JP2004301648A - Analysis apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a rapid, versatile and compact analysis apparatus which uses a microplate and has high measuring accuracy. <P>SOLUTION: The analysis apparatus comprises the microplate having a plurality of sample wells and a detector for receiving lights from the sample wells. The microplate moves in a field of view of the detector at a constant speed. The microplate can preferably move and undergo reciprocative motion. Each group of a plurality of the sample wells, disposed in a line, is preferably separated. The sample wells are irradiated by the monochromatic light collected and repeat light emission and quenching at regular intervals. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to an analyzer using a microplate, and is particularly useful for biochemical analysis at a cell level.
[0002]
[Prior art]
In recent years, biochemical analysis or analysis at the cell level has been emphasized, and analysis has been attempted by various measuring methods. Among them, photoluminescence, chemiluminescence, and absorbance analysis are particularly frequently used as excellent analytical methods such as rapidity, stability, and accuracy of measurement. Photoluminescence and chemiluminescence measure the amount of excitation light generated by the absorption of light applied to a specimen or by a chemical reaction, while absorbance analysis measures the ratio of incident light to transmitted light. As a result, the specific element in the sample can be detected.
[0003]
In the analysis at the biochemical cell level, a large number of samples must be measured at the same time, and a standard sample container known as a microplate is used to enable automation such as screening. The microplate has a structure in which a plurality of sample wells are arranged to accommodate a plurality of samples and can be fixed to the device.
[0004]
In order to utilize each of the above-mentioned measuring methods using such a microplate, various analyzers are actually put into practical use. For example, an apparatus called a microplate reader as shown in FIG. 8 is put into practical use. .
A combination of a halogen lamp 51 and an interference filter 52 is used as a monochromatic light source, and the light from the light source condensed by a convex lens 53 is turned on and off by a mechanical chopper 54 to irradiate the microplate 2. 55, a motor for rotating the chopper; 56, a fan for cooling the lamp 51; 57, an optical fiber for guiding monochromatic light; here, the light transmitted through the microplate 2 is received by the photodiode 3. An example is shown. The analyzer further includes a light source driving power supply unit 58, a chopper driving unit 59, a microplate driving unit 60, a detector signal amplification processing unit 61, an arithmetic processing unit 62, and a display unit 63. Analysis.
[0005]
9 relates to a high-throughput photodetection device and method used for biochemical cell-level analysis. FIG. 9 shows a switch mechanism and an optical relay structure in which different light sources and detectors are selected according to the application. Alternatively, a device has been proposed in which a switch mechanism and an optical path enable top / bottom illumination and top / bottom detection, or a combination thereof (see, for example, Patent Document 1).
[0006]
[Patent Document 1]
JP 2002-509235 A [Problems to be Solved by the Invention]
However, the following problems may occur in the analyzer described in the related art.
Normally, when a solution sample is put into a microplate and measurement is performed using a single or a plurality of detectors, in a type in which the microplate is moved, it is necessary to wait until the liquid level becomes constant. It is difficult to obtain accurate measurement values due to the generation of stray light and fluctuations in the effective length of the sample layer when measuring with the liquid surface oscillating, but a long waiting time makes it difficult to measure the reaction state of the sample. This may cause a change in the sample itself. In addition, in the above-described type and the type in which the detector is moved (including the case where the light source is provided), accurate measurement is difficult unless the position of the detector and the position of the well are accurately matched. Although it is possible to maintain the measurement accuracy even with rough alignment, the number of samples is reduced or the size of the apparatus is increased. Therefore, there is a demand for a compact analyzer that can quickly measure a sample after it is put into a sample well.
[0007]
In addition, the halogen lamp used in the light source unit generally requires (1) about 30 minutes to be in a stable light emitting state after the power is turned on, and (2) the life as a light source is short, about 1500 hours, and is a regular. (3) The surface temperature is high (about 300 ° C) because the filament is heated to emit light, and it is necessary to provide a cooling space or forced cooling means around it, and the light source section becomes large. (4) It is difficult to perform ON-OFF operation in a fast cycle, so that a chopper is required and miniaturization is difficult.
[0008]
Therefore, an object of the present invention is to solve the above-mentioned problems and to provide a compact and highly versatile analyzer having high measurement accuracy in an analyzer using a microplate.
[0009]
[Means for Solving the Problems]
Means for Solving the Problems The present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, have found that the above-described object can be achieved by an analyzer described below, and have completed the present invention.
[0010]
The present invention is an analysis apparatus including a microplate having a plurality of sample wells and a detector that receives light from the sample well as a component, wherein the microplate moves at a substantially constant speed in a field of view of the detector. It is characterized by. When the microplate is moved at a constant speed, the surface of the sample in the well can maintain a stable state, so that a plurality of samples can be measured at high speed and with high accuracy.
[0011]
Here, it is preferable that the microplate can reciprocate in the field of view of the detector. By averaging the detector output, it is possible to measure a plurality of samples at high speed and with high accuracy. In addition, by repeating the reciprocating movement, continuous measurement with a fixed time interval can be performed.
[0012]
Further, it is preferable that the microplate has a configuration capable of being separated into a plurality of sample well groups arranged on a line. By using such a well unit, it is possible to prevent an operation error, a complicated operation, and an increase in the size of the apparatus due to automation, and the speed and versatility of the present invention can be more effectively utilized.
[0013]
Further, a device for condensing and irradiating light from a light source unit to the sample well and detecting absorbance by the detector, wherein the light source unit is a light source for generating monochromatic light, and emits and quenches at a constant period Is repeated. Such a device can be applied very well to such demands in that it can be applied in a wide range simply by replacing the light source unit, the detection unit or the optical filter.
[0014]
At this time, it is preferable that the light source unit is made of a direct transition solid state light emitting device. By using such an element, a signal having a large output from the detector and having a small noise component can be obtained.
[0015]
Further, it is preferable that the wavelength range of the light from the light source section is approximately 420 to 670 nm. For detection of a color reaction in a biological reaction, the wavelength range is preferable in terms of detection sensitivity or selectivity, and an element having a large light emission amount and a high modulation frequency can be used.
[0016]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
The present invention is an analysis apparatus including a microplate having a plurality of sample wells and a detector that receives light from the sample well as a component, wherein the microplate moves at a substantially constant speed in a field of view of the detector. It is characterized by. That is, the present inventor has found that in a measurement using a microplate having a sample well, the sample surface in the well maintains an extremely stable state when the microplate is moved at a constant speed. Thus, a plurality of samples can be measured at high speed and with high accuracy.
[0017]
FIG. 1 is an analysis apparatus illustrating an embodiment of the present invention, and an apparatus having a structure in which a microplate 2 provided with a plurality of wells 1 moves at a constant speed will be described as an example. A method of calculating a measurement value from an output when the microplate 2 passes through the detector 3 is adopted. Hereinafter, an operation method of the present apparatus will be exemplified.
(1) Insert a sample into well 1.
(2) A plurality of wells 1 are arranged in a microplate 2 (only when the wells and the plate are separate, and when the wells and the plate are integrated, it is unnecessary). In FIG. 1, eight wells 1 are arranged at equal intervals in the direction a.
(3) The microplate 2 is set on the tray 4 at the position of the front face A of the apparatus.
(4) When the measurement is started, the microplate 2 moves along with the tray 4 along the guide 5 into the apparatus at a constant speed.
(5) The microplate 2 moves from the initial setting position to the position of the detector 3 provided inside the apparatus by substantially the same distance, and then reverses and moves to the initial setting position.
(6) Remove the microplate 2 and set another microplate 2 ', and repeat (4) and (5).
By the above operation, a state where measurement can be performed at a constant speed with respect to the detector 3 can be created. At this time, by arranging the same number of the detectors 3 at the same interval as the number of the wells 1 in the a direction, the wells 1 in the a direction can be measured simultaneously.
[0018]
At this time, the sample surface inside the well 1 has a substantially constant shape in the moving direction (⇒) as shown in FIG. 2A (in the figure, the n _1st well and the n _2nd well in the a direction). Indicates the state of the well). Therefore, as shown in FIG. 2B, an output having substantially the same waveform can be obtained from the detector 3 at substantially constant speed movement.
[0019]
The position of each well 1 at the time of movement is determined by a slit 6 provided at a position adjacent to the microplate 2 by a position sensor 7 disposed at a position corresponding to a sensor mounting portion 8 on which the detectors 3 are arranged. By detecting, detection is possible. The position sensor 7 detects the interruption of the reflected light at the slit 6 using a reflection sensor such as a proximity sensor including a light source and a sensor, or transmits the light at the slit 6 using a transmission sensor such as a photo interrupter. There is a method of detecting the shredding of the image.
[0020]
Further, the output of the detector 3 is subjected to signal processing in synchronization with the output of the position sensor 7, and the output from the start to the end of the sample measurement for each well is averaged, as shown in FIG. 2 (C). It is possible to obtain a pulse signal for each well and to perform highly accurate measurement. Specifically, after the amplification / synchronous rectification processing, the data is AD-converted, and a measurement value is calculated by an arithmetic processing unit (not shown) and displayed on a display unit (not shown).
[0021]
As described above, the analyzer refers to an absorbance measurement method of irradiating a sample with light from a light source unit and detecting a difference from light transmitted through the sample, and a light in a specific wavelength range (for example, light in an ultraviolet region). Either a photoluminescence method that detects light of a specific wavelength that is different when the sample is irradiated, or a chemiluminescence method that detects light generated by the reaction of a sample with a specific reagent, such as a luminol reaction It is possible.
[0022]
Any material and shape of the well can be selected in relation to the reagent to be used and the specific wavelength used in each measurement method. For example, in the absorbance measurement using light in the visible region, a flat bottom plate or the like is used, in the photoluminescence method, a flat quartz plate or the like is used, and in the chemiluminescence method, the bottom is used. Often, a flat or round shape is used.
[0023]
The detector can be arbitrarily selected in relation to the specific wavelength used. That is, it is determined by the relationship with the luminescent reagent, the absorption wavelength of the reaction product, or the like, and the detector itself has selectivity, or the detector has no selectivity but the optical filter ensures the selectivity. In general, solid-state detectors are often used. For example, in the case of absorbance measurement using light in the visible region, a photodiode or a CCD (electronic coupling device) is used, and in the photoluminescence method, a photodiode or a photocell is used. In a chemiluminescence method, a photodiode or a photocell is often used. Further, the size of the apparatus can be reduced by using small elements, and this is particularly effective in the present invention in which a plurality of elements are arranged.
[0024]
Various mechanisms can be applied to the movement of the microplate. As an example, a movement mechanism using an actuator is shown in FIG. With the microplate 2 having 96 wells arranged on the tray 4, the microplate 2 is moved along the guide 5 by an actuator 9 provided on the lower surface of the tray 4. The moving speed is controlled by the actuator 9 and passes through the sensor mounting portion 8 at a constant speed (for example, 10 to 50 m / sec).
[0025]
The position of each well can be detected by the slit as described above, but can also be detected from the detector output. That is, normally, the detector output can detect the difference between the light from the sample in the well and the light from other parts, as shown in FIG. Utilization makes it possible to detect the position of a well, that is, the position of a sample. Therefore, it is not necessary to provide a slit in a tray like the apparatus illustrated in FIG. 1 and provide a position sensor.
[0026]
Here, it is preferable that the microplate can reciprocate in the field of view of the detector. The present inventor has found that in the above measurement, when the microplate is reciprocated at a constant speed and the output when detected by the fixed detector is averaged, there is no difference from the measured value on the stationary surface. Thus, a plurality of samples can be measured at high speed and with high accuracy. When high-speed measurement is performed, it is possible to make one round trip in 1 second or less, and it is possible to process a large number of samples quickly. In addition, by repeating the reciprocating movement, continuous measurement with a fixed time interval can be performed.
[0027]
That is, in the analyzer of FIG. 1, the measured value is obtained by using both outputs when the microplate 2 passes through the detector 3 in the direction of the arrow (⇒) and when the microplate 2 is inverted and passes through the detector 3 again. Is calculated.
First, signal processing is performed by synchronizing the output of the detector 3 with the output of the position sensor 7, and the signal of the forward path and the signal of the return path are added, thereby averaging as shown in FIG. The obtained signal can be obtained, and the distribution state of the sample in each well can be measured. It is suitable for the measurement of a sample without physical or chemical change in a short time, and is particularly effective when the thickness (depth) of the sample is related to the output of the detector as in the measurement of absorbance.
[0028]
Further, by comparing the averaged signal of only the forward path and the averaged signal of only the return path, it is possible to track a change in the sample after a predetermined time. Further, by repeating the reciprocating movement at a constant time interval and tracking the average value, it is possible to continuously measure the change of the sample in a predetermined time. It is suitable for tracking the physical and chemical changes of a sample in a short time, and is particularly effective when measuring a biological reaction using a chemiluminescence method.
[0029]
Further, it is preferable that the microplate is configured to be separable into a plurality of sample well groups (hereinafter referred to as “well units”) arranged on a line. As a general microplate, a type in which many wells are inserted one by one into one microplate and a type in which many wells are integrally formed in one plate are known. In the type in which the wells are inserted one by one into the microplate, operation errors of the wells, such as oblique insertion, are likely to occur, and the operation tends to be complicated. Further, the automation leads to an increase in the size of the apparatus. On the other hand, in the type in which 96 wells are formed in one microplate, which is widely used at present, there is a high possibility that reagents or samples are mixed into adjacent wells when the reagents or samples are inserted into the wells. It will be. By using a well unit in which a predetermined number of wells are arranged on a line, the speed and versatility of the present invention can be more effectively utilized.
[0030]
FIG. 4A shows an example of the well unit 10 as a minimum unit. Eight wells are arranged in a row to form a well unit 10. As shown in FIG. 4 (B), if 12 well units 10 are arranged, it is possible to form the above-mentioned 96 wells in one microplate 2, and the specifications of the analyzer or the unit can be freely determined by the analyst. By making the number selectable, an operation error can be reduced and a highly versatile device can be provided. Of course, when measuring with one or a small number of well units, it is needless to say that the moving distance of the microplate 2 can be shortened, and more rapid measurement is possible.
[0031]
Further, a device for condensing and irradiating light from a light source unit to the sample well and detecting absorbance by the detector, wherein the light source unit is a light source for generating monochromatic light, and emits and quenches at a constant period Is repeated. In the measurement of absorbance in a biological reaction, since a reagent to be used is different depending on the type of a sample, a highly versatile apparatus that can be used in various bands is required. The device having the configuration as in the present invention is a device which can be very well applied to such requirements in that it can be applied in a wide range only by replacing the light source unit, the detection unit or the optical filter.
[0032]
In particular, by using a monochromatic light source unit, measurement with extremely high sensitivity can be performed on a trace amount of a specific substance present in a sample, and the influence of a disturbing component can be significantly reduced. In addition, by performing a modulation process at a constant period based on the extinction state of the light source unit, so-called drift factors such as an offset in an optical system including a detector or an amplified signal processing unit can be eliminated, and a stable operation can be performed. Measurement becomes possible. In the present apparatus, these elements work in combination, and it is possible to obtain a highly accurate measurement value.
[0033]
Further, by condensing the light from the light source and narrowing the optical path width, more detailed sample information can be obtained at each part in the well. And high-precision measurement is possible. Particularly in the case of biological reactions, the reactivity often differs depending on the measurement site, and more accurate information can be extracted by such measurement using a beam.
[0034]
Furthermore, the modulation frequency can be arbitrarily selected according to the response measure of the element used for the light source unit and the detector, but when a light emitting diode is used, a frequency of several tens of kHz can be selected. The number of measurement signals during the passage through the well can be increased. Therefore, an advantageous effect that information of a large number of measurement sites in a well can be obtained in combination with the measurement effect by the beam can be obtained, which is very suitable for measurement of a biological reaction. In addition, information on the influence of stray light due to the background and minute changes on the liquid surface can be obtained accurately, which is highly effective in eliminating such influence.
[0035]
FIG. 5 shows a configuration example of a detection system in the absorbance analyzer. In a device in which a plurality of light source units 11 and a plurality of detectors 3 arranged in a row corresponding to the number of wells are arranged coaxially, a state at the moment when the wells 1 move and are arranged on the same optical axis. The light from each light source unit 11 modulated at a constant period is condensed by the same number of equally spaced lenses 12, irradiates each well 1, and the transmitted light is received by each detector 3. Thus, a change in each well 1 can be detected. Here, the position of the well 1, that is, the position of the tray 4 can be detected by receiving the light from the position detecting light source 7 ′ via the slit 6 by the position detecting sensor 7 ″. It is possible to select an arbitrary element in relation to a specific wavelength used.In general, a solid-state light-emitting element is often used, for example, in a visible region, a light-emitting diode or a semiconductor laser is used, In the near infrared region, a glow lamp or a halogen lamp is used, and in the near ultraviolet region, a light emitting diode, a mercury lamp, or the like is often used.
[0036]
FIG. 6 shows an example of the overall configuration of an absorbance analyzer using the detection system. That is, the eight light source units 11 arranged at the same pitch as each well 1 emit light of a specific wavelength (for example, 450 nm blue light) required for measurement while controlling the modulation timing by the oscillator 14 and the light source driving unit 15. Etc.) to emit light. The light from the light source is modulated at a constant frequency. When a blue diode is used, it is preferable to select a frequency of 1 to 10 kHz. The weak signal from each detector 3 is amplified by the amplifier 16 and rectified by the electronic switch 20 in the signal processing unit 17 in synchronization with the modulation timing to eliminate the influence of the stray light due to the background. , Are processed by the data processing unit 17 via the AD converter 16, and each measured value is displayed and output.
[0037]
FIG. 7 shows the main points of the electronic circuit. The pulsating output from the amplifier 16 is averaged by the low-pass filter 22 via the electronic switch 20 and the differential amplifier 21, so that the influence of stray light due to the background can be eliminated. The cut-off frequency of the low-pass filter 22 is preferably practically about 1/10 or less of the modulation frequency. Reference numeral 23 denotes a position sensor signal processing unit. The position sensor output is guided to a comparator 24 via a low-pass filter 22 after differential amplification in the same manner as described above, and is compared with a comparison voltage Ref to be converted into a digital signal. And is used as a signal indicating the timing of AD conversion of a detector signal obtained by measuring a sample in the well 1. By applying such an electronic circuit to the present invention, particularly high-frequency signals can be quickly processed, and a large number of samples can be measured at high speed.
[0038]
At this time, it is preferable that the light source unit is made of a direct transition solid state light emitting device. By using such an element, it is possible to realize a light source that can easily perform modulation at a constant cycle and that is clearly switched between ON and OFF, and can obtain a signal with a large output from the detector and a small noise component. it can. Further, the size of the apparatus can be reduced by using small elements, and this is particularly effective in the present invention in which a plurality of elements are arranged. Specifically, for the wavelength range of 450 to 470 nm, which is often used in biological reactions, examples of usable elements include LEDs made of gallium nitride or indium gallium nitride.
[0039]
Further, it is preferable that the wavelength range of the light from the light source section is approximately 420 to 670 nm. For example, among biological reactions, a method using tetramethylbenzidine as a reagent is excellent for an antigen-antibody reaction, and a wavelength range of about 450 nm is most preferable for detection of a color reaction in terms of detection sensitivity or selectivity. . At this time, a light source using a gallium nitride-based light source is particularly useful among the direct transition solid state light emitting devices. The amount of light emission is large and the modulation frequency can be set high, which is suitable for the configuration of the present invention.
[0040]
As described above, the technique of the present invention can be applied to such a plurality of samples in a wide range, and can provide an analyzer having high versatility and high measurement accuracy.
[0041]
【The invention's effect】
As described above, in the analyzer to which the present invention is applied, a plurality of samples can be measured at high speed and with high accuracy.
[0042]
In particular, by allowing the microplate to reciprocate in the field of view of the detector, it is possible to measure a plurality of samples at high speed and with high accuracy by averaging the output of the detector. In addition, by repeating the reciprocating movement, continuous measurement with a fixed time interval can be performed.
[0043]
Furthermore, the microplate is configured to be separable for each of a plurality of sample well groups arranged on a line, thereby preventing operation errors, complicated operations, and an increase in size of the apparatus due to automation. Versatility and versatility can be more effectively utilized.
[0044]
In addition, in an absorbance measuring device that repeatedly emits and quenches monochromatic light to be condensed and irradiated on a sample well at a fixed period, it can be applied to a wide range.
[0045]
At this time, by using a direct transition solid state light emitting element for the light source section, a signal having a large output from the detector and a small noise component can be obtained.
[0046]
For detection of a color reaction in a biological reaction, it is preferable that the wavelength range is approximately 420 to 670 nm from the viewpoint of detection sensitivity or selectivity, and an element having a large light emission amount and a high modulation frequency can be used.
[Brief description of the drawings]
FIG. 1 is an explanatory diagram showing a first configuration example of an analyzer according to the present invention.
FIG. 2 is an explanatory diagram illustrating a sample state and a detector output in the first configuration example.
FIG. 3 is an explanatory diagram illustrating details of a measurement unit in the first configuration example.
FIG. 4 is an explanatory view illustrating a well unit according to the present invention.
FIG. 5 is an explanatory diagram illustrating a measuring section of a second configuration example of the analyzer according to the present invention.
FIG. 6 is an explanatory diagram illustrating the entire device in a second configuration example.
FIG. 7 is an explanatory diagram illustrating a main part of an electronic circuit in a second configuration example.
FIG. 8 is an explanatory diagram showing a configuration example of one of the analyzers according to the related art.
FIG. 9 is an explanatory diagram showing another configuration example of the analyzer according to the conventional technique.
[Explanation of symbols]
Reference Signs List 1 well 2 microplate 3 detector 7 position sensor 10 well unit 11 light source unit 12 lens 14 oscillator

Claims (6)

A microplate having a plurality of sample wells, an analyzer including, as a component, a detector receiving light from the sample well, wherein the microplate moves at a substantially constant speed in a field of view of the detector. Analysis equipment. The analyzer according to claim 1, wherein the microplate is reciprocally movable in a field of view of the detector. 3. The analyzer according to claim 1, wherein the microplate is configured to be separable into a plurality of sample well groups arranged on a line. A device for collecting and irradiating light from a light source unit to the sample well and detecting absorbance by the detector, wherein the light source unit is a light source for generating monochromatic light, and repeats light emission and quenching at a constant cycle. The analyzer according to claim 1, wherein: The analyzer according to claim 4, wherein the light source unit includes a direct transition solid state light emitting device. The analyzer according to claim 4, wherein a wavelength range of light from the light source unit is approximately 420 to 670 nm.

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