JPH0325354A - Automatic fluorescent analyzing instrument - Google Patents

Abstract

PURPOSE:To eliminate the need for a precise adjustment and to improve the accuracy of measurement by providing a detecting means which detects the transmitted light quantity of the exciting light transmitted through reaction cells and detecting the fluorescent light intensity at the center of the reaction cells based on the transmitted light quantity thereof. CONSTITUTION:This instrument is constituted by providing a photometric control part 11, etc. and the position of a reaction disk 2 is adjusted prior to the start of each measurement cycle. The transmitted light quantity of the exciting light past between the reaction cells in the initial time is detected when the cycle is started. The transmitted light quantity transmitted through the cells 3 as the cells 3 move is then successively measured. When the light quantity decreases to a prescribed quantity, for example, to 2/3 of the above-mentioned light quantity, this decrease is regarded as the timing for starting the measurement and thereafter the transmitted light quantity and the fluorescent light quantity are alternately detected and stored time sequentially at least before the cells 3 cross the photometric path. The position decreased to 2/3 of the initial value is in succession detected by tracing the stored transmitted light quantity back to the past. The central position of the cell is specified on the basis of this position. The fluorescent light quantity corresponding to this position is thus detected. Namely, the fluorescent light intensity of the cell center is detected at all times.

Description

Detailed Description of the Invention [Objective of the Invention] (Industrial Field of Application) The present invention aims to identify specific components by irradiating a reaction solution with excitation light of a predetermined wavelength and detecting the amount of fluorescence generated. This invention relates to an automatic fluorescence analyzer for measuring the concentration of .

(Prior art) For example, using human serum as a sample, a desired reagent is added to it to cause a reaction, and the reaction state is optically detected while moving a reaction cell containing this reaction solution. In this manner, automatic chemical analyzers are known that measure the concentration of a specific portion of a reaction solution by detection using a so-called colorimetric method.

On the other hand, similar to this automatic chemical analyzer, a moving reaction cell is irradiated with excitation light of a specific wavelength at a photometry position, and the amount of fluorescence generated is detected to identify or isolate each reaction solution. Automated fluorescence analyzers have recently appeared that measure concentration.

In such an automatic fluorescence analyzer, a plurality of reaction cells are arranged around the periphery of a circular reaction disk, and are rotated by, for example, one rotation plus one pitch per cycle of measurement, so that each reaction cell can be detected in each cycle. Cells are now deferred.

Further, a photometry section is provided at a predetermined position on the rotation path of the reaction disk for irradiating each moving reaction cell with excitation light and detecting fluorescence generated by a fluorescent substance in the reaction solution. As a result, the amount of fluorescent light of the reaction solution in each reaction cell can be detected for each cycle, and by selecting excitation light of a specific wavelength, the concentration of a specific component can be measured.

When detecting the amount of fluorescent light in a reaction solution in this way, it is important to set the photometry timing appropriately, and to ensure that the center of the reaction cell shape and the optical axis of the photometry optical path from the photometry section exactly match. must be maintained. FIG. 6 shows the configuration of the photometry section 1 in a conventional automatic fluorescence analyzer, in which a large number of reaction cells 3 are arranged around the periphery of a reaction disk 2, and the reaction disk 2 is fixed to a shaft 4 and held against a gear 4a. The driving force from the pulse motor 6 is transmitted through the gear 6a. A cut plate 7b is attached to the shaft of the pulse motor 6.
A cutting plate 7a is provided on the shaft 4, and a photointerrupter 8a, 8b is arranged facing each cutting plate 7a, 7b. 9 is a constant temperature bath for keeping the reaction cell 3 at a constant temperature, and 10 is a bearing. In each cycle, the reaction disk 2 rotates one revolution plus one
Each time the cutting plate 7b moves by a pitch, the cutting plate 7b moves forward by one pitch, so that the center of the reaction cell 3 and the optical axis L of the optical path coincide, and one measurement signal is generated.

The measurement signal is output to the photometry control section 11. Photometry control section
1 is an interface 12, a central processing element (μ1 CPU
) 13, ROM 14, RAM 15, etc., reads the amount of fluorescent light from the reaction cell 3 in response to input of measurement signals, and outputs it to the main controller 16, which centrally manages the automatic fluorescence analyzer.

The main control unit 16 includes an interface 17, a central processing element (
It is composed of μ1 (CPU) 18, ROM 19, RAM 20, etc. A common memory (consisting of a RAM) 21 is also provided, and is configured to store a command to start measurement from the main control section 16 and the amount of fluorescent light of each reaction cell measured by the photometry control section 1l. 22 is a reaction disk controller.

Figure 7(a). (b) shows the measurement signal and the intensity of fluorescence detected at the timing of generation of this measurement signal. When the measurement signal shown in FIG. 7(a) is generated, the intensity of fluorescence detected from the reaction cell 3, ie, the amount of fluorescence light, is detected at the rising timing of this signal. By adjusting this measurement signal so that it is generated when the center of the reaction cell 3 and the optical axis L of the optical path coincide, the peak value of the fluorescence intensity shown in FIG. 7(b) can be detected. Become. However, Fig. 8(a).
When a measurement signal is generated with the optical axis L shifted from the center of the reaction cell 3 as shown in (b), the respective fluorescence intensities are shifted left and right from the peak value and are detected. Variations in fluorescence intensity result in a decrease in measurement accuracy. Therefore, during measurement, the measurement timing must be adjusted so that the measurement signal rises when the center of the reaction cell and the optical axis of the optical path coincide.

(Problem to be Solved by the Invention) However, in conventional automatic fluorescence analyzers, precise adjustment is required so that the measurement signal rises when the center of the reaction cell and the optical axis of the optical path coincide. There are problems that require a lot of effort. For example, the cut plate that generates the measurement signal is attached to a shaft that advances the reaction cell one pitch per revolution, so that the measurement signal that is generated will evenly divide the reaction disk.

Therefore, since it is necessary to attach the reaction cells at positions that evenly divide the reaction disk, a high degree of attachment precision is required for the support for attaching the reaction cells to the reaction disk.

The present invention has been made in response to the above-mentioned problems.
The object of the present invention is to provide an automatic fluorescence analyzer that can improve measurement accuracy without requiring precise adjustment.

[Structure of the Invention] (Means for Solving the Problems) In order to achieve the above object, the present invention sequentially moves a plurality of reaction cells each containing a reaction solution at a constant speed at least within the photometric optical path, and performs photometry. In an automatic fluorescence analyzer that measures the concentration of a specific component by irradiating excitation light of a specific wavelength onto a reaction cell passing through an optical path and detecting the amount of fluorescence generated, the excitation light of a specific wavelength is applied to the reaction cell. a first detection means for detecting the amount of fluorescence generated by irradiation; a second detection means for detecting the amount of transmitted excitation light; The amount of transmitted excitation light that passes through the reaction cell is sequentially stored, and after a predetermined difference occurs between both amounts of transmitted light, the amount of transmitted light and the amount of fluorescent light are alternately stored in chronological order until at least the reaction cell crosses the photometric optical path. The amount of transmitted light stored in chronological order is searched for a position where a predetermined difference has occurred between the initial value and the amount of fluorescent light corresponding to a predetermined part of the reaction cell is detected. It is characterized by comprising means.

(Function) Prior to the start of each measurement cycle, the reaction disk is adjusted to a position such that excitation light is passed between adjacent reaction cells, and when a measurement cycle is started, these are initially The amount of transmitted excitation light passing between reaction cells is detected. Next, as the reaction cell moves, the amount of transmitted light that passes through the reaction cell is sequentially measured, and when the amount of transmitted light decreases by a predetermined amount, for example, 2/3, from the amount of transmitted light, this is regarded as the timing to start measurement, and from then on, at least no reaction occurs. Until the cell crosses the photometric optical path, the amount of transmitted light and the amount of fluorescent light are alternately detected and stored in time series. Next, by looking back at the amount of transmitted light stored in chronological order, a position where a predetermined difference has occurred from the initial value, that is, a position where the amount of transmitted light has decreased by 2/3, is found, and based on this position, the reaction cell is The center position of is specified, and the amount of fluorescent light corresponding to this center position is detected.

This allows the fluorescence intensity at the center of the reaction cell to be detected at all times, eliminating the need for precise adjustment and improving measurement accuracy.

(Example) Embodiments of the present invention will be described below with reference to the drawings.

FIG. 1 is a structural diagram showing an embodiment of the automatic fluorescence analyzer of the present invention. A plurality of reaction cells 3 are arranged around the circumference of the circular reaction disk 2, and these reaction cells 3 are rotated by a pulse motor 6 as shown in FIG. It is controlled to advance one pitch.

The pulse motor 6 is controlled by drive pulses from the reaction disk controller 22. Also, the controller 22
A home signal indicating that the reaction cell 3 is located at a predetermined specific position (home position) is detected and added to the cell by the combination of the cut plate 7a and the photointerrupter 8a.

A sampler 24 holding a sample container 24a containing a large number of samples to be analyzed is arranged around the reaction disk 2, and is rotated by a sampling arm 25 having a sampling probe 25a at the tip. It is configured such that a sample is dispensed to a predetermined reaction cell 3 by vertical movement. Further, around the other side of the reaction disk 2, there is arranged a reagent storage 26 that holds reagent containers 26a containing a large number of reagents to be mixed with a sample and subjected to a predetermined analysis, and has a reagent probe 27a at the tip. The reagent arm 27 is configured to rotate and move up and down, thereby dispensing a reagent to a predetermined reaction cell 3.

Furthermore, around the other side of the reaction disk 2, there is a stirring bar 28 at the tip.
A stirring arm 28 is arranged and has a rotation. It is configured to stir the sample and reagent in a predetermined reaction cell 3 by vertical movement. A photometry section 30 for optically measuring the concentration of a specific component in the reaction liquid contained in the reaction cell 3 is provided at the path of the reaction cell 3 after stirring has been completed. In the case of an automatic fluorescence analyzer, the concentration of a specific component is measured by irradiating a reaction cell with excitation light of a specific wavelength and detecting the amount of fluorescent light generated.

In addition, an elevator 41 having a cleaning nozzle 41a at the tip is arranged at the path position of the reaction cell 3 where photometry around the other parts of the reaction disk 2 has been completed, and by moving up and down, the reaction cell 3 directly below which has been photometered is disposed. It is constructed so that it can be cleaned and reused. Furthermore, a sampling pump 25b and a reagent pump 27b are connected to the sampling arm 25 and the reagent arm 27, respectively,
It is configured to inhale a predetermined amount of sample and reagent and supply it to the sampling probe 25a and the reagent probe 27a.

The details of the photometry section 30 are constructed as shown in FIG. Light generated from a halogen lamp 31 serving as a light source is alternately switched between a night vision state and a non-night vision state by a chopper 32, and then only a specific wavelength for use as excitation light is selected and passed by a filter 33a. . For example, if a wavelength of 4,921 m is selected as a specific wavelength, the direction of this excitation light is changed by mirrors 34a, 34b, and 34c and enters the grating 35, and the direction of the excitation light is further changed and output via the convex lens 36a. After that, the reaction cell 3 is irradiated. Due to the presence of the fluorescent substance in the reaction solution, fluorescence, for example light with a wavelength of 520 nm, is generated from the reaction solution and enters the photometry section 30 again. That is, the light is incident on the grating 35 via the convex lens 36a, and after its path is separated from the excitation light by the grating 35 due to the difference in wavelength from the excitation light, it is again directed to the mirror 34c. The course direction of the light is changed by 34b, and the light enters a photomultiplier 39 via a filter 38. The photomultiplier 39 detects the amount of incident fluorescent light.

On the other hand, a photodiode 37 is arranged at a position opposite to the photometry section 30 of the reaction cell 3, and this photodiode 3
Excitation light that has passed through the reaction cell 3 via the convex lens 36b and filter 33b is incident on 7, and the amount of the transmitted light is detected. That is, this embodiment is characterized in that the photodiode 37 is arranged to detect the amount of excitation light transmitted through the reaction cell 3 in this manner.

As shown in FIG. 3, after the transmitted light of the excitation light is detected as a voltage by the photodiode 37, the preamplifier 4
2 and a main amplifier 43, and then input to a multiplexer 44. On the other hand, photo multiplier 39
The detected fluorescence is amplified by a preamplifier 46 and a lock-in amplifier 47, and then input to a multiplexer 44. be done.

The multiplexer 44 to which the transmitted light and fluorescence are input in this way selects the input voltage designated by the μ-CPU 13 of the photometry control unit 11 and converts the ADC (analog, digital,
converter) 45.

The ADC 45 converts the input voltage into a digital voltage in response to a conversion start signal from the μ-CPU 13 of the photometry control section 11. The ADC 45 is connected to the μ-CPU of the photometry control unit 11 during conversion.
BUSY signal is output to 13, and R is output when the conversion is completed.
Output the EADY signal to indicate the READY of the μ-CPU 13.
The digital value converted by the signal is output to the μ-CPU I3.

The photometry control section 11 and the main control section 16 have the same configuration as in FIG. 6. The main control section 16 centrally manages each unit that constitutes the apparatus, and performs various control operations as necessary. The main controller 16 adjusts the positional relationship so that excitation light passes between adjacent reaction cells 3 when the reaction disk 2 is stopped. The main control unit 16 also controls the reaction disk 2.
When rotating, a measurement signal is sent to the photometry control section l1 via the common memory 21. Based on this, the photometry control section 11 causes the photometry section 30 to start photometry, and operates to store the detected amount of fluorescent light in the common memory 21 for each reaction cell 3. The start and end of photometry is performed by storing a measurement start code from the main control unit 16 in the operation instruction area of the common memory.

Next, the operation of this embodiment will be explained.

It is assumed that the position of the automatic fluorescence analyzer is adjusted so that excitation light passes between each reaction cell 3 when the reaction disk 2 is stopped. The main control section 16 controls each unit configured as shown in FIG. 1 using a timing chart as shown in FIG. The main control unit 16 rotates the reaction disk 2 by one rotation plus one pitch per cycle, and performs sampling operations, reagent dispensing operations, stirring operations, and cleaning of the sample to be measured at the operating position of each unit while the reaction disk 2 is stopped. Perform operations, etc. Further, photometry is performed while the reaction disk 2 rotates one rotation plus one pitch.

Under the control of the main control unit 16, the photometry control unit 11 first detects the amount of transmitted excitation light that has passed between each adjacent reaction cell 3 while the reaction disk 2 is stopped at the initial stage, and stores it in the RAM 15. Remember. Next, when the reaction cell 3 is rotated one rotation and one pitch by the main control section 16, the photometry control section 11
sequentially stores the amount of transmitted excitation light transmitted through the reaction cell 3 that has started moving by reading the output of the ADC 45,
This continues until the amount of transmitted light attenuates by a predetermined amount from the initial amount of transmitted light, for example, until it becomes ⅔ or less. As a result, the tube wall of the reaction cell 3 is detected and the timing for starting measurement is obtained.

FIG. 4(a) is a characteristic diagram showing this situation, where the intensity of transmitted light P detected when the reaction disk 2 is stopped at 1=0
1 is detected as t1, which decreases to approximately 2Pl/3 as the reaction cell 3 rotates, and this is considered to be the tube wall of the reaction cell 3. That is, as shown in FIG. 4(b), since the reaction cell 3 begins to enter the measurement optical path at an angle greater than the critical angle θ,
It is totally reflected at the interface between the glass of the reaction cell 3 and the measurement liquid and does not reach the photodiode 37, so the intensity of the transmitted light is P.
It decreases to 273 or less of 1. Note that 3a indicates a liquid to be measured; for example, in the case of water, the refractive index is 1.33, and in the case of air, the refractive index is 1. Further, when the reaction cell 3 is made of glass, this refractive index is 1.41.

In such a case, θ is approximately 70.6 as calculated by the following formula:
It becomes 1°.

Sinθj! ．． 33/1.41. −, θ≧70.61
0 Subsequently, as the reaction cell 3 further rotates, the intensity of the transmitted light from the tube wall toward the center again becomes a constant value P2
(tz).

Furthermore, since the reaction cell 3 begins to leave the measurement optical path at an angle greater than the critical angle θ, it is totally reflected at the interface between the glass of the reaction cell 3 and the measurement liquid 3a and does not reach the photodiode 37, resulting in the intensity of the transmitted light. A position t3 where the height decreases to 2/3 or less of P1 is detected.

When the photometry control unit 11 detects the tube wall of the reaction cell 3, the amount of transmitted light and the amount of fluorescent light are alternately read and stored in chronological order at least until the reaction cell 3 crosses the optical axis L of the optical path. The number N of reaction cells 3 whose amount of transmitted light is read at this time is expressed by the following equation.

N= (A+B/2)/(CX (D+E)X2} However, N: Number of transmitted light amount or fluorescence light amount data read until the reaction cell crosses the optical path, A: Angle of the reaction cell viewed from the center of the reaction disk , B: Angle of the gap between reaction cells as seen from the center of the reaction disk (rad), C: Rotation speed of the reaction disk (rad/s), D = Multiplexer input voltage switching time, E: ADC conversion time, Next The photometry control unit 11 traces back the amount of transmitted light stored in chronological order and searches for a position where a predetermined difference from the initial amount of transmitted light has decreased to, for example, 2P1/3 as described above, thereby determining the amount of light on the tube wall of the reaction cell 3. Find the location of the amount of transmitted light and set its data position as the i-th from the front.Next, extract the i/2nd from the front from among the fluorescent light amounts stored in chronological order to determine the amount of fluorescent light to be detected.Or The amount of fluorescence to be detected can also be determined by averaging the i/2-th neighboring data from among the amounts of fluorescence stored in time series.

By repeating the same procedure as many times as there are reaction cells 3 on the reaction disk 2, the amount of fluorescence can be measured for each reaction cell. When the measurement is completed, the photometry control section 1
1 stores an end code in the operation instruction area of the common memory 21 and notifies the main control unit 16 that the instructed operation has ended. Further, the measured amount of fluorescence light is stored in the common memory 21.

In this way, according to this embodiment, the tube wall of the reaction cell is detected, and based on this, the amount of fluorescent light corresponding to a predetermined part of the reaction cell is detected, so that the fluorescence intensity of the cell at the center of the reaction cell is always determined. can be detected accurately. Therefore, the purpose can be achieved because it is possible to suppress variations without requiring precise adjustment as in the prior art.

[Effects of the Invention] As described above, according to the present invention, a detection means for detecting the amount of transmitted excitation light that has passed through or transmitted through a reaction cell is provided,
Since the fluorescence intensity at the center of the reaction cell can be detected based on the amount of transmitted light, precise adjustment is not required and measurement accuracy can be improved.

[Brief explanation of drawings]

Fig. 1 is a block diagram showing an embodiment of the automatic fluorescence analyzer of the present invention, Fig. 2 is a block diagram showing the main parts of Fig. 1, and Fig. 3 is a block diagram showing other main parts of Fig. 1. Figure 4(a). (
b) is a characteristic diagram and a schematic diagram explaining the measurement principle of the present invention;
5 is a timing chart showing the operation of the main parts of FIG. 1, FIG. 6 is a configuration diagram showing a conventional example, and FIG. 7(a). (b
) is a signal waveform and characteristic diagram explaining the conventional measurement principle, and FIGS. 8(a) and 8(b) are schematic diagrams explaining the conventional drawbacks. 2...Reaction disk, 3...Reaction Cenobe 11...
- Photometry control section, 16... Main control section, 21... Common memory, 30... Photometry section, 37... Photodiode, 39... Photo multiplier. (01 { 30:l・1 summer (b) Figure 4

Claims (1)

[Claims] A plurality of reaction cells each containing a reaction solution are sequentially moved at a uniform speed at least within the photometric optical path, and excitation light of a specific wavelength is irradiated to the reaction cells passing through the photometric optical path, and the amount of generated fluorescence is detected. In an automatic fluorescence analyzer that measures the concentration of a specific component by irradiating a reaction cell with excitation light of a specific wavelength, a first detection means detects the amount of fluorescence generated, and a second detection means detects the amount of transmitted excitation light. 2 detection means;
At the initial stage, the transmitted light of the excitation light passing between the reaction cells is stored, and the transmitted light amount of the excitation light transmitted through the reaction cells is sequentially stored, and after a predetermined difference occurs between the two transmitted light amounts, at least the reaction cell is moved to the photometric optical path. means for storing the amount of transmitted light and the amount of fluorescent light alternately in chronological order until the amount of transmitted light crosses the 1. An automatic fluorescence analyzer, characterized in that it is equipped with means for detecting the amount of fluorescent light corresponding to a predetermined portion of a reaction cell.