JP2010117250A - Method for stirring analyte solution and method for analyzing analyte - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for stirring an analyte solution capable of uniform reaction and improvement in the reaction efficiency by vigorously moving an analysis target substance by effectively stirring the analyte solution in an analysis of the analyte solution using an analyte chip, and quantitative analysis in a short time, and a method for analyzing an analyte. <P>SOLUTION: An analysis chip to which an analyte solution containing a target substance is applied is set in a reaction device provided with a piezoelectric element as a vibration source. The piezoelectric element is operated and its vibration is propagated to the analyte solution so that the analyte solution is effectively stirred well. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  In the present invention, an analysis chip to which a sample solution containing a test substance is applied is installed in a reaction apparatus equipped with a piezoelectric element as a vibration source, and the piezoelectric element is operated to transmit vibration to the sample solution. The present invention relates to a sample solution stirring method and a sample analysis method for stirring a sample solution.

  The analysis chip has a substrate on which selective binding substances such as genes, proteins, lipids and sugars are immobilized, and the selective binding substance on the substrate and the test substance are reacted as a normal solution, and the reaction results It is used to analyze the presence / absence, state or amount of the test substance. As this substrate, glass, metal and resin substrates are generally used.

  As an embodiment of the analysis chip, there is an analysis chip called a microarray in which molecules such as DNA are arranged on a substrate at a high density for the purpose of simultaneously measuring hundreds to tens of thousands of gene expressions. By using a microarray, systematic and comprehensive gene expression analysis in animal models of various diseases and cell biology phenomena can be performed. Specifically, it becomes possible to clarify the function of the gene, that is, the protein encoded by the gene, and to specify the time when the protein is expressed and the place where it acts. Disease genes and therapies by analyzing changes in gene expression at the cellular or tissue level of an organism by microarrays and combining this with physiological, cell biological and biochemical event data to build a gene expression profile database It is considered possible to search for related genes and therapeutic methods.

  Currently, problems in the reaction using the analysis chip include that the reaction for binding the test substance in the sample solution to the selective binding substance immobilized on the analysis chip does not proceed sufficiently, and uneven reaction occurs. . In such a state, the concentration of the test substance cannot be compared quantitatively, and the performance of the microarray cannot be fully exhibited. As these causes, it is conceivable that the mobility of the test substance in the sample solution is insufficient in the reaction between the test substance and the selective binding substance immobilized on the analysis chip.

  Therefore, a reaction method has been proposed for improving the reaction efficiency and making the reaction uniform. Specifically, with regard to a nucleic acid hybridization method and apparatus, a method and apparatus for circulating a sample solution using a pump has been proposed in order to efficiently combine a selective binding substance and a test substance ( Patent Document 1). However, when using the proposed method and apparatus, a pump for feeding a minute liquid and a heater for heating the liquid are required, and the apparatus becomes very complicated.

  There has also been proposed a device that emits sound waves toward a sample solution and generates a localized flow in the sample solution to stir the sample liquid (see Patent Document 2). However, this proposed device stirs a liquid in an open system and is considered difficult to apply to an analysis chip.

Also disclosed is a method of stirring by facilitating molecular motion in a fine droplet and an apparatus used therefor (see Patent Document 3). However, the proposed stirring method and apparatus stir the inside of a very small amount of droplets, and cannot be said to be a technique for stirring a liquid having a certain volume such as an analysis chip. Therefore, none of the methods is always satisfactory as a means for solving the above problems.
JP2007-209236 JP-A-2005-257406 JP 2001-327846 A

  The object of the present invention has been made in view of the above, and when analyzing a sample solution using an analysis chip, the sample substance is vigorously moved by efficiently stirring the sample solution to react. It is an object of the present invention to provide a sample solution agitation method and a sample analysis method that enable uniform analysis and improvement in reaction efficiency and enable quantitative analysis in a short time.

  In view of the above-mentioned problems, the present inventors have conducted intensive studies. As a result, when analyzing a sample solution using an analysis chip, the analysis chip to which the sample solution is applied is used as a vibration source and installed in a device equipped with a piezoelectric element. Then, it is found that the reaction between the test substance in the sample solution and the selective binding substance fixed to the analysis chip can be performed quickly and uniformly by propagating vibration to the sample solution and stirring the sample solution. The inventors have found that a test substance in a sample solution can be quantitatively detected, and have completed the present invention.

  That is, the sample solution agitation method of the present invention is configured by installing an analysis chip to which a sample solution containing a test substance is applied in a reaction apparatus equipped with a piezoelectric element as a vibration source and operating the piezoelectric element. In this method, the sample solution is stirred by propagating vibration to the sample solution.

  The sample analysis method of the present invention includes a step of applying a sample solution containing a test substance to an analysis chip to which a selective binding substance is fixed, and a piezoelectric element using the analysis chip to which the sample solution is applied as a vibration source. And a selective coupling fixed to the analysis chip by agitating the sample solution by causing the vibration to propagate to the sample solution by operating the piezoelectric element of the vibration source which is a vibration generating mechanism. The method includes a step of reacting and binding a test substance in a sample solution to a test substance, and a step of detecting a test substance bound to the selective binding substance.

  The sample analyzer of the present invention comprises an analysis chip provided with a substrate for applying a sample solution containing a test substance, and a reaction device in which the analysis chip is installed and used. The apparatus is an apparatus including a piezoelectric element as a vibration source that vibrates the analysis chip and propagates the vibration to the sample solution to stir the sample solution.

  According to a preferred aspect of the sample analyzer of the present invention, the analysis chip includes a substrate and a cover member that can be in close contact with a part of the substrate, and has a gap between the cover member and the substrate. The cover member is provided with at least one through hole communicating with the gap.

  According to a preferred aspect of the sample analyzer of the present invention, beads are enclosed in the gap of the analysis chip.

  By analyzing the test substance in the sample solution using the analysis chip according to the method for stirring the sample solution of the present invention, the molecular motion of the test substance in the sample solution can be greatly accelerated compared to the conventional stirring method. . As a result, the reaction efficiency is greatly improved as compared with the conventional analysis method, and the reaction unevenness is remarkably reduced. Therefore, by agitating the sample solution by the sample solution agitation method of the present invention, the target reaction can be completed in a short time, and the S / N ratio and detection sensitivity are greatly improved, and quantitative Evaluation is possible.

  Hereinafter, the method for stirring the sample solution and the method for analyzing the sample of the present invention will be described more specifically.

  The analysis chip referred to in the present invention is an inorganic material such as glass, ceramics and silicon, metals such as stainless steel and gold (plating), or polymethyl methacrylate (PMMA), polyethylene terephthalate, cellulose acetate, polycarbonate, polystyrene, polydimethyl. Hundreds to tens of thousands of selective binding substances for proteins, nucleic acids, sugars, lipids, cells, low-molecular compounds, etc., which are test substances, are fixed to a substrate made of a polymer material such as siloxane and silicon rubber. . The sample solution containing the test substance is applied to the analysis chip on which the selective binding substance is fixed, the test substance is bound to the selective binding substance, and the amount and property of the test substance can be measured.

  The selective binding substance used in the present invention refers to a substance that can selectively bind to a test substance directly or indirectly. Examples of such selectively binding substances include nucleic acids, proteins, saccharides and other antigenic compounds. The nucleic acid includes deoxyribonucleic acid (DNA), ribonucleic acid (RNA), peptide nucleic acid (PNA), complementary DNA (cDNA), complementary RNA (cRNA) and the like. Proteins include antibodies and fragments thereof, antigens and enzyme substrates. Sugars include oligosaccharides and polysaccharides. Other antigenic compounds include peptides and small molecule compounds. Particularly preferred selective binding substances are nucleic acids and proteins (particularly antibodies, antigens, etc.). In this respect, examples of the analysis chip particularly preferably used in the present invention are a DNA microarray (also referred to as a DNA chip) or a protein analysis chip. Such a selective binding substance may be commercially available, or may be synthesized or prepared from a natural source such as a living tissue or a cell.

  The sample solution referred to in the present invention refers to a solution containing a test substance to be analyzed. Here, the test substance includes proteins such as antibodies, antigens and peptide aptamers, nucleic acids such as DNA, RNA and nucleic acid aptamers, lipids such as simple lipids and complex lipids, saccharides such as polysaccharides and oligosaccharides, cells, low Examples thereof include at least one selected from molecular compounds and complexes thereof. The test substance is preferably labeled with a fluorescent compound or a radioactive compound before being applied to the analysis chip. In addition, a method in which a test substance is biotinylated in advance, applied to an analysis chip and bound to a selective binding substance, and detected by a method of binding fluorescently labeled avidin or streptavidin is also preferably used. .

  “Apply” as used in the present invention refers to bringing a sample solution into contact with a selective binding substance by hanging a sample solution on a reaction part of an analysis chip. By binding the test substance in the sample solution to the selective binding substance of the analysis chip, it is possible to determine what kind of test substance is contained in the sample solution. The application can be performed by an arbitrary method depending on the shape of the analysis chip to be used.

  After the sample solution is applied to the analysis chip, the analysis chip to which the sample solution is applied is installed in the reaction apparatus. The reaction apparatus used in the present invention refers to an apparatus used when a test substance in a sample solution applied to an analysis chip is combined with a selective binding substance of the analysis chip, and includes a vibration source. The vibration source is capable of propagating vibration to the specimen solution and can control vibration frequency, amplitude, and acceleration relatively easily. In the present invention, a piezoelectric element is used. Furthermore, the reaction apparatus is equipped with a mechanism for heating and cooling the analysis chip to a predetermined temperature, and a mechanism for swinging the analysis chip by swiveling rotation, reciprocating rotation, figure-eight rotation, gravity rotation, etc. Is preferred.

  The piezoelectric element used in the present invention is a kind of ferroelectric, and refers to an element that generates a voltage when a force such as vibration or pressure is applied, and expands or contracts when a voltage is applied. The piezoelectric element can be slightly expanded and contracted by controlling the voltage, and the degree of stirring can be adjusted. A method in which vibration is propagated to the sample solution by applying a voltage to the piezoelectric element while the piezoelectric element is in contact with the sample solution applied to the analysis chip, or a vibration plate is in contact with the piezoelectric element Thus, a method is preferably used in which the vibration plate is brought into contact with the analysis chip to which the sample solution is applied and a voltage is applied to the piezoelectric element so that the vibration is propagated to the analysis chip through the vibration plate and the sample solution is stirred. . In the case of stirring by bringing the piezoelectric element into contact with the sample solution, the method using the vibration plate is preferable in consideration of the necessity of cleaning for each use.

  Examples of the piezoelectric element used in the present invention include ceramics such as barium titanate ceramics and solid solution ceramics of lead zirconate and titanate, ammonium dihydrogen phosphate, potassium dihydrogen phosphate, crystal, and Rochelle. Examples thereof include organic polymer compounds such as salts, polyvinylidene fluoride, polyvinyl chloride, and nylon 11.

  By the above stirring method, the sample solution containing the test substance can be analyzed using the analysis chip. At this time, the shape of the analysis chip may be a flat plate structure or an uneven structure.

  Next, the analysis chip used in the present invention will be described with reference to the drawings.

  FIG. 1 is a perspective view schematically showing an example of a substrate constituting an analysis chip used in the present invention, and FIG. 2 is a longitudinal sectional view schematically showing an example of an analysis chip used in the present invention. FIG. 3 is a perspective view schematically showing an example of an analysis chip used in the present invention, and FIG. FIG. 4 is a partial cross-sectional view showing an example of a cover member constituting the analysis chip used in the present invention.

  1 and 2, at least a part of the substrate 1 has an uneven structure. As the shape of the concave and convex portions of the concavo-convex structure, the convex portion is particularly preferably a columnar structure such as a prism, a cylinder, and a truncated cone. Moreover, it is preferable that the shape of the upper surface of a convex part is circular or square, such as a tri-octagon. The concave portions or the convex portions have completely or substantially the same structure, and are preferably alternately arranged regularly. In the case of such a regular arrangement, the shape of the concave portion is determined according to the shape of the convex portion. In the case of an analysis chip having a concavo-convex structure, the selective binding substance 2 is fixed on the upper surface of the convex portion.

  Furthermore, in the analysis chip, as shown in FIG. 2, it is also a preferable aspect that the cover member 3 that covers the concave portion and the convex portion of the substrate 1 is bonded with an adhesive member 4. At this time, it is necessary to provide a space between the upper surface of the convex portion of the substrate 1 and the cover member 3 so that the selective binding substance and the test substance can be combined. The size of such a space portion is preferably 1 to 500 μm in the height direction, for example. When the size of the space is smaller than 1 μm, the chance that the test substance comes into contact with the fixed selective binding substance is extremely reduced, and the signal after hybridization is significantly reduced. On the other hand, if the size of the space exceeds 500 μm, a large amount of liquid is required, and when analyzing a small amount of sample, the concentration of the sample solution becomes thin, the hybridization reactivity decreases, and the signal at the time of detection is weak. Become. Here, hybridization means that nucleic acid molecules having complementary sequences form a complex, and in the present invention, it has a sequence complementary to a selective binding substance immobilized on an analysis chip. Detection and quantification of each test substance can be performed using the property of specifically binding to the test substance.

  Furthermore, in consideration of measuring the signal (fluorescence) after hybridization, the cover member 3 is preferably removable after hybridization, so that double-sided tape, PDMS (polydimethylsiloxane) or the like is used as a fixing means. The adhesive member 4 is preferably used.

  Moreover, as shown in FIGS. 2 to 4, the cover member 3 is preferably provided with one or more through holes 5 communicating with the space portion. The through-hole 5 is for injecting a liquid such as a sample solution or a binding buffer, and at the same time, for maintaining the pressure inside the substrate 1 at atmospheric pressure. It is preferable that there are a plurality of through holes 5 with respect to one gap, and among these, the number of the through holes 5 is 3 to 6, so that the sample solution can be easily filled. When the cover member has a plurality of through holes, the diameters thereof may be the same or different, but one of the plurality of through holes functions as an apply port and the other through hole functions as an air outlet. In this case, from the viewpoint of easy application of the sample solution and hermetic retention of the sample solution, it is preferable that only the apply port has a wide hole diameter necessary for the application and other through holes have a narrower hole diameter. Specifically, the size of the through-hole 5 for applying the specimen solution is in the range of 0.01 mm to 2.0 mm in diameter, and the diameter of the other through-hole 5 is preferably 0.01 mm to 1.0 mm. preferable.

  Further, the cover member can be provided with a liquid level holding chamber 7 that preferably communicates with the through hole 5. By providing the liquid level chamber 7, it is possible to suppress the rise of the liquid level of the sample solution applied from the through hole 5 and filled in the space, and to easily and reliably seal the through hole 5 with the sealing member. It is possible to prevent the inflow of air into the sample solution and the outflow of the sample solution. The size of the liquid level holding chamber 7 is preferably in the range of 1.0 mm to 10 mm in diameter.

  The cover member 3 as described above is used in, for example, an injection molding method, a hot embossing method, and a cutting method in the case of resin, a sand blast method in the case of glass or ceramic, and a semiconductor process in the case of silicon. It is preferably produced by a method or the like.

  Furthermore, as shown in FIG. 2, fine particles (beads) 6 can be enclosed in the space between the substrate 1 and the cover member 3. When the sample solution is applied to the space and the vibration is propagated to the sample solution, the fine particles 6 move violently in the sample solution, and the stirring efficiency is remarkably increased. As a result, hybridization promoting effect is brought about. Here, as the material of the fine particles 6, for example, glass, ceramics (for example, yttria-stabilized zirconia), metals such as stainless steel, polymers such as nylon and polystyrene, and magnetic materials are preferably used. Among these, ceramic fine particles are particularly preferably used because they are physically and chemically stable and have a large specific gravity. The size of the fine particles 6 is preferably such that the diameter of the fine particles 6 is larger than the shortest distance between the upper surface of the convex portion and the cover member in a substrate having an uneven structure such as the substrate 1.

  Furthermore, in the present invention, by combining the method of rotating the substrate 1 to drop the fine particles 6 in the direction of gravity, the method of shaking the substrate, the method of moving the fine particles 6 by magnetic force using magnetic fine particles, and the like, The stirring efficiency can be further improved.

  Next, the reaction apparatus used in the present invention will be described. 5 and 6 are cross-sectional views schematically showing a sample analyzer in a state where an analysis chip is installed in the reaction apparatus used in the present invention.

  When the hybridization reaction of the substrate 1 having the cover member 3 is performed, a reaction apparatus as shown in FIG. 5 can be preferably used. That is, the substrate 1 to which the cover member 3 is bonded is set in the reaction device 8 a, and the switch 11 a is inserted into the piezoelectric element 10 a installed on the diaphragm 11 with the diaphragm 11 in close contact with the cover member 3. By applying a voltage from 12a, the piezoelectric element 10a is vibrated, and the specimen solution can be agitated by propagating the vibration to the specimen solution filled in the space portion of the cover member 3 and the substrate 1 via the diaphragm 11. In the reaction, the reaction can be performed at a desired temperature by using the temperature adjusting mechanism 14a.

  When the substrate 1 does not have the cover member 3, a hybridization reaction can be performed by a reaction apparatus as shown in FIG. That is, the substrate 1 to which the sample solution is applied is set in the reaction device 8a, and the gap cover glass 9 provided with the piezoelectric element 10b is brought into close contact with the substrate 1 so that the sample solution is sealed. By applying a voltage from the power source 12b to the piezoelectric element 10b, the piezoelectric element 10b is vibrated, and the vibration is propagated to the specimen solution sealed in the space portion of the gap cover glass 9 and the substrate 1, thereby stirring the specimen solution. In the reaction, the reaction can be performed at a desired temperature using the temperature control mechanism 14b.

  Next, the present invention will be described in further detail with reference to examples. However, the present invention is not limited to the following examples.

Example 1
(Preparation of analysis chip substrate)
As the substrate, the outer shape is 76 mm in length, 26 mm in width, and 1 mm in thickness, and a recess having a length of 39.4 mm, width of 19.0 mm, and depth of 0.15 mm is provided in the central portion of the substrate. A polymethyl methacrylate (PMMA) resin substrate (hereinafter referred to as “substrate A”) provided with 9248 convex portions having a height of 1 mm and a height of 0.15 mm was used. In this substrate A, the difference in height between the upper surface of the convex portion and the upper surface of the flat portion (the convex portion has an average height value) was 3 μm or less. Further, the variation in the height of the upper surface of the convex portion (the difference between the height of the upper surface of the highest convex portion and the height of the upper surface of the lowest convex portion) was 3 μm or less. Further, the pitch of the convex portions (the distance from the central portion of the convex portion to the central portion of the adjacent convex portion) was set to 0.5 mm.

  The substrate A was immersed in a 10N aqueous sodium hydroxide solution at a temperature of 70 ° C. for 12 hours. This substrate A was washed with pure water, 0.1N HCl aqueous solution and pure water in this order to generate carboxyl groups on the substrate surface.

(Immobilization of selective binding substances)
On the substrate A prepared as described above, an oligonucleotide was immobilized as a selective binding substance (probe DNA) under the following conditions. As an oligonucleotide, an oligonucleotide set for DNA microarray “Homo sapiens (Human) AROS V4.0 (60 bases each)” manufactured by Operon was used. This oligonucleotide was dissolved in pure water to a concentration of 0.3 nmol / μL to obtain a stock solution. When spotting this stock solution onto the substrate, PBS (8 g NaCl, 2.9 g Na ₂ HPO ₄ · 12H ₂ O, 0.2 g KCl, and 0.2 g KH ₂ PO ₄ were combined. A solution made up to 1 L by dissolving in pure water and adding hydrochloric acid to adjust the pH to 5.5) diluted 10-fold to a final concentration of probe DNA of 0.03 nmol / μL, and a substrate made of PMMA In order to condense the carboxyl group generated on the surface with the terminal amino group of the probe DNA, 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) was added to make this final concentration 50 mg / mL. This solution was spotted on the upper surfaces of all the convex portions of the substrate A using an arrayer (spotter) (manufactured by Nippon Laser Electronics; Gene Stamp-II). Next, the substrate on which the solution was spotted was placed in a sealed plastic container and incubated for about 20 hours under the conditions of a temperature of 37 ° C. and a humidity of 100%. Finally, the substrate was washed with pure water and centrifuged by a spin dryer and dried.

(Attaching the cover member to the chip substrate for analysis)
A cover member was attached to the substrate A on which the selective binding substance was immobilized as follows. As the cover member, a PMMA flat plate having a length of 41.4 mm, a width of 21 mm, and a thickness of 1 mm was produced by cutting to obtain a cover member. The produced cover member was provided with a through hole 5 and a liquid level holding chamber 7 as shown in FIG. The diameter of the through hole 5 was 0.8 mm, the diameter of the liquid level holding chamber was 2.0 mm, and it was installed at the four corners of the cover member.
Then, a double-sided tape with a length of 41.4 mm, a width of 21 mm, a width of 1 mm, and a thickness of 25 μm is cut into a size that borders the cover member, and laminated and pasted so that the thickness (clearance) is 50 μm. After that, the cover member was attached to the substrate A.

(Fine particle encapsulation in analysis chip)
120 mg of zirconia fine particles having a diameter of 180 μm were enclosed in a space formed by the substrate A and the cover member (concave portion of the concavo-convex structure on the surface of the substrate A) on the substrate A to which the cover member was attached as described above. The fine particles were sealed from the through hole (through hole 5 illustrated in FIG. 2 or 4) of the cover member. The analysis chip obtained as described above was designated as “Analysis chip 1”.

(Preparation of test substance)
As a test substance, general aRNA (antisense RNA) was used as a test substance of a microarray. 5 μg of Cy3-labeled aRNA was obtained from 5 μg of commercial human cell-derived total RNA (Human Reference RNA manufactured by CLONTECH) using an aRNA preparation kit manufactured by Ambion. In this Example 1, and the following Example 2 and Comparative Example 1, unless otherwise specified, the test substance solution at the time of hybridization was 1% by weight of BSA, 5 × SSC, unless otherwise specified. , 0.01 wt% salmon sperm DNA, 0.1 wt% SDS diluted with each solution (each concentration is the final concentration) was used.

(Hybridization)
The test substance (Cy3-labeled aRNA) prepared by the above method was mixed with a solution (1 wt% BSA, 5 × SSC, 0.01 wt% salmon sperm DNA, 0.1 wt% SDS as described above (each All the concentrations were dissolved in a final concentration)) to prepare 170 μL of a sample solution containing 200 ng of Cy3-labeled aRNA. Using a micropipette, the surface of the analysis chip A on which oligonucleotides were spotted (the substrate A and the space between the cover members) was applied. After the four through-holes provided in the cover member are closed with Kapton tape, the analysis chip 1 is connected to the reaction apparatus A having a piezoelectric element and a diaphragm as shown in FIG. Was set in contact with each other. The set temperature of the reaction apparatus was 37 ° C., the piezoelectric element was vibrated by turning on the power of the vibration source piezoelectric element, the vibration was propagated to the sample solution through the vibration plate, and the reaction was performed for 4 hours.

(Measurement of fluorescence signal value)
After removing the cover member and the double-sided tape of the analysis chip 1, the substrate A was washed and dried. The substrate A after the above treatment was set on a DNA chip scanner (GenePix 4000B, manufactured by Axon Instruments), and the test substance that had undergone the hybridization reaction was set with a laser output of 33% and a photomultiplier voltage setting of 500. The label signal value (fluorescence intensity) and background noise were measured. Of the 9248 spots, 32 were used as negative control spots for background fluorescence measurement, and the true signal value of each spot was calculated by subtracting the background signal value from each signal value.

  Table 1 shows the sum of the fluorescence signal values, the average value of the positive control, and the CV value of the positive control. Since the signal intensity was strong, while the CV value was small, the level was sufficiently satisfactory for quantitative evaluation.

(Example 2)
(Immobilization of selective binding substance to substrate)
As the substrate B, a coated slide glass for DNA microarray (Matsunami Glass) was used. On this substrate B, an oligonucleotide was immobilized as a selective binding substance (probe DNA) under the following conditions. As the oligonucleotide, the oligonucleotide set “Homo sapiens (Human) AROS V4.0 (60 bases each)” for DNA microarray manufactured by Operon was used. This oligonucleotide was dissolved in a spotting solution for DNA microarray (Matsunami Glass) so that the final concentration was 0.03 nmol / μL. A total of 9248 spots (68 spots in the minor axis direction and 136 spots in the major axis direction) were spotted on this solution using an arrayer (spotter) (manufactured by Nippon Laser Electronics; Gene Stamp-II). The analysis chip obtained as described above was referred to as “analysis chip 2”.

(Hybridization)
A test substance (Cy3-labeled aRNA) prepared in the same manner as described in Example 1 was dissolved in the above solution to prepare a 60 μL sample solution containing 200 ng of Cy3-labeled aRNA, and an analysis chip using a micropipette. Two oligonucleotides were applied to the spotted surface. As shown in FIG. 6, the analysis chip 2 is set in a reaction device B having a piezoelectric element, and a sample solution is applied to a gap cover glass (Matsunami Glass) with a piezoelectric element connected to the reaction device attached inside. The piezoelectric element and the specimen solution were brought into contact with each other. The set temperature of the reaction apparatus was 37 ° C., the piezoelectric element was vibrated by turning on the power of the piezoelectric element of the vibration source, the vibration was propagated to the sample solution, and the reaction was performed for 4 hours.

(Measurement of fluorescence signal value)
The analysis chip 2 was washed and dried. In the state where the substrate B after the above treatment is set on a DNA chip scanner (GenePix 4000B manufactured by Axon Instruments), the laser output is 33%, and the voltage setting of the photomultiplier is 500, The label signal value (fluorescence intensity) and background noise were measured. Of the 9248 spots, 32 were used as negative control spots for background fluorescence measurement, and the background signal value was subtracted from each signal value to calculate the true signal value of each spot.

  Table 1 shows the sum of the fluorescence signal values, the average value of the positive control, and the CV value of the positive control. Both signal intensity and CV values were sufficient to make a quantitative assessment.

(Comparative Example 1)
As the analysis chip, the “analysis chip 2” used in Example 2 was used, and instead of using the reaction apparatus B at the time of hybridization, it was allowed to stand at a temperature of 37 ° C. using a hybridization incubator (Tytec). Then, the same operation as in Example 2 was performed except that the reaction was performed.

  Table 1 shows the sum of the fluorescence signal values, the average value of the positive control, and the CV value of the positive control. Compared with Example 1 and Example 2, the signal intensity in Comparative Example 1 was considerably low, while the CV value was high, indicating that the reaction efficiency was considerably poor and the reaction unevenness was large.

(Comparative Example 2)
The “analysis chip 2” used in Example 2 was used as the analysis chip, and instead of using the reaction apparatus B at the time of hybridization, a hybridization oven equipped with a reciprocating shaker (Tytec) was used. The same operation as in Example 2 was performed except that the reaction was performed by reciprocating motion at a temperature of ° C. Table 1 shows the sum of the fluorescence signal values, the average value of the positive control, and the CV value of the positive control. Compared with Example 1 and Example 2, the signal intensity in Comparative Example 2 was low, while the CV value was quite high, indicating that the reaction efficiency was poor and the reaction unevenness was very large.

  By stirring the sample solution applied to the analysis chip by the sample solution stirring method of the present invention, the reaction efficiency between the selective binding substance immobilized on the analysis chip and the test substance in the sample solution can be increased. In addition, since the occurrence of uneven reaction can be suppressed, the test substance can be detected quickly and quantitatively. In particular, in a microarray characterized by a substrate having a concavo-convex structure, the microarray inherently has good characteristics by the method for stirring a sample solution according to the present invention that improves measurement value variation and enables highly reliable measurement. It is possible to further utilize characteristics such as a high S / N ratio and high detection sensitivity. As described above, the sample solution agitation method and sample analysis method of the present invention are very useful industrially because they can improve the precision of microarrays that are frequently used in fields such as biology, medicine, and microbiology. .

FIG. 1 is a perspective view schematically showing an example of a substrate constituting an analysis chip used in the present invention. FIG. 2 is a longitudinal sectional view schematically showing an example of the analysis chip used in the present invention. FIG. 3 is a perspective view schematically showing an example of the analysis chip used in the present invention. FIG. 4 is a partial cross-sectional view showing an example of a cover member constituting the analysis chip used in the present invention. FIG. 5 is a cross-sectional view schematically showing a sample analyzer in a state where an analysis chip is installed in the reaction apparatus used in the present invention. FIG. 6 is a cross-sectional view schematically showing another sample analyzer in a state where an analysis chip is installed in the reaction apparatus used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Substrate 2 Selective binding substance 3 Cover member 4 Adhesive agent 5 Through hole 6 Fine particles (beads)
7 Liquid level holding chambers 8a and 8b Reactor 9 Gap cover glass 10a and 10b Piezoelectric element 11 Vibration plates 12a and 12b Power supplies 13a and 13b Switches 14a and 14b Temperature control mechanism

Claims (5)

  An analysis chip to which a sample solution containing a test substance is applied is placed in a reaction apparatus equipped with a piezoelectric element as a vibration source, and the piezoelectric element is operated to vibrate the sample solution and stir the sample solution. To stir the sample solution.   The analysis chip includes a substrate and a cover member that can be in close contact with a part of the substrate, and has a gap portion between the cover member and the substrate, and the cover member has at least one through-hole communicating with the gap portion. The method for stirring a sample solution according to claim 1, which is provided.   The method for stirring a sample solution according to claim 2, wherein beads are enclosed in the voids of the analysis chip.   A step of applying a sample solution containing a test substance to an analysis chip to which a selective binding substance is fixed; a step of installing the analysis chip to which the sample solution is applied as a vibration source in a reaction apparatus equipped with a piezoelectric element; A step of causing vibration to propagate to the sample solution by actuating the piezoelectric element of the vibration source, stirring the sample solution, and reacting the selective binding substance fixed on the analysis chip with the test substance in the sample solution; A method for analyzing a sample, the method comprising at least a step of detecting a test substance bound to a selective binding substance.   An analysis chip including a substrate for applying a sample solution containing a test substance, and a reaction device installed and used inside the analysis chip, the reaction device vibrates the analysis chip into a sample solution. A specimen analyzer comprising a piezoelectric element as a vibration source for agitating a specimen solution by propagating vibration.

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