obtained.
The corrector 17 can also calculate a corrected wavelength component absorbance by subtracting the wavelength component absorbance of light absorbed by the added pigment from the detected wavelength components. In that case, even when a part of the absorption wavelength of the added pigment is within a band of an absorption wavelength of the analyte, the corrector 17 can calculate a corrected wavelength component absorbance of the analyte.
The controller 19 shown in
In the present embodiment, light of a wavelength absorbed by pigment, which is used for calculating an optical path length, passes through the same path in the same cell as light for measuring the absorbance of an analyte, so that an optical path length passing through the test object 3, itself, can be obtained. In addition, upon measurement of the test object 3, measurement of optical path length is performed. Thus, in the measurement in each spectrophotometer tool, the absorbance can be corrected by the optical path length in which light passes through the test object 3. In the method of the present embodiment, before spectrophotometric measurement, pigment can be dissolved in the test object 3 at a predetermined concentration. Therefore, the method thereof can be applied to the spectrophotometer tool 2 having any configuration other than that shown in
Additionally, the present embodiment uses the spectrophotometer tool 2 in which a pigment is applied in such a manner that the pigment dissolved in the test object 3 has a predetermined concentration.
The spectrophotometer tool 2 has a structure such that between a transparent substrate 25 and a transparent cover 26 there is interposed a spacer 27 with the recess 21, the pathway 22, the hollow cavity 23, and the air pathway 24 formed therein. At a part of the cover 26 overlapping the recess 21 is formed a hole 21A. In the spectrophotometer tool 2, a pigment layer 28 is formed on an inner surface of the recess 21 as the pigment holding portion. The pigment layer 28 is formed by applying a predetermined amount of a pigment that is soluble in the test object 3 and absorbs light with a wavelength other than the wavelength of light absorbed by the analyte in the test object 3. The pigment to be applied is selected according to the analyte.
The configuration of the pigment holding portion is not restricted to the recess 21 as long as the pigment can be dissolved in the test object 3 at a predetermined concentration. The pigment holding portion may be provided, for example, by arranging a product prepared by mixing a predetermined amount of a pigment in a porous material allowing light transmission through the test object 3 and drying the mixture in the pathway 22 leading to the hollow cavity 23. In this case, without forming the recess 21, the test object 3 may be injected in the pathway 22 with the pigment holding portion arranged therein using a dispensing nozzle or may be suctioned from the air pathway 24 to allow the pigment to be dissolved in the test object 2 at a predetermined concentration and introduced into the hollow cavity 23.
The hollow cavity 23 is covered with the transparent cover 26 to maintain the surface of the test object 3 at a constant level. The hollow cavity 23 is configured to be sandwiched between the two planes. In the case of spectrophotometric measurement using transmitted light, the cover 26 and at least a part of the substrate 25 facing the hollow cavity 23 are transparent. In a spectrophotometric measurement using reflected light, one of the planes sandwiching the hollow cavity 23 therebetween, for example, the cover 26 on the upper side is transparent, and, the other plane, for example, the substrate 25 reflects light. When using reflected light, light may be applied from the substrate 25 on the lower side and reflected on the cover 26 on the upper side.
The spectrophotometer tool 2 has a plate-like shape as a whole, in which the rectangular substrate 25 and cover 26 are bonded to each other with the spacer 27 interposed therebetween. The substrate 25 and the cover 26, respectively, are made of, for example, transparent PET, PMMA, PS, glass, or vinylon.
The pigment layer 28 is formed by spottedly applying a pigment-containing solution on the inner surface of the recess 21 and then drying the solution. The pigment-containing solution is prepared by dissolving a pigment in a solvent at a predetermined concentration. The amount of the pigment-containing solution is accurately measured such that the amount of the pigment of the pigment layer 28 is equal to a predetermined amount, and spottedly applied onto the inner surface of the recess 21 to be dried. The pigment layer 28 may include a reagent added to the test object.
When a predetermined amount of the test object 3 is injected (or spottedly applied) in the recess 21 of the spectrophotometer tool 2 thus formed, the pigment applied in the recess 21 is dissolved in the test object 3 to have a predetermined concentration. The test object 3 containing the dissolved pigment is introduced into the hollow cavity 23 through the pathway 22. After that, the spectrophotometer tool 2 is supported in the sample chamber 12 shown in
Use and function of the spectrophotometer tool 2 shown in
In the modified example shown in
With the use of the spectrophotometer tool 2 described above, spectrophotometric measurement can be easily performed by the method of the present embodiment only by accurately measuring the amount of the test object 3 to inject in the spectrophotometer tool 2. By preparing the spectrophotometer tool 2 with a pigment applied according to the analyte, analysis for various analytes can be easily performed by absorption spectrophotometry. Hereinafter, a description will be given of operation of the spectrophotometer 1 according to the present embodiment.
The controller 19 confirms, by a signal of a sensor (not shown), that the spectrophotometer tool 2 is appropriately supported in the sample chamber 12 and the chamber is closed to prevent light from the outside from coming in. Then, the controller 10 controls the light emitter 11 in order to apply light with a plurality of wavelengths to the spectrophotometer tool 2 and simultaneously controls the light receiver 13 to receive the light (step S11). The detector 14 detects wavelength component absorbances from the light with the plurality of wavelengths received by the light receiver 13 (step S12).
The optical path length calculator 16 of the calculator 15 extracts an absorbance (wavelength component absorbance)of a predetermined wavelength absorbed by the pigment (step S13) and compares the absorbance and a predetermined value of the wavelength component absorbance to calculate an optical path length (step S14). The corrector 17 corrects, using the optical path length calculated by the optical path length calculator 16, the absorbance of a wavelength component other than the wavelength of light absorbed by the pigment in the wavelength component absorbances detected by the detector 14 to calculate a corrected wavelength component absorbance in the reference optical path length (step S15). The output device 18 outputs the corrected wavelength component absorbance to the outside (step S 16). The analyzer receives the corrected wavelength component absorbance and uses, for example, the relationship between the absorbance, in the reference optical path length, of a wavelength of light absorbed by the analyte and the concentration of the analyte to determine a concentration of the analyte from the corrected wavelength component absorbance.
After calculating the optical path length by the optical path length calculator 16 (step S24), the corrector 17 subtracts the wavelength component absorbance of light absorbed by the added pigment from the detected wavelength component absorbance (step S25). Then, the corrector 17 corrects the difference of the subtraction using the optical path length calculated by the optical path length calculator 16 to calculate a corrected wavelength component absorbance in the reference optical path length (step S26). As in
As described hereinabove, according to the spectrophotometer 1 of the present embodiment, by dissolving a pigment in the test object 3 at a predetermined concentration, absorbance correction can be performed by an optical path length in which light passes through the test object 3, upon measurement in each analysis tool. In addition, with the use of the spectrophotometer tool 2 of the present embodiment, by injecting an accurately measured amount of the test object 3, the pigment can be dissolved in the test object 3 at a predetermined concentration. Hereinafter, a description will be given of specific examples of combinations of analyte and added pigment.
Regarding the items of No. 1: ALT (alanine transaminase), AST (aspartate aminotransferase), and UN (urea nitrogen), mainly by the reaction of serum with a reagent, analysis is performed to monitor a concentration of a reduced form (NADH or NADPH) of nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) changing with concentrations of the substances of the item. In the Analyte of No. 1, NAD(P)H is the short form of “NADH or NADPH”. The dominant wavelength of “NADH or NADPH” is 340 nm.
Similarly, regarding the items of No. 2: CK (creatine kinase), CRE (creatinine), LD (lactate dehydrogenase), GLU (glucose), TG (triglyceride), T-CHO (total cholesterol), and HDL-C (HDL cholesterol), a concentration of the reduced form (NADH or NADPH) of NAD or NADP (dominant wavelength: 340 nm) is analyzed. In the items of No. 1 and No. 2, possible correction wavelength range is 400 to 800 nm. In the case of the items of Nos. 1 and 2, an added pigment can be malachite green or Brilliant Blue FCF having an absorption wavelength of 630 nm.
In the items of No. 3: AMY (amylase) and ALP (alkaline phosphatase), the amount of change in the absorbance of p-nitrophenol (pNP) is measured. The dominant wavelength of pNP is 405 nm. Also in the absorbance measurement of pNP, malachite green or Brilliant Blue FCF can be used.
The item of No. 4: GGT (γ-glutamyltransferase) is quantified by measuring a concentration of 5-amino-2-nitrobenzoic acid. The dominant wavelength of GGT is also 405 nm, and malachite green or Brilliant Blue FCF can also be used.
The item of No. 5: T-BIL (biliverdin), which is an intermediate of a biodegradation product of hem in hemoglobin or the like, is a green tetrapyrrole compound. Biliverdin has a dominant wavelength of 450 nm. As a pigment to be added, there can be used indocyanine green, malachite green, or Brilliant Blue FCF.
Regarding TP (total protein) of No. 6, a concentration of a protein-copper ion complex is measured. The protein-copper ion complex has a dominant wavelength of 550 nm, and indocyanine green can be used an added pigment.
In quantification of Ca (calcium) of No. 7 or Mg (magnesium) of No. 8, measurement is performed to monitor a change in color tone due to a complex reaction product with o-cresolphthalein complexon (OCPC) produced by chelation. In Nos. 7 and 8, the dominant wavelength is 570 nm, and the added pigment can be Brilliant Blue FCF or indocyanine green.
UA (urea acid) of No. 9 is quantified by measuring a concentration of a condensation reaction product of 4-AA with Trinder's reagent (a condensation reaction product of 4-aminoantipyrine with Trinder's reagent) produced using Trinder's reagent. The condensation reaction product of 4-AA with Trinder's reagent has a dominant wavelength of 630 nm, and the possible correction wavelength range is 400 to 570 nm. Thus, as a pigment added for analysis, there can be used Mordant Blue 29, phloxine B, phloxine BP, or Food Red No. 2.
ALB (albumin) of No. 10 is quantified by measuring a concentration of an Alb-BCG (albumin-bromocresol green) conjugate. The dominant wavelength of Alb-BCG is 630 nm, and the possible correction wavelength ranges are 400 to 570 and 700 to 800 m. The analysis of Alb-BCG can use indocyanine green or phloxine B as an added pigment.
Regarding IP (inorganic phosphate) of No. 11, measurement is performed to determine a concentration of phosphomolybdic acid. The dominant wavelength of phosphomolybdic acid is 730 nm, and the possible correction wavelength range is 400 to 660 nm. Malachite green or Brilliant Blue FCF can be used as an added pigment.
As shown in
The controller 31 is composed of a central processing unit (CPU) and the like and executes processing for the spectrophotometric measurement described above according to a control program 39 stored in the external memory 33.
The main memory 32 is composed of a random-access-memory (RAM) and the like. The control program 39 stored in the external memory 33 is loaded into the main memory 32, which is used as a work area for the controller 31.
The external memory 33 is composed of a nonvolatile memory, such as flash memory, hard disk, digital versatile disc-random access memory (DVD-RAM), or digital versatile disc rewritable (DVD-RW). The external memory 33 pre-stores the control program 29 for causing the controller 31 to execute the above-described processing. In addition, according to instruction of the controller 31, the external memory 33 supplies data stored in the control program 29 to the controller 31 and stores data supplied from the controller 31.
The operation device 34 is composed of a keyboard with a pointing device, such as a mouse or a touch panel, and the like, and an interface device connecting the keyboard with the pointing device and the like to the internal bus 30. Input operation relating to analytes and pigments to be added is received via the operation device 34.
The display device 35 is composed of a liquid crystal display (LCD) or an organic EL display, a speaker, and the like and displays data calculated regarding spectrophotometric measurement, such as cell lengths, a corrected absorbance of an analyte, and a concentration of the analyte.
The input/output device 36 is composed of a serial interface or a parallel interface. The input/output device 36 is connected to the light emitter 11 and the light receiver 13. In addition, a sensor for detecting the spectrophotometer tool 2 placed in the sample chamber 12, a sensor for detecting the closing of the sample chamber 12, and the like are connected to the input/output device 36. The controller 31 sends a command to the light emitter 11 and the light receiver 13 via the input/output device 36 and receives a signal from the light receiver 13.
Regarding processings by the detector 14, the calculator 15, the output device 18, the controller 19, and the like in the spectrophotometer 1, the control program 39 uses the controller 31, the main memory 32, the external memory 33, the operation device 34, the display device 35, the input/output device 36, a transmitter/receiver 37, and the like as resources to perform processing, whereby the processings thereby are executed.
In addition, the hardware structure and flowcharts described above are merely examples and thus can be arbitrarily changed and corrected.
A central section for executing control processings, which includes the controller 31, the main memory 32, the external memory 33, the operation device 34, and the internal bus 30, can be formed by using not an exclusive system but a general computer system. For example, a computer program for executing the above-described operation may be stored in a computer readable memory medium (such as flexible disc, CD-ROM, or DVD-ROM) to be distributed. Then, the computer program may be installed in a computer to form the spectrophotometer 1 executing the above-described processings. Alternatively, the computer program may be pre-stored in a memory of a server on a communication network such as the Internet and, for example, may be downloaded by a general computer system to form the spectrophotometer 1.
In addition, when function of the spectrophotometer 1 is executed by role-sharing of an operating system (OS) and an application program or cooperation therebetween, only the application program may be stored in a memory medium or memory.
Furthermore, it is also possible to superimpose the computer program on a carrier wave to distribute via a communication network. For example, the computer program may be posted on a bulletin board system (BBS) on the communication network to distribute the computer program via the network. Then, the spectrophotometer 1 may be formed such that the above-described processings can be executed by booting the computer program to execute as in other application programs under control of the OS.
The present invention is not restricted at all to the above-described embodiments and specific examples. The invention encompasses various modifications and changes without departing from the description provided in the claims and within a range where those skilled in the art can easily conceive the invention.
The entire contents of this patent application are incorporated herein by reference.
Having described and illustrated the principles of this application by reference to one (or more) preferred embodiment(s), it should be apparent that the preferred embodiment(s) may be modified in arrangement and detail without departing from the principles disclosed herein and that it is intended that the application be construed as including all such modifications and variations insofar as they come within the spirit and scope of the subject matter disclosed herein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 2011-150436 | Jul 2011 | JP | national |
