JPS59184844A - Photometer for fluorescent light and scattered light - Google Patents

Abstract

PURPOSE:To shorten an analysis time by holding a reaction cubet containing a liquid to be inspected, on the circumference at a constant angle pitch, and irradiating a photometric flux from an optical fiber for holding it at an unequal angle pitch. CONSTITUTION:A reaction cubet 1 containing a liquid to be inspected is held at a constant angle pitch on a turntable 2. A rotor 3 is provided on the lower side of the table 2, a spectroscope 5 is fixed to the rotor 3, and a white light from a light source 4 is brought to spectrum to a single color light by an interference filter, and projected to the cubet 1 through optical fibers 6 (6-1-6-n) placed at an unequal angle pitch. The fibers 6 are divided into an excitation light leading- out group G1 and an irradiating light leading-out group G2. A fluorescent light and a scattered light generated by an excitation luminous flux are led into a photodetector 8L through optical members 12, 13.

Description

DETAILED DESCRIPTION OF THE INVENTION (a) Industrial field of application The present invention can be incorporated into an automatic analyzer that performs multi-item analysis of a large number of test liquids in biochemical analysis, etc. A fluorescence/scattered light photometer that separately and continuously measures the light intensity of secondary light emitted from the test liquid, that is, fluorescent light and scattered light, and obtains quantitative analysis data. It is something.

(B) Prior Art Most of the automatic analyzers used in recent biochemical analyzes are of a separate and independent type, and employ a batch process in their analysis process.

This process consists of dispensing the collected sample into a reaction cuvette [2] While transporting the sample along a predetermined route, add and stir the reagent determined for each analysis item to prepare the test solution; A commonly used method is to perform quantitative analysis by measuring the absorbance of monochromatic light of a specific wavelength irradiated on the sample solution during the reaction process or after the reaction is completed using a photoelectric spectrophotometer. As an automatic seed analyzer FL, multiple monochromatic lights with different wavelengths are sequentially irradiated onto the test liquid, and B! 'i set and gill 2
We use the so-called sequential multi-method, which allows the selection of data for specific analysis items from a large number of measured data.
There are a variety of things on offer.

However, if the sample has fluorescence, it is possible to perform a more accurate quantitative analysis using fluorescence analysis, which has a higher detection sensitivity than the absorbance analysis described above. Scattered light analysis, which compares and measures the light intensity of scattered light by

To avoid this, there is an accessory device on the market that can be incorporated into the above-mentioned automatic analyzer to attach an excitation spectrometer to a spectrophotometer for absorbance measurement.
Both fluorescence analysis and scattered light analysis are performed simultaneously or selectively on the test liquid f in separate reaction cuvettes in a sequential multi-method.

Fluorescence and scattering photometers that can be used are not yet available.

(c) Purpose In view of the current VC mentioned above, the purpose of the present invention is to provide a system that can be incorporated into an automatic analyzer, has a simple configuration, and can generate the required data with high precision in a short time for multi-item analysis. An object of the present invention is to provide a light/scattered light photometer as described above.

@Construction As schematically shown in FIGS. 1 and 2, the fluorescence/scattering photometer according to the present invention rotates the reaction cuvette 7)'(1) containing the test liquid. A turn f-pull (2) is held equidistantly with respect to the center (0) and is rotatably supported on a support (not shown), and a groove is located under H1j of this table (2). The rotor (3) has a rotor (3) rotatably provided at the center thereof, and a light 1j (4) consisting of, for example, a tungsten-logen lamp is provided in the central part S of the rotor (3).
A spectrometer (5) is provided which separates the white light into a plurality of monochromatic lights having different wavelengths, and the monochromatic light from the spectrometer (5) is separately guided to the reaction cuvette (1). Unfortunately, between the spectrometer (5) and the reaction cuvette (1), several optical fibers (6) were connected to the excitation light output group for fluorescence generation and the irradiation light output group for scattering light generation. divided,
In each group, the equi-orientation pitch (aO
) are provided on the rotor (3) at an unequal angular pitch in which the angular pitch is successively increased by a certain angle (Δα) from It is fixed along the circumference of the reaction cuvette (1) and has a slit (7) at a position corresponding to each reaction cuvette (1), and only the excitation light beam and the irradiation light beam that pass through the slit (7) reach the reaction cuvette (1). A rough slit member (7) is provided so as to pass through the center of the
Furthermore, the optical fiber (6) is fixed to the rotor (3) in the vicinity of the bottom surface of the reaction cuvette (1) in accordance with the angular position of the optical fiber (6), and the excitation light beam passing through the reaction cuvette (1) The generated fluorescence and the scattered light generated in the irradiation light beam passing through the reaction cuvette (1) are introduced into photodetectors (8L) and (8S) separately fixed near the center of the rotor (3). An optical member (2) consisting of a condensing lens for the fluorescent light (98 fluorescent light filter tact, a reflecting mirror 0υ), a condensing lens (9) for the scattered light, a reflecting mirror (from lυ) In addition to providing an optical member α, which is different from the above, and fixed to the rotor (3) on the outer circumference of the reaction cuvette (1) in accordance with the angular position of the optical fiber (6), the reaction cuvette ( The means for preventing the transmitted light flux of 1) from being irradiated onto each of the detection cuvettes (1) is constructed by providing a means 0.υ. ), sequential fluorescence photometry and scattered light photometry are performed on a plurality of monochromatic excitation light beams and monochromatic irradiation light beams having different wavelengths, respectively.

(E) Embodiment FIGS. 1 and 2 show an embodiment of the device according to the present invention.
The configuration will be explained with reference to the drawings. Fig. 1 is a schematic diagram 1 showing the main parts of this apparatus with the turntable (2) holding the reaction cuvette (1) removed, and Fig. 2 is a schematic side sectional view thereof. .

A turntable (2), which is designed to hold the reaction cuvette (1) containing the test liquid at a constant angular pitch (G0) on the same circumference of the periphery, is attached to a support (not shown). The rotor (3) is rotatably mounted on the lower side of the table (2) concentrically with the table (2). A spectrometer (5) is fixed to the center of the rotor (3), and a light source (in this example, a tungsten halogen lamp with high brightness is used) is attached to the center of the spectrometer (5).
4) is passed through a plurality of interference filters (5).
') have different wavelengths λ1, λ2...--λ
Each of these monochromatic lights is separated into n monochromatic colors by a spectrometer (5
) and the reaction cuvette (1), an optical fiber (6) installed in the rotor (3) is passed radially and parallel to the table (2), and the optical fiber (6) is inserted into the reaction cuvette (1) as a photometric light beam. As shown in Figure 1, the optical fiber (6) is divided into an excitation light output group (G1) for generating fluorescent light and an irradiation light output group (G2) for generating scattered light. The optical fibers (6) are connected to each group (G1).
, (G2) are arranged at an unequal angular pitch in which the angular pitch is sequentially increased by a constant angle (Δα) to be described later from the equiangular angular pitch (G0) of the reaction cuvette (1).

Place the reaction cuvette (
A slit member (7) is fixed along the circumference of the inner side 1 of the slit member (7).
A slit (7) is provided at a position corresponding to each reaction cuvette (1), and only the excitation light flux and irradiation light flux that pass through this slit (7) reach the reaction cuvette (1).
The distance is 11 so that it passes through the center of 1).

In order to introduce the fluorescence generated by the excitation light flux passing through the center of the reaction cuvette (1) to the photodetector (8L) fixed near the center of the rotor (3), a photodetector (8L) is placed on the bottom of the reaction cuvette (C1). A condensing lens (9) and a fluorescence filter u are placed on the nearby odor rotor (3) to match the angular positions of the optical fibers (6) of the excitation light deriving group (G1).
An optical member (14) consisting of Q1 and a reflecting mirror (b) is identified.

In addition, in order to introduce the scattered light generated by the irradiation light beam passing through the center of the reaction cuvette (1) to a separate photodetector (8B) fixed near the center of the rotor (3), However, the optical member σ■ consisting of the condenser lens <9), the reflector 0], and l is fixed in a fixed position in accordance with each angular position of the optical fiber (6) of the irradiation light deriving group (G2). ing.

In this device, the photodetectors ωL) and (8B) have the following:
The following two conditions are established so that a plurality of fluorescent lights and scattered lights generated by each monochromatic light flux passing through the test liquid in the reaction cuvette (1) do not enter at the same time.

One of the conditions is that the incident range of the excitation monochromatic beam and the irradiation monochromatic beam be centered on the axis of the reaction cuvette (1) so that effective and accurate photometry can be carried out for the test liquid in the reaction cuvette (1). The optical path area is limited to a diameter d (for example, about 2 rim), and a slit (7
) is such that the gap S is S≦d, and the other 1
One condition is that the optical fiber (6) is
) and the irradiation light guide group (G2') are arranged on the rotor (3) at the above-mentioned unequal pitch.

This second condition will be explained regarding the optical fiber (6) of the excitation light deriving group (G1).

In Figure 1, a plurality of optical fibers (6
), the one whose angular position with respect to the rotation center (0) coincides with that of the reaction cuvette (1) depending on the respective rotation stop positions of the turntable (2) and rotor (3) is designated as (6).
-1), and the one next to it is +1-iK in the counterclockwise direction.
(6-2), (6-3)... (6-n) and these optical fibers (6-1), (6-2)
It is assumed that monochromatic light having wavelengths λ1, λ2, .

Then, using the optical fiber (6-1) as a reference, each of the optical fibers (6-2), (6-3), etc.
・(6-n) The center angle (radian) of the rotor (3) that can be tilted with respect to the rotation center (0) is G1, G2, αn-1,
The reference optical fiber (6-1) of the reaction cuvette (1)
) and the center of the object having the same angular position as the reference position (A)
,! : k to do, α1=α0×1+αn-1=αOX'
(n-1) +-7dX(n-1). [Here, G0 is the arrangement angle pitch (radian) of the reaction cuvette (1) described above, and R is the arrangement angle pitch (radian) of the reaction cuvette (1).
) reference position! (A) and the rotation axis of the rotor (3). ] In other words, the optical fiber (6) is arranged at a constant angular pitch (G0) of the reaction cuvette (1).
They are arranged at an unequal angular pitch in which the angular pitch is +11th thicker by Δa).

This means that the optical fiber (6) of the irradiation light deriving group (G2)
The same is true for the reference position (in the reference position)), which is only flattened by an angle β (equal to an integer multiple of the equidistant angular pitch α0 of the reaction cuvette) with respect to the reference position)) VC.

The photodetectors (8L) and (88) are connected to the reference plane αn.
-1 It is fixed to the lower part of the spectrometer (5) at the same angular position as the optical fiber which makes a central angle of approximately -Y- counterclockwise from the eye bar (6-1).

For example, the total number of reaction cuvettes (1) held equidistantly on the turntable (2) at angular edges Ca0) is 9.
0'', it is G0-4''. Reaction cuvette (1
) The wavelength of the monochromatic light projected onto the sample solution is usually 8-
Although it takes about 10 beams, this device uses excitation light,
Due to the provision of two groups of irradiation light, this n is set to 5 types, n = n=
It is 5. If d--2mn%R = 200m shoulder, then ±=124 and 91° per vehicle.

2 In this case, the angular position of the original fiber (6-3) located at the center is G2 # 9.1°, so for the photodetectors (8L) and (8B) occupying the same angular position as n, The monochromatic light beam of wavelength λ1 from the optical fiber (6-1) passes through the reaction cuvee 7) 11), and finally exits through the reflection @Oυ. The incident angle of the fluorescent light and scattered light is approximately 9
, 1°. Therefore, since it is impossible for the photodetector (8L) (88) to receive light at an incident angle larger than this, there will be no shortage of detected light amount.

The excitation monochromatic light beam and the irradiation monochromatic light beam passing through the center of the reaction cuvette (1) act on the test liquid, respectively, and generate fluorescent light and scattered light, but a considerable amount of light is
Transmit that. In order to ensure that any transmitted light does not disturb accurate photometry, a rotor (3) K is mounted on the outer circumference of the reaction cuvette (1), and the transmitted light flux is transferred to a detector (8).
L), (8B'') As a means for preventing K from entering, nine optical traps 04 are installed corresponding to the angular position of the optical fiber (6).

Next, we will explain the operation of this device first.
j Take up the light.

The reaction cuvette (1) located at the reference position (A) is given the number ■, and the reaction cuvettes (1) arranged counterclockwise from the standard position (A) are given the numbers ■, ■, ■, etc.

In the state shown in Figure 1, a single color of light with a wavelength of λ1 is excited by the interference filter (5') of the spectrometer (5) through the optical fiber (6-, 1) and the slit (7). A light beam is projected onto the test liquid in the reaction cuvette (1) of N021 as shown in the figure, and a part of the transmitted light beam is captured and absorbed by the optical trap, but the excitation light beam with wavelength λ1 is reflected in the test liquid. The fluorescence of wavelength λ' generated by passing through the center is focused: y,
The light is focused by <a (nl), then the fluorescent light beam is collected by a fluorescent filter OO that passes only the wavelength of the fluorescent light, and finally reflected by the reflecting mirror α〃.As shown by the dotted line, the photodetector (8] -) is emitted and receives light ζ not 1.6
(11 lights are carried out. Then, in the spectrometer (5), the 1112 interference filters (5 filters have a length of λ2s)
The monochromatic light ``6, ``''λ'' is connected to the original fiber (
6-2), (6-3)・-・-(6-n) as Hete pJ,
Q) i4 likelihood bundle is NO, 2, NO, 3, etc.
Into the reaction cuvette (1) of N O,n (- 几zo i is projected, but both of these two 1 and the fibers are written as iii). )
The slit (7nl) corresponding to
The slit member (7') prevents the reaction cuvette (1) VC from reaching the reaction cuvette (1). From this state, the rotor (3) moves clockwise d-.

When the rotating ring is rotated by Δα, that is, T facian, the optical fiber (6-2) matches the angular position of the slit (7) corresponding to the N092 reaction cuvette (1), so the above-mentioned result is achieved. Similarly, the photodetector (8L) performs photometry of only the fluorescent light beam generated by the excitation light beam of wavelength λ2 of the sample solution (1) of the NO12 reaction cuben. Regarding rotation in the direction of force by Δα-T radians, N Q, 3, N014,...
For the test liquid in the reaction cuvette (1) of NO, n, the detector (8L) measures the fluorescence luminous flux generated by the excitation luminous flux of wavelengths λ5, λ4, ... λn.
This is done sequentially.

Then, the rotor (3) continues to rotate clockwise,
When the optical fiber (6-1) is aligned with the reaction cuvette (1) of NO, 1 (slab corresponding to the adjacent reaction cuvette (1) of NO, N) (7), the reaction cuvette (1) of NO, H is aligned. The excitation light beam of wavelength λ1 of the test liquid is not measured due to the generated fluorescent light beam, and from that point on, as the rotor (3) rotates by Δα in the same direction, N081
, NO, 2, . . . ----NQ, n The order is not Vc.

By rotating the rotor (3) clockwise in this way, the fluorescence light flux generated by all the monochromatic excitation light fluxes of different wavelengths for each specific sample of the test liquid in each reaction cuvette (1) is measured. , the data necessary for fluorescence analysis can be obtained.

Regarding the scattered light photometry operation, the above-mentioned fluorescence di: ] light is the light 7 a(c(6-1) ~(6n)'e). Since it is not fluorescent light but scattered light that is generated by irradiating the test liquid, the condensing lens (9) and the reflection tax 0.
The difference is that the scattered light flux is emitted to the photodetector (8th) at 2t only by passing through the σ, and the light is received by the 2" LVC, but in other respects the operation is the same. .

Needless to explain, the above-mentioned fluorescence photometry and scattered light photometry perform simultaneous TK on the test liquid in each reaction cuvette (1). Both methods allow you to obtain the data you need for analysis.

In the above explanation, the operation has been explained assuming that the reaction cuvette (1) contains 2"L of test liquid after the addition of reagents has been completed, or the turntable (2) is intermittently When dispensing the sample solution, dispensing and adding reagents while rotating, and cleaning the reaction cubera (after photometry) in (1), the photometry range is maintained equidistantly on the table (2). limited to about 60% of the reaction cuvette (1)
It is normal to Therefore, in this case, the phase angle (b) between the excitation light derivation group (G1) and the irradiation light derivation group (G2) of the optical fiber (6) shown in FIG. Starting from the reference position (A), the reaction cuvette (1) held on the table (2) is rotated clockwise by a rotation angle of 200°, for example, necessary to photometrically scan the reaction cuvette (1) held on the table (2) in the photometric range. During this time, fluorescence and scattered light photometry are performed in parallel, and then the rotor (3) is rotated counterclockwise so that the angular position of the optical fiber (6-1) matches the reference position (A). Just let it come back. It is only necessary to select data corresponding to the analysis item from a large number of selected data.

By the way, if you want to selectively perform either fluorescence analysis or scattered light analysis on separate reaction cuvettes (1) on the test liquid, the method shown in the schematic cut-out diagram in Figure 3 Another example device may be used. This other embodiment of the device directs monochromatic light from a spectrometer (5) into a reaction cuvette (
1) Group (G1) derived as excitation light for fluorescence generation
and a group (G2) derived as irradiation light for scattering light generation.
The optical fibers (6-1) to (6-n) are arranged on the rotor (3) in accordance with the angular positions of the divided optical fibers (6-1) to (6-n), and are arranged in the rotor (3) to detect fluorescent light or scattered light from the reaction cuvette (1). Each of the optical members μs and G3 for introducing the light into the separate photodetectors (8L) and (8B) is composed of a condenser lens (9), a fluorescent filter, and a reflecting mirror (11), As only the fluorescence filter Q0 is shown in FIG. 4, it is provided so that it can be inserted into and removed from the optical member Q21 through the holder α0.0 with respect to the α optical axes. , a spectrometer (5) fixed to the center of the rotor (3), each consisting of a setter that is rotatable and fixed at a predetermined rotational angle position, An optical fiber (6-1 ) to (6-It) 1.Solution, the arrangement angle pitch is matched.

Therefore, the holders (15, Q5j are placed at the reference positions (Af, crsi) of the respective fluorescence filters OQ and the reference positions (A'), (A'), (1) of the reaction cuvette (1) shown in FIG.
When the spectrometer (5) is set so that it is directly below the cuvette (E), fluorescence photometry can be performed simultaneously on the test liquids in two reaction cuvettes (1).

Then, holder 0■ (from the set position mentioned above,
Rotate the former counterclockwise and the latter clockwise to connect the optical fiber of the rotor (3) (6-1).
) to (6-n) are self-installed.When set to hold the spectrometer (5 units ζ 1'L (" t7. reaction of cubera)
This can be done simultaneously for the test liquid in (1). In this way, in this other example device, the fluorescence j1 of the test liquid
4+j light or scattered light photometry can be performed selectively and efficiently.

Also, the light source (4) is strong! ! Although not shown, optical fibers (
A half mirror 1 reflecting mirror is provided between 6) and the slit member (75), separate reference photodetectors are provided above the photodetectors (8L) and (88), and a double beam system is adopted.
It is desirable to cancel the shadows caused by the fluctuation of the light source (4).

In addition, in the apparatus of the first embodiment described above, as shown in FIG. Although the optical system is adopted, a forward scattering and condensing system in which the condensing lens (9) is provided diagonally downward outside the reaction cup (1) may also be used. (1) Monochromatic light flux, -tn of irradiated monochromatic light flux, transmitted light flux f: Detector (8L)
, (8B), instead of providing the above-mentioned optical trap circle, a reaction cuvette (reaction cuvette)
Illiσii K 'II and a mirror finish is applied to the outside of the slight part facing the slit (7) to create a turn-chip.
Is there a proper way to accurately remove the bubbles when setting to pull (2)? , just leave it there.

(c) Effect 31: , The light-induced light scattering light probability C j Every time you move,
Fluorescence photometry and scattered light IPjj light were performed on the 1φ liquid in each reaction cuvette (this, ij, and ij respectively).
12< is shi'1] (in case of , use 2 digits at a time) i
:]('j Next, I'm going to go to the next page. Since there is no photometry on the scales, it is detected that jL< EL (beautiful, ¥) 7 j + 1 dog 1 i knee + te, y, , evening', i, is 6 digits 0

[Brief explanation of the drawing]

Figure 1: The light of the sky 1j, i. 1・ri4+ :)
II, 2nd pot, 1 hazono pattern (3)... rotor,
(4)...-γ(: 1))F, (5)...Spectrometer, (6)...Optical fiber, (7)...
・Suri Soto, (1)...Suri Sotoberi,
(8L)...Keikoit! Photodetector for li light, (8th)...Photodetector for photometry of scattered light, (9)...Condensing lens, 4...Fluorescence filter, (1υ...Reflection;
tax, u4, 罎...optical abduction, CIIυ...
Optical drag, (J■, Oa... holder for fluorescent filter, (A)
, (B)...Reference position, (G1)...Excitation light 2n3 output optical fiber group, (
G2)...Illustrated Kouji - Izuyo Kouai (Group. Proxy Ben): 11 ± l'il Miya Takeo -, 1, \;
・-1111 and-5・ Figure 1 Figure 4

Claims (1)

[Scope of Claims] 1. A turntable which holds reaction cuvettes containing a test solution equidistantly disposed about the center and is supported by a support, and a turntable arranged concentrically with the cuvette on the underside of the turntable. a rotor that is rotatable; a spectrometer that is fixed to the center of the rotor and separates white light from a light source into a plurality of monochromatic lights having different wavelengths; and an equiangular pitch of the reaction cuvettes. The monochromatic light from the spectrometer is arranged radially in an unequal angle viate whose angular pitch is increased by a constant angle, and the monochromatic light from the spectroscope is guided into the reaction cuvette as excitation light for fluorescence generation. The optical fibers are divided into groups to be led out as irradiation light for light generation, and the slits are fixed along the circumference adjacent to the nVC inside the reaction cuvettes on the bottom surface of the turntable, and a slit is formed at a position corresponding to each reaction cuvette. a slit member configured to allow only the excitation light beam or irradiation light beam passing through the slit to pass through the center of the reaction cuvette; and a reaction cuvette fixed to the rotor near the bottom of the reaction cuvette. - an optical member that introduces fluorescence generated by the excitation light beam passing through the reaction cuvette or scattered light generated by the irradiation light beam passing through the reaction cuvette to a photodetector separately fixed near the center of the rotor; A fluorescence/scattering device comprising a filter provided on the rotor on one or more circumferences of the reaction cuvette itself, and means for preventing the transmitted light flux from the reaction cuvette from entering the respective detectors. Photometer. 2. Optical members for introducing the light generated in the reaction cuvette as a photometric light flux into separate photodetectors are divided into a member consisting of a condenser lens, a fluorescence filter, and a reflecting mirror for fluorescent light, and a member for scattering light. 2. The fluorescence/scattered light photometer according to claim 1, each comprising a condenser lens and a separate member comprising a reflecting mirror. 3. An optical member that introduces the light generated in the reaction cuvette as a photometric light beam into separate photodetectors is a condenser lens,
The fluorescence/scattering device according to claim 1, which comprises an optical filter and a reflecting mirror, and wherein only the fluorescence filter can be inserted into and removed from the optical axis of the optical member via its holder. Photometer.