CN102023208B - Microbead analysis method and microbead analyser - Google Patents

Abstract

The invention provides a microbead analysis method and a microbead analyser. The microbead is in the shape of a cylinder, and is provided with a top surface and a bottom surface, which are disposed in an opposite as well as parallel manner, and a side surface extends from the top surface and the bottom surface. The microbead can be used to hold a recognization pattern disposed at least one of the top surface and the bottom surface and the material fixed on the surface of the microbead, which has close affinity with the analyzed material. The microbead analysis method can be implemented in such a way that the fluorescence emitted from the surface of the microbead caused by the interaction between the analyzed material and the material having close affinity with the analyzed material on the areas including the area on the top surface and the bottom surface, on which is the recognization pattern not formed, and the side surface, can be detected.

Description

Microballon analytical approach and microballon analyzer

Technical field

The present invention relates to microballon analytical approach and microballon analyzer.More specifically, for example the present invention relates to carry by use the microballon analysis that the microballon of pattern formed thereon carries out, this pattern is for the image recognition of each microballon.

Background technology

For example, particulate vector (so-called " microballon ") is for the biochemical analysis of nucleotide and protein.For example, in nucleotide analysis, carry the microballon of surveying nucleotide chain (it has with the base sequence of target nucleotide chain complementation and is fixed in bead surface) for the target nucleotide chain of emanating in target nucleotide chain and the interaction of surveying nucleotide chain.Or, in protein analysis, the microballon that similarly there is the antibody with respect to target protein in the bead surface of being fixed on by the use target protein of emanating.

If carry out mark with fluorescent material in advance, target nucleotide chain or the target protein of can optical detection in bead surface, catching and separating.In addition the fluorescence intensity of measuring in bead surface, makes it possible to the target material that quantitative measurement separates.In the time that target material is nucleotide chain, if use the intercalator of emitting fluorescence in the time being incorporated in the combination chain forming in the interaction between nucleotide chain at target nucleotide chain and detection, the target nucleotide chain can optical detection separating.

Recently, use the biochemical analysis of above-mentioned microballon aspect processing power, further to improve, and developed various technology further to improve the processing speed of analysis.

For example, Jap.P. No.3468750 (hereinafter, be called patent documentation 1) disclose " detect the method for the multiple analytes in sample; its analytical reactions product by multiple analytes is identified multiple analytes; the method comprises: a) make various types of fluorescent grains contact with sample; each fluorescent grain has different fluorescence signals and different analytical reactions products; the specifically bonding of an analyte in each analytical reactions product and sample, each fluorescent grain has at least one and is marked with from the teeth outwards the nano particle of fluorescent dye; B) sample is added to labelled reagent; C) analyze the fluorescent grain of indicating analytical reactions product and analyte bonding by certification mark; Meanwhile, d) according to the function of the different fluorescence signals relevant from each fluorescent grain, determine the fluorescent grain with each analyte bonding " (referring to its claim 23).

According to prior art, in " suspension array technology (the Suspension Array Technology) " being provided by Luminex company, two kinds of fluorescent colorant mark microballons that change to some extent by being used in the color aspect of launched light, can identify nearly 100 kinds of microballons.By fix different detection nucleotide chain or antibody on 100 kinds of microballons, can in once analyzing, analyze 100 kinds of different target nucleotide chains or target protein by " suspension array technology " simultaneously.

Above-mentioned patent documentation 1 has also been recorded " limiting fluorescent grain by the size and dimension of microballon extraly " (referring to its claim 25), and disclose and can use the size and dimension of microballon as the additional parameter for identifying microballon (for example,, referring to the 0037th section of patent documentation 1 etc.).

Accordingly, at " Multifunctional encoded Particles for high-throughputbiomolecule analysis " (Science, 2007, the 315th volume, No. 5817,1393-6 page (hereinafter, being called non-patent literature 1)) in disclose and formed the technology that makes it possible to microballon to carry out the some coding of image recognition.Can prepare many kinds (more than 1,000,000 kind) microballon by above-mentioned technology.The document has been recorded by prepare microballon by photoetching in pipeline, and this microballon carries the lip-deep point code of half of the oval surface that is formed at microballon and is fixed on second half lip-deep detection nucleotide chain (referring to Fig. 1 of non-patent literature 1).

Summary of the invention

For the microballon that carries the identification icon such as a coding, detect from as be fixed on the target material bonding of the mark bead surface or be combined in the process of fluorescence of fluorescent material transmitting of the intercalator bonding in target material, worry that identification icon self can emitting fluorescence.Measuring according to the fluorescence intensity in bead surface in the process of target amount of substance, the above-mentioned fluorescence from identification icon becomes noise fluorescence (causing the factor of measuring error).

In addition,, because form the different multiple identification icon of pattern form on each microballon, the glimmering light intensity of the noise producing on each microballon can change according to the difference of pattern form.In this case, need to after the impact of proofreading and correct the noise fluorescence on each microballon, calculate target amount of substance, this causes analysis speed to reduce again.

In non-patent literature 1, disclosed microballon has the oval surface that two halves separate: be used to form the region of a coding and the region for fixing detection nucleotide chain.If can only detect fluorescence from survey nucleotide chain fixed area, can detect from serving as a mark and the fluorescence of the fluorescent material of target nucleus glycosides chain bonding with microballon, and not be subject to the impact from the noise fluorescence of identification icon.But the document does not have any description about this possibility.

But, in order to manufacture this microballon that the some coding forming discretely forms region and surveys nucleotide chain fixed area that has, need very complicated preparation process.Particularly, need to carry out following steps: will form the monomer flow in region for the preparation of a coding and in pipeline, be fed in the situation that they contact with each other for the preparation of the fluid that detects nucleotide chain, and the monomer flow that forms region for some coding with UV irradiation is to carry out photoetching (referring to Fig. 1 of non-patent literature 1).A coding is formed to region to be separated and has also caused the problem that increases the overall dimensions of microballon with detection nucleotide chain fixed area.

Therefore, need following microballon analytical approach: its high precision detects from serving as a mark and the fluorescence of the fluorescent material of target material bonding etc., is not subject to the impact from the noise fluorescence of identification icon, and does not also need complicated preparation process.

According to embodiments of the invention, a kind of microballon analytical approach is provided, it is for being formed the microballon of cylinder body shape, described microballon have face with each other and almost parallel place top surface and basal surface, and the side surface extending from described top surface and described basal surface, and described microballon carries at least one the lip-deep identification icon being formed in described top surface and described basal surface, and the lip-deep and amalyzing substances that is fixed on described microballon has the material of compatibility, the region of described method from the region that does not form identification icon and described side surface including described top surface and described basal surface detect due to described amalyzing substances and with described amalyzing substances have compatibility described material interaction and from the fluorescence of described bead surface transmitting.

Above-mentioned analytical approach comprises the steps:

(1) described microballon is mixed with described amalyzing substances;

(2) obtain the transmission image of the described microballon that comprises described identification icon, and detect described identification icon by described transmission image; And

(3) obtain the fluoroscopic image of described microballon, and detect the described fluorescence from the region the region and the contour area that do not form described identification icon in described top surface and the described basal surface of described microballon by described fluoroscopic image.

By detecting the fluorescence from above-mentioned zone, can detect the high strength fluorescence of getting rid of the noise fluorescence obtaining from identification icon.

In above-mentioned microballon analytical approach, in the time described microballon being placed on to the Surface Contact of a surface measuring on substrate and in described top surface and the described basal surface of described microballon and described measurement substrate, obtain described transmission image and described fluoroscopic image by image acquiring device, described image acquiring device is arranged on the position of facing with described measurement substrate surface.

In the time that the microballon of arrangement described above is maintained on measurement substrate, the transmission image obtaining by image acquiring device comprises reliable identification icon.

Preferably, when on the surface of measurement substrate that described microballon is placed on to surface roughening, or when on the surface that described microballon is placed on to described measurement substrate in liquid, obtain described transmission image and described fluoroscopic image.

Measure on substrate by as mentioned above microballon being placed on, can obtain the transmission image and the fluoroscopic image that do not produce interference fringe.

In addition, when the image of can be in taking in the described top surface of described microballon and described basal surface, obtain described transmission image, can, in the time taking the image of described side surface of described microballon, obtain described fluoroscopic image.

According to another embodiment of the present invention, a kind of microballon analyzer is also provided, and it is for analyzing microballon, and described microballon carries identification icon in its surface, described microballon analyzer comprises: image acquiring device, and it is for obtaining transmission image and the fluoroscopic image of described microballon; For detected the device of described identification icon by described transmission image; With for detecting at described fluoroscopic image from the device of fluorescence in region of described identification icon that does not form described microballon.

Above-mentioned microballon analyzer also comprises: measure substrate, it is for placing described microballon, and the surface of wherein said measurement substrate and described image acquiring device are placed as and face with each other, and described measurement substrate is surface roughening.

According to another embodiment of the present invention, a kind of microballon analyzer is provided, it is for analyzing the microballon that is formed cylinder body shape, described microballon has and faces with each other and top surface that almost parallel is placed and basal surface and the side surface that extends from described top surface and described basal surface, and described microballon carries at least one the lip-deep identification icon being formed in described top surface and described basal surface.Described microballon analyzer comprises: for the image acquiring device of transmission image, it is for obtaining the transmission image of described microballon; With the image acquiring device for fluoroscopic image, it is for obtaining the fluoroscopic image of described microballon.The described image acquiring device for transmission image obtains the described top surface of described microballon and a surperficial transmission image of described basal surface, and the described image acquiring device for fluoroscopic image obtains the fluoroscopic image of the described side surface of described microballon.

Above-mentioned microballon analyzer can comprise pipeline.In this case, the described image acquiring device for transmission image and the described image acquiring device for fluoroscopic image obtain the image of the described microballon flowing at described pipeline.

According to another embodiment of the present invention, a kind of microballon analyzer is provided, and it is for analyzing microballon, and described microballon carries identification icon in its surface, described microballon analyzer comprises: image acquisition section, and it obtains transmission image and the fluoroscopic image of described microballon; Part I, it detects described identification icon by described transmission image; And Part II, it detects from the fluorescence in region of described identification icon that does not form described microballon in described fluoroscopic image.

According to another embodiment of the present invention, a kind of microballon analyzer is provided, it is for analyzing the microballon that is formed cylinder body shape, described microballon has and faces with each other and top surface that almost parallel is placed and basal surface and the side surface that extends from described top surface and described basal surface, and described microballon carries and is formed at least one lip-deep identification icon in top surface and described basal surface described in it.Described microballon analyzer comprises: for the image acquisition section of transmission image, it obtains the transmission image of described microballon; With the image acquisition section for fluoroscopic image, it obtains the fluoroscopic image of described microballon.The described image acquisition section for transmission image obtains the described top surface of described microballon and a surperficial transmission image of described basal surface, and the described image acquisition section for fluoroscopic image obtains the fluoroscopic image of the described side surface of described microballon.

In the present invention, " identification icon " is the given shape being formed on microballon, and " identification icon " can be identified by the general image recognition device such as CCD camera and image analysis software.Identification icon is the shape for distinguishing each microballon, and concrete shape, size and other features are not subject to concrete restriction.The typical case of identification icon comprises so-called some coating, bar code etc.

In the present invention, " amalyzing substances " comprises the far-ranging compound of analyzing in the biochemical analysis of use microballon, for example nucleic acid, protein and peptide.For example, amalyzing substances can comprise little molecule in the body such as steroid hormone and catecholamine, cell within a cell organ, virus or various cells such as microbial cell or mammalian cell etc. such as microsome and mitochondria.

" there is the material of compatibility with amalyzing substances " and comprise far-ranging can interact with amalyzing substances and can with the compound of amalyzing substances bonding with compatibility, for example nucleic acid, protein, peptide, sugar chain and various synthetic compound and native compound.

Amalyzing substances and comprise " nucleic acid-nucleic acid ", " protein (or peptide)-protein (comprising antibody) ", " protein (or peptide)-nucleic acid (fit) " and " protein-sugar chain " with the example that amalyzing substances has a combination of the material of compatibility.For the combination of " nucleic acid-nucleic acid ", " interaction " is between the nucleic acid of base sequence with mutual complementation, to form two strands.For example, for example, or for the combination of " protein-protein ",, " interaction " is protein-protein bonding, the bonding between receptor protein and ligandin matter or the bonding between antigen protein and antibody.

Sugar chain comprises the monose chain of mutual bonding and the monose chain with lipid or protein modification, also comprises oligosaccharides, glycolipid and glycoprotein etc.Nucleic acid comprises DNA and RNA, also has the nucleic acid analog that the structure by changing ribose district and phosphate backbone district obtains (such as LNA (lock nucleic acid) and PNA) etc.

According to embodiments of the invention, provide high precision test example as from serving as a mark and the microballon analytical approach of the fluorescence of the fluorescent material of target material bonding, the method is not subject to the impact from the noise fluorescence of identification icon, and do not need complicated preparation process.

According to the following detailed description for the best mode for carrying out the invention shown in accompanying drawing, above-mentioned and other object of the present invention, feature and advantage will become clearer.

Brief description of the drawings

Fig. 1 is for the schematic diagram in the example of the microballon that microballon analytical approach is used is according to an embodiment of the invention described, wherein figure (A) is vertical view, and figure (B) is side view;

Fig. 2 A to Fig. 2 C is the schematic diagram of the identification icon for the code area that is formed at microballon is described;

Fig. 3 A to Fig. 3 C is the schematic diagram for the lip-deep detecting material that is fixed on microballon is described;

Fig. 4 A and Fig. 4 B are that explanation carries the transmission image of microballon of identification icon formed thereon and the photo of the example of fluoroscopic image (not being to draw), and wherein Fig. 4 A is transmission image, and Fig. 4 B is fluoroscopic image;

Fig. 5 is for the schematic diagram of the schematic structure of microballon analyzer (the first example) is according to an embodiment of the invention described;

Fig. 6 is for the schematic diagram of the structure of the measurement substrate of microballon analyzer is according to an embodiment of the invention described;

Fig. 7 is for illustrating that fluorescence detection device carries out the schematic diagram in the region of fluoroscopic examination;

Fig. 8 is for the schematic diagram of the schematic structure of microballon analyzer (the second example) is according to an embodiment of the invention described; And

Fig. 9 A and Fig. 9 B are that explanation carries the transmission image of microballon of identification icon formed thereon and the photo of other examples of fluoroscopic image (not being to draw), and wherein Fig. 9 A is transmission image, and Fig. 9 B is fluoroscopic image.

Embodiment

Hereinafter, embodiments of the invention will be described with reference to the drawings.The embodiments described below are exemplary embodiment of the present invention, and should be appreciated that scope of the present invention not limits thus.To the present invention be described according to following order:

1. microballon

(1) identification icon

(2) detecting material

(3) method of generation microballon

(i) film forms step

(ii) forming step

(iii) separating step

(iv) detecting material fixing step

2. microballon analytical approach (the first example)

(1) microballon analytical approach general introduction

(2) microballon analyzer (the first example)

(3) detailed process of microballon analytical approach

(i) course of reaction

(ii) keep-process

(iii) testing process

(a) detect identification icon

(b) detect fluorescence

3. microballon analytical approach (the second example)

(1) microballon analyzer (the second example)

(2) detailed process of microballon analytical approach

(i) pipe feed process

(ii) testing process

(a) detect identification icon

(b) detect fluorescence

1. microballon

(1) identification icon

In microballon analytical approach according to an embodiment of the invention, use to carry the identification icon being formed on microballon and be fixed on to there is the microballon of the material of compatibility with amalyzing substances on microballon.Fig. 1 shows the example of the microballon using in microballon analytical approach according to embodiments of the invention.Fig. 1 (A) is schematic plan, and Fig. 1 (B) is schematic side elevation.

In Fig. 1, microballon represents by Reference numeral 1, and is formed cylinder body shape, and this cylinder body shape has and faces with each other and top surface 11 that almost parallel is placed and basal surface 12 and the side surface 13 that extends from top surface and basal surface.Observe and be circular top surface 11 and basal surface 12 although below the microballon of describing as example is had from top, and microballon 1 entirety is cylindrical, but can be in shape triangular prism, four jiaos of prisms or other polygonal prisms for the microballon of embodiments of the invention.But, in order to form the transmission image that comprises identification icon according to method described below, top surface 11 and basal surface 12 need to be formed as to cylinder body shape, make top surface and basal surface toward each other and place substantially in parallel.

The diameter D of the thickness H of microballon 1 and top surface 11 (or basal surface 12) determines arbitrarily, but preferably, makes whole microballon 1 be formed as plate-like shape by making thickness H be less than diameter D.

On microballon 1 at least one surface in top surface 11 and basal surface 12, (in Fig. 1, being top surface 11) has code area 111, is formed for the pattern of the image recognition to each microballon on code area 111.Region on top surface 11 except code area 111 is the noncoding region 112 that does not form identification icon.Can on basal surface 12, form code area 111, or can all form code area 111 on both at top surface 11 and basal surface 12.

Fig. 2 A to Fig. 2 C is the schematic plan of the identification icon for the code area 111 that is formed at microballon 1 is described.Here, microballon 1 is interpreted as comprising the 1A, the 1B that carry respectively different identification icons and one group of microballon of these 3 kinds of microballons of 1C.

The multiple through holes 2 that are penetrated into basal surface 12 from the top surface 11 of microballon are formed at Fig. 2 A (referring to the dot pattern shaded circles in figure) to the code area 111 of each microballon 1A, the 1B shown in 2C and 1C.In the matrix that can be listed as the five-element five in code area 111, form 25 through holes 2 at most altogether, these through holes have formed the identification icon for identifying microballon 1.For microballon 1A, 1B or 1C, identify each microballon 1A, 1B or 1C according to the position that is formed with through hole 2 in 25 positions.

Specifically, for the microballon 1A shown in Fig. 2 A, on 9 positions in 25 possible positions, form through hole 2.In the drawings, form the position of through hole 2 and represented by dot pattern shaded circles, and the position that does not form through hole is represented by the white point with black circle.For the microballon 1B shown in Fig. 2 B, form through hole 2 nine positions equally, but the position of pattern is different from the pattern position in microballon 1A.Therefore,, according to the position of formed through hole 2, can identify respectively microballon 1A and microballon 1B.

Or, for the microballon 1C shown in Fig. 2 C, form through hole 10 positions.Therefore,, according to the quantity of formed through hole 2, can make microballon 1C be different from microballon 1A or microballon 1B.

The quantity of the through hole 2 forming in code area 111 is arbitrarily in 0 to 25 scope, can form through hole in any position selected from 25 positions.As mentioned above, by quantity and the position of any change through hole 2, can in the code area of microballon 1 111, form different patterns.Because carry out check pattern by pattern recognition device, so can identify 25 power kind microballons up to 2.

Above-mentioned identification icon is example.If the identification icon forming on the microballon for embodiments of the invention is the known discernible pattern of pattern recognition device, the shape and size of this pattern being not particularly limited.

(2) detecting material

The material that has a compatibility with amalyzing substances is fixed on the surface of microballon 1.Fig. 3 A to Fig. 3 C shows on the surface that is fixed on microballon 1 and has the schematic diagram of the material of compatibility with amalyzing substances.Hereinafter, amalyzing substances will be called as " target material ", and the material with amalyzing substances with compatibility will be called as " detecting material ".

To be fixed on the surface of the microballon 1A shown in Fig. 3 A by the represented detecting material of Reference numeral PA.Detecting material PA is the compound suitable with target material, for example, have the nucleic acid of specific base sequence, the protein with specific amino acid sequence or peptide or sugar chain.Detecting material PA is at least fixed on the top surface 11 including code area 111 and noncoding region 112 and on the side surface 13 of microballon 1A, detecting material can also be fixed on basal surface 12 in addition.If also formed identification icon on basal surface 12, detecting material PA is fixed on the code area and noncoding region of basal surface 12.

In the time that target material is nucleic acid, detecting material PA has and the nucleotide chain of the base sequence of the base sequence complementation of target nucleotide chain.By hybridizing (forming double-stranded) with detecting material PA, the target nucleotide chain in seizure and separating sample on microballon 1A in this way.In this case, if detecting material PA has the base sequence with at least a portion complementation of the base sequence of target nucleotide chain, and detecting material PA can form two strands under specific cross reaction conditions, the base quantity (length) of detecting material PA is arbitrarily, and base quantity is not subject to concrete restriction.Conventionally, the base quantity of detecting material PA is several to several (dozen) individual bases, preferably approximately 10 to 30 bases.

In the time that target material is protein, detecting material PA be peptide (for example, the partial amino-acid series of protein ligands), antibody or can with the interactional material of target protein (for example, receptor protein).In the interaction of target protein and detecting material PA, the target protein in seizure and separating sample on microballon 1A in this way.

On the other hand, will be separately fixed on the surface of the microballon 1B shown in Fig. 3 B or 3C or 1C by Reference numeral PB or the represented detecting material of PC.Detecting material PB or PC are the compounds suitable with target material, for example, have the nucleic acid of specific base sequence, the protein with specific amino acid sequence or peptide or sugar chain.For microballon 1B or 1C, detecting material is at least fixed on the top surface 11 including code area 111 and noncoding region 112 and on side surface 13 equally.

The lip-deep detecting material PA that is fixed on microballon 1A is the material from different target materials with compatibility with the lip-deep detecting material PB and the PC that are fixed on microballon 1B and 1C.Therefore, can on the surface of microballon 1A, 1B and 1C, catch respectively and separate different target material (TA, TB and TC).In this case, for example, detecting material PA, PB and PC have not homotactic nucleotide, protein or peptide, or the different antibody of antigenicity.

Detecting material PA, PB and PC respectively with the interaction of target material TA, TB and TC in, maintain microballon 1A, 1B and the 1C emitting fluorescence of the target material capturing.For example, fluorescence is from serving as a mark and the fluorescent material of target material bonding or from being combined in intercalator transmitting between detecting material and target material.Microballon analytical approach can be by side by side detect fluorescence and by using pattern recognition device identification to be formed at the identification icon on each microballon, analyze target substances of multiple types simultaneously according to an embodiment of the invention.

(3) method of generation microballon

For example, can prepare the microballon using in microballon analytical approach according to an embodiment of the invention according to the following step.

(i) film forms step

First, on substrate, form film (for the material of microballon).For example, for substrate be glass substrate or silicon substrate.Material for substrate is not subject to concrete restriction, can use the material of common used suitable selection in photoetching technique.

On substrate, form the film of polymkeric substance, silicon dioxide or metal (aluminium, chromium, gold, silver etc.).According to the material thickness of film, can form film by known method, for example, use the coating of rotary coating machine or slit coater or by spraying or for example, by vapour deposition (physical vapour deposition (PVD) (PVD) or chemical vapor deposition (CVD)).Suitably select the thickness of film according to the desired thickness (in Fig. 1, referring to Reference numeral H) of the microballon forming.

Can preferably use for example, photoetching resist such as epoxy radicals resist (SU-8), polyimide-based resist, acrylic acid resist or phenolic aldehyde base resist as membraneous material.By using polymkeric substance photoetching resist film, can be to prepare microballon than silica membrane and the lower cost of metallic film, and obtain and there is more low-gravity microballon.Microballon is mixed with the sample that comprises target material, and be dispersed in liquid phase in analytic process.If the proportion of microballon is larger, be difficult to keep for a long time disperse state stable in liquid phase.

Using SU-8 is particularly advantageous as polymkeric substance.SU-8 is chemical amplifying type epoxy radicals minus photoetching resist.SU-8 is that the conduct of American I BM research and development is for utilizing ultra-thin resist film formation technology and photoetching technique to form the material of fine structure by combination.

Carry out the formation of film by rotary coating, can easily adjust the thickness of SU-8 film.In addition, SU-8 height printing opacity, and to various solvents, bronsted lowry acids and bases bronsted lowry and the hot tolerance with higher degree.Therefore, by using SU-8 can prepare easily the microballon with various thickness.Can also in the step of preparing microballon and use microballon to analyze, obtain stable performance.

(ii) forming step

Subsequently, by photoetching make formed film shaped be given shape.In the time using such as the resist of SU-8 as microballon material, heat as required film for solidifying (prebake).Then make film exposure by the complete shape with microballon with the photomask (being called hereinafter, " mask " for short) of drawing identification icon thereon.The substrate that irradiation is crossed is dipped into developer solution, to remove the film in unwanted region.Use again washing fluid (isopropyl alcohol: IPA) to rinse substrate, to remove unwanted region completely.Then, carry out substrate after bake and bank up with earth, the microballon shape producing on film is remained on substrate.

Then,, by carry out the shape of designing mask according to the required form of prepared microballon identification icon, can be formed in the microballon on substrate with any identification icon shape.Can realize irradiation by using maskless exposure machine.

If use silicon dioxide or metal as microballon material, by normally used resist rotary coating on the surface of film, and prebake substrate as required.Then by making substrate exposure to above-mentioned similar mask.The substrate of exposure is immersed to developer solution, to remove the resist in unwanted region.Use again washing fluid (being mainly ultrapure water) for several times to rinse substrate, to remove unwanted region, and after cure substrate.After film being carried out to patterning by etching, remove resist completely.Like this, on substrate, on remaining film, there is microballon shape.

(iii) separating step

By be shaped after film from substrate separation.For example, can, by substrate is immersed to alkalescence or acid separation solution, carry out separating film.Or, can be by carry out ultrasonic processing and submergence simultaneously, accelerate to separate.

Separate microballon there is the cylinder body shape obtaining from film, its have face with each other and almost parallel arrange two surfaces.By adjusting the thickness of the film that forms, can select arbitrarily to face with each other and two surfaces that almost parallel is arranged between distance (, microballon thickness (referring to the Reference numeral H in Fig. 1)).

(iv) detecting material fixing step

Subsequently, make the surface of the microballon obtaining after separating film through modified with functional group, for fixing detecting material.

For example, the functional group of modification can be hydroxyl, amino, carboxyl, isothiocyanate group, epoxy radicals or maleimide base.In common manufacture DNA and protein-chip, carry out modified with functional group and introduced connector, with fixed nucleotide chain or peptide on substrate surface.Also can use in the present invention similar method.

To describe as a specific example and use hydroxyl modified bead surface.In this case, first make bead surface through aminopropyl triethoxysilane processing, can make microballon modify, then microballon is immersed and is dissolved with in the dimethyl formamide (DMF) of gamma-valerolactone, to react.Or, by after making the processing of bead surface process glycidoxypropyl methoxy silane, microballon is immersed to tetraethylene glycol and adds in the liquid mixture of a small amount of concentrated sulphuric acid, to react, can modify.

Finally, detecting material is fixed in the bead surface of modified with functional group.For example, when fixed nucleic acid or peptide are during as detecting material, along with by nucleotide or Freamine Ⅲ (hereinafter, be referred to as " monomer solution ") be dropwise added in bead surface, detecting material can be fixed on microballon by progressively synthesizing (step synthesis).

According to required nucleotide or amino acid sequence, by repeatedly carrying out one after the other dropwise adding the synthetic circulation there is bonding reaction in the part that will become microballon region of film by containing corresponding group or amino acid whose monomer solution, can carry out the progressively synthetic of nucleic acid or peptide.

For example, in the time of fixed nucleic acid, first dropwise add by suction pipe the monomer solution that contains nucleosides, then, make 5-ethylmercapto group tetrazole solution by dropwise adding and react.After rinsing and being dried, by dropwise adding oxidizing solution and reacting with oxidizing solution, make nucleosides tris phosphite be transformed into nucleoside phosphorylase three esters.After flushing, dropwise add mixing acetic anhydride/tetrahydrofuran solution, to react, by unreacted hydroxy-end capped (the be capped) that introduce by modified with functional group.After rinsing and being dried, dropwise add the dichloromethane solution that contains dichloroacetic acid, to remove dual-methoxy trityl protecting group the nucleosides 5 '-hydroxyl connecting from microballon.After rinsing and being dried; repeat (a) nucleosides bonding, (b) flushing, (c) oxidation, (d) flushing and (e) remove dual-methoxy trityl protecting group and the step of (f) rinsing; finally, make nucleic acid base deprotection.Can fixedly there is in this way the nucleic acid of required base sequence.

Or; for example; in the time of fixing peptide; prepare peptide by repeating following steps: on microballon, contain the amino acid whose monomer solution that makes alpha-amido and side chain functionalities be subject to suitably protection by condensation and carry out condensation by dropwise adding, and the final various protecting groups of removing on the shielded peptide obtaining.Can fixedly there is in this way the peptide of amino acid needed sequence.

By dropwise add the solution that contains previously prepared nucleic acid or peptide on microballon, in the reacting of introduced functional group and previously prepared nucleic acid or peptide, can realize the fixing of nucleic acid or peptide.

By noting solution or put note by ink-jet with suction pipe or micro-dispenser point, can realize and dropwise add monomer solution or previously prepared nucleic acid or peptide solution.Can after detecting material fixing step, carry out above-mentioned separating step.In this case, after being formed on the surface that microballon on substrate forms the film in region in detecting material is fixed on to forming step, separating film, to produce microballon.

2. microballon analytical approach (the first example)

(1) microballon analytical approach general introduction

In microballon analytical approach according to an embodiment of the invention, on the surface of above-mentioned microballon 1, form identification icon, the microballon that is fixed with detecting material by use detects fluorescence and identification icon.Analyze target substances of multiple types like this, simultaneously.

Specifically, for example, in the situation that completing the many conditions of mononucleotide (SNP) analysis by use microballon 1, the base sequence of supposing detecting material PA is and a base sequence that SNP is corresponding, and the base sequence of detecting material PB and PC is corresponding with other SNP respectively base sequence.The one group of microballon being made up of microballon 1A, 1B and 1C is mixed with fluorescence labeling sample nucleic acid, with each detecting material generation hybridization reaction.After reaction, by measuring fluorescence intensity and detecting the lip-deep identification icon of each microballon, determine the amount of each SNP contained in sample nucleic acid.

Can by use each have be formed at lip-deep identification icon and the microballon with the specific corresponding fixing detecting material of microballon, in analysis, by detecting fluorescence and identification icon, can analyze in this way target substances of multiple types simultaneously.But said system has problem, that is, detecting in the process of fluorescence from serving as a mark with the fluorescent material of target material bonding and intercalator, identification icon self can produce noise fluorescence.

Fig. 4 A and 4B show the example of transmission image and the fluoroscopic image of the microballon that carries identification icon formed thereon.Fig. 4 A and Fig. 4 B show separately by carry fixing detecting material and with fluorescent material directly in addition the microballon 1 of mark be placed on sample arm, make microballon disperse after from above the image of shooting.Fig. 4 A is transmission image, and Fig. 4 B is fluoroscopic image.

In the fluoroscopic image shown in Fig. 4 B, can confirm, be formed at the stronger fluorescence of identification icon transmitting on microballon 1.Note, detect from serving as a mark and the fluorescence of the fluorescent material of detecting material bonding along the profile (being discussed in more detail below) of microballon reliably.

According in the process of the fluorescence intensity measurement target amount of substance in bead surface, the fluorescence producing due to identification icon becomes noise fluorescence, that is, and and measuring error factor.In addition, on each microballon, form the different identification icon of pattern form (referring to microballon 1A, 1B and 1C in Fig. 2 A to Fig. 2 C), due to pattern form difference, so the glimmering light intensity of the noise producing may exist difference on each microballon.In this case, need to after correction noise fluorescence is on the impact of each microballon, calculate target amount of substance, this can cause analysis speed to reduce again.

In the transmission image shown in Fig. 4 A, can confirm to have produced interference fringe (Newton ring).When the thickness in the gap (air layer) that between forms in the time that two elements (being microballon and sample arm in this case) are together with being attached to is mutually equal to or less than particular value, produce above-mentioned interference striped.Consequent interference fringe makes pattern recognition device be difficult in transmission image, detect identification icon, and causes the variation of the fluorescence intensity that produces fluoroscopic image.

For example, in order to detect from serving as a mark and the fluorescence of the fluorescent material of target material bonding, noise fluorescence and the interference fringe in order to prevent from producing due to identification icon simultaneously, microballon analytical approach adopts the following step according to an embodiment of the invention.Hereinafter, be used as example with microballon 1, describe the concrete steps in microballon analytical approach according to an embodiment of the invention with reference to Fig. 5 and Fig. 6, Fig. 5 and Fig. 6 show the first example of the schematic construction of the microballon analyzer using in microballon analytical approach.

(2) microballon analyzer (the first example)

First, will the schematic construction of microballon analyzer be described.Fig. 5 shows as obtaining and is placed on the transmission image light source 32 used of measuring the microballon 1 on substrate 31, and for obtaining fluoroscopic image light source used 33.For example, use Halogen lamp LED or mercury vapor lamp as light source 32, and for example use semiconductor laser as light source 33.Scioptics 34 will be applied to microballon 1 from light source 32 and the light of reflection, transmission is collected in image acquiring device 35.Equally, for example, scioptics 34, by the Ear Mucosa Treated by He Ne Laser Irradiation by from light source 33 and from for example serving as a mark with the phosphor collection of fluorescent material transmitting that is fixed on the target material bonding microballon 1 in image acquiring device 35.Image acquiring device 35 can be the area image sensor such as CCD or cmos element, and image acquiring device 35 obtains transmission image and the fluoroscopic image of microballon 1, and outputs to respectively pattern recognition device 36 and fluorescence detection device 37.Analytical equipment 38 demonstrates the Comprehensive analysis results that is detected the identification icon that obtains and fluorescence intensity output by pattern recognition device 36 and fluorescence detection device 37.Although Fig. 5 shows the structure of independent pattern recognition device 36, fluorescence detection device 37 and the analytical equipment 38 installed, said apparatus also can be for example combined with multi-purpose computer, program and display.

Can make to measure the surface roughening of substrate 31, to prevent interference fringe (referring to Fig. 6).In the time existing thickness to be equal to or less than the gap (air layer) of particular value between the microballon 1 of measuring substrate 31 and support on it, will produce interference fringe.Measure the irregular structure 311 that forms on the surface of substrate 31 by microballon 1 is remained on teat and maintain recess and microballon 1 between gap be not less than particular value, prevented interference fringe.By surface sand-blasting or adopt from the teeth outwards anti-Newton ring film, can realize the surface roughening processing of measuring substrate 31.For example, can be used as anti-Newton ring film by the coating solution of the inorganic pigment that coating contains dispersion on substrate one or two surface is carried out to the film that surface roughening was obtained.For anti-Newton ring film preferably at the film of excellence aspect light transmission, it has by coating and contains the beaded glass or the prepared irregular surface film of resin particle that are dispersed in bonding agent.

(3) detailed process of microballon analytical approach

(i) course of reaction

First, microballon 1 is mixed with the sample that contains target material, makes to be fixed between detecting material in bead surface and target material and react, with by target material capture in bead surface.

After with fluorescent material labels targets material, or exist fluorescence intercalator after fluorescence intercalator is bonded in the complex that the interaction between target material and detecting material forms, mix microballon 1 and sample.

(ii) keep-process

Then, collect as required and rinse microballon 1, to remove the material (pollutant) except the target material of microballon absorption, then, microballon 1 is placed in the mode of disperseing on the measurement substrate 31 of microballon analyzer (referring to Fig. 6).

Be dispersed in the microballon 1 of measuring on substrate 31 and be maintained on measurement substrate 31, arrange with the mode of the Surface Contact of measuring substrate on the surface (top surface 11 or basal surface 12) in two surfaces that microballon 1 faces with each other with it and almost parallel is placed.By upwards supporting microballon 1 the party, can, by being mounted to the image acquiring device 35 in the face of measuring substrate, obtain the image of the identification icon forming on the code area of top surface 11 and/or basal surface 12.

By making the thickness H of microballon 1 be less than the diameter D of top surface (or basal surface 12), and make whole microballon 1 more approach plate-like shape, microballon 1 can be arranged in more reliably and measure on substrate 31.

(iii) testing process

(a) detect identification icon

Subsequently, be maintained on the microballon 1 of measuring on substrate 31 being applied to from the light of light source 32 and 33, and take transmission image and the fluoroscopic image of microballon 1 by image acquiring device 35.

If being maintained at, measures on substrate 31 microballon 1, because microballon in keep-process 1 is aligned to its top surface 11 or basal surface 12 and the Surface Contact of measuring substrate, so comprise more reliably identification icon in the transmission image obtaining by image acquiring device 35.Can also, by forming irregular surface 311 on the surface measuring substrate 31, obtain the fluoroscopic image that there is no the transmission image of interference fringe and there is no fluorescence intensity change.

Export the transmission image obtaining by image acquiring device 35 to pattern recognition device 36.Pattern recognition device 36 detects identification icon in transmission image, and exports identification icon to analytical equipment 38 as electronic signal.Can detect identification icon by general image routine analyzer or its update routine.

By microballon 1 being placed on to the method for measuring on substrate 31 in liquid, also can prevent interference fringe.When measuring substrate 31 and being maintained at while existing thickness to be equal to or less than the gap (air layer) of particular value between the microballon 1 of measuring on substrate 31, produce interference fringe.Therefore,, if microballon 1 is placed in liquid on the surface of measuring substrate,, not measuring the possibility that forms air layer between substrate 31 and microballon 1, therefore prevented generation interference fringe.

Specifically, the microballon floating on a liquid 1 is dropwise added to and is measured on substrate, measure on substrate so that microballon is placed on.If worry that the liquid dropwise adding disappears due to dry, can again dropwise add as required liquid, so that the microballon being placed in liquid is placed on measurement substrate 31 always.For liquid preferably there is the liquid with microballon 1 with identical refractive index, and the buffer solution preferably using in course of reaction or there is the more buffer solution of high salt concentration.

(b) detect fluorescence

The fluoroscopic image being obtained by image acquiring device 35 is outputed in fluorescence detection device 37.Fluorescence detection device 37 detects fluorescence from the presumptive area of fluoroscopic image, and exports the fluorescence intensity that converts electronic signal to analytical equipment 38.

Fig. 7 shows the fluoroscopic examination region (referring to the Reference numeral L in figure) of being detected by fluorescence detection device 37.

As mentioned above, by microballon 1 being arranged to the mode with the Surface Contact of its top surface 11 or basal surface 12 and measurement substrate, microballon 1 is maintained to be measured on substrate 31.Therefore, as shown in the figure, the fluoroscopic image obtaining by the image acquiring device 35 being mounted in the face of measuring substrate surface, is the captured image of direction from top surface 11 or basal surface 12.

In above-mentioned fluoroscopic image, fluorescence detection device 37 detects from the fluorescence that does not carry the region of identification icon in top surface 12 or side surface 13.By detecting from by the fluorescence in the excluded region, code area 111 of formation identification icon, can detect fluorescence (referring to Fig. 4 B) in the case of not having to produce the noise fluorescence from the identification icon in code area 111.

In fluoroscopic image, fluorescence detection device 37 also detects the fluorescence from the contour area of microballon 1 simultaneously.The contour area of microballon 1 is launched stronger fluorescence (referring to Fig. 4 B) by assembling from the fluorescence of side surface 13.Therefore,, by side by side detecting the fluorescence from contour area, can improve S/N and compare and the signal of exporting to analytical equipment 38 is amplified.

For example, be identified in the following manner being undertaken by fluorescence detection device 37 the region L of fluoroscopic examination.

First, along with taking out arbitrary hue sequence from fluoroscopic image or by being recalculated by color information, fluoroscopic image being calculated as to monochrome image binaryzation.Then by using certain luminance as threshold value, fluoroscopic image two-value is turned to 0 or 1.Subsequently, calculate brightness and become 1 or become 0 point from 1 from 0, obtained border (profile).

Then, with reference to the size of pre-stored code area 111, the noncoding region 112 in the region in identification border.And, defining the contour area including border, but profile width W extends to and comprises noncoding region 112 width that does not comprise the region of code area 111, and the contour area obtaining is defined as fluoroscopic examination region L.

If but fluoroscopic examination region L does not comprise code area 111 region that comprises contour area, fluoroscopic examination region L is not particularly limited, and as shown in the figure, fluoroscopic examination region can be the region that comprises whole contour areas and part noncoding region 112, comprise whole contour areas and all noncoding regions 112 region or comprise partial contour region and the region of part noncoding region 112.Not only can pass through said method, the method that can also be applicable to the shape (polygonal prism, for example triangular prism or four jiaos of prisms) of the microballon using by other defines fluoroscopic examination region L.

As mentioned above, in microballon analytical approach according to an embodiment of the invention, by detecting from the noncoding region of microballon and the fluorescence of contour area, can detect the high strength fluorescence not having by the noise fluorescence producing in the identification icon forming in code area.Therefore,, by said method, can under high sensitivity, analyze target material, and carry out high-precision quantitative measurement.

Describe the situation that obtains fluoroscopic image from the direction of top surface 11 or basal surface 12, but the invention is not restricted to this.

3. microballon analytical approach (the second example)

(1) microballon analyzer (the second example)

Fig. 8 shows the second example for the microballon analyzer of embodiments of the invention.With microballon analyzer heavy phase in above-mentioned the first example with member by the description of reference numerals with identical.

In the microballon analyzer of the second example, although can obtain as shown in Figure 5 and Figure 6 the image of measuring the microballon 1 on substrate 31, can obtain as shown in Figure 8 the image of the microballon 1 in pipeline 61.

Pipeline 61 is not subject to concrete restriction, but preferably use by can visible light transmissive and the pipeline 61 that forms by etching of the transparent material (for example, glass, quartz or transparent resin) of fluorescence, by pipeline 61 or the transparent material pipe of molded formation.The structure of pipeline 61 and be not subject to concrete restriction such as the device of the pump of liquid being sent into pipeline 61, but the device of installing in pipeline 61 for the direction with respect to light source and the fixing microballon 1 of image acquiring device as described below preferably.

The example of stationary installation comprises not allowing column structure, the bowl configurations with the pit more shallow and wider than microballon than the thickness H of microballon or the structure similar to so-called flow cytometer of multiple pillars that interval that microballon passes through places, one of them connects one microballon is fed in pipeline 61, to arrange microballon 1.

Measure on substrate 31 being applied on the microballon 1 in pipeline 61 from the light of light source 32 and 33 or being applied to, obtain transmission image and fluoroscopic image by image acquiring device 65 and 66.Image acquiring device 65 and 66 can be the area image sensor such as CCD or CMOS.Similar with the microballon analyzer in the first example, the light that scioptics 63 and 64 are collected is preferred for Image Acquisition.

Can be installed separately image acquiring device 65 for obtaining transmission image and for obtaining the image acquiring device 66 of fluoroscopic image.For example, face top surface 11 or the basal surface 12 of microballon 1 for the image acquiring device 65 of transmission image, image acquiring device 65 obtains the light reflecting from top surface 11 or basal surface 12, or by the light of top surface 11 or basal surface 12 transmissions, thereby obtain transmission image from top surface 11 or basal surface 12.Or, the side surface 13 for the image acquiring device 66 of fluoroscopic image in the face of microballon 1, image acquiring device 66 obtains the fluorescence of launching from side surface 13, and obtains fluoroscopic image from side surface 13.

The position of the image acquiring device 65 and 66 of installing is not subject to concrete restriction.For example, if by for example being obtained from the transmission image of top surface 11 or basal surface 12 with from the fluoroscopic image of side surface 13 by mirror-reflection, can be arranged on not in the face of top surface 11 is not also in the face of the position of basal surface 12 for the image acquiring device 65 of transmission image, can be arranged on the not position in the face of side surface 13 for the image acquiring device 66 of fluoroscopic image.

By below by using microballon analyzer in the second example to carry out the detailed process of descriptive analysis method.

(2) detailed process of microballon analytical approach (the second example)

For example, by the method similar to " (1) course of reaction " in " 2. microballon analytical approach (the first example) ", target material is bonded to microballon 1, and microballon 1 is for detection of operation.

(i) pipe feed process

Microballon 1 is fed by pipeline 61 with the state floating on a liquid, makes microballon 1 through obtained the position of the image of microballon 1 by image acquiring device 65 and 66.Although liquid preferably has the refractive index identical with microballon 1, but this liquid does not preferably change the character of target material or can not decompose target material and the applicable liquid that keeps target material, for example, the buffer solution using in the time catching target material or there is the more buffer solution of salinity.In addition the bubble such as air had better not be introduced in pipeline 61.

(ii) testing process

(a) detect identification icon

To be applied to from the light of light source 32 and 33 in pipeline 61 through on the microballon 1 of image space, by image acquiring device 65 and 66 acquisition transmission image and fluoroscopic images.If for the image acquiring device 65 of transmission image directly or for example, by optical devices (lens 63 or catoptron) in the face of forming the region of identification icon, the transmission image that obtained comprises identification icon reliably.

The same with the situation of the microballon analyzer in the first example, export obtained transmission image to pattern recognition device 36, analytical equipment 38 detects identification icon.

(b) detect fluorescence

For the image acquiring device 66 of fluoroscopic image directly or for example, by optical devices (lens 64 or catoptron) side surface 13 in the face of microballon 1, but in the face of top surface 11 or basal surface 12.Because do not face the carrying surface of the identification icon of formation to some extent for the image acquiring device 66 of fluorescence, do not detect the fluorescence from identification icon, so can detect fluorescence in the situation that there is no noise fluorescence.

Fig. 9 A and 9B are the fluoroscopic images of the microballon 1 taken from side surface 13, and Fig. 9 A is the image obtaining with complementary target material (coupling completely), and Fig. 9 B is the image obtaining with incomplementarity target material (mismatch).

Because at the contour area of microballon 1 (, the side surface of microballon 1) the middle fluorescence of collecting from fluorescent material, and this fluorescence is high strength transmitting, so as shown in Fig. 9 A and 9B, exist obviously different between the situation of mating completely and the situation of mismatch from the fluoroscopic image of side surface 13.Therefore, by taking fluoroscopic image from side surface 13, can improve S/N ratio, and strengthen the signal of exporting to analytical equipment 38 from fluorescence detection device 37.

For high precision detects identification icon, preferably make optical axis for the image acquiring device 65 of transmission image perpendicular to top surface 11 or basal surface 12, and from obtaining transmission image perpendicular to the direction of top surface 11 or basal surface 12, and, in order to improve S/N ratio, preferably make optical axis for the image acquiring device 66 of fluoroscopic image perpendicular to side surface 13, and from obtaining fluoroscopic image perpendicular to the direction of side surface 13.For example, the optical axis of image acquiring device 65 or 66 is optical axises of the lens of composition light receiving unit.

As mentioned above, the analyzer of microballon according to an embodiment of the invention and the microballon analytical approach that have higher S/N ratio can be evaluated and tested fluorescence with high sensitivity.In addition, because if obtain fluoroscopic image from the side surface 13 of microballon 1, need on top surface 11 or basal surface 12, not form noncoding region 112, so can reduce the size of microballon 1 and improve processing power (the processing capacity of time per unit).

According to an embodiment of the invention microballon analytical approach accurately test example as from serving as a mark and the fluorescence of the fluorescent material of target material bonding, and be not subject to the impact from the noise fluorescence of identification icon, this microballon analytical approach can contribute to further to improve processing power and the processing speed of the various biochemical analysises that undertaken by use microballon.

The application comprises and submits the formerly patented claim JP 2009-213360 and submit the theme that formerly disclosed content is relevant in patented claim JP 2010-148819 in Japan in the Japanese Patent Room on June 30th, 2010 in Japan in the Japanese Patent Room on September 15th, 2009, and the full content of above-mentioned application is incorporated herein by reference.

It will be appreciated by those skilled in the art that in the scope or its full scope of equivalents of appended claims, can carry out various amendments, combination, restructuring and change according to design requirement and other factors.

Claims (4)

1. a microballon analytical approach, it is for being formed the microballon of cylinder body shape, described microballon has and faces with each other and the top surface of parallel placement and basal surface and the side surface that extends from described top surface and described basal surface, and described microballon carries at least one the lip-deep identification icon being formed in described top surface and described basal surface and the lip-deep and amalyzing substances that is fixed on described microballon and has the material of compatibility, and described method comprises the steps:

Region from the region that does not form identification icon and described side surface including described top surface and described basal surface detect due to described amalyzing substances and with described amalyzing substances have compatibility described material interaction and from the fluorescence of described bead surface transmitting

Wherein, described method also comprises the following steps:

Described microballon is mixed with described amalyzing substances;

Acquisition comprises the transmission image of the described microballon of described identification icon, and detects described identification icon by described transmission image; And

Obtain the fluoroscopic image of described microballon, and detect the described fluorescence from the region the region and the contour area that do not form described identification icon in described top surface and the described basal surface of described microballon by described fluoroscopic image,

Wherein, when on the surface of measurement substrate that described microballon is placed on to surface roughening or when the Surface Contact of a surface on the surface that described microballon is placed on to described measurement substrate in liquid and in described top surface and the described basal surface of described microballon and described measurement substrate, obtain described transmission image and described fluoroscopic image by image acquiring device, described image acquiring device is arranged on the position of facing with described measurement substrate surface.

2. microballon analytical approach according to claim 1,

Wherein, in the time taking the image of in the described top surface of described microballon and described basal surface, obtain described transmission image, in the time taking the image of described side surface of described microballon, obtained described fluoroscopic image.

3. a microballon analyzer, it is for analyzing microballon, and described microballon carries identification icon in its surface, and described microballon analyzer comprises: image acquiring device, it is for obtaining transmission image and the fluoroscopic image of described microballon;

For detected the device of described identification icon by described transmission image; With

For detecting at described fluoroscopic image from the device of fluorescence in region of described identification icon that does not form described microballon.

4. microballon analyzer according to claim 3, also comprises

Measure substrate, it is for placing described microballon,

The surface of wherein said measurement substrate and described image acquiring device are placed as and face with each other, and described measurement substrate is surface roughening.