JP5651485B2 - Nucleic acid analyzer - Google Patents

Description

  The present invention relates to a nucleic acid analyzer used for detecting and analyzing biomolecules immobilized on fine particles.

  New techniques for determining the base sequences of DNA and RNA have been developed.

  Traditionally, a method using electrophoresis has been used to determine a base sequence. A cDNA fragment sample synthesized by performing a reverse transcription reaction from a DNA fragment or RNA sample for sequencing in advance is prepared, and a known Sanger method is used. After the dideoxy reaction, electrophoresis was performed, and molecular weight separation development patterns were measured and analyzed.

  On the other hand, recently, a method for fixing a large number of DNA fragments as samples on a substrate and determining the sequence information of many fragments in parallel has been developed, and the base analysis speed has been dramatically improved.

  For example, in Non-Patent Document 1, by using microparticles as a carrier for supporting a DNA fragment and fixing microparticles to which nucleic acids are bound on a glass substrate at random, a plurality of samples are developed in a planar shape, and a two-dimensional image is obtained. Many samples are analyzed in parallel by measuring using a sensor.

  In recent years, in order to further improve the throughput of base decoding and the accuracy of analysis results, a technique for limiting the site to which the carrier is immobilized has been developed.

  For example, in Patent Document 1, a scaffold used for fixing the carrier is regularly placed on the substrate so that the carrier to be analyzed has a configuration suitable for the pixel arrangement of the two-dimensional image sensor.

JP 2008-190937 A

Science 2005, VOL. 309, pp.1728-1732 P.N.A.S. 2006, vol. 103, pp 19635-19640 Nanotechnology, 2007, vol. 18, pp 044017-044021

  The immobilization of the carrier to be analyzed occurs due to the reaction between the functional group on the substrate surface and the functional group on the carrier, and contact between the two is essential for immobilization.

  However, in Patent Document 1, there is a possibility that when fine particles are put on a substrate, a site where the fine particles do not contact may appear.

  The present invention has been made to solve this problem, and an object of the present invention is to provide a nucleic acid analyzer that makes a carrier contact with a substrate with as high a probability as possible.

  The nucleic acid analyzer of the present invention is a nucleic acid analyzer for analyzing a target substance on a substrate, a substrate on which a carrier to which the target substance is bound is fixed, a first channel between the substrate, a reaction And a second flow path formed in the sealing member and continuous with the first flow path, and the structure of the first flow path and the second flow path is The formed angle is 5 degrees to 90 degrees.

  According to the present invention, since the local concentration of the carrier on the substrate surface is improved, an efficient immobilization reaction can be performed. As a result, the immobilization reaction time can be shortened and the amount of sample used can be reduced.

It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the apparatus using the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of a structure of the fixed reaction board | substrate used for the reaction device for nucleic acid analysis of this invention. It is a figure for demonstrating an example of the production method of the fixed reaction board | substrate used for the reaction device for nucleic acid analysis of this invention.

  Embodiments of the present invention will be described with reference to the drawings.

[Example 1: Preparation of concentration gradient using inertial force of carrier]
The nucleic acid analysis reaction device of the present invention will be described with reference to FIGS. 1A to 1C. By sticking a channel substrate (sealing member) 102 having a recess on the fixed substrate 101, at least two openings of the inlet 103 and the outlet 104 are provided, and the solution is supplied to the fixed substrate 101. A flow path system is formed. A solution containing a carrier (also referred to as microparticles or beads) 105 on which a nucleic acid is immobilized is fed by a pump 106 from an inflow port.

  Here, the fine flow path has a flow path (fine flow path) 107 parallel to the plane including the fixed substrate 101 and a flow path 108 intersecting the plane including the fixed substrate 101, and at the intersecting bent portion 109. The carrier 105 is concentrated to the outer periphery of the flow path by its own inertial force. The inside of the flow path parallel to the substrate is in a laminar flow state, and is supplied to the surface of the fixed substrate 101 which is an immobilization reaction field while maintaining the concentration gradient formed by the bending portion 109.

  As a material of the fixed substrate 101, a substrate made of an inorganic material such as a glass substrate, a sapphire substrate, a silicon substrate, a substrate made of a metal such as stainless steel, an organic material such as a polymethyl methacrylate resin, a polycarbonate resin, or a cycloolefin resin. A substrate can be used.

  The material of the flow path substrate 102 is not particularly limited, but a combination of a material that can be processed to 1 mm or less and a processing method is desired. For example, injection molding and nanoimprint processing on thermoplastic resins such as polymethyl methacrylate resin and cycloolefin resin, optical imprint using UV curable resin, processing combining photolithography and etching on silicon, quartz, glass, etc. There is a processing method in which a thermosetting resin such as PDMS is poured into a mold and cured.

  When the fixed substrate 101 and the flow path substrate 102 are bonded to each other, and between glass and PDMS and PDMS, the two can be firmly bonded by bonding after performing oxygen plasma treatment or excimer UV irradiation. it can. Moreover, when one side is a thermoplastic resin board | substrate, it can weld by heating to a glass transition temperature.

The flow path 108 that intersects the plane including the fixed substrate 101 formed inside the flow path substrate 102 is sufficiently fine and long so that the internal flow becomes a laminar flow. Specifically, the channel cross-sectional area of 4 mm ² or less and the length of 1 mm or more are desirable, but the present invention is not limited to this as long as the Reynolds number of the internal fluid is 2300 or less and the environment is dominated by laminar flow.

  The length of the fine channel 107 parallel to the fixed substrate 101 is preferably short. The embodiment of the present invention is preferably 100 mm or less, and more preferably 10 mm or less. Further, the fine flow path 107 parallel to the substrate is not essential, and a structure in which the fixed reaction unit 110 and the vertical flow path 108 are directly connected may be used. By shortening the length of the fine channel 107 parallel to the substrate, the time for the solution to reach the fixed substrate 101 from the bent portion can be shortened, and the elimination by the diffusion of the carrier concentration gradient can be prevented.

  The intersecting angle between the plane including the fixed substrate 101 and the flow path 108 intersecting with the plane may be 1 degree or more, more preferably 5 degrees to 175 degrees, and still more preferably 90 degrees. By applying a sudden change in direction at the bent portion, the difference between the direction of inertial force applied to the carrier and the direction of fluid flow increases, and a larger carrier concentration gradient can be generated.

  It is desirable that the flow path 108 intersecting the plane including the fixed substrate 101 exists on both the inflow side and the outflow side of the reaction part, but it may be one.

  A pump for feeding the carrier solution is not particularly specified, but a pump having a mechanism capable of feeding back and forth is preferable. By performing the reciprocating liquid supply, the solution having a higher carrier concentration can be supplied to the surface of the fixed substrate 101 at the bent portion 109 (discharge port side) even during the return path. As a result, the utilization efficiency of the carrier 105 can be improved. Even when the reaction takes time and the concentration gradient of the carrier is eliminated by diffusion, the carrier concentration in the vicinity of the fixed substrate 101 can always be kept high by continuously performing the reciprocating solution.

  In the present invention, the binding between the carrier 105 and the fixed substrate 101 is not limited, but examples include affinity of biotin and avidin, electrostatic interaction, hydrophobic interaction, covalent bond, and physical capture. it can. Each method is an establishment-dependent phenomenon that occurs when the carrier and the substrate exist at a distance that can affect the carrier. By improving the carrier concentration in the effective range, the carrier fixing rate and the carrier per unit area of the substrate Both the fixing amount can be improved.

[Example 2: Formation of concentration gradient by electric field]
When the carrier 201 has a zeta potential, the electrode pair 203 is installed at a position orthogonal to the flow of the fluid in the microchannel 202 instead of the microchannel bend 109 of the elements constituting the first embodiment, and the voltage is applied. By applying this, a concentration gradient of the carrier can be created. This embodiment will be described with reference to FIGS. 2A and 2B.

  For example, when the carrier is a fine particle and has a negative zeta potential, by applying a positive charge to the electrode pair 203 on the fixed substrate side and a negative charge to the distal electrode, the carrier on the positive electrode side (that is, the substrate surface side) The concentration can be increased. The carrier solution having a concentration gradient formed by this electric field is supplied to the immobilization reaction substrate 204 in the same manner as in Example 1 so that an efficient immobilization reaction can be performed.

  The position of the electrode pair 203 does not have to be in the flow path, and may be installed along the flow path so that an electric field orthogonal to the fluid flow can be formed. By disposing the electrode outside the fine channel 202, it is possible to create a carrier concentration gradient while preventing electrolysis of the carrier dispersion.

  The voltage applied to the electrode may be a constant voltage, or may change with time. When the concentration of the carrier 201 contained in the carrier solution is dilute, the carrier concentration can be increased by applying a strong electric field to attach the carrier around the electrode and separating it from the solution. This process can be used for the immobilization reaction even if only a dilute carrier solution is obtained.

  In addition to the intensity of the electric field, the positive and negative directions of the charge may be changed. In this case, the carrier 201 may have either a positive or negative zeta potential, and can cope with a wide variety of carriers and solution environments (pH, salt concentration, etc.) by switching the voltage applied to the electrode pair 203. Can do.

  As shown in FIG. 2B, when the carrier solution passing through the fine channel 202 includes the carrier 205 bound to the nucleic acid and the carrier 206 not bound to the nucleic acid, a positive charge is applied to the fixed reaction substrate 204 side. Thus, since the nucleic acid has a negative charge, the carrier 205 bound to the nucleic acid can be preferentially concentrated. That is, the nucleic acid fixation concentration per unit area of the fixation reaction substrate 204 can be improved.

[Example 3: Creation of concentration gradient by magnetic field]
When the magnetic carrier 301 is used, a magnetic force source 302 (permanent magnet or electromagnet) can be installed instead of the electrode pair 203 of the elements constituting the second embodiment, and a concentration gradient of the carrier can be created by a magnetic field. This embodiment will be described with reference to FIG. By providing this magnetic force source 302 on the fixed reaction substrate 304 side of the fine channel 303, the carrier concentration on the surface side of the fixed reaction substrate 304 can be increased. The carrier solution having a concentration gradient formed by this magnetic field is supplied to the fixed reaction substrate 304 in the same manner as in Example 1, and an efficient immobilization reaction can be performed.

  The strength of the magnetic field in the fine channel 303 may be constant, but it is desirable to have a mechanism that can be arbitrarily adjusted. That is, if a permanent magnet is used, the distance from the flow path can be made variable, or a magnetic shield can be inserted between the fine flow path 303 and the magnetic force source 302. When the magnetic source is an electromagnet, the intensity of the magnetic field can be controlled by controlling the amount of current in addition to the case of the permanent magnet described above. As a result, an appropriate magnetic force can be set according to the density of the magnetic carrier 301.

  The position of the magnetic source 302 may be directly below the fixed reaction substrate 304, but it is more preferable to install it on the fine flow path 303. In the case where the magnetic source 302 is provided directly below the fixed reaction substrate 304, it is necessary to generate a uniform magnetic field over a wide area in order to avoid uneven spots on the carrier fixation density. Thus, the area requiring a uniform magnetic field can be reduced, leading to simplification of the magnetic source and cost reduction.

Example 4
In the present embodiment, an example of a preferable configuration of a nucleic acid analyzer using a nucleic acid analysis device will be described with reference to FIG.

  The nucleic acid analyzer of the present embodiment includes means for supplying a nucleotide having a fluorescent dye, a nucleic acid synthase, and a nucleic acid sample to the nucleic acid analysis device, means for irradiating the nucleic acid analysis device with light, and on the nucleic acid analysis device. And a luminescence detection means for measuring the fluorescence of the fluorescent dye incorporated into the nucleic acid chain by the nucleic acid extension reaction caused by the coexistence of the nucleotide, the nucleic acid synthase, and the nucleic acid sample. More specifically, the device 405 including the flow path substrate 401, the immobilization substrate 402, the injection port 403 that is a solution exchange port, and the discharge port 404 is installed. Note that PDMS (Polydimethylsiloxane) is used for the flow path substrate 401. Laser light 409 and 410 oscillated from a YAG laser light source (wavelength 532 nm, output 20 mW) 407 and a YAG laser light source (wavelength 355 nm, output 20 mW) 408, only the laser light 409 is circularly polarized by a λ / 4 plate 411, and a dichroic mirror. After adjusting the two laser beams to be coaxial by 412 (reflecting 410 nm or less), the light is condensed by a lens 413 and then irradiated to the device 405 through a prism 414 at a critical angle or more.

  Hereinafter, the case where gold fine particles having a diameter of about 50 nm are used as a carrier will be described as an example. In this case, localized surface plasmons are generated in the gold fine particles existing on the surface of the immobilized substrate 402 by laser irradiation, and the target substance phosphor captured by the DNA probe bound to the gold fine particles exists in the fluorescence enhancement field. Will do. The phosphor is excited by laser light, and part of the enhanced fluorescence is emitted through a detection window 406 provided in a part of the flow path substrate 401. Further, the fluorescence emitted from the detection window 406 is converted into a parallel light beam by the objective lens 415 (× 60, NA 1.35, working distance 0.15 mm), and the background light and the excitation light are blocked by the optical filter 416 to form an image. An image is formed on the two-dimensional CCD camera 418 by the lens 417.

  In the case of the sequential reaction method, as a nucleotide with a fluorescent dye, 3′-position at the position of 3′OH of ribose as disclosed in PNAS 2006, vol. 103, pp 19635-19640 (Non-patent Document 2). An O-allyl group can be used as a protecting group, and the O-allyl group linked to a fluorescent dye via the allyl group at the 5-position of pyrimidine or at the 7-position of purine can be used. Since the allyl group is cleaved by light irradiation (for example, a wavelength of 355 nm) or contact with palladium, the quenching of the dye and the control of the extension reaction can be simultaneously achieved. Even in the sequential reaction, it is not necessary to remove unreacted nucleotides by washing. Furthermore, since no washing step is required, the extension reaction can be measured in real time. In this case, it is not necessary to add a 3′-O-allyl group as a protecting group at the 3′OH position of ribose in the nucleotide, and the dye is formed via a functional group that can be cleaved by light irradiation (for example, wavelength 355 nm). Nucleotides linked to can be used.

  The example of the nucleic acid analyzer described above can also be applied when semiconductor fine particles are used as the carrier. For example, when Qdot (R) 565 conjugate (manufactured by Invitrogen) is used as the semiconductor fine particles, it can be sufficiently excited with a YAG laser light source (wavelength 532 nm, output 20 mW) 407. This excitation energy emits fluorescence by moving to Alexa 633 (manufactured by Invitrogen), which is not excited by light of 532 nm. In other words, dyes associated with unreacted nucleotides are not excited, and are only captured by DNA probes and close to the semiconductor fine particles to emit light, so that the captured nucleotides can be identified by fluorescence measurement. Is possible.

  As described above, by assembling a nucleic acid analyzer using the nucleic acid analysis device of this example, it is possible to increase the immobilization efficiency of the carrier bound to the nucleic acid, shorten the immobilization time, and reduce the amount of sample used. .

Example 5
An example of a preferable configuration of the fixed substrate used in the present invention will be described with reference to FIG.

  Adhesive pads 502 are regularly formed on the smooth support substrate 501, for example, in a lattice shape as shown in FIG. 5. The adhesive pad 502 and the fine particles 503 are bonded by chemical bonds via linear molecules 505. It is preferable that the functional group 506 at the end of the linear molecule 505 and the bonding pad 502 are bonded by chemical interaction. At that time, it is preferable that the functional group 506 has a weak interaction with the smooth support substrate 501 and a strong interaction with the adhesive pad 502. From such a viewpoint, quartz glass, sapphire, a silicon substrate, or the like can be used as the smooth substrate. The adhesive pad 502 can be made of a material selected from gold, titanium, nickel, and aluminum. The functional group 506 must be selected in consideration of the combination of the smooth support substrate 501 and the adhesive pad 502. For example, a sulfohydryl group, an amino group, a carboxyl group, a phosphate group, an aldehyde group, or the like can be used. The linear molecule 505 plays a role of connecting the fine particles 503 and the adhesive pad 502, and the length thereof is not greatly limited. However, in the case of a low molecule, a linear molecule having about 3 to 20 carbon atoms is preferable. The functional group 507 at the end of the linear molecule 505 provides adhesion with the fine particles 503. In the case where a polymer is used as the linear molecule 505, a polymer having a plurality of side chains and a side chain having a functional group 506 and a side chain having a functional group 507 can be used. As the fine particles 503, metal fine particles or semiconductor fine particles can be used. For example, gold fine particles having a diameter of 5 nm to 100 nm are commercially available and can be utilized. As the semiconductor fine particles, compound semiconductors such as CdSe having a diameter of about 10 nm to 20 nm are commercially available and can be utilized. The functional group that can be used as the functional group 507 varies depending on the type of fine particles, but for example, when gold fine particles are used, a sulfohydryl group, an amino group, and the like are preferable. When semiconductor fine particles are used, fine particles whose surface is modified with streptavidin are commercially available, and biotin can be used as the functional group 507. The nucleic acid molecule 504 can be a DNA or RNA nucleic acid molecule. The end of the nucleic acid molecule is modified in advance in the same manner as the functional group 507 and reacted with the fine particles 503. Alternatively, a nucleic acid having a region complementary to the nucleic acid molecule to be analyzed may be bound to the microparticle 503 via the functional group 507, and the nucleic acid molecule 504 may be bound to the microparticle 503 by hybridization. By using oligo dT as a nucleic acid having this complementary region, a wide range of mRNA can be captured. The nucleic acid molecule 504 immobilized on one fine particle 503 is preferably a single molecule. In order to fix one molecule of the nucleic acid molecule 504 to one particle 503, it is preferable that the particle size of the particle 503 is smaller. When one nucleic acid molecule is immobilized on the surface of the fine particle, the state of charge on the fine particle changes, and an effect of inhibiting the fixation reaction of other unimmobilized nucleic acid molecules occurs. This is because this effect increases as the particle size decreases. As a result of extensive studies by the inventors, it has been found that the fine particle size is preferably about 20 nm or less. In addition, the binding reaction between the fine particles 503 and the nucleic acid molecules 504 is performed in a liquid phase, and the concentration of the nucleic acid molecules 504 is reduced to about 1/10 or less with respect to the fine particles 503 so that the diameter of the fine particles 503 is 1 μm. Even in such a case, the nucleic acid molecule 504 immobilized on one fine particle 503 can be made into one molecule. On the other hand, as the size of the fine particles 503 increases, the fixation density of the nucleic acid molecules 504 decreases. In the case of identifying bright spots by simple fluorescence detection, it is preferable that the bright spots are separated by about 1 μm in consideration of the diffraction limit. Therefore, it is suitable that the size of the fine particles 503 is 1 μm or less.

  As a method for forming the adhesive pad 502 on the smooth support substrate 501, a thin film process already in practical use for semiconductors can be used. For example, after forming a thin film by vapor deposition / sputtering through a mask or vapor deposition / sputtering, it can be produced by dry or wet etching. Regular arrangement can be easily realized by using a thin film process. The interval between the pads can be set arbitrarily, but when optical measurement is performed as the detection means, it is preferably 500 nm or more in consideration of the diffraction limit of light detection.

  After the bonding pad 502 is formed on the smooth support substrate 501, linear molecules 505 that connect the fine particles 503 and the bonding pad 502 are supplied, and the linear molecules 505 are fixed on the bonding pad 502. At this time, for the purpose of preventing non-specific adsorption on the smooth support substrate 501, before supplying the linear molecules 505, a material having a strong adhesive force with the smooth support substrate 501 is reacted on the smooth support substrate 501. The method is effective. For example, a silane coupling agent can be used. Next, the fixed substrate and the flow path substrate are attached to each other, the fine particles 503 having the nucleic acid molecules 504 fixed on the surface thereof are supplied onto the substrate, and the fine particles 503 are fixed on the adhesive pad 502, and can be used for analysis.

  When the fine particles 503 are fixed on the adhesive pad 502, there is a possibility that a plurality of fine particles 503 are fixed to one adhesive pad 502. If a plurality of nucleic acids are fixed, information on different types of nucleic acid fragments will overlap, and accurate nucleic acid analysis will not be possible. Therefore, one fine particle 503 must be fixed to one adhesive pad 502. Therefore, the inventors have repeatedly conducted fixing experiments under various conditions, and as a result of intensive studies, if the condition that the diameter d of the adhesive pad 502 is smaller than the diameter D of the fine particles 503 is established, one adhesive pad 502 is obtained. It was found that one fine particle 503 can be fixed to each other. If fine particles 503 having a size equal to or larger than that of the bonding pad 502 are fixed, unreacted linear molecules are covered with the fixed fine particles and cannot react with other fine particles. Is done. In order to immobilize one molecule of the nucleic acid molecule 504 on one fine particle 503, the diameter D of the fine particle 503 is preferably 20 nm or less, and therefore, the diameter d of the adhesive pad 502 is preferably 20 nm or less. Furthermore, as a result of intensive studies, when the fine particle 503 has a charge on its surface, an electrostatic repulsive force acts between the fine particles, so that the diameter d of the adhesive pad 502 is larger than the diameter D of the fine particle 503. It was found that even when the size was large, the number of fixed fine particles per adhesive pad was one. Therefore, when the surface charge of the fine particles 503 is small and the electrostatic repulsion force is weak, the diameter d of the adhesive pad 502 is preferably smaller than the diameter D of the fine particles 503, and the surface charge of the fine particles 503 is large and the electrostatic repulsion force. It is clear that the diameter d of the adhesive pad 502 does not necessarily have to be smaller than the diameter D of the fine particles 503 when the resistance is strong.

  Several methods are conceivable for detecting information about a nucleic acid sample using the nucleic acid analysis device of this example, but a method using a fluorescence detection method is preferred from the viewpoint of sensitivity and simplicity. First, fine particles supplemented with nucleic acid molecules 504 as a sample are supplied to a nucleic acid analysis reaction device prepared by bonding a flow path substrate and a fixed substrate, and the fine particles are immobilized on the surface of the fixed substrate. Next, a nucleotide having a fluorescent dye and a nucleic acid synthase are supplied. A nucleic acid elongation reaction is caused on the device, and the fluorescence of the fluorescent dye incorporated into the nucleic acid chain during the elongation reaction is measured. In this case, a so-called sequential extension reaction method in which one type of nucleotide is supplied, unreacted nucleotides are washed, fluorescence observation, different types of nucleotides are supplied, and the subsequent steps are repeated can be easily realized. By quenching the fluorescent dye after the fluorescence observation or using a nucleotide having the fluorescent dye attached to the phosphate site, a continuous reaction can be caused to obtain the base sequence information of the nucleic acid sample. On the other hand, by supplying four types of nucleotides having different fluorescent dyes, a continuous nucleic acid extension reaction is performed without washing, and a so-called real-time reaction method is realized by continuously performing fluorescence observation. You can also. In this case, if a nucleotide with a fluorescent dye attached to the phosphate moiety is used, the phosphate moiety is cleaved after the extension reaction, so that the fluorescence can be measured continuously without quenching to obtain the base sequence information of the nucleic acid sample. it can.

  By using fine particles having a diameter of about 100 nm or less, such as gold, silver, platinum, and aluminum, that can excite localized plasmons in the visible region, fluorescence can be enhanced and observed. For example, Nanotechnology, 2007, vol. 18, pp 044017-044021 (Non-Patent Document 3) reports the phenomenon of fluorescence enhancement by surface plasmons of gold fine particles. The fluorescence of the fluorescent dye attached to the nucleotide can be enhanced and measured, and the signal / noise can be increased.

  When semiconductor fine particles are used as the fine particles, the semiconductor fine particles are excited with light from an external light source, and the excitation energy is transferred to the fluorescent dyes associated with the incorporated nucleotides. Can also be observed. In this case, the excitation light source may excite only the semiconductor fine particles, which is preferable in that one kind of light source may be used.

  When fine particles made of a polymer material are used as the fine particles, the particle sizes of the fine particles can be made uniform, and the size of the particle size can be selected widely from several tens of nm to several μm. In addition, it is preferable in that the amount of functional groups introduced for the fixation reaction of the nucleic acid molecule 504 immobilized on the surface of the fine particles can be made uniform by surface-modifying the functional group of the polymer material on the scaffold. In particular, when only one molecule of the nucleic acid molecule 504 is immobilized on the surface of the fine particle, the reproducibility of the immobilization rate is very high, which is preferable.

  As in this configuration example, by restricting the adhesion part of the carrier on the substrate, the area on the substrate to which the carrier can be coupled is reduced, and the coupling efficiency of the carrier is reduced. By combining with a flow path substrate that causes a gradient in the carrier concentration in the flow path, the concentration of the carrier on the surface of the substrate can be increased and a decrease in the efficiency of the immobilization reaction can be suppressed.

Example 6
An example of a method for manufacturing the fixed substrate shown in Example 5 will be described with reference to FIG. An electron beam positive resist 602 is applied on a smooth support base 601 by a spin coating method. As the smooth support substrate, a glass substrate, a sapphire substrate, a silicon wafer or the like is used. When the device is used, when it is necessary to irradiate excitation light from the back surface opposite to the surface on which the fine particles are arranged, a quartz substrate or a sapphire substrate having excellent light transmittance may be used. Examples of the positive resist for electron beam include polymethyl methacrylate and ZEP-520A (manufactured by Nippon Zeon Co., Ltd.). After aligning using the position of the marker on the substrate, electron beam direct drawing exposure is performed to form a through hole in the resist. For example, a through hole having a diameter of 15 nm is formed. Through-holes depend on the number of nucleic acid molecules that can be analyzed by parallel processing, but forming at a pitch of about 1 μm takes into account the simplicity of manufacturing, high yield, and the number of nucleic acid molecules that can be analyzed by parallel processing. Then it is suitable. The through hole formation region also depends on the number of nucleic acid molecules that can be analyzed by parallel processing, but greatly depends on the position accuracy and position resolution on the detection device side. For example, when reaction sites (fine particles) are formed with a pitch of 1 μm, 1 million reaction sites can be formed if the through-hole formation region is 1 mm × 1 mm. After forming the through hole, a material constituting the bonding pad 603, for example, gold, titanium, nickel, aluminum, is formed by sputtering. When a glass substrate or sapphire substrate is used as the smooth support base and gold, aluminum, or nickel is used as the bonding pad material, titanium or chrome is used to reinforce the bonding between the substrate material and the bonding pad material. It is preferable to put a thin film. Next, the linear molecule 604 is reacted with the bonding pad 603. When the bonding pad 603 is gold, titanium, aluminum, or nickel, it is preferable to use a sulfohydryl group, a phosphate group, a phosphate group, or a thiazole group as the functional group 605 at the end of the linear molecule, respectively. For example, biotin can be used for the functional group 606 on the opposite side of the linear molecule. After reacting the linear molecules with the bonding pad, the resist is peeled off. After removing the resist, non-specific adsorption preventing treatment is applied to the surface of the smooth substrate other than that on which the bonding pads are formed. In order to prevent adsorption to nucleotides with fluorescent dyes, coating with nonspecific adsorption preventing molecules 607 having a negatively charged functional group is performed. For example, epoxy silane is applied to the surface by spin coating, and after heat treatment, it is treated with a weakly acidic solution (pH 5 to pH 6) to open the epoxy group and introduce OH group to the surface. Adsorption prevention effect can be brought about.

DESCRIPTION OF SYMBOLS 101 Fixed substrate 102,401 Channel substrate 103 Inlet 104 Outlet 105,201 Carrier 106 Pump 107,202,303 Fine channel 108,111 Channel 109 Bending part 110 Fixed reaction part 203 Electrode pair 204,304 Fixed reaction board 205 Carrier 206 conjugated with nucleic acid 206 Carrier 301 not bonded with nucleic acid 301 Magnetic carrier 302 Magnetic source 305 Magnetic shield 402 Immobilized substrate 403 Inlet 404 Outlet 405 Device 406 Detection window 407, 408 YAG laser light source 409, 410 Laser light 411 λ / 4 plate 412 Dichroic mirror 413 Lens 414 Prism 415 Objective lens 416 Optical filter 417 Imaging lens 418 Two-dimensional CCD camera 501 Smooth support substrate 502 Adhesive pad 503 Fine particle 504 Nucleic acid molecule 505, 604 Linear molecule 5 6,507,605,606 functional group 601 smooth supporting substrate 602 electron beam positive resist 603 bond pad 607 nonspecific adsorption inhibitory molecule

Claims (2)

In the nucleic acid analyzer that analyzes the target substance on the substrate,
A substrate on which a carrier bonded with a target substance is fixed, and a sealing member that forms a flow path and a reaction region between the substrate and the substrate;
Means for fixing the carrier are regularly arranged in the reaction region on the substrate,
Having a concentration control means for increasing the concentration of the carrier in the flow path as it approaches the surface of the substrate;
The concentration control means is an electric field generating mechanism that applies an electric field along the flow path,
The electric field generating mechanism is formed by arranging an electrode pair at a position orthogonal to the fluid flow in the flow path,
The electric field generated by the electric field generation mechanism is variable depending on the concentration of the carrier, and the polarity of the electrode pair is also variable.
The flow path has an inflow side fine flow path connected to the reaction area inlet side and an outflow side fine flow path connected to the reaction area outlet side,
The nucleic acid analyzer according to claim 1, wherein the carrier concentration deviation occurs in the inflow side microchannel and the outflow side microchannel. The nucleic acid analyzer according to claim 1,
A nucleic acid analysis device in which a nucleic acid to be detected is fixed to the solid phase substrate, a means for supplying a nucleotide having a fluorescent dye and a nucleic acid sample to the nucleic acid analysis device, and light to the nucleic acid analysis device. Luminescence detection that measures fluorescence of a fluorescent dye incorporated into a nucleic acid chain by a nucleic acid extension reaction caused by the presence of the nucleotide, the nucleic acid synthase, and the nucleic acid sample on the nucleic acid analysis device A nucleic acid analyzer for obtaining base sequence information of the nucleic acid sample.

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