JP6606786B2 - Biomolecule extraction chip and method for producing biomolecule extraction chip - Google Patents

Description

  The present invention relates to a biomolecule extraction chip and a method for manufacturing a biomolecule extraction chip. In particular, a part of the nanowire formed in the microchannel of the biomolecule extraction chip is embedded in the microchannel, so that the nanowire can be microchanneled even if the flow rate of the sample is increased. The present invention relates to a biomolecule extraction chip that can efficiently extract biomolecules without peeling from the substrate, and a method for manufacturing the biomolecule extraction chip.

  The exosome encapsulates microRNA (micro ribonucleic acid; hereinafter sometimes referred to as “miRNA”), which is a novel biomarker for early cancer / disease diagnosis, and is contained in exosomes in body fluids and culture supernatants. The analysis of the included miRNA is actively performed. In order to effectively search for unknown biomarkers by analyzing exosome-derived miRNA and to perform minimally invasive diagnosis, efficient extraction of exosomes from a small sample, that is, a large amount of information obtained from a small amount of solution is important. is there.

  As an existing exosome separation method, an ultracentrifugation method, an agglutination reagent method, and the like are known. The most commonly used ultracentrifugation method requires a sample amount of several tens of mL and a separation time of 4 to 5 hours, and also efficiently separates exosomes with a recovery rate of about 5 to 25%. There is a problem that it is difficult.

  On the other hand, the agglutination reagent method is a simple method in which the agglutination reagent is dropped on the target sample and allowed to stand, but the separation requires a long period of time (0.5 hours to overnight). There are problems such as use of a machine, change in particle size, decrease in the number of particles, and decrease in the amount of marker protein.

  In order to solve the above problems, the present inventors adsorb exosome-derived miRNA by adsorbing exosomes from a small amount of body fluid or culture supernatant using nanowires formed in the microchannel of the chip. A method for highly efficient extraction has been announced (see Non-Patent Document 1).

S. Ito et al. , “NANOWIRE DEVICES FOR EXOSOMAL MICRORNA EXTRACTION”, 17th International Conference on Miniaturized Systems for Chemistry 18: Nr. 18th Annual Science for Chemistry and Life Sciences. Su Zhang et al. "Growth and replication of ordered ZnO nanoarrays on general flexible substrates", J. et al. Mater. Chem. , 2010, 20, 10606-10610 Jung Kim et al. , “Nanoire-integrated microfluidic devices for Facial and Reagent-free mechanical cell lysis”, Lab Chip, 2012, 12, 2914-2921.

  However, in the chip described in Non-Patent Document 1, nanowires are grown from the surface in the microchannel. Therefore, if the sample input speed is increased in order to improve the throughput of sample recovery, there is a problem that the nanowires are separated from the surface of the microchannel.

  By the way, in order to efficiently replicate ZnO nanowires used in chips such as solar cells, LEDs (light emitting diodes), and UV (ultraviolet) leathers, as shown in FIG. 1, (1) nanowires grown from the substrate surface are embedded. (2) The resin layer I embedded with nanowires is prepared by peeling the resin from the substrate surface, and (3) nanowires embedded in the resin layer I are grown, 4) The resin II was filled so as to embed nanowires grown from the surface of the resin layer I, and (5) the nanowires were embedded by peeling the resin II so as to break the nanowires from the surface of the resin layer I. The resin layer I and the resin layer II are prepared, and (6) the procedures (3) to (5) are performed using the resin layer I and the resin layer II. A method of efficiently producing a resin in which a part of a nanowire is embedded by a process of repeating is known (see Non-Patent Document 2).

  However, the method described in Non-Patent Document 2 is for producing a resin in which a part of the nanowire is embedded twice based on the resin in which a part of the nanowire is embedded. Therefore, at the time of the above-mentioned step “(5) producing the resin layer I and the resin layer II in which the nanowire is embedded by peeling the resin II so as to break the nanowire from the surface of the resin layer I”, The surfaces of the two obtained “resin layers I” and “resin layer II” must have the same shape, that is, flat surfaces. Therefore, the method described in Non-Patent Document 2 has a problem that it is difficult to design a microchannel or the like and embed a part of the nanowire in the microchannel.

  The present invention has been made in order to solve the above-described conventional problems, and as a result of extensive research, a substrate including a stepped portion is produced by making full use of electron beam lithography and photolithography techniques. When the substrate on which nanowires are grown is used as a template and transferred to the substrate of the biomolecule extraction chip, the stepped portion becomes a microchannel, and a part of the transferred nanowire is embedded in the microchannel and embedded. The inventors have newly found that a biomolecule extraction chip in which a part of the nanowire is embedded in a microchannel can be produced by growing the nanowire, and the present invention has been completed.

  That is, an object of the present invention may be described as a biomolecule extraction chip (hereinafter referred to as “chip”) in which a part of the nanowire formed in the microchannel is embedded in the microchannel. And a method of manufacturing the chip.

  The present invention relates to a chip and a method for manufacturing the chip, which will be described below.

(1) A biomolecule extraction device including a substrate, a microchannel formed on the substrate, and a nanowire formed in the microchannel, and a part of the nanowire is embedded in the microchannel Chip.
(2) The biomolecule extraction chip according to (1), wherein the surface of the nanowire is positively charged.
(3) The biomolecule extraction chip according to (1), wherein the surface of the nanowire is formed of a material having an isoelectric point lower than the isoelectric point of nucleic acid.
(4) The above-mentioned (1), wherein a sample input part is formed at one end of the microchannel, a residue or sample recovery part is formed at the other end, and the nanowire is formed between the sample input part and the residue or sample recovery part The chip for biomolecule extraction according to any one of (1) to (3).
(5) a step of forming a step on the substrate to form a microchannel by transferring;
A step of growing a nanowire on the step and producing a mold;
Transferring the template to the substrate of the biomolecule extraction chip;
A step of growing nanowires embedded in a microchannel of a biomolecule extraction chip;
A method for producing a biomolecule extraction chip in which a part of a nanowire is embedded in the microchannel.

  In the chip of the present invention, a part of the nanowire formed in the microchannel is embedded in the microchannel, so that the nanowire is separated from the microchannel even if the inflow rate of the sample is increased. It becomes difficult. Therefore, the extraction time of the biomolecule can be shortened.

  Further, in the manufacture of the chip of the present invention, a substrate including a step portion produced by using electron beam lithography and photolithography techniques is used as a mold. Accordingly, the stepped portion, that is, the shape such as the width and depth of the microchannel after transfer, the number, etc. can be arbitrarily designed.

  Furthermore, nanowires can be repeatedly grown on the step portion of the mold used in the chip manufacturing method of the present invention. Therefore, the chip can be repeatedly manufactured using the same mold.

FIG. 1 is a diagram illustrating a conventional method for efficiently producing a resin in which a part of a nanowire is embedded. FIG. 2 is a diagram showing an example of the chip 1 of the present invention. FIG. 3 is a diagram showing an example of a manufacturing process of the chip 1 of the present invention. FIG. 4 is a drawing-substituting photograph, and FIG. 4 (1) is a field emission scanning electron microscope (FESEM) photograph of a nanowire grown on the step 14 of the substrate 11 which is a mold in Example 1. FIG. 4 (2) is an enlarged photograph of FIG. 4 (1). FIG. 5 is a drawing-substituting photograph, FIG. 5 (1) is an FESEM photograph of the step 14 of the substrate 11 which is a mold after the polydimethylsiloxane 2 is peeled off in Example 1, and FIG. 5 (2) is FIG. ) Is an enlarged photo. FIG. 6 is a drawing-substituting photograph, FIG. 6 (1) is a FESEM photograph of the microchannel 3 of polydimethylsiloxane 2 after the template is transferred in Example 1, and FIG. 6 (2) is FIG. 6 (1). It is an enlarged photo. FIG. 7 is a drawing-substituting photograph. FIG. 7A is a FESEM photograph of the microchannel 3 after the nanowire 17 is grown for 3.5 hours after the polydimethylsiloxane 2 is peeled off in Example 1. FIG. (2) and (3) are enlarged photographs of FIG. 7 (1). FIG. 8 is a drawing-substituting photograph, and FIG. 8 (1) is an FESEM photograph of the microchannel 3 after the nanowire 17 is grown for 10 hours after the polydimethylsiloxane 2 is peeled off in Example 1. FIG. ) Is an enlarged photograph of FIG.

  Hereinafter, a biomolecule extraction chip in which a part of the nanowires formed in the microchannel is embedded in the microchannel and a method for manufacturing the chip will be described in detail. In the present invention, “a part of the nanowire is embedded in the microchannel” means that at least particles or a catalyst serving as a nucleus for growing the nanowire are placed on the surface in the microchannel. It means that it is embedded in the inner wall inside the microchannel.

  FIG. 2 shows an example of the chip of the present invention. 2 includes a substrate 2, a microchannel 3 formed on the substrate 2, a sample insertion portion 4 formed at one end of the microchannel 3, and a residue formed at the other end of the microchannel 3. Or the sample collection | recovery part 5 is included. And in the flow path (dotted line part in a figure) between the sample injection | throwing-in part 4 and a residue or the sample collection | recovery part 5, the nanowire partially embedded in the flow path is provided. In addition, when a sample is directly input into the microchannel 3 and a residue or sample is directly recovered from the microchannel 3, the sample input unit 4 and the residue or sample recovery unit 5 may not be provided.

  For example, when separating exosomes using the chip 1 of the present invention, a sample solution containing exosomes (culture fluid, body fluid such as serum and urine) is sent from the sample input unit 4 using a syringe pump or the like. Then, the solution after separating the sample from the residue or the sample collection unit 5 is collected. Then, after the sample solution is flowed, the exosome adsorbed on the nanowire may be analyzed. Since the exosome is negatively charged, the surface of the nanowire is preferably positively charged. For example, a nanowire having a coating layer formed of a positively charged material such as ZnO nanowire or nickel oxide may be used. In addition, when nanowires whose surface is positively charged are used, biomolecules such as proteins that are positively charged can be extracted by applying an electric field so that the recovery part becomes negative.

  For example, when nucleic acid is extracted from a sample selected from cells, viruses, and fungi using the chip 1 of the present invention, a suspension in which the sample is suspended is loaded into the sample loading unit 4. Specific examples of cells, viruses, and fungi include cells having a cell membrane structure. Staphylococcus, Bacillus subtilis, Escherichia coli, Salmonella, Pseudomonas aeruginosa, Vibrio cholerae, Shigella, Bacillus anthracis, Mycobacterium tuberculosis And bacteria such as Clostridium botulinum, tetanus, and streptococci, and blood cells such as granulocytes, lymphocytes, reticulocytes, erythrocytes, leukocytes, and platelets. Examples of viruses include norovirus, rotavirus, influenza virus, adenovirus, coronavirus, measles virus, rubella virus, hepatitis virus, herpes virus, HIV and the like. Examples of the fungi include mushrooms, molds, yeasts, etc., and specifically include ringworm fungi, Candida, Aspergillus, filing yeast, and the like. In addition to exosomes, mitochondria and extracellular vesicles can also be mentioned as samples.

Then, by applying an electric field so that the sample input unit 4 is negative and the residue or sample recovery unit 5 is positive, the sample is crushed with the nanowire, and the nucleic acid extended from the residue or sample recovery unit 5 can be recovered. it can. In addition, when recovering nucleic acid using the chip 1 of the present invention, a residue extraction unit is provided at the other end between the sample input unit 4 and the portion of the microchannel 3 provided with the nanowire (dotted line portion in FIG. 1). The nucleic acid may be taken out from the residue or the sample collection unit 5 and the crushed cell residue may be taken out from the residue extraction unit. When nucleic acid is extracted, it is preferable that the surface of the nanowire has no isoelectric point lower than the isoelectric point of the nucleic acid because the nucleic acid leaked from the pores of the cells and the like is preferably not adsorbed to the nanowire by electrostatic interaction. Preferably formed from a material. As will be described later, after the nanowire is grown, the surface of the nanowire may be covered with a material such as SiO ₂ or TiO ₂ .

By incorporating the chip 1 of the present invention into a known analyzer, an analyzer can be configured. Moreover, by using together with a known analyzer, the extracted biomolecule can be analyzed as it is. Further, an analysis unit for analyzing the biomolecule extracted on the chip 1 may be formed. For example, when nucleic acid is extracted using the chip 1 of the present invention, the nucleic acid extracted from the sample elongates when flowing between nanowires,
(1) By making the chip 1 into a shape that can be incorporated into a known nucleic acid sequencer, nucleic acid extraction and nucleic acid sequence analysis can be carried out with one apparatus.
(2) The nucleic acid sequence can be analyzed by separately introducing the extended nucleic acid into a nucleic acid sequencer.
(3) By forming an analysis unit such as an electrode for performing nucleic acid analysis on the chip 1, a new analysis device capable of performing nucleic acid extraction and nucleic acid sequence analysis with a single device can be produced.

  The chip 1 of the present invention can be manufactured by transferring a template produced using electron beam lithography and photolithography technology to a resin or the like, and further growing nanowires embedded in the transferred resin or the like. FIG. 3 shows an example of a manufacturing process of the chip 1 of the present invention.

(1) The Cr layer 12 is deposited on the mold substrate 11 by sputtering.
(2) A positive photoresist 13 is applied with a spin coater. Next, a pattern corresponding to the microchannel 3 of the chip 1 when transferred is produced by photolithography.
(3) The Cr layer 12 other than the pattern corresponding to the microchannel 3 is etched with a Cr etchant solution.
(4) By etching the substrate 11 only selectively, the substrate 11 other than the pattern corresponding to the microchannel 3 is etched to form the step 14 corresponding to the microchannel 3. The depth of the micro flow path 3 can be adjusted by adjusting the depth of the step 14.
(5) The Cr layer 12 and the positive photoresist 13 on the step 14 are etched with a Cr etchant solution.
(6) Particles or catalyst 15 for producing nanowires are applied on the substrate 11 by pulsed laser deposition (PLD).
(7) After performing the O ₂ plasma, a resist 16 for electron beam lithography is applied, and a place where the nanowire is grown on the step 14 is drawn by electron beam lithography. The drawing of electron beam lithography may be formed in a pattern in which nanowires are to be grown. When nanowires are grown at a predetermined interval, dots at predetermined intervals are used. When nanowires are to be grown at random, from any location on the step 14. All that is on the step 14 may be drawn so that the nanowire can grow. After drawing by electron beam lithography, the resist is developed and removed.
(8) The nanowire 17 is grown from the place where the resist is removed and the particles or the catalyst 15 are exposed, and a template used for manufacturing the chip 1 of the present invention is manufactured.
(9) A material for the substrate 2 of the chip 1 is applied to a mold.
(10) The substrate 1 is peeled from the mold to form the chip 1 in which a part of the nanowire 17 is embedded in the microchannel 3.
(11) The nanowire 17 embedded in the microchannel 3 is further grown. After the growth of the nanowire 17, the sample input unit 4, the residue or sample recovery unit 5, and a residue extraction unit as necessary are formed. Note that the sample input unit 4, the residue or sample recovery unit 5, and the residue extraction unit may be simultaneously designed when the microchannel 3 is designed by photolithography, and may be formed by transfer.

  When a plurality of chips 1 are manufactured, the above steps (8) to (11) may be repeated using the mold produced in the above step. The above process is merely an example of the manufacturing process of the chip 1, and a step 14 is formed on the substrate 11, and the nanowires 17 grown on the step 14 are transferred to the substrate 2 of the chip 1. There is no particular limitation as long as a part of the nanowire 17 can be embedded in the microchannel 3 of the chip 1.

  In the above steps, the template substrate 11 is not particularly limited as long as the step 14 can be formed by etching. For example, when the step 14 is formed by dry etching, silicon, quartz glass, Pyrex (registered trademark) glass, or the like can be used.

  Also, when the step 14 is formed by wet etching, silicon, quartz glass, Pyrex (registered trademark) glass, or the like may be used as in dry etching.

  The positive photoresist 13 is not particularly limited as long as it is generally used in the semiconductor manufacturing field, such as TSMR V50 and PMER. In addition, a negative type photoresist may be used instead of the positive type, and there is no particular limitation as long as it is generally used in the semiconductor manufacturing field, such as SU-8 and KMPR.

The etchant solution for the Cr layer 12 is not particularly limited as long as it can etch Cr, such as H ₂ O: Ce (NH ₄ ) ₂ (NO ₃ ) ₆ : HClO ₄ . Further, the present invention is not limited to Cr, and a material other than Cr may be deposited and used in combination with an etchant that can etch the material.

As a method for selectively etching only the substrate 11, when dry etching is performed on the substrate 11, a known dry method such as sulfur hexafluoride (SF ₆ ), carbon tetrafluoride (CF ₄ ), trifluoromethane (CHF ₃ ), or the like. An etching gas may be used. When the substrate 11 is wet-etched, hydrogen fluoride (HF) may be used when the substrate 11 is glass, and potassium hydroxide (KOH), sodium hydroxide (NaOH), or the like may be used when silicon is used.

  Examples of the particles 15 for producing the nanowire 17 include ZnO. Examples of the catalyst 15 for producing the nanowire 17 include gold, platinum, aluminum, copper, iron, cobalt, silver, tin, indium, zinc, and gallium.

  The resist 16 for electron beam lithography is not particularly limited as long as it is generally used in the semiconductor field, such as ZEP520 and PMMA. The resist removing solution is not particularly limited as long as it is a common removing solution in the semiconductor field, such as dimethylformamide and acetone.

On the step 14, the nanowire 17 may be grown from the particles or the catalyst 15 by a known method. For example, when ZnO fine particles are used as the particles 15, they can be produced using the hydrothermal synthesis method described in Non-Patent Document 3. Specifically, a heated substrate in a precursor solution in which zinc nitrate hexahydrate (Zn (NO ₃ ) ₂ .6H ₂ O) and hexamethylenetetramine (C ₆ H ₁₂ N ₄ ) are dissolved in deionized water. By immersing 11, the ZnO nanowire 17 can be grown from the portion where the ZnO particles 15 of the step 14 are exposed.

When the catalyst 15 is used, the nanowire 17 can be produced by the following process.
_{(A) SiO 2, Li 2} O, MgO, Al 2 O 3, CaO, TiO 2, Mn 2 O 3, Fe 2 O 3, CoO, NiO, CuO, ZnO, Ga 2 O 3, SrO, In 2 O ₃ , SnO ₂ , Sm ₂ O ₃ , EuO and other materials are used to form the core nanowire by physical vapor deposition such as pulse laser deposition and VLS (Vapor-Liquid-Solid) method.
(B) A coating layer around the core nanowire by using a general vapor deposition method such as sputtering, EB (Electron Beam) vapor deposition, PVD (Physical Vapor Deposition), ALD (Atomic Layer Deposition) using SiO ₂ , TiO ₂ or the like. Form. In addition, when growing a nanowire using the catalyst 15 on the level | step difference 14, the coating layer of said (b) is not essential, You may grow and transfer only a core nanowire.

  In the present invention, the length of the nanowire embedded in the microchannel 3 after the transfer is determined according to the length of the nanowire 17 grown on the step 14. Therefore, when it is desired to increase the sample inflow rate, the growth of the nanowire 17 on the step 14 may be adjusted as appropriate so that the length of the embedded nanowire becomes longer. For example, when all of the electron beam lithography resist 16 on the step 14 is drawn, the nanowire-producing particles or the catalyst 15 are exposed on the step 14. When transferred to the substrate 2 for producing the chip 1 in this state, the particles on the step 14 or the catalyst 15 are transferred so as to be embedded in the microchannel 3. By growing the nanowire 17 from the embedded particle or catalyst 15, the nanowire 17 partially embedded in the microchannel 3 can also be produced.

  The substrate 2 for producing the chip 1 of the present invention is not particularly limited as long as the template can be transferred. For example, thermoplastic resins such as polyethylene, polypropylene, polyvinyl chloride, polyvinylidene chloride, polystyrene, polyvinyl acetate, polytetrafluoroethylene, ABS (acrylonitrile butadiene styrene) resin, AS (acrylonitrile styrene) resin, acrylic resin (PMMA), etc. A thermosetting resin such as phenol resin, epoxy resin, melamine resin, urea resin, unsaturated polyester resin, alkyd resin, polyurethane, thermosetting polyimide, silicone rubber; In addition, when analyzing the target biomolecule using the chip 1 as it is, a resin having light permeability and non-affinity with the biomolecule is desirable. For example, cycloolefin polymer (COP), polydimethylsiloxane (PDMS) , Polymethyl methacrylate (PMMA), polycarbonate (PC), plastic made of hard polyethylene, silicon and the like.

  The growth of the nanowire 17 embedded in the microchannel 3 after peeling off the substrate 2 may be performed in the same procedure as described above. In addition, when growing the nanowire 17 using the catalyst 15, the nanowire 17 may not have a branched chain and may have a branched chain.

  The diameter of the nanowire 17 grown in the microchannel 3 may be appropriately adjusted according to the purpose. For example, when adsorbing an exosome, if it is too thin, the exosome may be destroyed. Therefore, it may be larger than the exosome, for example, 100 nm or more. Moreover, when crushing cells, etc., the diameter may be changed according to the size of cells, viruses, fungi, etc., the strength of the cell membrane or cell wall, etc., and the prokaryotic cells are 1 μm to 10 μm, true Nucleus cells are 5 μm to 10 μm, viruses are several tens to several hundreds of nm, blood cells are 7 μm to 15 μm in size, so the diameter of the nanowire may be 10 nm or more, 15 nm or more, 20 nm or more, or 25 nm or more. Good. In addition, the diameter of the nanowire needs to be smaller than the cell or the like that is the object of crushing, for example, 200 nm or less, 150 nm or less, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, or 50 nm or less. Also good.

  When the nanowire 17 is formed using the particles 15, the diameter of the nanowire 17 can be appropriately adjusted by changing the size of the particles applied to the step 14 or by forming a coating layer on the produced nanowire 17. The coating layer may be formed with the same material and coating method as the coating layer when the catalyst 15 is used, and the deposition time may be adjusted as appropriate. Moreover, also when growing using the catalyst 15, a diameter can be suitably adjusted by changing the vapor deposition time at the time of forming a coating layer.

  The length of the nanowire 17 may be adjusted as appropriate in consideration of the depth of the microchannel 3 and the like. The length of the nanowire 17 can be adjusted by changing the immersion time in the precursor solution when formed using the particles 15. Moreover, when forming using the catalyst 15, it can adjust suitably by changing the growth time by VLS after forming core nanowire.

  The depth and width of the microchannel 3 formed on the chip 1 are not particularly limited and may be adjusted according to the target biomolecule. However, if the space volume of the microchannel 3 is too small, the pressure loss increases. Therefore, for example, the depth and width may be 1 μm or more. Further, the number of micro flow paths 3 formed on the chip 1 is not particularly limited, and a plurality of micro flow paths 3 may be provided on the chip 1. The width and depth of the microchannel 3 can be adjusted by the width and height of the step 14, and the number of the microchannel 3 can be adjusted by the number of the steps 14.

  If the sample input unit 4, residue or sample recovery unit 5, and residue extraction unit are not designed together with the micro flow path 3, the template is transferred to the substrate 2 and then formed using a biopsy trepan, ultrasonic drill, or the like. Good.

  The present invention will be described in detail with reference to the following examples, which are provided merely for the purpose of illustrating the present invention and for reference to specific embodiments thereof. These exemplifications are for explaining specific specific embodiments of the present invention, but are not intended to limit or limit the scope of the invention disclosed in the present application.

<Example 1>
[Chip fabrication]
The chip of the present invention was produced by the following procedure.
(1) An 800 nm thick Cr layer 12 is deposited on a silicon (111) substrate 11 (Electronic End Materials Corporation) using an RF (radio frequency) sputtering apparatus (SVC-700LRF, Sanyu Denshi). It was.

(2) A positive photoresist 13 (TSMR V50, Tokyo Ohka Kogyo Co.) was applied by a spin coater. Thereafter, a pattern corresponding to the microchannel 3 having a length of about 11 mm having a circle of about 1 mm in width and a diameter of about 2 mm at both ends was produced by photolithography. After pattern formation, the resist was developed with SMR-V50 EL (Tokyo Ohka Kogyo Co.).

(3) In a Cr etchant solution (H ₂ O: Ce (NH ₄ ) ₂ (NO ₃ ) ₆ : HClO ₄ = 85: 10: 5 (mass% ratio)), other than the pattern corresponding to the microchannel 3 The Cr layer on the silicon was etched.

(4) Using a reactive ion etching apparatus (RIE-10NR, Samco Co.), the substrate 11 other than the pattern corresponding to the microchannel 3 was etched by a depth of about 1 μm with CF ₄ gas.

(5) Using the same Cr etchant solution as in (3) above, removing the Cr layer 12 and the positive photoresist 13 on the pattern corresponding to the microchannel 3, the step 14 (depth 1 μm) ( Corresponding to microchannel 3 after transfer).

(6) Using ZnO pellets (purity 4N, manufactured by Kojundo Chemical Laboratory Co., Ltd.) on the silicon substrate 11, using a pulse laser deposition apparatus (PLD-YA-8670, manufactured by Shonan Kogyo Co., Ltd.) The coating was applied to a thickness of 100 nm.

(7) A positive electron beam resist 16 (ZEP520A, Zeon Corp.) was applied to a thickness of about 500 nm by a spin coater. Next, by electron beam lithography (SPG-724, Sanyu Electron Co.), a pattern of spots on which nanowires 17 are grown (a circle having a diameter of about 500 nm, a distance between an arbitrary spot and an end of an adjacent spot is about 1 μm) (Lattice). After the drawing, the positive electron beam resist 16 was developed using ZEP 520 A7 to remove the spot type positive electron beam resist 16, thereby producing a spot pattern reaching the ZnO particle layer on the step 14.

(8) 0.04461 g of zinc nitrate hexahydrate (Zn (NO ₃ ) ₂ .6H ₂ O, 98%, manufactured by Sigma Adrich), hexamethylenetetramine ((C ₆ H ₁₂ N ₄ ), 99 ⁺ %, Sigma Adrich Co.) was added to 0.02103 g and 50 mL of deionized water to prepare a precursor solution of 3 mM zinc nitrate hexahydrate and hexamethylenetetramine. Next, the substrate 11 was placed in the precursor solution and immersed in a 95 ° C. oven for 10 hours to grow the nanowire 17 from the ZnO particle layer, thereby producing a template. 4 (1) is a FESEM photograph of nanowires grown on the step 14 of the substrate 11, and FIG. 4 (2) is an enlarged photograph of the photograph of FIG. 4 (1). As is clear from the photographs of FIGS. 4A and 4B, it was confirmed that the nanowires grew upward from the spot on the step 14. Note that the length of the nanowire shown in FIG. 4B was about 2 μm from the layer of the positive electron beam resist 16 on the step 14.

(9) Polydimethylsiloxane 2 (PDMS, Silpot 184, Dow Corning Toray) to be the substrate 2 of the chip 1 is made of prepolymer (Silpot 184, Dow Corning Toray) and a curing agent (Catalyst Solpot 184, Dow Corning Toray). The mixture was mixed at a ratio of 10: 1, allowed to stand for 5 minutes, and then degassed in vacuum for 30 minutes. Thereafter, the produced PDMS was poured into the mold produced in the above (8), defoamed in vacuum for 1 hour, and heated at 75 ° C. for 2 hours.

(10) The polydimethylsiloxane 2 poured in (9) from the mold was peeled off by hand. FIG. 5 (1) is a FESEM photograph of the step 14 of the substrate 11 after the polydimethylsiloxane 2 is peeled, and FIG. 5 (2) is a photograph further enlarging the photograph of FIG. 5 (1). As is clear from the photographs in FIGS. 5 (1) and (2), the nanowire 17 after the polydimethylsiloxane 2 was peeled was cut from the root portion. On the other hand, FIG. 6 (1) is a FESEM photograph of the microchannel 3 portion of the polydimethylsiloxane 2 to which the template has been transferred, and FIG. 6 (2) is a photograph further enlarging the photograph of FIG. 6 (1). . As is clear from the photographs in FIGS. 6 (1) and (2), a part of the nanowire 17 was embedded in the microchannel 3.

(11) Nanowires 17 were further grown from the cross section of the nanowires 17 embedded in the polydimethylsiloxane 2 by the same procedure as in the above step (8). FIG. 7 (1) is a FESEM photograph of the microchannel 3 after 3.5 hours, and FIGS. 7 (2) and (3) are photographs obtained by further enlarging the photograph of FIG. 7 (1). FIG. 8 (1) is a FESEM photograph of the microchannel 3 after 10 hours, and FIG. 8 (2) is a photograph further enlarging the photograph of FIG. 8 (1). As is apparent from the photographs shown in FIGS. 6 to 8, it was confirmed that the nanowire 17 transferred into the microchannel 3 of the polydimethylsiloxane 2 was further grown. In addition, the diameter of each nanowire 17 shown in FIG. 8 (2) was about 100 nm.

  In the biomolecule extraction chip of the present invention, since a part of the nanowire is embedded in the microchannel, it is difficult for the nanowire to be separated from the microchannel even if the flow rate of the sample is increased. Therefore, since the speed of extracting biomolecules from a sample can be improved, it is useful for rapid biomolecule extraction in medical institutions, universities, companies, research institutions, and the like.

Claims (5)

  A biomolecule extraction chip comprising a substrate, a microchannel formed on the substrate, and a nanowire formed in the microchannel, and a part of the nanowire is embedded in the microchannel.   The biomolecule extraction chip according to claim 1, wherein the surface of the nanowire is positively charged.   The biomolecule extraction chip according to claim 1, wherein a surface of the nanowire is formed of a material having an isoelectric point lower than that of a nucleic acid.   The sample input part and the residue or sample recovery part are formed at one end of the microchannel, and the nanowire is formed between the sample input part and the residue or sample recovery part. The biomolecule extraction chip according to any one of the above. Forming a step on the substrate to form a microchannel by transferring,
A step of growing a nanowire on the step and producing a mold;
Transferring the template to the substrate of the biomolecule extraction chip;
A step of growing nanowires embedded in a microchannel of a biomolecule extraction chip;
A method for producing a biomolecule extraction chip in which a part of a nanowire is embedded in the microchannel.