JP2010066146A - Microarray measuring method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for measuring a microarray substrate with positional variations of a simple substrate of a spot. <P>SOLUTION: The method for measuring a microarray substrate is composed of steps: adding a first substrate chemically reacting with a first reagent previously fixed to a spot to emit light; photographing the light of the spot to create a primary photometry image; creating a primary photometry image with the spot position specified from the primary photometry image; adding a specimen and a second reagent onto the microarray substrate after removing the first substance to bond to and react with each other and then removing the specimen not bonded to an antibody previously fixed to the spot and the second agent; adding a second substrate chemically reacting with the second reagent to emit light; photographing the light of the spot to create a secondary photometry image; and specifying the spot position in the secondary photometry image using the primary photometry analysis image and measuring the amount of luminescence of the spot. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for measuring a microarray having spot-based position variations.

  Microarrays are used for analysis of biomolecules. Its use is diverse, including gene analysis and functional analysis of proteins expressed from genes. For example, in the case of gene analysis using a microarray, it is possible to comprehensively analyze gene expression in individual cells, tissues or individuals.

  The microarray is also called a DNA chip. A small spot made of glass or silicon is formed on a small substrate of 1 x 3 inches made of glass or silicon by applying droplets of the gene solution in nanoliter units at a high density to form microscopic spots. A method for immobilizing gene fragments on the substrate is disclosed (for example, see Patent Document 1).

  By performing various chemical reactions such as blocking, washing, and detection reaction on the substrate containing the immobilized product, it is possible to perform comprehensive gene analysis and functional analysis of the expressed protein. The chemical reaction on the substrate is performed by dropping a reaction solution onto the substrate, and the detection is performed by using a scanner or a CCD (Charge Coupled Devices) to detect the luminescence generated by the chemical reaction or the fluorescence generated by the chemical reaction and physical force. This is done by reading with a camera. By analyzing the read image, the presence or absence of the target sample and the target sample amount can be indirectly grasped from the light emission amount.

In order to automatically analyze an image read by a CCD camera or a scanner, a technique for correcting the positional deviation of the substrate and the positional deviation of the spot is required. In order to correct the positional deviation of the spot, a method is disclosed in which a spot that always emits light and a spot that does not always emit light are provided as a reference pattern area at the time of sample analysis, the spot position is determined by the light emission pattern, and correction is performed ( For example, see Patent Document 2).
Japanese National Patent Publication No. 10-503841 JP 2003-307518 A

  However, when the spot is formed on the microarray substrate, the positioning accuracy is not sufficient, and if the position variation of the spot alone occurs, there is a positional deviation between the predetermined spot measurement range and the actual spot emission region. As a result, there has been a problem that accurate measurement of the amount of luminescence cannot be performed.

  Therefore, an object of the present invention is to provide a microarray measurement method that can accurately measure the amount of light emission even if the light emission region is displaced due to the variation in the position of a single spot.

  In order to achieve this object, the microarray measurement method of the present invention is a microarray measurement method for measuring the amount of light emitted from a plurality of spots provided on a microarray substrate. A first substrate addition step of adding a first substrate that emits light upon chemical reaction onto the microarray substrate; a primary photometry step of photographing a light of the spot to generate a primary photometric image; and the primary A first image analysis step for generating a primary photometric analysis image specifying the position of the spot from the photometric image; and after removing the first substrate, adding a specimen and a second reagent on the microarray substrate A sample addition step for performing a binding reaction and removing the sample and the second reagent that did not bind to the antibody immobilized in advance on the spot; A second substrate addition step of adding a second substrate that emits light upon chemical reaction with a drug onto the microarray substrate; a secondary photometry step of photographing the spot light to generate a secondary photometry image; It is characterized by comprising a second image analysis step for specifying the position of the spot in the secondary photometric image using the primary photometric analysis image and measuring the light emission amount of the spot. .

  The microarray measurement method of the present invention is the microarray measurement method for measuring the amount of light emitted from a plurality of spots provided on a microarray substrate, wherein the first substrate that chemically reacts with the first reagent immobilized in advance on the spots is provided. A first substrate adding step to be added on the microarray substrate; a primary photometric step of irradiating the spot with excitation light and photographing a fluorescence from the spot to generate a primary photometric image; and the primary photometry A first image analysis step for generating a primary photometric analysis image specifying the position of the spot from the image, and after removing the first substrate, adding a specimen and a second reagent on the microarray substrate and binding A sample addition step for reacting and removing the sample that did not bind to the antibody immobilized in advance on the spot and the second reagent; A second substrate addition step of adding a second substrate that chemically reacts with a drug onto the microarray substrate, and irradiating the spot with excitation light and photographing the fluorescence from the spot to generate a secondary photometric image A secondary photometry step, and a second image analysis step of specifying the position of the spot in the secondary photometry image using the primary photometry analysis image and measuring the light emission amount of the spot. It is characterized by.

  In the microarray measurement method of the present invention, in the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate, the light transmitted through the substrate positioning mark provided on the microarray substrate was photographed. A first fixing error detecting step for detecting a fixing position error of the microarray substrate using an image, and a first substrate that emits light by chemically reacting with a first reagent fixed in advance on the spot on the microarray substrate A first substrate addition step for adding to the light source, a primary photometry step for photographing the light of the spot to generate a primary photometric image, and a primary photometric analysis image for specifying the position of the spot from the primary photometric image A first image analysis step to generate a sample and a second sample on the microarray substrate after removing the first substrate The sample was added and allowed to bind, and the sample that did not bind to the antibody previously immobilized on the spot and the second reagent were removed, and the light transmitted through the substrate positioning mark was photographed. A second fixing error detecting step for detecting a fixing position error of the microarray substrate using an image; and a second substrate that chemically reacts with the second reagent to emit light, and is added to the microarray substrate. Substrate addition step, secondary photometry step of photographing the spot light and generating secondary photometry image, primary photometry using the confirmation mark and the substrate position determination mark provided on the microarray substrate An image misalignment correcting step for correcting a misalignment that occurs relatively between the analysis image and the secondary photometry image, and using the primary photometry analysis image, Serial identifies the position of the spot, in which is characterized in that is constituted by a second image analyzing step of measuring a light emission amount of the spot.

  In the microarray measurement method of the present invention, in the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate, the light transmitted through the substrate positioning mark provided on the microarray substrate was photographed. A first fixing error detection step for detecting a fixing position error of the microarray substrate using an image, and a first substrate that chemically reacts with a first reagent previously fixed to the spot are added to the microarray substrate. A first substrate adding step, a primary photometric step of irradiating the spot with excitation light, photographing fluorescence from the spot to generate a primary photometric image, and a position of the spot from the primary photometric image. A first image analysis step for generating the identified primary photometric analysis image; and after removing the first substrate, the microphone A sample addition step for adding a specimen and a second reagent on the array substrate to cause a binding reaction, and removing the specimen and the second reagent that did not bind to the antibody immobilized in advance on the spot; and determining the substrate position A second fixing error detecting step for detecting a fixing position error of the microarray substrate using an image obtained by photographing the light transmitted through the mark, and a second substrate chemically reacting with the second reagent on the microarray substrate. Provided on the microarray substrate, a second substrate addition step for adding to the substrate, a secondary photometry step for irradiating the spot with excitation light, photographing fluorescence from the spot to generate a secondary photometry image, and the microarray substrate An image misalignment correction step for correcting a relative misalignment between the primary photometric analysis image and the secondary photometric image using the confirmation mark and the substrate position determination mark. And a second image analysis step of identifying the position of the spot in the secondary photometry image using the primary photometry analysis image and measuring the light emission amount of the spot. It is a thing.

  As described above, according to the microarray measurement method of the present invention, the step of adding the first substrate that emits light by chemically reacting with the first reagent immobilized in advance on the spot, and photographing the light of the spot A step of generating a primary photometric image, a step of generating a primary photometric analysis image specifying the position of the spot from the primary photometric image, and after removing the first substrate, a specimen is formed on the microarray substrate And a second reagent are added to cause a binding reaction, and the step of removing the specimen and the second reagent that did not bind to the antibody immobilized on the spot in advance, and a chemical reaction with the second reagent to emit light Adding a second substrate to generate a secondary photometric image by photographing the spot light, and using the primary photometric analysis image, the position of the spot in the secondary photometric image. Was identified by performing the steps of measuring the amount of light emission of the spot, even if the position variation of a single spot is generated, it is possible to perform measurement of accurate emission amount.

  Embodiments of a detection area position detection method according to the present invention will be described below in detail with reference to the drawings.

(Embodiment 1)
In the microarray measurement method of the present embodiment, light emission or fluorescence due to a chemical reaction at a plurality of spots provided on a microarray substrate is photographed with a CCD camera, and the amount of a specimen is measured from the light emission amount. Used for things. This microarray measurement method is realized in a microarray measurement apparatus that includes a stage for fixing a microarray substrate, a CCD camera with the stage in the field of view, and a signal processing apparatus for processing an image captured by the CCD camera.

  The microarray substrate is provided with a plurality of spots. The first reagent that chemically reacts with the first substrate and emits light is evenly immobilized on all spots on the microarray substrate. In addition, an antibody for immobilizing the specimen and the second reagent that emits light by chemical reaction with the second substrate is immobilized on each spot. Here, as these antibodies, different antibodies may be immobilized for each spot so that a plurality of specimens can be measured. Depending on the purpose of measurement, the type of antibody can be freely changed. The creation of the microarray substrate will be described in detail later.

  Here, the microarray measurement method of the present embodiment will be described with reference to FIG. FIG. 1 is a flowchart showing the steps of the microarray measurement method in the present embodiment.

  First, a first substrate addition step of adding a first substrate to the microarray substrate fixed on the stage is performed (S101). This first substrate is a solution and is added so that it is spread evenly on all spots on the microarray substrate. When this first substrate is developed, a chemical reaction occurs with the first reagent, and light emission is started at each spot.

  Next, a primary photometric step of photographing the microarray substrate with a CCD camera is performed (S102). This CCD camera is, for example, a monochrome CCD camera. The CCD camera is exposed for a predetermined time (for example, 1 minute), and the amount of light received by the CCD element during the exposure is integrated and output as the luminance value of each pixel. An image in which the luminance value is expressed in multiple values is taken as a primary photometric image.

  FIG. 2A shows a primary photometric image output from the CCD camera. In FIG. 2A, 201 is a spot. Since the first reagent is fixed uniformly to all spots, each spot emits light uniformly in the primary photometric image. Since each spot emits light, the luminance value corresponding to the spot is larger than the surroundings. Reference numerals 202 and 203 denote spots where positional deviation occurs alone.

  Next, a first image analysis step is performed to analyze the primary photometric image and specify the center coordinates of each spot and the boundary between the spots (S103). The image obtained as a result is a primary photometric analysis image, which is shown in FIG. In FIG. 2B, 204 represents the center coordinate point of each spot, and 205 represents the boundary (spot size) of each spot.

  Next, a sample addition step is performed (S104). In this step, first, the microarray substrate is washed to wash away the first substrate. Next, a sample that reacts with the antibody at each spot and a second reagent that specifically adsorbs to this sample are added excessively on the microarray substrate, and the antibody immobilized on each spot in advance and the antigen in this sample are combined. Antigen-antibody reaction and specific adsorption. After the antigen-antibody reaction is completed, the microarray substrate is washed to remove the remaining antigen and the second reagent that cannot react with the antibody.

  This specimen addition step is a process for reacting a component in the specimen with an immobilized product directly or indirectly, a reaction process with a labeled product, or a washing process, and a preparation process for generating secondary photometry. These steps differ depending on the conditions such as what the immobilized substance is, what the detection target is, and what reaction method is used for light emission. For example, even when an antibody is immobilized, the reaction process differs depending on whether an antibody already labeled with an enzyme is immobilized or an unlabeled antibody is immobilized. The reaction process varies depending on the difference in the label such as biotin or biotin. In addition, processing differs depending on whether the measurement method is a sandwich method or a competitive method. Here, an example in which an antibody is immobilized on a substrate and a sandwich assay is performed is shown. However, it is not limited to this reaction system.

  Next, a second substrate addition step of adding a second substrate to the microarray substrate is performed (S105). At this time, since the amount of antigen specifically adsorbed to each spot differs depending on the specimen, the emission luminance of each spot varies. In addition, the 2nd substrate added here does not raise | generate a chemical reaction with a 1st reagent, but reacts only with a 2nd reagent, and light-emits.

  Next, a secondary photometry step of photographing the microarray substrate with a CCD camera is performed (S106). Here, as in the primary photometric step, the CCD camera is exposed for a predetermined time to generate a secondary photometric image. At this time, the amount of light emission is different for each spot, and in some cases, there is a spot that hardly emits light. FIG. 2C shows a schematic diagram of the secondary photometry image.

  Next, the secondary photometric image is analyzed, and a second image analysis step for specifying the light emission amount of each spot is performed (S107). First, as shown in FIG. 2D, the secondary photometric image is superimposed on the primary photometric analysis image obtained in the first image analysis step. Then, the center coordinates of the spot where the amount of light emission should be detected and the boundary between the spots are known. In each spot, the luminance values of the respective pixels within the boundary of the spot are integrated to obtain the light emission amount. Finally, this luminescence amount is applied to a calibration curve prepared in advance to determine the component amount in the sample.

  In the first substrate addition step and the second substrate addition step, when a fluorescent substance is generated by a chemical reaction, the fluorescent substance is excited during the execution of the primary photometry step and the secondary photometry step. The excitation light is irradiated from the outside.

Here, luminescent substrates that can be used as the first substrate and the second substrate are shown below. Luminol, isoluminol, luciferin (firefly luciferin, coelenterazine, Cypridina luciferin), lucigenin, lophine, acridinium ester, gallic acid, indole derivatives, Disodium 3- (4-methoxyspiro {1,2-dioxetane-3,2'- (5'-chloro) tricyclo [3.3.1.1 ^3,7 ] decan} -4-yl) phenyl phosphate, Disodium 2-chloro-5- (4-methoxyspiro {1,2-dioxetane-3,2 '- (5'-chloro) tricyclo [3.3.1.1 ^{3, 7]} decan} -4-yl) phenyl phosphate , 3- (2'-Spiroadamantane) -4-metho y-4- (3''-phosphoryloxy) phenyl-1,2-dioxetane (AMPPD), Rumijen PPD, Galacton (registered trademark) and the like.

  In addition, fluorescent substrates that can be used as the first substrate and the second substrate are shown below. cascade Blue, Alexa-405, Alexa-350, AMCA, Pacific Blue, Marina Blue, Cy2, BODIPY FL, Fluorescein, Alexa-488, Oregon Green-488, Alexa-500, Oregon Green-14, Oregon Green-14 514, Cascade Yellow, Alexa-532, Alexa-555, Cy3, Alexa-546, PE-R, Tetramethylrhodamine, Rhodamine Red, Alexa-568, Texas Red, Alexa-594, Alexa-633a, 64C Alexa-660, Cy3.5, Cy5.5, Alexa 680, Alexa-700, Cy7, Alexa-750, SYBR Green I, SYBR Green II, SYBR Gold, EtBr, MDPF, SYPRO Orange, SYPRO Ruby, SYPRO Rose, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red, SYPRO Red Q Diamond, Pro-Q Emerald 300, Pro-Q Sapphire 365, Pro-Q Sapphire 488, ELF39, ELF97, DDAO, Amplex Gold, Amplex Red, Tyramine, p-HydropropylP) D-galact side, 4-Metylumbellipherylphosphate, 3- (p-Hydroxyphenyl) propionic acid, Pyrene, BBTP (2'- [2-benzothiazoyl] -6'-hydroxybenzothiazole phosphate) and the like.

  In addition, in the selection of the two types of substrates for addition and the two types of immobilization reagents, the added first substrate and second substrate are different from each of the immobilized two reagents. It is necessary to choose one that only reacts and does not interfere with each other.

  Here, a procedure for forming a spot by immobilizing an antibody on a microarray substrate will be described. FIG. 3 is a diagram showing a manufacturing process of a measurement microarray substrate.

  First, a coating substrate 301 is prepared. As the coating substrate 301, a glass substrate or a plastic substrate such as polystyrene can be used. In addition, a surface chemical treatment (silanization, amination, aldehyde formation, thiolation, epoxidation, active esterification), gold coating, aluminum coating, nitrocellulose, vinylidene fluoride, polylysine, etc. It may be used after being subjected to a treatment such as a hydrophilic polymer coating such as a conductive polymer coating, a polyacrylamide gel or an agarose gel, a photoreactivity, or a porous structure.

  Next, a mixed solution 305 containing two reagents involved in light emission or fluorescence is prepared. The prepared solution is stirred and gently applied until uniform. As a reagent 303 directly involved in primary photometry and a reagent directly involved in secondary photometry, fluorescent proteins (EBFP, ECFP, TagCFP, AmCyan, Midoriishi-Cyan, CFP, TurboGFP, AcGFP, TagGFP, Azami-Green) can be used. , ZsGreen, EmGFP, EGFP, GFP2, HyPer, TagYFP, EYFP, Venus, YFP, PhiYFP, PhiYFP-m, TurboYFP, ZsYellow, Kusabila-Orange, TurboRFP, DsR , JRed, KillerRed, HcRed, Keima-Red, PS-C P2, Dendra2, Kaede, Kikume Green-Red, EosFP, PA-GFP, Dronpa-Green, KFPRed (after activation), luciferase (non-secretory type, firefly-derived, Renilla-derived, click beetle-derived, railworm Origin, Iriomote botal, Secretory type, Copepoda, Sea firefly, Alkaline phosphatase (bovine, bovine kidney, bovine live, calf intestine, escherichia coli, human plasticine, tein, pincentain rabbit intestine, shrimp, Atlantic Phosphatase), peroxidase (horseradish, arthromyces ramosus, glycine max), glucose oxidase (Aspergillus genus, Penicillium genus), beta-galactosidase (aspergillus oryzae, bovine liver, bovine testes, escherichia coli, kluyveromyces lactis, saccharomyces fragilis), β -Glucuronidase (bovine liver, escherichia coli, Helix aspersa, Helix pomatia, patella vulgata), aequorin and other luminescence and fluorescence reactions can be involved Protein, fluorescently labeled DNA probes, luminescent labeled DNA probes, fluorescently labeled RNA probes, luminescent labeled RNA probes can be used. As reagents indirectly involved in secondary photometry, antibodies, antigens, DNA probes, RNA probes, proteins, peptides, organic molecules, sugar chains, and cells can be used.

  Of the two reagents, one reagent is a reagent 303 for specifying the application state of all the spots 201 and is a reagent 303 that directly participates in primary photometry. The other reagent is a reagent 304 for measuring the amount of the sample, and is a reagent 304 that directly or indirectly participates in secondary photometry. In addition, the reagent 303 that is directly involved in the primary photometry is adjusted so that the same concentration is applied to each spot in order to display almost the same luminance in any spot 201 during the primary photometry.

  The adjusted mixed liquid 305 is immobilized on the substrate using a coating machine. There are various coating methods. For example, the mixed liquid 305 is applied to the pins 306 of the coating machine, and the mixed liquid 305 is pressed against the substrate surface to apply the mixed liquid 305 to the array region 302 of the substrate. Apply to. Typical coating methods include photolithography, micro stamping, mechanical micro spotting, micro pipetting, micro dispensing, jet printing, electrospray deposition, and nano lithography. The applied reagent is fixed in a matrix in the array region 302 of the substrate. Since the fixed spot 201 fixes the uniformly mixed coating solution 305, the two reagents are fixed uniformly in each spot, so the light emission shape of each spot indicated by each of the two reagents Will be similar. Therefore, the detection area of the secondary photometric image can be defined based on the analysis data of each spot of the primary photometric image.

  When the immobilization on the substrate is completed, a blocking agent is added onto the immobilization substrate. Blocking agents that can be used at this time include skim milk, BSA, fish gelatin, lectin, serum, casein, synthetic polymers, blocking agents derived from plant components, etc., which can be used in combination or mixed with a surfactant. Can be used. By performing blocking, it is possible to suppress non-specific reactions that occur outside the immobilization region on the substrate.

  When blocking is complete, the blocking agent on the substrate is eliminated. In order to completely remove the blocking agent, the washing operation is repeated several times.

  Next, in some cases, the substrate is cleaned once with a buffer involved in primary photometry. This operation is performed when the type and pH of the washing operation and the buffer in which the substrate is dissolved are greatly different. Further, this operation is an operation called buffer exchange, and when a substrate is added, it is possible to perform a quick light emission reaction. In this way, a microarray substrate is produced.

  As described above, in this embodiment, first, a chemical reaction in which all the spots on the microarray substrate emit light is caused to analyze the positions of the spots, and then the actual measurement is performed. Even if positional deviation occurs in spot units, the range of the spot to be measured can be specified, so that it is possible to accurately measure the light emission amount.

  Note that it is also possible to detect an antibody application error on the microarray substrate after the first image analysis step. FIG. 4 is a diagram showing an example of a coating error that can be discriminated from the primary photometric image.

  If the application is successful, all the spots 201 in the area 401 emit light at the primary photometric stage. If the application is successful as shown in FIG. 4 (a), the same concentration of primary photometric reagent is applied to all spots 201. Therefore, each spot has a substantially similar circular shape and a spot size. Can be obtained.

  Further, FIG. 4B is a diagram of application that corresponds to a pass / fail boundary. Here, variation in shape at the time of application such as a spot 402 having a small shape, a misalignment spot 403, and an elliptical spot 404 is shown. Whether such a variation is acceptable or not is determined based on whether or not the spot is within a preset variation range. The setting contents of the range are the application position, the shape of the circle, and the luminance value. Application pass / fail judgment can be made by setting the ranges of these analysis parameters.

  FIG. 4C is a diagram when it is determined that an application error has occurred. Spot shapes due to application errors include a spot 406 whose shape is too small, a spot 408 whose shape is too deformed, a spot 405 whose shape is too large, and a spot 407 whose position is too shifted. As the luminance value of the application error, there arises a problem that the luminance value increases or decreases with the shape change and cannot be identified due to the shape change exceeding the detection limit on the software. The error spot determination criteria may be set such that, for example, the spot size diameter is within 14 to 16 pixels and the spot position error is within 10%. The spot determined to be an error spot and the spot determined to be difficult to measure the exact light emission amount due to the influence of the error spot are not measured in the second image analysis step. In this way, it is possible to eliminate measurement defects due to spot coating errors on the microarray substrate.

(Embodiment 2)
The present embodiment is different from the first embodiment in that a confirmation mark and a substrate position determination mark are provided on the microarray substrate, and the positional displacement of the microarray substrate is detected using this mark.

  First, the confirmation mark and the substrate position determination mark will be described with reference to FIG. FIG. 5 is a diagram showing the installation positions of the confirmation mark 501 and the substrate position determination mark 502.

  The installation position of the confirmation mark 501 may be provided at the apex position of the outer peripheral portion of the array region 302 as shown in FIG. By installing the confirmation mark 501 at each apex location, the number of installations can be minimized. Further, if the field of view of the CCD camera is adjusted so that all confirmation marks 501 are present in the measurement image, one spot can be measured without being missed.

  If the array region 302 is symmetric even if it is reversed upside down, such as a square or a rectangle, or reversed right and left, the substrate is placed near any one of the four confirmation marks 501. If the position determination mark 502 is provided, it is possible to determine that the fixing direction of the microarray substrate is wrong.

  Here, the confirmation mark 501 and the substrate position determination mark 502 need only be able to react with the first substrate and the second substrate, and two or more types of reagents that react with the first substrate and the second substrate, respectively. May be mixed.

  Further, in order to increase the application area of the array region 302, as shown in FIG. 5B, the installation position of the confirmation mark 501 may be provided at each vertex position inside the array region 302, and the substrate at that time The position determination mark 502 may be installed at a position adjacent to the confirmation mark 501 as in FIG.

  Next, the microarray measurement method of the present embodiment will be described with reference to FIG. FIG. 6 is a flowchart showing the steps of the microarray measurement method in the present embodiment. 6 differs from FIG. 1 described in the first embodiment in that a first fixed error detection step (S201) is performed between the primary photometry step (S102) and the first image analysis step (S103). The second fixed error detection step (S202) and the image shift correction step (S203) are provided between the provided point and the secondary photometry step (S106) and the second image analysis step (S107). . The other steps are the same as those in the first embodiment, and thus the same reference numerals are given and description thereof is omitted.

  First, the first fixed error detection step will be described. This step detects whether the microarray substrate is fixed to the stage by mistake in the up / down / left / right orientation, and if it is determined that the orientation is wrongly fixed, notifies the user of the microarray substrate fixation error. Is.

  For example, when the substrate position determination mark 502 on the microarray substrate is provided so as to exist at the upper left of the primary photometry image, the substrate position determination mark 502 in the primary photometry image is other than the upper left of the image. When the position is detected, it is determined that the microarray substrate is fixed in the wrong direction, and the user is notified.

  Next, the second fixed error detection step will be described. This step is necessary when the sample addition step (S104) is performed with the microarray substrate removed from the stage. When the sample addition step is completed and the microarray substrate is fixed to the stage again, the user is notified that there is an error in the fixing direction. Since the specific method is the same as the first fixed error detection step, the description thereof is omitted.

  Next, the image misalignment correction step will be described. In this step, the positional deviation between the primary photometric analysis image and the secondary photometric image is corrected using the confirmation mark 501 and the substrate position determination mark 502. The positional deviation may be vertical, horizontal, or rotational, and the vertical, horizontal, or horizontal deviation is detected by detecting the relative position of the confirmation mark 501 between the primary photometric analysis image and the secondary photometric image. If the image correction is performed so as to shift the image, the detailed description is omitted. Here, two forms for correcting the rotational deviation will be described below. The following example shows a case where the secondary photometric image is relatively rotated with respect to the primary photometric image.

  The first is a correction method using midpoint coordinates. FIG. 7 is a diagram showing a method for correcting the substrate position of the secondary photometric image and determining the detection area using the midpoint coordinates. First, the central coordinates of the confirmation mark 501 and the central coordinates of the substrate position determination mark 502 are calculated from the primary photometric analysis image 701 and the secondary photometric image 702.

  Next, the center coordinates of the confirmation mark 501 closest to the substrate position determination mark 502 and the central coordinates of the confirmation mark 501 positioned diagonally are connected by a straight line, and the midpoint coordinates on the straight line are calculated. Then, the image 704 with the midpoint position 704a in the secondary photometric image is moved relative to the image 703 with the midpoint position 703a in the primary photometric analysis image, and the midpoint positions are superimposed.

  Next, the midpoint position is fixed with respect to the image 705 in which the midpoint positions of the primary photometric analysis image and the secondary photometric image are matched, and the center of the confirmation mark 501 of the primary photometric analysis image is centered on this point. A secondary photometric correction image 706 is obtained by correcting the secondary photometric image by rotating the image so that the center coordinate of the confirmation mark 501 of the secondary photometric image matches the coordinate.

  In the second image analysis step, the detection area of the secondary photometry correction image is defined based on the position data of the secondary photometry correction image and the primary photometry analysis image that have been subjected to position correction. Measure.

  The second correction method uses origin coordinates. FIG. 8 is a diagram illustrating a method for correcting the substrate position of the secondary photometric image and determining the detection area using the origin coordinates. First, the center coordinates of the confirmation mark 501 and the center coordinates of the substrate position determination mark 502 are calculated from the primary photometry analysis image 801 and the secondary photometry image 802. Next, the center coordinate of the confirmation mark 501 closest to the substrate position determination mark 502 is set as the origin coordinate.

  Next, the image 804 with the origin position 804a in the secondary photometry image is moved to the image 803 with the origin position 803a in the primary photometry analysis image, and the origin coordinate positions are superimposed. Then, the origin coordinate position is fixed with respect to the image 805 in which the origin positions of the primary photometry analysis image and the secondary photometry image are matched, and the center of the confirmation mark 501 acquired from the primary photometry analysis image is centered on this point. The image is rotated so that the center coordinates of the confirmation mark 501 acquired in the secondary photometric image and the center coordinates of the substrate position determination mark 502 coincide with the coordinates and the center coordinates of the substrate position determination mark 502. In order to simplify this process, the image may be rotated and moved until the center coordinate of the confirmation mark 501 located diagonally with the origin coordinate coincides. When the primary photometric analysis image and the secondary photometric image completely overlap, the substrate position correction is completed.

  The detection area of the secondary photometry image is defined based on the position data of the image 806 obtained by correcting the position of the substrate with respect to the origin position and the position data of the spot 201 obtained by the primary photometry.

  If the analysis is performed by this method, one of the center coordinates is used as the origin coordinate, so that the number of processes can be simplified as compared with the analysis method using the midpoint coordinates.

  As described above, in the present embodiment, since the mounting position shift of the microarray substrate to the stage is corrected using the mark provided on the microarray substrate, it is possible to prevent measurement failure due to the mounting position shift. It becomes.

  The confirmation mark in the present embodiment is a case where a spot that emits light at the time of primary and secondary photometry is used. In addition to this, transmitted light is provided on the microarray substrate itself, and the transmission region is used as a confirmation mark. It can also be used. This transmissive region has the same shape as the confirmation mark 501 and the substrate position determination mark 502 shown in this embodiment, and is arranged at the same position.

  FIG. 9 is a flowchart showing the steps of a microarray measurement method when a light transmission region is provided on the microarray substrate. A difference from the above-described method is that a first fixed error detection step of irradiating the microarray substrate with light from the opposite side of the CCD camera, photographing the transmitted light at that time, and determining an error in fixing the microarray substrate (S301) Is performed before the first substrate addition step (S101), and the second fixed error detection step (S302) for determining the microarray substrate fixing direction error using the transmitted light is also referred to as the sample addition step (S104). This is a point that is performed during the second substrate addition step (S105).

  In the microarray substrate orientation error determination performed in these steps and the image misalignment correction step (S303), the operations are the same except that the transmission region is used instead of the light emitting spot, and thus detailed description is omitted. To do.

  In this embodiment, the image taken by the CCD camera is corrected to eliminate measurement errors due to misalignment, but the position of the array area reflected on the CCD camera by finely adjusting the stage for fixing the microarray substrate You may make it eliminate gap.

  A model experiment was conducted using HRP as a reagent directly involved in primary photometry and IgE as a reagent indirectly involved in secondary photometry.

  As the substrate, a substrate (GenTel, PATH Protein Microarray Slide) whose array region was treated with nitrocellulose was used. In the coating solution, Human IgE antibody (Yamasa Shoyu Co., Ltd.) and HRP (Sigma Aldrich) were added. HRP was added as a primary photometric reagent, and IgE antibody was added as a secondary photometric reagent. At the time of coating, the coating solution was adjusted so that the HRP amount per spot could be coated by 10 fmol. The reagent composition of the confirmation mark for preventing the shooting area from being lost was also the same as the reagent composition of the spot. Samples were prepared so that IgE antibody was applied in an amount of 0.3, 0.1, 0.03, 0.01, 0.003, and 0.001 fmol for each spot. As a control, a spot on which a mixture of IgE antibody and BSA was immobilized was prepared. BSA was added as a structure stabilizer in the low concentration region of IgE antibody. The prepared mixed solution was applied to the substrate surface using MicroCaster (Whatman). The coated sample was left to stand overnight at 25 ° C. in a tapper with a wet cloth.

  Next, a solution obtained by adding 25 mM Tris-HCl, 0.45 M NaCl, and 0.1% Tween 20 to 3% skim milk was added onto the nitrocellulose substrate 101 as a blocking agent. The blocking treatment was performed for 30 minutes while stirring with a shaker. By performing blocking, adsorption outside the immobilization region can be suppressed.

  Next, the blocking agent was removed from the substrate, and the washing operation was repeated three times, and then the substrate was fixed to the stage for photographing. Thereafter, a luminol solution (Roche, BM Chemiluminescence Blotting Substrate), which is a substrate for primary photometry, was added. By adding luminol, primary light emission occurs. The emission brightness for one minute was photographed with a CCD camera and used as a primary photometric image.

  Next, after removing the luminol solution from the substrate, the washing operation was repeated three times, and an anti-IgE antibody (BECKMAN COULTER, Cell Lab Mouse Anti-Human IgE) was added as a secondary antibody. After the addition, the reaction was carried out while shaking on a shaker at 25 ° C. for 1 hour. The secondary antibody used was biotinylated.

  Next, the washing operation was repeated three times, and then luciferase (Kikkoman Corporation, Intelligent AB) was added. After the addition, the reaction was carried out while shaking on a shaker at 25 ° C. for 1 hour. The luciferase used was streptavidinated.

  Next, in order to remove unreacted substances, the washing operation was repeated three times. Thereafter, buffer exchange was performed once with a solution having the same composition as that of the substrate solution for secondary photometry, except that only the substrate was removed.

  Thereafter, the substrate was fixed on the stage for photographing, and then a luciferin solution (Kikkoman Corp., Intelligent AB) was added as a substrate solution for secondary photometry. Secondary luminescence is generated by adding luciferin. The light emission luminance for 5 minutes was photographed with a CCD camera to obtain a secondary photometric image.

  FIG. 10 is a primary photometric image and a secondary photometric image of a model experiment using HRP and IgE described above. FIG. 10A shows a primary photometric image 1001 using HRP and IgE for a model experiment, and FIG. 10B shows a secondary photometric image 1002 using HRP and IgE for a model experiment. Here, the row of sample 1003 is 0.3 fmol of IgE, the row of sample 1004 is 0.1 fmol of IgE, the row of sample 1005 is 0.03 fmol of IgE, the row of sample 1006 is 0.01 fmol of IgE, and the sample 1007 The row is coated with 0.003 fmol of IgE, and the row of sample 1008 is coated with 0.001 fmol of IgE.

  First, from the primary photometric image 1001, the confirmation mark 1009, the substrate position determination mark 1010, and the center coordinates and spot size of each spot were obtained. Next, a confirmation mark 1009 positioned next to the substrate position determination mark 1010 and a confirmation mark 1011 positioned diagonally are connected by a straight line to obtain a middle point coordinate.

  Next, the center coordinates of the confirmation mark 1012 and the substrate position determination mark 1013 of the secondary photometric image 1002 were obtained. Next, a check mark 1012 positioned next to the substrate position determination mark 1013 and a check mark 1014 positioned diagonally are connected by a straight line to obtain a middle point coordinate. Thereafter, the central coordinate data of the secondary photometric image 1002 was moved to a position that matches the central coordinate data of the primary photometric image 1001. Next, the midpoint position was fixed, and the secondary photometric image 1002 was rotated around the midpoint position until the position of the confirmation mark and the confirmation mark for determining the substrate position coincided. When 1001 and 1002 completely matched, the spot analysis position of the secondary photometry image 1002 was determined based on the spot analysis data of the primary photometry image 1001.

  The measurement result obtained from the analysis position thus obtained (hereinafter referred to as the present measurement method result) is obtained by visually identifying each spot position using only the secondary photometric image and determining its luminance value. In this technique (hereinafter referred to as a manual method result) and the technique described in the background art, only the secondary photometric image is used and each spot is regarded as being in a fixed position with reference to the confirmation mark. The luminance information of the position was compared with the result obtained as the luminance value of each spot (hereinafter referred to as the conventional method result).

FIG. 11A is a diagram showing measurement results of a model experiment using HRP and IgE described above. Among these, diamonds indicate the results of this measurement method, squares indicate the results of the manual method, and triangles indicate the results of the conventional method. The horizontal axis represents the concentration of immobilized IgE, and the vertical axis represents the light emission amount obtained by integrating the luminance values of the spots. As shown in FIG. 11 (a), the measurement method result was able to obtain characteristics closer to the manual method result than the conventional method result. When this was compared with the determination coefficient R ² of the regression method, the result of this measurement method was R ² = 0.9915, the result of the conventional method was R ² = 0.9784, and the result of the manual method was R ² = 0.9927. This also shows that the microarray measurement method of the present invention is more useful than the conventional method.

Further, FIG. 11B is a diagram showing the measurement results of the model experiment using ALP instead of the above-described HRP. The model experiment using IgE antibody and ALP was carried out in the same procedure as described above, except that HRP was replaced with ALP, and luminol used in the substrate reaction for primary photometry was replaced with Lumigen PPD (Wako Pure Chemical Industries, Ltd.). , Lumi-PhosPlus). In FIG. 11B, the horizontal axis and the vertical axis are the same as those in FIG. 11A, and the rhombus indicates the result of this measurement method and the square indicates the result of the manual method. At this time, when confirmed by the determination coefficient R ² of the regression method, the result of this measurement method was R ² = 0.9779, and the result of the manual method was R ² = 0.9688.

  From this, it can be seen that even if ALP is used, the same result as HRP can be obtained. Even if an enzyme other than HRP and ALP is used, it can be immobilized on the substrate without losing enzyme activity. It is inferred that similar results can be obtained.

Furthermore, FIG. 12 shows the measurement results in the case where BSA was added and only HRP was added without adding BSA in the model experiment described above. The horizontal axis and the vertical axis are the same as those in FIG. 11A. The rhombus is the one to which BSA is added, and the square is the one to which BSA is not added. Here, the square measurement results are the same as the measurement method results shown in FIG. The rhombus is a result of manual method in which the spot position is confirmed visually. At this time, since the determination coefficient R ^{2 of the} rhombus was 0.9823, it was proved that a good result was obtained even when HRP was used instead of BSA.

  Thus, conventionally, in the low concentration region of IgE, BSA was necessary to prevent the adsorption of IgE on the surface of the sample tube when adjusting the coating solution. However, HRP is used instead of BSA as in the present invention. It was also found that the adsorption of IgE in the low concentration range could be prevented by adding. It can be inferred that the same effect can be obtained with a polymer in which amino acids having a hydrophobic group such as ALP are bound.

  FIGS. 13A and 13B show images of the model experiment described above. FIG. 13A shows a primary photometric image, and FIG. 13B shows a secondary photometric image. Here, a spot 1301, a spot 1302, and a spot 1303 represent spots shifted in the vertical direction (vertical direction in the drawing). FIG. 14 shows the relative ratio of the light emission amounts of these spots.

  FIG. 14 shows the ratio of the light emission amount of the result of the present measurement method and the light emission amount of the result of the conventional method with respect to the light emission amount of the manual method result as a reference of 100%. In FIG. 14, the horizontal line bar graph shows the result of this measurement method, and the shaded bar graph shows the result of the conventional method. As shown in FIG. 14, the measurement method results show that the emission amount within ± 10% of the manual method results can be measured with respect to the shifted spot, and the luminance value within an appropriate range with respect to the shifted spot. It can be seen that is measured.

  According to the microarray measurement method of the present invention, even if a positional shift occurs on a microarray substrate in units of spots, the measurement can be performed by specifying the position of the spot. It is useful as a measurement method in the above.

Flowchart showing the steps of the microarray measurement method in the first embodiment The figure which shows the example of the image which pinpoints a spot position Diagram showing the outline of microarray substrate creation The figure which shows the example of application error judgment The figure which shows the example of arrangement | positioning of a confirmation mark and a board | substrate position determination mark Flowchart showing steps of the microarray measurement method in the second embodiment Diagram showing the procedure for correcting misalignment The figure which shows the procedure of another form which correct | amends position shift The flowchart which shows the process of another form of the microarray measuring method in Embodiment 2. The figure which shows the primary photometry and secondary photometry image obtained by the model experiment Diagram showing measurement results of model experiment The figure which shows the measurement result which compares the presence or absence of BSA Diagram showing images obtained from model experiments Diagram showing comparison of measurement accuracy

Explanation of symbols

S101 First substrate addition step S102 Primary photometry step S103 First image analysis step S104 Specimen addition step S105 Second substrate addition step S106 Secondary photometry step S107 Second image analysis step S201, S301 First fixed error Detection step S202, S302 Second fixed error detection step S203, S303 Image shift correction step 201 Spot 501 Confirmation mark 502 Substrate position determination mark

Claims (8)

In the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate,
A first substrate addition step of adding a first substrate that emits light upon chemical reaction with a first reagent immobilized in advance on the spot onto the microarray substrate;
A primary photometric step of photographing the spot light to generate a primary photometric image;
A first image analysis step of generating a primary photometric analysis image specifying the position of the spot from the primary photometric image;
After removing the first substrate, the specimen and the second reagent are added to the microarray substrate to cause a binding reaction, and the specimen and the second reagent that have not been bound to the antibody immobilized in advance on the spot. A sample addition step to be removed;
A second substrate addition step of adding a second substrate that emits light upon chemical reaction with the second reagent onto the microarray substrate;
A secondary photometry step of photographing the spot light to generate a secondary photometry image;
A microarray measurement method comprising: a second image analysis step of specifying a position of the spot in the secondary photometric image using the primary photometric analysis image and measuring a light emission amount of the spot. In the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate,
A first substrate addition step of adding on the microarray substrate a first substrate that chemically reacts with a first reagent previously immobilized on the spot;
A primary photometric step of irradiating the spot with excitation light and photographing a fluorescence from the spot to generate a primary photometric image;
A first image analysis step of generating a primary photometric analysis image specifying the position of the spot from the primary photometric image;
After removing the first substrate, the specimen and the second reagent are added to the microarray substrate to cause a binding reaction, and the specimen and the second reagent that have not been bound to the antibody immobilized in advance on the spot. A sample addition step to be removed;
Adding a second substrate that chemically reacts with the second reagent onto the microarray substrate;
A secondary photometric step of irradiating the spot with excitation light and photographing the fluorescence from the spot to generate a secondary photometric image;
A microarray measurement method comprising: a second image analysis step of specifying a position of the spot in the secondary photometric image using the primary photometric analysis image and measuring a light emission amount of the spot. On the microarray substrate, a substrate position determination mark for detecting a fixed position error of the microarray substrate is arranged,
A first fixed error detection step of detecting a fixed position error of the microarray substrate using the substrate position determination mark between the primary photometry step and the first image analysis step;
2. A second fixed error detection step for detecting a fixed position error of the microarray substrate using the substrate position determination mark between the secondary photometry step and the second image analysis step. Or the microarray measuring method of 2.
Said On the microarray substrate, a confirmation mark for detecting a displacement of the microarray substrate is further arranged.
Between the primary photometric analysis image and the secondary photometric analysis image using the substrate position determination mark and the confirmation mark between the second fixed error detection step and the second image analysis step. The microarray measurement method according to claim 3, further comprising an image misalignment correction step for correcting a misalignment that occurs relative to the image. In the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate,
A first fixed error detection step of detecting a fixed position error of the microarray substrate using an image obtained by photographing light transmitted through a substrate position determination mark provided on the microarray substrate;
A first substrate addition step of adding a first substrate that emits light upon chemical reaction with a first reagent immobilized in advance on the spot onto the microarray substrate;
A primary photometric step of photographing the spot light to generate a primary photometric image;
A first image analysis step of generating a primary photometric analysis image specifying the position of the spot from the primary photometric image;
After removing the first substrate, the specimen and the second reagent are added to the microarray substrate to cause a binding reaction, and the specimen and the second reagent that have not been bound to the antibody immobilized in advance on the spot. A sample addition step to be removed;
A second fixed error detection step of detecting a fixed position error of the microarray substrate using an image obtained by photographing light transmitted through the substrate position determination mark;
A second substrate addition step of adding a second substrate that emits light upon chemical reaction with the second reagent onto the microarray substrate;
A secondary photometry step of photographing the spot light to generate a secondary photometry image;
An image misalignment correcting step of correcting a misalignment that occurs relatively between the primary photometric analysis image and the secondary photometric image using the confirmation mark provided on the microarray substrate and the substrate position determination mark;
A microarray measurement method comprising: a second image analysis step of specifying a position of the spot in the secondary photometric image using the primary photometric analysis image and measuring a light emission amount of the spot. In the microarray measurement method for measuring the light emission amount of a plurality of spots provided on the microarray substrate,
A first fixed error detection step of detecting a fixed position error of the microarray substrate using an image obtained by photographing light transmitted through a substrate position determination mark provided on the microarray substrate;
A first substrate addition step of adding on the microarray substrate a first substrate that chemically reacts with a first reagent previously immobilized on the spot;
A primary photometric step of irradiating the spot with excitation light and photographing a fluorescence from the spot to generate a primary photometric image;
A first image analysis step of generating a primary photometric analysis image specifying the position of the spot from the primary photometric image;
After removing the first substrate, the specimen and the second reagent are added to the microarray substrate to cause a binding reaction, and the specimen and the second reagent that have not been bound to the antibody immobilized in advance on the spot. A sample addition step to be removed;
A second fixed error detection step of detecting a fixed position error of the microarray substrate using an image obtained by photographing light transmitted through the substrate position determination mark;
Adding a second substrate that chemically reacts with the second reagent onto the microarray substrate;
A secondary photometric step of irradiating the spot with excitation light and photographing the fluorescence from the spot to generate a secondary photometric image;
An image misalignment correcting step of correcting a misalignment that occurs relatively between the primary photometric analysis image and the secondary photometric image using the confirmation mark provided on the microarray substrate and the substrate position determination mark;
A microarray measurement method comprising: a second image analysis step of specifying a position of the spot in the secondary photometric image using the primary photometric analysis image and measuring a light emission amount of the spot. The image misalignment correcting step includes:
Connect the center coordinates of the confirmation marks located diagonally with a straight line,
Calculate the midpoint position on the straight line,
Moving the secondary photometric image so that the midpoint position of the confirmation mark in the secondary photometric image matches the midpoint position of the confirmation mark in the primary photometric analysis image;
Based on the position where the midpoints match,
The shift is corrected by rotating the secondary photometric image until the central coordinate of the confirmation mark in the secondary photometric image matches the central coordinate of the confirmation mark in the primary photometric analysis image. Item 7. The microarray measurement method according to any one of Items 4 to 6. The image misalignment correcting step includes:
The origin coordinates are the center coordinates of the confirmation mark adjacent to the substrate position determination mark,
Moving the secondary photometric image so that the origin coordinate obtained from the secondary photometric image matches the origin coordinate obtained from the primary photometric analysis image;
Positioned diagonally to the origin coordinate in the secondary photometry image with respect to the center coordinate of the confirmation mark located diagonally to the origin coordinate in the primary photometry analysis image with reference to the position where the origin coordinate coincides The microarray measurement method according to any one of claims 4 to 6, wherein the shift is corrected by rotating the secondary photometric image until the center coordinate position of the confirmation mark to be matched coincides.