JP4474226B2 - Sample analysis method and analyzer - Google Patents

Description

  The present invention relates to a technique for analyzing a sample on a substrate. For example, it relates to protein chip technology.

  Design No. 1211341 discloses a spot seal used to spot a liquid sample on a spot object. In the spot seal, circular holes are arranged in 4 rows and 12 columns. A spot seal is affixed to a spot target portion such as a slide glass, and a liquid sample is dropped into the hole portion. Then, the spot seal is peeled off, and a slide glass or the like on which the sample is spotted is set in an inspection / measurement device.

  In addition, Japanese Patent No. 12111698 discloses a spot seal in which four seals overlap for spotting a liquid sample on a spot object. The shape of each seal is the same, and the size and location of the holes are the same.

Design No. 1211341 Design No. 12111698

  The inventor of the present application has come up with the idea of carrying out an analysis on a substrate using the spot seal disclosed in Design No. 1213441. Specifically, for example, a spot seal is attached to the substrate, a measurement sample is dropped into the spot hole, and the measurement sample is fixed to the substrate. Subsequently, the analysis reagent is dropped into the same spot hole. By repeating this process, an interaction reaction between the measurement sample and the analysis reagent is performed.

  However, the inventor of the present application has found that although the above method is easy, the analysis accuracy is not so good. And as a result of intensive studies, it was found that there is a difference in reaction between the center portion of the spot and the peripheral portion, and the measurement accuracy is lowered. It was also found that the liquid sample tends to accumulate at the boundary between the spot seal and the substrate (that is, at the outer periphery of the spot hole). A lot of measurement samples and analytical reagents accumulate around the spot hole, and the reaction occurs actively in the spot periphery.

  Therefore, an object of the present invention relates to uniforming the reaction at the center and the periphery of the spot.

  The present invention relates to covering a first solution spotted on a substrate with a larger reaction hole, supplying a second solution to the reaction hole, and causing the first solution and the second solution to react. Thereby, reaction of a 1st solution and a 2nd solution can be made substantially uniform in a spot center part and a peripheral part.

  The first solution includes a primary antibody-containing solution in the case of immunoassay and an antigen-containing solution. The second solution includes a secondary antibody-containing solution that recognizes an antigen and a tertiary antibody-containing solution that specifically binds to the secondary antibody and is subjected to labeling for detection.

  According to the present invention, analysis can be easily performed on a substrate with high accuracy.

  The above and other novel features and effects of the present invention will be described below with reference to the drawings. In addition, this invention is not limited to a following example, A various change is possible and these changes are also included by this invention.

  In this embodiment, a case where analysis is performed by reacting a sample with a reagent on a solid substrate using a spot seal and a reagent seal will be described. Reactions that can be analyzed on solid substrates include interactions involving in vivo molecules such as proteins, low molecular weight compounds, nucleic acids, lipids, carbohydrates, and living cells, such as antigen-antibody reactions, ligand-receptor reactions, enzyme-substrate reactions There are catalytic reactions.

  The instrument utilized in a present Example is shown to Fig.1 (a)-(c).

  A spot seal 101 shown in FIG. 1A is a seal for spotting a solution at a predetermined position on a solid substrate. The thing of the substantially the same magnitude | size as a solid substrate is preferable. In this embodiment, the hydrophobic sheet is 26 mm long × 76 mm wide. On top of that, spot holes 103 having a diameter of 2 mm are arranged at intervals of 5 mm in 3 vertical rows and 5 horizontal rows, and two spot groups each consisting of a total of 15 spot holes 103 are provided. Two spot groups can be used for measuring different types of samples.

  A reagent seal 102 shown in FIG. 1B is a seal for supplying a reagent to a predetermined position on the solid substrate, and includes a reagent hole 104. The thing of the same magnitude | size as a solid substrate and a spot seal | sticker is preferable. In this embodiment, the hydrophobic sheet is 26 mm long × 76 mm wide. On top of that, there are provided two reagent holes 104 having a size capable of including 15 spot holes 103 in one spot group of the spot seal. The reagent holes 104 have a rectangular shape of 15 mm in length × 24 mm in width, and two of them are arranged side by side at intervals of 1.5 mm. Here, the size of the reagent hole 104 is preferably larger than the spot hole 103 of the spot seal. This is because it is larger than the spot diameter of the sample immobilized on the solid substrate. The distance between the outer periphery of the reagent hole 104 and the outer periphery of the spot depends on the sample used, the solution composition, the protocol, the spot shape, and the like. For example, when measuring by diluting a sample or reagent such as protein antigen or antibody in a solution containing 30% glycerol and 10% BSA, the outer periphery of the reagent hole 104 is at least 0.6 mm or more from the outer periphery of the spot. It is preferable that they are separated. This numerical value is a region where these sample solutions are repeatedly added to the inside of the spot hole 103 of the same size, the reaction is performed, the signal intensity data of the cross section of the spot is obtained, and the area exceeding the average value of the signal intensity inside the spot Can be calculated based on the distance from the outer periphery of the spot.

  The spot seal 101 and the reagent seal 102 can be formed by processing a water-resistant material or a material having a low hygroscopic property (for example, a hydrophobic sheet). The back surface is made of an adhesive material such as a seal and can be directly attached to a solid substrate. It is also possible to use a material that has affinity for a solid substrate and can be brought into close contact with. For example, when the solid substrate is a glass slide glass, a silicone polymer such as PDMS (polydimethylsiloxane) can be used.

  Of course, the spot seal 101 and the reagent seal 102 are not limited to the above-described structures, and their shapes may be changed according to the purpose of use.

  For example, there may be only one spot group, or three or more spot groups. One spot group may include one spot or a plurality of spots. The shape of the spot hole 103 is usually circular, but is not limited thereto, and may be an ellipse, a triangle, a quadrangle, or a polygon. There is no particular limitation on the size, and when it is desired to keep the sample to be used in a very small amount, for example, a circle having a diameter of 0.5 mm is desirable. The arrangement pattern of the hole for the spot is not particularly limited, but it is desirable to arrange the spot hole away from the adjacent spot by a distance such as 2 mm, for example, when the solid substrate is washed.

Although the shape of the reagent hole 104 is, for example, a reagent hole including only one spot hole, the same shape as the spot hole and a large size is desirable. In the case of a reagent hole that includes a plurality of spot holes, it is desirable that the inner periphery of the reagent hole is separated from the outer periphery of all the spot holes by a distance that does not affect the measurement. Shapes can be used. It is desirable that the arrangement pattern of the reagent holes matches the arrangement pattern of the spot group. For example, the reagent holes may be arranged in 2 rows × 3 rows. If an automatic spotter capable of spotting a very small amount of sample is used, the reagent holes may be arranged in 10 columns × 10 columns.

  A solid substrate 105 shown in FIG. 1 (c) is for fixing a target sample in a spot shape thereon, and capturing and detecting a substance that specifically binds to the sample on the solid substrate. For example, the slide glass is 26 mm long × 76 mm wide.

  For the type of solid substrate, use a membrane such as nitrocellulose or PVDF, silicon such as glass or wafer, resin such as plastic, metal, etc., if necessary, modified to fix the sample. it can. The types of modification include poly-L-lysine and aminosilane that can immobilize the target molecule by physical adsorption, functional groups such as aldehyde group and epoxy group that can immobilize the target molecule by covalent bond, and Avidin, Ni-NTA, or the like that can be immobilized using affinity for the target molecule can be used. Further, it can be used for a substrate composed of a thin layer of a hydrophilic porous matrix such as polyacrylamide gel or agarose gel.

  FIG. 2 shows the analysis procedure of this embodiment. In this embodiment, a pipetter 201, a sealed container 204, a gas phase incubator, and a detection device are used in addition to the above-described instruments.

  The pipetter 201 is for spotting a sample in an arbitrary amount on a solid substrate.

  The hermetic container 204 seals the solid substrate together with a small amount of water droplets 203, thereby maintaining the humidity inside the container for a long time and preventing evaporation of the sample on the solid substrate.

  The gas phase incubator is for maintaining a temperature suitable for the sample and the solid substrate.

The detection apparatus is for measuring a solid substrate, and uses a detection system and apparatus suitable for analysis. From the numerical values obtained here, qualitative and quantitative analysis of the substance immobilized on the solid substrate is performed. For example, when the label is a fluorescent material, a fluorescence detection scanner or a fluorescence microscope is used. If the label is a radioisotope, autoradiography is used, and if the label is an enzyme, the corresponding substrate, luminescence, coloring reagent, etc. are used in combination. Or SPR (Surface for detection)
It is also possible to use a Plasmon Resnance device. SPR is a phenomenon in which a surface plasmon wave, which is an excited state of a metal surface, is resonantly excited, and can be used to detect a change in mass due to the binding / dissociation of two molecules. There is an advantage that a label is not required for sensing mass changes. Furthermore, the sample captured on the solid substrate can be used for analyzing with a mass spectrometer. In this case, the sample can be identified by mass spectrometry after adding a matrix for absorbing excessive energy to the sample captured on the solid substrate as necessary, ionizing the molecules by laser irradiation.

  Hereinafter, with reference to FIG. 2, an analysis work process using the spot seal and the reagent seal will be described. In this embodiment, the work is performed in the order of sample spot (A), sample fixation (B), reagent addition (C), sample-reagent reaction (D), and detection (E).

  First, in the spot (A) of the sample, a spot seal 101 is attached to the solid substrate 105, and a sample 202 such as a primary antibody or antigen is manually dropped using a micropipette 201. Thereby, a plurality of reagents such as measurement samples can be spotted at predetermined positions on the solid substrate.

  Next, in the sample fixation (B), the solid substrate is held in the sealed container 204 together with the water droplet 203, and the binding reaction of the sample onto the solid substrate is performed. Thereby, since the evaporation of the plurality of samples spotted on the solid substrate can be prevented, the plurality of samples can be reliably fixed to the solid substrate.

  The spot (A) of the sample and the fixation (B) of the sample can be performed by a commercially available automatic spotter (contact type, non-contact type) or the like instead of using the spot seal 101.

Next, in addition of the reagent (C), after the spot seal 101 is peeled off, the reagent seal 102 is stuck on the solid substrate. At this time, the reagent hole 104 of the reagent seal 02 covers the sample fixed on the solid substrate. In the present embodiment, 15 samples spotted in 3 rows × 5 rows on a solid substrate related to one spot group in the spot seal 101 are covered with one reagent hole 104. To do. Then, a reagent 205 such as a secondary antibody or a tertiary antibody is added inside the two reagent holes 104. As a result, 15 samples related to one spot group can be reacted with the same reagent under substantially the same and substantially the same conditions. Further, by supplying different reagents to the reagent holes 104, different reactions can be performed substantially simultaneously on the same solid substrate.

  In the sample-reagent reaction (D), the solid substrate is held in a sealed container together with water droplets, and the spot-fixed sample-reagent reaction is performed. Thereby, since the evaporation of the reagent on the solid substrate can be prevented, the reagent and the sample can be reacted reliably.

  Finally, in detection (E), the solid substrate is detected by the detection device, and an image 206 is obtained. Thereby, reaction of a sample and a reagent can be confirmed.

[Experiment]
The effect of this example was verified for protein quantification based on antigen-antibody reaction. Antigen-antibody reaction is quantification of tumor markers human CEA (Carcinoembryonic antigen carcinoembryonic antigen) and CRP (C-reactive protein C-reactive protein).

  In this experiment, a dissolution solution, a blocking agent, and a cleaning solution are used.

  The lysis solution is used to dissolve the sample and the reagent, and a buffer solution is used to keep the sample stable by avoiding a sudden pH change. In addition, a humectant such as glycerol is added as required in an amount of about 20 to 40% in order to prevent evaporation of the sample and reagent and to keep it stable. In order to prevent non-specific adsorption, an appropriate concentration of a blocking agent is added to the lysis solution used for lysis other than the substance first immobilized on the solid substrate.

The blocking agent is for blocking unreacted regions on the solid substrate. It is possible to prevent other substances from adsorbing to a region where the target substance is not immobilized on the solid substrate and causing an increase in non-specific signal. The blocking agent is selected according to the properties of the solid substrate, sample, and reagent. For example, inactive proteins such as BSA (Bovine Serum Albumin) and skim milk, PEG (Polyethylene Glycol), PVA
Use polymers such as (Polyvinyl Alcohol).

  The washing solution is for efficiently removing unreacted samples and reagents. For example, a solution obtained by adding a surfactant such as Tween 20 or Triton X to the above buffer solution as necessary is used.

  The experimental procedure is as follows. First, the spot seal 101 was attached to the solid substrate 105, the primary antibody was dropped onto each spot, and the primary antibody was immobilized on the solid substrate (immobilization of the primary antibody on the solid substrate). Next, an unreacted region on the solid substrate was blocked (blocking). Next, antigens of different concentrations were added to each spot, and the antigen to be measured was supplemented by the primary antibody (antigen capture by the primary antibody). Next, the spot seal 101 was peeled off and the reagent seal 102 was affixed, and a secondary antibody that recognizes the antigen was dropped into the reagent hole and allowed to react (reaction with the secondary antibody). Next, a tertiary antibody that specifically binds to the secondary antibody and was labeled for detection was dropped into the reagent hole and allowed to react (reaction with the labeled tertiary antibody). Next, the reagent seal 102 was peeled off, and the solid substrate was detected by a detection scanner (detection by a fluorescence scanner). And it compared with the case where only the seal | sticker for spots was used (result of experiment).

[Immobilization of primary antibody on solid substrate]
In this experiment, Proteochip Type A (Proteogen, Soul, Korea) was used as a solid substrate.
Proteochip is a protein chip in which a protein binding reagent 'ProLinker', which is a kind of calix crown derivative, is bound on a slide glass (size 26mm x width 76mm). It can be fixed with high density and uniformity while keeping. As spot seals, spot holes with a diameter of 2 mm are arranged on a hydrophobic sheet of the same size as a solid substrate, 26 mm long × 76 mm wide, at intervals of 5 mm in 3 vertical rows × 5 horizontal rows, for a total of 15 A spot group provided with two spots was used. One spot group was used for CEA measurement, and the other spot group was used for CRP measurement.

A spot seal 101 was pasted on ProteoChip Type A in advance. Mouse-derived anti-human CEA monoclonal antibody (Fitzgerald, MA, USA) and mouse-derived anti-human CRP monoclonal antibody (Fitzgerald, MA, USA), PBS containing 30% Glycerol
(pH = 7.4) was used to dilute to 100 μg / ml. These were spotted 1.5 μl at a time using a micro pipettor 201.

Proteochip Type A was placed in a closed container 204 containing water drops 203 at the corners and incubated overnight at 37 ° C. to immobilize the primary antibody on Proteochip Type A. After fixation, Proteochip
Type A was immersed in 50 ml of washing solution and shaken for 10 minutes to remove unreacted antibodies. After washing, excess water was removed with filter paper.

〔blocking〕
Proteochip Type A was immersed in PBS (pH = 7.4) containing 50 ml of 3% BSA, and gently shaken for 1.5 hours. After discarding the blocking solution, 50 ml of washing solution was added and shaken for 10 minutes. After washing, excess water was removed with filter paper.

[Capture antigen by primary antibody]
Human recombinant CEA and CRP were diluted to 1 ng / ml to 1 μg / ml using PBS (pH = 7.4) containing 30% Glycerol and 10% BSA. These antigen solutions and negative controls were spotted by 1.5 μl each using a micropipettor 201 to the spots where the corresponding primary antibodies were immobilized. Proteochip Type A was placed in a sealed container 204 with a water drop 203 in the corner and incubated at 37 ° C. for 1 hour to react the antigen with the primary antibody. After removing the antigen solution by aspiration, Proteochip Type A was immersed in 50 ml of washing solution and shaken for 15 minutes to remove unreacted antigen. After removing excess moisture by blowing nitrogen gas, the spot seal was peeled off.

[Reaction with secondary antibody]
The reagent seal was affixed so that the reagent hole was in the same position as the spot area of Proteochip Type A. In this experiment, 15 spot holes in one group of spot seals were formed on a hydrophobic sheet having a size of 26 mm × 76 mm in width, which is the same size as that of the spot seal. A reagent hole measuring 15 mm in length and 24 mm in width,
Two of them arranged side by side at intervals of 1.5 mm were used.

Rabbit-derived anti-human CEA polyclonal antibody (Fitzgerald, MA, USA) and goat-derived anti-human CRP polyclonal antibody (Fitzgerald, MA, USA) were treated with 30% Glycerol,
Each was diluted to 100 μg / ml with PBS (pH = 7.4) containing 10% BSA. And 200 microliters of these was added inside each reagent hole.

  Proteochip Type A was placed in a sealed container 204 with water drops 203 in the corners and incubated at 37 ° C. for 30 minutes to react with the antigen. After removing the antibody solution by aspiration, Proteochip Type A was immersed in 50 ml of washing solution and shaken for 15 minutes to remove unreacted antibody. After washing, excess water was removed by blowing nitrogen gas.

[Reaction with labeled tertiary antibody]
Cy5-labeled goat-derived anti-rabbit IgG polyclonal antibody (Zymed) for CEA detection
laboratories Inc, San Francisco, Calif.), and Cy5-labeled donkey-derived anti-goat IgG polyclonal antibody (Chemicon International. Inc, Temecula, Calif.) for CRP detection in PBS containing 30% Glycerol and 10% BSA (pH = 7. 4) was used to dilute to 2 μg / ml. And 200 microliters of these was added inside each reagent hole.

  Proteochip Type A was placed in a sealed container 204 with water drops 203 in the corners and incubated at 37 ° C. for 30 minutes to react with the secondary antibody. After removing the antibody solution by aspiration, Proteochip Type A was immersed in 50 ml of washing solution and shaken for 15 minutes to remove unreacted antibody. After washing, excess water was removed by blowing nitrogen gas, and the reagent seal was peeled off.

[Detection by fluorescence scanner]
Proteochip Type A was scanned using a fluorescence detection scanner Scan Array Express (Packard BioScience, Billerica, Mass.). QuantumArray (Packard BioScience, Billerica, Mass.) Was used for analysis of scanned images.

〔results of the experiment〕
3A to 3C show the measurement results of CEA and CRP in this example. FIG. 3A is an image diagram of a scanned image obtained by fluorescence detection, and FIGS. 3B and 3C are graphs in which the signal intensities of CEA and CRP are quantified, respectively.

  Using a reagent seal with reagent holes of a size that includes multiple spots, adding reagents that can be used in common to multiple spots, simplifying the experimental operation by eliminating spot operations, Work time was shortened.

  In addition, by using a reagent seal that can divide the solid substrate into two regions, two types of proteins can be measured on a single solid substrate under different conditions. Each protein has different optimum conditions for functioning, for example, suitable pH, cofactor, salt concentration and the like. Therefore, in order to realize multi-item and highly integrated protein analysis which is a merit of protein chips, it is desirable to employ different conditions for each protein on one protein chip for measurement. By using the reagent seal, it is possible to use different blocking solutions, washing solutions and the like for each protein in addition to the reagents, and this is a technology that contributes to the establishment of a multi-item, highly integrated protein chip technology.

  FIG. 4 shows measurement results when the entire reaction process is performed using only spot seals. FIG. 4 (a) is a fluorescence scan image and shows the variation in signal intensity inside the spot. FIG. 4B is a graph in which the signal intensity at AA ′ in FIG. FIG. 5 shows the measurement results in this example. FIG. 5A is a fluorescence scan image. FIG. 5B is a graph in which the signal intensity at BB ′ in FIG.

  By using a reagent seal having a reagent hole larger than the spot hole, it was possible to reduce variations in signal intensity inside the spot, which was a problem when only the spot seal was used. Specifically, when only the spot seal was used, the signal intensity variation within the spot was 55% in terms of CV, but by using the reagent seal in combination, it could be reduced to 36%, improving the measurement accuracy. I succeeded in making it happen. If only the spot seal is used, the spot seal is affixed to the solid substrate so that the inside of the spot becomes concave and the liquid sample is likely to remain around the spot. In addition, it is considered that one of the causes is that the tip of the pipetter contacts the solid substrate during spot operation, and molecules previously immobilized on the portion peel off.

  This example relates to a two-layer seal in which the spot seal and the reagent seal in Example 1 are overlapped. Hereinafter, only main differences from the first embodiment will be described with reference to FIGS.

  In the two-layer seal 601 shown in FIG. 6A, a reagent lower layer member 603 having a reagent hole 606 larger than the spot hole is laminated and pasted on the spot upper layer member 602 having spot holes. Is. A spot hole is disposed at a position included in the reagent hole. If necessary, another member may be arranged on the spot upper layer member 602, between the spot upper layer member 602 and the reagent lower layer member 603, and below the reagent lower layer member 603, so that two or more layers are formed. . For example, a second reagent member may be arranged between the spot upper layer member 602 and the reagent lower layer member 603 to form three layers.

  FIG. 6B shows the spot upper layer member 602. Those having substantially the same size as the solid substrate and the lower layer member for reagent 603 are preferable. In this embodiment, the hydrophobic sheet is 26 mm long × 76 mm wide. On top of that, spot holes 103 having a diameter of 2 mm are arranged at 5 mm intervals in 3 vertical rows and 5 horizontal rows, and two spot groups each consisting of 15 spot holes 604 are provided. It can be used for measuring different types of samples. Further, a step 605 having substantially the same shape as the reagent hole of the reagent lower layer member 603 is provided on the bottom side corresponding to the two spot groups. The step 605 is fitted into the reagent hole 606 when the spot upper layer member 602 and the reagent lower layer member 603 are overlapped.

FIG. 6C shows the reagent lower layer member 603. The solid substrate and the spot upper layer member 602 are preferably approximately the same size, and in this embodiment, the hydrophobic sheet is 26 mm long × 76 mm wide. Further, two reagent holes 606 having a size capable of including 15 spot holes 604 in one spot group of the spot upper layer member 602 are provided. The reagent holes 606 have a rectangular shape of 15 mm in length and 24 mm in width, and are arranged side by side at intervals of 1.5 mm.

  FIG. 6D shows the usage state of the two-layer seal 601. In the analysis, a two-layer seal 601 is attached to the solid substrate 105. At this time, the step 605 of the spot upper layer member 602 is also in close contact with the solid substrate 105. And a sample is dripped at the hole for spots, and a sample is fixed on a solid substrate. Thereafter, the spot upper layer member 602 is peeled off, and the reagent lower layer member 603 left on the solid substrate is used in the reaction step with the reagent.

  In this embodiment, the spot upper layer member 602 and the reagent lower layer member 603 are combined in advance at appropriate positions, and the reagent lower layer member 603 left on the solid substrate after the spot upper layer member 602 is peeled off Can be used as is. For this reason, it is possible to avoid positional deviation between the spot hole 604 and the reagent hole 606. Further, the operation of arranging the reagent seal again can be omitted.

  Although the step 605 is used in this embodiment, for example, a soft sheet having no step may be used as the spot upper layer member 602.

  This embodiment relates to an integrated seal in which the spot seal and the reagent seal in Embodiment 1 are integrated. Hereinafter, only main differences from the first embodiment will be described with reference to FIGS.

  The integrated seal 701 shown in FIG. 7A has spot holes arranged in 3 rows × 5 rows at an interval of 5 mm, and two spot groups each consisting of a total of 15 spot holes. A cut and a crease are provided around each spot group. When a part of the integrated seal 701 including the spot group is bent according to this fold, a rectangular reagent portion 702 of 15 mm length × 24 mm width is formed.

FIG. 7B shows a use state of the integrated seal 701. In the analysis, the integrated seal 701 is attached to the solid substrate 105, the sample is dropped into the spot holes, and the sample is immobilized on the solid substrate. Then, a part of the integrated seal 701 is bent according to the crease, and the formed reagent portion 702 is used for a reaction step with the reagent.

  In this embodiment, since the reagent portion 702 formed on the solid substrate after a part of the integrated seal 701 is bent can be used as it is, the reagent seal is re-arranged after avoiding the problem of displacement. This operation can be omitted. In addition, since the integrated seal can be manufactured by providing a cut and a crease on a single layer of seal, the manufacturing process is easy.

In this embodiment, a part of the integrated seal 701 is bent according to the crease to form the reagent part 702. For example, a part of the integrated seal 701 is cut off to form the reagent part 702. Also good.

  It is expected to be used especially in fields where large-scale quantification / analysis of biomolecules is required, such as clinical testing and drug discovery. Also, it is expected to be used as an analysis system combined with a detection device.

A spot seal is shown. The reagent seal is shown. A solid substrate is shown. The concrete work process of the analysis procedure in Example 1 is shown. It is an image figure of the measurement result (scanned image) of CEA and CRP in Example 1. It is the graph which digitized the signal intensity of CEA. It is the graph which digitized the signal intensity of CRP. It is a fluorescence scan image at the time of using only the seal | sticker for spots. It is the graph which digitized the signal intensity in AA 'of Drawing 4 (a). It is a fluorescence scan image at the time of using Example 1. FIG. It is the graph which digitized the signal intensity in BB 'of Fig.5 (a). 2 shows a two-layer seal in Example 2. The upper layer member for spots is shown. The lower member for reagents is shown. The use state of a two-layer seal is shown. The integrated seal in Example 3 is shown. The state of use of the integrated seal is shown.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 101 ... Spot seal, 102 ... Reagent seal, 103 ... Spot hole, 104 ... Reagent hole, 105 ... Solid substrate, 201 ... Micro pipettor, 202, 205 ... Sample, reagent, 203 ... Water drop, 204 ... Sealed container , 206 ... Measurement image, 601 ... Two-layer seal, 602 ... Spot upper layer member, 603 ... Reagent lower layer member, 604 ... Spot hole, 605 ... Step, 606 ... Reagent hole, 701 ... Integrated seal, 702 ... Reagent site.

Claims (8)

A method for analyzing a sample, comprising:
Place a spot member with a spot hole on the substrate,
Supply the first solution to the spot hole,
A reaction member having a reaction hole larger than the spot hole is disposed on the substrate, and the first solution spotted on the substrate is covered with the reaction hole,
Supplying the reaction hole second solution, reacting the first solution and the second solution,
The spot member and the reaction member are directly or indirectly laminated so as to be peelable,
In a state where the spot member and the reaction member are laminated, the first solution is supplied to the spot hole,
A method of supplying the second solution to the reaction hole after peeling off the spot member from the reaction member. A method for analyzing a sample, comprising:
Place a spot member with a spot hole on the substrate,
Supply the first solution to the spot hole,
A reaction member having a reaction hole larger than the spot hole is disposed on the substrate, and the first solution spotted on the substrate is covered with the reaction hole,
Supplying the reaction hole second solution, reacting the first solution and the second solution,
The spot member and the reaction member are fixed directly or indirectly,
In a state where the spot member and the reaction member are fixed, the first solution is supplied to the spot hole,
A method of supplying the second solution to the reaction hole after the spot member is substantially removed from the substrate. A spot member that can be placed on a substrate and has a spot hole;
A reaction member having a reaction hole larger than the spot hole, which can be disposed on the substrate,
The spot member and the reaction member are directly or indirectly laminated,
The spot member is peelable from the reaction member;
An analytical instrument with a hole for spot and a hole for reaction. A spot member that can be placed on a substrate and has a spot hole;
A reaction member having a reaction hole larger than the spot hole, which can be disposed on the substrate,
The spot member and the reaction member are fixed directly or indirectly,
The spot member fits in the reaction hole;
An analytical instrument capable of substantially removing the spot member from the reaction hole. The analytical instrument according to claim 3 or 4,
The spot member comprises a plurality of spot holes,
An analytical instrument, wherein the reaction member includes a reaction hole having a size that covers a plurality of spot holes. The analytical instrument according to claim 3 or 4,
The spot member comprises a plurality of spot holes,
An analytical instrument in which the reaction member includes a plurality of reaction holes. The analytical instrument according to claim 3 or 4,
An analytical instrument in which the spot member and the reaction member are seals that can be attached to a substrate. The analytical instrument according to claim 3 or 4,
The spot member is a member for supplying the first solution containing the protein to a predetermined position on the substrate,
An analytical instrument, wherein the reaction member is a member for supplying a second solution containing a reagent that reacts with a protein to a predetermined position on a substrate.

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