WO2014136913A1

WO2014136913A1 - Device for separating and extracting biomolecules, method for manufacturing same, and method for separating and extracting biomolecules

Info

Publication number: WO2014136913A1
Application number: PCT/JP2014/055867
Authority: WO
Inventors: 隆雄安井; 剛柳田; 範匡加地; 川合　知二; 馬場　嘉信
Original assignee: 国立大学法人名古屋大学; 国立大学法人大阪大学
Priority date: 2013-03-06
Filing date: 2014-03-06
Publication date: 2014-09-12
Also published as: JP2016103979A

Abstract

Provided is a device for efficiently separating and extracting biomolecules having a short fragment length within a short period of time. The device for separating and extracting biomolecules comprises a separation part, which contains nanowires having a branched chain with one or more branches, formed on a microchannel that is provided on a substrate.

Description

Biomolecule separation and extraction device, method for producing the same, and biomolecule separation and extraction method

The present invention relates to a device for separating and extracting biomolecules, a method for producing the same, and a method for separating and extracting biomolecules.

In recent years, due to advances in molecular biology, genetic analysis such as gene diagnosis such as gene deletion and drug susceptibility SNP (Single Nucleotide Polymorphism), diagnosis of infectious diseases due to pathogenic bacteria, and allergen diagnosis in hospitals and other medical institutions Diagnosis based on this is spreading. Therefore, there is a need for a method for extracting a nucleic acid, which is a gene, from a cell or the like by an efficient and simple operation and analyzing the gene. For the analysis of the extracted nucleic acid, an enzymatic method, a chemical decomposition method, and the like are known, and an automatic sequencing kit using these principles is currently on the market.

By the way, ultra-high-speed analysis of DNA base sequences, ultra-trace and ultra-high-speed detection of viruses, ultra-sensitive and ultra-high-speed detection of allergens such as pollen, etc. are indispensable technologies for realizing a safe, secure, and healthy society. It is. However, the conventional technique requires an amplification step using PCR or the like of the DNA to be analyzed, and the DNA analysis cannot be performed quickly. As a technique for solving this problem, a single molecule analysis technique (Non-patent Document 1 “M. Tsutsui et al.,“ Identifying single nucleotides by tunneling current ”, Nat. Nanotech. , 2010, 5, 286-290.).

In addition, a DNA extension technique for extending a long-chain DNA into a single DNA for use as a sample for single-molecule analysis like the technique described in Non-Patent Document 1 is also known (Non-Patent Document 2 “Yasui”). Et al., “DNA analysis using nanostructures”, NEW GLASS, 2009, Vol. 24, No. 1, p22-27 ”). The conventional DNA stretching technique combines a nanochannel on quartz glass with electron beam lithography and photolithography to form a bamboo-like shape made of SiO ₂ having a diameter of about 500 nm and a height of about 4000 nm, called a nanopillar. A thing is formed by a fine processing technique (top-down method). And it is the technique of obtaining the extended | stretched DNA which can be used for single molecule analysis by allowing the long-chain DNA extracted from the cell etc. to pass through between nano pillars. However, since the nanopillar is formed in a top-down manner, it is difficult to reduce the diameter of the nanopillar. As a result, it is difficult to separate and extract a nucleic acid having a short fragment length.

On the other hand, the present inventors used nanowires thinner than the nanopillars described in Non-Patent Document 2 in which nanowires are grown randomly or radially by bottom-up using a catalyst in a microchannel provided on a substrate, It has been found that nucleic acids can be extracted directly from cells and the like, and a patent application has been filed (see Patent Document 1 “Japanese Patent Application No. 2012-236451”).

By the way, in the field of nanowires, it is known that a branched chain of nanowires can be further formed from a core nanowire (Non-Patent Document 3 “Seung Hwan Ko et al.,“ Nanoforest of Hydrologically Thermal ZnONanohilophia ”. Efficiency Dye-Sensitized Solar Cell “Nano Lett. 2011, 11, 666-671” and Non-Patent Document 4 “Maththew J. Bierman1 et al.,“ Dislocation-DrivenNowirwNowerWhirthow ”. No. 5879, 23 May 2008, pp. 1060-1063)).

However, the invention described in Patent Document 1 is intended to extract nucleic acids directly from cells and the like themselves without going through a pretreatment step such as cell lysis, and is more than nucleic acids extracted from cells. There is a problem that short nucleic acid fragments cannot be selectively separated and extracted.
In addition, the nanowire described in Non-Patent Document 3 is intended to increase the efficiency of solar cells, and does not pay attention to application to separation and extraction of nucleic acids. Furthermore, in the technique of Non-Patent Document 3, since nanowires are grown by a hydrothermal method, it is difficult to form nanowires having branched chains at desired spatial positions, particularly at specific positions such as microchannels. It is.
Non-Patent Document 4 merely discloses that a nanowire having a branched chain can be formed. Therefore, even in the technique described in Non-Patent Document 4, no attention is paid to the application of nanowires to separation and extraction of nucleic acids, and the density of nanowires cannot be controlled.
The present invention has been made in order to solve the above-mentioned conventional problems, and as a result of intensive research, the method described in Patent Document 1 is further improved so that the inside of the microchannel provided on the substrate can be improved. By forming a nanowire having a branched chain on the surface, and further controlling the number of branch chain forming steps for forming the branched chain, the density of the nanowire formed in the microchannel can be controlled, and separated and extracted. The present inventors have newly found that the size of a biomolecule can be changed, thereby completing the present invention.

That is, an object of the present invention is to provide a device for separating and extracting biomolecules, a method for producing the same, and a method for separating and extracting biomolecules.

The present invention relates to a device for separating and extracting biomolecules, a method for producing the same, and a method for separating and extracting biomolecules as described below.

(1) A biomolecule separation / extraction device in which a separation part including a nanowire having a branched chain having one or more branches is formed on a microchannel provided on a substrate.
(2) The device for separating and extracting a biomolecule according to (1), wherein the branched chain is a nanowire grown from a catalyst deposited on the nanowire.
(3) The biomolecule according to (1) or (2), wherein the microchannel is formed of two intersecting microchannels, and the separation part is formed only in one microchannel. Device for separation and extraction.
(4) The living body according to any one of (1) to (3), wherein a sample input unit for supplying a sample solution and a separated sample recovery unit for recovering a separated sample are provided in the microchannel. Device for separation and extraction of molecules.
(5) forming a microchannel on a substrate, depositing a catalyst on the microchannel, forming a nanowire from the catalyst, depositing a catalyst on the nanowire, and forming a nanowire from the catalyst Including the steps of:
A method for producing a device for separating and extracting biomolecules, comprising the steps of depositing a catalyst on the nanowire and forming the nanowire from the catalyst one or more times.
(6) For separation and extraction of biomolecules according to (5), further comprising the steps of forming a sample input part for supplying a sample solution to the microchannel and a separated sample recovery part for recovering a separated sample. Device manufacturing method.
(7) A step of putting a sample into one end of a microchannel of a biomolecule separation / extraction device in which a separation part including a nanowire having a branched chain with one or more branches is formed on a microchannel provided on a substrate When,
Applying an electric field to the microchannel;
Separating biomolecules in the separation unit;
A method for separating and extracting biomolecules.
(8) The separation unit of the biomolecule separation / extraction device in which a separation unit including a nanowire having a branched chain having a branch number of 1 or more is formed on one of the intersecting microchannels provided on the substrate. Introducing a sample into one end of a microchannel that is not formed;
Applying an electric field to the microchannel where the separation part is not formed;
Stopping the application of the electric field to the microchannel where the separation part is not formed, and applying the electric field to the microchannel where the separation part is formed;
Separating biomolecules in the separation unit;
A method for separating and extracting biomolecules.

The device for separating and extracting a biomolecule of the present invention forms a nanowire having a branched chain in a microchannel on a substrate, and controls the number of the branch chain forming steps, thereby controlling the nanowire formed in the microchannel. The density can be controlled. Therefore, separation and extraction according to the size of the biomolecule to be separated and extracted are possible. Moreover, the device for separating and extracting biomolecules of the present invention can inject and move a sample solution containing biomolecules to a separation part where nanowires are densely applied by applying an electric field. Therefore, compared with the conventional separation / extraction method using nanostructures produced by the top-down method, separation / extraction method using gel, and separation / extraction method using polymer solution, the time for separating and extracting biomolecules is reduced. It can be shortened.

1 is a conceptual diagram illustrating an example of a biomolecule separation / extraction method using the biomolecule separation / extraction device of Embodiment 1. FIG. 6 is a conceptual diagram illustrating an example of a biomolecule separation and extraction method using the biomolecule separation and extraction device of Embodiment 2. FIG. It is a figure which shows an example of the preparation procedures of the device for isolation | separation extraction of a biomolecule. 2 is an optical photograph of the device produced in Example 1. It is the microscope picture which expanded the vicinity where the microchannel of Drawing 4A intersects. 4 is an FESEM photograph in which a separation portion of the device manufactured in Example 1 is enlarged. 4 is an FESEM photograph in which a separation portion of the device manufactured in Example 2 is enlarged. 4 is an FESEM photograph in which a separation portion of the device manufactured in Example 3 is enlarged. It is the FESEM photograph which expanded the isolation | separation part part of the device produced in Example 4. FIG. 6 is an FESEM photograph in which a separation portion of the device produced in Example 5 is enlarged. It is a graph which shows that T4 and (lambda) DNA were isolate | separated by the isolation | separation part by the isolation | separation experiment of Example 6. FIG. It is a graph which shows that 38 kbpDNA and 10 kbpDNA were isolate | separated by the isolation | separation part by the isolation | separation experiment of Example 7. FIG. It is a graph which shows that 10 kbpDNA and 1 kbpDNA were isolate | separated by the isolation | separation part by the isolation | separation experiment of Example 8. FIG.

Hereinafter, a device for separating and extracting a biomolecule according to an embodiment of the present invention, a method for manufacturing the same, and a method for separating and extracting a biomolecule will be described in detail.

First, in the present embodiment, “branched chain” means a nanowire branched from a formed nanowire. Therefore, the nanowire branched from the nanowire first formed on the substrate is the “branched chain” of the present embodiment, and the nanowire further branched from the “branched chain” is also the “branched chain” of the present embodiment. In the present embodiment, “stem” or “nanowire to be a trunk” means a nanowire formed first on a substrate.

In the present embodiment, the “number of branches” means the number of repeated branches from the formed nanowires. For example, a nanowire with a branch number of 1 means a nanowire in which a branched chain is formed on a trunk nanowire, and a nanowire with a branch number of 2 means that a branched chain is further formed on a branched chain branched from the trunk nanowire. Means nanowire.

Furthermore, in the present embodiment, the “branched chain forming step” means a step for forming a branched chain. In this embodiment, as will be described later, a catalyst is deposited on the formed nanowire, and the nanowire is formed from the catalyst. Therefore, for example, when the branch chain forming process is performed twice, the catalyst is deposited on the main nanowire in the first branch chain forming process, but the catalyst may not be deposited on all the nanowires. As a result, the formed nanowire includes (1) a nanowire in which a branched chain is formed on the trunk, and (2) a trunk in which a branched chain is not formed. In the second branched chain formation step, the catalyst is deposited on the trunk and the branched chain of (1) and the trunk on which the branched chain of (2) is not formed, but the catalyst is still not deposited. It may contain a trunk. As a result, (a) a nanowire in which a branched chain is further formed on a branched chain branched from the trunk, (b) a nanowire in which a branched chain is formed from the trunk, and (c) a nanowire in which the trunk is mixed can be formed. There is sex. Further, in the nanowire in which a branched chain is further formed on the branched chain branched from the trunk of (a), the branched chain formed on the branched chain branched from the trunk (a-1) in one nanowire. And (a-2) a branched chain branched from the trunk may be formed. Therefore, in the present embodiment, when the “branched chain forming step” is described as n times, it is sufficient that the formed nanowire includes at least a nanowire having n branches, and includes a nanowire having a branch number smaller than n. It may be. Similarly, when “branch number n” is described, it is only necessary to include nanowires having n branches, and nanowires having a branch number smaller than n may be included. In the following description, when nanowires having 0 to n branches are described without distinction, they may be simply referred to as nanowires.

The “biomolecule” in the present embodiment means a biomolecule such as DNA, RNA, protein and the like, and is not particularly limited as long as it can be separated and extracted by the nanowire of the present embodiment.

FIG. 1 is a conceptual diagram showing an example of a biomolecule separation and extraction method using the biomolecule separation and extraction device (hereinafter also simply referred to as “device 1”) according to Embodiment 1 of the present invention. is there. A first microchannel 20 is formed on the substrate 10 of the device 1, and a sample input unit 21 and a separated sample collection unit 22 are formed in the first microchannel 20. In addition, the first microchannel 20 is formed with a separation unit 30 in which nanowires having branched chains are in a dense state. The sample solution 40 containing biomolecules is input to the sample input unit 21 and an external electric field is applied to the first microchannel 20, whereby the input sample solution 40 moves toward the separated sample recovery unit 22. The sample solution 40 is size-separated by the separation unit 30 and collected by the separated sample collection unit 22. For example, the device 1 continues to flow the sample solution 40 containing biomolecules, and the biomolecules of a predetermined size or larger are captured by the separation unit 30 and do not flow downstream, but only the biomolecules of a predetermined size or smaller are selectively used. It is effective when separating and extracting.

FIG. 2 shows a device for separating and extracting biomolecules according to Embodiment 2 of the present invention (hereinafter, simply referred to as “device 2”. Note that when

devices

1 and 2 are described without distinction, “ It is a conceptual diagram which shows an example of the isolation | separation extraction method of the biomolecule using the "device". As shown in FIG. 2, a first microchannel 20 and a second microchannel 50 that intersects the first microchannel 20 are formed on the substrate 10 of the device 2. A separated sample collection unit 22 and a separation unit 30 are formed in the first microchannel 20. In the second microchannel 50, a sample input unit 21 and a sample recovery unit 51 for recovering the input unseparated sample solution are formed. The sample solution 40 containing the biomolecule is introduced into the sample introduction unit 21 and an external electric field is applied to the second microchannel 50, whereby the introduced sample solution 40 is supplied to the sample recovery unit 51 as shown in FIG. Move in the direction of. Next, when the application of the external electric field to the second microchannel 50 is stopped and the external electric field is applied to the first microchannel 20, as shown in FIG. 2B, the first microchannel 20 and the second microchannel Only the sample solution 40 corresponding to the volume of the portion where the paths 50 intersect moves in the direction of the separation unit 30. The sample solution 40 is size-separated by the separation unit 30 and can be collected from the separated sample collection unit 22 of the first microchannel 20. Since the device 2 can switch the micro flow path to which an external voltage is applied at regular intervals and flow the sample solution 40 having a predetermined volume to the separation unit 30 for separation and extraction, the biomolecule contained in the sample solution 40 It is also useful for statistically examining the size distribution of

The substrate 10 used for forming the device of the present embodiment includes PDMS (poly (dimethylsiloxane)), PMMA (poly (methyl methacrylate)), PC (polycarbonate), plastic made of hard polyethylene, silicon, glass, and the like. There is no particular limitation as long as a path can be formed and nanowires can be grown.

The first microchannel 20 and the second microchannel 50 can be manufactured by using a photolithography technique described later. The width and depth of the first microchannel 20 and the second microchannel 50 are not particularly limited as long as the sample solution 40 containing biomolecules can flow and nanowires having branched chains can be grown sufficiently. .

In the device 1, if the sample solution 40 can be directly input to the first microchannel 20 on the substrate 10 and the separated sample separated by the separation unit 30 can be directly recovered, the sample introduction unit 21 and the separation completed There is no particular need to provide the sample collection unit 22. Further, in the device 2, if the sample solution 40 can be directly input into the second microchannel 50, and the unseparated sample solution and the separated sample solution can be directly recovered, the sample input unit 21, the separated There is no need to provide the sample collection unit 22 and the sample collection unit 51 in particular. When these are provided, the size, shape, etc. are not particularly limited as long as they are provided so as to be connected to the microchannel, and may be formed by an ultrasonic drill, a sand blaster, or the like.

The device of the present embodiment can be manufactured using electron beam lithography and photolithography techniques. FIG. 3 is a diagram showing an example of the manufacturing procedure of the device of this embodiment, and shows an example in which the branch chain forming step is performed three times. In FIG. 3, the device separation portion 30 is shown in an enlarged manner. A device manufacturing procedure will be described below with reference to (1) to (13) of FIG.

(1) A Cr layer is deposited on the substrate 10 by sputtering.
(2) A positive photoresist is applied by a spin coater, and then, by photolithography, the first microchannel 20 in the case of the device 1 and the first microchannel 20 and the second microchannel 50 in the case of the device 2 Create a pattern. After developing the resist, the Cr layer of the first microchannel 20 or the first microchannel 20 and the second microchannel 50 is etched with a Cr etchant solution.
(3) The first micro-channel 20 or the first micro-channel 20 and the second micro-channel 50 are produced by a method of selectively etching only the substrate 10, and then the resist on the substrate 10 is removed. . Thereafter, if necessary, through holes for the sample loading unit 21, the separated sample collection unit 22, and the sample collection unit 51 are formed by an ultrasonic drill or the like. Furthermore, in the case of the device 2, a through hole for forming an electrode (hereinafter referred to as “electrode hole”) may be described at the end of the first microchannel 20 on the opposite side of the separated sample collection unit 22 as necessary. Is formed by an ultrasonic drill or the like.
(4) A nanowire growth catalyst is deposited on the surface of the microchannel forming the separation part 30 by sputtering. The catalyst is preferably deposited only in the microchannel, but even if deposited on the substrate and nanowires are formed from the deposited catalyst as shown in the figures after (5) of FIG. 3, Since it can be removed in a process described later, there is no particular problem.
(5) Nanowires are formed from the catalyst deposited in (4) above.
(6) A catalyst is deposited on the nanowire formed in (5) above by sputtering. In the step (5), nanowires are formed in random directions. Therefore, in the step (6), when the catalyst is sputtered, the catalyst is deposited around the side surface of the nanowire in the direction in which the catalyst flies.
(7) A nanowire is formed from the portion where the catalyst is deposited in the above (6), and a nanowire having 1 branch is formed.
(8) A catalyst is deposited on the nanowire having 1 branch formed in the above (7) by sputtering.
(9) A nanowire is formed from the portion where the catalyst is deposited in (8) above, and a nanowire having two branches is formed.
(10) A catalyst is deposited on the nanowire having two branches formed in (9) by sputtering.
(11) A nanowire is formed from the portion where the catalyst is deposited in the above (10), and a nanowire having 3 branches is formed.
(12) Etching Cr stacked on a substrate portion other than the microchannel with a Cr etchant solution to remove nanowires formed in the portion other than the microchannel.
(13) If necessary, the sample input part 21 formed in (3) above, the separated sample recovery part 22, and the through hole part for the sample recovery part 51, and in the case of the device 2, the first microchannel Electrodes are formed in the 20 electrode holes. Then, a cover glass covers the surface on which the first microchannel 20 or the first microchannel 20 and the second microchannel 50 are formed.
The above procedure shows an example in which the number of branch chain forming steps is three. However, the deposition of the catalyst on the nanowire and the formation of the nanowire from the catalyst take into consideration the size of the biomolecule to be separated, etc. What is necessary is just to repeat the frequency | count desired as the frequency | count of 1 or more.

The positive photoresist in the above procedure is not particularly limited as long as it is generally used in the semiconductor manufacturing field, such as TSMR V50, PMER. In addition, a negative type photoresist may be used instead of the positive type, and in this case, 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 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 . In this embodiment, depositing on the substrate in step (1) 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 the substrate 10, for example, when quartz glass is used for the substrate, it may be etched with CF ₄ gas, SF ₆ gas or the like using a reactive ion etching apparatus. When a material other than quartz glass is used as the substrate, the material may be etched by a method that can selectively etch the material.

The catalyst for growing the nanowire is not particularly limited as long as it is a liquid metal, and examples thereof include gold, platinum, aluminum, copper, iron, cobalt, silver, tin, indium, zinc, and gallium. Further, as will be described later, the nanowire of the present embodiment grows in a bottom-up manner, but the diameter of the nanowire serving as a trunk is almost the same as that of one molecule of the catalyst. What is necessary is just to select a catalyst suitably.

The nanowire of the separation part 30 is formed by growing from the catalyst deposited in the microchannel as described above by the bottom-up method, and first, the nanowire as a trunk is formed. The formation of the nanowire is not particularly limited as long as it is a general physical vapor deposition method such as pulsed laser deposition or VLS (Vapor-Liquid-Solid) method.

The core nanowire is a nanowire having the same diameter as one catalyst molecule and grows from the separation part 30 with the catalyst facing upward. In order to control the diameter of the nanowire, it is necessary to select a catalyst molecule having a desired size and to grow a nanowire serving as a trunk at a temperature and pressure at which the catalyst does not disappear. Although it varies depending on the catalyst, the temperature is preferably 400 to 900 ° C. and the pressure is preferably 0.1 to 10 Pa.

The branched chain can be produced by depositing a catalyst on the formed nanowires by sputtering and forming nanowires from the catalyst as shown in the procedure for producing the separation and extraction device. The material, conditions, and the like that form the branched chain may be the same as those of the nanowire serving as the trunk. Further, the lengths of the nanowires and the branched chains that are trunks are not particularly limited, and may be appropriately adjusted in consideration of the depth of the separation unit 30 and the number of branch chain forming steps. The length of the branched chain can be adjusted by changing the deposition time.

The material forming the _{_{nanowire, 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 the like.

The diameter of the nanowire formed depends on the size of the catalyst. Therefore, the diameter of the nanowire does not change even if the number of branch chain forming steps changes. Therefore, by adjusting the vapor deposition time and the number of branch chain forming steps in each branched chain forming step, and further by changing the time for sputtering the catalyst and adjusting the amount of catalyst to be deposited, the desired density can be separated. The portion 30 can be formed.

On the other hand, after forming a nanowire with n branches through a desired number of branch chain formation steps, a coating layer may be formed around the nanowire with n branches. The coating layer is not particularly limited as long as it is a general vapor deposition method such as sputtering, EB vapor deposition, PVD, or ALD. As coating conditions, it is necessary to carry out under a temperature condition in which the nanowire does not melt, and it is desirable to carry out at 800 ° C. or less although it depends on the nanowire material. What is necessary is just to adjust the thickness of a coating layer suitably by changing vapor deposition time. For example, when nucleic acid is separated and extracted as a biomolecule, it is preferable that the nucleic acid is not adsorbed on the surface of the nanowire by electrostatic interaction. Therefore, the coating layer is preferably formed from a material having an isoelectric point lower than that of nucleic acid, and among these materials, SiO ₂ and TiO ₂ are preferable.

In this embodiment, a nanowire having a branched chain having n branches is formed in a dense state in the separation unit 30, thereby forming a filter made of nanowires having a branched chain. As a result, similar to the principle of chromatography, small-sized biomolecules pass through the separation unit 30 quickly, but large-sized biomolecules take time to pass through the separation unit 30 or are moved by the nanowire filter. Is prevented. As a result, biomolecules contained in the sample solution 40 can be separated according to size. As described above, the nanowire having a branched chain according to the present embodiment increases the number of times the branched chain forming step is performed, thereby further forming a branched chain from the branched chain, and a space through which the biomolecule in the separation unit 30 passes. Becomes dense. Also, increasing the amount of catalyst deposited can increase the nanowire density per unit volume, resulting in a dense space. Furthermore, the diameter of each nanowire can be adjusted by changing the thickness of the coating layer of the nanowire. Therefore, in the nanowire having a branched chain formed in the separation unit 30 of the present embodiment, the number of nanowire branches, the nanowire density per unit volume, and the thickness of the coating layer of the nanowire so that an intended sample size can be separated. May be adjusted as appropriate. Moreover, the separation part 30 may be formed from nanowires with n branches, or a plurality of separation parts 30 may be provided, and each may be formed from nanowires with different branch numbers n.

The electrode to be provided in the sample input part 21, the separated sample recovery part 22, the through hole for the sample recovery part 51, and the electrode hole is not particularly limited as long as it is a commonly used electrode material such as platinum or gold. There is no.

Next, a method for separating and extracting biomolecules using the device of this embodiment will be described. For example, when a nucleic acid is used as a biomolecule, the sample solution 40 is prepared by extracting the nucleic acid from cultured cells or the like by a known method and adding it to the solution. The solution is not particularly limited as long as it is a solution capable of suppressing nucleic acid degradation, such as TE buffer. When the device 1 is used, the sample solution 40 is loaded into the sample loading unit 21 and a voltage is applied so that the sample loading unit 21 is negative and the separated sample collection unit 22 is positive. Then, the nucleic acid in the sample solution 40 is subjected to size separation while passing through the separation unit 30, and then collected from the separated sample collection unit 22.

On the other hand, when the device 2 is used, the sample solution 40 is loaded into the sample loading unit 21, and a voltage is applied so that the sample loading unit 21 is negative and the sample collection unit 51 that collects an unseparated sample is positive. The application of voltage is stopped at an arbitrary timing when the sample solution 40 passes through a portion intersecting the first microchannel 20. Next, a voltage is applied so that the separated sample recovery unit 22 is positive and the end of the first microchannel 20 opposite to the separated sample recovery unit 22 is negative. Then, the sample solution 40 corresponding to the volume of the portion where the first microchannel 20 and the second microchannel 50 intersect moves in the direction of the separated sample collection unit 22, and the nucleic acid in the sample solution 40 is separated by the separation unit 30. The size separation is performed while passing the sample, and thereafter, the sample is collected from the separated sample collection unit 22. When the device 2 is used, the separation and extraction of the nucleic acid contained in the same sample solution 40 are repeatedly performed by repeatedly switching the voltage application to the first microchannel 20 and the second microchannel 50. Can do. The voltage to be applied is not particularly limited as long as the nucleic acid can migrate, and may be, for example, 1 V / mm or more.

In the above example, a negatively charged nucleic acid is separated and extracted as a biomolecule. For example, when a positively charged biomolecule such as a protein is separated and extracted, voltage application is applied to the nucleic acid. By reversing the case, separation and extraction are possible. Further, when a coating layer is provided on the nanowire, it is desirable to provide the coating layer with a positively charged material such as nickel oxide so that positively charged biomolecules are not adsorbed on the nanowire.

Hereinafter, the present invention will be specifically described with reference to examples. However, these examples are provided merely for the purpose of explaining 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.

[Production of devices]
<Example 1>
The device of the present invention was produced by the following procedure. First, a Cr layer with a thickness of 250 nm was deposited on quartz glass (Crystal Base Co.) using an RF sputtering apparatus (SVC-700LRF, Sanyu Denshi). Next, a positive type photoresist (TSMR V50, Tokyo Ohka Kogyo Co.) was applied by a spin coater. Thereafter, intersecting microchannel patterns having a width of 25 μm were produced by photolithography. After developing the resist, the Cr layer was etched for 5 minutes with a Cr etchant solution (H ₂ O: Ce (NH ₄ ) ₂ (NO ₃ ) ₆ : HClO ₄ = 85: 10: 5 (mass% ratio)). Two microchannels intersecting each other with a depth of 2 μm were produced by CF ₄ gas using a reactive ion etching apparatus (RIE-10NR, Samco Co.). Next, the resist was peeled off using a resist removing solution (NMD3, Tokyo Ohka Kogyo Co.). A sample insertion part, a separated sample recovery part, a sample recovery part, and an electrode hole having a diameter of 1.5 mm were produced by an ultrasonic drill (SOM-121, Shinoda Co.). Thereafter, a gold catalyst was deposited over the entire microchannel by sputtering, and pulsed laser deposition was performed using SnO ₂ as a material at room temperature for 10 Pa for 60 minutes, thereby producing a nanowire serving as a trunk. Next, a gold catalyst was deposited on the prepared nanowires by sputtering, and a branched chain forming step was performed by performing pulse laser deposition for 60 minutes at room temperature at 10 Pa using SnO ₂ as a material, thereby producing nanowires having 1 branch. . Finally, the device of the present invention was manufactured by covering the surface on which the microchannel was formed with a quartz cover glass having a thickness of 130 μm. The diameter of the produced nanowire was about 10 nm.

FIG. 4A is an optical photograph of the device produced in Example 1, showing the overall appearance of the device. FIG. 4B is an enlarged micrograph of the vicinity where the microchannels of FIG. 4A intersect, and the width L of the first microchannel and the second microchannel is about 25 μm. In addition, although only a part is shown in FIG. 4B, the length of the separation portion was 4 mm. Moreover, the depth of the microchannel was 2 μm. FIG. 5A is an FESEM photograph in which a separation portion of the device manufactured in Example 1 is enlarged.

<Example 2>
A nanowire device having 2 branches was prepared in the same procedure as in Example 1 except that the branch chain forming step in Example 1 was repeated twice. FIG. 5B is an FESEM photograph in which a separation portion of the device produced in Example 2 is enlarged.

<Example 3>
A nanowire device having 3 branches was prepared in the same procedure as in Example 1 except that the branch chain forming step in Example 1 was performed 3 times. FIG. 5C is an FESEM photograph in which a separation portion of the device manufactured in Example 3 is enlarged.

<Example 4>
A nanowire device having 4 branches was prepared in the same procedure as in Example 1 except that the branch chain formation step in Example 1 was performed 4 times. FIG. 5D is an FESEM photograph in which a separation portion of the device manufactured in Example 4 is enlarged.

<Example 5>
A nanowire device having 5 branches was prepared in the same procedure as in Example 1 except that the branch chain forming step in Example 1 was performed 5 times. FIG. 5E is an enlarged FESEM photograph of the separation portion of the device produced in Example 5.

As is clear from the photographs of FIGS. 5A to 5E, as the number of branch chain forming steps was increased, it was possible to form a separation part in which nanowires having branched chains were densely packed.

[Preparation of sample solution]
T4, λDNA, 38 kbpDNA, 10 kbpDNA (all manufactured by Nippon Gene) were prepared as DNA samples, and the DNA samples of the combination used in the examples described later were put into a sample tube, and TE buffer (10 mmol / l Tris-HCl was used. (PH 8.0), 1 mmol / l EDTA (pH 8.0); manufactured by Nippon Gene Co., Ltd.) and 10 mM YOYO-1 (manufactured by Life Technologies Japan Co., Ltd.) were added to adjust the concentration to be described later. The sample solution was stained with nucleic acid. DNA and YOYO-1 were mixed so that dye: basepair = 1: 15.

[Nucleic acid separation experiment]
<Example 6>
The device (number of branches) prepared in 0.9 μl of the DNA sample solution prepared so that the concentration of T4 (about 166 kbp) was 20 ng / μl and the concentration of λDNA (about 48.5 kbp) was 50 ng / μl was prepared in Example 1 above. The sample was introduced into the sample introduction part of 1). A voltage of 20 V / mm was applied to the platinum electrode inserted into the sample input part and the sample recovery part so that the sample input part was negative and the sample recovery part was positive. At the timing when the sample passes through the crossing part of the micro flow path, the voltage application to the sample input part and the sample recovery part is stopped, and the electrode hole is connected to the platinum electrode inserted into the separated sample recovery part and the electrode hole. Is negative, and the separated sample recovery part is positive, and a voltage of 6.67 V / mm is applied for about 2 seconds.

FIG. 6 is a graph showing that T4 and λDNA were separated in the separation part in the separation experiment of Example 6. In the figure, the horizontal axis represents the distance from the end of the separation unit on the side where the sample solution flows, and the vertical axis represents the fluorescence intensity at the distance. As is clear from FIG. 6, when a sample solution containing T4 and λDNA is allowed to flow into a separation part containing a nanowire with one branch, T4 and λDNA can be accurately obtained with a moving distance of less than 150 μm for only about 2 seconds. Could be separated.

<Example 7>
Using 38 kbp DNA with a concentration of 39 ng / μl and 10 kbp DNA with a concentration of 10 ng / μl as a sample, applying the device prepared in Example 2 (number of branches 2) to the separated sample collection part and the platinum electrode inserted into the electrode hole The sample was separated in the same manner as in Example 6 except that was set to about 3 seconds.

FIG. 7 is a graph showing that 38 kbp DNA and 10 kbp DNA were separated in the separation part by the separation experiment of Example 7. As is apparent from FIG. 7, when 38 kbp DNA and 10 kbp DNA sample solution are allowed to flow into the separation part containing nanowires with 2 branches, 38 kbp DNA and 10 kbp DNA are accurately separated with a moving distance of less than 300 μm for only about 3 seconds. I was able to.

<Example 8>
Samples were separated in the same manner as in Example 6 except that 10 kbp DNA with a concentration of 15 ng / μl and 1 kbp DNA with a concentration of 20 ng / μl were used and the device (number of branches 4) prepared in Example 4 was used.

FIG. 8 is a graph showing that 10 kbp DNA and 1 kbp DNA were separated in the separation part by the separation experiment of Example 8. As is clear from FIG. 8, when 10 kbp DNA and 1 kbp DNA sample solution are allowed to flow into the separation part containing nanowires having 4 branches, 10 kbp DNA and 1 kbp DNA are separated accurately with a moving distance of less than 150 μm for only about 2 seconds. I was able to.

From the above experimental results, it was revealed that samples with different sizes can be efficiently separated in a short time by changing the number of nanowire branches in the separation part. In the above example, in order to show that the separation can be performed in a shorter time, it was shown that the separation unit can be separated by the moving DNA, but by continuing to apply voltage, it is possible to recover the sample that has passed through the separation unit. Of course it is possible.

A sample can be separated with high accuracy in a short time by using a biomolecule separation / extraction device in which a separation part including a nanowire having a branched chain is formed on a microchannel provided on a substrate. In addition, by adjusting the number of branches, it is possible to separate and extract biomolecules of a desired size, which is useful for easy and rapid separation and extraction of biomolecules at medical institutions, universities, companies, research institutions, etc. It is.

Claims

A device for separation and extraction of biomolecules, in which a separation part including nanowires having a branched chain with one or more branches is formed on a microchannel provided on a substrate.
The device for separating and extracting a biomolecule according to claim 1, wherein the branched chain is a nanowire grown from a catalyst deposited on the nanowire.
The biomolecule separation / extraction device according to claim 1 or 2, wherein the microchannel is formed of two intersecting microchannels, and the separation unit is formed only in one microchannel. device.
The biomolecule separation according to any one of claims 1 to 3, wherein a sample input unit for supplying a sample solution and a separated sample recovery unit for recovering a separated sample are provided in the microchannel. Extraction device.
Forming a microchannel on a substrate; depositing a catalyst on the microchannel; forming a nanowire from the catalyst; depositing a catalyst on the nanowire and forming a nanowire from the catalyst; Including,
A method for producing a device for separating and extracting biomolecules, comprising the steps of depositing a catalyst on the nanowire and forming the nanowire from the catalyst one or more times.
The method for producing a device for separating and extracting a biomolecule according to claim 5, further comprising a step of forming a sample input unit for introducing a sample solution into the microchannel and a separated sample recovery unit for recovering the separated sample. .
A step of introducing a sample into one end of a microchannel of a biomolecule separation and extraction device in which a separation part including a nanowire having a branched chain having a branch number of 1 or more is formed on a microchannel provided on a substrate;
Applying an electric field to the microchannel;
Separating biomolecules in the separation unit;
A method for separating and extracting biomolecules.
On one microchannel of the intersecting microchannels provided on the substrate, a separation unit of a biomolecule separation / extraction device in which a separation unit including a nanowire having a branched chain having a branch number of 1 or more is formed. Introducing a sample into one end of a non-microchannel,
Applying an electric field to the microchannel where the separation part is not formed;
Stopping the application of the electric field to the microchannel where the separation part is not formed, and applying the electric field to the microchannel where the separation part is formed;
Separating biomolecules in the separation unit;
A method for separating and extracting biomolecules.