JP2009198259A - Chemical substance recovering kit, biomolecule inspection system, and biomolecule inspection method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a chemical substance recovering kit capable of efficiently recovering a chemical substance. <P>SOLUTION: The chemical substance recovering kit includes a substrate 10 provided with a flow channel 111 of a solution, the solution filter 30 arranged to the outlet 22 of the flow channel 111 of the substrate 10 and an absorbing layer 40 for absorbing the solution permeated through the solution filter 30. A probe biomolecule 15 is fixed to the inside of the flow channel and a plurality of beads 5a, 5b and 5c are interrupted in the flow channel, so that the probe biomolecule is fixed to the respective surfaces of a plurality of the beads. The solution filter includes a membrane filter containing a polycarbonate. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

  The present invention relates to a chemical substance inspection technique, and more particularly to a chemical substance recovery kit, a biomolecule inspection system, and a biomolecule inspection method.

Various genetically modified crops have been developed, and the development of testing methods for detecting the presence of recombinant genes is required. There is also a demand for the development of a method for detecting allergen-causing proteins contained in foods, or the development of a method for detecting specific proteins in the medical field (see, for example, Patent Document 1). Usually, the biomolecule contained in the solution to be examined is very small or at a low concentration. Therefore, development of a technique that can efficiently recover chemical substances such as biomolecules is desired.
Japanese Patent Laid-Open No. 2004-250385

  An object of the present invention is to provide a chemical substance recovery kit, a biomolecule inspection system, and a biomolecule inspection method that can efficiently recover a chemical substance.

  In order to achieve the above object, the first feature of the present invention is that (a) a substrate provided with a solution flow path, (b) a solution filter disposed at the outlet of the substrate flow path, and (c) The gist of the invention is a chemical substance recovery kit including an absorption layer that absorbs a solution that has passed through a solution filter.

  The second feature of the present invention is (a) a column provided with an injection hole and a discharge hole, (b) a solution filter that is disposed in the injection hole and allows the solution to pass therethrough, and (c) is disposed in the column. The gist of the present invention is a chemical substance recovery kit including an absorption layer that absorbs a solution and (d) a gas filter that is disposed in the discharge hole and transmits gas inside the absorption layer.

  The third feature of the present invention is that (a) a substrate provided with a solution flow path, (b) a solution filter disposed at the outlet of the substrate flow path, and (c) a solution that passes through the solution filter. The gist of the present invention is a biomolecular test system comprising an absorption layer that absorbs selenium and (d) a detection mechanism that detects a labeling reagent contained in the solution absorbed in the absorption layer.

  The fourth feature of the present invention is that (a) preparing a substrate provided with a flow path, (b) fixing a probe biomolecule in the flow path of the substrate, and (c) Flowing a solution containing the target biomolecule and capturing the target biomolecule with the probe biomolecule; (d) modifying the captured target biomolecule with the enzyme; and (e) a flow path provided on the substrate. Placing a solution filter at the outlet; (f) flowing a solution containing the enzyme substrate into the flow path to react the enzyme and the substrate; and (g) dissolving the solution containing the enzyme and substrate reaction product. The gist of the present invention is a biomolecule testing method including a step of introducing a solution containing a reaction product into an absorbing layer through a filter.

  According to the present invention, it is possible to provide a chemical substance recovery kit, a biomolecule inspection system, and a biomolecule inspection method that can efficiently recover a chemical substance.

  Next, embodiments of the present invention will be described with reference to the drawings. In the following description of the drawings, the same or similar parts are denoted by the same or similar reference numerals. The embodiments shown below exemplify apparatuses and methods for embodying the technical idea of the present invention, and the technical idea of the present invention specifies the arrangement of components and the like as follows. Not what you want. The technical idea of the present invention can be variously modified within the scope of the claims.

  The chemical substance recovery kit according to the embodiment includes a substrate 10 provided with a solution flow path 111 and an outlet of the flow path 111 of the substrate 10, as shown in FIG. 1 and FIG. And a solution filter 30 disposed in 22 and an absorption layer 40 that absorbs the solution that has passed through the solution filter 30.

  The substrate 10 is made of, for example, a cyclo-olefin polymer (COP) resin, a polydimethylsiloxane (PDMS) resin, a resin such as acrylic resin or polycarbonate, a transparent material such as glass, or a low protein adsorption material. The channel 111 is provided, for example, in a tunnel shape inside the substrate 10 and extends in parallel with the front surface and the back surface of the substrate 10. For example, the channel 111 has a width of 2 mm and a depth of 200 μm. The inlet 21 of the channel 111 is provided on the front surface of the substrate 10, and the outlet 22 of the channel 111 is provided on the back surface of the substrate 10. The substrate 10 is manufactured by a resin molding technique using a mold. The substrate 10 may be a combination of a plurality of members.

  The solution filter 30 is preferably in close contact with the back surface of the substrate 10. It is detachably disposed on the back surface of the substrate 10 so as to cover the outlet 22 of the flow path 111. The diameter of the solution filter 30 is longer than the diameter of the outlet 22 of the flow path 111, for example, 5.6 mm. As the solution filter 30, for example, a polycarbonate type membrane filter (manufactured by Advantech Toyo Co., Ltd., catalog number 16180001) that is light transmissive and has low autofluorescence can be used.

  A column 50 that holds the absorption layer 40 is disposed below the solution filter 30. The column 50 is made of a material such as a cyclic polyolefin (COP) resin, a resin such as an acrylic resin or polycarbonate, or glass. The material of the column 50 may be a transparent material or an opaque material. The absorption layer 40 is light transmissive, for example, and is made of a polymer water-absorbing resin (manufactured by Kenyu Corporation, portable mini toilet / purple, product number 3AP-100). The absorption layer 40 is filled in, for example, a space having a diameter of 4 mm and a thickness of 3 mm inside the column 50, and the density is 40 mg / ml.

  The column 50 is provided with a gas filter 60 that allows the gas inside the absorption layer 40 to pass therethrough. The diameter of the gas filter 60 is, for example, 3.97 mm, and may be light transmissive. As the gas filter 60, a porous sheet made of ultra high molecular weight polyethylene having a pore diameter of 30 μm, a polycarbonate type membrane filter, or the like can be used. A washer or the like may be disposed as a weight between the absorption layer 40 and the gas filter 60. As a material of the gas filter 60, ashless pulp (manufactured by Advantech Toyo Co., Ltd., catalog number 48079000) can also be used. When using a reflector, it is desirable that the gas filter 60 be a light transmissive material.

  As shown in FIG. 3, a damming portion 112 is provided in the flow path 111 of the substrate 10. Further, a plurality of beads 5a, 5b, 5c,... For example, the gap above the damming portion 112 is a gap provided in the flow path 111 so as to be narrower than the diameter of each of the plurality of beads 5a, 5b, 5c. As shown in FIG. 4, probe biomolecules 15 for capturing target biomolecules are fixed to the surfaces of the plurality of beads 5a, 5b, 5c. As the probe biomolecule 15, nucleic acids such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and peptide nucleic acid (PNA), or proteins such as antibodies, enzymes, receptors, and ligands can be used.

  As shown in FIGS. 1 and 2, an injection funnel portion 201 is provided on the surface of the substrate 10 so as to surround the inlet 21 of the flow path 111. The injection funnel 201 is made of, for example, a cyclic polyolefin (Cyclo-olefin polymer, COP) resin, polydimethylsiloxane (PDMS) resin, a resin such as acrylic resin or polycarbonate, a transparent material such as glass, or a low protein adsorption material. Consists of. The injection funnel 201 may be formed integrally with the substrate 10. Further, the injection funnel 201 is removable from the surface of the substrate 10 as shown in FIG.

  In addition to the components of the chemical substance recovery kit, the biomolecule testing system according to the embodiment has a suction pump that sucks the gas in the absorption layer 40 through the gas filter 60 shown in FIG. In addition, the biomolecule inspection system further includes a detection mechanism 70 that detects a reagent contained in the solution absorbed in the absorption layer 40. For example, when the reagent is a fluorescent reagent, the detection mechanism 70 is a fluorescent microscope that detects the fluorescence of the fluorescent reagent. In addition, the biomolecule testing system according to the embodiment may further include a temperature adjustment mechanism that controls the temperature of the substrate 10, an ultrasonic generator for stirring the solution in the flow path 111, and the like.

  Next, a biomolecule test method using the biomolecule test system according to the embodiment will be described.

  (a) As shown in FIG. 6, prepare the substrate 10 on which the solution filter 30, the column 50, the absorption layer 40, and the gas filter 60 are not arranged, and connect a suction pump to the outlet 22 of the flow path 111 of the substrate 10. To do. Next, as shown in FIG. 4, a plurality of beads 5a, 5b, 5c... With the probe biomolecule 15 immobilized on the surface are prepared, and the damming portion 112 of the channel 111 is dammed as shown in FIG. stop.

  (b) A test target solution containing the target biomolecule captured by the probe biomolecule 15 is supplied to the injection funnel 201, and the test target solution is drawn from the outlet 22 of the flow path 111 with a suction pressure of 20 kPa using a suction pump. Suction. At this time, the target biomolecule contained in the solution to be examined is captured by the probe biomolecule 15 immobilized on the surface of each of the plurality of beads 5a, 5b, 5c.

  (c) Next, a solution containing an antibody that specifically binds to the target biomolecule and is labeled with biotin is supplied to the injection funnel 201, and contains a biotin-labeled antibody from the outlet 22 of the channel 111 using a suction pump. Aspirate the solution with a vacuum of 20 kPa. At this time, the biotin-labeled antibody contained in the solution binds to the target biomolecule captured by the probe biomolecule 15 immobilized on the surface of each of the plurality of beads 5a, 5b, 5c.

  (d) A solution containing avidin labeled with an enzyme such as horseradish peroxidase (HRP) is supplied to the funnel 201 for injection, and contains an enzyme-labeled avidin from the outlet 22 of the channel 111 using a suction pump. Aspirate the solution with a vacuum of 20 kPa. At this time, the enzyme-labeled avidin contained in the solution binds to the biotin-labeled antibody bound to the target biomolecule captured by the probe biomolecule 15 immobilized on the surface of each of the plurality of beads 5a, 5b, 5c ... To do.

(e) Next, as shown in FIG. 2, the solution filter 30, the column 50, the absorption layer 40, and the gas filter 60 are disposed at the outlet of the flow path 111. Thereafter, a solution containing an enzyme substrate such as 10-acetyl-3,7-dihydroxyphenoxazine and hydrogen peroxide (H ₂ O ₂ ) is supplied to the injection funnel 201. Then, the gas in the flow path 111 and the absorption layer 40 is sucked from the gas filter 60 with a suction pressure of 20 kPa using a suction pump, and the enzyme substrate-containing solution is introduced into the absorption layer 40. At this time, the enzyme-labeled avidin immobilized on the surface of each of the plurality of beads 5a, 5b, 5c ... reacts with the enzyme substrate, for example, 7-hydroxy-3H-phenoxazin-3-one (7-Hydroxy-3H- Enzymatic reaction products such as phenoxazin-3-one, common name: Resorufin are produced. The enzyme reaction product is transported to the absorption layer 40 by the solution and collected in the absorption layer 40.

  (f) 7-hydroxy-3H-phenoxazin-3-one emits fluorescence having a wavelength of 587 nm when irradiated with excitation light having a wavelength of 563 nm. Therefore, when the absorption layer 40 is irradiated with excitation light from the detection mechanism 70 such as a fluorescence microscope, 7-hydroxy-3H-phenoxazin-3-one emits fluorescence. Thereafter, the concentration of the target biomolecule contained in the test target solution is calculated based on the fluorescence intensity of the absorption layer 40, and the biomolecule test method according to the embodiment is terminated. During fluorescence observation, a reflector or the like may be disposed on at least a part of the column 50.

  Previously, there was an attempt to detect 7-hydroxy-3H-phenoxazin-3-one in the flow path by performing ELISA (Enzyme-Linked Immuno Sorbent Assay) in the flow path on the substrate . However, since 7-hydroxy-3H-phenoxazin-3-one produced by the enzyme reaction flows away with the solution in the flow path, it was difficult to detect. On the other hand, according to the biomolecule inspection method according to the embodiment, the solution containing 7-hydroxy-3H-phenoxazin-3-one is absorbed by the absorption layer 40 made of a polymer water-absorbing resin or the like. Therefore, 7-hydroxy-3H-phenoxazin-3-one can be easily recovered.

  When the solution filter 30 is not provided at the outlet 22 of the flow path 111, the solution that has exited from the outlet 22 of the flow path 111 may wrap around the back surface of the substrate 10 and may be difficult to introduce into the absorption layer 40. On the other hand, if the solution filter 30 made of a porous material or the like having many pores smaller than the diameter of the outlet 22 is disposed at the outlet 22, the phenomenon that the solution wraps around the back surface of the substrate 10 does not occur, which is efficient. In addition, the solution can be collected in the absorption layer 40.

  Further, by irradiating the absorption layer 40 with fluorescence, the fluorescence of 7-hydroxy-3H-phenoxazin-3-one can be easily detected. Furthermore, since the absorption layer 40 is solid, it is easy to handle, and no special optical system is required for fluorescence observation. When fluorescence of 7-hydroxy-3H-phenoxazin-3-one is detected, it can be determined that the target biomolecule was present in the solution to be examined.

  In addition, although the amount of the test target solution is sufficient, if the concentration of the target biomolecule contained in the test target solution is low, conventionally, in order to increase the detection sensitivity, the target biomolecule is concentrated before the test target solution is inspected. It was. In contrast, according to the biomolecule inspection method according to the embodiment, the process of increasing the concentration of the target biomolecule in the solution to be inspected is unnecessary if the number of probe biomolecules 15 is sufficient.

For example, when a 30 μl test target solution with a concentration of bacteria as a target biomolecule of 10 ⁵ cells / ml was flowed to the flow path 111, a 300 μl test target solution with a concentration of bacteria of 10 ⁴ cells / ml was flowed. In some cases, the number of bacteria captured by the probe biomolecule 15 is approximate. Therefore, according to the biomolecule testing method according to the embodiment, the target biomolecules are concentrated in the step of capturing the target biomolecules on the plurality of beads 5a, 5b, 5c. . Therefore, it is not necessary to concentrate the target biomolecule before supplying it to the channel 111. Needless to say, the target biomolecules are concentrated on the plurality of beads 5a, 5b, 5c... So that the subsequent enzyme reaction can proceed efficiently and the enzyme reaction product can be efficiently recovered.

(First embodiment)
A suction pump was connected to the outlet 22 of the flow path 111 of the substrate 10 shown in FIG. Next, beads having a diameter of 30 μm were prepared, and a rabbit anti-Listeria antibody was bound to the surface of the beads. Next, the beads were diluted 1/20 with 1% BSA-PBS, and flowed from the injection funnel part 201 to the flow path 111 while sucking at a suction pressure of 20 kPa using a suction pump, and dammed to the damming part 112 . Next, 30 μl of 1% BSA-PBS was introduced into the channel 111. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

A listeria-killed test solution containing 10 ⁷ cells / ml, 10 ⁶ cells / ml, 10 ⁵ cells / ml, or 0 cells / ml diluted with 1-fold concentration of PBS is 20 kPa using a suction pump. 30 μl was introduced from the injection funnel portion 201 into the flow path 111 while suctioning at a suction pressure of. Due to the introduction, dead Listeria are specifically bound to the rabbit anti-Listeria antibody on the bead surface. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

  Next, a biotin-labeled anti-Listeria antibody-containing solution suspended in 1% BSA-PBS and having a concentration of 4 μg / ml is sucked from the injection funnel 201 into the channel 111 while sucking with a suction pump at a pressure of 20 kPa. 30 μl was introduced. By the introduction, the biotin-labeled anti-Listeria antibody binds to the killed Listeria captured by the rabbit anti-Listeria antibody on the bead surface. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

  Funnel section for injection while sucking HRP-labeled streptavidin (PIERCE, catalog number 29994) suspended at 1% BSA-PBS with a suction pressure of 20 kPa using a suction pump 30 μl was introduced from 201 into the channel 111. By introduction, HRP-labeled streptavidin binds to the biotin-labeled anti-Listeria antibody bound to the killed Listeria captured by the rabbit anti-Listeria antibody on the bead surface. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

  Next, as shown in FIG. 2, the solution filter 30, the absorption layer 40, and the gas filter 60 were arranged at the outlet 22 of the flow path 111. A polycarbonate filter was used for each of the solution filter 30 and the gas filter 60. The material of the absorption layer 40 was 40 mg / ml polymer absorbent. Further, 8 μl of an enzyme reaction stop solution (Molecular Probes, Amplex (registered trademark) Red / UltraRed Stop reagent, catalog number A33855) having a concentration of 5.5 mg / ml was added to the absorption layer 40.

  Sodium phosphate (pH 7.4) with a concentration of 50 mMol / l, hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenoxazine (trade name: Amplex (registered trademark) Red ) To prepare an enzyme substrate solution. The concentration of hydrogen peroxide in the enzyme substrate solution is 2 mmol / l, and the concentration of 10-acetyl-3,7-dihydroxyphenoxazine is 100 μmol / l.

  Next, 30 μl of the enzyme substrate solution was introduced from the injection funnel portion 201 into the flow path 111 while sucking the gas from the gas filter 60 with a suction pressure of 20 kPa using a suction pump. Introducing 10-acetyl-3,7-dihydroxyphenoxy in the presence of HRP labeled HRP-labeled streptavidin bound to biotin-labeled anti-Listeria antibody bound to killed Listeria killed by rabbit anti-Listeria antibody on the bead surface Sadin and hydrogen peroxide react to change to 7-hydroxy-3H-phenoxazin-3-one. The solution containing 7-hydroxy-3H-phenoxazin-3-one flows through the flow path 111, passes through the solution filter 30, and is absorbed by the absorption layer 40. The reaction time was about 20 minutes.

  Next, three places in the absorption layer 40 were irradiated with excitation light having a wavelength of 563 nm and observed with a fluorescence microscope (AX70, manufactured by Olympus Corporation). As shown in FIG. 8, when the concentration of the killed Listeria was the highest, the strongest fluorescence was detected, and a decrease in fluorescence intensity was confirmed as the concentration decreased. In the first example, in order to prevent reagent contamination, the reagent-containing solution was dispensed into eight continuous tubes. In addition, four types of Listeria dead bacteria-containing test solutions with different concentrations were tested simultaneously using four biomolecular test systems.

(Second embodiment)
A suction pump was connected to the outlet 22 of the flow path 111 of the substrate 10 shown in FIG. Next, the channel 111 was washed with 60 μl PBST. Thereafter, beads having a diameter of 40 μm were prepared, and a rabbit anti-Listeria antibody was bound to the bead surface. Next, 40 μl of a diluted solution obtained by diluting the beads to 1/20 with 15% glycerol-1% BSA-PBS is flowed from the injection funnel 201 to the channel 111 using a suction pump, and the beads are dammed to the damming portion 112. stopped. Next, 30 μl of 1% BSA-PBS was introduced from the injection funnel 201 into the channel 111. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

Flow through the funnel section 201 for injection containing a Listeria bacterium-containing test solution diluted in Fraser medium at a concentration of 10 ⁶ cells / ml, 10 ⁵ cells / ml, 10 ⁴ cells / ml, or 0 cells / ml. 30 μl was introduced into the channel 111. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST. Four types of Listeria dead bacteria-containing test target solutions with different concentrations are simultaneously tested using four biomolecular test systems.

  Next, 30 μl of a biotin-labeled anti-Listeria antibody-containing solution suspended in 1% BSA-PBS and having a concentration of 4 μg / ml was introduced into the channel 111 from the injection funnel 201. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

  30 μl of HRP-labeled streptavidin suspended in 1% BSA-PBS and having a concentration of 1 μg / ml was introduced from the injection funnel 201 into the channel 111. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST. Thereafter, the inside of the injection funnel 201 was wiped with a clean room swab (ASONE # 1-6918-04, clean foam stick, model number TX752B, head size (mm) 3.4 × 3.4 × 20.0, total length 69 mm). The operations of washing with PBST and wiping with a cotton swab were repeated twice.

  Next, as shown in FIG. 2, the solution filter 30, the absorption layer 40, and the gas filter 60 were arranged at the outlet of the flow path 111. For each of the solution filter 30 and the gas filter 60, a polycarbonate filter (ADVANTEC, catalog number 16180001), which hardly generates fluorescence, was rounded to a diameter of 5.6 mm was used. As the material of the absorption layer 40, a 40 mg / ml polymer water-absorbing resin (Kenyu Corporation, portable mini toilet / purple, product number 3AP-100) was used. Further, 8 μl of enzyme reaction stop solution having a concentration of 5.5 mg / ml was added to the absorption layer 40.

  Hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenoxazine were added to sodium phosphate (pH 7.4) at a concentration of 50 mmol / l to prepare an enzyme substrate solution. The concentration of hydrogen peroxide in the enzyme substrate solution is 2 mmol / l, and the concentration of 10-acetyl-3,7-dihydroxyphenoxazine is 100 μmol / l. Next, 30 μl of the enzyme substrate solution was introduced into the channel 111 while sucking gas from the gas filter 60. A solution containing 7-hydroxy-3H-phenoxazin-3-one produced by the enzyme reaction was collected in the absorption layer 40. The reaction time was about 25 minutes.

  Next, three places in the absorption layer 40 were irradiated with excitation light having a wavelength of 563 nm and observed with a fluorescence microscope. As shown in FIG. 9, the strongest fluorescence was detected when the concentration of Listeria killed was the highest, and a decrease in fluorescence intensity was confirmed as the concentration decreased. The display scale of the fluorescence microscope is 2000-3000, and the exposure time is 100 ms. U-DM-CY-2 was used for the filter of the fluorescence microscope. Next, the fluorescence intensity was digitized from the image obtained with the fluorescence microscope as shown in FIG.

  In the second example, in order to prevent reagent contamination, the reagent-containing solution was dispensed into eight continuous tubes. Further, by wiping the inside of the funnel portion 201 for injection with a cotton swab, the non-specifically adsorbed HRP-labeled antibody decreased, and the background noise decreased.

(Third embodiment)
7-Hydroxy-3H in the same manner as in the second example except that a polyethylene filter (custom product manufactured by Watson, 4 mm diameter filter chip, ultrahigh molecular weight polyethylene porous sheet, pore size 30 mm) was used as the gas filter 60. -Phenoxazin-3-one was recovered in the absorbent layer 40. The reaction time was about 36 minutes.

  Next, three places in the absorption layer 40 were irradiated with excitation light having a wavelength of 563 nm and observed with a fluorescence microscope. As shown in FIG. 11, the strongest fluorescence was detected when the concentration of Listeria killed was the highest, and a decrease in fluorescence intensity was confirmed as the concentration decreased. The display scale of the fluorescence microscope is 2000-3000, and the exposure time is 100 ms. U-DM-CY-2 was used for the filter of the fluorescence microscope. Next, the fluorescence intensity was digitized from the images obtained with the fluorescence microscope as shown in FIGS.

(Fourth embodiment)
In the third example, 30 μl of the Listeria killed bacteria-containing test solution having a concentration of 10 ⁶ cells / ml, 10 ⁵ cells / ml, 10 ⁴ cells / ml, or 0 cells / ml was introduced into the channel 111. In contrast, in the fourth example, 30 μl or 300 μl of the Listeria killed bacteria-containing test target solution having a concentration of 10 ⁵ cells / ml or 0 cells / ml was introduced into the channel 111. Other procedures were performed in the same manner as in the third example, and 7-hydroxy-3H-phenoxazin-3-one was recovered in the absorption layer 40.

  Next, three places in the absorption layer 40 were irradiated with excitation light having a wavelength of 563 nm and observed with a fluorescence microscope. As shown in FIGS. 14 and 15, it was confirmed that even when the concentration was the same, the fluorescence intensity became stronger as the amount of the listeria dead bacteria-containing test target solution was larger. The display scale of the fluorescence microscope is 2000-3000, and the exposure time is 100 ms. U-DM-CY-2 was used for the filter of the fluorescence microscope. Next, the fluorescence intensity was digitized from the image obtained with the fluorescence microscope as shown in FIG.

(Fifth embodiment)
Nonspecific adsorption of biomolecules into the injection funnel portion 201 and the flow path 111 of the substrate 10 shown in FIG. 6 was examined. First, a suction pump was connected to the outlet 22 of the flow path 111 of the substrate 10. Next, the injection funnel 201 and the flow path 111 were washed with 60 μl of PBST and sucked with a suction pressure of 20 kPa using a suction pump. Next, 30 μl of HRP-labeled streptavidin-containing solution with a concentration of 1 μg / ml suspended in 1% BSA-PBS was flowed without placing beads in the channel 111. The HRP-labeled streptavidin-containing solution was also sucked at a suction pressure of 20 kPa using a suction pump. Thereafter, the injection funnel 201 and the channel 111 were washed twice with 30 μl PBST, and the injection funnel 201 and the channel 111 were further washed once with 60 μl PBST.

  Next, as shown in FIG. 2, the solution filter 30, the absorption layer 40, and the gas filter 60 were arranged at the outlet of the flow path 111. Also, hydrogen peroxide and 10-acetyl-3,7-dihydroxyphenoxazine were added to sodium phosphate (pH 7.4) with a concentration of 50 mmol / l to prepare an enzyme substrate solution. The concentration of hydrogen peroxide in the enzyme substrate solution is 2 mmol / l, and the concentration of 10-acetyl-3,7-dihydroxyphenoxazine is 100 μmol / l. Next, 30 μl of the enzyme substrate solution was introduced into the flow path 111 from the injection funnel 201 while sucking the gas from the gas filter 60 and absorbed into the absorption layer 40.

  Next, three places in the absorption layer 40 were irradiated with excitation light having a wavelength of 563 nm and observed with a fluorescence microscope. The display scale of the fluorescence microscope is 2000-5000, and the exposure time is 100 ms. U-DM-CY-2 was used for the filter of the fluorescence microscope. Although there was no biomolecule to capture HRP-labeled streptavidin in channel 111, when HRP-labeled streptavidin was flowed as shown in FIG. 17, the fluorescence of 7-hydroxy-3H-phenoxazin-3-one confirmed. Therefore, it was shown that HRP-labeled streptavidin may adsorb nonspecifically inside the injection funnel 201 and the channel 111 of the substrate 10.

  Next, a separate substrate 10 shown in FIG. 6 is prepared, and an HRP-labeled streptavidin-containing solution is caused to flow from the injection funnel portion 201 to the flow path 111 under the same conditions. Washed. Then, wipe the inside of the funnel part 201 for injection with a cotton swab, wash the funnel part 201 and the channel 111 for injection twice with 30 μl of PBST, and further wash the funnel part 201 and the channel 111 for injection once with 60 μl of PBST. Washed. Again, wipe the inside of the injection funnel part 201 with a cotton swab, wash the injection funnel part 201 and the flow path 111 twice with 30 μl PBST, and further wash the injection funnel part 201 and the flow path 111 with 60 μl PBST. Washed twice.

  After that, as shown in FIG. 2, the solution filter 30, the absorption layer 40, and the gas filter 60 are arranged at the outlet of the flow path 111, and the gas from the gas filter 60 is sucked in, while the funnel portion for injection 201 passes the flow path 111. 30 μl of the enzyme substrate solution was introduced into and absorbed by the absorption layer 40. Thereafter, when the absorption layer 40 was observed with a fluorescence microscope under the same conditions as when the funnel portion 201 for injection was not wiped with a cotton swab, as shown in FIG. 18, when HRP-labeled streptavidin was flowed, it did not flow The difference in the fluorescence intensity in the case became smaller. Therefore, it was shown that the HRP-labeled streptavidin adsorbed nonspecifically inside the injection funnel portion 201 can be effectively removed by wiping the injection funnel portion 201 with a cotton swab.

  Next, a separate substrate 10 shown in FIG. 6 was newly prepared, and an HRP-labeled streptavidin-containing solution was allowed to flow through the flow path 111 from the injection funnel 201 under the same conditions. Thereafter, as shown in FIG. 5, the funnel portion 201 for injection was removed. Next, the channel 111 was washed twice with 30 μl PBST, and the channel 111 was washed once with 60 μl PBST.

  Thereafter, the solution filter 30, the absorption layer 40, and the gas filter 60 are arranged at the outlet of the channel 111, and 30 μl of the enzyme substrate solution is introduced into the channel 111 from the injection funnel 201 while sucking the gas from the gas filter 60. And absorbed by the absorption layer 40. Then, when the absorption layer 40 was observed with a fluorescence microscope under the same conditions as when the funnel portion 201 for injection was not removed, as shown in FIG. 19, when HRP-labeled streptavidin was flowed and when it was not flowed The difference in the fluorescence intensity was almost eliminated. Therefore, it was shown that by removing the funnel portion 201 for injection, HRP-labeled streptavidin adsorbed nonspecifically inside the funnel portion 201 for injection can be effectively removed.

  Next, a separate substrate 10 shown in FIG. 6 was newly prepared, and an HRP-labeled streptavidin-containing solution was allowed to flow through the flow path 111 from the injection funnel 201 under the same conditions. Thereafter, the injection funnel part 201 and the flow path 111 are washed with 30 μl of hydrogen peroxide obtained by diluting the stock solution having a hydrogen peroxide concentration of 30 to 35.5% to 1/100, and then the injection funnel part 201 with 30 μl PBST. The channel 111 was washed twice, and the injection funnel 201 and the channel 111 were washed once with 60 μl of PBST.

  After that, as shown in FIG. 2, the solution filter 30, the absorption layer 40, and the gas filter 60 are arranged at the outlet of the flow path 111, and the gas from the gas filter 60 is sucked in, while the funnel portion for injection 201 passes the flow path 111. 30 μl of the enzyme substrate solution was introduced into and absorbed by the absorption layer 40. Thereafter, when the absorption layer 40 was observed with a fluorescence microscope under the same conditions as when the funnel portion 201 for injection was removed, as shown in FIG. 20, the fluorescence when HRP-labeled streptavidin was flowed and when it was not flowed The difference in strength was slight. Therefore, it is shown that the HRP-labeled streptavidin adsorbed nonspecifically inside the injection funnel 201 and the channel 111 can be effectively removed by washing the injection funnel 201 and the channel 111 with hydrogen peroxide. It was.

(Other embodiments)
As mentioned above, although this invention was described by embodiment, it should not be understood that the description and drawing which form a part of this indication limit this invention. For example, as shown in FIG. 21 which is a top view and FIG. 22 which is a side view, the support member 100 may be provided with a plurality of columns 50a, 50b and 50c. As shown in FIG. 22, solution filters 30a, 30b, and 30c are arranged in the injection holes 141a, 141b, and 141c of the columns 50a, 50b, and 50c, respectively, and the gas filters 60a, 60b, and 60c are arranged in the discharge holes 142a, 142b, and 142c, respectively. 60c is arranged respectively. The columns 50a, 50b, 50c are filled with polymer water-absorbing resin beads constituting the absorption layers 40a, 40b, 40c, respectively. By disposing a substrate provided with a plurality of flow paths above the support member 100, a plurality of biomolecule testing methods according to the embodiment can be simultaneously performed. Further, as shown in FIG. 23, silicon rubber layers 91 and 92 may be provided on the front and back surfaces of the support member 100, respectively. By arranging the silicon rubber layers 91 and 92, the stability of the support member 100 can be increased.

  Further, in the embodiment, an example in which 7-hydroxy-3H-phenoxazin-3-one is recovered in the absorption layer 40 shown in FIG. 2 is shown, but the chemical substance recovery kit according to the embodiment includes various substances. It can be used for recovery. For example, as shown in FIG. 24, when the antibody is immobilized on the bead 5a and a solution containing the antigen is passed through the flow path of the substrate 10, only the antigen is captured by the antibody on the bead 5a. Thereafter, by arranging an absorption layer at the outlet of the flow path of the substrate 10 and flowing an acidic solution or the like through the flow path of the substrate 10, the purified antigen can be recovered in the absorption layer. As shown in FIG. 25, when a 10-fold amount of a solution containing an antigen at the same concentration is flowed, 10-fold amount of the antigen is captured by the antibody on the bead 5a. Therefore, after that, if an absorption layer is disposed at the outlet of the flow path of the substrate 10 and an acidic solution or the like is allowed to flow through the flow path of the substrate 10, the concentrated and purified antigen can be recovered in the absorption layer.

  Alternatively, the chemical substance recovery kit according to the embodiment can also be used for antibody recovery. For example, a bead having an NHS group (N-Hydroxy succinimide ester) that reacts with an amino group to form a covalent bond is blocked in the flow path of the substrate, and an antigen protein is bound onto the bead. Thereafter, when a solution containing the antibody is passed through the flow path of the substrate, only the antibody binds to the antigen on the bead. Thereafter, the purified antibody can be recovered by flowing an acidic solution or the like through the flow path of the substrate and absorbing the solution flowing through the flow path into the absorption layer.

  The chemical substance recovery kit according to the embodiment can also be used for recovering nucleic acids. For example, when a solution containing biotin-labeled double-stranded DNA is flowed through the flow path of the substrate after blocking in the flow path of the substrate coated with streptavidin, only the biotin-labeled double-stranded DNA binds to streptavidin on the beads. Thereafter, an alkaline eluate is introduced into the flow path, and the solution containing the single-stranded DNA is absorbed into the absorption layer, whereby the purified single-stranded DNA can be recovered.

  As indicated above, various alternative embodiments, examples and operational techniques will be apparent to those skilled in the art from this disclosure. Therefore, the technical scope of the present invention is defined only by the invention specifying matters according to the scope of claims reasonable from the above description.

  The chemical substance recovery kit, biomolecule test system, and biomolecule test method of the present invention include testing for microorganisms such as Listeria, Salmonella, 0-157, coliforms, yeast, Campylobacter, Staphylococcus aureus, and Pseudomonas, and food poisoning bacteria, Testing for microbial toxins such as Staphylococcus aureus toxin and Bacillus diarrheal toxins such as Cereus, recombinant soybeans, genetically modified organisms such as recombinant corn, eggs, peanuts, almonds, soybeans And allergic raw materials such as hazelnuts, bovine spongiform encephalopathy (BSE) measures such as cattle, pigs, sheep, goats, chickens, ducks, turkeys and ducks (Mycotoxin) testing, alachlor, aldicarb, atrazine, triazine, and urine Inspection of residual pesticides such as herbicides, determination of vitamin B groups such as folic acid, vitamin B21, and biotin, polycyclic aromatics, petroleum-based fuel, DDT (Dichloro-diphenyl-trichloroethane), chlordane, polychlorinated biphenyl (PCB) ), And chemical pollutants such as phencyclidine (PCP) and environmental hormones, and cedar pollen.

1 is a top view of a biomolecule inspection system according to an embodiment of the present invention. It is sectional drawing of the biomolecule test | inspection system which concerns on embodiment of this invention. It is an expansion perspective view of the board | substrate which concerns on embodiment of this invention. It is a schematic diagram of the bead which concerns on embodiment of this invention. 1 is a first cross-sectional view of a biomolecule test chip according to an embodiment of the present invention. It is a 2nd sectional view of the biomolecule inspection chip concerning an embodiment of the invention. It is a 3rd sectional view of the biomolecule inspection chip concerning an embodiment of the invention. It is a fluorescence observation image of the absorption layer which concerns on the 1st Example of embodiment of this invention. It is a fluorescence observation image of the absorption layer which concerns on the 2nd Example of embodiment of this invention. It is a graph which shows the fluorescence intensity of the absorption layer which concerns on the 2nd Example of embodiment of this invention. It is a fluorescence observation image of the absorption layer which concerns on the 3rd Example of embodiment of this invention. It is a graph which shows the fluorescence intensity of the absorption layer which concerns on the 3rd Example of embodiment of this invention. It is a table | surface which shows the fluorescence intensity of the absorption layer which concerns on the 3rd Example of embodiment of this invention. It is a fluorescence observation image of the absorption layer which concerns on the 4th Example of embodiment of this invention. It is a fluorescence observation image of the absorption layer which concerns on the 4th Example of embodiment of this invention. It is a graph which shows the fluorescence intensity of the absorption layer which concerns on the 4th Example of embodiment of this invention. It is a 1st fluorescence observation image of the absorption layer which concerns on the 5th Example of embodiment of this invention. It is a 2nd fluorescence observation image of the absorption layer which concerns on the 5th Example of embodiment of this invention. It is a 3rd fluorescence observation image of the absorption layer which concerns on the 5th Example of embodiment of this invention. It is a 4th fluorescence observation image of the absorption layer which concerns on the 5th Example of embodiment of this invention. It is a top view of the biomolecule test chip concerning other embodiments of the present invention. It is a 1st side view of the biomolecule test chip concerning other embodiments of the present invention. It is a 2nd side view of the biomolecule test chip which concerns on other embodiment of this invention. It is a 1st sectional view of the biomolecule test chip concerning other embodiments of the present invention. It is a 2nd sectional view of the biomolecule test chip concerning other embodiments of the present invention.

Explanation of symbols

5a, 5b, 5c… beads
10 ... Board
15 ... probe biomolecules
21 ... Entrance
22 ... Exit
30, 30a, 30b, 30c… Solution filter
40, 40a, 40b, 40c… absorption layer
50, 50a, 50b, 50c… Column
60, 60a, 60b, 60c… Gas filter
70 ... Detection mechanism
91, 92… Silicone rubber layer
100 ... Support member
111 ... Flow path
112 ... Damping part
141a, 141b, 141c… Injection hole
142a, 142b, 142c… discharge hole
201 ... funnel for injection

Claims (40)

A substrate provided with a solution flow path;
A solution filter disposed at an outlet of the flow path of the substrate;
A chemical substance recovery kit comprising: an absorption layer that absorbs the solution that has passed through the solution filter.   2. The chemical substance recovery kit according to claim 1, wherein a probe biomolecule is fixed in the flow path.   The chemical substance recovery kit according to claim 1, wherein a plurality of beads are dammed in the flow path.   4. The chemical substance recovery kit according to claim 3, wherein probe biomolecules are immobilized on the surfaces of the plurality of beads.   The chemical solution recovery kit according to any one of claims 1 to 4, wherein the solution filter is a membrane filter containing polycarbonate.   6. The chemical substance recovery kit according to claim 1, wherein the solution filter is disposed so as to cover an outlet of the flow path.   The chemical substance recovery kit according to claim 1, wherein the absorption layer includes a polymer water-absorbing resin.   The chemical substance recovery kit according to any one of claims 1 to 7, further comprising a column for holding the absorption layer.   The chemical recovery kit according to claim 8, wherein the column is provided with a gas filter that allows gas in the absorption layer to pass therethrough.   The chemical substance recovery kit according to claim 9, wherein the gas filter is a membrane filter containing polycarbonate.   The chemical recovery kit according to claim 9, wherein the gas filter is made of pulp.   The chemical recovery kit according to claim 9, wherein the gas filter is made of ashless pulp.   The chemical substance recovery kit according to any one of claims 1 to 12, further comprising an injection funnel portion disposed at an inlet of the flow path provided on the substrate.   14. The chemical substance recovery kit according to claim 13, wherein the injection funnel is removable from the substrate. A column provided with an injection hole and a discharge hole;
A solution filter disposed in the injection hole and permeable to the solution;
An absorbent layer disposed within the column and absorbing the solution;
A chemical substance recovery kit, comprising: a gas filter that is disposed in the discharge hole and transmits gas inside the absorption layer.   The chemical solution recovery kit according to claim 15, wherein the solution filter is a membrane filter containing polycarbonate.   The chemical substance recovery kit according to claim 15 or 16, wherein the absorption layer contains a polymer water-absorbing resin.   The chemical substance recovery kit according to any one of claims 15 to 17, wherein the gas filter is a membrane filter containing polycarbonate.   The chemical substance recovery kit according to any one of claims 15 to 17, wherein the gas filter is made of pulp.   The chemical recovery kit according to any one of claims 15 to 17, wherein the gas filter is made of ashless pulp. A substrate provided with a solution flow path;
A solution filter disposed at an outlet of the flow path of the substrate;
An absorption layer that absorbs the solution that has passed through the solution filter;
And a detection mechanism for detecting a reagent contained in the solution absorbed in the absorption layer.   The biomolecule test system according to claim 21, wherein a probe biomolecule is fixed in the flow path.   The biomolecule testing system according to claim 21, wherein a plurality of beads are dammed in the flow path.   The biomolecule test system according to claim 23, wherein probe biomolecules are immobilized on the surfaces of the plurality of beads.   The biomolecule testing system according to any one of claims 21 to 24, wherein the solution filter is a membrane filter containing polycarbonate.   The biomolecule test system according to any one of claims 21 to 25, wherein the solution filter is disposed so as to cover an outlet of the flow path.   27. The biomolecule testing system according to any one of claims 21 to 26, wherein the absorption layer includes a polymer water-absorbing resin.   The biomolecule test system according to any one of claims 21 to 27, further comprising a column for holding the absorption layer.   29. The biomolecule testing system according to claim 28, wherein the column is provided with a gas filter that allows the gas inside the absorption layer to pass therethrough.   30. The biomolecule testing system according to claim 29, wherein the gas filter is a membrane filter containing polycarbonate.   30. The biomolecule testing system according to claim 29, wherein the gas filter is made of pulp.   30. The biomolecule test system according to claim 29, wherein the gas filter is made of ashless pulp.   The biomolecule inspection system according to any one of claims 21 to 32, wherein the reagent is a fluorescent reagent, and the detection mechanism detects the fluorescent reagent.   The biomolecule testing system according to any one of claims 26 to 32, further comprising a suction pump that sucks the gas in the column through the gas filter.   The biomolecule testing system according to any one of claims 21 to 34, further comprising an injection funnel disposed at an inlet of the flow path provided on the substrate.   36. The biomolecule testing system according to claim 35, wherein the injection funnel is removable from the substrate. Preparing a substrate provided with a flow path;
Immobilizing probe biomolecules in the flow path of the substrate;
Flowing a solution containing a target biomolecule through the flow path, and capturing the target biomolecule with the probe biomolecule;
Modifying the captured target biomolecule with an enzyme;
Disposing a solution filter at an outlet of a flow path provided in the substrate;
Flowing a solution containing the enzyme substrate into the flow path to react the enzyme and the substrate;
Introducing a solution containing the reaction product of the enzyme and the substrate into an absorption layer that absorbs the solution containing the reaction product via the solution filter.   38. The biomolecule testing method according to claim 37, wherein the reaction product is a fluorescent substance.   39. The biomolecule inspection method according to claim 38, further comprising a step of irradiating the absorption layer with excitation light and detecting fluorescence of the fluorescent substance.   40. The biomolecule inspection method according to claim 39, further comprising a step of measuring the target biomolecule based on the detected fluorescence intensity.