JPH03120445A - Automatic fluorescence intensity measuring instrument - Google Patents

Abstract

PURPOSE:To efficiently automate calibration and to make continuous measure ment by measuring and storing the fluorescence intensity of a reference sample, automatically setting the luminous intensity of a reference light source by refer ring to this value and calibrating the characteristics of a fluorescence detector from this intensity. CONSTITUTION:The fluorescence emitted by the reference sample 261 is made incident to the fluorescence detector (photomultiplier) 32 and the fluorescent output component thereof is stored in a memory 106. For example, an LED is used for the light source 29. A subtractor 108 subtracts the same output to the sample 261 stored in the memory 106 from the output of the detector 32 to the light source 29 and inputs the result thereof to the power source 107 to control the quantity of the light emitted by the light source 29. The output of the detector 32 to the light source 29 is made equal to the same output as to the sample 261 and, therefore, if the luminance of the light source 29, i.e., the voltage of the power source 107 is fixed in this state, the light source 29 can thereafter be used as a reference light source in place of the sample 261. The fluctuation in the sensitivity of the detector 32 is, therefore, rapidly and automatically compensated by the reference light source 29.

Description

[Detailed Description of the Invention] [Industrial Application Field] The present invention relates to a fluorometer, and is particularly suitable for use in automatic analyzers for clinical tests, and is capable of automatically calibrating quickly, accurately, and
The present invention also relates to an automatic fluorescence measuring device that can perform measurements continuously.

[Prior Art] A fluorometer measures the intensity of weak fluorescence (secondary light) obtained by irradiating a sample with excitation light. However, since the intensity of the fluorescence depends on the intensity of the excitation light,
As described in Japanese Patent No. 125043, in addition to the fluorescence detector described above, a photodetector for monitoring excitation light was provided to calibrate the sensitivity.

[Problems to be Solved by the Invention] In the fluorometer, in addition to fluctuations in the excitation light, fluctuations in the sensitivity and dark current of the fluorescence detector cause measurement errors. Furthermore, fluctuations in the sensitivity and dark current of the photodetector for monitoring the excitation light described above also cause measurement errors. Conventionally, each calibration means has been appropriately adjusted for these measurement error factors.
It is known. However, the dark current and fluorescence current of the excitation photodetector are extremely weak, so in order to accurately detect and calibrate them, it is necessary to use a low-pass filter with a time constant of about 1 second, for example. However, there was a problem in that the calibration time was prolonged. Improvements have been strongly desired, especially in automatic analyzers for clinical tests that need to rapidly measure many samples in succession.

SUMMARY OF THE INVENTION An object of the present invention is to provide an automatic fluorescence measurement device that efficiently and automatically performs the above calibration.

[Means for Solving the Problems] In order to achieve the above object, the present invention provides a reference light source with stable characteristics, such as a light emitting diode, and thereby automatically calibrates the characteristics of a fluorescence detector. . Therefore, first, the fluorescence intensity of the reference sample is measured and memorized, and this value is referred to to automatically set the luminosity of the reference light source.Next, the fluorescence detector output for the reference light source and the above The sensitivity of the fluorescence detector is automatically corrected by comparing the fluorescence intensity of the reference sample.

Further, the ratio of the luminous intensity of fluorescence generated by the sample to the luminous intensity of the excitation light is calculated to standardize the fluorescence luminous intensity, thereby removing the influence of fluctuations in the luminous intensity of the excitation light.

Furthermore, the dark currents of the fluorescence detector and the excitation light detector are stored and automatically excluded from the measurement results.

[Function] The automatic fluorescence measurement device of the present invention configured as described above outputs fluorescence measurement data standardized by excitation light intensity.

Furthermore, the measurement error 7 due to the dark current of the excitation light and fluorescence detectors is removed.

Furthermore, sensitivity fluctuations of the fluorescence detector are accurately corrected using a stable reference light source calibrated in advance using a reference sample.

Further, the above-mentioned measurement, error correction, calibration operation, etc. are automatically and quickly performed by a computer device equipped with at least a memory and an arithmetic unit.

[Example code] Embodiments of the present invention will be described below with reference to the drawings.

First, the principles of the fluorescence measuring method and its calibration method according to the present invention will be explained with reference to FIGS.

FIG. 2 is a diagram illustrating the fluorescence measurement method according to the present invention.

In FIG. 3A, a fluorescence detector (photomultiplier) 32 is shielded from light and is outputting dark current because a voltage is applied from a power source 38. This dark current value is stored in memory 101. 1! The output voltage adjustment terminal 381 of the source 38 is connected to a reference potential. Similarly, the value of the dark current of the excitation photodetector 24 is also stored in the memory 102.

FIG. 2(b) is a diagram showing a method for measuring the fluorescence emitted by the sample 26. The sample 26 emits fluorescence when irradiated with excitation light from the light source 17, and this fluorescence is received by the photomultiplier 32. The photomultiplier 32 superimposes a fluorescence current corresponding to the fluorescence on the dark current and outputs it, so the dark current stored in advance is read out from the memory 101, subtracted by the subtracter 103, and the above Only the fluorescent current component is input to the divider 105. The light output component of the light source 17 is detected by the excitation light detector 24 via the half mirror 23, and the dark current component stored in the memory 102 in advance is subtracted by the subtracter 103.
to be applied. Due to the above operation, the output of the divider 105 becomes a value obtained by standardizing the fluorescence output with the excitation light output, so the light source 1
It is unrelated to the output fluctuation of 7.

Therefore, by the measurements shown in FIG. 2(b).

It becomes possible to measure fluorescence without the effects of dark current and excitation light fluctuations.

However, the photomultiplier 32 is the element whose sensitivity, dark current, etc. are most likely to fluctuate among the fluorescence measurement systems mentioned above, and although the influence of dark current can be removed by the above method, separate countermeasures must be taken to deal with fluctuations in sensitivity. There is a need.

FIG. 3 is a diagram showing a sensitivity calibration method of the photomultiplier 32 according to the present invention. In FIG. 3A, fluorescence emitted by a reference sample 261 is incident on the photomultiplier 32, and the fluorescence output component is stored in the memory 106. Fluorescence output components of other samples can be calibrated using this fluorescence output component as a reference.

However, this measurement requires time to replace the sample, so
It cannot be done frequently, so Fig. 3(b)
As shown in FIG. 2, a light source 29 serving as a reference whose output is equivalent to the output of the reference sample 261 is prepared. As the light source 29, for example, an economically stable light emitting diode such as an LED is used. The subtracter 108 extracts the reference sample 2 stored in the memory 106 from the output of the photomultiplier 32 for the light source 29.
The same output for 61 is subtracted, and the result is input to power supply 107 to control the amount of light emitted from light source 29. Due to such a feedback path, the output of the photomultiplier 32 to the light source 29 becomes equal to the same output to the reference sample 261, so if the brightness of the light M29, that is, the voltage of the electric power WX107 is fixed in this state, the light source will be 29 can be used instead of the reference sample 261.

FIG. 4 is a diagram showing the method of the present invention for compensating for sensitivity fluctuations of the photomultiplier 32 after the brightness of the light source 29 is fixed as described above. The light from the light source 29 whose brightness has been set to a predetermined value using the method shown in FIG. The output voltage for the reference sample thus obtained is subtracted, and the remaining amount is applied to the power supply 38 to control its output voltage.

As shown in FIG. 5, the current amplification factor of the photomultiplier 32 changes depending on the value of the applied voltage.
The sensitivity of the photomultiplier 32 can be controlled by changing the voltage of the power supply 38 shown.

Therefore, the photomultiplier 32 shown in FIG.
The feedback path for the reference sample 216 operates such that the output of the photomultiplier 32 to the light source 29 always matches the output to the reference sample.
This allows the light source 29 to quickly and automatically compensate for variations in the sensitivity of the photomultiplier 32 without using the light source 29.

FIG. 1 is a diagram showing an embodiment of the present invention in which the methods shown in FIGS. 2 to 4 described above are implemented by a computer 41. The outputs of the photomultiplier 32, excitation light detector 24, etc. are taken into the computer 41 via preamplifiers 33, 34 and AD converters (analog-to-digital converters) 35, 36, respectively. The computer 41 also lights up the light source 29 via the interface 39, and also controls the drive mechanism 3a and other mechanical systems 40 for transporting the sample, reference sample, etc.
etc.

A shutter 46 driven by a solenoid 45 is provided in front of the light source 17, and when measuring each dark current shown in FIG. 29 is cut off, and the dark currents of the photomultiplier 32 and excitation photodetector 24 are stored in the memory 411 in the computer 41 via preamplifiers 33, 34, AD converters 35, 36, etc., respectively. At this time, the computer 41 sends a predetermined reference signal to the DA converter 37 and sets the output voltage of the flt source 38 to a reference voltage value.

When measuring the reference sample shown in FIG. 3(a), the computer 41 moves the drive mechanism 3att 111g to set the reference sample, opens the shutter 46, irradiates the reference sample with light from the light source 17, and irradiates the reference sample with its fluorescence. Strength photo multiplier 3
2, the arithmetic unit 412 in the computer 41 subtracts the dark current value stored in the memory 411, and stores the result in a predetermined area in the memory 411. At this time as well, the power supply 38 is generating the reference voltage value.

Next, when setting the output of the light source 29 as shown in FIG. The current of the light source 29 is controlled so that the residual error converges toward zero, and when the residual error converges within a predetermined range, the value is stored in the memory 411. From now on, light source 2
9 is driven by the above-mentioned current value stored in the memory 411 and is used as a reference light source.

After the above preparatory work, fluorescence measurement of the sample is automatically performed. That is, the N movement mechanism 3 is moved to insert the sample, the shutter 46 is opened, and the excitation light intensity is transmitted to the excitation light detector 2.
At the same time, the fluorescence of the sample 26 is detected by the photomultiplier 32 and sent to the computer 41.
After subtracting each dark current component stored in advance in the memory 411 from both signals by the calculation unit 412,
The fluorescence signal component is divided by the excitation light component to generate a standardized fluorescence signal, which is sent to the display 43, printer 44, etc.

As the above fluorescence measurement is repeated, the sensitivity of the forchiplier 32 gradually changes. Therefore, the sensitivity of the photo multiplier 32 shown in FIG. Compares the output of the output with the reference sample signal stored in the memory 411, drives the power supply 38 based on the difference signal, and drives the power supply 38 when the output of both falls within a predetermined error range. Memory value 41
1, and this value is used to drive the power supply 38 until the first sensitivity correction.

Similarly, correction of dark current can also be performed at any time.

FIG. 6 shows data obtained by comparing the stability of the automatic fluorescence intensity meter of the present invention described above with that of a conventional one. The relative fluorescence sensitivity of the conventional device is approximately 4% after 4 hours as shown in A.
descend. On the other hand, in the apparatus of the present invention, as shown in B, the stability was reduced to 1% or less in 4 hours, and the stability was improved by approximately 4 times.

This effect uses a light source 29 and a photomultiplier 32.
This is due to sensitivity correction.

FIG. 7 is a diagram showing an outline of an automatic sample transport device used in an automatic analyzer for clinical tests b1, in which the drive mechanism 3a, reaction disk 2, reaction container 1. This corresponds to the part such as sample 26. In Figure 7, a sample disk 7 with a sample container 8 mounted on it and a reagent disk 10 with a reagent container 12 mounted on it are placed coaxially on the right side, and a reaction disk 2 equipped with a reaction container 1 on the left side. It is located. The reagent disk 10 and sample disk 7 are placed in a cold storage tank 13.

The reaction disks 2 are housed in a constant temperature bath 5, and each disk is rotated under the control of a computer 41 to transport predetermined sample containers, reagent containers, reaction containers, etc. to predetermined positions. The transported sample and reagent are sequentially taken out into a predetermined reaction container by the pipetting mechanism 14. In this way, each time a reaction container containing a predetermined sample, reagent, etc. is sent into the fluorometer 6,
The above measurements are made and data are taken. The reaction vessel after the measurement is cleaned in the cleaning section 4, and the probe 15 of the pipetting mechanism 14 is cleaned by the cleaning device when returning from the reaction vessel. 16 before proceeding to the next operation.

FIG. 8 is a timing chart illustrating the relationship between the above conveyance operation and the sensitivity correction of the photomultiplier 32 performed using the light source 29. FIG. 8 will be explained from top to bottom. The measurement cycle for one sample is 18 seconds. First, the reaction disk 2 rotates one pitch to transport the cleaned reaction container to a predetermined position.Then, the sample disk is rotated to transport a predetermined sample. During this time, 1 next to the pipetting machine
4 is cleaned by the cleaning device 16, then rotated to the sample side to take out the sample, and rotated to the reaction vessel side to introduce the sample. Although not shown in FIG. 8, necessary types of reagents are similarly taken out into the reaction container. The probe 15 of the pipetting mechanism 14 is lifted upward while its arm is rotating, and performs suction on the sample side and discharge operation on the reaction vessel side. When the discharge of the sample and reagent is completed, the shutter 46 opens and fluorescence measurement is performed. will be carried out. Furthermore, the sensitivity of the photo multiplier 32 is corrected by turning on the light source 29 during the previous time period. When the light source 29 is turned on, the preamplifier 33 generates an output.

The value of the output voltage is input into the computer 41 by operating the AD converter 35, and compared with the output value for the reference sample described above. If it is insufficient, the output voltage of the power supply 38 is increased by one step and the above comparison is performed.

This is repeated until the output of the power supply 38 is fixed when the output of the preamplifier matches the output value for the reference sample. The reference sample is contained in one of the sample containers, for example. Further, the excitation light intensity from the light source 17 is measured by operating the AD converter 36 while the shutter 46 is open, and the dark current of the excitation light detector 24 is measured while each light source is cut off during the measurement cycle. Measure between.

[Effects of the Invention] As described in detail above, when the present invention is applied, fluorescence measurement data standardized by excitation light intensity can be obtained, so that the influence of excitation light fluctuations can be removed.

Furthermore, measurement errors due to the dark currents of the excitation light and fluorescence detectors can be eliminated.

Furthermore, sensitivity fluctuations of the fluorescence detector can be accurately corrected using a stable reference light source calibrated in advance using a reference sample.

Further, the above-mentioned measurement, error correction, calibration operation, etc. can be performed automatically and quickly using a computer device equipped with at least a memory and an arithmetic unit.

[Brief explanation of the drawing]

FIG. 1 is a diagram showing an example of the automatic fluorescence measuring device of the present invention, FIG. ) is a diagram showing the principle of the present invention, which detects the fluorescence of a sample and its excitation light to obtain a fluorescence intensity output standardized by the excitation light intensity, and Figure 3 (a) is a diagram showing a method for measuring the fluorescence intensity of a reference sample. , FIG. 3(b) is a diagram illustrating a detailed explanation of the present invention for setting the brightness of a reference light source to a predetermined value, and FIG. 4 is a diagram showing a method of the present invention for correcting the sensitivity of a fluorescence detector using a reference light source. , Fig. 5 is a characteristic diagram of a photomultiplier for fluorescence detection, Fig. 6 is a diagram showing the stability over time of the fluorescence measuring device according to the present invention compared with that of a conventional device, and Fig. 7 is a diagram showing the characteristics of a photomultiplier for fluorescence detection. Explanatory diagram of the conveying device, No. 8
The figure is a time chart explaining the operation of the fluorescence measuring device of the present invention. 1... Reaction container, 2... Reaction disk, 3a...
Drive mechanism, 4...Cleaning section, 5...Thermostat, 6...
Fluorometer, 7... Sample disk, 8... Sample container, 10... Reagent disk, 13... Heat insulating tank, 14...
... Pipette extension mechanism, 15... Probe, 16...
-Cleaning mechanism, 17... light source. 23...Half mirror 24...Excitation light detector, 26
...Sample, 261...Reference sample, 29...Light source,
32... Photo multiplier, 33... Preamplifier, 35... AD converter, 38... Power supply, 39...
・Interface, 41... Computer, 411
...Memory, 412...Calculation unit, 42...Keyboard, 43...Display, 44...Printer, 4
5...Solenoid, 46...Shutter, 101...
・Memory, 103...subtractor, 105...divider,
107...Power supply.

Claims (1)

[Claims] 1. A fluorescence photometry device that measures the intensity of fluorescence obtained by irradiating a sample with excitation light, which comprises a reference light source, a reference value storage device, a comparison means, and a fluorescence detector for driving the fluorescence detector. 1 power supply and a control value storage means for the first power supply, the output of the fluorescence detector for the reference light source and the reference value stored in the reference value storage device are compared by the comparison means, and the control value is obtained. controlling the voltage of the first power supply by the first comparison signal;
An automatic fluorescence photometry device characterized in that the first comparison signal is stored in a control value storage means of the first power source. 2. The automatic fluorescence measurement device according to claim 1, wherein a fluorescence intensity signal obtained by inputting fluorescence generated by a reference sample into the fluorescence detector is stored in the reference value storage device. 3. Claim 1 or 2, comprising: a second power source for driving the reference light source; and a control value storage means for the second power source, the output of the fluorescence detector for the reference light source and the reference value storage device. A second signal is obtained by comparing the reference value stored in the
1. An automatic fluorescence photometry apparatus characterized in that the voltage of the power supply is controlled, and the second comparison signal is stored in a control value storage means of the second power supply. 4. In any one of claims 1 to 3, the device comprises the excitation light detection means and the division means, and the output of the fluorescence detector and the output of the excitation light detection means are input to the division means. An automatic fluorescence photometry device characterized by: 5. In any one of claims 1 to 4, first dark current storage means for storing the dark current of the excitation light detection means;
a subtraction means for subtracting the dark current value stored in the first dark current storage means from the output of the excitation light detection means; a second dark current storage means for storing the dark current of the fluorescence detector; and a second dark current storage means for storing the dark current of the fluorescence detector; 1. An automatic fluorescence measurement device comprising: subtraction means for subtracting a dark current value stored by the second dark current subtraction means from the output of the device. 6. In any one of claims 1 to 5, at least a step of lighting the reference light source, and a step of comparing the output of the fluorescence detector and the reference value stored in the reference value storage device by the comparing means. , comprising a storage device for storing a procedure for controlling the voltage of the first power supply using the first comparison signal, and a procedure for storing the first comparison signal in the control value storage means of the first power supply. An automatic fluorescence measurement device featuring: 7. In any one of claims 1 to 6, at least a step of transporting the reference sample, a step of storing the fluorescence intensity of the reference sample as a reference value, and a step of turning on the reference light source and performing the step of transporting the reference sample with respect to the reference light source. a step of comparing the output of the fluorescence detector and the reference value to obtain the second comparison signal;
The voltage of the second power supply is controlled by the second comparison signal, and the voltage of the second power supply is controlled by the second comparison signal.
1. An automatic fluorescence photometry device characterized by comprising a storage device for storing a procedure for storing a comparison signal of the above in a control value storage means of a second power supply.