JP2002116148A - Fluorescent plate analyzer - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a fluorescent plate analyzer being inexpensive and compact and facilitating the maintenance of a source of excitation light. SOLUTION: A blue LED 8 is provided in an irradiation and detection optical system 6 as the source of excitation light. A well 4 located in an irradiation position A is irradiated with light from the LED 8, as the excitation light, via a collimator lens 10, a band-pass filter 12, a dichroic mirror 14 and an objective lens 16. With fluorescence emitted from this sample, a photomultiplier tube 24 is irradiated via the objective lens 16, the dichromic mirror 14, a spectroscopic filter 18, a condensing lens 20 and a pinhole slit 22, and the existence or absence of fluorescent materials in the well 4 located in the irradiation position A and the concentrations are measured.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a reaction plate holding table for holding a reaction plate having a plurality of sample holding positions, and an irradiation optical system for irradiating the sample holding positions of the reaction plate with excitation light. And a detection plate for detecting fluorescence from a sample holding position. The fluorescence plate analyzer is
It is used in the fields of biochemistry, biology, molecular biology, clinical field, etc., and is also applied to DNA analyzers.

[0002]

2. Description of the Related Art Conventionally, in a fluorescence plate analyzer, a sample labeled with a fluorescent substance is accommodated in a plurality of wells of a reaction plate such as a microtiter plate, and the wells are irradiated with excitation light to emit fluorescence from the wells. Measure. As an excitation light source for the irradiation optical system for irradiating the well with excitation light,
Gas lasers and semiconductor lasers are used.

[0003]

However, there is a problem that the gas laser has a short life and is expensive. further,
When a gas laser is used as the excitation light source, the size of the light source and the necessity of waste heat treatment increase the size of the apparatus and increase the installation space. In addition, since the semiconductor laser has a large temperature dependence of the oscillation frequency and the output, a temperature control mechanism is required, and there is a problem that the temperature control mechanism increases the size and complexity of the device.

[0004] As described above, the conventional fluorescent plate analysis apparatus has a problem that the excitation light source makes the apparatus expensive, large-sized, and complicated in maintenance. Accordingly, an object of the present invention is to provide a fluorescent plate analyzer that is inexpensive and compact, and that allows easy maintenance of the excitation light source.

[0005]

According to the present invention, there is provided a reaction plate holding table for holding a reaction plate having one or more sample holding positions, and a reaction plate for irradiating the sample holding position of the reaction plate with excitation light. A fluorescent plate analysis apparatus including an irradiation optical system and a detection optical system for detecting fluorescence from a sample holding position, wherein the irradiation optical system includes an LED as an excitation light source.

In the fluorescence plate analyzer according to the present invention, an LED (Light Emitting Diode, LED) is used as an excitation light source for irradiating the sample holding position of the reaction plate with excitation light.
Light emitting diode). LEDs are smaller, cheaper, and easier to maintain than gas lasers. Further, light having a stable oscillation frequency and output can be obtained without temperature control unlike a semiconductor laser.

[0007]

BEST MODE FOR CARRYING OUT THE INVENTION The irradiation optical system preferably includes a plurality of LEDs as excitation light sources arranged one-dimensionally or two-dimensionally corresponding to the sample holding position of the reaction plate. As a result, it becomes possible to simultaneously irradiate a plurality of sample holding positions with the excitation light, and to shorten the measurement time.

[0008]

FIG. 1 is a schematic perspective view showing one embodiment of the present invention. For example, a reaction plate 2 such as a microtiter plate is provided. A plurality of reaction wells (sample holding positions) 4 for accommodating a sample are formed in the reaction plate 2. The reaction plate 2 is held on a reaction plate holder (not shown), and is moved in a two-dimensional direction (see an arrow in the figure) by an actuator provided on the reaction plate holder.

An irradiation and detection optical system 6 is provided on the bottom side of the reaction plate 2 (the side opposite to the surface on which the wells 4 are formed). The irradiation and detection optical system 6 is provided with an excitation LED 8 as an excitation light source. As the LED 8, for example, a blue LED having an oscillation frequency of 480 nm can be used. A collimator lens 10 for converting light from the LED 8 into parallel light is provided. In the optical path of the light that has passed through the collimator lens 10 from the LED 8 side, a band-pass filter 12 having a transmission wavelength range of about 480 nm and a half-value width of 10 nm is provided. A dichroic mirror 14 that transmits light having a wavelength of 505 nm or less and reflects light having a longer wavelength is provided in the optical path of the light transmitted through the bandpass filter 12.
Is transmitted through the dichroic mirror 14. On the optical path of the transmitted light of the dichroic mirror 14, an objective lens 16 formed of a condenser lens is provided. Objective lens 1
The light from 6 is condensed at the irradiation position A. The irradiation optical system constituting the present invention includes an LED 8, a collimator lens 10, a band pass filter 12, a dichroic mirror 14, and an objective lens 16.

The irradiation and detection optical system 6 is further provided with a spectral filter 18 for transmitting light of a predetermined fluorescence wavelength on the optical path of the fluorescence condensed by the objective lens 16 from the well 4 side and reflected by the dichroic mirror 14. Have been. A condensing lens 20 is provided on the optical path of the fluorescence transmitted through the spectral filter 18. A pinhole slit 22 and a photomultiplier tube 2 are provided in the optical path of the light from the condenser lens 20.
4 are provided. The light transmitted through the spectral filter 18 is condensed by a condenser lens 20 into a hole of a pinhole slit 22 and passes through the hole to a photomultiplier tube 24.
Is irradiated. The detection optical system of the present invention includes an objective lens 1
6, dichroic mirror 14, spectral filter 18,
It comprises a condenser lens 20, a pinhole slit 22, and a photomultiplier tube 24.

Next, the operation of this embodiment will be described. PCR (Polymerase Chain Reactio) labeled with a fluorescent substance
n) a product, an antigen or antibody labeled with a fluorescent substance used in a fluorescent immunoassay (fluorescent immunoassay), or a double-stranded DNA in which an oligonucleotide labeled with a fluorescent substance and a deoxyribonucleic acid (DNA) fragment are hybridized. For example, a sample subjected to a reaction treatment in advance is accommodated in each well 4 of the reaction plate 2.

The reaction plate 2 is moved by an actuator provided on the reaction plate holding table so that the well 4 to be measured matches the detection position A. The light from the LED 8 emitted at a wavelength of 480 nm is
To make parallel light. The parallel light is split by the band-pass filter 12 and transmitted to the dichroic mirror 14 while transmitting only light in the wavelength range around 480 nm. The light from the bandpass filter 12 is applied to the dichroic mirror 1
4 is transmitted. The light transmitted through the dichroic mirror 14 is condensed by the objective lens 16 on the well 4 located at the irradiation position A, and irradiated as excitation light. The fluorescent substance in the sample in the well 4 located at the irradiation position A is excited by the irradiation of the excitation light to emit fluorescence.

The fluorescent light emitted from the sample is transmitted to the objective lens 16.
And scattered light from the well 4 is reflected toward the spectral filter 18 while being removed by the dichroic mirror 14. The light from the dichroic mirror 14 is separated by the spectral filter 18 and a predetermined fluorescence wavelength is transmitted to the condenser lens 20 side. The light transmitted through the spectral filter 18 is condensed by the condenser lens 20 into the hole of the pinhole slit 22, and passes through the hole of the pinhole slit 22 to irradiate the photomultiplier tube 24.

By detecting the electric signal from the photomultiplier tube 24, the presence or absence and the concentration of the fluorescent substance in the well 4 located at the irradiation position A are measured. Well 4 to be measured
Is measured, the next well 4 to be measured is moved to the detection position A by the actuator provided on the reaction plate holding table.
The reaction plate 2 is moved so that In this embodiment, since the LED 8 is used as the excitation light source, it is cheaper and more compact than a conventional fluorescent plate analyzer using a gas laser or a semiconductor laser, and the maintenance of the excitation light source can be facilitated. .

FIG. 2 is an exploded perspective view showing an irradiation optical system of another embodiment. The blue LEDs 8 are arranged to correspond to the arrangement of the wells of the reaction plate to form an LED array 8a. Opposite to the LED array 8a, there is provided a lens array 26 made of a transparent substrate on which a plurality of condenser lenses are formed corresponding to the arrangement of the LEDs 8. On the opposite side of the lens array 26 from the LED array 8a, a planar spectral filter 28 for dispersing light emitted from the LEDs 8 and transmitted through the lens array 26 is provided.
The spectral filter 28 has light of a wavelength that excites the fluorescent substance,
For example, when fluorescein or rhodamine 110 is used as a fluorescent substance for labeling, it transmits 480 nm light. In this embodiment, the reaction plate 2 is arranged on the side of the spectral filter 28 opposite to the LED array 8a and the lens array 26 during measurement.

FIG. 3 shows a schematic configuration diagram of the irradiation optical system and the detection optical system of this embodiment. An irradiation optical system 30 including an LED array 8a, a lens array 26, and a spectral filter 28 shown in FIG. 2 is provided on a bottom surface side of the reaction plate 2 held on a reaction plate holding table (not shown). A condensing lens 32 for condensing light from the reaction plate 2 is provided on the side of the reaction plate 2 opposite to the irradiation optical system 30.
Is provided. Condenser lens 3 from reaction plate 2 side
A spectral filter 34 is provided in the optical path of the light that has passed through 2. The spectral filter 34 includes two types of spectral filters that transmit light of different wavelengths.
One of the spectral filters transmits light of a certain fluorescent wavelength, and the other spectral filter transmits light of a fluorescent wavelength different from the fluorescent wavelength. The spectral filter 34 is a spectral filter switching mechanism (not shown).
Is moved in the direction of the arrow in FIG.

On the opposite side of the spectral filter 34 from the condenser lens 32, a CCD (Charge Cou
pled Device) 36 is disposed. Reaction plate 2
From the condenser lens 32 to the spectral filter 34.
Is imaged on the CCD 36 via the. In this embodiment, the condenser lens 32, the spectral filter 34 and the CCD 3
8 constitutes a detection optical system 38.

In this embodiment, a reaction plate 2 containing a sample in a well 4 is placed on a reaction plate holding table, and LE
By simultaneously turning on all the LEDs 8 of the D array 8a, all the wells 4 of the reaction plate 2 can be measured at the same time, so that the measurement time can be reduced. Further, since there is no need to provide a drive mechanism such as an actuator for moving the reaction plate 2, the configuration of the reaction plate holding base is simplified, and the cost of the apparatus can be reduced. By switching the spectral filter 34, two types of fluorescent wavelengths can be measured.

In the above embodiment, the blue light source is used as an excitation light source for irradiating the sample holding position of the reaction plate with excitation light.
Although the ED is used, the present invention is not limited to this, and an LED that emits light of another color, that is, light of another wavelength, depending on the fluorescent substance used can be used. Further, the irradiation optical system is not limited to the above-described embodiment, and LEDs may be arranged in a line to form a one-dimensional array, and irradiation may be performed by irradiating excitation light for each column or row. An LED may be provided corresponding to the arrangement of the wells in the region including the well, and the region may be irradiated with excitation light for measurement. Further, the detection optical system is not limited to the above-described embodiment, and may detect the fluorescence for each column or row, or may detect the fluorescence for each region including a plurality of wells. Good. Further, the configurations of the irradiation optical system and the detection optical system may be combined. Needless to say, when the configuration of the irradiation optical system or the detection optical system is changed, the configuration of the reaction plate moving mechanism of the reaction plate holding table is also changed.

The reaction plate 2 used in the above embodiment is not limited to a microtiter plate. For example, the reaction plate 2 may have one or more convex sample dots formed on the surface of a flat substrate, The fluorescent plate analyzer of the present invention can be applied to any plate capable of holding a sample, such as a plate having one or more concave pits for accommodating the sample on its surface. Although the irradiation and detection optical system 6 is provided on the bottom surface of the reaction plate 2 in the above embodiment, the present invention is not limited to this, and the surface of the reaction plate 2 where the wells 4 are formed is provided. An irradiation and detection optical system 6 may be provided.

[0021]

According to the fluorescent plate analyzer of the present invention,
Since an LED is used as an excitation light source for irradiating the sample holding position of the reaction plate with excitation light, it is cheaper and more compact than conventional fluorescent plate analyzers using gas lasers or semiconductor lasers. Maintenance can be facilitated.

[Brief description of the drawings]

FIG. 1 is a schematic configuration perspective view showing one embodiment.

FIG. 2 is an exploded perspective view showing an irradiation optical system according to another embodiment.

FIG. 3 is a schematic configuration diagram showing an irradiation optical system and a detection optical system of the embodiment.

[Explanation of symbols]

 2 Reaction plate 4 Well 6 Irradiation and detection optical system 8 Blue LED 8a LED array 10 Collimator lens 12 Bandpass filter 14 Dichroic mirror 16 Objective lens 18, 28, 34 Spectral filter 20, 32 Condenser lens 22 Pinhole slit 24 Photoelectron increase Doubler 26 Lens array 30 Irradiation optical system 36 CCD 38 Detection optical system

Claims (2)

[Claims] 1. A reaction plate holding table for holding a reaction plate having one or more sample holding positions, an irradiation optical system for irradiating a sample holding position of the reaction plate with excitation light, and a sample holding position. A fluorescence plate analysis device comprising: a detection optical system for detecting fluorescence from a fluorescent plate; and the irradiation optical system includes an LED as an excitation light source. 2. The fluorescent plate according to claim 1, wherein the irradiation optical system includes a plurality of LEDs as excitation light sources arranged one-dimensionally or two-dimensionally corresponding to a sample holding position of the reaction plate. Analysis device.