JP2021061757A - Inspection method of analysis chip, inspection device and production method of analysis chip - Google Patents

Abstract

To provide an inspection method of an analysis chip capable of determining surely, quality of spot liquid applied to each spot part of a chip, an inspection device and a production method of an analysis chip.SOLUTION: An inspection method of an analysis chip of the invention is a method for inspecting an analysis chip obtained by applying in array, a mixed reagent of a compound including a selection binding substance and a deliquescent substance to a region which requires the application of the reagent on a carrier surface, and stabilizing the selection binding substance, and the method comprises: a deliquescing step for supplying to a stabilized region of the selection binding substance on the carrier surface, air having a vapor pressure higher than a balanced vapor pressure of a saturated solution of the deliquescent substance on a temperature of the carrier surface, for deliquescing the deliquescent substance; an image acquiring step for acquiring an image including the stabilized region on which the deliquescent substance is deliquesced by image acquiring means for acquiring an image; and a determining step for determining whether or not, the mixed reagent is present on the region which requires the application of the reagent, on the basis of the image acquired by the image acquiring means.SELECTED DRAWING: Figure 4

Description

The present invention relates to an analysis chip inspection method, an inspection device, and an analysis chip manufacturing method.

Research on the analysis of genetic information of various organisms has begun. Information on a large number of genes including human genes, their base sequences, proteins encoded by the gene sequences, and sugar chains secondarily produced from these proteins is being rapidly clarified. The functions of macromolecules such as genes, proteins, and sugar chains whose sequences have been clarified can be investigated by various methods. Mainly, nucleic acids can be investigated for the relationship between various genes and their biological function expression by utilizing the complementarity between various nucleic acids / nucleic acids such as Northern blotting or Southern blotting. For proteins, the function and expression of proteins can be investigated by utilizing the reaction between proteins / proteins represented by Western blotting.

As a method for analyzing a large number of gene expressions at once, there is a DNA microarray method (analysis chip method). This method is basically the same as the above-mentioned conventional method in that it is a nucleic acid detection / quantification method based on a nucleic acid / nucleic acid hybridization reaction. This analytical chip method can be applied to the detection and quantification of proteins and sugar chains based on specific reactions between proteins / proteins, sugar chains / sugar chains, and sugar chains / proteins. In this technique, a large number of DNA fragments, proteins, and sugar chains are aligned and fixed at high density on a flat glass substrate or a resin substrate on which an uneven pattern is formed. As a specific usage of the analysis chip method, for example, a sample in which an expression gene of a cell to be studied is labeled with a fluorescent dye or the like is hybridized on a convex portion formed on a substrate, and nucleic acids (DNA) complementary to each other are used. Alternatively, a typical method is to bind RNA) to each other and read the site at high speed with a high-resolution fluorescence detector (scanner). There is also a method of detecting a response such as a current value based on an electrochemical reaction. In this way, the amount of each gene in the sample can be estimated quickly. Further, the field of application of the analysis chip is used not only for gene expression analysis for estimating the amount of expressed gene, but also as a means for detecting single nucleotide polymorphism (SNP) of a gene.

A typical method for producing an analysis chip is a spotting method. Typically, a solution (spot solution) containing pre-synthesized DNA is applied (spotted) to the tip surface of a convex portion on a substrate with a metal pin (needle) such as stainless steel, or an inkjet. There is a method of applying to the tip surface of the convex part by the method. Hundreds to tens of thousands of types of DNA are usually applied to the substrate on the convex portions formed in the form of a matrix array. When a metal pin is used, the pin tip does not come into contact with the substrate, and in the case of the inkjet method, the nozzle is blocked by dust clogging or the like, so that spot defects (spot omission) may occur. At the manufacturing site, it is necessary not to overlook the analysis chip in which the spot defect has occurred, to appropriately detect the defect without over-detection, and to eliminate the analysis chip having the spot defect to ensure the quality. Patent Documents 1 to 3 disclose a quality inspection method for an analysis chip.

JP-A-2007-717777 Japanese Unexamined Patent Publication No. 2005-10138 Japanese Unexamined Patent Publication No. 2003-130875

In Patent Documents 1 to 3, a DNA solution applied to a place other than the actual spot portion is irradiated with ultraviolet rays and observed to confirm the presence or absence of an abnormality in the inkjet nozzle, or a fluorescent molecule or dye is added to the spot solution. Is disclosed. However, Patent Documents 1 to 3 have the following problems (1) and (2).
(1) It is an indirect test and lacks certainty because the actual spot part is not observed. (2) When fluorescent molecules or dyes are added to the spot solution, the fluorescence emitted by them is detected at the time of detection after hybridization. Being noisy

Further, in addition to Patent Documents 1 to 3, it is also known that a crystal of a precipitated inorganic salt component or the like is imaged in a state where the spot solution is dried, and the image is processed to inspect the quality. However, since the direction and size of salt crystal growth change depending on the delicate conditions of temperature and humidity when the spot liquid dries, it may be difficult to observe the salt depending on the state of the crystals. In addition, when the spot liquid is applied to the upper surface of the convex portion, crystals are concentrated on the edge of the upper surface of the convex portion, and although the selective binding substance is actually immobilized on the upper surface of the convex portion, it is erroneously regarded as defective. In some cases, it was judged.
Therefore, it is required to reliably inspect the spot defect (spot omission) without generating noise when detecting the selectively binding substance represented by DNA on the substrate.

The present invention has been made in view of the above problems, and is an inspection method for an analysis chip, an inspection device, and a method for manufacturing an analysis chip, which can reliably determine the quality of the spot liquid applied to each spot portion of the chip. The purpose is to provide.

In order to solve the above problems, in the method for inspecting an analytical chip according to the present invention, a mixed reagent of a compound containing a selective binding substance and a deliquescent substance is applied in an array to a required region on the surface of a carrier, and the selective binding substance is applied. This is an inspection method for an analytical chip in which the substance is immobilized, and air having a water vapor pressure higher than the equilibrium water vapor pressure of a saturated aqueous solution of a deliquescent substance at the surface temperature of the carrier is applied to the immobilized region of the selective binding substance on the surface of the carrier. A deliquescent step of supplying and deliquescent the deliquescent substance, an image acquisition step of obtaining an image including the immobilized region deliquescented by the deliquescent substance by an image acquisition means for acquiring an image, and an image acquisition means acquired by the image acquisition means. It is characterized by including a determination step of determining whether or not the mixed reagent is present in the required region from the image.

Further, in the method for inspecting an analysis chip according to the present invention, in the above invention, the determination step is obtained by binarizing the image based on a first threshold value set for brightness, and from the binarized image. , The number of pixels of one value is counted as the first count value, the first count value is compared with the second threshold value set for the first count value, and the mixed reagent is compared. Is determined to be present in the required region.

Further, in the method for inspecting an analysis chip according to the present invention, in the above invention, the determination step binarizes the image based on a third threshold value smaller than the first threshold value, and determines the fixation region. The number of pixels of one of the values in between is counted as the second count value, the second count value is compared with the fourth threshold value set for the second count value, and the mixture is described. It is characterized in that it is determined whether or not the reagent is present between the immobilized regions.

Further, the method for inspecting an analysis chip according to the present invention is characterized in that, in the above invention, the deliquescent substance is a salt.

Further, the method for inspecting an analysis chip according to the present invention is characterized in that, in the above invention, the selective binding substance is DNA, RNA or protein.

Further, the inspection device for the analysis chip according to the present invention applies a mixed reagent of a compound containing a selective binding substance and a deliquescent substance to a required region on the surface of a carrier in an array, and analyzes the selective binding substance immobilized. In the chip inspection device, air having a water vapor pressure higher than the equilibrium water vapor pressure of the saturated aqueous solution of the deliquescent substance at the surface temperature of the carrier is supplied to the immobilized region of the selective binding substance on the surface of the carrier. The humidifying unit that humidifies the surface of the carrier, the image acquisition unit that obtains an image of the surface of the carrier to which the humidified air is supplied including the immobilized region, and the image acquisition means that acquire the image are used. It is characterized by comprising an analysis unit for determining whether or not the mixed reagent is present in the required region from the obtained image.

Further, the method for producing an analysis chip according to the present invention includes a coating step of applying a mixed reagent of a compound containing a selective binding substance and a deliquescent substance to a required region on the surface of a carrier in an array, and the selection in the mixed reagent. The inspection step comprises a fixation step of fixing the binding substance to a required region of the carrier surface and an inspection step of determining whether or not the mixed reagent is present in the required region, and the inspection step is performed on the carrier surface. To the immobilized region of the selective binding substance, air with a water vapor pressure higher than the equilibrium water vapor pressure of the saturated aqueous solution of the deliquescent substance at the surface temperature of the carrier is supplied, and the deliquescent step of deliquescent the deliquescent substance and an image are shown. An image acquisition step of obtaining an image including the immobilized region in which the deliquescent substance is deliquescented by the image acquisition means to be acquired, and whether or not the mixed reagent is present in the required region from the image acquired by the image acquisition means. It is characterized by including a determination step for determining whether or not.

According to the present invention, in the spot inspection of a selective binding substance typified by DNA on a substrate at the time of manufacture, the spot defect is reliably inspected without over-detection or oversight without causing noise during use. It has the effect of being able to.

FIG. 1 is a plan view schematically showing an analysis chip according to an embodiment of the present invention. FIG. 2 is a cross-sectional view taken along the line AA of the analysis chip shown in FIG. FIG. 3 is a flowchart illustrating a method for manufacturing an analysis chip according to an embodiment of the present invention. FIG. 4 is a diagram showing a configuration of a spot inspection device for an analysis chip according to an embodiment of the present invention. FIG. 5 is a flowchart illustrating a spot inspection in the method for manufacturing an analysis chip according to an embodiment of the present invention. FIG. 6 is a diagram showing an example of an image of a normally applied spot liquid obtained by binarizing an image. FIG. 7 is a diagram showing an example of an image of the spot liquid when the coating is removed, which is obtained by binarizing the image. FIG. 8 is a diagram showing an example of an image of the spot liquid when the coating is leaked, which is obtained by binarizing the image. FIG. 9 is a diagram showing an image obtained by cutting out a part of the convex portion group (A) of the acquired substrate image. FIG. 10 is a diagram showing a binarized image of the convex portion group (A) shown in FIG. FIG. 11 is a graph showing the number of pixels (the number of white pixels) of the white portion in each spot of the binarized image shown in FIG. FIG. 12 is a graph showing the number of white pixels in the good spot to which the spot liquid was normally applied and the defective spot to which the spot liquid was not properly applied in the binarized image shown in FIG. FIG. 13 is a diagram showing a binarized image of the convex portion group (A) in the second embodiment. FIG. 14 is a diagram illustrating a configuration of a spot inspection device for an analysis chip according to a comparative example. FIG. 15 is a diagram showing a substrate image of the convex portion group (A) in Comparative Example 1. FIG. 16 is a diagram showing a binarized image of the convex portion group (A) in Comparative Example 1. FIG. 17 is a graph showing the number of white pixels at each spot of the binarized image shown in FIG. FIG. 18 is a graph showing the number of white pixels in the good spot to which the spot liquid was normally applied and the defective spot to which the spot liquid was not properly applied in the binarized image shown in FIG. FIG. 19 is a diagram showing a substrate image of the convex portion group (B) in Comparative Example 2. FIG. 20 is a diagram showing a binarized image of the convex portion group (C) in Example 3. FIG. 21 is a diagram showing a binarized image of the convex portion group (C) in Comparative Example 3.

Hereinafter, embodiments for carrying out the present invention will be described in detail together with drawings. The present invention is not limited to the following embodiments. In addition, each of the figures referred to in the following description merely schematically shows the shape, size, and positional relationship to the extent that the content of the present invention can be understood. That is, the present invention is not limited to the shape, size, and positional relationship exemplified in each figure. Further, in the description of the drawings, the same parts are designated by the same reference numerals.

In the analysis chip targeted by the present invention, a reagent (hereinafter referred to as a spot solution) containing a selective binding substance such as DNA, protein, or sugar chain and a deliquescent substance is applied to the surface of a carrier typified by glass or plastic. It is fixed.

The analysis chip according to the present invention is used for dropping a sample onto the reaction part of the analysis chip and measuring the presence / absence, amount, properties, etc. of the test substance. Specific examples thereof include a biochip that measures the presence or absence and amount of a test substance by the reaction between the selectively binding substance immobilized on the surface of the carrier and the test substance. More specifically, an analysis chip in which a nucleic acid is immobilized on a carrier surface, a protein chip in which a protein typified by an antibody is immobilized on a carrier surface, a sugar chain chip in which a sugar chain is immobilized on a carrier surface, and a cell as a carrier. Examples thereof include cell chips immobilized on the surface.

(Embodiment)
The analysis chip according to the embodiment of the present invention will be described with reference to FIGS. 1 and 2. FIG. 1 is a plan view schematically showing an analysis chip according to an embodiment of the present invention. FIG. 2 is a cross-sectional view taken along the line AA of the analysis chip shown in FIG. The analysis chip 1 shown in FIGS. 1 and 2 includes a substrate 10 having a plurality of uneven portions 11. The substrate 10 corresponds to the carrier described above. The substrate 10 is a flat plate having a rectangular outer edge on the main surface. Here, the main surface means the surface having the largest area among the six surfaces constituting the flat plate. As the material of the substrate 10, a general slide glass or a plastic smooth plate having the same size is preferably used. Further, it is preferable that the surface of the substrate 10 is a color or material that absorbs light, such as black, in order to suppress reflection of light from the substrate 10 during observation (imaging).

A plurality of (four in the example of FIG. 1) uneven portions 11 are formed on one main surface of the substrate 10. A plurality of (32 in the example of FIG. 1) convex portions 12 are formed on the concave-convex portion 11. A plurality of (100 in the example of FIG. 1) convex portions 13 are formed in the convex portion group 12 so as to project convexly from the bottom surface of the concave-convex portion 11. Here, the convex portion 13 is a place (or region) where the test substance and the selective binding substance specifically bind to each other. The upper surface of the convex portion 13 is flat, and a spot liquid (mixing reagent) containing a selective binding substance is applied to the upper surface. The selective binding substance is fixed to the upper surface of the convex portion 13 in the spot liquid. Hereinafter, the upper surface of the convex portion 13 coated with the spot liquid may be referred to as a spot. Further, applying the spot liquid to the upper surface of the convex portion 13 may be referred to as spotting. The upper surface of the convex portion 13 corresponds to an immobilization region of the selective binding substance in the spot liquid, and is a region necessary for the spot liquid to be applied.

The selective binding substance in the present invention means various substances that can be selectively bound to the test substance directly or indirectly. Typical examples of selectively binding substances that can bind to a test substance include nucleic acids, proteins, peptides, sugars, and lipids.

Among the selective binding substances, the nucleic acid includes DNA and RNA, and may be PNA or LNA. As the DNA, chromosomal DNA, viral DNA, DNA such as bacteria and mold, cDNA reverse transcribed from RNA, fragments which are a part thereof, and the like can be used, but the DNA is not limited thereto. Further, as RNA, messenger RNA, ribosomal RNA, small RNA, microRNA, fragments which are a part thereof and the like can be used, but the RNA is not limited thereto. Nucleic acid also includes chemically synthesized DNA or RNA. Since a single-stranded nucleic acid having a specific base sequence selectively hybridizes and binds to a single-stranded nucleic acid having a base sequence complementary to the base sequence or a part thereof, the selective binding property referred to in the present invention. Corresponds to the substance. The nucleic acid may be derived from a natural product such as a living cell, or may be synthesized by a nucleic acid synthesizer. Preparation of DNA or RNA from living cells is performed by a known method, for example, for extraction of DNA by the method of Blin et al. (Blin et al., Nucleic Acids Res. 3: 2303 (1976)), and of RNA. Extraction can be carried out by the method of Fabalolo et al. (Favalolo et al., Methods Enzymol. 65: 718 (1980)) and the like. Nucleic acids to be immobilized include chain or cyclic plasmid DNA or chromosomal DNA, DNA fragments obtained by cleaving these with restriction enzymes or chemically, DNA synthesized with enzymes in vitro, or chemically synthesized oligonucleotides. Etc. can also be used.

Examples of the protein include an antibody and an antigen-binding fragment of an antibody such as a Fab fragment and an F (ab') 2 fragment, and various antigens. An antibody or an antigen-binding fragment thereof selectively binds to a corresponding antigen, and the antigen selectively binds to a corresponding antibody, and thus falls under the category of "selective binding substance".

Examples of sugars include sugar chains such as various monosaccharides, oligosaccharides, and polysaccharides.
The lipid may be a complex lipid as well as a simple lipid.
Furthermore, substances having antigenicity other than the above nucleic acids, proteins, sugars and lipids can be immobilized. Further, as a selective binding substance, cells may be immobilized on the surface of the carrier.
Particularly preferred of these selective binding substances include DNA, RNA, proteins, peptides, sugars, sugar chains, and lipids.

The spot solution contains a deliquescent substance in addition to the selective binding substance. There are many deliquescent substances, such as lithium chloride, sodium chloride, potassium chloride, magnesium chloride, magnesium bromide, magnesium perchlorate, magnesium nitrate, magnesium nitrite, magnesium acetate, magnesium sulfate, urea, 1- Examples thereof include ethyl-3- (3-dimethylaminopropyl) carbodiimide and phosphoric acid. Of these, sodium chloride, potassium chloride, magnesium chloride, and urea do not adversely affect selective binding substances and are less toxic, and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) has a carboxyl group and amino. Since it is a group condensing agent, it can be preferably used.

A solution obtained by mixing EDC and sodium chloride as a spot solution can be preferably used. The concentration of EDC is 10 mM to 200 mM, preferably 50 to 100 mM. The preferred range of sodium chloride solution is 40-120 mM.
The spot solution may also contain other salts for buffer adjustment. As an example, a phosphate buffer solution, a citrate buffer solution, a citrate phosphate buffer solution, a boric acid buffer solution, and a tartaric acid buffer solution can be used.

The number of the convex portion group 12 can be set to an arbitrary number such as 2, 4, 8 or the like. Further, the plurality of convex portions 13 provided in the convex portion group 12 are arranged in a matrix. When applying the spot solution to the convex portion 13, a sample is placed in a microtiter plate or the like, and the sample is dispensed into each convex portion 13 using, for example, a multi-pipette such as 4 stations, 6 stations, 8 stations, or 12 stations. In this case, the number of protrusions 13 is preferably a multiple of the number of multipipettes, for example, a multiple of 4, a multiple of 6, a multiple of 8, and a multiple of 12.
Further, a plurality of uneven portions 11 are arranged according to the size of the substrate 10.

When the analysis chip 1 is used, the entire substrate 10 or the uneven portion 11 is sealed with a cover or the like (not shown). The cover used at this time may be any material such as glass, various polymers (for example, polystyrene, polymethylmethacrylate, polycarbonate, polyolefin), and silicone.

Subsequently, an example of how to use the analysis chip 1 will be described. First, the gene is extracted from the sample collected from the sample. Then, the fluorescent label is bound to the extracted gene (test substance). The sample to which the fluorescent label is bound is reacted with the spot solution (selective binding substance) on the DNA chip. In the reaction treatment, for example, the test substance in the sample is reacted with the selective binding substance immobilized on the tip surface of the convex portion 13 by stirring the substance at 32 ° C. for several hours. In the stirring process, the sample is stirred by moving the analysis chip 1 by rotation, vibration, or a combination thereof, or by using beads for stirring enclosed in a cover covering the analysis chip 1. ..

The stirring device for stirring the analysis chip 1 is not particularly limited as long as it can give a centrifugal acceleration of 1 × g or more by the combination of the rotation speed of the horizontal circular motion and the radius of gyration. In the commercially available product, a plate shaker can be preferably used, for example, "BioShake 5000 elm", "BioShake 3000-Telm", "BioShake 3000 elm" (all manufactured by Q Instruments), "monoshake", "teleshake". , "Teleshake 1536" (above, manufactured by Thermo Scientific), "MS3 Basic", "MS3 Digital", "VXR basic Vibrax" (registered trademark), "VORTEX 3" (above, manufactured by IKA), "microplate" Examples include "Shaker N-704" (manufactured by Nisshin Rika), "Plate Shaker KM-M01" (manufactured by Kajix), and "Plate Mixer P-10" (manufactured by Juji Field).

As the solution (sample) containing the test substance used in the present invention, body fluids such as blood, serum, plasma, urine, stool, cerebrospinal fluid, saliva, and various tissue fluids, various foods and drinks, and dilutions thereof should be used. However, it is not limited to these.

The test substance used in the present invention includes nucleic acids (target nucleic acids) to be measured, for example, genes such as pathogens and viruses, genes causing genetic diseases, and a part thereof, various biological components having antigenicity, pathogens, and the like. Antibodies against viruses and the like can be mentioned, but the present invention is not limited to these. For example, when the test substance is a nucleic acid, it is hybridization, and when it is a protein, it is an antigen-antibody reaction.

The sample used in the present invention is preferably a solution in which the presence / absence, amount, properties, etc. of the test substance can be confirmed. Specific examples thereof include, but are not limited to, solutions containing nucleic acids, antibodies, sugar chains and the like collected, extracted and purified from blood, tissues, cells and the like.

The nucleic acid to be the test substance may be a nucleic acid extracted from blood or cells labeled with a fluorescent substance or the like, or may be amplified by a nucleic acid amplification method such as PCR using the nucleic acid as a template. When the nucleic acid amplification product is used as a test substance, the amplified nucleic acid can be labeled by performing amplification in the presence of nucleoside triphosphate labeled with a fluorescent substance or the like. When the test substance is an antigen or antibody, the test substance antigen or antibody may be directly labeled by a conventional method, or the test substance antigen or antibody may be bound to a selective binding substance. After that, the carrier can be washed, and the labeled antibody or antigen that reacts with the antigen or antibody can be reacted to measure the label bound to the carrier. When an unamplified nucleic acid is used as a test substance, for example, the phosphate group at the 5'end of the nucleic acid is removed by alkaline phosphatase, and the test substance labeled with a fluorescent substance is reacted with the selective binding substance. A method of measuring a bound label, or a method of capturing a test substance with a selective binding substance (capture probe) and then binding a detection probe labeled with a fluorescent substance or the like to the test substance to measure the label of the detection probe. (Sandwich hybridization method) is preferably used.

After the reaction treatment, the analysis chip 1 is subjected to a washing treatment to remove the test substance that has not reacted with the selective binding substance from the analysis chip 1. After the cleaning treatment, the analysis chip 1 is centrifugally dried using a general centrifuge dedicated to chips and slide glasses.

The analysis chip 1 that has been washed and dried reads an image using a high-resolution fluorescence detection device or the like, and quantifies the signal intensity (fluorescence intensity). Preferably used high-resolution fluorescence detectors include, but are not limited to, 3D-Gene® (registered trademark) Scanner (Toray), SureScan microarray scanner (Agilent Technologies), GenePix (Filgen), and the like. ..

Subsequently, a method for manufacturing the analysis chip 1 will be described with reference to FIGS. 3 to 8. FIG. 3 is a flowchart illustrating a method for manufacturing an analysis chip according to an embodiment of the present invention. ..

In manufacturing the analysis chip 1, first, the substrate 10 is prepared, and then the surface treatment of the substrate 10 is performed (step S101). In the surface treatment, a functional group is generated on the surface of the substrate so that the selectively binding substance can be immobilized on the substrate 10. For example, a carboxyl group is generated on the surface of the substrate by hydrolysis treatment.

After the surface treatment, the spot liquid is applied to each convex portion 13 (spot) (step S102). For example, the spot liquid is applied to the upper surface of the convex portion using a plurality of pins. Specifically, the spot liquid is adhered to the tip of the pin, and the adhered spot liquid is brought into contact with the upper surface of the convex portion 13 to be applied.

After applying the spot liquid to each convex portion 13, a cross-linking reaction treatment is carried out (step S103). By the cross-linking reaction, the functional group generated on the upper surface of the convex portion 13 and the selective binding substance are crosslinked, and the selective binding substance is fixed to the spot.

After the cross-linking reaction treatment, an inspection (spot inspection) is performed on the coating state of the spot liquid at each spot (step S104). The spot inspection will be described later.

After the spot inspection, the analysis chip 1 determined to be appropriately coated with the spot liquid on the upper surface of the convex portion 13 in the spot inspection is cleaned and then assembled (step S105). In the assembling process, a cover is adhered to the analysis chip 1 as needed, and beads are filled in the cover.

After the assembly process, the shipping process is performed (step S106). In the shipping process, product packaging and product inspection are carried out. After the shipping process, the analysis chip 1 is shipped.

Here, the spot inspection (step S104) in the above-mentioned manufacturing method will be described. FIG. 4 is a diagram showing a configuration of a spot inspection device for an analysis chip according to an embodiment of the present invention.

The spot inspection device 100 shown in FIG. 4 includes an image acquisition unit 110 that acquires an image of the substrate 10 including the convex portion 13, an analysis unit 120 that analyzes the image acquired by the image acquisition unit 110, and an analysis unit 120 that analyzes the image acquired by the image acquisition unit 110. A humidifying unit 130 that supplies humidified air to the chip 1 is provided. Each unit of the spot inspection device 100 is driven under the control of a control unit (not shown).

The image acquisition unit 110 includes a light source unit 111, a light source power supply 112, a lens barrel 113, and an image pickup unit 114.

The light source unit 111 is configured by using an LED, a laser light source, a halogen lamp, or the like. The light source unit 111 emits illumination light by the electric power supplied from the light source power supply 112. The illumination light emitted by the light source unit 111 is incident on the lens barrel 113.

The lens barrel 113 is provided with an objective lens, a relay lens, and the like, and emits illumination light supplied from the light source unit 111 toward the analysis chip 1, and also takes in the light from the analysis chip 1 and guides it to the image pickup unit 114. It glows.

The imaging unit 114 receives the light guided by the lens barrel 113 and converts it into an electric signal. The imaging unit 114 outputs the converted electrical signal to the analysis unit 120. The imaging unit 114 is configured by using a CMOS (Complementary Metal Oxide Semiconductor) image sensor, a CCD (Charge Coupled Device) image sensor, a photomultiplier tube (PMT), or the like.

The analysis unit 120 generates an image of the analysis chip 1 based on the electric signal generated by the imaging unit 114, or binarizes the image to determine the application status of the spot liquid at the spot. The analysis unit 120 includes a general-purpose processor such as a CPU (Central Processing Unit), a dedicated processor such as a GPU (Graphics Processing Unit), a programmable logic device such as an FPGA (field-programmable gate array), a display, and an input means (for example, a keyboard). Etc. are used.

The humidification unit 130 includes a bottle 131, a water bath 132, an air pump 133, an air supply tube 134, a humidification tube 135, and a thermometer 136.

The bottle 131 contains water 131a and air stone 131b.
The water bath 132 accommodates the liquid adjusted to the set temperature and the bottle 131.

Between the air pump 133 and the air supply tube 134, there is a flow meter 133a that measures the flow rate of the gas output from the air pump 133, and a flow rate adjusting valve 133b that adjusts the flow rate of the gas output from the air pump 133. Is provided.

In the humidification unit 130, the temperature of the water 131a in the bottle 131 is adjusted by the water bath 132. Further, in the humidification unit 130, air is sent from the air pump 133 to the water 131a via the air supply tube 134. The end of the air supply tube 134 on the bottle 131 side is arranged in the vicinity of the air stone 131b, and the air discharged from the air supply tube 134 is taken into the air stone 131b. When air is taken into the air stone 131b, minute bubbles are discharged from the air stone 131b, and the bubbles rise in the bottle 131 as humidified air and flow into the humidifying tube 135. The tip of the humidifying tube 135 is arranged toward the analysis chip 1, and the air that has passed through the tube is supplied to each uneven portion 11 (convex portion 13) of the analysis chip 1. At this time, the user can confirm the temperature of the water 131a with the hygrometer 136.

Here, as described above, in addition to the case where the pin tip does not touch the convex portion 13 and the spot solution is not applied to the upper surface of the convex portion, the spot solution is applied to the convex portion 13 by the subsequent treatment. May spill from the upper surface of the convex portion 13. It is necessary to carry out an inspection to see if these phenomena are occurring. For the inspection, generally, a crystal of a precipitated inorganic salt component or the like is imaged in a dry state of the spot solution, and the image is processed. However, as described above, since the direction and size of salt crystal growth change depending on the delicate conditions of temperature and humidity when the spot liquid dries, it may be difficult to observe the salt depending on the state of the crystals. Further, when the spot liquid is applied to the upper surface of the convex portion 13, crystals are concentrated on the edge of the upper surface of the convex portion 13, and the selective binding substance is actually immobilized on the upper surface of the convex portion 13. It may be misjudged as defective.

Therefore, in the present invention, an image is acquired in a state where the spot solution is a liquid by the deliquescent action of the deliquescent substance contained in the spot solution. Then, since the liquid spot liquid is photographed, the problem of directionality does not occur as in the case of salt crystals. Further, the precipitates near the edge of the upper surface of the convex portion 13 are also deliquescent to become a liquid and become uniform on the upper surface of the convex portion 13, so that over-detection of spot defects due to deviation does not occur.

In order to deliquesce the deliquescent material, air having a water vapor pressure higher than the equilibrium water vapor pressure of the saturated aqueous solution of the deliquescent material at the surface temperature of the carrier is introduced into the immobilized region of the selective binding material on the carrier surface, that is, the spot solution. It may be supplied to the coated area. For example, in the case of sodium chloride (NaCl), the water vapor pressure corresponds to 20 ° C. and the humidity corresponds to 75% RH. As a method of supplying air at this vapor pressure, a method of blowing air that has passed through water as shown in an embodiment to a carrier (a method of using a humidification unit 130), a method of covering the entire device with a cover and a humidifying function therein. Alternatively, there is a method of sending air having a temperature and humidity prepared and controlled by an air conditioner having a humidifying and dehumidifying function.

FIG. 5 is a flowchart illustrating a spot inspection in the method for manufacturing an analysis chip according to an embodiment of the present invention. In the spot inspection, first, humidified air is supplied to the region of the substrate 10 to which the spot liquid is applied (step S201). The humidifying unit 130 described above supplies humidified air to the area of the substrate 10 to which the spot liquid is applied. Here, the humidified air (air common to the substrate 10 by the humidifying unit 130) is air having a water vapor pressure higher than the equilibrium water vapor pressure of the saturated aqueous solution of the deliquescent substance at the surface temperature of the carrier.

After supplying the humidified air or maintaining the supply, the image acquisition unit 110 acquires an image of the substrate 10 (step S202). The image pickup unit 114 outputs the captured image data to the analysis unit 120.

When the analysis unit 120 acquires the image data, it generates a substrate image and binarizes the substrate image (step S203). The analysis unit 120 generates a binarized image obtained by binarizing the brightness of the substrate image with a preset first threshold value as a boundary. In this binarized image, one value is assigned white and the other value is assigned black.

The analysis unit 120 digitizes the spot based on the binarized image (step S204). The analysis unit 120 extracts each area of each spot and counts the number of pixels to which white is assigned (the number of white pixels) in each spot.

After that, the analysis unit 120 determines the quality of each spot based on the counted number of white pixels and the second threshold value set in advance (step S205). If the number of white pixels is less than the threshold value, the analysis unit 120 determines that the target convex portion 13 is a good spot (OK) to which the spot liquid is appropriately applied. On the other hand, if the number of white pixels is equal to or greater than the threshold value, the analysis unit 120 determines that the convex portion 13 is a defective spot (NG) to which the spot liquid is not properly applied. The analysis unit 120 determines the quality of each spot. The analysis unit 120 determines whether or not all the spots are good or bad, and then proceeds to step S106.

Here, the difference between the binarized images depending on the quality of the spot will be described with reference to FIGS. 6 to 8. FIG. 6 is a diagram showing an example of an image of a normally applied spot liquid obtained by binarizing an image. FIG. 7 is a diagram showing an example of an image of the spot liquid when the coating is removed, which is obtained by binarizing the image. FIG. 8 is a diagram showing an example of an image of the spot liquid when the coating is leaked, which is obtained by binarizing the image.

When the spot liquid is properly applied, the humidified air spreads the spot liquid on the tip surface of the convex portion 13. Specifically, the deliquescent substance in the spot liquid is deliquescent and spreads on the tip surface of the convex portion 13. As a result, most of the obtained binarized image is black and the number of white pixels is reduced, as in the binarized image G11 (see FIG. 6).

On the other hand, when the spot liquid is not properly applied and the coating is missing, the spot liquid does not exist on the tip surface of the convex portion 13. As a result, most of the obtained binarized image becomes white and the number of white pixels increases, as in the binarized image G12 (see FIG. 7).

Further, when the spot liquid is applied but a part of the spot liquid leaks from the convex portion 13, only a part of the spot liquid is present on the tip surface of the convex portion 13 and the rest is below the convex portion 13 (unevenness). It exists at the bottom of the portion 11 (see FIG. 8). As a result, the obtained binarized image has a larger number of white pixels than the case where the spot liquid is appropriately applied (see FIG. 6) as in the binarized image G13, and the binarized image obtained has a larger number of white pixels as in the region G14. The area around the spot becomes black (see FIG. 8).

In the present embodiment, the number of white pixels (second threshold value) for determining that the spot liquid is appropriately applied is set based on the number of white pixels of the binarized image G13.

Returning to FIG. 5, if there is a spot determined to be NG (step S206: Yes), the analysis unit 120 shifts to step S207. On the other hand, if there is no spot determined to be NG (step S206: No), the analysis unit 120 proceeds to step S211.

In step S207, the analysis unit 120 binarizes the substrate image again. In step S207, the analysis unit 120 binarizes the substrate image by a third threshold. The third threshold value is set to a value at which the value of the luminance value is made smaller than the first threshold value so that the spot liquid falling between the spots (the bottom of the uneven portion 11) can be identified.

In step S208, the analysis unit 120 determines whether or not there is a spot liquid between the spots. The analysis unit 120 counts, for example, the number of white pixels between spots, compares the count value between the spots with a preset fourth threshold value, and determines whether or not a spot liquid exists between the spots. To judge. The fourth threshold value is set based on the number of white pixels between the spots when the spot liquid does not fall between the spots (the bottom of the uneven portion 11). When the number of white pixels is equal to or greater than the fourth threshold value (step S208: Yes), the analysis unit 120 determines that there is a spot liquid between the spots, and proceeds to step S209. On the other hand, when the number of white pixels is less than the fourth threshold value (step S208: No), the analysis unit 120 determines that there is no spot liquid between the spots, and proceeds to step S210.

In step S209, the analysis unit 120 determines that the analysis chip 1 is NG after applying the spot liquid. The NG after applying the spot liquid corresponds to the case where the spot liquid spills from the upper surface of the convex portion 13 due to the subsequent treatment even though the spot liquid is applied to the convex portion 13.

In step S210, the analysis unit 120 determines that the analysis chip 1 is NG before spot liquid application. The NG before spot liquid application corresponds to the case where the pin tip does not touch the convex portion 13 and the spot solution is not applied to the upper surface of the convex portion 13.

Further, in step S211 the analysis unit 120 determines that the analysis chip 1 is a non-defective product.

By the above processing, the spot inspection on the analysis chip 1 is carried out. The analysis unit 120 may record or display the determination result (OK, NG before spot liquid application, NG after spot liquid application) of the analysis chip 1.

In the above-described embodiment, the analysis chip 1 after the spot liquid is applied is imaged with a substrate image in a state where humidified air is supplied, and binarized to determine whether or not the spot liquid is properly applied. I tried to do it. According to the present embodiment, when spot inspection of a selective binding substance typified by DNA on a substrate at the time of manufacture is performed, the spot defect is surely detected without over-detection or oversight without causing noise during use. Can be inspected.

In the above-described embodiment, an example in which the analysis unit 120 determines the coating state of the spot liquid has been described, but a substrate image or a binarized image is displayed on the display so that the user can visually determine the application state. May be good.

Further, in the above-described embodiment, the configuration in which the spot liquid is applied to the convex portion 13 formed on the substrate 10 has been described as an example, but the spot liquid is applied to a predetermined region of the substrate on which the convex portion 13 is not formed. The spot inspection described above can be applied to the configured configuration.

Hereinafter, the details of the present invention will be further described with reference to Examples. It should be noted that the present invention is not limited to the present embodiment.

<Substrate (carrier)>
A substrate corresponding to the substrate 10 was produced. The outer shape of the substrate is 75.0 × 25.4 × 1.0 (mm), and there are four uneven portions (corresponding to the uneven portion 11) on the surface of the substrate. Each uneven portion has a 4 × 8 convex portion group (corresponding to the convex portion group 12). Each convex portion group has 10 × 10 = 100 convex portions (corresponding to the convex portion 13), and the entire substrate has 4 × 4 × 8 × 10 × 10 = 12800 convex portions. The numerical values indicating the size and distance of each part are all indicated in units of mm. The substrate material is polymethylmethacrylate (PMMA) containing carbon black. A selective binding substance (nucleic acid) or the like is immobilized on the upper surface of the convex portion of the substrate.

<Surface treatment of substrate>
The substrate was immersed in a 10 N aqueous sodium hydroxide solution at 65 ° C. for 12 hours. Then, pure water, 0.1N HCl aqueous solution, and pure water were washed in this order. In this way, the side chain of PMMA on the surface of the substrate was hydrolyzed to generate a carboxyl group.

なお、ＥＤＣは１−エチル−３−（３−ジメチルアミノプロピル）カルボジイミド、ＮａＣｌは塩化ナトリウム、ＤＭＦはＮ，Ｎ−ジメチルホルムアミドを示す。 <Preparation of DNA solution>
A 60-mer DNA (corresponding to a selective binding substance) having an amination at the 5'end was synthesized, and a spot solution having the composition shown in Table 1 below was prepared.

EDC indicates 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide, NaCl indicates sodium chloride, and DMF indicates N, N-dimethylformamide.

<Application of DNA solution and immobilization of DNA>
The solution shown in Table 1 was applied to the entire upper surface of the 12800 convex portions of the substrate by a spotting robot. When the substrate was observed with a microscope after the coating work was completed, the spot liquid after coating was dry on the upper surface of the convex portion (temperature and humidity 23 ° C., 50% RH). Next, the substrate and a small amount of water were placed in a closed plastic container, placed in an oven at 37 ° C., and left to stand for 20 hours. During this period, the humidity in the closed plastic container is 100% RH, so that the spot liquid becomes a liquid by the action of deliquescent EDC and NaCl. In this state, a condensation reaction (crosslinking reaction) between the 5'terminal amino group of DNA and the carboxyl group on the surface of the substrate proceeds, and the DNA is immobilized on the substrate. At this time, if the amount of the spot liquid on the upper surface of the convex portion is too large, or if the convex portion has a shape defect, the spot liquid spills from the upper surface of the convex portion and mixes with the spot liquid on the upper surface of the adjacent convex portion. It becomes defective.
After 20 hours at 37 ° C., the liquid was taken out from a closed plastic container (37 ° C., 100% RH) in an atmosphere of 23 ° C. and 50% RH, and the spot liquid on the upper surface of the convex portion was quickly dried.

(Example 1)
In Example 1, a spot inspection was performed using a spot inspection device 100 having a humidification unit 130 as illustrated in FIG.
An image of a CMOS image sensor (resolution 2M, 2048 × 1088pixel) equipped with a telecentric lens (2x optical magnification, WD66.7) after fixing the substrate produced by the above method and illuminating the substrate with blue LED illumination. Was acquired. The acquired image was saved in a personal computer.

A bottle having a capacity of 2000 ml was prepared as a humidifying device (humidifying unit: see FIG. 4), two holes having a diameter of 5 mm were made in the lid of the bottle, and one hole having a diameter of 6 mm was further made. Insert a 750 mm silicone tube (inner diameter 3 x outer diameter 5 mm) and a 700 mm silicone tube (inner diameter 3 x outer diameter 5 mm) into a 5 mm diameter hole in the lid, and insert the 750 mm silicone tube inside the bottle. A 100 mm long air stone was connected to the end face to enter. A hole having a diameter of 6 mm was inserted so that the tip of the thermometer was on the inside.

1200 mL (about 100 mm from the bottom) of water was injected into the bottle, and the length of the silicon tube was adjusted so that the air stone completely sinks to the bottom of the bottle when the lid is closed. The end face of the 700 mm silicon tube was adjusted to a position about 40 mm away from the water surface so as not to come into contact with water. The length of the thermometer was adjusted so that the temperature measuring part came into contact with water.

A 1.5 W air pump was connected to the outer end of the 700 mm silicone tube outside the bottle, and a flow control valve and flow meter were installed at appropriate positions between the bottle and the pump.
As a result, the air whose flow rate is adjusted by the flow rate adjusting valve after the pump is operated is discharged as minute bubbles from the air stone near the bottom of the bottle to become humidified air, and then discharged from the 700 mm silicon tube. It has become a humidifying device (humidifying unit) that can locally supply humidified air to the target location.

Next, the bottle was placed in a water bath set at 40 ° C. and allowed to stand until the water temperature in the bottle reached 40 ° C. The air flow rate was adjusted to 1 L / min by the flow rate adjusting valve, the tube was fixed so that the distance between the discharge side tube of the humidifier and the substrate was about 20 mm and the angle was about 45 degrees, and an image was acquired at the same time. At this time, it was observed that high-humidity air was discharged from the discharge tube and the deliquescent substances contained in the spot liquid on the substrate surface were deliquescent.

FIG. 9 is a diagram showing an image obtained by cutting out a part of the convex portion group (A) of the acquired substrate image. FIG. 10 is a diagram showing a binarized image of the convex portion group (A) shown in FIG. 9 among the binarized images obtained by binarizing the data of the substrate image. In the substrate image and the binarized image, numbers indicating the positions of the spots are assigned. For example, the upper left spot is represented by 1 (0,1) and the lower right spot is represented by 100 (9,10). From the binarized image shown in FIG. 10, it was found that the spot liquid was not properly applied at the spot numbers 59, 60, 79, 80, 89, 90, 99 and 100.

FIG. 11 is a graph showing the number of pixels (the number of white pixels) of the white portion in each spot of the binarized image shown in FIG. FIG. 11 shows the result of counting the number of white pixels of each spot from the binarized image shown in FIG. As can be seen from FIG. 11, the size of the number of white pixels is clearly separated between the spot number of the above-mentioned defective spot and the spot number of the other good spot. The average number of white pixels in the good spots was 195, while the average number of white pixels in the bad spots was 708.

FIG. 12 is a graph showing the number of white pixels in the good spot to which the spot liquid was normally applied and the defective spot to which the spot liquid was not properly applied in the binarized image shown in FIG. FIG. 12 is a graph in which the average value of the number of white pixels in each of the good spot and the bad spot and the error bar 3 times the standard deviation are superimposed. As shown in FIG. 12, the error bars of the good spot and the bad spot do not overlap, and the result is that the good / bad spot can be completely distinguished with a standard deviation of 3 times. It is considered that this is because by supplying high-humidity air to the spot solution and deliquescent the deliquescent substance, the numerical value on the upper surface of the convex portion to which the spot solution could be applied has a small variation and shows a stable value. This showed that accurate judgment can be made even using a computer.

(Example 2)
In Example 2, the image of the convex portion group (A) acquired in Example 1 (see FIG. 9) is used so that the bottom surface of the uneven portion (concavo-convex portion 11: see FIG. 1) of the substrate can be observed. The threshold value for binarization was set smaller than 1, and binarization was performed. FIG. 13 is a diagram showing a binarized image of the convex portion group (A) of the substrate image.
In FIG. 13, traces of the spot liquid were confirmed on the bottom surface of the uneven portion between the spot numbers 59 and 60, between 79 and 80, and between 89, 90, 99 and 100. From this result, it was presumed that the reason why the spot liquid was not properly applied to these spots in Example 1 was that the spot liquid spilled from the convex portion after being applied.

(Comparative Example 1)
In Comparative Example 1, a spot inspection was performed using a spot inspection device having no humidifying unit. FIG. 14 is a diagram illustrating the configuration of the spot inspection device 200. The spot inspection device 200 shown in FIG. 14 is different from the spot inspection device 100 described above in that it does not have a humidifying unit 130, and has the same configuration other than that.

FIG. 15 is a diagram showing an image obtained by cutting out an image of the same convex portion group (A) as in the first embodiment among the acquired substrate images. FIG. 16 is a diagram showing a binarized image of the convex portion group (A) shown in FIG. 15 among the binarized images obtained by binarizing the data of the substrate image. The substrate image shown in FIG. 9 and the substrate image shown in FIG. 15 are different depending on whether or not they are humidified. Therefore, even in Comparative Example 1, spot numbers 59, 60, 79, 80, 89, 90, 99, and 100 are defective spots.

FIG. 17 is a graph showing the number of pixels (the number of white pixels) of the white portion in each spot of the binarized image shown in FIG. FIG. 17 shows the result of counting the number of white pixels of each spot from the binarized image shown in FIG. As can be seen from FIG. 17, the variation in the number of white pixels of the spot numbers of the good spots became larger than in the case of the first embodiment. The average number of white pixels in the good spot was 416, and the average number of white pixels in the bad spot was 698.

FIG. 18 is a graph showing the number of white pixels in the good spot to which the spot liquid was normally applied and the defective spot to which the spot liquid was not properly applied in the binarized image shown in FIG. FIG. 18 is a graph in which the average value of the number of white pixels in each of the good spot and the bad spot and the error bar 3 times the standard deviation are superimposed. As shown in FIG. 18, the error bars of the good spot and the bad spot overlapped, and the result was that the good / bad spot could not be distinguished by the standard deviation of 3 times. This result indicates that erroneous determination can occur by 0.1% or more. In this case, 10 or more erroneous determination spots are included in each analysis chip, which is considered unacceptable.

(Comparative Example 2)
In Comparative Example 2, among the substrate images acquired in Comparative Example 1, an image obtained by cutting out an image of the convex portion group (B) different from Comparative Example 1 (convex portion group (B)) is used, and the others are compared. A spot inspection was performed in the same manner as in Example 1. FIG. 19 is a diagram showing the substrate image. In spot numbers 2, 43 and 44, a small amount of NaCl crystals were observed when carefully observed, but since the color tone was lighter than that of the upper surface of the other convex parts, the spot solution was found in the visual inspection and the image inspection using a computer. It was in a state where there was a possibility of making a wrong judgment if it was not applied. The presence of crystals may be confirmed by sufficiently magnifying and observing the acquired substrate image, but considering that the number of convex parts existing on one substrate is 12,800, when it occurs frequently. Is not a realistic inspection method.

(Example 3)
In Example 3, among the substrate images acquired in Example 1, an image obtained by cutting out an image of the convex portion group (C) different from that in Example 1 is used, and the other parts are spot-inspected in the same manner as in Example 1. Was done. FIG. 20 is a diagram showing a binarized image obtained by binarizing the data of the substrate image. As shown in the binarized image shown in FIG. 20, since the substrate image is taken in a state where the NaCl crystals are deliquescent, it is found that the image can more accurately determine the presence or absence of the spot liquid. It was. As described in Comparative Example 3, for example, in spot number 78, it was correctly determined that the spot liquid was applied.

(Comparative Example 3)
In Comparative Example 3, among the substrate images acquired in Comparative Example 1, an image of a convex portion group (C) (the same convex portion group as in Example 3) different from Comparative Example 1 (convex portion group (A)) is displayed. Using the cut-out image, spot inspection was performed using a spot inspection device without a humidification unit in the same manner as in Comparative Example 1. FIG. 21 is a diagram showing a binarized image of the convex portion group (C) among the binarized images obtained by binarizing the data of the substrate image. From the binarized image shown in FIG. 21, it was determined that no crystals were seen on the upper surface of the convex portion at the spot number 78 and the spot liquid was not applied. On the other hand, in Example 3 (see FIG. 20) described above, it was determined that the spot liquid was applied to the same spot number 78, and the two results did not match. Therefore, when hybridization was actually carried out using this substrate and fluorescence was observed, it was confirmed that fluorescence was observed at spot number 78 and that the spot solution was applied to spot number 78. That is, it was revealed that the presence or absence of the spot liquid was correctly determined in Example 3, but was erroneously determined in Comparative Example 3.

The method for inspecting the analysis chip, the inspection device, and the method for manufacturing the analysis chip of the present invention can reliably determine the quality of the spot liquid applied to each spot of the chip. Therefore, the present invention is very useful industrially because it can accurately determine the quality of the analysis chip.

1 Analysis chip 10 Substrate 11 Concavo-convex part 12 Convex part group 13 Convex part 100 Spot inspection device (with humidification unit)
110 Image acquisition unit 120 Analysis unit 130 Humidification unit 200 Spot inspection device (without humidification unit)

Claims (7)

A method for inspecting an analytical chip in which a mixed reagent of a compound containing a selective binding substance and a deliquescent substance is applied in an array to a required region on the surface of a carrier and the selective binding substance is immobilized.
A deliquescent step in which air having a vapor pressure higher than the equilibrium vapor pressure of a saturated aqueous solution of a deliquescent substance at the surface temperature of the carrier is supplied to the immobilized region of the selective binding substance on the surface of the carrier to deliquesce the deliquescent substance. ,
An image acquisition step of obtaining an image including the immobilized region in which the deliquescent substance is deliquescented by the image acquisition means for acquiring an image, and an image acquisition step.
A determination step for determining whether or not the mixed reagent is present in the required region from the image acquired by the image acquisition means, and a determination step.
A method for inspecting an analytical chip, which comprises. The determination step is
The image is binarized based on a first threshold set for brightness.
From the binarized image, the number of pixels of one value is counted as the first count value, and
The first count value is compared with the second threshold value set for the first count value to determine whether or not the mixed reagent is present in the required region.
The method for inspecting an analysis chip according to claim 1, wherein the analysis chip is inspected. The determination step is
The image is binarized based on a third threshold that is smaller than the first threshold.
The number of pixels of one value between the fixed regions is counted as a second count value, and the number of pixels is counted.
The second count value is compared with a fourth threshold set relative to the second count value to determine if the mixed reagent is present between the immobilized regions.
The method for inspecting an analysis chip according to claim 2, wherein the analysis chip is inspected. The deliquescent substance is a salt,
The method for inspecting an analysis chip according to any one of claims 1 to 3, wherein the analysis chip is inspected. The selective binding substance is DNA, RNA or protein.
The method for inspecting an analysis chip according to any one of claims 1 to 4, wherein the analysis chip is inspected. An analytical chip inspection device in which a mixed reagent of a compound containing a selective binding substance and a deliquescent substance is applied in a sequence to a required region on the surface of a carrier and the selective binding substance is immobilized.
A humidifying unit that supplies air with a water vapor pressure higher than the equilibrium water vapor pressure of a saturated aqueous solution of a deliquescent substance at the surface temperature of the carrier to the immobilized region of the selective binding substance on the surface of the carrier.
An image acquisition unit that acquires an image of the surface of the carrier to which humidified air is supplied, including the immobilized region, by an image acquisition means for acquiring an image.
An analysis unit for determining whether or not the mixed reagent is present in the required region from the image acquired by the image acquisition means, and an analysis unit.
An analysis chip inspection device characterized by comprising. A coating step of applying a mixed reagent of a compound containing a selective binding substance and a deliquescent substance to a required region on the surface of a carrier in an array.
A fixation step of fixing the selective binding substance in the mixed reagent to a required region on the surface of the carrier, and
A test step to determine if the mixed reagent is present in the required region, and
Including
The inspection step
A deliquescent step in which air having a vapor pressure higher than the equilibrium vapor pressure of a saturated aqueous solution of a deliquescent substance at the surface temperature of the carrier is supplied to the immobilized region of the selective binding substance on the surface of the carrier to deliquesce the deliquescent substance. ,
An image acquisition step of obtaining an image including the immobilized region in which the deliquescent substance is deliquescented by the image acquisition means for acquiring an image, and
A determination step for determining whether or not the mixed reagent is present in the required region from the image acquired by the image acquisition means, and a determination step.
A method for manufacturing an analytical chip, which comprises.

Images