JPS6218862B2 - - Google Patents

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a fluorescence polarization measuring device, and more particularly to a device having a function suitable for measuring changes in a reaction over time and extrapolating a value after a certain period of time from the measured value. .

In reactions that change over time, direct measurement values may not be obtained at the intended measurement time.

For example, if you want to find the measured value just before the reaction stops,
When the sample must be removed from the measuring section to stop the reaction, or when two or more phenomena must be measured at the same time on the same sample, certain components in the reaction solution may be lost, but There are cases where measured values at intermediate times are required. In these measurements, it is common practice to make approximations from measured values over a limited period of time to calculate values at the intended measurement time.

In particular, in the case of fluorescence polarization measurement according to the present invention, the vertical polarization component and the horizontal polarization component must be measured separately for the same sample, and it is impossible to measure both components at the same time at the desired time. Can not. Therefore, it is necessary to approximate (extrapolate) the measured values to find the value at the desired time.

The conventional method has been to extrapolate a straight line ruler to the data recorded on an analog recorder to make a straight line approximation. However, changes in substances over time, especially changes in biologically related substances, involve integrated changes in multiple substances rather than linear changes in a single substance, so the observed reaction process progresses in a curved manner. There are many cases.

When the reaction progresses in a curved manner, linear approximation causes errors. Furthermore, even if a curve ruler is used, it is very difficult to match the curvature of the reaction curve with the ruler. Furthermore, manual approximation by a measurer is inaccurate because the approximations vary depending on the individual.

The present invention solves the above-mentioned drawbacks of the prior art, performs an approximation based on the reaction, and determines the degree of polarization in fluorescence polarization measurement from the value obtained by the approximation.

An object of the present invention is to provide an apparatus that effectively approximates the reaction process of biologically related substances and obtains a degree of fluorescence polarization with minimal error from the extrapolated value obtained by the approximation. It is in.

The present invention experimentally confirms that reactions of biological substances proceed in a linear or curved manner, and performs approximations based on this reaction, and in the case of only curve approximation, curvature is emphasized and errors are magnified. In order to solve this problem, we combined linear approximation and curve approximation. With these approximations,
Find the desired degree of fluorescence polarization.

FIG. 1 shows a functional system diagram of an embodiment according to the present invention.

White light emitted from a light source 1 is separated into monochromatic light by an excitation side spectrometer 2, polarized by an excitation side polarizer 3, and irradiated onto a measurement cuvette 4. The sample in the measurement cuvette is excited and emits fluorescence. The fluorescent light passes through a fluorescent polarizer 7 and is guided to a fluorescent spectroscope 8. The light incident on the fluorescence-side spectroscope is split into monochromatic light having a designated fluorescence wavelength, and only this monochromatic light reaches the photodetector 9.

When the sample is placed in the measurement cuvette, the cuvette is set at the photometry position, and the measurement is started, the controller 1
The pulse motor 13 is operated by the control signal from the pump 2, and the excitation side polarizer is set so as to pass only the vertical component of the excitation light.

On the other hand, the fluorescent side polarizer is driven by the pulse motor 14 operated by a command signal from the control section. In this drive, 90° rotation is intermittently repeated at regular time intervals.

That is, the fluorescent light emitted from the excited sample is separated into a light component orthogonal to the polarized excitation light and a light component parallel to the polarized excitation light before reaching the photodetector.

The optical signal detected by the photodetector is converted into an electrical signal, passed through a preamplifier 10, and then sent to an A/D converter 1.
1 is converted into a digital signal.

The perpendicular component light and the parallel component light are each converted into digital signals by an A/D converter and then stored in the memory section 15. After the measurement has been carried out for a certain period of time, the measurement is carried out according to a command from the control section. The sample contained in the measurement cuvette 4 passes through a filter section 6, where components such as particles are separated, and only the solution is collected in the measurement cuvette 5. The photometric cuvette 2 containing the liquid is moved to the photometric position in place of the photometric cuvette 1,
A measurement of the fluorescence intensity of the superfluous liquid is made.

Here, the reaction is considered to have progressed until the middle time (T ^f shown in FIGS. 2 and 3). However, it is impossible to actually perform measurements at time T ^f . Therefore, as stated in the claims of the present invention, approximation is performed based on measured values within a certain limited time, and the time T ^f
Perform extrapolation for .

Specifically, the arithmetic unit 14 creates an approximate expression using the vertical component optical signal data and the parallel component optical signal data stored in the memory unit.

Measurement examples in this measurement are shown in FIGS. 2 and 3. The solid line indicates actually measured data, and the dotted line indicates a straight line or curve obtained by an approximate formula.

Reactions do not necessarily proceed linearly, but often follow a curve. Therefore, in the present invention, both vertical and parallel components are subjected to linear approximation, curve approximation is performed, and linear approximation is performed for one component, and curve approximation is performed for the other component. The approximation was made based on actual measurements. In particular, as shown in FIG. 3, it is observed as actual measurement data that the parallel component optical signal fits into a straight line approximation, and the perpendicular component optical signal fits into a curved line approximation. In these actual measurement examples,
Performing only linear approximation or curve approximation for both components will increase the error in the extrapolated value.

Furthermore, in the present invention, the fluorescence polarization degree: P is calculated using the following calculation formula.

P=(I⊥(T)-I⊥(F))-(I(T)-I(F))×G/(I⊥(T)-I⊥(F))+(I(T)- I
(F))×G where I⊥(T): Approximate value of vertical component at T ^f I(T): Approximate value of parallel component at T ^f I⊥(F): Fluorescence polarization of perpendicular component of supersolution Intensity I(F): Fluorescent polarization intensity of parallel component of hypersolution G: Device constant obtained when the excitation side polarizer is horizontal.

The degree of fluorescence polarization determined by the above formula: P is I
If ⊥(T) and I(T) have an error, an error will naturally occur. Furthermore, if the extrapolation approximations, I⊥(T) and T(T), approximate values that are far from the actual reaction, the value of P will also not correspond to the actual reaction.

The three types of approximate values obtained and the degree of fluorescence polarization P for each are printed on the display section 17 in the present invention.

The present invention provides the following effects.

1. Take advantage of the advantages of conventional manual approximation (linear extrapolation using a straight line ruler), reduce errors when linearly approximating a curved reaction curve, and improve the accuracy of extrapolated values. .

2. By performing not only extrapolation approximation using only linear approximation, but also approximation using curve approximation and a combination of linear approximation and curve approximation, approximation that immediately responds to the actual reaction becomes possible.

3. The complexity of having to make approximations by hand for each measurer is eliminated, and individual differences among measurers are eliminated.

4. As described in the embodiments of the present invention, the present invention is extremely effective when one component changes linearly and the other component changes curved.

5 Calculate and display the value of the degree of fluorescence polarization: P from the extrapolated values obtained by linear approximation, curve approximation, and a combination of both to provide researchers with numerical information on the reaction process. What you can do is
It becomes possible.

[Brief explanation of the drawing]

FIG. 1 is a functional system diagram of an embodiment of the present invention, and FIGS. 2 and 3 are explanatory diagrams of an approximation method of an embodiment of the present invention. 1... Light source, 3, 7... Polarizer, 4, 5... Cubeette, 9... Detector, 16... Arithmetic unit.

Claims (1)

[Claims] 1. A time-varying substance is placed in a photometric cuvette, and polarized excitation light is irradiated onto the suspended sample solution in the cuvette and the filtrate of the sample solution, so that the fluorescent light emitted by the substance is collimated. In a device that separates the polarized light component and the vertically polarized light component and measures the light intensity of both components, an approximate straight line is created for one component from the measured values of both components within a certain limited time, and an approximate straight line is created for the other component. is equipped with a function to create an approximate curve and read out the values after a certain period of time, and calculates the degree of fluorescence polarization from the values after the certain period of time for both components of the sample solution and the light intensity of both components of the filtrate. A fluorescence polarization measuring device characterized by determining the .