JP2021144007A - Multichannel light detection device - Google Patents

Abstract

To provide a light emission intensity measurement device that reduces an error of detection sensitivity of light intensity between measurement channels.SOLUTION: A device 10, which has a plurality of measurement channels, and measures light intensity of chemoluminance, comprises: a measurement base 13 that has a sample holding member capable of arranging each of a plurality of samples to each of a plurality of measurement channels; light detection means that has a plurality of light detectors 19 measuring light intensity of the plurality of samples arranged in the sample holding member for each measurement channel; correction information storage means 22 that stores correction information for each measurement channel; and means 21 that acquires light intensity correcting light emission intensity from the plurality of light detectors corrected by a computation with reference to the correction information for each measurement channel. The correction information is based on a relationship between first light intensity of a calibration sample or prescribed light source arranged in the sample holding member and measured by the plurality of light detectors and second light intensity of the calibration sample or prescribed light source measured by other light detection device.SELECTED DRAWING: Figure 1

Description

The present invention relates to techniques for measuring emission signals of biological, chemical, medical, pharmaceutical and environmental samples.

In the fields of biology, chemistry, medicine, pharmacy and the environment, there have been many needs for measuring a variety of luminescent samples present in a sample. Bioassays are roughly classified into two types according to the luminescence signal, a method using a fluorescent protein and a method using chemiluminescence (including bioluminescence from luciferase or the like). Chemiluminescence is extremely weak compared to fluorescence, and it is desirable to use a highly sensitive measuring device.

On the other hand, the present inventor has previously tried an analog multi-channel optical detector (Patent Application 2013-268325 (Patent 6161072)), but since the measurement result was a fluctuating current waveform in the first place, the sensitivity of each channel. It was difficult to adjust and standardize. For the same reason, the sensitivity was extremely poor and the signal-to-noise ratio (S / B ratio) was only doubled. Since it is an analog type, the sensitivity cannot be adjusted between each channel, so the reliability of the measured values obtained from it is low.

The present inventor arranges each sample in the light receiving part of many channels of the measurement base, and can simultaneously measure the chemiluminescence of each sample by a photomultiplier tube (photomultiplier tube) connected to each channel, and has high sensitivity. A luminescence measurement system has been developed (see, for example, Patent Document 1).

Japanese Patent No. 6161072

In a measuring device such as Patent Document 1 in which a large number of channels are measured by a photodetector, even if the photodetector of each channel, for example, the applied high voltage and gain of a photomultiplier tube is adjusted to adjust the sensitivity, the interchannels still remain. There is a problem that the detection sensitivity of is different.

An object of the present invention is to provide a light emission intensity measuring device capable of reducing an error in the detection sensitivity of light intensity between measurement channels.

According to one aspect of the present invention, it is a device for measuring the light intensity of chemical emission having a plurality of measurement channels, and a sample holding member capable of arranging a plurality of samples for each of the plurality of measurement channels. A measurement base having a measurement base, a light detection means having a plurality of light detectors for measuring the light intensity of the plurality of samples arranged on the sample holding member for each measurement channel, and correction information for each measurement channel are stored. The correction information is provided with a means for storing correction information and a means for acquiring the light intensity obtained by calculating the light emission intensity from the plurality of light detectors by referring to the correction information for each measurement channel. A sample for calibration or a predetermined light source is arranged on the sample holding member, and the first light intensity measured by the plurality of light detectors and the sample for calibration or the predetermined light source are measured by another light detection device. The above device is provided based on the relationship with the measured second light intensity.

According to the above aspect, the detection sensitivity of the light intensity between the measurement channels is obtained by acquiring the light emission intensity corrected by calculation with reference to the correction information for each measurement channel from the plurality of photodetectors. It is possible to provide a light emission intensity measuring device in which the error of the above is reduced.

It is a block diagram which shows the schematic structure of the multi-channel optical measuring apparatus which concerns on one Embodiment of this invention. It is a partially enlarged view of a sample stage. It is a figure which shows the example which acquires the correction information. It is a figure which shows the example of the correction information.

Hereinafter, an embodiment of the present invention will be described with reference to the drawings. Elements common to a plurality of drawings are designated by the same reference numerals, and the repetition of detailed description of the elements will be omitted.

FIG. 1 is a block diagram showing a schematic configuration of a multi-channel optical measuring device according to an embodiment of the present invention. FIG. 2 is a partially enlarged view of the sample stage. Referring to FIGS. 1 and 2, the multi-channel optical measuring device 10 has a main body 11 and a personal computer (PC) 12 connected by a USB (universal serial bus) or the like. Body 11 has a sample stage 13, a light detector 14, and a control unit 15. The sample stage 13 is provided with a sample holding member 16 on which an 8-unit PCR tube PT for accommodating a sample can be placed. Specifically, the sample holding member 16 is provided with an opening 16a on the top surface into which the tip of the 8-unit PCR tube PT is inserted. The bottom surface 16c of each opening 16a, 8 single light receiving portion 17 _{1-17 8} for receiving the light emission of the sample is provided for each measurement channel. In the sample holding member 16, in the space where the tip of the 8-unit PCR tube PT is accommodated, each measurement channel is separated by a partition portion 16b, and the chemiluminescence of the sample of each measurement channel is transmitted to the light receiving portion 17 _{1 of the other measurement channel.} It is configured so as not to enter the -17 _8. Light receiving portion 17 _{1-17 8} receives is output to the optical detector 14 via the optical waveguide 18 _{1-18 8} provided for each measurement channel. The multi-channel optical measuring device 10 is not limited to eight measurement channels, and may have a plurality of measurement channels other than the eight channels.

The light conduction path 18 is a light-transmitting plastic material, and the outside thereof is covered with clad steel in order to minimize light loss. An optical fiber or the like can be used in addition to the plastic material. Further, when the photodetector 19 can be directly arranged on the light receiving unit 17, the light conduction path 18 may be omitted.

The photodetector unit 14 is provided with a photodetector 19 and a counter circuit 20 for each measurement channel, and the output of each counter circuit 20 is sent to the control unit 15.

The photodetector 19 converts the light sent from the optical conduction path 18 into an electric signal (photocurrent). As the photodetector 19, for example, a semiconductor optical detection element such as a photodiode, an avalanche photodiode, a phototransistor, or a photoconductor, a photomultiplier tube (PMT), or the like can be used. The photodetector 19 is preferably a PMT in terms of sensitivity. On the output side of the semiconductor photodetector, the photocurrent is converted into a voltage signal. An amplifier (not shown) that amplifies the voltage signal may be provided.

The counter circuit 20 measures the light intensity from the voltage signal (output signal) from the photodetector 19. When the light incident on the photodetector 19 is weak, this output signal becomes a pulse. The counter circuit 20 has, for example, a discriminator (wave height discriminator), a waveform shaper, and a counter in order to count the number of pulses from this pulsed output signal. The discriminator sets the lower limit of the output signal as a predetermined voltage value, removes a signal having a voltage smaller than that, and selects only pulses having a voltage value equal to or higher than the predetermined voltage value. The waveform shaper shapes the waveform of the selected pulse into a pulse width in the detection range of the counter downstream thereof. The counter is, for example, a gate circuit and counts pulses exceeding a predetermined voltage value. The counter circuit 20 outputs the number of pulses counted in a predetermined time period to the control unit 15 as light intensity. The unit of this light intensity is, for example, a relative emission unit (Relative Luminescence Unit (RLU)).

The control unit 15 receives the number of pulses in parallel from the counter circuit 20 of each measurement channel, performs parallel-serial conversion and the like, and outputs the pulse number to the PC 12 by an interface, for example, USB.

The PC 12 has an arithmetic control unit 21, a storage unit (memory) 22, a display unit 23 such as a display, and a user interface (not shown) such as a mouse and a keyboard. The arithmetic control unit 21 is, for example, a CPU, a GPU, or the like. The calculation control unit 21 acquires the light emission intensity corrected by calculation with reference to the correction information 24 stored in the storage unit 22 for the light intensity (number of pulses) of each channel.

The storage unit 22 is a semiconductor memory such as a ROM (read-only memory) or a RAM (random access memory), and may further have a hard disk drive. The storage unit 22 stores the correction information 24 for each measurement channel. The correction information 24 for each measurement channel is information for calibrating the light intensity of each measurement channel measured by the multi-channel optical measurement device 10 so as to be comparable to each other, and is also obtained by the multi-channel optical measurement device 10. This is information for converting the light intensity expressed by the relative light emission unit into the absolute light intensity.

FIG. 3 is a diagram showing a configuration example for acquiring correction information. With reference to FIG. 3 together with FIG. 1, a multi-specimen measuring device 100 is used as an example in order to acquire correction information for each channel of the multi-channel optical measuring device 10. The multi-sample measuring device 100 has a chamber 101 configured to prevent outside light from entering. The sample stage 102 is arranged on the bottom surface in the chamber 101. A CCD camera 103 is arranged above the chamber 101 as an example of one photodetector. The chemiluminescence of a large number of samples on the sample stage 102 can be measured simultaneously by the CCD camera 103. As an alternative to the CCD camera 103, a microchannel plate type PMT (two-dimensional PMT) may be used.

The main body 11 of the multi-channel optical measuring device 10 is installed on the sample stage 102 in the chamber 101 of the multi-sample measuring device 100. On the sample stage 13 of the main body 11, an 8-unit PCR tube containing a sample Smp for calibration is placed. The chemiluminescence of the sample Smp for calibration is sent from the light receiving unit 17 arranged on the lower surface of the 8-unit PCR tube to the photodetector unit 14, and the light intensity is measured for each channel as a relative light emission unit, and output to the PC 110. ..

On the other hand, in parallel, the multi-specimen measuring device 100 measures the chemiluminescence of the sample Smp for calibration from above by the CCD camera 103 arranged above the sample stage 13, for example, the chemiluminescence of the sample Smp for calibration. The luminous flux of is converted into absolute light intensity (photons / sec) as light intensity and output to the PC 110.

In the normal use of the multi-channel optical measuring device 10, the sample stage 13 is a dark room due to the lid portion, but the lid portion is not used in this process.

In the PC 110, the correlation between the light intensity measured in each measurement channel by the multi-channel light measuring device 10 and the light intensity measured by the multi-sample measuring device 100 is acquired by the following formula. _{The relationship between the light intensity A i} (relative emission unit) of each channel of the multi-channel light measuring device 10 _{and the light intensity B i} (photon / sec) of the sample corresponding to each measurement channel measured by the multi-sample measuring device 100 is as follows. It is represented by the formula (1).
_{log 10 A i = m i ×} log 10 B i + b i ··· (1)
However, b _i and m _i is a real number, i (= 1~8) represent the channel number.
Thus m _i and b _i of each channel obtained is correction information 24. The correction information 24 is acquired prior to the measurement of the desired sample and stored in the storage unit 22 of the multi-channel optical measuring device 10.

The arithmetic control unit 21 of the multi-channel optical measurement device 10 refers to the correction information 24 stored in the storage unit 22 and uses the following equation (2) from _{the light intensity SA i (relative light emission unit) of each measurement channel.} Therefore, it can be converted into a standardized light intensity (light intensity SB _{i) expressed in absolute light intensity.
SB i = 10 ^ [(log} 10 SA i -b i) / m i] ··· (2)

Thereby, the sensitivity to chemiluminescence between each channel of the multi-channel light measuring device 10 can be matched, and the error of the light intensity between each channel can be reduced. Further, the light intensity of the relative light emission unit acquired in each channel can be expressed by the absolute light intensity. The absolute light intensity is expressed in photons / second as a unit.

As the calibration sample Smp, it is preferable to use a plurality of samples having emission intensities in order to acquire correction information. Further, when the emission intensity of the sample Smp for calibration changes with time, it is preferable that the multi-channel optical measuring device 10 and the multi-sample measuring device 100 simultaneously measure the sample Smp a plurality of times at the same time.

[Example of correction information 1]
As the multi-sample measuring device 100, a "IVIS Lumina II system" (hereinafter referred to as "IVIS") manufactured by PerkinElmer Co., Ltd. was used. As a sample for calibration, a standard stock solution (100 μg / mL) of placental alkaline phosphatase (PLAP) attached to the trade name “Great EscAPe ™ SEAP Chemiluminescence Kit 2.0” manufactured by Clontech Inc. in the United States was used at three different concentrations (100 μg / mL). 20, 5 and 1 μg / mL). For each of these three types of PLAP diluents, transfer 10 μL of PLAP diluent to an 8-unit PCR tube, mix 8 10 μL substrates included in the kit at the same time using an 8-channel micropipette, and immediately use a multi-channel optical measuring device. It was placed on the sample stage 13 of 10. After the addition of the substrate, the measurement time was set to 2 minutes, and the sample of each concentration was repeatedly measured 7 times to obtain 21 data in each measurement channel. The above formula (1), was determined parameters b _i and m _i by the least squares method for each measurement channel. In addition, IVIS has a function of acquiring an image of a sample by one CCD and converting the light emission intensity of a light emitting part into a light intensity expressed by an absolute light intensity.

FIG. 4 is a table showing correction information for each channel. Referring to FIG. 4, m _i and b _i between channels show different values. However, it can be seen that the correlation coefficient r ² can show a value very close to 1. This is the equation obtained by substituting the obtained m _i and b _i (1) is the light intensity B obtained by multi-analyte measuring device 100 from the light intensity A multichannel optical measuring device 10 indicating that it can better predict There is. As a result, it can be seen that the error in the light intensity between the channels can be reduced, that is, the detection sensitivities between the channels can be matched.

Examples of other correction information acquisition and application methods of the multi-channel optical measuring device according to the embodiment of the present invention are as follows.

[Example 2 of correction information]
On the sample stage 13, a sample for calibration similar to that in Example 1 of the above correction information is arranged in each of the plurality of measurement channels, and the light intensity A _i (i = 1 to 8) is determined by the light detector of each measurement channel. In addition to measuring, the light intensity of the calibration sample placed on one of the measurement channels, for example, channel 1 Ch1, is measured using a calibrated light detector (for example, IVIS). Obtain the reference light intensity B _ref. B _ref is represented by absolute light intensity. Correction information seeking b ₁ and m ₁ by the least squares method for the first channel Ch1 by the equation (1) based on light intensity A ₁ measured by the photodetector of the reference light intensity B _ref and the first channel Ch1 Let it be 24.

The correction information 24 is acquired prior to the measurement of the desired sample and stored in the storage unit 22 of the multi-channel optical measuring device 10. The arithmetic control unit 21 of the multi-channel optical measurement device 10 also uses the correction information 24 stored in the storage unit 22 as correction information for measurement channels other than the first channel Ch1, and the light intensity SA _i (relative emission unit) of the sample. By using the correction coefficient a of the correction information from)), it is possible to convert each measurement channel into a standardized light intensity (light intensity SB _{i) expressed in absolute light intensity by the following equation (3).}
SB _i = 10 ^ [(log ₁₀ SA _i −b ₁ ) / m ₁ ] ・ ・ ・ (3)
Here, i = 1-8.

The correction information 24 may be acquired not only in one measurement channel but also in a plurality of measurement channels by the above method and used as correction information for other measurement channels. Further, a plurality of brightnesses are used by using a light source whose brightness can be changed instead of the sample for calibration (for example, a stabilized light source for a photoelectron multiplying tube manufactured by Hamamatsu Photonics Co., Ltd., model L11494-525 (peak emission wavelength: 522 nm)). Based on the reference light intensity B _ref and the light intensity A _{1 measured by the light detector of the first channel Ch 1} , the correction information 24 may be obtained by obtaining _{b 1} and m 1 for the first channel Ch 1 by the above equation (1). , seeking b _i and m _i in a plurality of measurement channels may be as correction information 24 of the other measurement channels may be as correction information 24 of the corresponding measured channel seeking b _i and m _i in each measurement channel ..

[Example 3 of correction information]
On the sample stage 13, a light source (for example, the model L11494-525) whose brightness (or emitted light beam) is measured in advance by another light detection device is arranged in each of the plurality of measurement channels to obtain a plurality of brightness. _{The light intensity A i} (i = 1 to 8) is measured by the light detector of each measurement channel, and _{the reference light intensity B ref} obtained from the known brightness of the light source is used (absolute light intensity). Correction information seeking b _i and m _i by the least squares method for each measurement channel Chi by the equation (1) based on light intensity A _i measured by the photodetector of the reference light intensity B _ref and the measurement channel Chi Let it be 24.

The correction information 24 including the correction coefficient a is acquired prior to the measurement of the desired sample and stored in the storage unit 22 of the multi-channel optical measuring device 10. The arithmetic control unit 21 of the multi-channel optical measurement device 10 refers to the correction information 24 stored in the storage unit 22, and corrects the correction information b from _{the light intensity SA i (relative emission unit) of the sample of each measurement channel.} the use of _i and m _i, can be converted to a standardized light intensity expressed in absolute light intensity for each measurement channel by the formula (2) (light intensity SB _i).

Although the preferred embodiment of the present invention has been described in detail above, the present invention is not limited to the specific embodiment, and various modifications and changes can be made within the scope of the present invention described in the claims. It is possible.

10 Multi-channel optical measuring device 11 Main unit 12 Personal computer (PC)
13 Sample stage 14 Photodetector 16 Sample holding member 17 _{1 to} 17 ₈ Light receiving unit 19 Photodetector 20 Counter circuit 21 Arithmetic control unit 22 Storage unit 24 Correction information

Claims (9)

A device that measures the light intensity of chemiluminescence that has multiple measurement channels.
A measurement base having a sample holding member capable of arranging a plurality of samples for each of the plurality of measurement channels.
A photodetector having a plurality of photodetectors for measuring the light intensity of the plurality of samples arranged on the sample holding member for each measurement channel, and
A correction information storage means for storing correction information for each measurement channel, and
It is provided with a means for acquiring the light intensity corrected by calculation with reference to the correction information for each measurement channel for the light emission intensity from the plurality of photodetectors.
The correction information includes a first light intensity measured by the plurality of light detectors by arranging a sample for calibration or a predetermined light source on the sample holding member, and the sample for calibration or a predetermined light source. The device based on the relationship with the second light intensity measured by the light detection device. The device according to claim 1, wherein the second light intensity is a light intensity measured by the other light detection device in which the sample for calibration is arranged on the plurality of sample holding members. The device according to claim 2, wherein the other photodetector can measure the light intensity of chemiluminescence of the calibration sample arranged in each of the plurality of measurement channels with one photodetector. The device according to any one of claims 1 to 3, wherein the second light intensity represents a luminous flux. In the correction information, the relationship between the first light intensity A (relative light emission unit) and the second light intensity B (photons / sec) is expressed by the following equation.
_{B i = 10 ^ [(log} 10 A i -b i) / m i]
However, b _i and m _i is a real number, i is representative of the channel number, of the claims 1-4, apparatus according to any one claim. The correction information includes a first light intensity measured in at least one of the plurality of measurement channels and a second light intensity measured by another light detection device using the calibration sample or the predetermined light source. Including the correction coefficient obtained based on the relationship with
The method according to claim 1, wherein the means for acquiring the corrected light intensity obtains the light intensity corrected by calculation with reference to the correction coefficient for the emission intensity of a channel other than at least one of the plurality of measurement channels. Device. The device according to any one of claims 1 to 6, wherein the predetermined light source is preliminarily calibrated for brightness or light flux emitted by the other photodetector. The device according to any one of claims 1 to 7, wherein the plurality of photodetectors are photomultiplier tubes. The apparatus according to any one of claims 1 to 7, wherein the plurality of photodetectors are semiconductor photodetectors.

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