JP2008256378A - Specimen detection method and biochip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To perform detection reaction with high accuracy, in a short time, by improving the contact efficiency of the specimen in a sample liquid with a detection reagent. <P>SOLUTION: The detection method of the specimen in the sample liquid by using the detection reagent 14 that specifically reacts with the specimen, to obtain data with respect to the structure of the specimen, includes a step of dropping the sample liquid containing the specimen on a substrate 16, to bring it into contact with the detection reagent 14 and a step of applying shearing force with respect to the liquid droplet interface of the sample liquid, by allowing a liquid to flow along the liquid droplet interface of the sample liquid. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a specimen detection method and a biochip, and more particularly to a method for obtaining biological information by reaction of biological substances.

  In recent years, many studies have been made to detect biologically related substances collected from living bodies by reacting them outside the living body. For example, as in Patent Document 1, in a DNA chip in which artificially prepared DNA is immobilized on a substrate surface, it is detected which base sequence DNA on the chip specifically binds to DNA collected from a living body. Thus, genetic information can be obtained. As described above, in general, detection is performed by bringing a detection reagent that reacts specifically with the specimen into contact with the sample liquid containing the specimen in a state where the reagent is immobilized on the substrate surface.

  At this time, the specimen in the sample liquid is diffused until it reaches the detection reagent on the substrate, and detection is performed by generating a conjugate of both. For this reason, it is necessary to hold the sample liquid on the substrate surface until a detectable amount of the conjugate is produced. On the other hand, depending on the purpose of detection and the type of detection reaction, there is a case where it is desired to perform the above reaction in a short time. In this way, the reaction time can be shortened by shortening the diffusion time for the reaction in which the process of diffusing the analyte in the sample solution to the detection reagent on the substrate is controlled (diffusion-controlled reaction). Is desired.

On the other hand, for example, in Non-Patent Document 1, a DNA chip including a detection unit in which a large number of columnar structures each having a detection reagent formed on the top surface are arranged and beads enclosed in the detection unit is provided. Proposed. Then, the sample solution is supplied to the gap between the columnar structures in the DNA chip, and the beads are moved by a shaker, so that the contact efficiency between the specimen in the sample solution and the detection reagent formed on the top surface of the columnar structure is increased. It is increasing.
JP 2000-72222 A The 86th Annual Meeting of the Chemical Society of Japan, 2J6-17

  However, in Patent Document 1, since the specimen in the droplet reaches the detection reagent on the substrate only by molecular diffusion, the reaction time may increase as the amount of the sample liquid increases.

  Further, in Non-Patent Document 1, the use of beads makes it possible to increase the contact efficiency between the sample in the sample liquid and the detection reagent, but the sample is adsorbed to the wall surface where the detection reagent is not formed. (Non-specific adsorption), the sample solution is not only wasted, but the detection accuracy may be reduced.

  The present invention has been made in view of such circumstances, and improves the contact efficiency between the sample in the sample liquid and the detection reagent, and can detect the sample with high accuracy and in a short time. The purpose is to provide a biochip.

  According to a first aspect of the present invention, there is provided a method for detecting a sample in a sample liquid using a detection reagent that specifically reacts with the sample and obtains information on the structure of the sample in order to achieve the object. A step of dropping a sample solution containing a liquid onto a substrate and bringing the sample solution into contact with the detection reagent; and a step of applying a shear force to the droplet interface by flowing the liquid along the droplet interface of the sample solution And a specimen detection method characterized by comprising:

  According to the first aspect, a shearing force is applied to the droplet interface by flowing the liquid along the droplet interface of the sample liquid containing the specimen. As a result, the inside of the droplet flows by receiving a shearing force, and the diffusion distance of the specimen to the detection reagent in the droplet can be shortened. Therefore, the contact efficiency between the specimen in the droplet and the detection reagent fixed in the space can be increased, and the detection reaction can be performed with high accuracy and in a short time. The specimen includes a detection target substance originally contained in the sample liquid or a substance that oscillates a detection signal (signal oscillation substance) by further reacting with a detection reagent after reacting with the specimen.

  A second aspect of the present invention is characterized in that in the first aspect, the liquid is a liquid that is incompatible with the sample liquid.

  When the liquid and the sample liquid are compatible with each other, not only the shearing force on the droplet cannot be effectively applied, but also the originally intended detection reaction may be hindered. According to claim 2, such a problem can be suppressed.

  A third aspect is characterized in that, in the first or second aspect, the detection reagent is fixed on the substrate.

  According to the third aspect, in order to improve the detection accuracy, even if the substrate is washed after contacting the sample in the sample liquid and the detection reagent fixed on the substrate, the reacted sample is removed from the substrate. Since it remains without peeling, the detection accuracy can be improved.

  A fourth aspect of the present invention is characterized in that, in any one of the first to third aspects, the detection reagent is a biological substance.

  A fifth aspect of the present invention is characterized in that, in any one of the first to fourth aspects, the biological substance includes one or more of amino acids, peptides, proteins, and nucleic acids.

  According to a sixth aspect of the present invention, there is provided a biochip for detecting an analyte contained in a sample liquid for achieving the object, wherein the biochip is formed on the substrate and holds a droplet containing the analyte. A space, communicating with one end of the space, supplying a liquid to the liquid droplets, communicating with the other end of the space, for discharging the liquid from the space A detection reagent for reacting specifically with the specimen and obtaining information on the structure of the specimen is fixed to a substrate surface on which the droplets in the space are arranged. A biochip is provided.

  In the present invention, a droplet refers to a sample liquid having a curved interface. Further, in order to stably hold the droplets even in the flow field, a method of making the outer periphery of the portion holding the droplets on the substrate difficult to get wet with the sample liquid is used.

  A seventh aspect according to the sixth aspect is characterized in that the supply flow path and the discharge flow path are micro flow paths having an equivalent diameter of 1 mm or less.

  According to the seventh aspect, when a liquid is allowed to flow through a supply channel having an equivalent diameter of 1 mm or less, a laminar flow is easily formed, whereby a shearing force can be effectively applied to the droplet interface. The equivalent diameter is more preferably 0.5 mm or less.

  An eighth aspect is characterized in that, in the sixth or seventh aspect, a height adjusting means for adjusting a height of the space from the substrate surface is provided.

  The liquid flowing along the droplets tends to flow in a region where the wall resistance is small, specifically, a region where the spatial volume between the wall surface and the droplet interface is large. According to the eighth aspect, since the volume of the space formed above the droplet interface can be adjusted, the flow of the liquid with respect to the droplet interface can be controlled.

  A ninth aspect according to any one of the sixth to eighth aspects, wherein the height H (mm) of the space in the cross section in the width direction of the space is a droplet on the width W (mm) of the space and the substrate. It is characterized in that it is larger than the difference from the fixed part length R (mm) for fixing.

  According to the ninth aspect, since the spatial volume formed above the droplet interface is larger than the spatial volume formed in the width direction of the droplet interface, the liquid is flown mainly along the droplet interface. be able to.

  A tenth aspect of the present invention is characterized in that, in any one of the sixth to ninth aspects, the detection reagent is a biological substance.

  An eleventh aspect is characterized in that in any one of the sixth to tenth aspects, the biological substance includes one or more of amino acids, peptides, proteins, and nucleic acids.

  According to the present invention, the contact efficiency between the specimen in the sample solution and the detection reagent can be improved, and the detection reaction can be performed with high accuracy and in a short time.

  Hereinafter, preferred embodiments of a specimen detection method and a biochip according to the present invention will be described in detail with reference to the accompanying drawings.

  FIG. 1 is a conceptual diagram illustrating an example of a configuration of a biochip 10 to which a specimen detection method according to the present invention is applied. In the figure, the biochip 10 is shown attached to the sensor (analyzer) 12. 2 is a cross-sectional view taken along line AA in FIG.

  In the present embodiment, when a droplet containing a specimen is placed on an antibody previously fixed on a substrate to cause an antigen-antibody reaction, a liquid that is inert and insoluble to the droplet (hereinafter simply referred to as “liquid”). Is applied to the droplet interface to reduce the diffusion distance of the specimen in the droplet.

  As shown in FIGS. 1 and 2, the biochip 10 mainly includes a substrate 16 on which a detection reagent 14 that specifically reacts with a specimen is fixed on the surface of a plate-like body, and is closely fixed to the surface of the substrate 16. As a result, the cover plate 20 having the recess 18 for forming the port 18A for storing the droplet on the detection reagent 14 is constituted.

  In the cover plate 20, grooves 22 and 23 communicate with both end sides of the recess 18. The other end of the groove 22 communicates with a supply port 24 that is a columnar cavity formed in the cover plate 20, and the other end of the groove 23 is a discharge port 26 that is a columnar cavity formed in the cover plate 20. Communicating with By closely fixing the substrate 16 to the recesses 18 and the grooves 22 and 23 formed on the surface of the cover plate 20, a port 18A, a channel 22A, and a channel 23A are formed, respectively.

  The upper part of the port 18A is a columnar cavity 30 into which a screw 28 (height adjusting means) is inserted. By adjusting the depth of the screw 28 inserted into the columnar cavity 30, the port 18A The volume of can be changed. As the screw 28, for example, a hexagonal screw or the like can be used.

  FIG. 3 is an enlarged cross-sectional view showing the vicinity of the port 18A. 3A is an enlarged perspective view of the vicinity of the port 18A, and FIG. 3B is a cross-sectional view taken along the line AA in FIG. 3A.

  As shown in FIG. 3, the port 18 </ b> A is formed in a size that allows a droplet to be placed on the detection reagent 14 formed on the surface of the substrate 16. In addition, in order to apply a shearing force to the surface of the droplet, the liquid is moved over the surface of the droplet D, not the space between the droplet D and the side wall surfaces 18C and 18D in the width direction of the port 18A. It is preferable to flow through.

  The liquid tends to flow in a region where the cross-sectional area is large, that is, a region where the wall resistance is small, on a plane (cross section in the width direction) orthogonal to the flow direction. Therefore, in the cross section in the width direction, the height H of the port 18A is larger than the difference (W−R) between the width W of the port 18A and the fixed length R at which the droplet D is fixed on the substrate 16. It is preferable to be formed. For example, when the droplet amount is about 10 μL, the width W can be set to 5 mm, the length L can be set to 5 mm, and the height H can be set to 3 mm. The cross-sectional shape of the port 18A is not limited to a rectangle as shown in FIG. 3B, and various shapes such as a trapezoid, a V-shape, and a semi-circle can be adopted.

  On the substrate 16 corresponding to the position of the port 18A, the detection reagent 14 is fixed. The detection reagent 14 is not limited as long as it can specifically react with the sample and obtain information on the sample, and various antibodies can be used, for example.

  As a method for fixing the detection reagent 14, for example, a method of fixing the detection reagent 14 by ejecting the detection reagent 14 from a nozzle of an ink jet apparatus and spraying it on the substrate 16 can be suitably employed. However, it is not limited to this fixing method.

The volumes of the supply port 24 and the discharge port 26 are not particularly limited as long as the pressure loss when the liquid flows is not excessively high, but is preferably 5 to 5000 mm ³ , for example. By using such a volume, each phenomenon occurring in the micro channel can be easily controlled.

  The planar sizes of the substrate 16 and the cover plate 20 are not particularly limited, but may be a portable size, for example, 40 × 40 mm. The thicknesses of the substrate 16 and the cover plate 20 are not particularly limited, but can be, for example, about 1 mm for the substrate 16 and about 5 mm for the cover plate 20 due to strength, economy, and the like.

The cross-sectional area of the grooves 22 and 23 formed on the surface of the cover plate 20 is not particularly limited. It is preferable to set. Therefore, the cross-sectional area of the grooves 22 and 23, as described above, preferably 1 mm ² or less, more preferably 0.0025~0.64mm ^2, 0.01~0.25mm ² being most preferred. The cross-sectional shape of the grooves 22 and 23 is not particularly limited, and various shapes such as a rectangle (square, rectangle), a trapezoid, a V shape, and a semicircle can be adopted. Further, the length l of the grooves 22 and 23 is not particularly limited, but may be, for example, about 10 mm.

  The material of the substrate 16 and the cover plate 20 is not particularly limited. However, it is preferable that the substrate 16 and the cover plate 20 are transparent because the state of the liquid droplet in the port 18A can be visually recognized. As such materials, various resin plates, more specifically, various resins such as polydimethyl sulfoxide (PDMS), polymethyl methacrylate (PMMA), polyvinyl chloride (PVC), ultraviolet curable resin, polycarbonate (PC), etc. A film, more specifically, polyethylene terephthalate (PET), polyethylene naphthalate (PEN), triacetyl cellulose (TAC), or the like can be employed.

  In addition to the above, the materials for manufacturing the substrate 16 and the cover plate 20 are metal, glass, ceramics, plastic, silicon, etc., depending on the requirements of heat resistance, pressure resistance and solvent resistance, ease of processing, etc. , And Teflon (registered trademark) can be suitably used, and polystyrene resin, PMMA resin, quartz glass, and Pyrex (registered trademark) glass are particularly preferable.

  As a method for manufacturing the cover plate 20 in which the minute ports 18A and the grooves 22, 23 and the like are formed as described above, a fine processing technique is preferably used. Examples of the microfabrication technology include the following.

(1) LIGA technology combining X-ray lithography and electroplating
(2) High aspect ratio photolithography using EPON SU8
(3) Mechanical micro-machining (micro-drilling that rotates a drill with a drill diameter of micro-order at high speed)
(4) High aspect ratio processing of silicon by deep RIE
(5) Hot Emboss processing method
(6) Stereolithography
(7) Laser processing method
(8) Ion Beam Method In the biochip 10 configured as described above, for example, as shown in FIG. 1, the light from the detection sensor 12 is irradiated to each of the droplets arranged on the substrate 16. By so setting, the conjugate produced in each droplet is optically analyzed.

  The liquid used in the present invention is not particularly limited as long as it is an inert and insoluble liquid (including a case where the solubility is low) with respect to the sample liquid containing the specimen, and depending on the type of the sample liquid, For example, water, liquid paraffin, phosphate buffered saline, etc. can be used. In the present invention, “inactive” means not causing a chemical reaction that inhibits the detection reaction. For this reason, the liquid which adsorbs or extracts unnecessary components other than the specimen in the sample liquid is not included in the inactive liquid and can be used in the present invention. The term “insoluble” means that the sample solution is not compatible with the sample solution. For example, the solution is insoluble when it is mixed with the sample solution in a beaker.

  Although it does not specifically limit as a test substance used for this invention, A biological substance, for example, an amino acid, a peptide, protein, a nucleic acid, etc. are contained.

  Next, a specimen detection procedure according to the present invention using the biochip 10 configured as described above will be described with reference to FIGS. FIG. 4 is a diagram for explaining the operation of the present invention, and FIG. 5 is a diagram for explaining a difference in liquid flow around the droplet.

  First, the sample solution is dropped into the port 18A to which the detection reagent 14 is fixed in advance, and is sealed with a screw 28. The appropriate amount of liquid is preferably 50 μL or less, and preferably 10 μL or less. Then, the specimen in the droplet D and the detection reagent 14 on the substrate 16 are brought into contact with each other to be reacted.

  At this time, as shown in FIG. 4A, the liquid (arrow F) is supplied to the droplet 18 from the supply port 24 through the flow path 22A to the port 18A by a pump or the like (not shown). Then, a liquid flow is formed so as to get over the interface of the droplet D. Thereby, a shearing force can be applied to the interface of the droplet D, and the inside of the droplet can be flowed (see FIG. 4B). Then, the liquid after flowing through the port 18A is discharged from the discharge port 26 through the flow path 23A.

  At this time, in the port 18A, as shown in FIG. 5A, the flow (dotted line) between the interface of the droplet D and the side wall surfaces 18C and 18D in the width direction is on the interface of the droplet D. The flow over the road (solid line) is larger.

  On the other hand, as shown in FIG. 5B, the interface of the droplet D and the side wall surfaces 18C and 18D in the width direction at the port 18A rather than the flow over the interface of the droplet D at the port 18A (dotted line) If the flow (solid line) between the two becomes large, the above effect becomes small, which is not preferable.

  Then, after sufficiently reacting the sample with the detection reagent, the supply of the liquid is stopped, and the signal oscillation substance is added to the droplet D as necessary, thereby detecting the sample by an optical method or by visual observation. At this time, a shearing force may be applied to the droplets after the addition of the signal oscillation substance by flowing a liquid again to increase the contact efficiency between the signal oscillation substance and the detection reagent after the reaction.

  As described above, according to the present embodiment, the shearing force is applied to the interface of the droplet D to flow in the droplet, thereby shortening the diffusion distance of the specimen in the droplet. Therefore, the contact efficiency with the detection reagent 14 on the substrate 16 can be improved, and detection can be performed with high accuracy in a short time.

  In addition, since a biochip in which a large number of detection reagents 14 are arranged is used, reproducibility can be examined and different types of analysis can be performed at once.

  Further, in the present embodiment, a liquid that is inactive and insoluble with respect to the sample liquid is used, but by flowing a liquid that adsorbs or extracts unnecessary components other than the specimen in the droplet along the droplet interface, It is also possible to selectively increase the analyte concentration in the droplet. Thus, the liquid flowing along the droplet interface can have various functions as long as the detection reaction is not hindered. Furthermore, a sound wave may be passed instead of the liquid as described above. Thereby, a surface acoustic wave can be propagated in the droplet.

  The preferred embodiments of the specimen detection method and the biochip according to the present invention have been described above, but the present invention is not limited to the above-described embodiments, and various aspects can be adopted.

  For example, in each of the above embodiments, a sample liquid containing a biological substance such as an antigen or an antibody is used as the sample liquid. However, the present invention is not limited to this and can be applied to analysis of other sample liquids.

  In each of the above embodiments, the present invention is applied to the case where the detection reagent 14 is fixed on the substrate in advance and the detection reaction is performed by dropping the sample liquid containing the specimen from the detection reagent 14. However, the present invention is not limited thereto. First, the present invention can be applied to the case where the detection reagent 14 is added after the sample liquid is dropped on the substrate.

  Further, after reacting the specimen in the sample solution with the detection reagent on the substrate 16, the substrate 16 can be once washed with water or the like (at this time, the conjugate of the specimen and the detection reagent is the substrate 16). It remains without peeling from the top). Then, by placing a droplet containing a detection signal oscillation substance on the detection reagent after reaction on the substrate 16 after washing, and applying a shearing force by flowing the liquid again, a signal oscillation substance is obtained. The contact efficiency between the reagent and the detection reagent after the reaction may be increased. Thereby, detection accuracy can be improved.

  In the above-described embodiment, the sample liquid is injected from the cylindrical cavity 30 and sealed by the seal member 28. However, a configuration in which these are not used may be used. For example, in a state where the substrate 16 and the cover plate are disassembled, first, after forming a droplet on the substrate 16, the droplet may be placed in the biochip by covering the cover plate.

  In the above embodiment, the biochip in which the flow path 22A, the port 18A, and the flow path 23A are provided on the same line has been described. However, the present invention is not limited thereto. Any may be used. In the embodiment of FIG. 1, the liquid can be supplied independently for each port 18A. However, the present invention is not limited to this, and the liquid is supplied to a plurality of ports 18A arranged in the same column or row. It is good also as a structure which shares the flow path to perform (for example, the structure which connects the flow path connected to each port 18A mutually, and makes it a common flow path).

  The reaction time required for detection of CRP (C-Reactive Protein) antigen was compared using the biochip 10 of FIG.

  As the substrate 16, a polystyrene substrate having a thickness of 1 mm was used. In addition, an antibody (detection reagent 14) that specifically binds to the CRP antigen is fixed to the bottom surface of the port 18A of the biochip 10, and a phosphate buffered saline solution (PBS, 5 μg / mL) containing CRP antigen (specimen). 1 μL of Phosphate Buffer Saline) was dropped.

  In addition, liquid paraffin was fed from the supply port 24 at a rate of 500 μl / min as a liquid flowing along the liquid droplet interface, and flowed through the liquid droplets (PBS). Then, a fluorescent signal was detected by reacting the labeling antibody.

  On the other hand, as a comparative example, the same procedure as in the above example was performed except that liquid paraffin was not poured and the sample was allowed to stand using a 96-well microplate.

  As a result, the time required for the antigen-antibody reaction was significantly shortened in the examples as compared with the comparative examples.

  From the above, it was found that by applying the present invention, the contact efficiency between the specimen in the sample solution and the detection reagent can be improved, and the detection reaction can be performed with high accuracy and in a short time.

It is a conceptual diagram explaining an example of a structure of the biochip to which the sample detection method according to the present invention is applied. It is the sectional view on the AA line of FIG. FIG. 2 is an enlarged sectional view showing the vicinity of a port 18A in FIG. It is a figure explaining the effect | action in this embodiment. It is a figure explaining the difference in the flow of the liquid around the droplet in this embodiment.

Explanation of symbols

  DESCRIPTION OF SYMBOLS 10 ... Biochip, 12 ... Analysis part (sensor), 14 ... Detection reagent, 16 ... Substrate, 18 ... Recess, 18A ... Port, 20 ... Cover plate, 22, 23 ... Groove, 22A, 23A ... Flow path, 24 ... supply port, 26 ... discharge port, 28 ... screw, 30 ... cylindrical cavity

Claims (11)

In a method for detecting a sample in a sample solution using a detection reagent that reacts specifically with the sample and obtains information on the structure of the sample,
Dropping a sample solution containing the specimen onto a substrate and bringing it into contact with the detection reagent;
Applying a shear force to the droplet interface by flowing a liquid along the droplet interface of the sample liquid;
A specimen detection method comprising:   The specimen detection method according to claim 1, wherein the liquid is a liquid that is incompatible with the sample liquid.   The specimen detection method according to claim 1, wherein the detection reagent is fixed on the substrate.   The specimen detection method according to claim 1, wherein the detection reagent is a biological substance.   5. The specimen detection method according to claim 1, wherein the biological substance includes one or more of amino acids, peptides, proteins, and nucleic acids. A biochip for detecting a specimen contained in a sample solution,
A space formed on the substrate for holding a droplet containing the specimen;
A supply channel for communicating with one end of the space and supplying a liquid to the droplet;
A discharge channel that communicates with the other end of the space and discharges the liquid from the space; and
A biochip characterized in that a detection reagent for reacting specifically with a specimen and obtaining information on the structure of the specimen is fixed on a substrate surface on which droplets in the space are arranged.   The biochip according to claim 6, wherein the supply channel and the discharge channel are microchannels having an equivalent diameter of 1 mm or less.   The biochip according to any one of claims 6 to 7, further comprising height adjusting means for adjusting a height of the space from the substrate surface. In the cross-section in the width direction of the space,
The height H (mm) of the space is larger than a difference between a width W (mm) of the space and a fixed portion length R (mm) for fixing a droplet on the substrate. The biochip according to any one of -8.   The biochip according to any one of claims 6 to 9, wherein the detection reagent is a biological substance.   The biochip according to any one of claims 6 to 10, wherein the biological substance includes one or more of amino acids, peptides, proteins, and nucleic acids.

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