JP3761726B2 - Microarray chip reading method and reading apparatus - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a method and apparatus for reading a microarray chip used for DNA analysis, immunological analysis, and the like, and more particularly to improvement of relative scanning between the microarray chip and excitation light.
[0002]
[Prior art]
In recent years, technologies in the field of genetic engineering have rapidly developed, and a human genome project has been developed with one purpose of decoding the base sequence of the human genome, which is thought to be as many as 100,000.
[0003]
On the other hand, enzyme immunoassay methods and fluorescent antibody methods using antigen-antibody reactions are used for diagnosis and research, and research to search for DNA affecting various genetic diseases is also progressing. Microarray technology has attracted attention as a method.
[0004]
In this microarray technology, as shown in FIG. 3, a large number of different known cDNAs (an example of specific binding substances) that have already been decoded are high as a matrix at predetermined intervals as dots on a membrane filter or glass slide. This technique uses a microarray chip (sometimes referred to as a DNA chip) pre-coated at a density (interval of several hundred μm or less). For example, DNA (biological organism) extracted from cells of a healthy person A labeled with a fluorescent dye a An example of a body-derived substance) and DNA extracted from cells of specimen B having a genetic disease, labeled with fluorescent dye b, are dropped onto the microarray chip with a pipette or the like, and DNA of each specimen and cDNA on the microarray chip And each fluorescent dye a on each cDAN on the microarray chip. b is relatively scanned with excitation light for exciting each cDNA, and each fluorescence for each cDNA is detected by a photodetector. Based on the detection result associated with the fluorescence emission position on the microarray chip, This is a technique for identifying which cDNA is hybridized between the two specimens and determining which DNA is hybridized between the two specimens and comparing the cDNA hybridized between the two specimens.
[0005]
By the way, the information on the light emission from the microarray chip is conventionally read as shown in FIG. 5 (1) by applying excitation light L having a beam diameter of 10 to 20 [mu] m in one direction of the microarray chip 10 (for example, the X direction shown in the figure). When main scanning is performed at a constant speed and the main scanning for one line is completed, the microarray chip 10 or excitation light is slightly moved (sub scanning) in the direction orthogonal to the X direction, and main scanning is performed again. By repeating scanning and sub-scanning, substantially the entire surface of the microarray chip 10 is scanned with the excitation light L. Then, light emission information from each portion of the microarray chip 10 scanned with the excitation light L is photoelectrically read, and based on the read light emission information, corresponding to the entire surface of the illustrated microarray chip 10 (FIG. 2), and a certain shape (rectangular in the figure) including the image portion 12 'corresponding to the dot 12 of the cDNA on the microarray chip 10 and the vicinity thereof based on the created digital image 10'. Area (image portion surrounded by a broken line in the figure) is set automatically or manually, and the digital image signal in the set area is summed (or integrated) to obtain an image signal for each area, Based on the obtained image signal, the presence or absence of light emission of the dot 12 of each cDNA on the microarray chip 10 is detected.
[0006]
[Problems to be solved by the invention]
By the way, in order to perform image quantification based on the digital image 10 'described above, it is necessary to create the digital image 10', so that not only the portion where the dots 12 actually exist, but also the image information ( Light emission information) needs to be read, and the excitation light L is scanned over substantially the entire surface of the microarray chip 10 as described above. Further, in order to obtain more accurate image information, it is necessary to use excitation light L having a minute beam diameter.
[0007]
However, in the case where excitation energy is applied while scanning at a constant speed as described above, the excitation light is made of high energy, or the scanning speed is slowed down in order to ensure a given energy per unit area or more, or Both of these measures need to be taken. Further, in order to scan with high density and high accuracy, it is necessary to use a high precision scanning system. Furthermore, in order to analyze the light emission information of the portion corresponding to the dot from the acquired digital image, it is necessary to use quantitative analysis software such as performing the above-mentioned enclosure on the digital image and performing integration processing. Since these structures are expensive, the entire reading apparatus is expensive.
[0008]
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a microarray chip reading method and reading apparatus that reduce costs and increase the reading processing speed.
[0009]
[Means for Solving the Problems]
According to the microarray chip reading method of the present invention, instead of scanning the entire surface of the microarray chip on which the test object is arranged at at least a part of a predetermined position with the excitation light with the excitation light, Scanning is intermittently performed with excitation light having a beam diameter larger than that of the test object so that only predetermined positions are irradiated with excitation light.
[0010]
That is, according to the microarray chip reading method of the present invention, a microarray chip in which objects to be detected that emit light upon being irradiated with excitation light are arranged at least in a part of a plurality of predetermined positions on a carrier. In the microarray chip reading method, the microarray chip and the excitation light are relatively moved so that the excitation light scans, and the emission from the detected object scanned by the excitation light is read.
The excitation light is enlarged or reduced on the microarray chip so that the beam diameter is larger than each detected object and does not irradiate two or more detected objects simultaneously. ,
The microarray chip and the excitation light are moved relative to each other intermittently from one predetermined position to another predetermined position so that the excitation light irradiates only the predetermined positions. It is.
[0011]
Here, the carrier is a membrane filter, a slide glass or the like.
[0012]
A number of predetermined positions set in advance means positions that are densely arranged two-dimensionally or one-dimensionally at a predetermined interval. For example, known in the carrier (membrane filter, slide glass, etc.) This corresponds to each position where a large number of specific binding substances and the like are applied in advance in a matrix at a predetermined interval in a high density (an interval of several hundred μm or less) as dots.
[0013]
The detection target that emits light upon irradiation with excitation light corresponds to, for example, a binding substance in which a biologically derived substance labeled with a fluorescent dye is hybridized to a specific binding substance, but is not limited thereto. Absent. A specific binding substance is a substance related to a wide range of living organisms. For example, hormones, tumor markers, enzymes, proteins, nucleic acids, antibodies, various substances that can be antigens, and various cDNAs and mRNAs. This means a substance that can immunologically bind to or hybridize with an organism-derived substance, and cDNA is particularly suitable. In addition, the substance derived from the organism of the specimen represents a substance capable of immunologically binding or hybridizing with a known specific binding substance arranged on a carrier, such as an antibody, an antigen, a substrate, DNA, mRNA, etc. In particular, DNA is suitable.
[0014]
The object to be detected is arranged at least at a part of a number of predetermined positions. When the object to be detected is arranged at all of the set many positions, a part of the many positions In this case, the detected object is placed on In addition, it does not exclude positively that reading is performed by applying the reading method of the present invention to a microarray chip in which a test object is not arranged at any of a large number of positions. When there is a purpose of reading in detecting that it is not arranged in any of the above, the reading method of the present invention may be applied.
[0015]
Reading the emitted light includes not only detecting the presence or absence of the emitted light but also detecting the emitted light intensity.
[0016]
The expansion or contraction of the beam may be performed using various known optical systems.
[0017]
The term “moving intermittently from one predetermined position to another predetermined position so that the excitation light only irradiates each predetermined position” means stopping at each predetermined position and irradiating the excitation light. While moving from one predetermined position to another predetermined position, it is not limited to not irradiating excitation light at all, but moving so that the irradiation time with respect to the time of irradiating the predetermined position is extremely short If it is. That is, in the region between one predetermined position and another predetermined position, the light emission information is not read, but only passes to move the excitation light between the next predetermined positions.
[0018]
In addition, when various kinds of known cDNAs as specific binding substances and unknown DNAs as organism-derived substances are applied, cDNAs hybridized with these unknown DNAs can be specified, so that various gene diseases can be identified. Work efficiency can be improved in the field of identifying affecting DNA.
[0019]
The microarray chip reader of the present invention includes a microarray chip in which objects to be detected that emit light upon being irradiated with excitation light are arranged at least in a part of a plurality of predetermined positions on a carrier. Scanning means for relatively moving the microarray chip and the excitation light so that the excitation light scans, and a photoelectric reading means for reading the light emission from the detected object scanned with the excitation light. In a microarray chip reader,
The excitation light is enlarged or reduced on the microarray chip so that its beam diameter is larger than each detected object and does not irradiate two or more detected objects simultaneously. A beam expansion / contraction optical system;
The scanning means relatively scans the excitation light or the microarray chip intermittently from one predetermined position to another predetermined position so that the excitation light irradiates only the predetermined positions. It is characterized by being.
[0020]
Various known configurations such as a photomultiplier tube can be applied as the photoelectric reading means, and various known configurations can also be applied to the beam expansion / contraction optical system.
[0021]
In order for the scanning means to relatively move the excitation light or the microarray chip so that the excitation light scans each predetermined position, the scanning means may store those predetermined positions in advance. Alternatively, the position indicating means for sequentially indicating the predetermined positions to the scanning means may be further provided, and the predetermined positions may be sequentially input from the position indicating means to the scanning means. Further, a position detecting means for sequentially detecting the position and inputting the detected predetermined position to the scanning means may be further provided so that each predetermined position detected by the position detecting means is sequentially input to the scanning means. Good.
[0022]
【The invention's effect】
According to the microarray chip reading method and reading apparatus of the present invention, the object to be detected may be arranged, and only a predetermined position set in advance is irradiated with excitation light. The time required for the excitation light scanning can be greatly shortened as compared with the case where the region where no object is arranged is scanned at high density.
[0023]
In addition, since it is necessary to acquire an image of the entire surface of the microarray chip as a digital image, in the present invention, compared with a conventional method or apparatus that requires high-density scanning with excitation light having a beam diameter smaller than the size of an object to be detected. Since it is sufficient to scan only a predetermined position set at a coarser interval than the conventional scanning density, the minimum movement pitch required for the scanning means is more relaxed than in the conventional case. Therefore, it is possible to suppress an increase in cost due to scanning with a high density guarantee.
[0024]
Furthermore, since each detected object is irradiated with excitation light having a beam diameter larger than that of each detected object on the microarray chip, the entire detected object can be irradiated with one irradiation. Luminescence information emitted from the entire object to be detected by irradiation can also be detected by one detection. Therefore, once a digital image is created as in the prior art, an area corresponding to the object to be detected is enclosed. By summing the detection results for each pixel in the enclosed area, a special process of obtaining light emission information emitted from the entire detected object becomes unnecessary.
[0025]
In addition, by making the beam diameter of the excitation light larger than the object to be detected, the applied excitation energy per unit area / unit time on the irradiated surface is lower than before, but as described above, the entire microarray chip As a result, the scanning time of the excitation light can be greatly shortened. Therefore, it is possible to prevent a decrease in applied energy by extending the time for the excitation light to stay at each predetermined position. Even when the residence time is extended in this way, the scanning time of the excitation light can be shortened for the entire microarray chip.
[0026]
In addition, while the beam diameter of the excitation light is larger than the detected object, it is set to a diameter that does not irradiate two or more detected objects at the same time. The reading result can be prevented.
[0027]
As described above, according to the microarray chip reading method and reading apparatus of the present invention, it is not necessary to use an expensive, high-density guaranteed scanning system, high-energy excitation light, and quantitative analysis software, thereby reducing costs. In addition, since the rough scanning may be performed, the scanning of the excitation light can be speeded up.
[0028]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, a specific embodiment of a reading apparatus for carrying out the microarray chip reading method of the present invention will be described with reference to the drawings.
[0029]
FIG. 1 is a diagram showing an embodiment of a microarray chip reader of the present invention, and FIG. 2 is a diagram showing an example of a microarray chip 10 to be read by the reader shown in FIG.
[0030]
FIG. 3 shows a microarray chip 10 ″ in which different cDNAs are respectively applied at predetermined positions preset in a high density in the form of a matrix on the slide glass 11. As shown in Fig. 3, the relationship between the applied position and its cDNA is known in advance, and the DNA of a subject having a genetic disease is labeled with a fluorescent dye. A microarray chip 10 shown in FIG. 2 is hybridized to the microarray chip 10 ″ and only the hybridized binding substance (object to be detected) 12 is left on the slide glass 11. For the sake of illustration, the position of the remaining combination 12 can be identified with the naked eye by comparing the microarray chip 10 ″ shown in FIG. 3 with the microarray chip 10 shown in FIG. Although described as possible, in an actual microarray chip, since cDNA is applied at a high density, it is difficult to distinguish these positions with the naked eye.
[0031]
The microarray chip reader 100 shown in FIG. 2 is a two-dimensionally movable stage 30 on which the microarray chip 10 shown in FIG. 2 is placed, an excitation light source 40 that emits excitation light L, and an output from the light source 40. A collimator lens 51 that converts the excitation light L into a parallel light beam, a polarization beam splitter 52 that transmits the excitation light L and reflects fluorescence K, which will be described later, and the excitation light L that has passed through the beam splitter 52 are placed on the stage 30. A condensing lens 60 for condensing on the microarray chip 10, a photomultiplier (PMT) 70 for photoelectrically detecting the fluorescence K emitted from the microarray chip 10, and an excitation light L to scan the microarray chip 10, A first step motor 21 for moving the microarray chip 10 in the Y-axis direction with respect to the excitation light L and a second step motor 22 for moving in the X-axis direction Position indicating means 80 for inputting the predetermined positions to be stopped to the first and second step motors 21 and 22 so that the excitation light L irradiates only predetermined positions on the microarray chip 10; It is the structure provided with.
[0032]
Here, the predetermined positions inputted to the first and second step motors 21 and 22 by the position indicating means 80 are all positions where the cDNA is applied on the microarray chip 10 ″ shown in FIG. Therefore, the combination 12 is not always present at the position.
[0033]
Specifically, the condensing lens 60 is set so that the excitation light L is condensed on the microarray chip 10 with a spot diameter larger than the size of the combination 12.
[0034]
Next, the operation of the embodiment of the reading apparatus 100 of the present embodiment will be described.
[0035]
First, the microarray chip 10 shown in FIG. 2 is placed at a predetermined position on the stage 30. At this time, each predetermined position where the cDNA is applied on the microarray chip 10 is associated with a position in the X-axis direction and a position in the Y-axis direction on the stage 30. This association is input from the position instruction means 80 to each of the step motors 21 and 22.
[0036]
On the other hand, the excitation light L is emitted from the excitation light source 40, and this excitation light L enters the collimator lens 51 to be a parallel light beam. The excitation light L converted into a parallel light beam passes through the polarization beam splitter 52 and is condensed on the microarray chip 10 placed on the stage 30 by the condenser lens 60.
[0037]
Each step motor 21, 22 drives the stage 30 in the XY plane so that the excitation light L irradiates the first predetermined position on the microarray chip 10 based on the indicated position input from the position indicating means 80. Stop at that position. The excitation light L irradiates the first predetermined position on the microarray chip 10 with a spot diameter wider than that of the combined substance 12. When the combined substance 12 exists at the first predetermined position irradiated with the excitation light L, As shown in FIG. 4, the combined substance 12 is entirely irradiated with the excitation light L, the fluorescent dye of the combined substance 12 is excited, and emits fluorescence K. When the binding substance 12 is not present at the first predetermined position irradiated with the excitation light L, the microarray chip 10 does not emit fluorescence K.
[0038]
When the combined substance 12 is present and the fluorescence K is emitted, the fluorescence K sequentially passes through the condenser lens 60 and the polarization beam splitter 52, enters the photomultiplier 70, and generates an electrical signal corresponding to the amount of light. It is converted and output to an external processing device. Information on a predetermined position irradiated with the excitation light L is input from the position instruction means 80 to the external output device, and the predetermined position is associated with the presence / absence of detection of the fluorescence K and the amount of light.
[0039]
When a predetermined time has elapsed from the irradiation of the excitation light L to the first predetermined position, the next predetermined position is input from the position indicating means 80 to the step motors 21 and 22, for example, only the step motor 22 is driven, and the stage 30 is moved to X After moving in the axial direction so that the excitation light L irradiates a predetermined position adjacent to the first predetermined position, it stops (see FIG. 4).
[0040]
Then, the excitation light L irradiates a predetermined position next to this, and as in the first predetermined position, when the binding substance 12 is present, fluorescence K is emitted and detected by the photomultiplier 70, and when it does not exist. Not detected.
[0041]
By repeating the above operation until all the predetermined positions on the microarray chip 10 are irradiated with the excitation light L, the processing apparatus associates all of the predetermined positions with the presence or absence of the emission of the fluorescence K and the light emission amount. Based on this correspondence, functional analysis of DNA of a subject with a genetic disease is performed.
[0042]
As described above, according to the microarray chip reading apparatus 100 of the present embodiment, the stage 30 is intermittently moved, and a predetermined position (cDNA is applied) where the conjugate 12 can exist. Since only the excitation position L is irradiated with the excitation light L, the time required for scanning the excitation light L can be greatly reduced as compared with the conventional case. Further, since the stage 30 may be moved intermittently at rough intervals, it can be realized by the inexpensive step motors 21 and 22, and an increase in cost can be suppressed.
[0043]
Further, since each conjugate 12 is irradiated with the excitation light L having a beam diameter larger than that of each conjugate 12 on the microarray chip 10, the entire conjugate 12 can be irradiated with one irradiation, and therefore Fluorescence K emitted from the entire conjugate 12 by this irradiation can also be recorded by one detection, and after creating a digital image once, an area corresponding to the conjugate 12 is enclosed and enclosed. By summing the detection results for each pixel in the region, a special process of obtaining the intensity or light quantity of the fluorescence K emitted from the entire combination 12 is not required.
[0044]
In the reader of this embodiment, the predetermined position on the microarray chip is the position where the cDNA is applied. However, the method and apparatus for reading the microarray chip of the present invention are limited to this mode. It is not a thing.
[Brief description of the drawings]
1 is a diagram showing an embodiment of a microarray chip reader according to the present invention. FIG. 2 is a diagram showing a microarray chip used in the reader shown in FIG. 1. FIG. 3 is a microarray chip shown in FIG. FIG. 4 is a diagram illustrating a scanning state of excitation light. FIG. 5 is a diagram illustrating a conventional scanning state and reading of excitation light.
10 Microarray chip
11 Slide glass
12 Conjugate (Detected object)
21, 22 Step motor
30 stages
40 Excitation light source
51 Collimator lens
52 Polarizing beam splitter
60 condenser lens
70 Photomultiplier
80 Position indication means
100 Reader L Excitation light K Fluorescence

Claims (6)

The microarray is arranged so that the excitation light scans a microarray chip in which objects to be detected that emit light upon irradiation with excitation light are arranged at least in a part of a plurality of predetermined positions on the carrier. In the microarray chip reading method of moving the chip and the excitation light relatively, and reading the light emission from the detected object scanned by the excitation light,
The excitation light is enlarged or reduced on the microarray chip so that the beam diameter is larger than each detected object and does not irradiate two or more detected objects simultaneously. ,
A microarray characterized by relatively moving the microarray chip and the excitation light intermittently from one predetermined position to another predetermined position so that the excitation light irradiates only the predetermined positions. Chip reading method. 2. The method for reading a microarray chip according to claim 1, wherein the test substance is a binding substance obtained by hybridizing a known specific binding substance with an organism-derived substance labeled with a fluorescent dye. 3. The microarray chip reading method according to claim 2, wherein the specific binding substance is cDNA, and the organism-derived substance is DNA. The microarray is arranged so that the excitation light scans a microarray chip in which objects to be detected that emit light upon irradiation with excitation light are arranged at least in a part of a plurality of predetermined positions on the carrier. In a microarray chip reader comprising: a scanning unit that relatively moves the chip and the excitation light; and a photoelectric reading unit that reads the emitted light from the detected object scanned with the excitation light.
The excitation light is enlarged or reduced on the microarray chip so that its beam diameter is larger than each detected object and does not irradiate two or more detected objects simultaneously. It further includes a beam expansion / contraction optical system,
The scanning means relatively scans the excitation light or the microarray chip intermittently from one predetermined position to another predetermined position so that the excitation light irradiates only the predetermined positions. A microarray chip reader, comprising: 5. The microarray chip reader according to claim 4, wherein the test object is a binding substance obtained by hybridizing a known specific binding substance to a biological substance labeled with a fluorescent dye. 6. The microarray chip reader according to claim 5, wherein the specific binding substance is cDNA, and the organism-derived substance is DNA.

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