JP4606926B2 - Sample inspection apparatus and manufacturing method thereof - Google Patents

Description

  The present invention relates to a sample inspection apparatus that performs analysis by detecting a luminescence reaction that occurs in a droplet containing a measurement object to be analyzed such as a specific gene or a specific protein, and a method for manufacturing the same.

  In recent years, DNA chips and DNA microarrays (hereinafter referred to as “DNA chips”) have been widely put into practical use as new tools for gene analysis for the purpose of disease diagnosis and prevention, drug discovery, and the like. As a DNA chip, generally, a surface for fixing a droplet of a DNA solution containing a DNA probe of a known gene sequence on a glass substrate (hereinafter referred to as a “sample mounting surface” if necessary) There are a variety of DNA probes that are spotted on top and fixed at high density, and DNA probes that are immobilized by synthesizing DNA on a glass substrate (on the sample mounting surface of the glass substrate). .

  Such a DNA chip realizes simultaneous analysis of multiple items (multiple types, different compositions) and many samples, and simultaneous analysis of the same items (same types and same compositions) and many samples, improving analysis efficiency Therefore, it is desired to reduce the analysis cost and further reduce the size of the analyzer. For this purpose, various studies have been made to further advance the miniaturization and densification of the reaction field (sample solution droplets) on the sample mounting surface.

  A DNA chip, for example, applies complementary DNA (cDNA) reversely transcribed from mRNA collected from a test object to an analysis unit to which a DNA probe is immobilized, and detects hybridization between the cDNA and the DNA probe on the substrate. Thus, the presence or absence of expression of the target gene can be detected. Moreover, according to such a DNA chip, for example, even a small amount of sample can be effectively analyzed. Furthermore, according to such a DNA chip, since various types of DNA probes can be immobilized on one substrate, it is possible to perform multi-item analysis on the same specimen in one DNA chip.

  In detecting hybridization as described above, a fluorescent label or the like is generally used. For example, when a gene to be detected is amplified by a PCR (polymerase chain reaction) method, the amplification product is labeled with a fluorescent substance. Thereby, the hybridization between the labeled amplification product and the immobilized DNA probe can be detected by fluorescence.

  As a detection means for detecting the light emission (or color development) described above, the light emission state (or color development state) in the DNA chip is recognized as a two-dimensional map, with the intention of obtaining a lot of information at one time. CCD (charge coupled device: charge coupled device) is generally used. A conventional detection apparatus equipped with a CCD as a light emission detection means has a CCD arranged in a dark room, and when used, a DNA microchip in which a detection target gene and an immobilized probe are hybridized is placed inside the apparatus. It arrange | positions and has the structure which detects the light emission (for example, refer nonpatent literature 1).

  In addition, with the intention of improving detection sensitivity, a fluorescence detection apparatus having a configuration in which a photodiode is used as a luminescence detection means and a fluorescence reaction tank serving as a reaction field for fluorescence reaction is formed on the photodiode is proposed. (For example, refer to Patent Document 1).

  Furthermore, by forming a matrix pattern made of metal or plastic polymer on the substrate (on the sample mounting surface of the substrate), a convex portion showing non-affinity for a liquid solvent such as a sample is provided, and adjacent reactions are performed. A reaction field array having a configuration for isolating the fields has been proposed (see, for example, Patent Document 2). In this reaction field array, when the above-mentioned convex portions are formed, the reaction field (liquid droplets of the sample solution) on the substrate is miniaturized and arranged in a large number on the substrate to arrange the sample solution on the substrate. It is intended to prevent the occurrence of mixing (contamination) between adjacent reaction fields due to droplet diffusion.

In addition, as a device for performing genetic testing at low cost and simply, a sample plate in which a plurality of reaction vessels in which a biological sample is stored is arranged one-dimensionally or two-dimensionally, and a plurality of pixels in which an optical sensor is formed are one. A photosensor array substrate on which a pixel selection circuit for selecting a pixel for selecting a pixel for reading a signal and a photosensor array arranged in a two-dimensional manner corresponds to the reaction tank and the pixel in a one-to-one vertical relationship. As described above, a member for fixing the sample plate and the photosensor array substrate is selected, and light generated by the reaction between the biological sample and the reagent is selected in the reaction tank corresponding to the selected pixel. There has been proposed a biological specimen inspection apparatus having a configuration in which a signal is received from the selected pixel and read out from the selected pixel (for example, a patent) Document reference 3). This apparatus is intended for a configuration aiming at miniaturization, high sensitivity, and low cost in order to perform genetic testing inexpensively and easily.
Matsunaga (supervised), Genome Engineering Research Group "DNA chip application technology", CMC Co., Ltd., July 31, 2000, p.45-p.49 JP 2002-350346 A JP-A-11-99000 JP 2003-329681 A

  However, in the prior art including the above-described technology, the volume of the droplets dropped on the sample mounting surface of the DNA chip is further reduced, and the number of the droplets is further increased, thereby miniaturizing the reaction field, If the density is further increased, the analysis efficiency is further improved, the analysis cost is further reduced, and the analyzer is further miniaturized, the analysis accuracy is likely to be lowered, which is extremely difficult to realize. It was.

More specifically, the volume of the analysis sample droplet (reaction field) is set to 1000 pL or less, and the density of the volume (reaction field) of the analysis sample in which the sample droplet is placed (unit of the sample mounting surface) When the number of droplets arranged per area) is 10,000 / cm ² or more, the occurrence of contamination between droplets is likely to occur, and the analysis accuracy is likely to be lowered.

In particular, the volume of the droplet is preferably 35 pL or less (more preferably 1.2 pL or less), and the density of the reaction field is preferably 100,000 / cm ² or more (more preferably 1,000,000 / cm ² or more). When trying to miniaturize and increase the density of the reaction field on the sample mounting surface at a high level, which has been extremely difficult to achieve in the past, a large number of analytical samples at once can be accurately and accurately It was extremely difficult to analyze quickly and in a small analysis space.

  For example, in the DNA chip and the detection device described in Non-Patent Document 1, the sample mounting surface of the DNA chip is made of a hydrophilic material (for example, glass). For this reason, there is a limit to sufficiently preventing the spread of the droplets dropped on the sample placement surface, so that the reaction field on the sample placement surface aiming at the extremely high level described above. When trying to reduce the size and increase the density, it is extremely difficult to sufficiently prevent the occurrence of so-called contamination, in which adjacent droplets dropped on the sample mounting surface mix together. It has been extremely difficult to ensure sufficient analysis accuracy.

  In addition, in the DNA chip and the detection device described in Non-Patent Document 1, it is difficult to accurately position the CCD arranged above the sample mounting surface of the DNA chip, so that the analysis accuracy can be further improved. There was a limit. More specifically, it is extremely difficult to make the distance of the CCD exactly constant for all droplets dropped on the sample mounting surface, and the droplet dropping position (fixed position) on the sample mounting surface. It was extremely difficult to eliminate the variation in the distance between each droplet and the CCD due to the difference.

  Furthermore, in the case of the DNA chip and the detection device, when the light emission or color development signal is weak, it is difficult to receive the signal by the CCD, and there is a problem that the analysis sensitivity is lowered. Further, when compared with the size of the DNA chip, it is necessary to design the dark room of the apparatus to be extremely large, so there is a limit when attempting to downsize the apparatus.

  Moreover, the detection apparatus described in Patent Document 1 has a configuration in which a container made of a transparent material standing on a transparent substrate is used as a fluorescence reaction tank, or a recess formed by processing the surface of a surface transparent substrate is a fluorescence reaction tank. And further refine the reaction field (droplets) and increase the density at the level described above to further improve the analysis efficiency, further reduce the analysis cost, and further improve the analysis equipment. However, there was a limit in simultaneously reducing the size. That is, it is not easy to form minute scale concave portions capable of accommodating droplets having a volume of 1000 pL or less (preferably 35 pL or less, more preferably 1.2 pL or less) on the surface of the transparent substrate. In addition, it is not easy to sufficiently reduce the variation in the volume of each recess. If the variation in volume of each recess cannot be sufficiently reduced, it is difficult to ensure sufficient analysis accuracy.

  Further, in the reaction field array described in Patent Document 2, when the sample droplet is aqueous, the convex matrix pattern (second region) formed on the substrate surface must be hydrophobic. Is formed from a resin material such as a photosensitive resin (see, for example, paragraph number 0029 and paragraph numbers 0035 to 0038 in Patent Document 2). The convex matrix pattern made of a resin material such as a photosensitive resin formed on the substrate is easy to peel off from the substrate, and it is difficult to obtain sufficient reliability. In particular, it is difficult to sufficiently prevent all or partial peeling of the convex matrix pattern from the substrate, and sufficient reliability is obtained when repeatedly used over a long period of time or when stored for a long period of time. It was difficult. The reason why this convex matrix pattern is easy to peel off from the substrate is not clearly clarified, but the convex matrix pattern is fixed to the substrate surface by weak bonds such as physical adsorption, intermolecular force, or hydrogen bond. The present inventors consider that this is one of the factors.

  Furthermore, in the reaction field array described in Patent Document 2, when the convex matrix pattern formed on the substrate surface is a metal convex matrix pattern formed by a film forming technique using a vapor deposition method, It is difficult to sufficiently prevent the above-described peeling, and it has been difficult to obtain sufficient reliability when repeatedly used for a long period of time or when stored for a long period of time. When a metal convex matrix pattern is used, a hydrophilic film made of a metal oxide is easily formed on the surface of the convex matrix pattern, and contamination occurs when water-based sample droplets are used. It becomes easy.

  Furthermore, in the reaction field array described in Patent Document 2, when a convex matrix pattern made of a resin material such as a photosensitive resin is formed on a substrate, the dimension setting of the thickness of the film made of the resin material, It was extremely difficult to achieve homogenization. For example, in the case where a film made of a resin material is formed on a substrate and a part thereof is removed by patterning by a technique such as etching to form a convex matrix pattern, the film thickness is removed when part of the film is removed. Unevenness easily occurs remarkably. When the film thickness unevenness becomes significant, the reaction field volume varies, making it difficult to obtain sufficient analysis accuracy.

Further, when the reaction field array described in Patent Document 2, greater height of the convex matrix pattern, it is extremely difficult to the density of the reaction field 10,000 / cm ² or more. For example, in the case of the reaction field array described in Patent Document 2, it is described that the occurrence of contamination can be prevented by adopting a configuration in which the height of the convex matrix pattern is 1 μm or more (for example, Patent In this case, the reaction field can be set to 1000 pL or less (for example, 800 pL) even if the volume of the reaction field can be set to 1000 pL or less (see, for example, 800 pL). It is considered that it is extremely difficult to set the density of the resin to 10,000 pieces / cm ² or more.

  Moreover, the biological sample test apparatus described in Patent Document 3 has a large reaction tank volume (for example, the reaction tank has a diameter of 2 mm and a depth of 2 mm, see paragraph No. 0016 in Patent Document 3). On the other hand, analysis objects such as genes and immobilized DNA are on the nanometer order. Thus, since the reaction field is extremely large with respect to the size of the gene to be analyzed and the immobilized DNA, a large amount of sample solution is required for one reaction tank. In addition, if there is a shortage of samples, hybrid formation could not be performed efficiently and the detection accuracy could be reduced.

  In addition, none of the above-mentioned problems occur only in the technical field related to the analysis using a DNA chip. The technical field performs qualitative or quantitative analysis of substances contained in the liquid phase, and the chemistry using the liquid phase as a reaction field. In the technical field of reaction development and reaction analysis related to reactions and biochemical reactions, when trying to make the reaction field on the sample mounting surface at a high level as described above fine and dense, It will occur.

The present invention has been made in view of the above problems, and in the case of miniaturizing and increasing the density of the reaction field (particularly, the volume of the reaction field is set to 1000 pL or less, and the density of the reaction field is 10,000 / It is an object of the present invention to provide a sample inspection apparatus that can ensure sufficient analysis accuracy even if it is equal to or greater than cm ^2, can be easily miniaturized, and can be used repeatedly, and a method for manufacturing the same. .

  As a result of intensive studies to achieve the above object, the inventors of the present invention include a droplet containing a measurement object to be analyzed, which is an analysis object, containing at least one kind of water or a water-soluble solvent. In the case of a droplet, a plurality of recesses are formed on the surface (sample mounting surface) of the substrate on which the droplet containing the measurement object to be analyzed is dropped using a hydrophobic monomolecular film made of organic molecules as a raw material. The inventors have found that it is extremely effective to achieve the above-described object by forming and forming the liquid droplets in the recesses, and the present invention has been achieved.

That is, the present invention comprises at least a substrate having a sample placement surface provided with two or more recesses for independently containing two or more droplets containing a measurement object to be analyzed,
Sample mounting surface has a first area covered by the first monolayer of hydrophobic, inside two or more second having a first and not covered in monolayer hydrophilic And at least divided into areas,
The bottom surface of the recess is formed by the second region, and the entire outer peripheral portion including the entire inner surface of the recess is formed by the first monomolecular film covering the first region,
Two or more photoelectric conversion units capable of independently photoelectrically converting light emitted from each of the two or more droplets accommodated in each of the two or more recesses are provided inside the substrate. For one-to-one correspondence
The first monomolecular film is formed using organic molecules, and is fixed to the sample mounting surface by a covalent bond ,
The bottom surface of the concave portion is further coated with a second monomolecular film made of a material having optical transparency to the light,
A thickness of the second monomolecular film is equal to or less than a thickness of the first monomolecular film covering the first region;
A hydrophilic fourth characteristic group capable of at least hydrogen bonding with the solvent contained in the droplet is bonded to the surface of the second monomolecular film,
The second monomolecular film is formed using an organic molecule, and is fixed to the sample mounting surface by a covalent bond ,
A sample analyzer characterized by the above is provided.

  Here, the “droplet” in the present invention refers to a droplet containing a substance to be analyzed and at least one solvent selected from water and a water-soluble solvent. As long as the above characteristics can be maintained, the droplets may contain other solutes and solvents as necessary depending on the purpose of analysis.

  Further, in the present invention, the state “two or more photoelectric conversion units are provided so as to correspond one-to-one with each of the two or more recesses” refers to the following state. That is, α recesses {recesses 1, recesses 2,..., Recesses i,..., Recesses α} (where i = 1, 2,..., Α; 1 ≦ α, Here, i indicates the number of the recess.) The same number of photoelectric conversion units {photoelectric conversion unit 1, photoelectric conversion unit 2,..., Photoelectric conversion unit i,. I = 1, 2,..., Α; 1 ≦ α, where i indicates the number of each of the two or more photoelectric conversion units, and the photoelectric conversion unit i indicates the same number as the recess i.) Is provided in the substrate, and a photoelectric conversion unit (photoelectric conversion unit i) for performing only the analysis of a droplet fixed to the concave portion (recess i) with respect to one concave portion (recess i). The state where one is arranged is shown.

  The formation position of the photoelectric conversion portion in the substrate (the formation position of the photoelectric conversion portion i corresponding to the recess i) is not particularly limited as long as light emitted from the droplet fixed to the corresponding recess (recess i) can be detected. However, from the viewpoint of sufficiently shortening the effective optical path from the concave portion to the photoelectric conversion portion (from the concave portion i to the photoelectric conversion portion i) to sufficiently secure analysis sensitivity and from the viewpoint of downsizing the apparatus, the photoelectric conversion portion is It is preferable that it is arranged almost directly below the corresponding recess.

  Furthermore, in the present invention, one photoelectric conversion unit may be composed of one photoelectric conversion element (for example, a photodiode), or one photoelectric conversion unit may be composed of two or more photoelectric conversion elements. Good. From the viewpoint of sufficiently reducing the size of the sample inspection apparatus, it is preferable that one photoelectric conversion unit is composed of one photoelectric conversion element. From the viewpoint of easily and sufficiently ensuring the analysis sensitivity of the apparatus, one photoelectric conversion unit is preferable. It is preferable that the conversion unit is composed of two or more photoelectric conversion elements.

  In the present invention, the “monomolecular film” refers to a film that is formed using organic molecules as a raw material and is fixed to the sample mounting surface by a covalent bond. Further, the monomolecular film may be composed of a single film (one layer) as long as it can simultaneously achieve the volume of the recess and the density of the recess. It may be a film made of a laminate in which a plurality of these films are laminated. In addition, when the second monolayer is formed after the first monolayer is formed, the first characteristic group may not be bonded to the surface of the monolayer. In that case, the first characteristic group may be bonded by surface treatment. Furthermore, the monomolecular film is a film in which the portion made of the single film and the portion made of the laminate are mixed as long as the volume of the concave portion and the density of the concave portion can be achieved simultaneously. It may be. Such a film can be used when it is necessary to divide a plurality of recesses on the sample mounting surface into two or more groups and to provide a difference in the volume of the recesses in each group.

  In the sample inspection apparatus of the present invention described above, since the monomolecular film formed on the sample mounting surface is formed using organic molecules, it has a very thin film thickness. Therefore, the volume of the concave portion formed on the sample mounting surface can be made extremely small, and minute droplets with a volume of 1000 pL or less can be easily accommodated. In addition to the state in which all of the droplets are contained in the recess, the “accommodated” state means that if the droplet is fixed in the recess, a part of the droplet protrudes from the opening of the recess. The state of being included is also included.

  In addition, the hydrophobic monomolecular film formed on the sample mounting surface is fixed on the sample mounting surface because the end of the organic molecule of the raw material is covalently bonded to the sample mounting surface. It is firmly fixed. Therefore, even if the distance between adjacent recesses on the sample mounting surface (the area of the first region covered with the monomolecular film) is very small, the monomolecular film separating the adjacent recesses is not placed on the sample mounting surface. It is firmly bonded on the surface, and its peeling is sufficiently prevented. Moreover, since the bottom surface (second region) of each recess has hydrophilicity, the liquid droplets stored in the recesses are securely fixed to the bottom surface of the recesses by hydrogen bonding, and from the recesses once stored It is sufficiently prevented from diffusing to the outside area.

Therefore, in the present invention, even if the distance between the adjacent recesses is very small and the density of the reaction field (droplets contained in the recesses) is 10,000 / cm ² or more, Is sufficiently prevented from mixing (contamination is surely prevented). Therefore, the sample inspection apparatus of the present invention can ensure sufficient analysis accuracy.

  Furthermore, since the hydrophobic monomolecular film formed on the sample mounting surface is easily formed with a uniform thickness, the variation in volume of each recess is sufficiently reduced, and each reaction field (in each recess) is reduced. The volume of the liquid droplets (contained) can be easily made constant. Therefore, also from this viewpoint, the sample inspection apparatus of the present invention can ensure sufficient analysis accuracy.

  In the present invention, the first region and the second region of the sample mounting surface are appropriately classified according to the fine two-dimensional arrangement pattern of the photoelectric conversion unit provided in the substrate, and the first region A monomolecular film can be easily formed selectively only in the region. Therefore, it is possible to easily form the recesses two-dimensionally on the sample mounting surface with an extremely fine pattern. For this reason, it becomes possible to detect efficiently the light emission reaction which occurs with the droplet accommodated in each recessed part in a photoelectric conversion part.

Therefore, according to the sample inspection apparatus of the present invention, since the concave portion having a very small volume is formed at a very high density on the sample mounting surface, the reaction field is made finer and the density is increased (particularly, the reaction field volume). Can be made 1000 pL or less and the density of the reaction field can be 10,000 pieces / cm ² or more). Moreover, according to the sample inspection apparatus of the present invention, sufficient analysis accuracy can be ensured. Furthermore, according to the sample inspection apparatus of the present invention, it is possible to perform analysis with a very small amount of sample, and the analysis cost can be reduced. In addition, since the concave portions can be formed with high density, the sample inspection apparatus of the present invention can efficiently perform many samples, multi-item analysis, multiple analysis of the same item, and the like at a time.

  Furthermore, since the sample inspection apparatus of the present invention has a configuration capable of easily and reliably miniaturizing and increasing the density of the reaction field as described above, it can be easily reduced in size.

  Further, as described above, in the sample inspection apparatus of the present invention, the monomolecular film is fixed to the first region of the sample mounting surface by covalent bonding. Therefore, even when the sample inspection apparatus is used repeatedly over a long period of time or when it is stored for a long period of time, peeling of the monomolecular film is sufficiently prevented, so that the sample inspection apparatus of the present invention has sufficient reliability. Obtainable.

  Further, the configuration of the concave portion is a configuration comprising a hydrophilic bottom surface (second region) and an entire inner surface formed of a hydrophobic monomolecular film (a configuration in which the first region is covered with a monomolecular film). ), The liquid droplet dropped on the bottom surface of the concave portion is fixed in a state of a substantially hemispherical shape or a substantially columnar shape having a substantially hemispherical tip. Therefore, compared to the geometric volume of the recess, the volume of the droplet actually contained in the recess (droplet fixed on the bottom surface of the recess) can be made very large. Therefore, even when a concave portion having a small geometric volume is formed, it is possible to use an amount of droplets that can provide sufficient analysis sensitivity. In addition, the liquid droplets dropped on the bottom surface of the recess are fixed in a substantially hemispherical shape or a substantially columnar shape with the tip portion being substantially hemispherical. Therefore, the droplets fixed on the bottom surface of the recess have a shape that is advantageous for condensing, and from this viewpoint, the sample analyzer of the present invention can easily obtain sufficient analysis sensitivity. It becomes.

  For example, when the bottom surface (second region) of the recess is approximated by a circle, the height (distance from the bottom surface of the recess to the apex of the droplet) is approximately equal to the radius r on the bottom surface of the recess. Can hold droplets. For example, the present inventors have confirmed that a droplet having a height of 1000 times or more the monomolecular film thickness can be fixed to a concave portion [the bottom surface of the concave portion (second region)]. For example, the present inventors have confirmed that a droplet having a height of about 10,000 times can be fixed to a concave portion [the bottom surface of the concave portion (second region)] in the examples described later. As described above, fixing a sufficient amount of droplets in a very small area as described above is extremely difficult to achieve with the above-described configuration of the prior art.

  For example, Patent Document 2 described above describes that the occurrence of contamination can be suppressed by setting the height of the convex matrix pattern to 1 μm or more (for example, claim 24 of Patent Document 2, Patent In the present invention, contamination can be sufficiently prevented even when the thickness of the monomolecular film corresponding to the height of the convex matrix pattern is 50 nm or less. The inventors confirmed that.

  The sample inspection apparatus of the present invention is extremely useful as an analysis apparatus such as a DNA chip and a DNA microarray and a manufacturing method thereof in various fields such as medical treatment, drug discovery, and analysis.

  Further, in the present invention, the presence of a monomolecular film can be confirmed by various analysis methods such as its molecular structure and constituent elements. For example, as will be described later in the examples, an infrared spectrometer, a nuclear magnetic resonance apparatus, an organic mass spectrometer, a secondary ion mass spectrometer, an X-ray photoelectron spectrometer, an X-ray reflectivity measuring apparatus, and a transmission It is possible to confirm the presence by performing analysis using at least one analyzer of the scanning electron microscope.

Here, in the sample inspection apparatus of the present invention, the reaction field (droplets contained in the recesses) is made finer and higher in density, preferably the reaction field volume is 1000 pL or less, and the reaction field From the viewpoint of setting the density to 10,000 / cm ² or more, the volume of the liquid droplets accommodated in the recesses is preferably 0.01 pL to 1000 pL, more preferably 0.01 pL to 35 pL, and More preferably, it is 01 pL-1.2 pL. In addition, when the volume of the droplet accommodated in the concave portion is less than 0.01 pL, a tendency that it becomes difficult to obtain sufficient analysis sensitivity increases.

Moreover, when the volume of the droplet accommodated in the concave portion exceeds 1000 pL, it tends to be difficult to make the reaction field finer and higher in density. Furthermore, when the volume of the droplets accommodated in the recesses is set to 0.01 pL to 35 pL, the density of the reaction field can be easily increased to 100,000 / cm ² or more. Further, when the volume of the droplets accommodated in the recesses is 0.01 pL to 1.2 pL, the density of the reaction field can be easily set to 1 million / cm ² or more.

As described above, in the sample inspection apparatus according to the present invention, the volume (geometric volume) of the recess can be made sufficiently smaller than the volume of the droplet accommodated in the recess. Therefore, in the present invention, when the volume of the liquid droplets accommodated in the recess is 0.01 pL to 1000 pL, the volume of the recess can be made sufficiently smaller than this. Specifically, in this case, the volume of the recess is preferably 2 × 10 ⁻⁶ pL to 1 pL.

From the same viewpoint as described above, when the volume of the liquid droplets contained in the recess is 0.01 pL to 35 pL, the volume of the recess is preferably 2 × 10 ⁻⁶ pL to ¹ × 10 ⁻¹ pL. Furthermore, from the same viewpoint as described above, when the volume of the liquid droplets contained in the recess is 0.01 pL to 1.2 pL, the volume of the recess is 2 × 10 ⁻⁶ pL to 2 × 10 ⁻³ pL. Is preferably 2 × 10 ⁻⁶ pL to 7 × 10 ⁻⁴ pL.

In the sample inspection apparatus of the present invention, the reaction field (droplets contained in the recesses) is made finer and higher in density, preferably the reaction field has a volume of 1000 pL or less, and the density of the reaction field. from the viewpoint of 10,000 / cm ² or more, it is preferable that the number of recesses formed per unit area of the sample mounting surface of 10,000 / cm ² or more.

  Here, in the present invention, the “unit area of the sample mounting surface” when determining the “number of recesses formed per unit area of the sample mounting surface” is the first unit in the sample mounting surface. It means a value calculated from the sum of the area of the area and the area of the second area. Therefore, even when the sample placement surface includes other regions other than the first region and the second region, the “unit area of the sample placement surface” is the sample placement surface excluding the other regions. It is calculated from the sum of the area of the first region and the area of the second region. In this specification, “the number of recesses formed per unit area of the sample mounting surface” is also referred to as “the density of the recesses on the sample mounting surface” as necessary.

In the present invention, from the viewpoint of setting the density of the reaction field to 100,000 pieces / cm ² or more, the number of recesses formed per unit area of the sample mounting surface is 100,000 pieces / cm ² or more. It is more preferable. Furthermore, from the viewpoint of setting the density of the reaction field to 1 million pieces / cm ² or more, the number of recesses formed per unit area of the sample mounting surface is 1 million pieces / cm ^{2 to} 8 million pieces / cm ² . More preferably.

Here, if the number of recesses formed per unit area of the sample mounting surface exceeds 8 million / cm ² , the size of the photoelectric conversion unit corresponding to each recess becomes small, and the analysis sensitivity of the photoelectric conversion unit The tendency to decrease is increased. In addition, when the photoelectric conversion unit becomes small in this way, the tendency of increasing the ratio of dark current generated in the photoelectric conversion unit increases. In this case, the volume of the liquid droplets stored in the recesses also tends to be small, the amount of fluorescent light emission is reduced, the analytical sensitivity is lowered, and the light receiving time (analytical time) is reduced to compensate for this decline. There is a tendency to take measures such as increasing the number of times or increasing the number of samplings.

In the present invention, the thickness of the monomolecular film is such that the reaction field can be miniaturized and densified, in particular, the volume of the concave portion of the sample mounting surface is in the range of 0.01 pL to 1 pL, and the sample The thickness of the recesses formed on the mounting surface is not particularly limited as long as the thickness allows the density to be 10,000 pieces / cm ² or more. For example, it may be equal to the size (length) of one organic molecule (single molecule) after covalently bonding to the sample mounting surface, or may have a thickness exceeding the size of one organic molecule. . However, from the viewpoint of more reliably obtaining the effects of the present invention, the thickness of the monomolecular film is preferably 0.5 nm to 50 nm, more preferably 0.5 nm to 10 nm, and 0.5 nm to More preferably, it is 5 nm.

More specifically, when the volume of droplets contained in the recesses is 0.01 pL to 1000 pL and the density of the recesses formed on the sample placement surface is 10,000 / cm ² or more. The volume of the recess is preferably 2 × 10 ⁻⁶ pL to 1 pL, the area of the bottom surface (second region) of the recess is preferably 4 μm ^{2 to} 17500 μm ^2, and the thickness of the monomolecular film is It is preferable to adjust so as to realize the size and area of the recess.

In addition, when the volume of the droplet accommodated in the recess is 0.01 pL to 35 pL and the density of the recess formed on the sample mounting surface is 100,000 / cm ² or more, the volume of the recess Is preferably 2 × 10 ⁻⁶ pL to ¹ × 10 ⁻¹ pL, the area of the bottom surface (second region) of the recess is preferably 4 μm ^{2 to} 1600 μm ^2, and the thickness of the monomolecular film is The thickness is preferably 0.5 nm to 50 nm.

Furthermore, when the volume of the liquid droplets stored in the recesses is 0.01 pL to 1.2 pL and the density of the recesses formed on the sample mounting surface is 1 million pieces / cm ² or more, the recesses it is preferred that the volume between ^{2 × 10 -6 pL~2 × 10 -3} pL, it is preferable that the area of the bottom surface of the recess (the second region) and 4μm ² ~155μm ^2, the thickness of the monolayer The thickness is preferably 0.5 nm to 10 nm. In this case, it is also preferable that the volume of the recess is further reduced to 2 × 10 ⁻⁶ pL to 7 × 10 ⁻⁴ pL, and the area of the bottom surface (second region) of the recess is in the same range as above. The thickness of the monomolecular film is preferably 0.5 nm to 5 nm.

Further, in the present invention, each of the volume of the droplet accommodated in the above-described recess, the density of the recess formed on the sample mounting surface, the volume of the recess, the area of the bottom of the recess, and the thickness of the monomolecular film From the viewpoint of easily and surely forming a sample inspection apparatus that satisfies the conditions and obtaining the effect of the present invention more reliably, the monomolecular film is formed as a substrate at a terminal portion bonded to an organic molecule, -OH, -NH ₂ , A substrate having a hydrophilic first characteristic group having at least one structure selected from the group consisting of N—H and —SH on the sample mounting surface is used as an organic molecule, and the first characteristic An organic molecule having a second characteristic group capable of undergoing a condensation reaction with a group at one end of the molecular chain and a hydrophobic third characteristic group at the other end of the molecular chain. A monomolecular film type that allows the first characteristic group and the second characteristic group to proceed with a condensation reaction with each other. It is preferably formed through the steps.

  As a result of intensive studies, the present inventors have used a substrate having the above-described configuration and an organic molecule having the above-described configuration to easily and reliably form a monomolecular film having an extremely thin and uniform thickness. Found to be effective to form. By forming the monomolecular film through this monomolecular film forming step, as described above, it is possible to easily and reliably form an extremely minute reaction field at a high density on the sample placement surface. Here, the substrate may not have the first characteristic group in advance on the sample mounting surface as long as the substrate has a configuration capable of binding the first characteristic group to the sample mounting surface. In the monomolecular film forming step, a substrate in which the first characteristic group is bonded to the sample mounting surface by a surface modification process may be used.

  Further, in the monomolecular film formation step, the condensation reaction proceeds in the container by bringing the organic molecule-containing liquid obtained by adding organic molecules into the aprotic solvent and the sample mounting surface of the substrate in contact with each other. The first step is included, and in the first step, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed in terms of a relative humidity value at 22 ° C., the relative humidity value is It is preferable to adjust so that it may be 35% or less.

  As a result of intensive research, the present inventors have conducted an organic molecule that undergoes a condensation reaction with the sample mounting surface having the above-mentioned first characteristic group by forming with a monomolecular film under the conditions of the above-mentioned first step. Thus, it was found that a monomolecular film having an extremely thin and uniform thickness can be easily and reliably formed even when an organic molecule having a very high reactivity and easily polymerized is used. . Here, the “aprotic solvent” used for the organic molecule-containing liquid in the first step refers to a solvent that can dissolve or disperse the organic molecules among the aprotic solvents. However, from the viewpoint of easily and accurately obtaining a monomolecular film having a uniform thickness, the aprotic solvent used in the first step is preferably a solvent capable of dissolving organic molecules.

  In the first step, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed in terms of relative humidity at 22 ° C. and exceeds 35%, the sample is placed during production. The polymerization reaction between organic molecules is more likely to proceed than the condensation reaction between the surface and organic molecules, and a lump made of a polymer made from organic molecules is easily grown to the micron order, and has an extremely thin and uniform thickness. There is a greater tendency for it to be difficult to obtain a monomolecular film. From the viewpoint of more reliably obtaining a monomolecular film having an extremely thin and uniform thickness, the water content in the gas phase is more preferably 25% or less when expressed in terms of relative humidity at 22 ° C. More preferably, it is 5% or less.

  Furthermore, a glove box is preferable as the container used in the first step. In the first step, the constituent gas constituting the gas phase with the moisture content adjusted to the above range is preferably at least one gas selected from the group consisting of a rare gas and a nitrogen gas. However, in the first step, if conditions under which the progress of the oxidation reaction of the organic molecule or the aprotic solvent and the progress of the deterioration due to the oxidation of the monomolecular film can be sufficiently suppressed, air is used. Also good. For example, air can be used by adjusting the temperature of the gas phase during the first step, the temperature of the organic molecule-containing liquid, the concentration of the organic molecules, the contact time between the organic molecules and the sample mounting surface, and the like.

  A detailed description of the organic molecules used as the raw material of the monomolecular film in the present invention will be described later.

Further, the present invention incorporates two or more photoelectric conversion units in a two-dimensionally arranged state, and at least one of two opposing main surfaces has —OH, —NH ₂ , A substrate forming step of forming a substrate to which a hydrophilic first characteristic group having at least one structure selected from the group consisting of ═N—H and —SH is bonded;
The main surface of the substrate to which the first characteristic group is bonded is used as a sample mounting surface for independently fixing two or more droplets containing the measurement object to be analyzed, and the sample mounting surface is a first area covered by the hydrophobicity of the first monomolecular film, inside the total peripheral portion not covered with the first monomolecular film is more than one second surrounded by the first region An area dividing step for dividing into at least areas;
By selectively forming the first monomolecular film only in the first region, two or more recesses for independently containing two or more droplets can be provided for each of the two or more photoelectric conversion units. A monomolecular film forming step provided to correspond one-to-one,
At least
The bottom surface of the recess is further coated with a second monomolecular film made of a light-transmitting material,
A thickness of the second monomolecular film is equal to or less than a thickness of the first monomolecular film covering the first region;
A hydrophilic fourth characteristic group capable of at least hydrogen bonding with the solvent contained in the droplet is bonded to the surface of the second monomolecular film,
The second monomolecular film is formed using an organic molecule, and is fixed to the sample mounting surface by a covalent bond,
In the monomolecular film formation process,
An organic molecule having a second characteristic group capable of undergoing a condensation reaction with the first characteristic group at one end of the molecular chain and having a hydrophobic third characteristic group at the other end of the molecular chain as a raw material for the first monomolecular film Use as
The organic molecule is placed on the sample so that the bottom surface of the obtained recess is formed by the second region, and the outer peripheral portion including the entire inner surface of the recess is formed by the first monomolecular film covering the first region. Contacting the surface, allowing the condensation reaction between the first characteristic group and the second characteristic group to proceed, and forming the first monomolecular film in a state of being covalently fixed to the sample mounting surface;
A method for manufacturing a sample inspection apparatus is provided.

  According to the method for manufacturing a sample inspection apparatus of the present invention, the above-described sample inspection apparatus of the present invention can be obtained easily and reliably.

According to the sample inspection apparatus of the present invention, recesses having a very small volume are formed at a very high density on the sample mounting surface. Therefore, miniaturization and high density of the reaction field (particularly, the volume of the reaction field is 1000 pL or less and the density of the reaction field is 10,000 / cm ² or more) can be easily and reliably achieved. .

  In addition, since the sample inspection apparatus of the present invention has a configuration in which the outer peripheral portion (first region) including the entire inner surface of the recess is covered with the monomolecular film, the distance between adjacent recesses can be greatly increased. Even if it is made small, mixing of the droplets accommodated in the recess is surely prevented. At this time, since the bottom surface (second region) of the recess has hydrophilicity, the liquid droplets accommodated in the recess are reliably fixed to the bottom surface of the recess by hydrogen bonding. That is, in the sample inspection apparatus of the present invention, the occurrence of contamination is reliably prevented even if the distance between adjacent recesses is very small. As a result, the sample inspection apparatus of the present invention can ensure sufficient analysis accuracy even when the reaction field is miniaturized and densified as described above.

  Furthermore, since the sample inspection apparatus of the present invention has a configuration capable of easily and reliably miniaturizing and increasing the density of the reaction field as described above, it can be easily reduced in size. In the sample inspection apparatus of the present invention, the monomolecular film is fixed to the first region of the sample placement surface by a covalent bond. Therefore, it is possible to sufficiently prevent all or partial peeling of the monomolecular film from the substrate. Therefore, the sample inspection apparatus of the present invention can obtain sufficient reliability even when used repeatedly over a long period of time or when stored for a long period of time.

  Further, according to the method for manufacturing the sample inspection device of the present invention, the sample inspection device of the present invention can be manufactured easily and reliably.

  Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the drawings. In the following description, the same or corresponding parts are denoted by the same reference numerals, and redundant description is omitted.

[First embodiment]
FIG. 1 is a perspective view showing a first embodiment of the sample inspection apparatus of the present invention. FIG. 2 is a partial sectional view schematically showing the basic configuration of the sample inspection apparatus shown in FIG. As shown in FIGS. 1 and 2, the sample inspection apparatus 100 is provided with a sample mounting surface provided with two or more recesses 3 for independently containing two or more droplets containing a measurement object to be analyzed. It has a configuration including at least a plate-like substrate 1 (semiconductor integrated circuit substrate) having F1, and a CPU 4, a memory unit 5, and a data input / output unit 6 provided integrally on a side surface of the substrate 1. Yes.

  As shown in FIG. 2, the sample mounting surface F1 has a first region F11 covered with the hydrophobic monomolecular film 2 and a hydrophilic area 2 that is not covered with the monomolecular film 2 and has a hydrophilic property. It is at least divided into the second region F12. The bottom surface of each recess 3 is formed by the second region F12, and the entire outer peripheral portion including the entire inner surface of each recess 3 is formed by the monomolecular film 2 covering the first region F11. Yes. Further, in the case of the sample inspection apparatus 100, all the regions other than the second region F12 in the sample mounting surface F1 are the first regions F11, and the first regions F11 are all monomolecular. The structure is covered with the film 2. Furthermore, the monomolecular film 2 is formed using organic molecules, and is fixed to the sample mounting surface F1 by a covalent bond.

  Further, inside the substrate 1, there are one pair of photoelectric conversion units 70 that can independently photoelectrically convert light emitted from droplets accommodated in the respective recesses 3 for each of the two or more recesses 3. 1 so as to correspond. Further, in the case of this sample inspection apparatus 100, one photoelectric conversion unit 70 has a configuration composed of one photodiode (photoelectric conversion element).

  Further, in the case of this sample inspection apparatus 100, the substrate 1 has a sample mounting surface F1, and has a protective layer 12 (uppermost layer) having light transmittance with respect to light emitted from the droplets, and the protective layer 12 And a semiconductor layer 7 including at least the photoelectric conversion unit 70.

  More specifically, as shown in FIG. 2, in the case of this sample inspection apparatus 100, the substrate 1 is located between the protective layer 12 and the semiconductor layer 7 from the semiconductor layer 7 side toward the protective layer 12 side. When viewed, the inorganic oxide layer 8, the interlayer insulating film 9, and the insulating film 10 are mainly stacked in this order. Further, a color filter 11 is further disposed in a region between the insulating film 10 and the protective layer 12 and immediately below each recess 3 (a region immediately above the photoelectric conversion unit). Further, a light shielding film 15 is further disposed between the interlayer insulating film 9 and the insulating film 10 and in a region other than the region directly below each recess 3 (a region other than the region immediately above the photoelectric conversion unit). Yes. Further, the first electrode 13 is embedded in the inorganic oxide layer 8, and the second electrode 14 is provided in a region between the inorganic oxide layer 8 and the interlayer insulating film 9 and in the vicinity of the first electrode 13. Is arranged.

The inorganic oxide layer 8 is a layer provided for forming the first electrode 13 and the second electrode 14. The inorganic oxide layer 8 is a layer disposed in an integrated state on the upper surface of the semiconductor layer 7 and has a light-transmitting property with respect to light emitted from the droplets stored in the recess 3. Note that a region made of silicon nitride may be formed in the lower region of the first electrode 13 of the inorganic oxide layer 8. The inorganic oxide constituting the inorganic oxide layer 8 is not particularly limited as long as it has light transmittance with respect to light emitted from the droplets accommodated in the recess 3. An example is SiO ₂ .

The interlayer insulating film 9 is a layer provided to prevent electrical contact between the light shielding film 15 and the second electrode 14. The insulator material constituting the interlayer insulating film 9 is not particularly limited as long as it has light transmittance with respect to light emitted from the droplets accommodated in the recess 3. An example is SiO ₂ .

  The insulating film 10 is a layer for separating the color filter 11 and the light shielding film 15 so as not to contact each other. The insulator material constituting the insulating film 10 is not particularly limited as long as it has light transmission properties with respect to light emitted from the droplets stored in the recess 3.

The protective layer 12 (the uppermost layer) is a layer that has the sample placement surface F1 and in which the recess 3 is formed. The protective layer 12 is a layer provided to secure the space and protect the color filter 11 when the color filter 11 is provided. The constituent material constituting the protective layer 12 is not particularly limited as long as it has a light-transmitting property with respect to light emitted from the liquid droplets stored in the recess 3. An example is SiO ₂ .

  The light shielding film 15 is a film provided to prevent light emitted from the liquid droplets stored in the recess 3 from entering the region of the semiconductor layer 7 other than the pn junction surface of the photoelectric conversion unit 70. The constituent material constituting the protective layer 12 is not particularly limited as long as it has light impermeability capable of shielding light emitted from the droplets accommodated in the recess 3. For example, a metal thin film (a metal thin film such as aluminum or chromium) can be used.

  The color filter 11 is a filter used for selectively allowing only light having a specific wavelength out of the light emitted from the liquid droplets stored in the recess 3 to enter the photoelectric conversion unit 70. The color filter 11 only needs to have a light-transmitting property with respect to light having a specific wavelength to be detected among the light emitted from the liquid droplets stored in the concave portion 3, and the constituent material thereof is also the above-described specific material. It is appropriately selected according to light having a wavelength.

The first electrode 13 is an electrode provided to correspond to the photoelectric conversion unit 70 on a one-to-one basis. The first electrode 13 is used for independently controlling the potential of the pn junction surface of the photoelectric conversion unit 70. Further, a resistor (not shown) is electrically connected to the first electrode 13 in order to independently control the potential of the pn junction surface of the photoelectric conversion unit 70. Note that the register is embedded in the substrate 1. The first electrode 13 controlled by the register changes the potential of the pn junction surface of the photodiode, and electrons generated by the photodiode move to the p ⁻ isolation region 76 and further to the vertical transfer portion n-type region 78. You will be able to move.

  The second electrode 14 is also an electrode provided so as to correspond to the photoelectric conversion unit 70 on a one-to-one basis. The second electrode 14 is an electrode for moving charges in the vertical transfer portion n-type region 78. Note that the second electrode 14 is not an essential component. For example, the first electrode 13 may also function as the second electrode.

  The semiconductor layer 7 has a configuration including an n-type silicon substrate 71 and a first p-well 72 disposed on the n-type silicon substrate 71. Furthermore, an n-type semiconductor region 74 is provided in a region of the upper surface of the first p-well 72 that is directly below the recess 3. A photodiode (photoelectric conversion element) including the n-type semiconductor region 74 and the first p well 72 functions as a photoelectric conversion unit 70 having a pn junction surface.

A p ⁻ isolation region 76 is provided on a region adjacent to the photoelectric conversion unit 70 on the upper surface of the first p well 72. Further, a vertical transfer portion n-type region 78 is provided above a region adjacent to the p ⁻ isolation region 76 on the upper surface of the first p well 72. Further, a second p well 77 is provided between the first p well 72 and the vertical transfer portion n-type region 78. Further, in the region on the upper surface of the first p well 72, the region between the units consisting of the photoelectric conversion unit 70, the p ⁻ isolation region 76, the vertical transfer unit n type region 78, and the second p well 77 is included. A p ⁺ separation region 73 is provided to separate the units.

The concentration of the introduced impurity in the first p well 72 and the second p well 77 is, for example, a relatively low concentration in the first p well 72 and a relatively high concentration in the second p well 77. The second p-well 77 is usually provided to assist in reducing the efficiency of charge transfer accompanying the downsizing of the n-type semiconductor region 74. Further, the p ⁺ isolation region 73 and the p ⁻ isolation region 76 are regions for normally separating the photodiode from the vertical transfer portion n-type region 78. The p ⁻ isolation region 76 is for performing charge transfer from the photoelectric conversion unit to the vertical transfer unit n-type region 78 when the potential is read by the first electrode 13. The p ⁺ -type region 75 on the n-type semiconductor region 74 is provided in order to prevent the incidence of ultraviolet rays on the photoelectric conversion unit when the incidence of ultraviolet rays occurs and the incidence of ultraviolet rays is inconvenient. .

  The vertical transfer unit n-type region 78 is parallel to the column direction of the photoelectric conversion unit 70 (two-dimensional array of 5 rows × 7 columns in the sample inspection apparatus 100 of FIG. 1) that is two-dimensionally arranged in β rows × γ columns. This is a columnar region that is arranged, one for each row. Note that “a state in which β rows × γ columns are two-dimensionally arranged” means that γ photoelectric conversion units 70 are linearly arranged at equal intervals in the row direction on the plane, and are in the same plane as the plane. A state is shown in which β photoelectric conversion units 70 are linearly arranged at equal intervals in the column direction (direction substantially orthogonal to the row direction). By controlling the potential of the pn junction surface by the first electrode 13 and the second electrode 14, the charges generated in the photoelectric conversion unit 70 are not mixed or dispersed, and the vertical transfer portion n-type region 78 moves in the vertical direction. It is moved in the (row direction). Note that the concave portions 3 corresponding to the photoelectric conversion units 70 are two-dimensionally arranged in β rows × γ columns on the sample mounting surface F1.

  Further, in the case of this substrate 1, a columnar shape arranged on the outer edge of the semiconductor layer 7 on the same plane as the γ vertical transfer portion n-type regions 78 so as to be orthogonal to the γ vertical transfer portion n-type regions 78. One horizontal transfer portion n-type region (not shown) is provided. The charges moving in the vertical direction in the vertical transfer portion n-type region 78 are moved in the horizontal direction (row direction) by moving to the horizontal transfer portion n-type region.

  Further, a vertical transfer unit operation signal input terminal (not shown) is connected to each of the γ vertical transfer unit n-type regions 78. The other end of the vertical transfer unit operation signal input terminal is electrically connected to the register (not shown) described above. Then, by applying a voltage to the first electrode 13 using the vertical transfer unit operation signal input terminal, charges generated on the pn junction surface of the photoelectric conversion unit 70 are moved to the vertical transfer unit n-type region 78, and then By applying a voltage to the second electrode 14, the electric charge can be moved in the vertical direction in the vertical transfer portion n-type region 78. By sequentially applying such a voltage to the first electrode and the second electrode, the charges generated at the pn junction surface of the photoelectric conversion unit 70 are moved in the vertical direction in the n-type region 78 of the vertical transfer unit. Can do.

  Further, a horizontal transfer unit operation signal input terminal (not shown) is connected to the horizontal transfer unit n-type region. The other end of the horizontal transfer unit operation signal input terminal is electrically connected to the register (not shown) described above. Then, by sequentially applying a voltage to the first electrode 13 and the second electrode 14 using the horizontal transfer unit operation signal input terminal, charges generated at the pn junction surface of the photoelectric conversion unit 70 are transferred to the horizontal transfer unit. It can be moved horizontally in the n-type region.

  Further, in the case of this substrate 1, an output amplifier (not shown) for amplifying and outputting the received light information and a signal output terminal (not shown) are further provided.

  The sample inspection apparatus 100 also includes an amplification transistor for amplifying the charge signal photoelectrically converted by the photodiode, a reset transistor for resetting the charge of the photodiode after the charge signal is read, and a metal wiring attached to them. , Power lines (both not shown) are provided.

  The memory unit 5 has a configuration in which information of analysis information {light information (wavelength or color, intensity, light amount, etc.) obtained by the luminescence reaction} obtained from the photoelectric conversion unit 70 is sequentially accumulated. The memory unit 5 stores reference data serving as a reference for performing analysis such as identification or quantification of a measurement object to be analyzed that exists in the recess based on analysis information obtained from the photoelectric conversion unit 70. ing. For example, when gene analysis is performed using a probe gene, it is information such as which type of probe gene is previously arranged in the concave portion 3 at which position among the plurality of concave portions 3 on the sample placement surface F1. . Further, for example, data for specifying the position of each concave portion 3 such as which concave portion 3 has detected light emission is also stored. Further, when the color filter 11 is used, reference data for specifying the wavelength or color is also stored. Further, in the memory unit 5, positioning of the droplet discharge means for dropping droplets into the recess 3 (for example, a droplet supply unit 40 provided in the sample inspection apparatus 101 of the second embodiment described later) with respect to the recess 3. The reference data for performing correctly may be stored. For example, reference data for moving the droplet discharge means relative to the substrate 1 such as the distance between the recesses 3 and the area of the bottom surface of the recesses 3. Further, the memory unit 5 may store a program for the CPU 4 to perform calculation processing (analysis calculation processing or calculation processing for positioning) based on the reference data. Further, the memory unit 5 may be provided with a function for storing the results performed by the CPU 4. Furthermore, the memory unit 5 may store programs and data necessary for exchanging data and information with the outside.

  The CPU 4 is connected to the memory unit 5 and the data input / output unit 6, compares the analysis information obtained from the photoelectric conversion unit 70 with the reference data stored in the memory unit 5, and performs these calculations. An arithmetic process is performed based on a program for performing the process. Then, an analysis result relating to analysis such as identification or quantification of the measurement object to be analyzed is obtained. Further, the CPU 4 may be configured to statistically process the validity and the analysis result of each concave portion 3 based on the calculation processing result, and make a determination such as estimation of the component composition of the measurement object. Further, the CPU 4 has a configuration for accurately controlling the positioning with respect to the recess 3 by controlling the droplet discharge means for dropping the droplet into the recess 3 based on the reference data for positioning stored in the memory unit 5. You may do it. Further, the CPU 4 may have a configuration for correcting data obtained from the photoelectric conversion unit 70. Further, the CPU 4 may be provided with a calculation function for causing a person to recognize analysis results, analysis results obtained by statistical means, and estimation and judgment obtained from these results. Further, the CPU 4 may be provided with a function for sensing an abnormality related to measurement, analysis, etc., and performing appropriate avoidance, and a function for warning the outside.

  The data input / output unit 6 is electrically connected to an information output device such as a monitor and an information input / output device such as a computer, and outputs data such as input of reference data to the memory unit 5 for outputting and analyzing analysis data. Exchange. The data input / output unit 6 may also serve to supply power as necessary.

  The formation position of the photoelectric conversion unit 70 in the substrate 1 is not particularly limited as long as the light emitted from the droplet fixed to the corresponding recess 3 can be detected. However, the effective optical path from the recess 3 to the photoelectric conversion unit 70 is sufficient. From the viewpoint of shortening and ensuring sufficient analysis sensitivity and reducing the size of the sample inspection apparatus 100, as shown in FIG. 2, the photoelectric conversion unit 70 is disposed almost directly below the corresponding recess 3. Preferably it is.

  Each concave portion 3 formed on the sample mounting surface F1 is for containing a droplet. As described above, since the recess 3 is formed using the monomolecular film 2 having an extremely thin film thickness, the recess 3 has an extremely small volume that can easily accommodate a minute droplet having a volume of 1000 pL or less. can do.

  A hydrophilic first characteristic group, which will be described later, is bonded to the bottom surface (second region F12) of each recess 3. Thereby, the droplet accommodated in the recess 3 is securely fixed to the bottom surface of the recess 3 by hydrogen bonding, and is sufficiently prevented from diffusing from the recess 3 once accommodated to the outside. The hydrophilic first characteristic group is also bonded to the first region F11 of the sample mounting surface F1 before the monomolecular film 2 is formed, and the monomolecular film 2 is formed on the first region F11. In the formation of the film, the condensation reaction between the organic molecules of the raw material and the hydrophilic first characteristic group proceeds, and the resulting monomolecular film 2 is fixed in a state of being covalently bonded to the sample mounting surface F1. Become.

The hydrophilic first characteristic group bonded to the sample mounting surface F1 preferably has active hydrogen capable of undergoing a condensation reaction with organic molecules. As the first characteristic group having an organic molecule and a condensation reactive active hydrogen, the terminal portion of the side to be bonded with an organic molecule by a condensation reaction of the _{above, -OH, -NH 2, = N} -H, and, -SH It is more preferable to have at least one structure selected from the group consisting of: In the first characteristic group other than the above, as the first characteristic group having an organic molecule and a condensation reactive active _{hydrogen, -SO 3 H, -SO 2 H} , -PO 3 H, -PO 3 H 2 and - More preferably, it has at least one structure selected from the group consisting of CO ₂ H.

Note that the first characteristic group having active hydrogen capable of undergoing a condensation reaction with an organic molecule is not only in a state where the whole is exposed from the sample mounting surface F1, but also includes only active hydrogen or -OH, -SH, = N containing active hydrogen. Only the portions of —H and —NH ₂ may be exposed from the sample mounting surface F 1, and a portion other than active hydrogen may be included in the substrate 1. For example, when a portion other than active hydrogen is included in the substrate 1, the portion other than active hydrogen may be bonded to a constituent element of the substrate 1. More specifically, for example, in a case where the sample placement surface F1 near the substrate 1 is made of a metal oxide having light transmission properties as the material, if the first characteristic group is -PO ₃ H , -PO ₃ H may be entirely exposed from the sample mounting surface F1, or only -OH of -PO ₃ H may be exposed, and the -PO _2- portion may be included in the substrate. Good. -PO ₂ contained within the substrate - the portion of -PO ₂ - may remain in the state, the oxygen bonded to P is bonded to a metal atom (metal ions) M of the metal oxides in bulk, for example, - It may be in a state having a structure such as POM.

  The covalent bond formed between the first region F11 of the substrate 1 and the monomolecular film 2 by the condensation reaction between the first characteristic group and the second characteristic group of the organic molecule described later is the sample mounting surface F1. Depending on the structure of the hydrophilic first characteristic group bonded to and the type of organic molecule used as the raw material of the monomolecular film, from the viewpoint of ease of production, -Si-O-, -Si-N-, And it is preferably a bond containing at least one structure selected from the group consisting of -Si-S-.

  Thus, the monomolecular film 2 is extremely firmly fixed on the sample mounting surface F1. Therefore, even if the distance between the adjacent recesses 3 on the sample mounting surface F1 (the area of the first region covered with the monomolecular film) is very small, the monomolecular film 2 that separates the adjacent recesses is The sample is firmly bonded on the sample mounting surface F1, and the separation thereof is sufficiently prevented.

Therefore, even when the distance between the adjacent recesses 3 is made very small and the density of the reaction field (droplets accommodated in the recesses) is 10,000 / cm ² or more, it is accommodated in the recesses 3. Mixing between the droplets is sufficiently prevented (contamination is surely prevented). Therefore, the sample inspection apparatus 100 can ensure sufficient analysis accuracy.

  In addition, the size (volume), shape, arrangement position, and arrangement interval of each recess 3 can be easily adjusted by the patterning method of the monomolecular film 2, and the viewpoint of obtaining the effect of the present invention more reliably. Can be set as follows.

That is, in the sample inspection apparatus 100, from the viewpoint of miniaturizing and increasing the density of the reaction field (droplets accommodated in the recesses), the reaction field volume is preferably set to 1000 pL or less, and the density of the reaction field is set to 1. From the viewpoint of setting it to 10,000 / cm ² or more, the volume of the droplet accommodated in the recess 3 is preferably 0.01 pL to 1000 pL, more preferably 0.01 pL to 35 pL, and 0.01 pL to More preferably, it is 1.2 pL.

  Further, as described above, by setting the configuration of the recess 3 to the above-described configuration, the liquid droplet dropped on the bottom surface of the recess has a substantially hemispherical shape or a substantially columnar shape having a substantially hemispherical tip. It will be fixed in the state of presenting. Therefore, compared with the geometric volume of the recessed part 3, the volume of the droplet actually accommodated in the recessed part 3 (droplet fixed on the bottom face of the recessed part 3) can be made very large. Therefore, even when the concave portion 3 having a small volume is formed, it is possible to use an amount of droplets that can provide sufficient analysis sensitivity. In addition, the liquid droplets dropped on the bottom surface of the recess 3 are fixed in a substantially hemispherical shape or a substantially columnar shape with the tip portion being substantially hemispherical. For this reason, the droplet fixed on the bottom surface of the recess 3 has a shape advantageous for condensing, and from this viewpoint, the sample analyzer 100 can easily obtain sufficient analysis sensitivity. Become.

For this reason, when the volume of the liquid droplets stored in the recesses is 0.01 pL to 1000 pL, the volume of the recesses 3 can be made sufficiently smaller than this. Specifically, in this case, the volume of the recess 3 is preferably 2 × 10 ⁻⁶ pL to 1 pL.

From the same viewpoint as described above, when the volume of the liquid droplets contained in the recess is 0.01 pL to 35 pL, the volume of the recess is preferably 2 × 10 ⁻⁶ pL to ¹ × 10 ⁻¹ pL. Furthermore, from the same viewpoint as described above, when the volume of the liquid droplets contained in the recess is 0.01 pL to 1.2 pL, the volume of the recess is 2 × 10 ⁻⁶ pL to 2 × 10 ⁻³ pL. Is preferably 2 × 10 ⁻⁶ pL to 7 × 10 ⁻⁴ pL.

From the viewpoint of miniaturizing and increasing the density of the reaction field (droplets contained in the recesses), preferably the volume of the reaction field is set to 1000 pL or less, and the density of the reaction field is set to 10,000 pieces / cm ² or more. Therefore, it is preferable that the number of the concave portions 3 formed per unit area of the sample mounting surface F1 is 10,000 / cm ² or more. Moreover, from the viewpoint of setting the density of the reaction field to 100,000 pieces / cm ² or more, the number of the concave portions 3 formed per unit area of the sample mounting surface F1 is more preferably 100,000 pieces / cm ² or more. preferable. Furthermore, from the viewpoint of setting the density of the reaction field to 1 million pieces / cm ² or more, the number of recesses formed per unit area of the sample mounting surface F1 is 1 million pieces / cm ^{2 to} 8 million pieces / cm ^2. More preferably.

  In order to sufficiently prevent the occurrence of contamination, the monomolecular film 2 preferably has sufficient hydrophobicity. Therefore, from this viewpoint, the monomolecular film 2 preferably has the following characteristics. That is, when a 5.3 μL water droplet is dropped on the surface of the monomolecular film 2 at 20 ° C., the contact angle of the water droplet with respect to the surface is preferably 80 to 180 °, and preferably 90 to 180 °. More preferably, it is more preferably 100 to 160 °. The critical surface energy of the monomolecular film is 72 mN / m or less at 20 ° C., preferably in the range of 8 to 72 mN / m, more preferably in the range of 8 to 25 mN / m. In addition, a contact angle can be measured with the measuring method prescribed | regulated to JISR3257: 1999, for example.

The thickness of the monomolecular film 2 is such that the reaction field can be miniaturized and densified, in particular, the volume of the concave portion of the sample mounting surface is in the range of 0.01 pL to 1 pL, and the sample mounting surface There is no particular limitation as long as the density of the recesses formed on F1 can be 10,000 pieces / cm ² or more. For example, it may be equal to the size (length) of one organic molecule (single molecule) after covalently bonding to the sample mounting surface F1, and has a thickness exceeding the size of one organic molecule. Also good. However, from the viewpoint of obtaining the effect of the present invention more reliably, the thickness of the monomolecular film 2 is preferably 0.5 nm to 50 nm, more preferably 0.5 nm to 10 nm, and 0.5 nm. More preferably, it is ˜5 nm.

More specifically, when the volume of the droplet accommodated in the recess 3 is 0.01 pL to 1000 pL and the density of the recess 3 is 10,000 / cm ² or more, the volume of the recess 3 is 2 X10 ⁻⁶ pL to 1 pL is preferable, the area of the bottom surface (second region F12) of the recess is preferably 4 μm ^{2 to} 17500 μm ^2, and the thickness of the monomolecular film 3 is the thickness of the recess 3 It is preferable to adjust so as to realize the size of the volume and the size of the area.

Further, when the volume of the droplets stored in the recess 3 is 0.01 pL to 35 pL and the density of the recesses formed on the sample mounting surface F1 is 100,000 / cm ² or more, the recess it is preferred to 3 of the volume and ^{2 × 10 -6 pL~1 × 10 -1} pL, the area of the bottom surface of the recess 3 (second area F12) preferably in the 4μm ² ~1600μm ^2, monomolecular The thickness of the film 2 is preferably 0.5 nm to 50 nm.

Furthermore, when the volume of the droplets accommodated in the recess 3 is 0.01 pL to 1.2 pL and the density of the recesses formed on the sample mounting surface F1 is 1 million pieces / cm ² or more. , it is preferred that the volume of the recess 3 and ^{2 × 10 -6 pL~2 × 10 -3} pL, the area of the bottom surface of the recess 3 (second area F12) preferably in the 4 [mu] m ² ～155Myuemu ^2, The thickness of the monomolecular film 2 is preferably 0.5 nm to 10 nm. In this case, it is also preferable to further reduce the volume of the recess 3 to 2 × 10 ⁻⁶ pL to 7 × 10 ⁻⁴ pL, and the area of the bottom surface (second region) of the recess 3 is the same as above. As a range, the thickness of the monomolecular film 2 is preferably 0.5 nm to 5 nm.

Moreover, from the viewpoint of preventing contamination more reliably, the width of the monomolecular film 2 formed between the adjacent recesses 3 is preferably 0.1 μm or more, and more preferably 1 to 100 μm. . Even when the width of the monomolecular film 2 is set, the volume of the recess 3 can be easily set in the range of 0.01 pL to 1 pL, and the density of the recess 3 can be easily set to 10,000 pieces / cm ² or more. When the member that separates the adjacent recesses 3 is only the monomolecular film 2, the interval between the adjacent recesses 3 is equal to the width of the monomolecular film 2, so the interval between the adjacent recesses 3 is 0.1 μm. It is preferable that it is above, and it is more preferable that it is 1-100 micrometers.

  The organic molecule has a second characteristic group capable of condensation reaction with the first characteristic group bonded to the sample mounting surface F1 at one end of the molecular chain, and a hydrophobic third characteristic group of the molecular chain. It is preferable to have it at the other end. As the condensation reaction between the second characteristic group and the first characteristic group bonded to the sample mounting surface F1 proceeds, the monomolecular film 2 is firmly fixed to the sample mounting surface F1 by covalent bonding. become.

  From the viewpoint of more reliably obtaining a monomolecular film having a thickness of 0.5 to 50 nm, the organic molecule preferably has any one of the following structures (i) to (iii).

  That is, (i) the organic molecule has a characteristic group represented by the following general formula (1) that can undergo a condensation reaction with the first characteristic group as the second characteristic group, and a methyl group as the third characteristic group , A halogen-substituted methyl group, a vinyl group, a cyclic ether group having 2 to 4 carbon atoms, a phenyl group, a halogen-substituted phenyl group, and a characteristic group selected from the group consisting of a cyano group, and It is preferable that a divalent organic group represented by the following general formula (2) is bonded between the two characteristic group and the third characteristic group.

−C _b E _2b − (2)
Here, in formula (1), Z ¹ is at least one selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. Or an atomic group. Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms. a represents an integer of 1 to 3. In formula (2), E represents at least one atom selected from the group consisting of H and F. b shows the integer of 2-22.

In addition, (ii) the organic molecule has a characteristic group represented by the following general formula (3), -O-, between carbons constituting the carbon skeleton of the divalent organic group represented by the general formula (2). It is preferable that at least one divalent characteristic group selected from the group consisting of —COO— and —C ₆ H ₄ — is further bonded. Here, in the following general formula (3), g and h each independently represent an integer of 1 to 3.

  Further, (iii) the organic molecule has, as the second characteristic group, a characteristic group represented by the following general formula (1) that can undergo a condensation reaction with the first characteristic group, and a methyl group as the third characteristic group , Halogen-substituted methyl group, vinyl group, cyclic ether group having 2 to 4 carbon atoms, phenyl group, halogen-substituted phenyl group, and two monovalent groups selected from the group consisting of cyano group And it is preferable to have the structure which the trivalent organic group represented by following General formula (4) couple | bonded between the 2nd characteristic group and the 3rd characteristic group.

Here, in formula (1), Z ¹ is at least one selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. Or an atomic group. Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms. a represents an integer of 1 to 3. In the formula (4), C _j L _2j is a characteristic group that is bonded to the second characteristic group. C _m G _2m and C _n J _2n are characteristic groups bonded to the third characteristic group. G, J and L may be the same or different and each represents at least one atom selected from the group consisting of H and F. j represents an integer of 1 to 18. m and n each independently represents an integer of 0 to 7.

  In the organic molecule having any one of the structures (i) to (iii), examples of the second characteristic group represented by (1) include a halogenated silyl group, an alkoxysilyl group, and an isocyanatesilyl group. Examples include various substituted silyl groups. In particular, a = 3 is preferable, and examples thereof include a trihalogenated silyl group, a trialkoxysilyl group, and a triisocyanatesilyl group.

  Examples of the halogen contained in the trihalogen silyl group include F, Cl, Br, and I. Of the trihalogensilyl groups, a chlorosilyl group is preferred. The alkoxy group in the trialkoxysilyl group preferably has 1 to 3 carbon atoms. Specific examples include a methoxysilyl group, an ethoxysilyl group, and a butoxysilyl group.

  Such organic molecules having various substituted silyl groups at their ends can form a covalent bond with the substrate surface as described above, and the formed monomolecular film 2 is firmly attached to the first region F11 by the covalent bond. Will be combined. For this reason, it can be said that the monomolecular film 2 is excellent in durability. Specifically, in the case of an organic molecule having a halogenated silyl group, a dehydrohalogenation reaction occurs between the active hydrogen contained in the first characteristic group in the first region F11 and an alkoxysilyl group is contained. If it is an organic molecule, a dealcoholization reaction occurs between the active hydrogen contained in the first characteristic group, and if it is an isocyanate silyl group, a deisocyanate reaction occurs between the active hydrogen contained in the first characteristic group. Each of the organic molecules and the substrate is covalently bonded by a siloxane bond (—Si—O—).

  Note that the covalent bond between the organic molecule and the substrate varies depending on the type of the first characteristic group. For example, when the first characteristic group is —NH, a —Si—N— bond is formed as the covalent bond, and —SH In this case, a —Si—S— bond is formed as a covalent bond.

Among the examples of the third characteristic group in the organic molecule having any one of the structures (i) to (iii), as the halogen-substituted methyl group, the monomolecular film 2 having sufficient hydrophobicity can be more reliably obtained. From the viewpoint of obtaining, CF ₃ —, CH ₂ Br—, and CH ₂ Cl— are preferable, and CF ₃ — is more preferable. The organic molecule whose third characteristic group is CF ₃ — has high orientation, and tends to increase the molecular density of the organic molecule when arrayed on the sample mounting surface F1 when the monomolecular film 2 is formed. Therefore, the monomolecular film 2 having hydrophobicity can be obtained more reliably.

Among the examples of the third characteristic group in the organic molecule having any one of the structures (i) to (iii), C ₂ H ₃ O— may be used as the cyclic ether group having 2 to 4 carbon atoms. preferable. When the third characteristic group is C ₂ H ₃ O—, the thickness of the monomolecular film 2 can be easily increased by utilizing the ring opening (addition) reaction of the epoxy group. At that time, it is possible to easily ensure sufficient uniformity of the film thickness.

For example, when the third characteristic group is C ₂ H ₃ O—, the ring-opening (addition) reaction of the epoxy group is caused to proceed by further bringing the alcohol into contact with the monomolecular film 2 formed at one end. It is possible to increase the thickness of the monomolecular film 2 by bonding a portion (hydrocarbon group) other than —OH of alcohol to the tip of the group. When the monomolecular film 2 having a relatively large film thickness is formed, if organic molecules having a size substantially equal to the film thickness is used, it is difficult to ensure sufficient film thickness uniformity. Therefore, the above method uses a relatively small organic molecule (an organic molecule whose third characteristic group is C ₂ H ₃ O—) and has a relatively large film thickness and high film thickness uniformity. Is useful in that it can be easily formed.

Furthermore, from the viewpoint of more surely obtaining the monomolecular film 2 having sufficient hydrophobicity among those listed as the third characteristic group in the organic molecule having any one of the structures (i) to (iii), halogen As the substituted phenyl group, C ₆ F ₅ — is preferable. The organic molecule whose third characteristic group is C ₆ F ₅ — has high orientation, and the molecular density of the organic molecule when arranged on the sample mounting surface F1 tends to be high when the monomolecular film 2 is formed. is there. Therefore, the monomolecular film 2 having hydrophobicity can be obtained more reliably.

  The organic molecule having the structure (i) preferably has a structure represented by any one of the following general formulas (20) to (29).

  Here, in formulas (20) to (29), Z is synonymous with Z in formula (1), a is synonymous with a in formula (1), and q is an integer of 2 to 22. . m and n are the conditions represented by the following formulas (I) to (III); 0 ≦ m ≦ 14 (I), 0 ≦ n ≦ 15 (II), 2 ≦ (m + n) ≦ 22 (III) ) Is an integer that simultaneously satisfies

  The organic molecule having the structure (ii) preferably has a structure represented by any one of the following general formulas (30) to (39).

Here, in the formulas (30) to (39), Z is synonymous with Z in the formula (1), a is synonymous with a in the formula (1), and A is represented by the general formula (3). Or at least one divalent characteristic group selected from the group consisting of —O—, —COO—, and —C ₆ H ₄ —. t shows the integer of 1-10. p shows the integer of 1-18. r and s are the conditions represented by the following formulas (IV) to (VI): 0 ≦ r ≦ 14 (IV), 0 ≦ s ≦ 15 (V), 2 ≦ (r + s) ≦ 22 (VI) ) Is an integer that simultaneously satisfies

  The organic molecule having the structure (iii) preferably has a structure represented by any one of the following general formulas (40) to (49).

  Here, in formulas (40) to (49), Z is synonymous with Z in formula (1), a is synonymous with a in formula (1), and t is an integer of 1 to 10. . p shows the integer of 1-18. r and s are the conditions represented by the following formulas (IV) to (VI): 0 ≦ r ≦ 14 (IV), 0 ≦ s ≦ 15 (V), 2 ≦ (r + s) ≦ 22 (VI) ) Is an integer that simultaneously satisfies

  Among the organic molecules represented by the general formulas (20) to (29), the viewpoint of ensuring sufficient uniformity of the monomolecular film 2 and the arrangement on the sample mounting surface F1 when the monomolecular film 2 is formed. From the viewpoint of sufficiently securing the molecular density of the organic molecules to be formed, the organic molecules represented by the general formula (20) and the general formula (21) are preferable.

Here, in the organic molecule represented by the general formula (20), the organic molecule represented by the following general formula (201) is preferable from the same viewpoint as described above.
CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (201)
Moreover, in the organic molecule represented by General formula (21), the organic molecule represented by following General formula (202) is preferable from a viewpoint similar to the above.
CH ₃ (CH ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (202)
Among the organic molecules represented by the general formula (201) and the general formula (202), the organic molecule represented by the general formula (201) is preferable from the viewpoint of obtaining sufficient hydrophobicity.

  Further, among the organic molecules represented by the general formulas (30) to (39), the viewpoint of sufficiently ensuring the uniformity of the monomolecular film 2 and the sample mounting surface F1 when the monomolecular film 2 is formed. From the viewpoint of sufficiently securing the molecular density of the organic molecules arranged in the organic molecules, the organic molecules represented by the general formula (30) and the general formula (31) are preferable.

Here, among the organic molecules represented by the general formula (30), organic molecules represented by the following general formulas (301) to (306) are preferable from the same viewpoint as described above.
CF ₃ (CF ₂ ) ₃ (CH ₂ ) ₂ O (CH ₂ ) ₁₅ SiCl ₃ (301)
CF ₃ COO (CH ₂ ) ₁₅ SiCl ₃ (302)
CF ₃ (CF ₂ ) ₃ (CH ₂ ) ₂ Si (CH ₃ ) ₂ (CH ₂ ) ₉ SiCl ₃ (303)
CF ₃ (CF ₂ ) ₇ Si (CH ₃ ) ₂ (CH ₂ ) ₉ SiCl ₃ (304)
CF ₃ (CH ₂ ) ₂ Si (CH ₃ ) ₂ (CH ₂ ) ₁₅ SiCl ₃ (305)
CF ₃ CH ₂ O (CH ₂ ) ₁₅ SiCl ₃ (306)
Moreover, in the organic molecule represented by General formula (31), the organic molecule represented by the following general formula (307)-(312) is preferable from a viewpoint similar to the above.
CH ₃ (CH ₂ ) ₅ O (CH ₂ ) ₁₅ SiCl ₃ (307)
CH ₃ COO (CH ₂ ) ₁₅ SiCl ₃ (308)
CH ₃ (CH ₂ ) ₅ Si (CH ₃ ) ₂ (CH ₂ ) ₉ SiCl ₃ (309)
CH ₃ (CH ₂ ) ₇ Si (CH ₃ ) ₂ (CH ₂ ) ₉ SiCl ₃ (310)
CH ₃ (CH ₂ ) ₂ Si (CH ₃ ) ₂ (CH ₂ ) ₁₅ SiCl ₃ (311)
CH ₃ CH ₂ O (CH ₂ ) ₁₅ SiCl ₃ (312)
Among the organic molecules represented by the above general formulas (201), (202), and (301) to (312), the monomolecular film 2 is selected from the viewpoint of sufficiently ensuring the uniformity of the monomolecular film 2. The organic molecule represented by the general formula (201) is most preferable from the viewpoints of sufficiently securing the molecular density of the organic molecules arranged on the sample mounting surface F1 and forming sufficient hydrophobicity.

  Since the monomolecular film 2 formed on the sample mounting surface F1 to which the above-described preferable first characteristic group is bonded using the above-described preferable organic molecules, it is easy to form with a uniform thickness. 3 can be sufficiently reduced, and the volume of each reaction field (droplet accommodated in each recess) can be easily made constant. Therefore, also from this viewpoint, the sample inspection apparatus 100 can ensure sufficient analysis accuracy.

  As other organic molecules described above, as long as the effects of the present invention can be obtained, JP-A-4-132737, JP-A-4-256466, JP-A-10-180179, and Organic molecules described in JP-A-4-359031 can be used.

The size of the plate-like substrate 1 is, for example, the number of recesses 3, the number of photoelectric conversion units 70 (or high electromagnetic conversion elements), the number of analysis samples (droplets) to be analyzed at once, and the number of analysis items. However, from the viewpoint of sufficiently reducing the size of the sample inspection apparatus 100, the area of the main surface including the sample placement surface F1 is 0.05 to 8 × 10 ⁻⁶ mm ² . It is preferable. Further, from the viewpoint of sufficiently miniaturizing the sample inspection apparatus 100, the thickness of the substrate 1 is preferably 0.05 to 1.5 mm, and more preferably 0.1 to 1.0 mm.

  In addition, as described above, the size (volume), shape, arrangement position, and arrangement interval of each recess 3 can be adjusted by the patterning method of the monomolecular film 2. Thereby, according to the fine two-dimensional arrangement pattern of the photoelectric conversion unit 70 provided in the substrate 1, the first region F11 and the second region F12 of the sample mounting surface F1 are appropriately classified, and the first The monomolecular film 2 can be easily selectively formed only in the region F11. Therefore, it is possible to easily form the recess 3 in a two-dimensional manner on the sample placement surface F1 with a very fine pattern. For this reason, it becomes possible for the photoelectric conversion unit 70 to efficiently detect the luminescence reaction that occurs in the droplets accommodated in the respective recesses 3.

Therefore, the sample inspection apparatus 100 has the recesses 3 having a very small volume formed on the sample mounting surface F1 with a very high density. Therefore, the reaction field is miniaturized and densified (particularly, the volume of the reaction field is reduced). It is possible to easily and reliably achieve 1000 pL or less and a reaction field density of 10,000 cells / cm ² or more. Further, the sample inspection apparatus 100 can ensure sufficient analysis accuracy. Furthermore, according to the sample inspection apparatus 100, it is possible to perform analysis with a very small amount of sample, and the analysis cost can be reduced. In addition, since the concave portions 3 can be formed with high density, the sample inspection apparatus 100 can efficiently perform many samples, multi-item analysis, multiple analysis of the same item, and the like at a time.

  Furthermore, since the sample inspection apparatus 100 has a configuration capable of easily and reliably miniaturizing and increasing the density of the reaction field as described above, the sample inspection apparatus 100 can be easily downsized. In the sample inspection apparatus 100, since the monomolecular film 2 is fixed to the first region F11 of the sample mounting surface F1 by covalent bonding, when the sample inspection apparatus 100 is used repeatedly over a long period of time, Even when stored for a long time, peeling of the monomolecular film 2 is sufficiently prevented. Therefore, the sample inspection apparatus 100 can obtain sufficient reliability.

  Further, according to the sample inspection apparatus 100, it is possible to analyze the presence / absence of the measurement target contained in the droplet and the content thereof. When performing biological analysis, the droplet may further include a probe that reacts with the measurement target to be analyzed. Furthermore, the probe may be fixed to the recess 3 in advance. The photoelectric conversion unit 70 can detect the reaction between the probe and the measurement object. Since the hydrophobic monomolecular film 2 is formed around the recess 3, even when the probe is immobilized on the recess 3, the solution containing the probe can be prevented from diffusing.

  Here, in the present invention, “probe” refers to a probe defined in JIS K 3600 2392. That is, a nucleic acid fragment used for searching for a target gene in a recombinant DNA experiment is shown. For example, it is designed and synthesized based on cDNA or protein amino acid sequence prepared from mRNA of the target gene.

  The probe preferably forms a hybrid with the measurement object. Examples of such probes include oligonucleotides, polynucleotides, cDNA, genomic DNA, single-stranded DNA, RNA, those labeled with these, antigens, antibodies, oligopeptides, and polypeptides.

  Among them, the probe is preferably at least one selected from the group consisting of a polynucleotide and a labeled polynucleotide. The at least one probe selected from the group consisting of a polynucleotide and a labeled polynucleotide may be a polymer synthesized by a pure chemical method or a polymer synthesized by a biochemical method. May be. More specifically, the at least one probe selected from the group consisting of a polynucleotide and a labeled polynucleotide is a heavy compound synthesized from at least one selected from the group consisting of deoxyribonucleotides and ribonucleotides. It is preferably a coalescence. That is, a polymer having a structure including at least one type of repeating unit selected from the group consisting of a repeating unit based on deoxyribonucleotides and a repeating unit based on ribonucleotides is preferable.

  For example, when the test target item is an enzyme, the other probe may be a substrate for the enzyme. When the test target item is a substrate, the probe may be an enzyme using the same as a substrate. May be. The type of measurement object is not particularly limited, and examples thereof include nucleic acids such as DNA and RNA, proteins, lipids, and the like, and examples of measurement objects to be analyzed include blood and cells. Examples include biological specimens, synthetic specimens such as nucleic acids and proteins, and the like.

Next, in the sample inspection apparatus 100, the light detection circuit operates, for example, as follows. First, charges (electrons) generated by photoelectric conversion at the pn junction surface according to the amount of light incident on the photodiode of each photoelectric conversion unit 70 are accumulated in the n-type region 74. The photodiode of each photoelectric conversion unit 70 receives light at a predetermined time. Next, a voltage is applied to the first electrode 13 to form a depletion layer (not shown) in the p ⁻ isolation region 76, and charges accumulated in the n type region 74 are transferred to the vertical transfer portion n type region 78. Move to. In FIG. 2, the first electrode 13 is shown only in the region above the vertical transfer portion n-type region 78, but the first electrode 13 is also partially formed on the p ⁻ isolation region 76. The above operations are performed in this area. This operation is performed simultaneously in the photodiodes of the photoelectric conversion units 70. A voltage is applied to the first electrode 13 and the second electrode 14 through a resistor. The resistor is designed to apply a voltage to the first electrode 13 and the second electrode 14 with a certain time interval.

  Further, the first electrode 13 and the second electrode 14 are designed to be alternately arranged along the vertical transfer portion n-type region 78, and as shown in FIG. 2, the first electrode 13 and the second electrode 14 are arranged. In many cases, a part of 14 is designed to overlap each other. By applying a voltage to the first electrode 13, charges generated by the photodiode are accumulated in a region below the first electrode 13 in the vertical transfer portion n-type region 78. Next, by applying a voltage to the second electrode 14 and releasing the voltage applied to the first electrode 13, the charge in the region of the first electrode 13 in the n-type region 78 of the vertical transfer portion 78. Is transferred to the region of the vertical transfer portion n-type region 78 below the second electrode 14. Next, by applying a voltage to the first electrode 13 and releasing the voltage applied to the second electrode 14, the charge in the region of the second electrode 14 in the vertical transfer portion n-type region 78 is It is transported not to the region of the vertical transfer portion n-type region 78 under the first electrode 13 but to the region of the vertical transfer portion n-type region 78 under the next first electrode 13. At this time, since the charge does not return to the region of the vertical transfer portion n-type region 78 below the first electrode 13, the first electrode 13 and the second electrode 13 in the region of the vertical transfer portion n-type region 78. In many cases, a technique of changing the impurity concentration in a part of the lower portion of each electrode 14 is employed.

  The charges transferred to the vertical transfer unit n-type region 78 can be sequentially transferred in the vertical direction by sequentially repeating the above operation. The charges that have reached the end of the vertical transfer unit n-type region 78 are then transferred to a horizontal transfer unit (not shown). The operation is the same as the method described above for the operation of the n-type region 78 in the vertical transfer section, and the charges transferred from the photodiode row are transported for each photodiode unit in the horizontal transfer section. Charge information is provided externally through an output amplifier. It is obvious that the register that performs horizontal transfer is faster than the register that performs vertical transfer.

  Further, the next light receiving operation may be performed on the photodiode during the charge transfer operation. In FIG. 2, the first electrode 13 and the second electrode 14 are illustrated as being two, but the number may be increased from one to three or four according to the handling of charge transport. By increasing the number, it becomes possible to transport charges more smoothly. By increasing the number to three or four, it is possible to eliminate regions having different impurity concentrations in a part of the vertical transfer portion n-type region 78 below the first electrode 13 and the second electrode 14 shown above. It becomes. Further, when the light emission amount is small, an appropriate charge can be obtained by increasing the light receiving time.

  Further, in order to remove the influence of the dark current generated in each recess 3 on the analysis result, a blank test is performed in which the above drive is performed before the droplet containing the measurement object is accommodated in each recess 3. The test result may be reflected in the obtained analysis result. This blank test may be performed only on the selected recesses 3 by selecting at least one recess 3 on the sample placement surface F1 as a representative of all the recesses 3. Further, from the viewpoint of further improving the analysis accuracy, this blank test may be performed individually for all the recesses 3.

  The above operation is an operation in a charge coupled device (CCD) system, and the operation method is naturally different in a metal-on-silicon (MOS) light receiving device. However, in the MOS method, the driving method is different from the above, but the light reception information from each photodiode can be read, and the necessary requirements of the present application are satisfied.

  Next, a preferred example of the method for manufacturing the sample inspection apparatus 100 (a preferred embodiment of the method for manufacturing the sample inspection apparatus of the present invention) will be described.

First, the substrate 1 is formed in the substrate forming step. As described above, the substrate 1 includes two or more photoelectric conversion units arranged in a two-dimensional array, and at least one of the two main surfaces facing each other has —OH, It is a substrate to which a hydrophilic first characteristic group having at least one structure selected from the group consisting of —NH ₂ , ═N—H, and —SH is bonded.

  The substrate 1 is a semiconductor integrated circuit substrate, and its manufacturing method is not particularly limited, and can be manufactured using a known thin film manufacturing technique. For example, the substrate 1 can be manufactured using, for example, a manufacturing technique employed in a MOS (metal-oxide film-semiconductor) process for manufacturing an IC (integrated circuit) from a silicon substrate.

  However, for the layer to be the uppermost layer (the protective layer 12 in the case of this substrate 1), the layer in which the first characteristic group is previously bonded to the surface (surface to be the sample mounting surface) is the uppermost layer. A layer (a protective layer 12 in the case of this substrate 1) may be formed in advance, and after forming a substrate including the uppermost layer not including the first characteristic group, its surface (surface serving as a sample mounting surface) ) May be combined with the first characteristic group having a structure having active hydrogen for the condensation reaction with the second characteristic group described above.

  Hereinafter, the above surface treatment is performed, and the first characteristic group having a structure having active hydrogen with respect to the condensation reaction with the second characteristic group is bonded to the surface of the uppermost layer (the surface on which the sample is placed). The case where it is made to explain is demonstrated.

For the method of providing the first characteristic group on the sample placement surface F1, a known surface treatment technique can be used. For example, a method of chemically oxidizing the surface of the uppermost layer, a method of performing a plasma treatment in the presence of oxygen, and a method of treating with ozone. Further, the surface of the uppermost layer (the surface on which the sample is placed) is, for example, SiCl ₄ , HSiCl ₃ , SiCl ₃ O— (SiCl ₂ —O) η-SiCl ₃ (where η is 0-6) Integer), Si (OH) ₄ , HSi (OH) ₃ , Si (OH) ₃ O— (Si (OH) ₂ —O) η-Si (OH) ₃ (where η is an integer of 0 to 6), etc. There is also a method of hydrophilization treatment.

  The oxidation treatment of the surface of the uppermost layer will be described more specifically. For example, the oxidation treatment of the surface of the uppermost layer can be performed by irradiating the surface of the uppermost layer with ultraviolet rays in the presence of an oxygen and hydrogen atom supply substance. Oxygen in the gas phase is decomposed by ultraviolet irradiation to generate ozone, and this ozone reacts with the hydrogen atom supply substance to generate active species having active hydrogen. Further, when the surface of the uppermost layer is irradiated with ultraviolet rays, the covalent bond between atoms of the material constituting the vicinity of the surface of the uppermost layer is broken, and a dangling bond is formed. When the active species containing active hydrogen acts on the dangling bonds, the surface of the uppermost layer to which the first characteristic group is bonded is obtained.

  As the hydrogen atom supply substance, for example, water, ammonia and the like can be preferably used from the viewpoint of availability and ease of handling. For example, when water is used as the hydrogen atom supply substance, the first characteristic group can exist as a characteristic group including at least a structure represented by —OH on the surface of the uppermost layer. When ammonia is used, the first characteristic group can exist as a characteristic group including at least a structure represented by -NH. In place of the ultraviolet irradiation treatment, a corona treatment, a plasma treatment, or the like can be employed.

When a semiconductor integrated circuit substrate such as the substrate 1 is employed, a silicon material is preferably used as a constituent material of the uppermost layer (here, the protective layer 12) from the following viewpoint. The layer containing a silicon material has a structure in which a hydroxyl group or a silanol group is bonded in advance to the surface due to the property of silicon that it is easily oxidized. That is, the layer containing a silicon material is a layer rich in the first characteristic group containing the above-described active hydrogen. Therefore, it is preferable that the uppermost layer is a layer containing a silicon material because it is advantageous for the condensation reaction with the second characteristic group of the organic molecule. When a semiconductor integrated circuit substrate such as the substrate 1 is employed, an inorganic oxide layer made of an inorganic oxide is preferably formed as the uppermost layer (here, the protective layer 12) from the following viewpoint. The inorganic oxide layer has a hydroxyl group or the like on its surface and is a layer rich in the first characteristic group containing active hydrogen. Therefore, it is preferable that the uppermost layer is an inorganic oxide layer because it is advantageous for the condensation reaction with the second characteristic group of the organic molecule. An example of the inorganic oxide layer is SiO ₂ .

  As described above, the substrate 1 is manufactured not only by the method of forming the monomolecular film 2 on the uppermost layer (here, the protective layer 12) of the substrate 1, but also by separately preparing the substrate to be the uppermost layer. In addition, it is possible to adopt a method in which the monomolecular film 2 is formed in advance on the surface of the substrate and bonded to the substrate on which the photoelectric conversion unit 70 and the like are formed. In this case, a substrate for forming a monomolecular film in advance (a substrate serving as the uppermost layer) has a first characteristic group in advance or has a surface to which the first characteristic group can be bonded by surface treatment. It has physical durability and chemical durability that can withstand the treatment in the single molecule formation step, and has light transmittance to light emitted from the droplets stored in the recesses. If it is a thing, it will not restrict | limit in particular.

  For example, glass substrate, quartz substrate, synthetic quartz substrate, silicon substrate, acrylic substrate, polycarbonate substrate, polystyrene substrate, vinyl chloride substrate, epoxy resin substrate, silicone resin (polydimethylsilicone) substrate, PMMA (poly Conventionally known substrates such as various polymer substrates such as (methyl methacrylate) substrates, ceramic substrates, and metal substrates can be used. Among these, a glass substrate or a quartz substrate is preferable because it has a structure with many hydroxyl groups on its surface. Even when these substrates are used as the uppermost layer, the first characteristic group may be bonded to the substrate surface by surface treatment. Further, a sheet-like substrate may be used as long as the first characteristic group can be provided and is transparent.

  Next, in the monomolecular film forming step, the monomolecular film 2 is formed on the sample mounting surface F1 of the substrate 1. That is, the recess 3 is formed on the sample placement surface F1.

  In the monomolecular film forming step, the main surface of the substrate 1 on which the first characteristic group is bonded is the sample mounting surface F1, and the sample mounting surface F1 is the first region F11 and two or more second regions. And forming the monomolecular film 2 selectively only in the first region F11, thereby providing two or more recesses 3 for independently containing two or more droplets. This is a step of providing one-to-one correspondence with each of the above photoelectric conversion units.

  Further, in the monomolecular film forming step, the second characteristic group capable of condensation reaction with the first characteristic group on the sample mounting surface F1 is provided at one end of the molecular chain, and the hydrophobic third characteristic group is provided on the molecular chain. Organic molecules at the other end are used as raw materials for the monomolecular film. The bottom surface of the obtained recess 2 is formed by the second region F12, and the outer peripheral portion including the entire inner surface of the recess 3 is formed by the monomolecular film 2 covering the first region F11. Is brought into contact with the sample mounting surface F1, and the condensation reaction between the first characteristic group and the second characteristic group proceeds to form a monomolecular film fixed to the sample mounting surface by covalent bonds.

  Here, from the viewpoint of the uniformity of the film thickness of the monomolecular film 2 and the viewpoint of the ease of manufacturing work, in the monomolecular film forming step, an organic molecule is added to the aprotic solvent in the container. It is preferable that the 1st process of advancing a condensation reaction by making the organic molecule containing liquid obtained and the sample mounting surface of a board | substrate contact is included. As described above, in this first step, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed in terms of a relative humidity value at 22 ° C., the relative humidity The value is adjusted to be 35% or less, preferably 25% or less, more preferably 5% or less.

  An example of a suitable method that can be employed in the first step (first embodiment of the method for manufacturing a sample inspection apparatus of the present invention) will be described.

  First, a resist pattern for covering the region to be the second region F12 of the sample mounting surface F1 is formed, and a region where the resist pattern is not formed on the sample mounting surface is defined as a first region. This resist pattern can be easily formed by a semiconductor thin film manufacturing technique. Next, an organic molecule is brought into contact with the sample mounting surface F1 after the resist pattern is formed, thereby selectively covering only the first region F11 with the monomolecular film, and then removing the resist pattern. Two or more recesses 3 are formed. Here, the resist pattern may be a positive resist pattern or a negative resist pattern.

  In addition, the following method is preferable as a method of bringing the organic molecule into contact with the sample mounting surface F1 in the first step. That is, first, an organic molecule is added to an aprotic solvent to prepare an organic molecule-containing liquid. Next, the organic molecule-containing liquid and the substrate after forming the resist pattern are placed in a container in which the moisture content in the gas phase inside the glove box or the like can be easily controlled within the above-described range, The condensation reaction described above is allowed to proceed.

Here, the aprotic solvent used for the preparation of the organic molecule-containing liquid can be appropriately determined according to the type of organic molecule as long as it does not dissolve the resist pattern, but the film thickness is thin (0.5 nm to 50 nm). Further, from the viewpoint of easily and reliably obtaining the monomolecular film 2 excellent in film thickness uniformity, a fluorinated solvent is preferable. As the fluorinated solvent, perfluorocarbon liquids and hydrofluoroether liquids manufactured by Sumitomo 3M are preferable, and specifically, various physical properties such as having a boiling point corresponding to the temperature conditions for performing the first step. From the viewpoint, trade names: “HFE-7200”, trade names: “PF-5080”, and “FC-77” manufactured by Sumitomo 3M Limited are preferable. The concentration of the organic molecule in the organic molecule-containing liquid is not particularly limited, but is, for example, about 3 × 10 ⁻² to 1 × 10 ⁻¹ M. The contact time between the sample mounting surface F1 and the organic molecule-containing liquid is not particularly limited. For example, it is several seconds to 10 hours, preferably 1 minute to 1 hour, and the temperature of the organic molecule-containing liquid is, for example, It is 10-80 degreeC, Preferably it is the range of 20-30 degreeC.

  The constituent gas constituting the gas phase with the water content adjusted to the above range is preferably at least one gas selected from the group consisting of a rare gas and a nitrogen gas. However, in the first step, if conditions under which the progress of the oxidation reaction of the organic molecule or the aprotic solvent and the progress of the deterioration due to the oxidation of the monomolecular film can be sufficiently suppressed, air is used. Also good.

  Next, the removal of the resist pattern after forming the monomolecular film can be performed by using, for example, acetone. Thereby, the 2 or more recessed part 3 can be formed.

  In the above method (the first embodiment of the manufacturing method of the sample inspection apparatus of the present invention), the method of forming the monomolecular film on the sample mounting surface F1 after forming the resist pattern is particularly limited to the above method. For example, methods such as a printing method, a transfer method, a screen method, a liquid discharge method, an ink jet method, and a stamp method can be employed.

  Next, an example of another suitable method that can be employed in the first step (second embodiment of the method for manufacturing a sample inspection apparatus of the present invention) will be described.

  First, a monomolecular film is formed on the sample mounting surface F1. In this case, the method for forming the monomolecular film is preferably performed under the same conditions as those in the first step, except that the monomolecular film is formed on the entire surface of the sample mounting surface F1. That is, in a container such as a glove box, a first step of causing a condensation reaction to proceed by bringing an organic molecule-containing liquid obtained by adding organic molecules into an aprotic solvent and a sample mounting surface of a substrate into contact with each other. It is preferably included. As described above, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed by converting it into a relative humidity value at 22 ° C., the relative humidity value is preferably 35% or less, preferably Is preferably adjusted to 25% or less, more preferably 5% or less.

  Here, in this manufacturing method, since a resist pattern is not used, the aprotic solvent used for the preparation of the organic molecule-containing liquid can be appropriately determined according to the type of the organic molecule, but the film thickness is thin (0.5 nm From the viewpoint of easily and reliably obtaining the monomolecular film 2 having excellent film thickness uniformity (˜50 nm), a solvent capable of sufficiently dissolving the organic molecules is preferable. For example, organic solvents such as hexadecane, chloroform, carbon tetrachloride, silicone oil, hexane and toluene can be used. Any one kind of these solvents may be used, or two or more kinds may be mixed and used.

Among these, a mixed solvent containing hexadecane, chloroform and carbon tetrachloride is preferable as the aprotic solvent. If an organic solvent is used in this way, for example, polymerization (polymerization) of organic molecules due to the presence of moisture can be sufficiently prevented. Therefore, the condensation reaction between the second characteristic group of the organic molecule and the first characteristic group of the sample placement surface F1 can be performed efficiently. As a result, organic molecules are bonded to the sample mounting surface F1 by covalent bonds {for example, siloxane bonds (-Si-O-)}, and the monomolecular film 2 is formed. The concentration of the organic molecule in the organic molecule-containing liquid is not particularly limited, but is, for example, about 3 × 10 ⁻² to 1 × 10 ⁻¹ M. The contact time between the sample mounting surface F1 and the organic molecule-containing liquid is not particularly limited. For example, it is several seconds to 10 hours, preferably 1 minute to 1 hour, and the temperature of the organic molecule-containing liquid is, for example, It is 10-80 degreeC, Preferably it is the range of 20-30 degreeC.

  Next, a photomask for selectively protecting the monomolecular film 2 covering the region to be the first region F11 on the sample mounting surface F1 from ultraviolet rays (ultraviolet irradiation) is prepared. This photomask covers a region to be the second region of the sample mounting surface when it is disposed between the ultraviolet light source and the sample mounting surface F1 on which the monomolecular film is formed. Only the molecular film is selectively irradiated with ultraviolet rays. Next, using this photomask, the sample mounting surface F1 after the monomolecular film 2 is formed through this photomask is irradiated with ultraviolet rays to form the second region F12 of the sample mounting surface F1. Only the monolayer covering the region is selectively removed. Thereby, two or more recessed parts are formed.

  For example, when a laser such as an excimer laser is used as a means for ultraviolet irradiation, a method of spot-irradiating ultraviolet rays onto a specific region of the monomolecular film without using a photomask can be employed. Further, instead of performing ultraviolet irradiation, only the monomolecular film covering the region to be the second region F12 of the sample mounting surface F1 is selectively removed by electron beam irradiation processing, corona processing, plasma processing, or the like. You may adopt the method of doing. These treatments are preferably carried out in the presence of oxygen. Thereby, the monomolecular film covering the region to be the second region F12 of the sample mounting surface F1 can be removed by oxidation.

  Next, when the first characteristic group is bonded to the second region F12 of the sample mounting surface F1 after the monomolecular film 2 is removed, the substrate 1 is completed. When the first characteristic group is not bonded to the second region F12, for example, the surface treatment described above is selectively performed only on the second region F12, so that the second region F12 A first characteristic group is attached.

  In this method (the second embodiment of the method for manufacturing the sample inspection apparatus of the present invention), the method for forming the monomolecular film on the entire surface of the sample mounting surface F1 is not particularly limited to the above-described method. A transfer method, a screen method, a liquid discharge method, an ink jet method, a stamp method, or the like can be employed.

  In the case where the monomolecular film 2 is formed as a film made of a laminate in which a plurality of single films are laminated, for example, the first monomolecular film is formed on the sample mounting surface F1 as described above. When the second monolayer is formed after the formation, the first characteristic group may not be bonded to the surface of the monolayer. In this case, the first surface treatment may cause the first monolayer. One characteristic group may be bonded.

  More specifically, when a characteristic group including a group having an unsaturated bond such as a vinyl group is bonded to the surface of the first monomolecular film formed on the sample mounting surface F1, moisture is absorbed. A first characteristic having at least an —OH structure by changing the portion of the group having an unsaturated bond by irradiating the surface of the monomolecular film with an energy beam such as an electron beam or an X-ray in an existing atmosphere. Groups can be introduced. In addition, when a characteristic group including a group having an unsaturated bond such as a vinyl group is bonded, for example, by immersing in a potassium permanganate aqueous solution, the portion of the group having an unsaturated bond is changed, and -COOH A first characteristic group having at least a structure can be introduced.

  The thickness of the monomolecular film 2 can be set as appropriate by selecting the type (length) of the organic molecule, making the above-described laminate, and the like. For example, the thickness of the third characteristic group of the organic molecule constituting the monomolecular film is It can also be adjusted by a method in which a molecule showing non-affinity is further bound to the tip. Specific examples are shown below.

  Further, when the organic molecule constituting the monomolecular film has a double bond or a triple bond at the tip of the third characteristic group as shown in the formula (22), after forming the monomolecular film, for example, A Grignard reagent (RMgX) may be further brought into contact with the monomolecular film. By this contact, the addition reaction between the third characteristic group and RMgX can proceed, and the hydrocarbon group (R-) of RMgX can be bonded to the tip of the third characteristic group. RMgX may be any of an alkyl group having 1 to 23 carbon atoms, a halogenated alkyl group, an alkenyl group, and a halogenated alkenyl group, and X is a halogen (F, Cl, Br, or I). .

  When the organic molecule constituting the monomolecular film has an epoxy group as the third characteristic group as shown in the formula (26), after the monomolecular film 2 is formed, alcohol (ROH) is added to the monomolecular film 2. May be further brought into contact with each other. By this contact, the ring opening (addition) reaction of the epoxy group proceeds, and the “R group” of alcohol (ROH) can be bonded to the third characteristic group. ROH may be any of an alkyl group having 1 to 23 carbon atoms, a halogenated alkyl group, an alkenyl group, and a halogenated alkenyl group, and X is a halogen (F, Cl, Br, or I). .

  In the monomolecular film forming step, the surface of the uppermost layer of the substrate 1 such as the protective layer 12 is preferably planarized in advance by a surface polishing process such as CMP (Chemical Mechanical Polishing Technology). .

  Next, an example of a method in which a single-stranded DNA (probe) having a sequence complementary to the detection target gene is immobilized in advance on the bottom surface of each recess 3 using the sample inspection apparatus 100 will be described below.

  First, single-stranded DNA is fixed to the bottom surface (second region F12) of each recess 3. This fixing method is not particularly limited, and a known technique used in DNA analysis can be applied. For example, a method of directly synthesizing DNA (oligonucleotide) on the bottom surface of the recess 3 (for example, Affymetrix method), a method in which a linker is previously bound to the bottom surface, and a single-stranded DNA is bound to this linker, a single-stranded DNA Examples include a method in which a functional group for immobilization on the surface is bonded to the end of the surface, and the functional group for immobilization is bonded to the surface. The linker is not particularly limited, and examples thereof include an amino group and a carboxyl group. The linker or the immobilized functional group can be bound to the probe or the surface by a conventionally known method.

  Next, a droplet containing a measurement object to be analyzed is placed in the recess 3. In this case, the target DNA itself contained in the droplet may be fluorescently labeled with Cy3 or the like, or a fluorescent intercalator such as SYBR-Green may be added to the droplet or the recess 3. Then, the recess 3 is irradiated with excitation light. At this time, when the single-stranded DNA immobilized on the bottom surface of the recess 3 and the target DNA are hybridized to form a double-stranded DNA, the fluorescent intercalator that has entered the target DNA or the fluorescent label of the target DNA Produces fluorescence.

  For example, when SYBR-Green is used, an SHG laser with a wavelength of 473 nm may be irradiated. The generated fluorescence is detected by the photoelectric conversion unit 70 (a photodiode in the case of the sample inspection apparatus 100). In the sample inspection apparatus 100, if different single-stranded DNAs are immobilized in the recesses corresponding to the respective photoelectric conversion units 70, it is possible to inspect a plurality of items with a single fluorescence detection. In addition, if the same single-stranded DNA is immobilized on each photoelectric conversion unit 70, data with high measurement accuracy (high reliability) can be obtained. As described above, since the sample inspection apparatus 100 can form the recesses 3 with extremely high density, a sample that can be analyzed at a time on the sample mounting surface F1 even if it is the same size as the conventional sample inspection apparatus. The number can be greatly improved.

  Examples of probes to be immobilized in the recesses 3 include single-stranded nucleic acids complementary to the detection target gene, but are not limited to these and include those described above. For example, when an antigen (or antibody) having a function of emitting fluorescence by an antigen-antibody reaction is used as a probe, a reaction with an antibody (or antigen) in a sample sample can be sensed. In addition to these, oligonucleotides, polynucleotides, RNA, DNA, labeled products thereof, antigens, antibodies, oligopeptides, amino acid sequences, polypeptides, proteins, enzymes, protein nucleic acids (PNA), and substances with the above names And the like.

  As a method for supplying a droplet containing a measurement object to be analyzed to the concave portion 3 of the sample inspection apparatus 101, there are various eases of supplying a small amount of droplets, accuracy of the amount of supplying droplets, and droplet supply speed. From the viewpoint, a method using an ink jet (for example, a method using a piezoelectric element or a method utilizing gas expansion by heating) is preferable. For example, even in the method using an ink jet, in the case of analysis that handles expensive and rare measurement objects such as those used for DNA gene analysis or protein analysis, the amount of liquid containing the measurement object is sufficiently reduced. There may be a need. In this case, without providing a liquid reservoir in the ink jet, each time the analysis is performed, the tip of the ink jet nozzle is inserted into the liquid containing the measurement object, and only the necessary amount of the liquid containing the measurement object is inserted into the tip of the nozzle. It is also possible to adopt a method of sucking and dropping a droplet from the nozzle to the recess 3. In addition, a method using a microdispenser or a micropipette can be employed.

[Second Embodiment]
Next, a second embodiment of the sample inspection apparatus of the present invention will be described. The sample inspection apparatus 101 according to the second embodiment discharges a droplet including a measurement target to be analyzed toward the concave portion 3 to the sample inspection apparatus 100 according to the first embodiment shown in FIGS. 1 and 2. The configuration further includes a droplet supply unit 40 (inkjet head) having the nozzles, and the configuration other than the droplet supply unit 40 is the same as that of the sample inspection apparatus 100 of the first embodiment. The CPU 4 is a control unit that adjusts the position of the nozzle with respect to the recess 3 by moving the nozzle of the droplet supply unit 40. That is, the droplet supply unit 40 is provided with a drive mechanism for arbitrarily changing the relative position with respect to the sample placement surface F1 under the control of the CPU 4.

  Hereinafter, the droplet supply unit 40 will be described. FIG. 3 is a perspective view showing a second embodiment of the sample inspection apparatus of the present invention.

  As shown in FIG. 3, the droplet supply unit 40 of the sample inspection apparatus 101 has β rows × γ columns (specifically, in the case of this droplet supply unit 40, 5 rows × 7 columns) on the sample mounting surface F1. ) In the two-dimensionally arranged recesses 3, the droplets can be simultaneously dropped onto the recesses 3 belonging to the same row. That is, five nozzles are linearly arranged in the droplet supply unit 40 so as to be parallel to the row of the recesses 3. The interval between the central axes of the five nozzles is adjusted to be the same as the interval between the centers of the bottom surfaces of the five recesses 3 belonging to the same row. As a result, the liquid droplets discharged from the respective discharge holes (not shown) of the five nozzles are dropped onto the center of the bottom surface of the recess 3.

  The CPU 4 is electrically connected to the droplet supply unit 40, and adjusts the position of the nozzle of the droplet supply unit 40 with respect to the recess 3 based on the reference data for positioning stored in the memory unit 5. To do. In addition, the control unit adjusts the amount of liquid droplets ejected from each nozzle. Specifically, the control unit aligns the position of each droplet supply unit 40 (the position of each nozzle) with one of the columns to which droplets are to be dropped, and then, from the discharge holes of each nozzle, each of the corresponding columns. A droplet is discharged toward the recess 3 (in the direction of arrow A shown in FIG. 3). Next, the control unit moves the position of each droplet supply unit 40 (the position of each nozzle) to another row where droplets should be dropped (for example, moved in the direction of arrow B shown in FIG. 3). Then, droplets are ejected into the recesses 3 of the corresponding row.

  Further, in the case of the sample inspection apparatus 101 shown in FIG. 3, in order to sufficiently ensure the accuracy of the dropping position of the droplets dropped from each nozzle of the droplet supply unit 40, description will be made with reference to FIGS. 4 to 6. The following configuration is provided.

  FIG. 4A is a perspective view schematically showing a basic configuration of a nozzle provided in the droplet supply unit 40 of the sample inspection apparatus 101 shown in FIG. FIGS. 4B and 4C are explanatory diagrams for explaining a method of aligning the droplet supply unit 40 shown in FIG. 4A with respect to the recess 3 in the sample inspection apparatus 101. FIG. .

  As shown in FIG. 4A, the nozzle 50 provided in the droplet supply unit 40 has a cylindrical shape. Further, the tip portion of the nozzle 50 has a substantially circular smooth surface. A discharge hole 501 for discharging a droplet is provided at the center of the tip of the nozzle 50. Further, a light emitting section 502 (for example, a light emitting diode) for alignment is provided at the tip of the nozzle 50. The light emitting unit 502 has a ring-shaped cross section (surface exposed at the tip of the nozzle 50). And the discharge hole 501 is arrange | positioned in the center (center of concentric circle) of this light emitting part 502 of a cross-section ring shape. In addition, the cross-sectional shape of this light emission part is not limited to the said ring shape.

  As shown in FIG. 4B, a light receiving unit 51 for receiving light emitted from the light emitting unit 502 at the tip of the nozzle 50 is provided at the end of the sample mounting surface F <b> 1 of the substrate 1. The light receiving unit 51 includes a photoelectric conversion element (for example, a photodiode) provided inside the substrate 1.

  Next, an example of the operation of the droplet supply unit 40 controlled by the CPU 4 will be described.

  When starting the dropping of the droplet, as shown in FIG. 4B, the CPU 4 drops the reference data for positioning stored in the memory unit 5 (for example, dropping the droplet, which is set in advance). Information on the relative position of the droplet supply unit 40 with respect to the immediately preceding sample placement surface F1 (distance between the sample placement surface F1 and the tip of the nozzle 50, coordinates of the tip of the nozzle 50 in a plane parallel to the sample placement surface F1) First, the droplet supply unit 40 is moved so that the tip of one of the plurality of nozzles 50 is directly above the light receiving unit 51 of the substrate 1.

  At this time, as shown in FIG. 4C, the CPU 4 issues a stop command to the drive mechanism of the droplet supply unit 40 when the light receiving unit 51 detects the light emission from the light emitting unit 502. You may determine the stop position of the part 40. FIG. In this case, when the light receiving unit 51 receives light emitted from the light emitting unit 502, the droplet supply unit 40 is stopped, and the alignment of the droplet supply unit 40 before droplet dropping is completed.

  The droplet supply unit 40 stores data on the distance from the light receiving unit 51 to the recess 3 where the supply of the droplet is started, and distance data between the recesses 3, and based on these data, the droplets are stored. By dropping the supply unit 40 and controlling its position, the dropping of the droplet can be started. Thereby, positioning of the droplet supply unit 40 can be performed with high accuracy. Further, at this time, when light emission from the light emitting portion 502 of the nozzle 50 is detected by the photoelectric conversion portion 70 of the concave portion 3 above each concave portion 3, the CPU 4 issues a droplet ejection command to each nozzle 50. Alternatively, the position of droplet discharge may be determined. In this way, the position of each nozzle 50 can be reliably determined with respect to each recess 3.

  It should be noted that the number of recesses 3 for detecting light emitted from the light emitting unit 502 of one nozzle 50 is set in advance (determination of whether the photoelectric conversion unit 70 of the recess 3 has detected or not detected light). By setting a reference light intensity threshold value), the distance between the sample mounting surface F1 and the tip of the nozzle 50 of the droplet supply unit 40 is more accurately adjusted when droplets are dropped. It is also possible. For example, as shown in FIG. 5A, when the droplet supply unit 40 is moved in the vertical direction and the parallel direction with respect to the sample mounting surface F1, the recess 3 that can detect the light emission from the light emitting unit 502 is formed. The height of the tip of the nozzle 50 of the droplet supply unit 40 can be determined by the number. That is, when the tip of the nozzle 50 is sufficiently away from the sample mounting surface F1, the number of the recessed portions 3 that detect light emission increases (in FIG. 5A, the recessed portion 3 that receives light is indicated by hatching). Then, when the tip of the nozzle 50 is moved closer to the sample mounting surface F1, the number of the concave portions 3 for detecting light emission gradually decreases, and finally, as shown in FIG. Light is received only by the light receiving unit. At this point, the height can be determined. Note that the distance between the surface and the tip of the nozzle 50 is not limited to the case where light is received by only one light receiving unit, and the number of the recesses 3 for detecting light emission from one nozzle 50 is set in advance, and the data is It can be arbitrarily determined by storing in the memory unit 5.

  If the difference in the intensity of light emitted from the light emitting portion 502 of the nozzle 50 is configured to be detected by the photoelectric conversion portion 70 of the concave portion 3, the height is also determined by the intensity of light emission detected by each concave portion 3. Can do. For example, the intensity of light emission detected by the recess 3 when the tip of the nozzle 50 is in an appropriate position is set. If the intensity is weaker than the set intensity, the tip of the nozzle 50 is brought closer to the sample placement surface F1 and set. If it is stronger than the strength, the control setting may be performed so that the tip of the nozzle 50 is separated from the sample placement surface F1. And when it becomes equivalent to setting intensity | strength, the position of the droplet supply part 40 can be fixed.

  An example of another configuration of the droplet supply unit 40 is shown in FIG. As shown in FIG. 6A, the nozzle 50 </ b> A of the droplet supply unit 40 has a discharge hole 701, and receives light emitted from the light emitting unit 702 for alignment and the light emitting unit 702 on the tip surface thereof. A light receiving portion 703 is provided. As shown in the perspective view of FIG. 6B, the substrate 1 includes a reflecting plate 71 such as an aluminum plate that can reflect the light emitted from the light emitting unit 702 at least at one place on the sample mounting surface F1. ing.

  Next, an example of the operation of the droplet supply unit 40 will be described. When the tip of the nozzle 50 </ b> A comes to a position corresponding to the reflecting plate 71, the light emitted from the light emitting unit 702 is reflected by the reflecting plate 71. Then, the light receiving unit 703 at the tip of the nozzle 50A detects the reflected light. In this way, it is possible to align the droplet supply unit 40 before the supply of droplets is started. If the distance from the reflecting plate 71 to the recess 3 where the supply of droplets is started and the distance between the recesses 3 are specified in advance, the droplet supply unit 40 is moved based on these data and the position thereof is determined. Can be controlled. Thereby, positioning of the droplet supply part 40 with respect to the recessed part 3 which drops a droplet can be performed with high precision.

  As mentioned above, although embodiment of this invention was described in detail, this invention is not limited to the said embodiment.

  For example, in the case of the sample inspection apparatuses 100, 101, and 102 described in the above-described embodiments, the case where one photoelectric conversion unit 70 is configured by one photoelectric conversion element (photodiode) has been described. The invention is not limited to this embodiment. That is, in this invention, one photoelectric conversion part may be comprised from one photoelectric conversion element, and one photoelectric conversion part may be comprised from two or more photoelectric conversion elements.

  In the case of the sample inspection apparatuses 100, 101, and 102 described in the above-described embodiments, the case where a photodiode is employed as the photoelectric conversion element provided in the photoelectric conversion unit 70 has been described. It is not limited to this embodiment. That is, in the present invention, the photoelectric conversion element that constitutes the photoelectric conversion unit has a configuration that receives light emitted from a droplet fixed to the recess and photoelectrically converts the light to generate electrons (for example, , Pn junction, and pin junction). For example, the photoelectric conversion element may be a phototransistor, an avalanche photodiode, a CdS cell, or a photo IC in addition to the photodiode. The photoelectric conversion element may be a solar cell (including a dye-sensitized solar cell).

  Furthermore, as an example in the case where one photoelectric conversion unit described above is composed of two or more photoelectric conversion elements, there is a configuration in which a plurality of the photoelectric conversion elements described above are arranged two-dimensionally. An image sensor is mentioned as what has this structure. More specifically, examples of the image sensor include a CCD (Charge Coupled Device) sensor and a MOS (Metal Oxide Semiconductor) sensor. These are classified according to the difference in reading method.

  More specifically, an IT-CCD (Inter Transfer-Charge Coupled Device) sensor, an FT-CCD (Flame Transfer-Charge Coupled Device) sensor, and an FIT-CCD (Frame Inter-Transcharge-Charge Sensor-Charge Charge Sensor). Examples thereof include a device sensor, an NMOS (N-Channel-Charge Coupled Device) sensor, and a PMOS (P-Channel-Charge Coupled Device) sensor. Furthermore, one photoelectric conversion unit may be composed of a plurality of image sensors. Furthermore, a configuration in which the above-described sensor is cooled may be employed from the viewpoint of preventing the occurrence of dark current and from the viewpoint of more reliably detecting minute light emission.

  Further, the sample inspection apparatus of the present invention is similar to the sample inspection apparatus 102 shown in FIG. 7 in that the CPU 4, the memory unit 5, and the data input / output unit 6 mounted on the sample inspection apparatus 100 shown in FIG. It may have a configuration that is not provided integrally. Further, at least one element of the CPU 4, the memory unit 5, and the data input / output unit 6 may be integrally provided on the substrate 1.

  Further, in each component of the substrate 1 shown in FIG. 2, all of the components belonging to the p-type semiconductor become components belonging to the n-type semiconductor, and all of the components belonging to the n-type semiconductor belong to the p-type semiconductor. It may be in the form of an element.

  In the present invention, the configuration of the substrate having the sample mounting surface has the sample mounting surface, and two or more recesses formed of a monomolecular film are formed on the sample mounting surface. With a photoelectric conversion unit provided in a one-to-one correspondence with a mechanism for outputting an electrical signal based on the charge generated by the photoelectric conversion element provided in the photoelectric conversion unit as analysis information There is no particular limitation as long as it is present, and the present invention is not limited to the configuration of the semiconductor integrated circuit substrate 1 employed in the sample inspection apparatuses 100, 101, and 102 described in the above embodiments.

  For example, in the semiconductor integrated circuit substrate 1 employed in the sample inspection apparatuses 100, 101, and 102, the structure of having a mechanism for outputting an electrical signal based on the electric charge generated by the photoelectric conversion element as analysis information has been described. In the present invention, a configuration having a signal detection transistor circuit that converts electrons (charges) generated by photoelectric conversion into voltage information has been described. In the present invention, the configuration of the substrate is such a photodiode or MOS sensor. The present invention is not limited to the configuration having a signal detection transistor circuit suitable for the above.

Furthermore, in the sample inspection apparatus of the present invention, at least one element of the light shielding film 15, the interlayer insulating film 9 and the color filter 11 in the substrate 1 is provided in each component of the substrate 1 shown in FIG. You may have the structure which is not. If the color filter 11 is not provided, the protective layer 12 may not be provided. In this case, the uppermost layer is not the protective layer 12 but a layer disposed on the uppermost part of the substrate 1 (for example, (This becomes the insulating film 10, the interlayer insulating film 9, or the inorganic oxide layer 8.)
Moreover, in the sample inspection apparatus of this invention, you may have the structure by which the bottom face of the recessed part was further coat | covered with the 2nd film | membrane which consists of a material which has the light transmittance with respect to light. In this case, a hydrophilic fourth characteristic group capable of at least hydrogen bonding with the solvent contained in the droplet is bonded to the surface of the second film. Since the droplet is selectively held on the surface of the second monomolecular film by hydrogen bonding with the fourth characteristic group, even in this case, the effect of the present invention can be obtained. However, in this case, the thickness of the second film is equal to or less than the thickness of the monomolecular film covering the first region.

  As a material constituting the second film, the second film has a light-transmitting property with respect to light emitted from the droplet accommodated in the recess 3, and has a fourth characteristic group capable of hydrogen bonding to the droplet. In addition, there is no particular limitation as long as it has a characteristic capable of forming a covalent bond with the first characteristic group of the sample mounting surface F1 of the substrate 1 by a condensation reaction.

From the viewpoint of more reliably obtaining the effects of the present invention, the fourth characteristic group includes —OH, —NH ₂ , ═N—H, —SH, —SO ₃ H, —SO ₂ H, —PO ₃ H, — It preferably has at least one structure selected from the group consisting of PO ₃ H ₂ , —CHO, and —CO ₂ H.

  In addition to the above structure, the fourth characteristic group includes a structure in which a metal atom (metal ion) M is cross-linked with oxygen (MOM), a structure of metal oxo (M = O), and a structure of metal hydroxo (M- O-H) may be used. Furthermore, the probe described above may be fixed to the surface of the second film.

  As a material constituting the second film, the above-mentioned fourth characteristic group is provided at one end of the molecular chain, and the above-described second characteristic group (characteristic group capable of condensation reaction with the first characteristic group) is used as the molecular chain. And an organic molecule having a structure in which a divalent organic group represented by the formula (2) described above is bonded between the fourth characteristic group and the second characteristic group. It is preferable.

In addition, as a material constituting the second film, a structure in which a divalent organic group represented by the above-described formula (2) is bonded between the fourth characteristic group and the second characteristic group. In the carbon atoms constituting the carbon skeleton of the divalent organic group represented by the formula (2), the characteristic group represented by the formula (3), -O-, -COO-, and It is preferably an organic molecule having a structure in which at least one divalent characteristic group selected from the group consisting of —C ₆ H ₄ — is further bonded.

  Further, the material constituting the second film has, as the second characteristic group, a characteristic group represented by the general formula (1) that can undergo a condensation reaction with the first characteristic group. Two monovalent groups selected from the group consisting of a methyl group, a halogen-substituted methyl group, a vinyl group, a cyclic ether group having 2 to 4 carbon atoms, a phenyl group, a halogen-substituted phenyl group, and a cyano group An organic compound having a structure in which a trivalent organic group represented by the formula (4) described above is bonded between two second characteristic groups and a fourth characteristic group It is preferably a molecule.

  EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated in more detail, this invention is not limited to these Examples at all.

(Preliminary experiment for confirmation of monolayer)
Using the same CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (manufactured by Shin-Etsu Chemical Co., Ltd.) as the organic molecules used in Example 1, a confirmation experiment of the monomolecular film formed on the substrate was performed. A substrate obtained by coating a glass substrate with aluminum vapor deposition was used for the infrared spectroscopic analysis sample, and a glass substrate was used for the other analysis samples.

CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ was dissolved in hydrofluoroether (manufactured by Sumitomo 3M: trade name “HFE-7200”) to prepare a 2 mass% organic molecule-containing liquid. Then, each substrate was immersed in this solution for 15 minutes. After the immersion, the substrate taken out of the solution was immediately washed with hydrofluoroether to remove CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl _{3 and the} like that were excessively attached to the substrate surface. Next, the substrate was naturally dried to remove the cleaning hydrofluoroether adhering to the substrate surface. The operations from the preparation of the organic molecule-containing liquid to the drying of the substrate were performed in a glove box in which dry nitrogen gas was introduced to create a dry atmosphere. The moisture content of the gas phase (nitrogen gas atmosphere) in the glove box was adjusted so that the relative humidity value was 5% or less when expressed in terms of the relative humidity value at 22 ° C.
By the above operation, in the organic molecule-containing liquid, the hydroxyl group or silanol group on the main surface of each substrate and the trichlorosilyl group at the end of CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (organic molecule) ( The condensation reaction (dehydrochlorination reaction) with -SiCl ₃ ) was allowed to proceed.

  The formed monomolecular film was analyzed by an infrared spectroscopic analyzer, a nuclear magnetic resonance apparatus, an organic mass spectrometer, a secondary ion mass spectrometer, an X-ray photoelectron spectrometer, an X-ray reflectivity measuring apparatus, and a transmission electron microscope. .

[Infrared spectroscopy]
Infrared spectroscopic analysis was performed by a highly sensitive reflection measurement method using a Fourier infrared analyzer (manufactured by Thermo Electron, Magna550). As shown in FIG. 12, the inverse target stretching vibration of the methylene group was observed at a wave number of 2930 cm ⁻¹ . Further, the target stretching vibration of the methylene group was observed at a wave number of 2888 cm ⁻¹ . This confirmed the presence of CH ₂ groups in the monomolecular film.

[ ¹ H-nuclear magnetic resonance analysis]
Nuclear magnetic resonance analysis was performed by measuring ¹ H nuclei using a nuclear magnetic resonance apparatus (Varian, UNITY INOVA600) capable of microanalysis using a nanoprobe. The measurement sample is obtained by polishing the substrate on which the monomolecular film is formed with an aluminum oxide abrasive, adding deuterated methanol and sodium hydroxide to the scraped powder, and heating the solution to remove the monomolecular component from the substrate. It was prepared by removing. As shown in FIG. 13, signals with an integrated intensity of 2H were observed at 0.72 ppm and 2.35 ppm. They were assigned to be a CH ₂ adjacent to the CH _2, CF ₂ group next to Si, respectively. Thus -CH ₂ CH ₂ monolayers - presence of was confirmed.

[ ¹⁹ F-nuclear magnetic resonance analysis]
¹⁹ F nuclei were measured using a nuclear magnetic resonance apparatus (Varian, UNITY INOVA600) capable of microanalysis using a nanoprobe in the same manner as described above. A sample prepared in the same manner as the measurement of ¹ H-nucleus was used. As shown in FIG. 14, a signal having an integrated intensity of 2F at -126.5 ppm, a signal having an integrated intensity of 2F at -123.5 ppm, -123.0 ppm and -121.9 ppm, and an integrated intensity at -122.2 ppm, respectively. Of 4F, a signal of 2F integrated intensity at -116.2 ppm, and a signal of 3F integrated intensity at -81.6 ppm. It was assigned to be _{CF 2, CF 2 CF 2 CF} 2 CF 2 CF 2, CF 2 next to the CH _2, CF ₃ next to each CF ₃ group. This confirmed the presence of CF ₃ (CF ₂ ) ₇ CH ₂ — in the monomolecular film.

[Organic mass spectrometry]
Organic mass spectrometry used a time-of-flight secondary ion mass spectrometer (TRIFT-II type time-of-flight secondary ion mass spectrometer manufactured by PHI-CEA) to analyze the substrate surface on which the monomolecular film was formed. As a result, mass numbers 31, 69, 119, and 169 were observed by positive ion analysis as shown in FIG. These mass numbers were assigned to be CF ⁺ , CF ₃ ⁺ , C ₂ F ₅ ⁺ , and C ₃ F ₇ ⁺ , respectively. Further, as shown in FIG. 16, mass numbers 19, 38, 69, 119, and 169 were observed by negative ion analysis. These mass numbers were assigned to be F ⁻ , F ₂ ⁻ , CF ₃ ⁻ , C ₂ F ₅ ⁻ and C ₃ F ₇ ⁻ , respectively. Thereby, CF ₃ CF ₂ CF _2-as a monomolecular film was confirmed.

[X-ray photoelectron spectroscopy]
Further, the C _1s bond energy of the monomolecular film was observed using an X-ray photoelectron spectrometer (SSX-100 type manufactured by SSI). As shown in FIG. 17, 285.8 eV, 287.3 eV, 289.2 eV, 291.1 eV, 292.5 eV, and 294.9 eV were observed. It was found that each energy value corresponds to CHx (+ SiCHx), CF ₂ CH ₂ , CF, CF ₂ CH ₂ , CF ₂ CF ₂ , and CF ₃ , respectively. These were found to be fragments of organic molecules, CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ .

[Thickness analysis]
The film thickness of the monomolecular film formed on the substrate was measured using an X-ray reflectivity measuring apparatus (manufactured by Rigaku Corporation). As a result, the film thickness was 2.26 nm.

  Further, using a transmission electron microscope (H-9000UHR manufactured by Hitachi, Ltd.), the epoxy resin was coated on the substrate on which the monomolecular film was formed, and the cross section was observed. As shown in FIG. 18, the thickness of the monomolecular film formed on the substrate was measured from the obtained image. As a result, the film thickness was 2 to 2.5 nm.

  From each of these analysis results, it was confirmed that the film formed on the substrate had a monomolecular film-like shape that was complementary to the substrate with a length of about one molecule.

Example 1
Hereinafter, a sample inspection apparatus having the same configuration as that of the sample inspection apparatus 102 shown in FIG. 7 was manufactured according to the procedure described with reference to FIGS.

A substrate 81 (semiconductor integrated circuit substrate, main surface size: 15 mm × 25 mm, thickness: 0.8 mm, two-dimensional at regular intervals) having the same structure as the substrate 1 shown in FIG. The number of photodiodes arranged: 120,000, the density of photodiodes arranged two-dimensionally at equal intervals: 37000 / cm ² ) was prepared.

  Next, the monomolecular film 83 was formed on the main surface of the substrate 81 (surface on the side on which the sample is placed) according to the following procedure based on the monomolecular film forming step according to the present invention.

  First, the main surface of the substrate 81 was washed with ultrapure water, and droplets of ultrapure water adhering to the surface were removed by warm air. Next, a positive resist (manufactured by Tokyo Ohka Co., Ltd., trade name “OFPR5000”) is applied to the main surface of the substrate 81 after cleaning using a spinner, and a mask pattern (the photodiodes in the substrate 81 is applied). Having a pattern corresponding to the arrangement condition).

  Next, the coated surface is subjected to ultraviolet exposure using a stepper (projection exposure apparatus), followed by development and cleaning, so that the main surface of the substrate 81 shown in FIGS. 8A and 8B is formed. A resist pattern based on the mask pattern was formed. FIG. 8A is a cross-sectional view schematically showing the resist pattern, and FIG. 8B is a case where the resist pattern shown in FIG. 8A is observed from the normal direction of the main surface of the substrate 81. FIG.

  In FIG. 8B, a plurality of rectangular patterns are resist patterns 82, and the size thereof is 60 μm long × 20 μm wide. In addition, the area | region where the resist pattern 82 is not formed of the main surfaces of the board | substrate 81 is an area | region used as 1st area | region (area | region where a monomolecular film is formed), and the area | region where the resist pattern 82 was formed is the 1st area | region. This is a region to be a region 2 (the bottom surface of the recess).

On the other hand, CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (manufactured by Shin-Etsu Chemical Co., Ltd.) as an organic molecule is dissolved in hydrofluoroether (manufactured by Sumitomo 3M Co., Ltd .: trade name “HFE-7200”). % Organic molecule-containing liquid was prepared. Then, the substrate 81 on which the resist pattern 82 was formed was immersed in this solution for 15 minutes. After the immersion, the substrate 81 taken out of the solution was immediately washed with hydrofluoroether to remove CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl _{3 and the} like that were excessively attached to the surface of the substrate 81. Next, the substrate 81 was naturally dried to remove the cleaning hydrofluoroether adhering to the surface of the substrate 81. The operations from the preparation of the organic molecule-containing liquid to the drying of the substrate were performed in a glove box in which dry nitrogen gas was introduced to create a dry atmosphere. At that time, when the moisture content in the gas phase (nitrogen gas atmosphere) in the glove box in contact with the organic molecule-containing liquid is expressed in terms of a relative humidity value at 22 ° C., the relative humidity value is 5% or less. Adjusted as follows.

Through the above operation, in the organic molecule-containing liquid, the hydroxyl group or silanol group on the main surface of the substrate 81 and the trichlorosilyl group at the end of CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ (organic molecule) ( The condensation reaction (dehydrochlorination reaction) with -SiCl ₃ ) was allowed to proceed. As a result, as shown in FIG. 9A, a monomolecular film 83 firmly fixed by a siloxane bond (—Si—O—) is formed in a region where the resist pattern 82 of the main surface of the substrate 81 is not formed. did.

  Next, the substrate 81 on which the monomolecular film 83 was formed was taken out of the glove box, and the main surface of the substrate 81 was observed with a microscope. As shown in the micrograph of FIG. 9B, it was confirmed that the monomolecular film 83 was formed in the region where the resist pattern 82 was not formed. Furthermore, from this microscopic observation, it was confirmed that the resist pattern 82 was kept in the same state as that immediately after the formation without peeling, partial lack, swelling or the like.

  Further, the substrate 81 was immersed in acetone (manufactured by Kanto Chemical Co., Inc.), and the resist pattern 82 on the main surface of the substrate 81 was completely dissolved and removed (see FIG. 10). Next, the substrate 81 was washed with ultrapure water and then dried. Next, the main surface of the substrate 81 after drying was observed with a microscope. As a result of microscopic observation, it was confirmed that the resist pattern 82 was completely removed from the surface of the substrate 81. In other words, it was confirmed that the surface of the region where the resist pattern 82 was formed was exposed, and the substrate 81 having the monomolecular film 83 formed on the surface could be obtained in the other regions.

As a result of microscopic observation, it was confirmed that the size of the bottom surface of each recess formed on the main surface of the substrate 81 was about 20 μm × about 60 μm, and a total of 120,000 pieces could be formed. That is, it was confirmed that 37,000 recesses (volume: 1.2 × 10 ⁻³ pL) could be formed per unit area (1 cm ² ) of the main surface of the substrate 81.

  Next, a liquid was dropped onto the substrate 81 thus manufactured, and it was confirmed whether or not the occurrence of contamination between the droplets accommodated in the respective recesses could be sufficiently prevented.

  First, a 2% by mass sodium chloride aqueous solution is applied to the entire main surface of the substrate 81 on which the monomolecular film 83 is formed, and a droplet (9 pL, height: about 10 μm) made of the sodium chloride aqueous solution 84 is applied to each recess. It applied (refer Fig.11 (a)). Next, after the substrate 81 was dried, the main surface of the substrate 81 was observed with a microscope. The result of the micrograph is shown in FIG. As is clear from the results shown in FIG. 11B, it is confirmed that columnar sodium chloride crystals 85 (bottom size: about 20 μm × about 60 μm) are selectively formed only in the recesses of the substrate 81. It was done. It was confirmed that all the sodium chloride crystals 85 were selectively formed only at the positions of the recesses.

Furthermore, it replaced with sodium chloride aqueous solution, and performed the same confirmation test using DNA aqueous solution containing SYBR-Green. As an aqueous DNA solution, an aqueous solution of a DNA probe (length: 40 bp) was used. As a result, it was confirmed that minute droplets made of the DNA aqueous solution could be regularly arranged only on the exposed portion of the substrate 1 without contamination. The DNA probes could be provided at a density of 37,000 pieces / cm ² .

In the above embodiment, CF ₃ (CF ₂ ) ₇ (CH ₂ ) ₂ SiCl ₃ was used as the organic molecule, but other organic molecules listed in the present specification similarly form a hydrophobic monomolecular film on the substrate. Can be formed.

  The sample inspection apparatus of the present invention is a technical field that requires analysis of a huge number of samples at different conditions such as reaction conditions and component composition conditions, for example, drug screening, drug analysis, It is useful as an analyzer in the technical field for performing protein analysis, single nucleotide polymorphism detection, and the like.

It is a perspective view which shows 1st embodiment of the sample inspection apparatus of this invention. It is a fragmentary sectional view which shows roughly the basic composition of the sample inspection apparatus shown in FIG. It is a perspective view which shows 2nd embodiment of the sample inspection apparatus of this invention. (A) is a perspective view schematically showing a basic configuration of a nozzle provided in the droplet supply unit 40 of the sample inspection apparatus 101 shown in FIG. 3, and (b) and (c) are sample inspection apparatuses 101. It is explanatory drawing for demonstrating the method of alignment of the droplet supply part 40 with respect to the recessed part 3 in FIG. (A) And (b) is explanatory drawing for demonstrating another method of alignment of the droplet supply part 40 with respect to the recessed part 3 in the sample inspection apparatus 101. FIG. (A) is a perspective view schematically showing another basic configuration of a nozzle provided in the droplet supply unit 40 of the sample inspection apparatus 101 shown in FIG. 3, and (b) is a recess in the sample inspection apparatus 101. 6 is an explanatory diagram for explaining still another method of aligning the droplet supply unit 40 with respect to 3. FIG. It is a perspective view which shows other embodiment of the sample inspection apparatus of this invention. (A) The cross section which shows typically an example of the resist pattern formed on the main surface (surface used as a sample mounting surface) of a board | substrate in the monomolecular film formation process at the time of manufacturing the sample analyzer of Example 1. It is a figure, (b) is a figure which shows the microscope picture at the time of observing the resist pattern shown to (a) from the normal line direction of the main surface of a board | substrate. (A) is sectional drawing which shows typically an example of the resist pattern and monomolecular film which were formed on the board | substrate in the monomolecular film formation process at the time of manufacturing the sample analyzer of Example 1, (b) It is a figure which shows the microscope picture at the time of observing the resist pattern shown to (a) from the normal line direction of the main surface of a board | substrate. FIG. 4 is a cross-sectional view schematically showing an example of a monomolecular film formed on a substrate in a monomolecular film forming step when manufacturing the sample analyzer of Example 1. (A) is sectional drawing which shows typically the state in which the droplet of sodium chloride aqueous solution was accommodated in the recessed part formed on the board | substrate of the sample analyzer of Example 1, (b) is (a). It is a figure which shows the microscope picture at the time of observing the shown board | substrate surface from the normal line direction of the main surface of the said board | substrate. It is the spectrum figure which analyzed the monomolecular film by infrared spectroscopy. It is the spectrum figure which analyzed the monomolecular film by < ¹ > H-nuclear magnetic resonance analysis. It is the spectrum figure which analyzed the monomolecular film by ¹⁹ F-nuclear magnetic resonance analysis. It is the spectrum figure which analyzed the monomolecular film by organic mass spectrometry (positive ion analysis). It is the spectrum figure which analyzed the monomolecular film by organic mass spectrometry (negative ion analysis). It is the spectrum figure which analyzed the monomolecular film by X-ray photoelectron spectroscopy analysis. It is a cross-sectional photograph by a transmission electron microscope.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Substrate (semiconductor integrated circuit board), 2 ... Monomolecular film, 3 ... Recess, 4 ... CPU, 5 ... Memory part, 6 ... Data input / output part, 7 ..Semiconductor layer 8 ... Inorganic oxide layer 9 ... Interlayer insulating film 10 ... Insulating film 11 ... Color filter 12 ... Protective layer 40 ... Drop supply Part, 50, 50A ... nozzle, 70 ... photoelectric conversion part, 72 ... p-well, 74 ... n-type region, 100, 101, 102 ... sample inspection device, F1 ... sample Placement surface, F11... First region, F12... Second region.

Claims (29)

At least a substrate having a sample mounting surface provided with two or more recesses for independently containing two or more droplets containing a measurement object to be analyzed;
The sample mounting surface has a first region covered with a hydrophobic first monomolecular film, and an inner periphery thereof is not covered with the first monomolecular film and the entire outer peripheral portion is the first region. And at least divided into two or more second regions surrounded by
The bottom surface of the recess is formed by the second region, and the outer peripheral portion including the entire inner surface of the recess is formed by the first monomolecular film covering the first region,
In the substrate, two or more photoelectric conversion units capable of independently photoelectrically converting light emitted from each of the two or more droplets accommodated in each of the two or more recesses are the two or more. Are provided so as to correspond one-to-one with each of the recesses of
The first monomolecular film is formed using an organic molecule, and is fixed to the sample mounting surface by a covalent bond ,
The bottom surface of the concave portion is further coated with a second monomolecular film made of a material having optical transparency to the light,
A thickness of the second monomolecular film is equal to or less than a thickness of the first monomolecular film covering the first region;
A hydrophilic fourth characteristic group capable of at least hydrogen bonding with the solvent contained in the droplet is bonded to the surface of the second monomolecular film,
The second monomolecular film is formed using an organic molecule, and is fixed to the sample mounting surface by a covalent bond ,
Sample inspection device characterized by
The sample inspection apparatus according to claim 1, wherein a volume of the concave portion is 2 × 10 ⁻⁶ pL to 1 pL. The sample inspection apparatus according to claim 1 or 2, wherein the number of the concave portions formed per unit area of the sample mounting surface is 10,000 pieces / cm ² or more. The sample inspection apparatus according to any one of claims 1 to 3, wherein a thickness of the first monomolecular film is 0.5 to 50 nm.
The first monomolecular film is:
As the substrate, a hydrophilic first having at least one structure selected from the group consisting of —OH, —NH ₂ , ═N—H, and —SH at a terminal portion bonded to the organic molecule. Using a substrate having a characteristic group on the sample mounting surface,
As the organic molecule, an organic molecule having a second characteristic group capable of condensation reaction with the first characteristic group at one end of the molecular chain and a hydrophobic third characteristic group at the other end of the molecular chain is used. And
The organic molecule is brought into contact with the sample mounting surface, and formed through a monomolecular film forming step in which a condensation reaction between the first characteristic group and the second characteristic group proceeds. The sample inspection apparatus according to any one of the above.
In the monomolecular film forming step, the condensation reaction is carried out by bringing an organic molecule-containing liquid obtained by adding the organic molecules into an aprotic solvent in a container and a sample mounting surface of the substrate. Includes a first step to proceed,
In the first step, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed in terms of a relative humidity value at 22 ° C., the relative humidity value is 35% or less. The sample inspection apparatus according to claim 5 to be adjusted. The organic molecule is
The second characteristic group has a characteristic group represented by the following general formula (1) capable of condensation reaction with the first characteristic group,
As the third characteristic group, a characteristic group selected from the group consisting of a methyl group, a halogen-substituted methyl group, a vinyl group, a cyclic ether group having 2 to 4 carbon atoms, a phenyl group, a halogen-substituted phenyl group, and a cyano group And
The structure according to any one of claims 1 to 6, which has a structure in which a divalent organic group represented by the following general formula (2) is bonded between the second characteristic group and the third characteristic group. The sample inspection apparatus according to one item.
−C _b E _2b − (2)
[In the formula (1), Z ¹ is at least one atom selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. Or Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms, and a represents an integer of 1 to 3. ,
In formula (2), E represents at least one atom selected from the group consisting of H and F, and b represents an integer of 2 to 22. ] The organic molecule has a characteristic group represented by the following general formula (3), -O-, -COO- between carbon atoms constituting the carbon skeleton of the divalent organic group represented by the general formula (2). And at least one divalent characteristic group selected from the group consisting of —C ₆ H ₄ —.
[In Formula (3), g and h show the integer of 1-3 each independently. ] The organic molecule is
The second characteristic group has a characteristic group represented by the following general formula (1) capable of condensation reaction with the first characteristic group,
The third characteristic group is a monovalent group selected from the group consisting of a methyl group, a halogen-substituted methyl group, a vinyl group, a cyclic ether group having 2 to 4 carbon atoms, a phenyl group, a halogen-substituted phenyl group, and a cyano group. Having two groups, and
Any one of Claims 1-6 which has the structure which the trivalent organic group represented by following General formula (4) couple | bonded between the said 2nd characteristic group and the said 3rd characteristic group. The sample inspection apparatus according to one item.
[In the formula (1), Z ¹ is at least one atom selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. Or Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms, and a represents an integer of 1 to 3. ,
In the formula (4), C _j L _2j is a characteristic group bonded to the second characteristic group, C _m G _2m and C _n J _2n are characteristic groups bonded to the third characteristic group, and G, J And L may be the same or different, and each represents at least one atom selected from the group consisting of H and F; j represents an integer of 1 to 18; Independently represents an integer of 0 to 7. ] The sample inspection apparatus according to claim 7, wherein the organic molecule has a structure represented by any one of the following general formulas (20) to (29).
[In the formulas (20) to (29), Z ¹ is at least selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. One atom or atomic group, Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms; Q represents an integer of 2 to 22, and m and n are respectively conditions represented by the following formulas (I) to (III);
0 ≦ m ≦ 14 (I)
0 ≦ n ≦ 15 (II)
2 ≦ (m + n) ≦ 22 (III)
An integer that satisfies ] The sample inspection apparatus according to claim 8, wherein the organic molecule has a structure represented by any one of the following general formulas (30) to (39).
[In the formulas (30) to (39), Z ¹ is at least selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. One atom or atomic group, Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms; of an integer, a is characteristic group represented by the general formula (3), -O -, - COO-, and, -C ₆ H ₄ - at least one divalent selected from the group consisting of A characteristic group, t is an integer of 1 to 10, p is an integer of 1 to 18, and r and s are conditions represented by the following formulas (IV) to (VI), respectively;
0 ≦ r ≦ 14 (IV)
0 ≦ s ≦ 15 (V)
2 ≦ (r + s) ≦ 22 (VI)
An integer that satisfies ] The sample inspection apparatus according to claim 9, wherein the organic molecule has a structure represented by any one of the following general formulas (40) to (49).
[In the formulas (40) to (49), Z ¹ is at least selected from the group consisting of F, Cl, Br, I, —OH, —SCN, —NCO, and an alkoxy group having 1 to 5 carbon atoms. One atom or atomic group, Z ² represents at least one atom or atomic group selected from the group consisting of H and an alkyl group having 1 to 5 carbon atoms; T represents an integer of 1 to 10, p represents an integer of 1 to 18, and r and s are the conditions represented by the following formulas (IV) to (VI), respectively;
0 ≦ r ≦ 14 (IV)
0 ≦ s ≦ 15 (V)
2 ≦ (r + s) ≦ 22 (VI)
An integer that satisfies ] The at least one structure selected from the group consisting of -Si-O-, -Si-N-, and -Si-S- is included in the covalent bond. The sample inspection apparatus as described in any one of Claims. The first characteristic group has at least one structure selected from the group consisting of —SO ₃ H, —SO ₂ H, —PO ₃ H, —PO ₃ H ₂ , —CO ₂ H, and —SH. The sample inspection apparatus according to any one of claims 5 to 13. The sample inspection apparatus according to claim 1, wherein the droplet further includes a probe that reacts with the measurement object to be analyzed. The sample inspection apparatus according to claim 15, wherein the probe is at least one selected from the group consisting of a polynucleotide and a labeled polynucleotide. The volume of the recess is 2 × 10 ^-6 pL ~ 2x10 ^-3 pL,
The number of the concave portions formed per unit area of the sample mounting surface is 1,000,000 / cm. ² ~ 8 million pieces / cm ² And
The first monomolecular film has a thickness of 0.5 nm to 10 nm;
The sample inspection apparatus according to claim 1, wherein a volume of a droplet accommodated in the concave portion is 0.01 pL to 1.2 pL.
The fourth characteristic group includes —OH, —NH ₂ , ═N—H, —SH, —SO ₃ H, —SO ₂ H, —PO ₃ H, —PO ₃ H ₂ , —CHO, and —CO. The sample inspection apparatus according to claim 1 , comprising at least one structure selected from the group consisting of ₂ H.
The sample inspection apparatus according to claim 1 or 18, wherein a probe that reacts with a measurement object to be analyzed is fixed on a surface of the second monomolecular film.
The sample inspection apparatus according to claim 19, wherein the probe is at least one selected from the group consisting of a polynucleotide and a labeled polynucleotide. The substrate is
An uppermost layer having the sample placement surface and having optical transparency to the light emitted from the droplet;
A semiconductor layer disposed in a state bonded to the back surface of the uppermost layer, and including the two or more photoelectric conversion units;
A laminate having at least
The sample inspection apparatus according to claim 1, wherein a photoelectric conversion element is provided in each of the two or more photoelectric conversion units. A droplet supply unit that is disposed above the sample mounting surface and has a nozzle that discharges a droplet including the measurement object to be analyzed toward the recess;
A controller that adjusts the position of the nozzle relative to the recess by moving the nozzle;
The sample inspection apparatus according to any one of claims 1 to 21, further comprising: The sample inspection apparatus according to any one of claims 1 to 22, wherein the photoelectric conversion element has a pn junction surface. In the monomolecular film forming step,
Forming a resist pattern for covering the region to be the second region of the sample mounting surface, and defining the region of the sample mounting surface where the resist pattern is not formed as the first region;
By contacting the organic molecules with the sample mounting surface after forming the resist pattern, the first monomolecular film is selectively covered only in the first region, and then the resist pattern is coated with the resist pattern. The sample inspection apparatus according to any one of Claims 5 to 23, wherein the two or more recesses are formed by removing.
In the monomolecular film forming step,
Forming the first monomolecular film on the sample mounting surface;
Using a photomask for selectively protecting the first monomolecular film covering the region to be the first region of the sample mounting surface from ultraviolet rays,
The sample mounting surface after the first monomolecular film is formed through the photomask is irradiated with the ultraviolet rays to cover the region to be the second region of the sample mounting surface. The sample inspection apparatus according to any one of claims 5 to 23, wherein the two or more concave portions are formed by selectively removing only the first monomolecular film.
Two or more photoelectric conversion units are built in a two-dimensional array, and at least one of the two main surfaces facing each other is —OH, —NH ₂ , ═N—H, and A substrate forming step of forming a substrate to which a hydrophilic first characteristic group having at least one structure selected from the group consisting of -SH is bonded;
The main surface of the substrate to which the first characteristic group is bonded is used as a sample mounting surface for independently fixing two or more droplets containing the measurement object to be analyzed, and the sample mounting surface is a first region which is covered by the first monolayer of hydrophobic, 2 or more of the total peripheral portion not internally coated on the first monolayer is surrounded by said first region Two or more for independently containing the two or more droplets by forming at least the first region and selectively forming the first monomolecular film only in the first region. A monomolecular film forming step in which the concave portion is provided so as to correspond to each of the two or more photoelectric conversion portions on a one-to-one basis;
At least
The bottom surface of the recess is further coated with a second monomolecular film made of a light-transmitting material,
A thickness of the second monomolecular film is equal to or less than a thickness of the first monomolecular film covering the first region;
A hydrophilic fourth characteristic group capable of at least hydrogen bonding with the solvent contained in the droplet is bonded to the surface of the second monomolecular film,
The second monomolecular film is formed using an organic molecule, and is fixed to the sample mounting surface by a covalent bond,
In the monomolecular film forming step,
An organic molecule having a second characteristic group capable of condensation reaction with the first characteristic group at one end of the molecular chain and having a hydrophobic third characteristic group at the other end of the molecular chain is the first single molecule. Used as raw material for membranes,
The bottom surface of the obtained recess is formed by the second region, and the outer peripheral portion including the entire inner surface of the recess is formed by the first monomolecular film covering the first region. An organic molecule is brought into contact with the sample mounting surface, a condensation reaction between the first characteristic group and the second characteristic group proceeds, and the first monomolecular film is fixed to the sample mounting surface by a covalent bond. Forming in the state
A method of manufacturing a sample inspection apparatus characterized by the above.
In the monomolecular film forming step, the condensation reaction is carried out by bringing an organic molecule-containing liquid obtained by adding the organic molecules into an aprotic solvent in a container and a sample mounting surface of the substrate. Includes a first step to proceed,
In the first step, when the moisture content in the gas phase in contact with the organic molecule-containing liquid is expressed in terms of a relative humidity value at 22 ° C., the relative humidity value is 35% or less. 27. A method of manufacturing a sample inspection apparatus according to claim 26, wherein the adjustment is performed. In the monomolecular film forming step,
Forming a resist pattern for covering the region to be the second region of the sample mounting surface, and defining the region of the sample mounting surface where the resist pattern is not formed as the first region;
By contacting the organic molecules with the sample mounting surface after forming the resist pattern, the first monomolecular film is selectively covered only in the first region, and then the resist pattern is coated with the resist pattern. 28. The method of manufacturing a sample inspection apparatus according to claim 26 or 27, wherein the two or more recesses are formed by removing.
In the monomolecular film forming step,
Forming the first monomolecular film on the sample mounting surface;
Using a photomask for selectively protecting the first monomolecular film covering the region to be the first region of the sample mounting surface from ultraviolet rays,
The sample mounting surface after the first monomolecular film is formed through the photomask is irradiated with the ultraviolet rays to cover the region to be the second region of the sample mounting surface. 28. The method of manufacturing a sample inspection apparatus according to claim 26, wherein the two or more recesses are formed by selectively removing only the first monomolecular film.