JP2005274545A - Concentration measuring method and concentration measuring device for minor component - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a concentration measuring method and concentration measuring device capable of easily measuring a minor component contained in a sample such as cholesterol contained in a body fluid with a simple device configuration and high sensitivity. <P>SOLUTION: A sample containing the minor component is placed on a hydrophobic porous membrane, and after drying the sample, the minor component is expanded by a solvent. The hydrophobic porous membrane with the minor component expanded thereon is brought into contact with a dyeing material, the intensity of dyeing in the resulting dyeing band of the minor component is optically measured, and the minor component is quantitatively determined based on the measurement result. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a concentration measuring method and a concentration measuring apparatus for quantifying a trace component in a sample such as cholesterol contained in a body fluid.

  Currently, in many clinical laboratories of hospitals, an automatic analyzer using a cholesterol oxidase method (COD method) is used to measure cholesterol concentration in serum. In this COD method, cholesterol oxidase is allowed to act on a sample to produce hydrogen peroxide, and by the action of peroxidase, for example, 3,5-dimethoxy-N-ethyl-N- (2-hydroxy-3-sulfopropyl) -aniline Sodium (DAOS) and 4-aminoantipyrine are oxidatively condensed to form a dye, and then the cholesterol in the sample is quantified by measuring the absorbance.

As another method for measuring serum cholesterol concentration, a so-called enzyme cycling method has been proposed (see Patent Documents 1 and 2). In this method, an enzyme cycling reaction is formed by cholesterol dehydrogenase, oxidized coenzyme, and reduced coenzyme, and the amount of cholesterol in the sample is quantified based on this reaction.
JP-A-8-103298 Japanese Patent Application Laid-Open No. 11-018798

Cholesterol is also contained in saliva and the like in addition to blood, and if this trace amount of cholesterol can be measured, a cholesterol test can be performed without collecting blood. However, the automated testing equipment using the COD method, which is normally used in clinical laboratories, is manufactured for the purpose of measuring cholesterol in the serum, so it is low in other body fluids such as saliva. It is difficult to measure the concentration of cholesterol with this device.

For example, it is required to quantify 1 mg / dL or less for salivary cholesterol, 5 mg / dL or less for urine cholesterol, and 50 mg / dL or less for cholesterol in serum HDL (high density lipoprotein) fractions. However, since it is optimized to quantitate cholesterol of about 100 mg / dL or more in the laboratory equipment usually used in clinical laboratories, it can cope with the low concentration measurement as described above. It is necessary to change the measurement system of the inspection device significantly.

In the COD method and enzyme cycling method, it is necessary to use relatively expensive enzymes and reagents such as enzymes, mediators and coenzymes that react specifically with cholesterol.
The present invention has been made in order to solve the above-described problems in the prior art. For example, a trace component contained in a sample, such as cholesterol contained in a body fluid, can be easily obtained with a simple apparatus configuration. An object of the present invention is to provide a concentration measuring method and a concentration measuring apparatus capable of measuring with high sensitivity.

  The present inventor uses a hydrophobic porous membrane formed using a specific resin, develops a sample containing cholesterol with a solvent in the hydrophobic porous membrane, then immerses it in an aqueous dye solution, and then polarizes the sample. By washing with a solvent, a sharp stained band is obtained from cholesterol separated from other components, and it is found that the concentration of cholesterol can be measured with high sensitivity by optically detecting this stained band to complete the present invention. It came.

  Furthermore, after developing a sample containing a trace component using a specific hydrophobic porous membrane in this way, contacting this hydrophobic porous membrane with a staining material to obtain a stained band, As a result, the present inventors have found that the concentration of not only cholesterol but also other trace components can be measured with high sensitivity.

  In the method for measuring the concentration of a trace component of the present invention, a sample containing the trace component is placed on a hydrophobic porous membrane, the sample is dried, the trace component is developed with a solvent, and then the trace component is developed. The obtained hydrophobic porous membrane is brought into contact with a staining substance, the intensity of staining in the staining band of the obtained trace component is optically measured, and the trace component is quantified based on the measurement result. It is said.

  The hydrophobic porous membrane used in the present invention is a porous membrane formed using a hydrophobic resin, and the hydrophobic resin is selected from polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, polyester and nylon. It is preferable that it is resin.

  In the concentration measurement method of the present invention, the sample is placed on a wide sample placement portion in the hydrophobic porous membrane, and then a solvent is brought into contact with the sample placement portion, so that it is narrower than the sample placement portion. The present invention is characterized in that the trace component is separated from other components by introducing the trace component into the development part formed in the width and developing in the longitudinal direction of the development part.

  In the concentration measurement method of the present invention, the hydrophobic porous membrane in which the trace component is developed is immersed in an aqueous dye solution, the trace component and the dye are brought into contact with each other, and then the hydrophobic porous membrane is washed with a polar solvent. Thus, a dyeing band of the trace component is obtained.

  The above-described concentration measurement method of the present invention is suitable for quantifying a trace amount of cholesterol contained in a sample. When quantifying cholesterol, it is preferable to use Nile Blue as a dye. Measure the absorbance of the cholesterol staining band obtained by staining with Nile Blue and measure the cholesterol concentration with high sensitivity based on this measurement result. can do.

The concentration measurement apparatus for trace components of the present invention is equipped with a hydrophobic porous membrane in which a sample containing trace components is placed, the sample is dried, and then the trace components are developed with a solvent.
And a means for optically measuring the intensity of staining in a staining band of the trace component obtained by bringing the hydrophobic porous membrane in which the trace component is developed into contact with the staining substance.

The concentration measuring device for trace components according to the present invention is characterized in that the hydrophobic porous membrane has a wide sample mounting portion for mounting the sample,
It has a developing part that extends from the sample mounting part and is formed to be narrower than the sample mounting part and that develops a trace component in the longitudinal direction and separates it from other components.

  The above-described concentration measuring apparatus of the present invention is suitable for quantifying a trace amount of cholesterol contained in a sample. When quantifying cholesterol, Nile Blue is used as a staining substance, and after immersing the hydrophobic porous membrane on which cholesterol has been developed in an aqueous solution of Nile Blue, a stained band is obtained by washing the hydrophobic porous membrane with a polar solvent. Preferably, the absorbance is measured using an absorptiometer as a means for optically measuring the intensity of staining in the staining band, and the cholesterol concentration can be measured with high sensitivity based on the measurement result.

According to the present invention, a trace component contained in a sample can be measured with high sensitivity with a simple apparatus configuration.

  Hereinafter, the present invention will be described in detail. FIG. 1 is a schematic view of a hydrophobic porous membrane used in a concentration measurement method according to an embodiment of the present invention. In this embodiment, the concentration of a trace amount of cholesterol contained in a sample such as saliva or urine is measured. The hydrophobic porous membrane 1 in FIG. 1 is made of polyvinylidene fluoride (PVFT), and has pores capable of developing cholesterol with an organic solvent by capillary action.

  In the present invention, a porous membrane formed using a hydrophobic resin is used as the hydrophobic porous membrane. Examples of the hydrophobic resin include polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, polyester, and nylon. When quantifying cholesterol, it is preferable to use polyvinylidene fluoride or polytetrafluoroethylene. In addition, the hydrophobic porous membrane in which the trace component is developed is immersed in an aqueous dye solution so that the trace component and the dye are brought into contact with each other, and then the hydrophobic porous membrane is washed with a polar solvent to dye the trace component. When obtaining a band, it is necessary to use a dye that can sufficiently remove the dye from the hydrophobic porous membrane by washing the dye used with a polar solvent.

  A sample containing cholesterol is placed on the sample placement unit 2 in FIG. 1 and dried to remove moisture, and then the end of the hydrophobic porous membrane 1 on the sample placement unit 2 side is immersed in an organic solvent. And expand. Cholesterol and some other components in the dried sample are introduced from the sample mounting unit 2 to the developing unit 3 and developed in the longitudinal direction of the developing unit 3. This separates cholesterol from other components in the sample.

  The organic solvent used for the development is appropriately selected depending on the combination of the resin that forms the hydrophobic porous membrane and the sample that contains the trace component that is the measurement target. In this embodiment, a mixed solvent of 2-propanol and acetonitrile is used.

  As shown in FIG. 1, it is particularly preferable that the hydrophobic porous membrane has a wide sample mounting portion 2 and a narrow developed portion 3 formed to be narrower than this. In the present embodiment, a hydrophobic porous membrane 1 having a fuse shape is used, and a thin developed portion 3 (width: 2 mm) extends in the longitudinal direction from a wide planar sample placement portion 2 (width: 5 mm). It has a shape that extends. Also, a wide surface is formed at the end of the developing unit 3 on the side opposite to the sample mounting unit 2 to constitute a solvent absorbing unit 4 that absorbs the solvent from the developing unit 3.

  The developing unit 3 is formed in a thin shape so as to separate a trace component that is a measurement target from other components and to obtain a sharp band that can be detected with high sensitivity. On the other hand, since the sample mounting portion 2 is formed wide, it is easy to spot the sample 5. After drying the sample 5 spotted on the sample 5 to remove moisture, the end portion is immersed in a developing solvent to narrow it. It can be introduced into the widened portion 3.

  By developing a sample containing cholesterol in this manner, cholesterol is separated from other components, and a sharp band enriched with cholesterol is formed in the developed part 3. Next, the hydrophobic porous membrane 1 in which cholesterol is developed is immersed in an aqueous Nile blue solution to stain this band. After soaking for a predetermined time, the hydrophobic porous membrane 1 is washed with a polar solvent such as water or methanol to remove the dye on the membrane, thereby obtaining a cholesterol staining band 6 as shown in FIG. .

After obtaining a band stained with Nile Blue, the reflected absorbance of this band is measured using an absorptiometer. As shown in FIG. 2, when scanning is performed with measurement light corresponding to the visible absorption wavelength of Nile Blue in the longitudinal direction of the development portion 3 in the vicinity of the staining band, a peak 7 having a sharp absorbance is obtained at the position of the staining band. The development and band staining were performed under the same conditions using a plurality of standard samples with known cholesterol concentrations in advance, and the integrated value (peak area) of the absorbance peak portion by the stained band obtained at the same location of the development part 3 ) And cholesterol concentration are prepared, and the concentration is quantified by comparing the peak area measured for the sample of unknown concentration with this.

As described above, it is required to quantify at 1 mg / dL or less for cholesterol in saliva and 5 mg / dL or less for cholesterol in urine, and 50 mg / dL for cholesterol in serum HDL (high density lipoprotein) fraction. Although it is required to quantify in the following, according to the present invention, such a highly sensitive cholesterol with a simple device configuration of a hydrophobic porous membrane and means for detecting a staining band such as an absorptiometer. Concentration measurement is possible. Furthermore, after the sample is placed on the hydrophobic porous membrane, the time required for color development on the membrane is, for example, about 30 minutes, and it is only necessary to dye the band using a relatively inexpensive dye, It can be measured efficiently with a simple operation. For this reason, it is suitable for automation of the apparatus and can be downsized as compared with the conventional cholesterol concentration measuring apparatus.

  In the present invention, the dye is used as a dyeing substance for dyeing the developed trace component, and the hydrophobic porous membrane is immersed in an aqueous dye solution, thereby bringing the dye and the trace component to be quantified into contact with each other and adsorbing them. Although it is preferable to obtain a dyed band by washing and removing the dye adhering to the hydrophobic porous membrane with a polar solvent, the dyeing substance and the trace component may be contacted by other methods. For example, a so-called enzyme antibody method may be applied to react an antibody labeled with an enzyme directly or indirectly to a trace component whose concentration is to be measured, and the enzyme may chemically develop a redox dye. . In the present invention, since a hydrophobic porous membrane is used as a base material on which a sample is developed, depending on the sample, a trace amount component is smaller than a paper chromatograph using a filter paper or a thin layer chromatograph using silica gel, alumina, or the like. Suitable for quantification.

  In this embodiment, an example of measuring the concentration of cholesterol has been shown. However, according to the present invention, free cholesterol, triglyceride and cholesterol ester can be separated and directly quantified, and all neutral lipids in saliva can be directly quantified. Can be used to test for hypercholesterolemia or hypertriglyceridemia. In a system using blood as a sample, HDL (high density lipoprotein) and LDL (low density lipoprotein) can be separated and quantified.

  In this way, when separating and quantifying multiple components, as shown in FIG. 3, between the two hydrophobic porous membranes 1a and 1b, these membranes have different characteristics for separating each component. The separation membrane 8 may be interposed and connected to form a developing membrane.

  In addition, if a hydrophobic membrane resin is appropriately selected and a water-soluble solvent is used as a developing solvent, trace components such as stress hormones contained in saliva can be separated, and then the enzyme antibody method, dot blot method, etc. It can be quantified using techniques.

  As described above, in the present invention, it is possible to separate and quantify trace components in various samples by appropriately selecting the material of the hydrophobic porous membrane, the developing solvent, the staining substance, the optical measurement system, etc. according to the purpose. is there. As the staining material, fluorescent dyes, compounds that develop color by chemical reaction, and the like can also be used. Further, as a means for optically measuring the staining intensity of the staining band, a spectrophotometer or the like may be used in addition to the absorptiometer depending on the dye used.

EXAMPLES Hereinafter, although an Example demonstrates this invention, this invention is not limited to these Examples.
[Example 1]
Saliva collected from the submandibular line and the parotid line on the PVDF porous membrane (length in the longitudinal direction: 30 mm, width of the sample mounting part: 5 mm, width of the developing part: 2 mm) shown in FIG. 1 to 3 μL was dried and dried, and then a 2-propanol / acetonitrile mixed solvent (volume ratio 3: 2) was immersed in the end of the sample mounting portion and developed for 7 minutes.

  Next, this PVDF porous membrane was dyed by immersing it in a 1% aqueous solution of Nile Blue dye (manufactured by Chroma) for 10 minutes. After staining, the porous membrane was washed with water and methanol for several minutes to obtain a sharp stained band of cholesterol on the downstream side of the developed portion.

  About the area | region containing this dyeing | staining band, reflection absorption was measured with the measurement light corresponding to the absorption wavelength of Nile blue using the commercially available absorptiometer. The concentration pattern obtained by the absorbance OD conversion had a sharp peak as shown in FIG.

  On the other hand, using a plurality of standard samples containing known amounts of cholesterol in advance, developing and staining in the same manner as described above, and measuring the amount of cholesterol based on the integrated value of the peak portion of the concentration pattern obtained by measuring reflection absorption As a result, a good linearity was established as shown in FIG. 4, and it was possible to quantify the detection limit to 0.03 mg.

With respect to the saliva sample collected from the submandibular line and the parotid line, the cholesterol level was measured based on the calibration curve. The average of 7 sets of measurements was 6.4 mg / dL for samples taken from the submandibular line and 4.6 mg / dL for samples taken from the parotid line.
[Comparative Example 1]
Using a normal thin layer chromatograph using silica gel, cholesterol solutions of various concentrations (chloroform: methanol = 2: 1) were spotted on the thin layer and developed with a developing solvent (chloroform: methanol = 89: 11). . Next, a ferric chloride solution (50 mg of ferric chloride, 5 ml of acetic acid, 5 ml of concentrated sulfuric acid, and 90 ml of distilled water) was sprayed on the surface of the thin layer, and the firing spots were colored at 110 ° C. When the reflection absorption of the obtained spot was measured, the detection limit was 0.3 mg.
[Comparative Example 2]
A normal thin layer chromatograph using silica gel developed a saliva sample in the same manner as in Example 1 to separate cholesterol, and then the stationary phase was stained with an aqueous dye solution (Nile Blue) and washed. As a result, the cholesterol staining band could not be obtained.

FIG. 1 is a schematic view of a hydrophobic porous membrane used in one embodiment of the present invention. FIG. 2 is a view showing a state where a cholesterol staining band is formed on the hydrophobic porous membrane of FIG. FIG. 3 is a schematic view of a hydrophobic porous membrane used in another embodiment of the present invention. FIG. 4 is a graph showing the relationship between the peak area by the stained band and the amount of cholesterol.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Hydrophobic porous membrane 2 Sample mounting part 3 Expanding part 4 Solvent absorption part 5 Sample 6 Staining band 7 Absorption peak 8 Separation membrane

Claims (14)

  A sample containing a trace component is placed on the hydrophobic porous membrane, the sample is dried, the trace component is developed with a solvent, and then the hydrophobic porous membrane on which the trace component is developed and the staining substance are combined. A method for measuring the concentration of a trace component, comprising: contacting the substrate, optically measuring the intensity of staining in the staining band of the trace component obtained, and quantifying the trace component based on the measurement result.   The method for measuring the concentration of trace components according to claim 1, wherein the hydrophobic porous membrane is a porous membrane formed using a hydrophobic resin.   The method for measuring the concentration of a trace component according to claim 2, wherein the hydrophobic resin is selected from polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, polyester and nylon.   The sample is placed on a wide sample mounting portion in the hydrophobic porous membrane, and then the solvent is brought into contact with the sample mounting portion, so that the trace component is transferred to the developing portion formed narrower than the sample mounting portion. The method for measuring a concentration of a trace component according to any one of claims 1 to 3, wherein the trace component is separated from other components by introducing the component in the longitudinal direction of the development portion.   A hydrophobic porous membrane in which a trace component is developed is immersed in an aqueous dye solution, the trace component and the dye are brought into contact with each other, and then the hydrophobic porous membrane is washed with a polar solvent to thereby stain the trace component. The method for measuring a concentration of a trace component according to any one of claims 1 to 4, wherein:   6. The method for measuring the concentration of a trace component according to claim 5, wherein the trace component for measuring the concentration is cholesterol.   The method for measuring the concentration of a minor component according to claim 6, wherein the dye is Nile Blue.   8. The method for measuring the concentration of a trace component according to claim 7, wherein the absorbance of the cholesterol staining band is measured, and cholesterol is quantified based on the measurement result. A hydrophobic porous membrane in which a sample containing a trace component is placed, and after drying this sample, the trace component is developed with a solvent;
Means for optically measuring the intensity of staining in a staining band of the trace component obtained by bringing the hydrophobic porous membrane in which the trace component is developed into contact with a staining substance Concentration measuring device.   The apparatus for measuring a concentration of a trace component according to claim 9, wherein the hydrophobic porous film is a porous film formed using a hydrophobic resin.   The concentration measuring apparatus for trace components according to claim 10, wherein the hydrophobic resin is selected from polyvinylidene fluoride, polytetrafluoroethylene, polyethylene, polypropylene, polyester and nylon. A hydrophobic porous membrane has a wide sample mounting portion for mounting the sample;
A developing portion that extends from the sample mounting portion and is formed to be narrower than the sample mounting portion and expands a trace component in the longitudinal direction to separate it from other components. The concentration measuring apparatus of the trace component in any one of 11.   The device for measuring a concentration of a trace component according to any one of claims 9 to 12, wherein the means for optically measuring the intensity of staining in the staining band of the trace component is an absorptiometer.   The trace component concentration measuring apparatus according to any one of claims 9 to 13, wherein the trace component for measuring the concentration is cholesterol.

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