JP2005106739A - Reaction container for microarrays - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To uniformize hybridization reactions by uniformizing the flow of a fluid in a reaction chamber in a reaction container for hybridization reactions of a nucleic acid probe of microarrays with a target nucleic acid contained in a sample. <P>SOLUTION: When a fluid containing a sample and a reaction solution is introduced to and delivered from to a reaction chamber via through holes provided at both the ends thereof to subject the introduced fluid to solution stirring, substitution and reaction in the reaction chamber, a flow limiting member having inner angles of less than 180° with through holes sandwiched therebetween is provided in order to limit the spread of the flow of a fluid introduced from these through holes to less than 180°. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to an improvement in a reaction vessel used for performing a specific binding reaction between a microarray and a target nucleic acid when a target nucleic acid or target protein is detected using a microarray to which a nucleic acid probe or a protein probe is immobilized.

  In order to detect a target nucleic acid having a specific base sequence such as a gene, a hybridization reaction with a probe nucleic acid having a base sequence complementary to the target nucleic acid has long been used. In recent years, many target nucleic acids have been detected at once using a microarray in which a large number of probe nucleic acids are implanted on a flat solid phase substrate surface such as a slide glass or a plastic chip.

  A hybridization reaction between a probe nucleic acid on a microarray using a glass substrate and a target nucleic acid in a sample is performed using a reaction vessel as shown in FIGS. In the figure, reference numeral 1 denotes a microarray using a slide glass substrate. As shown in FIG. 1B, many probe nucleic acids for capturing the target nucleic acid are implanted in a predetermined spot region 2 on the microarray surface. A resin lid 4 is disposed on the microarray 1 via a rubber O-ring 3 along the outer periphery thereof. Thus, a reaction chamber 5 defined by the microarray 1, the lid 4 and the O-ring 3 is formed. The lid 4 is formed with through holes 6a and 6b that communicate with both ends of the reaction chamber 5. The through holes introduce a sample and a reaction solution for a hybridization reaction into the reaction chamber, It is also used as an inlet and outlet for discharging from the reaction chamber. FIG. 1C shows a bottom view of the lid 4. As shown in the figure, the inlet and outlets 6 a and 6 b are formed close to the center of both short sides facing in the longitudinal direction in a substantially rectangular region defined by the O-ring 3.

  When detecting the target nucleic acid in the sample, a solution such as a buffer necessary for the sample and nucleic acid hybridization is taken in and out of the reaction chamber 5 through the inlets and outlets 6a and 6b, and uniformly on the spot region 2. Stir so that a correct reaction occurs. Thereby, the target nucleic acid contained in the sample is hybridized with the nucleic acid probe fixed to the spot region 2 of the microarray 1 and captured. Further, for example, by applying a known label such as a fluorescent label to the captured target nucleic acid by a known method such as sandwich hybridization, after performing post-treatment such as drying, the microarray 1 is removed, and a known detector such as a fluorescence detector is used. The label on the target nucleic acid is detected by the detector.

  In the case of a protein microarray that uses a protein probe such as an antibody instead of a probe nucleic acid to detect a target protein such as an antigen, the same reaction vessel as described above is used, and a specific binding reaction is performed in the same manner. .

  However, the conventional hybridization reaction vessel as described above cannot obtain a uniform reaction over the entire spot region 2 in the microarray 1, and the same probe is fixed by spotting. There is a problem in that the intensity of the detection signal obtained varies depending on the location of the spot region 2.

  As a result of analyzing this problem based on the knowledge of microfluidics, it was found that the flow in the reaction chamber 5 was not uniform when the sample and the reaction solution were taken in and out through the inlet and outlet 6a and 6b. . That is, as shown in FIG. 2, in the reaction chamber 2, a spindle-shaped fast flow region 11 connecting the introduction and discharge ports 6a and 6b and a slow flow stagnation region 12 in other peripheral regions are formed. In the stagnation region 12, the hybridization reaction is excessive, while in the fast flow region 11, the hybridization reaction is insufficient, or in the stagnation region 12, the washing is insufficient, while the fast flow region 11. Then, as a result of excessive cleaning, a weak detection signal is generated at the center of the spot region 2 and a strong detection signal is generated at the corners of the periphery. The analysis result by microfluidics was in agreement with the actual experimental result.

  The above situation is the same for the protein microarray. Furthermore, even in a region where the flow is fast, the length of each trajectory connecting the introduction port and the discharge port is different, which may include a subtle difference in flow velocity.

  The present invention has been made in view of the above circumstances, and performs a specific binding reaction between a microarray in which a nucleic acid probe or a protein probe is immobilized on a spot region on a plate-like substrate surface and a target nucleic acid or target protein contained in a sample. An object of the reaction container is to make the specific binding reaction in the spot region uniform by making the flow of fluid in the reaction chamber uniform.

The above problem is a reaction vessel for a microarray used to detect a target nucleic acid or target protein in a sample:
A microarray in which a nucleic acid probe or protein probe that specifically binds to the target nucleic acid or target protein is immobilized in a spot region on the surface of the plate-like substrate;
An elastic sealing member that surrounds at least a substantially rectangular region including the spot region and is liquid-tightly disposed on the surface of the microarray;
A lid that is coupled to the bottom surface of the elastic sealing member and forms a reaction chamber defined by the elastic sealing member between the microarray and the surface;
A first through hole provided to penetrate through the lid and communicate with one of the opposite ends of the reaction chamber in the longitudinal direction; and provided at the other end of the reaction chamber. A second through hole,
When the fluid containing the sample and the reaction solution is taken in and out of the reaction chamber through the first and second through holes, the introduced fluid is stirred and reacted in the reaction chamber, or the liquid is replaced. The introduction of the through-hole is less than 180 ° with the through-hole interposed so as to limit the spread of the flow of the fluid introduced from at least one of the first and second through-holes to less than 180 °. This is achieved by a microarray reaction vessel characterized by providing a flow restricting member having an inner angle.

Another object of the present invention is a reaction vessel for a microarray used for detecting a target nucleic acid or target protein in a sample:
A microarray in which a nucleic acid probe or protein probe that specifically binds to the target nucleic acid or target protein is immobilized in a spot region on the surface of the plate-like substrate;
An elastic sealing member that surrounds at least a substantially rectangular region including the spot region and is liquid-tightly disposed on the surface of the microarray;
A lid that is coupled to the bottom surface of the elastic sealing member and forms a reaction chamber defined by the elastic sealing member between the microarray and the surface;
A plurality of first through holes provided so as to pass through the lid and communicate with one end of both ends of the reaction chamber facing in the longitudinal direction, and the other end of the reaction chamber A plurality of second through holes provided,
A reaction for microarray, wherein a fluid containing a sample and a reaction solution is put into and taken out of the reaction chamber through the first and second through holes, and the introduced fluid is stirred and reacted in the reaction chamber. Accomplished by container.

  According to the reaction container for a microarray of the present invention, when the target nucleic acid or target protein is detected using the microarray, the flow of the fluid in the reaction chamber is made uniform so that the probe and the target are spread over the entire spot region. The specific binding reaction can be made uniform, and a uniform detection signal can be obtained.

  The present invention will be described below with reference to embodiments shown in the drawings.

  FIG. 3 is a plan view for explaining an embodiment of the present invention, and the same reference numerals are assigned to the same portions as those in FIG. FIG. 3 is a plan view of the lid body 4. As illustrated, an elastic sealing member (for example, an O-ring 5) made of an elastic material such as silicone rubber is provided along the outer periphery of the bottom surface of the rectangular lid 4. The O-ring 5 defines a substantially rectangular reaction chamber. Further, flow restricting members 7a, 7b, 7c and 7d made of Teflon seals having a certain thickness are provided at the corners of the substantially rectangular reaction chamber region defined by the O-ring 5. The flow limiting member is less elastic than the O-ring 5 and its thickness is slightly smaller than the thickness of the O-ring 5. When these flow restricting members are stacked on the microarray 1 shown in FIG. 1B, they are joined to the surface of the microarray in a watertight manner and limit the shape of the reaction chamber together with the O-ring 5. Here, the O-ring 5 makes the reaction chamber pressure resistant by elastic deformation. The flow restricting member 7 which is a low elastic sheet keeps the height of the reaction chamber constant. Between the flow restricting members 7a and 7b, a through hole 6a that penetrates the lid 4 is provided, and between the flow restricting members 7c and 7d, a through hole 6b that penetrates the lid 4 is provided. ing. The internal angle between the flow limiting members 7a and 7b and the internal angle between the flow limiting members 7c and 7d are 90 °. However, the effective range of the inner angle may be set to an appropriate value of less than 180 ° depending on conditions such as the type, amount, and flow rate of the reaction solution used, preferably 25 ° to 160 °, more preferably 60 °. ° to 120 °. The rest of the configuration is the same as that of the embodiment shown in FIGS. 1A to 1C, and the hybridization reaction container is formed by superposing the lid of FIG. 3 on the microarray surface of FIG. Is formed. On the other hand, the side wall portion of the reaction chamber has a straight or curved shape of 120 ° to 180 °, preferably 160 ° to 180 °. When this side wall part exceeds the effective range, an extra collision part is given to the fluid, and when the side wall part is below the effective range, a flow stop part as described above may be generated.

  In the above embodiment, both the microarray 1 and the lid 4 are formed of a glass slide, and the size is 25 mm × 75 mm. The rectangular reaction chamber defined by the O-ring 3 has a size of 20 mm × 75 mm and a thickness of about 100 μm. Further, the size of the spot region 2 in the microarray 1 is 17.5 mm × 17.5 mm, and the shorter one of the distances from the spot region 2 to the short side end of the microarray 1 is 23 mm. Furthermore, 8 spots x 8 spots (spot pitch 0.5 mm) blocks are arranged in 4 blocks x 4 blocks (block pitch 4.5 mm) in the entire spot area 2, and all spots have the same 35-mer. A total of 1024 spots of nucleic acid probes are spotted.

  According to the above embodiment, as a result of providing the flow restricting members 7a, 7b, 7c, 7d, the fluid such as the sample solution, the buffer solution, and the dry air injected from the through holes 6a or 7b is not limited to these restricting members 7a and 7b. Or limited by 7c and 7d, it does not spread 180 ° on both sides as in the prior art. Therefore, it collides with the O-ring wall at the periphery of the reaction chamber constituting the side wall of the reaction chamber not at 90 ° as in the prior art, but at a significantly smaller angle. Therefore, the flow does not stagnate at the collision portion, and a uniform flow is formed in the reaction chamber as shown in FIG. In particular, since the flow restricting member extends to the side wall of the reaction chamber, it has an action of accelerating the flow toward the side wall of the reaction chamber. Variation in flow rate can be reduced. As a result, in the spot region 2, the hybridization between the nucleic acid probe and the target nucleic acid in the sample is uniformly performed as a whole, and the uniformity of the final detection signal intensity can be improved.

  In the embodiment of FIG. 3, the flow restricting members 7a, 7b, 7c, and 7d are used. However, the same effect can be achieved by changing the shape of the O-ring 5 that limits the reaction chamber. Such a modification is shown in FIG.

  FIG. 5 (A) shows an example in which the O-ring 5 has a long and narrow octagonal shape, the lengths of the sides facing in the longitudinal direction are remarkably shortened, and the through holes 6a and 6b are provided inside thereof.

  FIG. 5B shows an example in which the O-ring 5 has a long and narrow hexagonal shape, and through holes 6a and 6b are provided on the inner side of the apex having an acute angle opposed in the longitudinal direction.

  FIG. 5C shows an example in which the O-ring 5 is formed into a long and slanted parallelogram, and through-holes 6a and 6b are provided on the inner sides of the opposing apexes.

  FIG. 5D shows an example in which the O-ring 5 has an elongated oval shape, and through holes 6a and 6b are provided on the inner sides of both ends in the major axis direction.

  Even in the modification shown in FIG. 5, the fluid introduced from the through-hole 6a or 6b is prevented from being spread by 180 ° by the O-ring 5 and is limited to a narrower range. The same effect as in the third embodiment can be obtained.

  FIG. 6 shows a microarray reaction vessel according to another embodiment of the present invention. In this embodiment, the shape of the reaction chamber is the same as that of the conventional example shown in FIG. 1, but the conventional example is provided with a pair of through holes 6a and 6b, whereas the two pairs of through holes 21a facing each other. , 21b and 22a, 22b. Even with such a configuration, the same effect that the flow of the fluid in the reaction chamber becomes uniform can be obtained, and thus the same effect that the detection signal becomes uniform can be obtained.

  In addition, although the said Example demonstrated the hybridization reaction container for detecting a target nucleic acid, those skilled in the art will understand that it is the same also about the reaction container for detecting a target protein. In the above-described embodiments, the microarray 1 and the lid 4 are both made of glass. However, these are any material (for example, plastic) or surface shape having optical transparency, heat resistance, and pressure resistance, for example, porous, It can be in the form of a microwell, a fiber, a pillar (a shape in which minute cylinders having the same height are arranged on the substrate surface), or a fine particle. In addition, either one of the microarray 1 and the lid 4 may be made of a material having poor light transmittance, and is mainly composed of a highly reflective member (metal, silicon oxide) or coated with any material. But you can. Moreover, it can also comprise so that joining of the microarray 1 and the cover body 4 may be peeled reversibly. For example, while the microarray 1 is made of metal such as stainless steel, the lid 4 is made of hard plastic, and the reaction is performed while maintaining the bonding by an appropriate pressing means, so that the pressure is released after the reaction and the microarray is made. Only 1 may be peeled off and transferred to a suitable measuring device for measurement. The pressing means that enables such reversible bonding can be any means that performs a reaction by pressing a conventional microarray onto a predetermined table. A reaction execution system having a pressing means and integrated with a liquid processing mechanism such as a pump is preferable. For example, as applied to an automatic slide processor LUCIDEA (trade name) sold by Amersham, The microarray 1 and the lid 4 can be designed. Further, the flow direction in the reaction chamber is not limited to one direction, and may be bidirectional so as to be advantageous for stirring or washing. In the case of bidirectional flow, it is more preferable that the flow limiting members formed in the first and second through holes are parallel to each other or have a uniform distance. Further, the inner angle by the sealing member or the flow restricting member may have multi-step corners that gradually increase within the above-described effective range. Further, the multi-step corners may be replaced with a continuous curve in which the vertex of each corner is the radius of curvature. When the shape of the flow restricting member is a curve, it is preferable to form the upstream flow restricting member on the circumference having the same radius from the downstream inlet or outlet. The fluid containing the sample and / or reagent may contain a granular solid phase such as beads or cells.

[Experimental example]
Using the example shown in FIG. 3 and the conventional example shown in FIGS. 1A to 1C, hybridization experiments were performed using the same protocol described below.

  A 75-mer DNA modified with Cy3 was used as the target nucleic acid, and a 35-mer DNA perfect-matched to the target was used as the probe. The target nucleic acid was used as a solution in which 1 pm was dissolved in 200 μL of a commercially available hybridization solution.

<Hybridization protocol>
1. Hybridization
Volume sucked and pumped during stirring for 30 minutes at 40 ° C: 30 μL
1. Target solution agitation (injection and drainage) rate: 20 μL / second Agitation cycle: 1 cycle / min Post-cleaning ・ Flashing twice with 1 × SSC (500μL), speed 20μL / sec
Washed with 1 × SSC, aspirated and pumped for 10 minutes at room temperature, 50 μL
Stirring (injection and discharge) speed: 20 μL / sec Stirring cycle: 0.5 cycle / min • Flash once with 0.1 × SSC (500 μL), speed 20 μL / sec
Washed with 0.1 x SSC, sucked and delivered for 5 minutes at room temperature, 30 μL
2. Speed of stirring (injection and discharge): 20 μL / sec. Stirring cycle: 1 cycle / min. • Flash once with 0.1 × SSC (500 μL), speed 20 μL / sec. Drying of slide 2 flushes with isopropyl alcohol (500 μL), speed 20 μL / sec 2 flushes with air (1000 μL), speed 20 μL / sec Air blow drying: 400 seconds After performing hybridization reaction according to the above protocol The fluorescence measurement of the microarray 1 was performed. When the fluorescence intensity measured for each spot was statistically processed to determine variation in signal intensity, the following results were obtained.

Signal from 1024 spots
Average value Standard deviation CV value (%)
Conventional example 37015 12724 34.4
The present invention 39207 2840 7.2

FIG. 1A is a cross-sectional view showing a conventional microarray reaction vessel. FIG. 1B is a plan view of the microarray in FIG. FIG. 1C is a bottom view of the lid body in FIG. FIG. 2 is an explanatory view showing the problems of the conventional microarray reaction vessel shown in FIG. FIG. 3 is a bottom view of the lid constituting the microarray reaction container according to the position example of the present invention. FIG. 4 is an explanatory diagram showing the operation of the embodiment shown in FIG. 5A to 5D are explanatory views showing a modification of the embodiment of FIG. FIG. 6 is a bottom view of a lid constituting a reaction container for microarray according to another embodiment of the present invention.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 1 ... Microarray, 2 ... Spot area | region, 3 ... O-ring, 4 ... Cover body, 5 ... Reaction chamber, 6a, 6b ... Through-hole, 7a-7d ... Flow limitation member

Claims (6)

A reaction vessel for a microarray used to detect a target nucleic acid or target protein in a sample:
A microarray in which a nucleic acid probe or protein probe that specifically binds to the target nucleic acid or target protein is immobilized in a spot region on the surface of the plate-like substrate;
An elastic sealing member that surrounds at least a substantially rectangular region including the spot region and is liquid-tightly disposed on the surface of the microarray;
A lid that is coupled to the bottom surface of the elastic sealing member and forms a reaction chamber defined by the elastic sealing member between the microarray and the surface;
A first through hole provided to penetrate through the lid and communicate with one of the opposite ends of the reaction chamber in the longitudinal direction; and provided at the other end of the reaction chamber. A second through hole,
When the fluid including the sample and the reaction solution is taken in and out of the reaction chamber through the first and second through holes, the introduced fluid is stirred and reacted in the reaction chamber, or the liquid is replaced. The introduction of the through-hole is less than 180 ° with the through-hole sandwiched between the first and second through-holes so as to limit the spread of the fluid flow introduced from at least one of the first and second through-holes A reaction vessel for microarray, comprising a flow restricting member forming an inner angle. 2. The microarray reaction container according to claim 1, wherein the elastic sealing member has a substantially rectangular ring shape, and the flow restricting member different from the elastic sealing member is provided at four inner corners thereof. 3. A microarray reaction vessel. 2. The microarray reaction container according to claim 1, wherein the elastic sealing member has a shape other than a rectangle so that the elastic sealing member also serves as the flow limiting member. Reaction vessel. The reaction container for microarray according to claim 3, wherein the elastic sealing member has an oval shape. The microarray reaction container according to any one of claims 1 to 3, wherein an inner angle of the flow limiting member is 180 ° to 25 °. A reaction vessel for a microarray used to detect a target nucleic acid or target protein in a sample:
A microarray in which a nucleic acid probe or protein probe that specifically binds to the target nucleic acid or target protein is immobilized in a spot region on the surface of the plate-like substrate;
An elastic sealing member that surrounds at least a substantially rectangular region including the spot region and is liquid-tightly disposed on the surface of the microarray;
A lid that is coupled to the bottom surface of the elastic sealing member and forms a reaction chamber defined by the elastic sealing member between the microarray and the surface;
A plurality of first through holes provided so as to pass through the lid and communicate with one end of both ends of the reaction chamber facing in the longitudinal direction, and the other end of the reaction chamber A plurality of second through holes provided,
A reaction for microarray, wherein a fluid containing a sample and a reaction solution is put into and taken out of the reaction chamber through the first and second through holes, and the introduced fluid is stirred and reacted in the reaction chamber. container.

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