JP2002286643A - Reader for light emitting image from inspection plate and optical system used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an apparatus system in which it is not required to scan the readout part of a reader on an inspection plate manufactured by using a DNA microarray or the like and by which a reaction result on the face, to be measured, of the inspection plate can be detected at a time as an image and to provide an optical system used for the apparatus system. SOLUTION: The optical system wherein a CCD image sensor and a fiber face plate are combined with an optical system constituting the readout part in the reader of a light emitting image which is used to detect the existence of the reaction of a probe array with a sample on a transparent substrate as the existence of a light emitting operation is used.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an apparatus for reading a luminescent image from a label obtained on a test plate obtained by a reaction or interaction between a probe for detecting a target substance in a sample and the target substance, and used therefor. Optical system.

[0002]

2. Description of the Related Art For analysis of a sample containing a target substance using a probe carrier such as a DNA microarray, a test plate is prepared by reacting a probe carrier with a sample, and a probe and a target in the test plate are prepared. In many cases, a method of detecting the presence or absence of a reaction with a substance as light emission from a labeling substance is employed. For such a reaction, a method of immobilizing a sample to be analyzed on a carrier and reacting a probe with the sample,
Conversely, there is a method in which a probe is immobilized on a carrier and a sample is reacted with the probe. The presence or absence of the reaction was determined by setting the reaction system so that only the conjugate (complex) between the probe and the target substance had the labeling substance, and detecting the luminescence from the labeling substance at the reaction position. Is generally detected.

[0003] Fluorescent dyes are widely used as such labeling substances, and an apparatus for detecting light emission from the fluorescent dye labeling substance is provided in an optical system of a confocal optical microscope. Using a combination of an optical filter and a dichroic mirror corresponding to the fluorescence wavelength,
A mechanism that guides fluorescence to be detected to MT (photomultiplier) is used. In such an apparatus, a spot size for irradiating light to induce fluorescence is also small, and thus an apparatus that scans each point on a DNA microarray finely is currently the mainstream (see FIG. 1).

[0004] As such an apparatus, a GenePix 4000 manufactured by Axon Instruments Inc. of Agilent Technologies, Inc. is used.
B and so on.

[0005]

In the case of an apparatus that scans the reading section of a reading apparatus on an inspection plate manufactured using a DNA microarray formed as a flat chip, the scanning is performed point by point. It takes several minutes to finish scanning the entire DNA microarray, and the XY stage and its driving system are indispensable.
It tends to be an expensive device with many parts.

An object of the present invention is to eliminate the need for scanning the reading section of a reading device on a test plate manufactured using a DNA microarray or the like, and to detect the reaction result on the surface to be measured of the test plate as an image at a time. An object of the present invention is to provide an apparatus system and an optical system used for the apparatus system.

[0007]

According to the present invention, there is provided an apparatus for detecting light emission from a plurality of measured portions disposed on a surface of a substrate according to the present invention, the device being in close contact with the measured portion and receiving light from the measured portion. A fiber face plate that has a light receiving surface for receiving light emission and transmits the received light to the other surface of the light receiving surface; and a fiber face plate that is transmitted from the measured section on the other surface of the fiber face plate. And a detecting means for receiving and detecting the reflected light.

In the case where the luminescence obtained from the measured portion is obtained by irradiating the fluorescent label with excitation light, the light enters the measured surface from the back side or the side of the front surface of the transparent substrate. An optical system for irradiating the surface to be measured with excitation light at an angle at which the light is reflected on the back surface or transmitted through the side surface may be further provided.

[0009] When a CCD image sensor is used as the light receiving means, it is preferable that the pixel pitch on the reading surface corresponds to the arrangement pitch of a plurality of measured parts arranged in the measured surface.

[0010] A light emission reading device according to the present invention is a device for reading light emission from a plurality of measured portions disposed on the surface of a substrate, the device being in close contact with the measured portion and receiving light from the measured portion. A fiber face plate that has a light receiving surface for receiving light emission and transmits the received light to the other surface of the light receiving surface; and a fiber face plate that is transmitted from the measured section on the other surface of the fiber face plate. A light emitting reading device comprising: a CCD image sensor for receiving received light and outputting information of the received light as electronic data; and a memory for storing electronic data output from the CCD image sensor. It is.

In the case where the light emission obtained from the measured portion is obtained by irradiating the fluorescent label with excitation light, this reading device is used for the surface of the transparent substrate from the back side or the side. An optical system for irradiating the measurement surface with excitation light at an angle of incidence on the measurement surface and reflection on the rear surface side or transmission on the side surface side, and excitation light generation means for supplying excitation light to the optical system are further provided. Things.

Further, the light-emitting reading device further includes a calling unit for electronic data relating to the amount of light stored in the memory, and a display unit for displaying a light-emitting image based on the electronic data called by the calling unit. can do.

The reading section of the light-emitting reading apparatus according to the present invention has an optical system having a configuration in which a CCD sensor is combined with a fiber face plate, and has a simple configuration and converts an optical image obtained from an inspection plate into a two-dimensional image. The light emission from the test plate can be measured in a shorter time by a simple operation excluding the scanning system. Further, since the optical system is simplified, the cost of the reading device itself can be reduced.

[0014] Even if the scanning system is simply eliminated by using a CCD sensor which is an array sensor, for example, if an imaging optical system is configured by an ordinary method, it is necessary to form an image on a reading surface of the CCD sensor. An optical lens and a holder that is a mechanical part for fixing the optical lens are required at a minimum. In addition, software for recognizing aberration, which is an essential problem due to the use of the optical lens, particularly distortion in the peripheral portion and the central portion of the image, and correcting the distortion is required. Furthermore, in order to match the focus, alignment in the Z direction is required at a minimum, and the number of components is large. In addition, when a fluorescent dye label is used, the amount of light is smaller than in the case of normal imaging, and the influence of aberration on measurement sensitivity may be large.

As described above, the conventional CCD camera system combining the optical system and the CCD image sensor has a problem that the measurement sensitivity and cost reduction cannot be sufficiently satisfied.

On the other hand, the optical system used in the present invention has a simple structure with a small number of components, such as a combination of a CCD image sensor and a fiber face plate, and has an aberration as in the case of using an optical lens system. In addition, since the reading operation is performed while the image contact surface of the fiber face plate is in close contact with the surface to be measured of the inspection plate, alignment in the Z direction for focus adjustment as in the case of using an optical lens system can be omitted.

[0017]

2 to 4 show embodiments of the present invention.

FIG. 2A shows a pixel 12 of the CCD sensor.
Above, a structure of a general CCD sensor provided with complementary color filters of cyan, yellow, green, and magenta is shown. The CCD sensor having such a configuration can be used as a component of the reading optical system. Further, a filter in which a microlens array is further provided above the complementary color filter can be used. Although this filter may be a three-primary-color filter, a configuration using a complementary color filter is preferable from the viewpoint of sensitivity. The filter can be appropriately selected depending on the type of light emission from the spot. In the example shown in FIG. 2, the arrangement pitch of the dot spots 13 of the probe DNA of the DNA microarray 14 corresponds to the arrangement pitch of the pixels of the CCD sensor. FIG. 2B shows a simpler monochrome CCD sensor, in which the arrangement pitch of the DNA spots 13 corresponds to the pixels of the CCD sensor at a ratio of 1: 1 and is arranged at a higher density. It can correspond to the spot of the probe DNA. More generally, m is large enough to surround a dot of DNA.
It is only necessary that × n pixels correspond to the arrangement pitch of the spots 13 of DNA. For a case where a plurality of fluorescent lights are detected from a spot, a thin cover-slip type filter 1 shown in FIG.
6 can be dealt with sequentially.

An image-forming surface of a fiber face plate is brought into close contact with a surface of a test plate having an array of spots obtained by performing a reaction between a probe and a sample to be tested for the presence or absence of a target substance. Cover and read the optical image. In the present specification, a fiber face plate is also called fiber optics, and is a plate-shaped optical element made of a number of optical fibers such as glass arranged in parallel with each other, and an image received on one surface is imaged on the other surface. A lens that has a function of transferring light to a lens and does not cause aberration such as a lens. The fiber face plate may be any one capable of transferring an image at the same magnification or one capable of changing the image magnification, and preferably has a high resolution.

In order to improve the reading efficiency of an optical image,
An encapsulant 15 having substantially no optical influence is dropped and filled between the surface to be measured and the image-contacting surface of the fiber face plate, and these are liquid-juncted without any gas phase. When measuring fluorescence, a generally used non-fluorescent glycerol diluent can be used. The liquid junction between the measured surface of the inspection plate and the tangent surface of the fiber face plate in this manner allows the element 2
2, the light quantity from the spot can be guided so as not to be wasted as much as possible. Light input to the fiber face plate is transmitted to the reading surface of the CCD sensor. That is, a light-emitting image composed of light-emitting spots on the surface to be measured is read at once by the CCD sensor as a two-dimensional image. From such a viewpoint, in order for the light transmitted through the fiber face plate 19 to be efficiently transmitted to the reading surface of the CCD sensor, the reading surface of the CCD sensor and the surface of the fiber face plate are filled and bonded with an optical adhesive. It is preferable that the gap be minimized even when a structure is provided or a gap is provided between these surfaces.

FIG. 5 shows the main components of the reader according to the present invention when measuring fluorescence. After the liquid junction is maintained by the mounting medium 15, the inspection plate is at a low angle such that the excitation light does not directly enter the CCD.
The beam 18 is irradiated from the back surface or the side surface under the condition that the beam 18 is reflected on the measured surface to the back or transmitted to the side. The beam 18 includes a wavelength necessary for exciting the fluorescent dye, and is emitted from a known excitation light generating unit via an optical system. As this beam, a laser beam or the like can be used.

Here, the reflection on the surface to be measured is substantially equal to the so-called total reflection condition. At this time, light called evanescent partially enters the interface, and is involved in excitation of fluorescence. I do. It is known that the condition for total reflection is represented by θc = arcsin (N ₂ / N ₁ ), where θc is the critical angle. The penetration depth dp of the evanescent wave at this time is dp = (λ / N ₁ ) / [2π ｛sin ² θi− (N ₂ /
N ₁ ) ₂ ｝ ^1/2 ] (J. Immunol. Me
thod., 74: 253-265 (1984). From this relational expression, the incident angle θi can be determined from the appropriate refractive index of the encapsulant, the refractive index (non-fluorescence) of the transparent substrate used for the inspection plate, and the like, and the spot thickness and the penetration depth dp Can be checked whether they are in a sufficient relationship.

FIG. 6 shows another embodiment of the reading apparatus according to the present invention. In FIG. 6, light emitted from a He-Ne laser line module 30 (which has a built-in unit for shaping the laser into a linear beam) passes through a slit and is bent by mirrors 32 and 33 for inspection. The surface to be measured of the inspection plate stored in the plate holder 34 is irradiated from the back surface side with respect to the surface to be measured. Mirror 3
4 and the inspection plate holder 34 have a fine adjustment mechanism to correspond to each pixel of the CCD sensor. The CCD camera unit 17 having a cooling function, which is a main part, can be superimposed on a position covering the surface to be measured of the inspection plate by tilting the L angle bar. The CCD camera unit sends the light quantity data stored therein to a computer 29 provided as necessary via an interface 35. The interface 35 uses a video terminal such as NTSC when a commercially available video board is inserted into the computer side. However, when a frame memory is provided inside the CCD camera unit 25 like a commercially available digital camera, the interface 35 is used. May be a DIV terminal or a USB terminal as well as a digital camera.

The image data captured by the computer can be displayed on a screen using commercially available image processing software,
For each dot area, the sum of electronic data corresponding to luminance, which is related to the quantitative value of each substance, can be obtained. In the present invention, since the pixel m × n region of the CCD is previously associated with the dot arrangement pitch of the DNA, there is no need to perform complicated processing with specially developed image processing software, and this can be easily performed. As for the laser light source, in order to use it as an excitation light source for multiple fluorescent substances, multiple laser sources such as a harmonic YAG laser are collected by a mirror, shaped into a linear beam, and then placed on the surface to be measured of the inspection plate. Irradiation may be performed.

Further, the present invention does not use the above-mentioned laser excitation light source and its optical system, uses radiation of a radioisotope label as an excitation light source instead of a fluorescent label, and converts it into fluorescence by a liquid scintillator. No problem at all.

The test plate to be inspected by the reader according to the present invention is obtained by reacting a sample to be tested for the presence or absence of a target substance with a probe carrier having probe spots arranged and fixed on a transparent substrate. The spots of the sample to be tested for the presence or absence of the target substance are obtained by reacting the probe with a sample-immobilized carrier in which the spots are arrayed and fixed on a transparent substrate. It can be detected.

A probe can be recognized by a specific target (target), and is often called a ligand. In addition, the probes include oligonucleotides, polynucleotides, peptides, or other polymers that can be recognized by a particular target. The term `` probe '' refers to a probe molecule itself having a probe function, such as an individual polynucleotide molecule, or a group of probe molecules having a probe function, such as a polynucleotide immobilized on a carrier surface in a dispersed state or the like. May mean. Also, a probe may be capable of binding, or become capable of binding, to a target as part of a ligand-antiligand pair. Probes and targets in the present invention can include bases as found in nature, or analogs thereof.

Further, as an example of a probe supplied on a carrier, a part of an oligonucleotide having a base sequence capable of hybridizing with a target nucleic acid may be bonded to a binding portion between a fluorescent dye and a carrier directly or via a linker. And the like. In such a configuration, the position of the binding portion between the fluorescent dye and the carrier in the molecule of the oligonucleotide is not particularly limited as long as the desired hybridization reaction is not impaired.

The probe used in the probe array manufactured by the method of the present invention is appropriately selected depending on the purpose of use. However, in order to suitably carry out the method of the present invention, , The probe is DNA, R
NA, cDNA, PNA, oligonucleotide, polynucleotide, other nucleic acid, oligopeptide, polypeptide, protein, enzyme, substrate for enzyme, antibody, epitope (antigen) for antibody, hormone, hormone receptor, ligand, ligand receptor, oligo sugar,
It is preferably one of polysaccharides, and if necessary, two or more of these can be used in combination.

On the other hand, in the method of the present invention, the probe has a structure capable of binding to the surface of the carrier, and the immobilization of the probe on the carrier is not limited to the most basic method of fixing by physical adsorption. Desirably, it is performed through a connectable structure. At that time, the structure of the probe that can be bonded to the carrier surface includes amino group, sulfhydryl group, carboxyl group, hydroxyl group, acid halide (haloformyl group;-
COX), halide compound (-X), aziridine, maleimide group, succinimide, isothiocyanate, sulfonyl chloride (-SO ₂ Cl), aldehyde (formyl group; -CHO), hydrazine, introducing an organic functional group such as iodoacetamide It is preferably formed by the following process.

In the method of manufacturing a probe array according to the present invention, in manufacturing a probe array, a plurality of types of separately prepared probes are used as a solution and discharged into each well on a carrier using a liquid discharging device. By supplying
This achieves various types of densification in the probe array.

The probe carrier having the probe immobilized on the carrier can be produced, for example, by the method described in JP-A-11-187900.

[0033]

According to the present invention, it is possible to provide an apparatus for reading a luminescent image from an inspection plate with a simple configuration and low manufacturing cost.

[Brief description of the drawings]

FIG. 1 is a diagram showing a configuration of a conventional reading device.

FIGS. 2A and 2B are diagrams showing the relationship between the arrangement of spots on an inspection plate and the arrangement of pixels of a CCD sensor.

FIG. 3 is a diagram illustrating a process of creating a liquid junction in the reading device.

FIG. 4 is a diagram showing an example in which a filter is additionally used outside the reading device.

FIG. 5 is a diagram showing an example of a reading device according to the present invention.

FIG. 6 is a diagram showing another example of the reading device according to the present invention.

[Explanation of symbols]

 Reference Signs List 1 inspection plate 2 objective lens 3 mirror 4 mirror 5 beam splitter 6 fluorescence filter 7 excitation filter 8 detector lens 9 laser source 10 pinhole 11 detector (PMT) 12 CCD pixel 13 inspection plate 14 glass substrate 15 encapsulant 16 filter 17 CCD Camera unit 18 Laser beam 19 Fiber face plate 20 Micro lens array 21 Colorless filter 22 CCD element 23 CCD package 24 Printed circuit board 25 Cooling unit 26 Signal cable

Claims (15)

[Claims] 1. An apparatus for detecting light emission from a plurality of measured parts arranged on a surface of a base, the apparatus being in close contact with the measured part and receiving light emitted from the measured part. A fiber face plate having a light receiving surface and transmitting the received light to the other surface side of the light receiving surface; and receiving and detecting light transmitted from the portion to be measured on the other surface side of the fiber face plate. A light detecting device having a detecting means for performing the detecting operation. 2. The light detecting device according to claim 1, wherein said light receiving means includes an output means for outputting information on the received light. 3. The light detection device according to claim 1, wherein the light receiving unit is a CCD image sensor that outputs information on the received light as electronic data. 4. The light emission from the measured part is obtained by irradiating the fluorescent label with excitation light, and the light enters the measured part from the back side with respect to the front surface of the transparent substrate, and the back side The photodetector according to any one of claims 1 to 3, further comprising an optical system for irradiating the measured portion with excitation light at an angle that reflects the light. 5. The photodetector according to claim 3, wherein a pixel pitch of the reading surface of the CCD image sensor corresponds to an arrangement pitch of the plurality of spots arranged on the measurement target. 6. The light-emitting device according to claim 1, wherein the light emission from the spot disposed on the measured portion is based on the binding between the probe and a target substance specifically recognized by the probe. Photodetector. 7. The photodetector according to claim 5, wherein said probe is a single-stranded nucleic acid probe. 8. The photodetector according to claim 6, wherein said single-stranded nucleic acid probe includes a single-stranded DNA probe. 9. The photodetector according to claim 6, wherein said single-stranded nucleic acid probe comprises an RNA probe. 10. The method according to claim 1, wherein the single-stranded nucleic acid probe is a single-stranded PNA.
7. The light detection device according to claim 6, further comprising a probe. 11. A device for reading light emitted from a plurality of measured parts arranged on a surface of a substrate, the light receiving unit being in close contact with the measured part and receiving light emitted from the measured part. A fiber face plate having a surface and transmitting the received light to the other surface of the light receiving surface; and receiving the light transmitted from the measured section on the other surface of the fiber face plate. A light emitting reading device comprising: a CCD image sensor for outputting the obtained light information as electronic data; and a memory for storing the electronic data output from the CCD image sensor. 12. A light emission from the measured part is obtained by irradiating the fluorescent label with excitation light, and the light is incident on the measured part from the back side or the side with respect to the surface of the substrate, 12. The apparatus according to claim 11, further comprising: an optical system for irradiating the measured part with the excitation light at an angle that reflects the light on the rear surface side or transmits the light on the side surface side, and excitation light generation means that supplies the excitation light to the optical system. Reader. 13. The electronic apparatus according to claim 11, further comprising: a calling unit for calling the electronic data relating to the amount of light stored in the memory; and a display unit for displaying the emission image based on the electronic data called by the calling unit. A reading device as described. 14. The light emission from a spot disposed on the surface to be measured is based on a reaction between a probe and a target substance specifically recognized by the probe.
14. The reading device according to any one of 13. 15. A detection method for detecting light emission from a plurality of measured parts disposed on a surface of a substrate, comprising: a light receiving surface for receiving light emitted from the measured part; A fiber face plate for transmitting the light to the other surface of the light receiving surface is brought into close contact with the portion to be measured, and on the other surface of the fiber face plate, light transmitted from the portion to be measured is received by light receiving means. A light detection method characterized by receiving and detecting.