JPS61272637A - Measuring instrument for polarized fluorescent light - Google Patents

Abstract

PURPOSE:To obtain an invariably accurate value by providing a cell which contains a sample, a polarizing filter for exciting light which illuminates the cell, and fixed polarizing filters which pass the vertical and horizontal components of fluorescent light from the sample, and computing the degree of polarization of the fluorescent light from photodetection signals which are obtained at the same time. CONSTITUTION:The sample cell 3 which is treated as specified is irradiated with light obtained by making a selection 11 of wavelength of a light source 1 and performing polarization 2. The vertical polarized component of fluorescent light emitted by a sample is passed through the filter 7 and photodetected 14 and the horizontal polarized component is passed through the filter 8 and photodetected 4; and selections 12 and 13 of wavelengths are made on respective optical paths to select the same fluorescent light wavelength. Signals which are detected 4 and 14 as to the same light source at the same time are amplified 5 and 15 and the degree of polarization of fluorescent light is computed 9 and displayed 6. Consequently, the reaction of variation in the intensity of the fluorescent light from the sample is tracked accurately and even when background fluorescent light varies, an accurate measured value is obtained.

Description

DETAILED DESCRIPTION OF THE INVENTION [Field of Application of the Invention] The present invention relates to a fluorescence photometry measurement device, and particularly to a fluorescence photometry measurement device suitable for quantitatively measuring a target substance in a biological fluid.

[Background of the invention]

Fluorescence photometry measurement devices are used to quantify trace amounts of antigens and antibodies in biological fluids using antigen-antibody reactions.

When a fluorescent molecule is excited by polarized light, the rotation of the molecule due to Brownian motion between absorption and emission of light dissolves the orientation of the excited molecule and reduces the degree of polarization of the fluorescent light. If the polarizing plate on the excitation light side is fixed vertically and the polarizing plate on the tip side is parallel or perpendicular to it and the fluorescence intensities obtained are ヮ and Io, respectively, then the degree of fluorescence polarization P is is expressed by the following equation.

When an antigen labeled with a fluorescent substance binds to the corresponding antibody, the molecule becomes significantly larger, the rotational Brownian motion of the fluorescent molecule is suppressed, and the degree of polarization becomes greater than before binding with the antibody. A device that utilizes the above principle is polarization fluoroimun analysis.
First, a fluorescently labeled antigen and an antibody are reacted, and when an unlabeled antigen (unknown substance) to be measured is added to this system, the antibody is consumed, and the amount of labeled antigen that binds to the antibody decreases, resulting in no binding. There will be more things. Since this substance is a low molecule, rotational Brownian motion occurs actively, and the degree of polarization decreases, so it is possible to find the unknown substance wax according to the degree of decrease. A difference occurs in the Brownian motion before and after JJ, which has a small molecular density of the fluorescently labeled antigen, binds to the antibody, and a higher sensitivity Ja' can be obtained. Fluorescein-based fluorescent substances are generally used as fluorescent substances in this method.

Conventional equipment for measuring the preemptive luminosity P measures the perpendicular component of the polarized light component (T) and one component (1,) ti: by rotating the polarizing plate, it is measured at the intersection of 77. There was a time difference in the 21tq constant. In order to determine the Il value of the beach quality, it was necessary to use each polarized light intensity at different measurement timings, so there was a drawback that it was not possible to obtain an accurate JJ value at a certain measurement point. For this reason, it has been difficult to accurately track the reaction in which the fluorescence intensity changes over time when measuring polarized light components. Furthermore, when the background fluorescence changes over time, it was not possible to distinguish between this background change and the fluorescence intensity derived from the target component.

[Invented on October 1] The object of the present invention is to accurately track reactions in which the fluorescence intensity from a sample changes, and to obtain accurate measurement values even when Pack Brown 1-II fluorescence fluctuates. The purpose of the present invention is to provide a light measuring device that can be used for commercial purposes.

“Summary of the Invention” The present invention provides a cell that can accommodate a sample, a polarizing filter that polarizes excitation light irradiated to the cell, a first fixed polarizing filter that passes a vertically polarized component of fluorescence from the sample, a second Ii' that passes the +lZ row polarized component of fluorescence from the sample;
It is characterized by being equipped with an I constant polarization filter and an arithmetic device that calculates the preemptive luminous intensity of the vertical component and the IP component light reception signal obtained at the same # and ¥.

[Embodiments of the invention]

FIG. 1 shows a schematic configuration of an embodiment of the present invention.

The sample cell 3 is used as a reaction vessel and can be positioned in the light path of the photometer. Sample cell: Before the sample cell is placed in the optical path, a predetermined amount of a reagent solution containing an antigen labeled with a fluorescein-based fluorescent substance is added, and a predetermined acidity of the reagent solution containing the corresponding antibody is added to generate an antigen-antibody reaction. Shinuru. Thereafter, a predetermined amount of the analyte (oil separation sample) is added using a sample addition device. This analyte contains unlabeled antigen.

The sample cell is then positioned in the optical path as shown in FIG.

This sample cell is irradiated with polarized light of a specific wavelength.

The light from the light source J passes through the excitation wavelength selector], 1k, and then passes through the polarizing filter 2 and is irradiated onto the cell 3.

At least the wall surfaces of the cell 3 in the excitation light incident direction and the two fluorescent light extraction directions are made of a transparent material.

The vertically polarized component of the fluorescence generated from the sample selectively passes through a vertically polarizing filter 7 that is fixedly installed, and is received by a photodetector 14. Further, the parallel polarized component of the fluorescence from the sample selectively passes through a fixedly installed parallel polarizing filter 8 and is received by the photodetector 4. Fluorescent wavelength selection ##12, 1.3 is arranged in the two-way fluorescent optical path,
The same fluorescent wavelength is selected. In Figure 1, two fluorescent wavelength selectors are provided, but the fluorescent light is taken out in one direction, and after selecting a specific wavelength light with one wavelength selector, the optical path is split into two. It is also possible to provide a polarizing filter for extracting different polarized light components in each branched optical path.

In either case, photodetectors 4 and 14 can simultaneously observe the fluorescence from the same light source. Each received light signal is sent to the amplifier 5.
．． The signal is amplified in step 15 and provided to calculation unit 9 to calculate the preemptive luminous intensity. The calculation result is displayed on the display device 6 as the concentration of the component to be detected or as the luminous intensity.

Table 1 shows 81 examples of results obtained using the conventional polarization component exchange method. In this example, we used a 20-fold dilution of Takajirirubin serum with physiological saline as a sample.
In Table 1, a reaction solution prepared for measuring digoxin is used. In addition, as anti-j) K, α-feto,
As the antibody, ferritin, cortisol, corticholesterin, uvamine, etc. can be used. FTTC or the like can be used as the fluorescent labeling substance.

The conditions of the measuring device in Table 1 are that the excitation wavelength is 490
nm, the fluorescence wavelength is 520 nm, the detector is the first
This is a photomultiplier tube. In Table 1, the tip luminosity is expressed as rn P in millidrops.

On the other hand, Table 2 shows examples of measurement results based on the present invention. The examples in Table 2 were measured under the same conditions as in Table 1, except that the m++ constant equipment was different. Comparing the results in Tables 1 and 2,
In the conventional method (Table 1), the B1P value fluctuates with the passage of time, but in the method of the present invention (Table 2), the inT' value is almost constant.

Conventional equipment required 30 seconds or more to measure each polarization component by rotating the polarizing plate, but Table 2 was created and even in rate measurement, it was possible to accurately measure 1F at a certain point in time. By calculating the P value, it was possible to determine the anti-J, L; strength change acid in the liquid in a short time. Table 1 shows the results. The rate measurement slope in Table 3: Table 3 (7m T') is 0.89 mP in 25 seconds, so it is 0.035611 IP/sec.

FIG. 2 shows an example of a rat line for measuring digoxin based on the measured values in Table 3.

The second embodiment makes it possible to carry out short-time fluorescence photometry measurement, which has been difficult up to now, and also makes it possible to measure fluorescent light by eliminating changes in background fluorescence intensity. - In the embodiment described above, the vertical component of the polarization component (■
By simultaneously measuring □) and parallel component (I//),
It is now possible to accurately track reactions in which the fluorescence intensity changes over time, and when the background fluorescence changes over time, it is possible to accurately track the reaction in which the fluorescence intensity changes over time. This has the effect of making it possible to distinguish between

〔Effect of the invention〕

As explained above, according to the present invention, signals of the parallel component and the perpendicular component of fluorescence can be obtained at the same timing, and these can be used to calculate the degree of polarization of the fluorescence, so that accurate measurement values can be obtained. is played.

[Brief explanation of drawings]

FIG. 1 is a schematic ellipse diagram of one embodiment of the present invention, and FIG. 2 is a diagram showing an example of a calibration curve for digoxin. 2.7.8...Polarizing filter, 3. Sample cell, 4°]
4... Photodetector, 9... Arithmetic unit.

Claims (1)

[Claims] 1. A cell capable of accommodating a sample, a polarizing filter that polarizes the excitation light irradiated to this cell, a first fixed polarizing filter that passes the vertically polarized component of the fluorescent light from the sample, and a polarizing filter that allows the vertically polarized component of the fluorescent light from the sample to pass A fluorescence polarization measurement device comprising: a second fixed polarization filter that passes a polarization component; and an arithmetic device that calculates a degree of polarization of fluorescence from the vertical component light reception signal and the parallel component light reception signal obtained at the same time.