JP4678544B2 - Optical quantification device - Google Patents

Description

  The present invention relates to a quantification apparatus for optically quantifying a pigment that precipitates and forms by a specific reaction.

  Conventionally, mass spectrometry using a large instrument, (high-speed) liquid chromatography, and the like are known as methods for quantifying trace amounts of chemical substances. In addition, bioassays that quantify and detect trace components using biological reactions are known. An ELISA method is known as one of bioassays. The ELISA method has a high sensitivity and is widely adopted as an official method in various inspection methods. Its feature is that the detection sensitivity is generally good and it can be quantified by adding control to the test group. However, there are drawbacks in that the operation is complicated and errors and variations due to the skill level of the measurer are large. In addition, a large amount of reaction reagent and solution are required for working hours as long as several hours.

  Therefore, the development of methods that can immediately determine the results at the site is progressing. For example, using an antigen-antibody reaction, an immunochromatography method that can specifically detect a test substance contained in the test liquid simply by dropping the test liquid, a glucose sensor that detects a test substance using an enzyme, SPR using surface plasmon resonance is exemplified. Among them, the immunochromatography method is easy to operate, and is applied to products for general consumers such as pregnancy test drugs.

  In the immunochromatography method, an antigen-antibody binding reaction is performed by infiltrating a reaction solution into a solid phase carrier on which an immune antibody or an immune antigen is immobilized by capillary action, and the binding amount is measured by some method. In the immunochromatography method, an antigen-antibody reaction unique to a test substance proceeds by sequentially dropping a plurality of treatment liquids containing a test liquid containing the test substance. The immunochromatography method incorporates a structure that allows the visualization reaction to progress such that the color tone changes as the antigen-antibody reaction progresses. The presence or amount of the test substance can be determined by examining the changing color tone. To detect. In the immunochromatography method, not only the presence of the test substance but also the amount of the test substance is detected as a change in color tone.

  Various reaction systems and labels are used in the visualization reaction for bringing about a color change to be finally detected. For example, a method of employing latex particles colored with an oil-soluble dye as a label is disclosed (Patent Document 1). This method is a method for performing qualitative determination based on changes in lightness and saturation before and after the reaction.

  Further, latex particles to which an antibody or antigen-binding fragment thereof is bound, a medium for suspending the latex particles, at least one aggregation inhibitor selected from the group consisting of saccharides and polyhydric alcohols, proteins, and buffers A latex composition for immunoassay having a pH of 9.0 to 9.8 is disclosed (Patent Document 2). This method performs qualitative judgment by visual observation.

  A magnetic inclusion particle comprising an organic polymer substance and a magnetic material dispersed in the organic polymer substance with a dispersion diameter of 1 to 30 nm, and having an average particle diameter of 50 to 300 nm and 0.01N A magnetic substance-encapsulated particle having a zeta potential measured in an aqueous sodium solution of −10 to −50 mV is disclosed (Patent Document 3). By applying an antigen or an antibody to the magnetic substance-encapsulated particles, it is applied to a label for an immunochromatography method. This is considered to be determined by measuring magnetism.

Furthermore, a measurement method of an immunochromatography method in which a substrate is colored using an enzyme is disclosed (Patent Document 4). As a method using an enzyme, there is a method in which a light source or an antibody involved in an antigen-antibody reaction is modified with an enzyme, and a substrate change due to the enzyme reaction using the enzyme is detected as a color tone change. This method can be measured with high accuracy even if the amount of the test substance to be detected is very small. Alkaline phosphatase is widely used as the enzyme employed. As a color development reaction applicable to a quantification method using alkaline phosphatase, a reaction between a substrate composed of bromochloroindolyl phosphate (BCIP) and a color former composed of nitro blue tetrazolium (NBT) is widely used. Since the amount of precipitated pigment produced also varies depending on the amount of alkaline phosphatase, the amount of alkaline phosphatase present can be quantified by quantifying the precipitated pigment produced by the reaction.
Japanese Patent No. 2955405 (Claim 1 etc.) JP 2007-315883 A (Claim 1 etc.) JP 2006-145256 A (Claim 1 etc.) Japanese Patent Laid-Open No. 10-300750 (Claim 1 etc.)

  However, the techniques of Patent Documents 1 to 3 only make qualitative judgments and are difficult to determine. The technique of Patent Document 4 analyzes a two-dimensional image obtained using a CCD camera. Therefore, there is a problem that the change in color tone is analyzed and the apparatus becomes complicated and the analysis is complicated.

  In addition, since the immunochromatographic reaction reacts while the reaction solution permeates (moves), it is difficult to average the solid phase carrier, and the background when detecting the precipitated dye increases and the SN ratio decreases. It is difficult to ensure sufficient measurement accuracy.

  This invention is made | formed in view of the said situation, and makes it the problem which should be solved to provide the optical quantification apparatus which can improve a S / N ratio and can quantify with high precision in the immunochromatography method which combined BCIP and NBT.

  The optical quantification device of the present invention according to claim 1 which solves the above-mentioned problem is characterized in that a precipitated dye produced by a reaction between a substrate composed of bromochloroindolyl phosphate and a color former composed of nitroblue tetrazolium is optically produced. An optical quantification device for quantification,
  An LED having an emission wavelength greater than 470 nm and less than 573 nm;
  An optical sensor sensitive to the emission wavelength;
  Conversion means for deriving a value corresponding to the amount of the precipitated pigment from the output signal of the photosensor;
  An optical system that irradiates the precipitation dye with light emitted from the LED and guides reflected light from the precipitation dye to the optical sensor;
  Have
  The light emission wavelength of the LED is 500 nm or more and 540 nm or less.

A feature of the optical quantification device of the present invention according to claim 2 that solves the above-mentioned problem is that, in claim 1 , the emission wavelength of the LED is 510 nm, 515 nm, 517 nm, 518 nm, 522 nm, or 523 nm.

In the optical quantification device according to claim 1 configured as described above, the range of wavelengths specifically absorbed by the precipitated dye produced by the reaction of BCIP and NBT is examined and discovered, and light of that wavelength is selected. By combining LEDs that can be released in an efficient manner, the amount of the precipitated pigment can be determined with high accuracy. Since the light emitted from the LED has almost a single wavelength, it is possible to selectively irradiate light having a wavelength that can be specifically absorbed by the precipitated dye, and noise is generated by irradiating light having an extra wavelength. Can be suppressed. In particular, by adopting this wavelength range, the quantitative accuracy of the precipitated pigment can be improved. Further, as defined in claim 2 , measurement at a high S / N ratio can be easily realized by adopting the wavelength of a commercially available LED.

  Hereinafter, the optical quantification device of the present invention will be described in detail based on embodiments. The optical quantification apparatus of this embodiment is an apparatus for optically quantifying the amount of precipitated pigment produced by a reaction using BCIP as a substrate and NBT as a color former. BCIP and NBT are combined for a reaction system employing alkaline phosphatase as a labeling enzyme. The antibody or antigen employed according to the test substance such as PCB is modified with alkaline phosphatase and immobilized on a solid phase carrier, and then the treatment liquid containing the test substance is flowed to advance the antigen-antibody reaction. Let Thereafter, BCIP acts as a substrate for alkaline phosphatase, and NBT acts as a color former. As a specific color reaction, BCIP is hydrolyzed by alkaline phosphatase to produce an intermediate, and this intermediate and NBT react to dimerize the intermediate. This dimer produces a blue to purple precipitate. Hereinafter, the generated precipitate (precipitated dye) is referred to as a precipitated dye. This precipitated dye can be deposited on a nitrocellulose film, a nylon film or the like, and is measured in a state where it is precipitated as a precipitated dye on them.

  The optical quantification apparatus of this embodiment is an apparatus that optically measures the amount of precipitated pigment. It quantifies by irradiating the precipitation pigment | dye with the light which LED light-emits, and measuring the magnitude | size of the reflected light. Light is absorbed by measuring and comparing the reflected light at the place where the precipitated dye is precipitated and the place where the surrounding dye is not precipitated, and by measuring and comparing the irradiated light and the reflected light. Can be calculated. In this manner, the precipitation pigment is irradiated with light emitted from the LED, and an optical system for measuring the reflected light is provided. For example, a dark box in which light from the outside is blocked can be formed, a sample to be measured can be placed therein, and an optical system formed by combining lenses or the like can be employed.

The emission wavelength of the LED is from 500 nm to 540 nm . In particular , the emission wavelength is more preferably 510 nm, 515 nm, 517 nm, 518 nm, 522 nm, or 523 nm. Since the emission wavelength has a certain range, the “emission wavelength” in this specification is specified by the peak position of the spectrum of the LED (the position where the emission intensity is highest). The emission wavelength of an LED is determined by the band gap of the pn bond formed by the semiconductor constituting the LED. The size of the band gap can be controlled by the type of material constituting the semiconductor and the type and amount of impurities added. As materials constituting the semiconductor, gallium phosphide (GaP), indium gallium nitride / gallium nitride / aluminum gallium nitride (InGaN / GaN / AlGaN), zinc selenide (ZnSe), aluminum indium gallium phosphide (AlGaInP), gallium arsenide phosphorus (GaAsP) can be exemplified.

  The optical sensor has sensitivity at the emission wavelength of the adopted LED, and is a sensor capable of taking out an output signal corresponding to the light intensity. Examples of the optical sensor include a photodiode, a CCD, a CMOS sensor, a photomultiplier tube, and a photoresistor such as CdS. Further, an area sensor, a line sensor, a photodiode array, or the like employing a CCD or a CMOS can be employed.

  The conversion means is means for deriving a value corresponding to the amount of precipitated pigment based on the output signal from the optical sensor. There is a method of calculating the amount of precipitated pigment by calculation after converting the output signal into digital data by an AD converter.

  As a specific measurement method, the intensity of the reflected light at the measurement site where the precipitated dye is supposed to adhere and the reflected light at the control site where no other precipitated pigment is assumed to be attached are measured. An example is a method of calculating the amount of precipitated pigment based on a calibration curve prepared in advance after calculating the intensity of reflected light at a measurement site using the intensity of reflected light as a background. In addition, the peak intensity can be calculated after continuously measuring the intensity of the reflected light at the site where the precipitated pigment is adhered and the control site in the vicinity thereof. Furthermore, the amount of precipitated pigment can be quantified only by measuring the reflected light at the measurement site without measuring the reflected light at the control site.

  The optical quantification apparatus of the present invention will be described in detail below based on examples. The drawings used in this example are schematic diagrams. For convenience of explanation, the scale, the ratio of the size of each member, and the like do not reflect the actual size. Some parts of the shape of the member are omitted. As shown in FIG. 1, the optical quantification apparatus 1 of this embodiment includes an LED 10, an optical system 20 (an irradiation lens 21 and a condensing lens 22), a photodiode 30, and a control means 40, and a test sample. This is an apparatus for quantifying the amount of precipitated dye 91 adhering to the piece 90. The light emitted from the LED 10 is condensed by the irradiation lens 21 and irradiated to the precipitation dye 91 that adheres to the test sample piece 90. The reflected light from the precipitation dye 91 is collected by the condensing lens 22 and collected on the photodiode 30. The control means 40 corresponds to the amount of the precipitated dye 91 from the LED control means for controlling the light emission of the LED 10, the measurement means for measuring the intensity of the light detected by the photodiode 30, and the measurement value measured by the measurement means. Conversion means for deriving the value. The test sample piece 90 is arranged so that the precipitation dye 91 is located at the focal point of the irradiation lens 21 and the condensing lens 22. The optical quantification apparatus 1 also measures the control parts 92 and 93 adjacent to the precipitated dye 91. The conversion means calculates a value corresponding to the amount of the precipitated dye 91 from the intensity of the reflected light at the precipitated dye 91 and the intensity of the reflected light at the control portions 92 and 93.

  In this example, the surface of the test sample piece 90 to which the precipitated dye 91 was adhered was continuously measured with a length of 12 mm using this apparatus. Two test sample pieces 90 were prepared (Samples 1 and 2). On this test sample piece 90, two precipitation pigments 91 precipitated in a line are attached in parallel in the measurement direction (interval is slightly over 5 mm, about 4 mm (A) and about 9 mm (B) from the measurement start site). The length of 12 mm was measured continuously in the parallel direction. The emission wavelength of the LED 10 is blue (product number: E1L53-3B0A2-02, manufactured by Toyoda Gosei, dominant emission wavelength is 465 nm to 470 nm), green (product number: UG5305S, manufactured by Stanley Electric, peak emission wavelength is 517 nm), yellow (product number: NSPY500S, manufactured by Nichia Corporation, dominant emission wavelength of 573 nm to 577 nm) and red (product number: NSPR510CS, manufactured by Nichia Corporation, dominant emission wavelength of 615 nm to 635 nm) were employed for testing. Here, the peak emission wavelength represents the wavelength with the highest output among the light emitted from the LED, and the dominant emission wavelength is a wavelength obtained by quantifying the color of the emitted LED light as seen by the human eye. Represents.

  The actual measurement results are shown in FIG. 2 (Sample 1) and FIG. 3 (Sample 2). Table 1 shows the degree of light absorption of the precipitated dye obtained from the intensity of the reflected light at the portion of the precipitated dye 91 and the control parts 92 and 93. Shown in 2 and 3, a predetermined coefficient is multiplied so that the background level is the same for each emission wavelength of the LED. In Table 1, the SN ratio increases as the peak intensity value (BG-P) obtained by subtracting the peak value (P) from the background value (BG) increases. Note that the background value in Table 1 creates an approximate line that seems to be the background excluding the peak, and the value of the position corresponding to the tip of the peak in the approximate line (the position corresponding to the length direction) The ground.

  As is clear from Table 1 and FIGS. 2 and 3, green (emission wavelength: 517 nm), blue, yellow, red from the larger BG-P value in any of the precipitated dyes A and B in Samples 1 and 2, respectively. It became clear that the SN ratio was excellent in this order. In particular, when an emission wavelength of 517 nm (green) is employed, the peak intensity is increased by about 20% compared to the case of blue. The green results are 30% (yellow) and 30 to 40% (red) compared to the yellow and red results, and the results of mutual comparison between blue, yellow and red are 10 to 20%. It was clarified that a peak intensity protruding around 517 nm (green) was exhibited only with a difference in degree. Further, it is considered that high peak intensities can be obtained even with widths of about ± 20 nm and ± 5 nm around 517 nm, which are closer to the green wavelength than blue or yellow.

  Here, the change in absorbance before and after the reaction of BCIP and NBT was measured. FIG. 4 shows the result of measuring the absorbance of a simple mixture before the reaction, and FIG. 5 shows the result of measuring the absorbance of the precipitated dye after the reaction. From the comparison of FIGS. 4 and 5, it was revealed that the absorbance increased between 500 nm and 700 nm due to the reaction. In particular, it was found to have a peak at about 580 nm. When this result is compared with the result of the S / N ratio described above, the S / N ratio of the green LED having the emission wavelength at the base of the peak is higher than that of the yellow LED having the emission wavelength at a wavelength with a large absorbance. It has been clarified that the LED has a factor affecting the SN ratio in addition to the absorbance. This is presumed to be due to the amount of precipitated pigment being quantified by reflected light.

It is the schematic of the optical quantification apparatus in an Example. It is a figure which shows the test result of the sample 1 in an Example. It is a figure which shows the test result of the sample 2 in an Example. It is a figure which shows the visible light absorption spectrum of the mixture of BCIP and NBT. It is a figure which shows the visible light absorption spectrum of the reaction material (precipitation dye) of BCIP and NBT.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 ... Optical quantitative determination apparatus 10 ... LED 20 ... Optical system (21: Lens for irradiation, 22: Lens for condensing) 30 ... Photodiode (light sensor) 40 ... Control means (including conversion means)
90: Test specimen 91: Precipitated dye 92, 93: Control site

Claims (2)

An optical quantification apparatus for optically quantifying a precipitated dye produced by a reaction between a substrate composed of bromochloroindolyl phosphate and a color former composed of nitroblue tetrazolium,
An LED having an emission wavelength greater than 470 nm and less than 573 nm;
An optical sensor sensitive to the emission wavelength;
Conversion means for deriving a value corresponding to the amount of the precipitated pigment from the output signal of the photosensor;
An optical system that irradiates the precipitation dye with light emitted from the LED and guides reflected light from the precipitation dye to the optical sensor;
I have a,
The emission wavelength of the LED optical quantification device according to claim der Rukoto than 540nm or less 500 nm. The optical quantification apparatus according to claim 1 , wherein an emission wavelength of the LED is 510nm, 515nm, 517nm, 518nm, 522nm or 523nm.

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