WO2006129845A1

WO2006129845A1 - Apparatus and method for fabricating nanoconstruct

Info

Publication number: WO2006129845A1
Application number: PCT/JP2006/311237
Authority: WO
Inventors: Satoshi Murata; Kotaro Somei
Original assignee: Tokyo Institute Of Technology
Priority date: 2005-06-03
Filing date: 2006-06-05
Publication date: 2006-12-07
Also published as: JP2006334741A

Abstract

A solution is dropped into a service port (20) in a microchannel (13) and then fed into a capillary pump (23) due to the capillary force. The temperature in a reaction chamber (22), in which a starting strand serving as a crystal seed for a nanoconstruct is fixed, is controlled with a heater and a temperature sensor. In fabricating the nanoconstruct, for example, solutions each containing a single DNA tile dissolved therein are successively dropped into the service port (20) and crystals of the nanoconstruct grow around the seed crystal in the solution flows.

Description

Nanostructure fabrication apparatus and method

Technical field

The present invention relates to a nanostructure production apparatus and method suitable for use in producing a nanostructure by self-assembling components such as DNA tiles, for example. This application claims priority on the basis of Japanese Patent Application No. 2005-164203 filed on June 3, 2005 in Japan, and this application is incorporated herein by reference. .

Background art

[0002] In recent years, DNA molecules have attracted attention as components of nanostructures. There are various motifs of the DNA molecules that constitute the constituent elements. For example, DNA tiles, DNA junctions, DNA polygons, and DNA polyhedra have been actively studied. Each of these DNA molecules has a different shape and number and position of sticky ends (single-stranded parts that do not form a double helix structure), and the structures formed by self-assembly are also different. Among them, the DNA tile is expected as a component that can be used to create various nanostructures because the specific binding of each DNA tile can be designed freely by designing the four sticky ends circled in Fig. 10. Yes.

By the way, the self-assembly process of DNA tiles corresponds mathematically to a one-dimensional cellular automaton model called a Wang tile, and it is possible to perform some kind of parallel computation using this algorithmic self-assembly (Algorithmic Self Assembly). -assembly). For example, if a DNA tile set with a sticky end corresponding to an XOR (exclusive OR) operation is used as a state transition rule, a two-dimensional crystal (nanostructure) of a DNA tile having a shellpin skifractal structure is obtained. (For example, refer nonpatent literature 1). At this time, by controlling the concentration of each DNA tile constant, if it is possible to keep the temperature optimal temperature for growth near the critical point, theoretically, extremely low defect sufficient 10 one ^6-10_ about ⁸ as calculated element It is said that nanostructures with a rate can be produced.

However, in the past, all DNA tiles were self-assembled in a single test tube. Since the nanostructures were produced by the batch reaction, it was difficult to control the environmental parameters such as the concentration and temperature of each DNA tile, and the produced nanostructures had many problems. Specifically, in the one-pot reaction, the concentration of each DNA tile decreases as the nanostructure crystals grow larger, so it is necessary to lower the temperature of the reaction solution as the concentration decreases. Is difficult, so the defect rate can only be reduced to about 1%. In addition, since many crystals other than the target nanostructure were formed in the test tube, they inhibited each other's growth, and it was not possible to produce a large nanostructure. Furthermore, in the one-pot reaction, crystals of nanostructures are produced in a solution, so that it is difficult to use them thereafter.

On the other hand, as another approach, stepwise self-assembly, in which two DNA tiles are placed in a test tube to advance self-assembly hierarchically, has been studied (for example, non-patent literature). 2). For example, as shown in FIG. 11A, by adding DNA tiles with index numbers 1 to 16 in a hierarchical manner, a fully-addressable nanostructure with 4 × 4 vertical and horizontal DNA tiles as shown in FIG. 11B can be obtained. It can be realized. In FIG. 11B, each DNA tile is conceptually shown as a square. In this method, self-assembly can be performed relatively reliably. However, the experimental operation is complicated, and it is difficult to control environmental parameters such as DNA tile concentration and temperature, as described above. There was a problem that nanostructures could not be produced.

Non-patent literature 1

Paul W. K. Rothemuna, Nick Papadakins, Eric Winfree, Algorithmic ¾elf— Assembly of DNA Sierpinski Triangles. ", PLoS Biology, 2 (12) e424, 2004

Non-Patent Document 2

T. Η. LaBean et al., "Stepwise DNA Self-Assembly of Fixed-size Nanostructures., Proc. FNANO'05, pp.179- 181

Disclosure of the invention

Problems to be solved by the invention

The technical problem of the present invention has been proposed in view of such a conventional situation, and it is possible to fabricate a complicated and large nanostructure at a desired place with almost no defects. An object of the present invention is to provide a device and a method for producing an effective nanostructure.

Means for solving the problem

[0004] In order to achieve the above-described object, a nanostructure fabrication apparatus according to the present invention is a nanostructure fabrication apparatus that creates a nanostructure in a microchannel, and the microchannel includes a plurality of microchannels. A supply means for supplying a solution containing self-assembling components of the nanostructure to at least a part of the microchannel, and provided downstream of the microchannel. And a reaction vessel in which the seed crystal of the nanostructure is immobilized.

In order to achieve the above-described object, a method for manufacturing a nanostructure according to the present invention is a method for manufacturing a nanostructure in a microchannel, and the microchannel includes: Divided into a plurality of regions, a solution containing self-assembled components of the nanostructure is supplied to at least a portion of the microchannel, and the solution is provided downstream of the microchannel. The nanostructure is produced in a reaction vessel in which a seed crystal of the nanostructure is fixed.

The invention's effect

[0005] According to the nanostructure manufacturing apparatus and method according to the present invention, a complex and large nanostructure can be manufactured at a desired place with almost no defects. Brief Description of Drawings

[0006] FIG. 1 is a diagram showing an external configuration of a microfluidic device in the present embodiment.

FIG. 2 is a diagram showing the shape of a microchannel.

FIG. 3 is a diagram showing the relationship between the position in the microchannel and the capillary force.

[FIG. 4] FIG. 4 is a diagram showing the flow of the solution when the solution is dropped into the service port of the microchannel.

FIG. 5 is a diagram showing a temperature profile of the microfluidic device when a voltage of 24 V is applied to the heater.

FIG. 6A and FIG. 6B are diagrams showing a conventional stepwise self-assembly using a one-pot reaction and a stepwise self-assembly using a microfluidic device. [FIG. 7] FIG. 7 is a diagram showing an example of a nanostructure produced when hierarchical algorithmic self-assembly is performed using a microfluidic device.

FIG. 8 is a diagram showing an example of a microchannel divided into a plurality of regions.

FIG. 9 is a diagram showing another example of a microchannel divided into a plurality of regions.

FIG. 10 is a diagram schematically showing an example of a DNA tile.

FIG. 11A and FIG. 11B are diagrams for explaining a method for producing a nanostructure by a conventional stepwise self-assembly.

BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, specific embodiments to which the present invention is applied will be described in detail with reference to the drawings. In this embodiment, the present invention is applied to a microfluidic device in which DNA tiles are self-assembled in a reaction field in a microchannel (diameter / zm to several hundreds of m) and a nanostructure is produced. It is applied.

First, the external configuration of the microfluidic device in the present embodiment is shown in FIG. As shown in FIG. 1, the microfluidic device 1 in the present embodiment is mainly formed by laminating a silicon substrate 11 on a glass substrate 10. A recess 12 is formed in the center of the silicon substrate 11, and the bottom surface of the recess 12 is a microchannel 13 to 13 for flowing a solution in which a DNA tile is dissolved (hereinafter, there is no need to distinguish between them). , "Microchannel 13"

13

Collectively. ) Is formed. In FIG. 1, the number of microchannels 13 is three, but it is needless to say that the number is not limited to this number. The microchannel 13 has an open surface so that a nanostructure crystal during or after fabrication can be observed with an AFM (Atomic Force Microscope). In order to suppress the evaporation of the solution due to such an open shape, the recess 12 is filled with oil such as mineral oil used in PCR (Polymerase Chain Reaction). In addition, in order to control the temperature during self-assembly, a heater 14 made of ITO (Indium Tin Oxide) and a temperature sensor 15 to 15 (hereinafter, it is necessary to distinguish between the glass substrate 10 and the silicon substrate 11). If there is no

14

Collectively referred to as “Sensor 15”. ) Is provided.

The shape of the microchannel 13 described above is shown in FIG. As shown in FIG. 2, the microchannel 13 includes a service port 20 for dropping a solution in which a DNA tile is dissolved, and a service port 20 described later. A stop valve 21 having a function to perform a reaction, a reaction chamber 22 that is a reaction field in which DNA tiles self-assemble, and a mechanical pump 23 are also configured. Among these, the reaction chamber 22 is fixed with an initial strand that becomes a seed crystal of the nanostructure. As a whole, the microchannel 13 is designed so that the flow path width gradually becomes narrower from the end of the service port 20 to the first pump 23, and as a result, the capillary force is increased. There is a flow of solution from port 20 to the cylinder pump 23. Hereinafter, the direction of the service port 20 is expressed as upstream, and the direction of the mechanical pump 23 is expressed as downstream.

FIG. 3 shows the relationship between the position in the microchannel 13 and the capillary force. As shown in FIG. 3, in the service port 20, in order to guide the solution to the chiral pump 23, the flow path width becomes narrower as it goes downstream, and the capillary force also becomes stronger accordingly. In the stop valve 21, the capillary force suddenly increases as the flow path width suddenly narrows. In the reaction chamber 22, the capillary force decreases slightly as the flow path width increases slightly. ing. In the first pump 23, the flow path width is narrowed again to the same width as the stop valve 21, and accordingly, the capillary force becomes as strong as the position of the stop valve 21.

The flow of the solution when the solution is dropped into the service port 20 in the microchannel 13 will be described with reference to FIG. In FIG. 4, the direction in which the capillary force acts is indicated by an arrow, and the strength of the capillary force is indicated by the size of the arrow. When the solution is dropped onto the service port 20 with a pipette (Fig. (A)), the solution flows downstream by capillary force (Fig. (B)). When reaching the final stop force 21 of the solution, the capillary force in the downstream direction of the capillary pump 23 is balanced with the capillary force in the upstream direction of the stop valve 21, and the solution stays in the peripheral region including the reaction chamber 22. (Figure (C)). Next, when the solution is dropped again with the pipette to the service port 20 (FIG. (D)), the boundary surface at the position of the stop valve 21 disappears, and the solution flows downstream by capillary force (FIG. (E )). Then, when the tail end of the solution reaches the stop valve 21, the solution stays in the peripheral region including the reaction chamber 22 ((F) in the figure). Thus, each time a solution is dropped into the service port 20, a flow of the solution from the service port 20 to the chiral pump 23 occurs. In the microfluidic device 1 in the present embodiment, a solution in which DNA tiles are dissolved is dropped into the service port 20 and fixed in the reaction chamber 22 in the flow of the solution in which the concentration of DNA tiles is constant. A nanostructure crystal is grown around the seed crystal. Thus, by growing the crystal in the flow of the solution, unnecessary by-products around the seed crystal are washed away, and a large nanostructure can be produced. In addition, crystal growth can be suppressed by growing the crystal along the flow of the solution. Furthermore, according to such a microfluidic device 1, conventional one-pot reactions such as dropping ligase into the service port 20 to connect DNA tiles, or dropping restriction enzymes to cleave specific sites, etc. Difficult reactions can be handled freely.

When nanostructures are produced by self-assembly of DNA tiles, not only the concentration of DNA tiles but also the temperature at the time of reaction is an important environmental parameter. In the microfluidic device 1 in this embodiment, The temperature of the reaction chamber 22 can be controlled by the heater 14 and the temperature sensor 15. Specifically, Figure 5 shows the temperature profile of the microfluidic device 1 when a voltage of 24 V is applied to the heater 14. As shown in FIG. 5, a uniform temperature field can be obtained in the three microchannels 13-13. Anti

13

As the temperature at the time, the highest temperature at which DNA tiles can be bonded to each other is preferable. This is because the bonding force between the non-complementary sticky ends is weaker than that between the complementary sticky ends, and thus defects can be suppressed by increasing the temperature as much as possible. This requires precise temperature control, but the microfluidic device 1 has a very large surface area per unit volume, making it extremely easy to achieve very precise temperature control with extremely high heat exchange efficiency. It can be carried out.

When performing algorithmic self-assembly using such a microfluidic device 1, a solution in which a plurality of DNA tiles (DNA tile sets) are dissolved is dropped onto the service port 20. As a result, the initial strand fixed in the reaction chamber 22 is used as a seed crystal to grow nanostructure crystals while keeping the concentration of the surrounding DNA tile constant, and the defect rate is extremely low as theoretically! Large nanostructures can be fabricated.

When performing stepwise self-assembly using microfluidic device 1. In this case, a solution in which one type of DNA tile is dissolved is dropped into the service port 20 sequentially. In this way, by dropping DNA tiles one by one, even if the sequence of the sticky ends of the DNA tile is extremely limited, a hierarchical nanostructure that is impossible with the conventional one-pot reaction is created. can do.

As an example, there are three types of white, black, and gray DNA tiles, as shown in Figure 6. White and black DNA tiles have sticky ends with exactly the same arrangement, and the gray DNA tile is a connector that connects them. Assume a situation where the sequence has sticky ends of the sequence. In addition, in FIG. 6, the arrangement of the sticky ends is conceptually shown by the shape (unevenness) of each piece of the DNA tile represented by a square. In the conventional one-pot reaction, as shown in Fig. 6A, only a nanostructure in which three types of DNA tiles are randomly assembled can be obtained. On the other hand, when the microfluidic device 1 according to the present embodiment is used, as shown in FIG. 6B, a nanostructure in which the DNA tiles of each color are assembled in layers can be produced. Before flowing the DNA tile of each color, it is preferable not to include the DNA tile! /, It is preferable to flow water to remove the extra DNA tile that has not been combined.

It is also possible to extend this and perform algorithmic self-assembly using the microfluidic device 1 in a hierarchical manner. In this case, for example, by evenly dropping a DNA tile set for even layers and a DNA tile set for odd layers corresponding to different state transition rules to the service port 20, the complex as shown in FIG. Patterned nanostructures can be fabricated. In particular, according to the microfluidic device 1, since the sequences of the sticky ends can be made the same in each DNA tile set, it is not necessary to increase the number of sticky end sequences.

Furthermore, in the microfluidic device 1, various proteins that specifically bind to a specific DNA sequence may be dropped onto the service port 20. Since each protein has a unique function, it is necessary to bind it to a nanostructure that also has DNA tiling power, so that a system with a useful function, for example, a group of enzymes necessary to degrade a specific molecule A DNA lattice or the like in which (protein group) is optimally arranged can be constructed.

In the microfluidic device 1, the solution conditions (metal ion concentration and other DN Various DNA effector elements whose shapes change depending on the presence of the A sequence may be dropped on the service port 20. By incorporating such a DNA actor element between DNA tiles, for example, it is possible to construct a filter made of a lattice (the size of the mesh is dynamically changed) that recognizes and passes only specific molecules.

In the microfluidic device 1, a metal colloid (for example, gold colloid) nanoparticle bonded with a DNA binding linker may be dropped onto the service port 20. By arranging them in a row, it becomes a nanowire and a wiring pattern can be formed.

In the microfluidic device 1, various chemically synthesized nanoelectronic devices (electronic elements having rectification and amplification effects) bonded with a DNA binding linker are dropped onto the service port 20. Anyway! Arranging them in a certain pattern makes it possible to construct an arithmetic circuit and a memory circuit.

In FIG. 2, the number of service ports of each microchannel 13 has been described as one. However, the present invention is not limited to such a configuration, and the microchannel is divided into a plurality of areas, and each area is divided into a plurality of areas. A service port may be provided. In this case, DNA tile formation from multiple single-stranded DNAs → Substructure A formation from two DNA tiles → Substructure B formation from two substructures A → Each target nanostructure Steps can be performed in each of the areas described above. Each region is controlled to an optimum temperature. In general, it is preferable to increase the initial stage of fabrication of nanostructures to a high temperature and to a low temperature as the stage progresses.

An example of such a microchannel is shown in FIG. The microchannel 30 shown in FIG. 8 is divided into first to fourth regions 31 to 31, and the first region 31 is heated to a high temperature and the second to third regions 31 to 31 are divided.

The 1 4 1 regions 31 and 31 are controlled at a medium temperature, and the fourth region 31 is controlled at a low temperature. Service port 3

2 3 4

A solution in which single-stranded DNA is dissolved is dropped into 2 and 32, and the service port 32 has a DN.

one two Three

The solution in which the A tile is dissolved is dropped, and the service port 32 has a sub-structure of the DNA tile.

Four

The solution in which the structure is dissolved is dropped. In such a microchannel 30, a DNA tile is formed from a plurality of single-stranded DNAs in the second region 31, and two in the third region 31.

2 3 DNA tiles form the DNA tile substructure, and in the fourth region 31 there are 2 D Sub-structural strength of NA tiles Nanostructures are formed and fixed by reaction chamber 33. Powerful components that cannot be crystallized are refluxed by the micropump 34 and decomposed into single-stranded DNA in the first region 31. The entire solution flow is generated by the micropump 35.

Another example of such a microchannel is shown in FIG. The microchannel 40 shown in FIG. 9 is divided into first to ninth regions 41 to 41, and the first, third, and sixth regions 41, 41,

1 9 1

The second, fourth, fifth, seventh and eighth regions 41, 41, 41, 41, 41 ί

3 6 2 4 5 7 8 The ninth region 41 is controlled to a low temperature. Also, service ports 42-42 have DN

9 1 6

The solution in which the tile is dissolved is dropped, and the solution in which the DNA tile is dissolved is dropped in the service port 42. In such a microchannel 40, the first, third, and sixth regions 41, 41, and 41 are completely decomposed into single-stranded DNA. The second, fourth, and seventh areas

1 3 6

In 41, 41, and 41, DNA tiles are formed from a plurality of single-stranded DNAs, and the fifth and eighth regions are formed.

2 4 7

In regions 41 and 41, the DNA tile substructure is formed from the two DNA tiles, and the ninth region

5 8

In region 41, two DNA tile substructures and nanostructures are formed.

9

Objects are fixed in the reaction chamber 43. The entire solution flow is generated by the micro pump 44.

As shown in Figs. 8 and 9, the DNA tile itself can also be fabricated in the microchannel, thereby making it possible to fabricate nanostructures with a lower defect rate.

In FIG. 8 and FIG. 9, the service port includes not only single-stranded DNA and DNA tile, but also enzyme groups such as ligase and restriction enzyme, protein, DNA activator element and various nanoparticles, It ’s okay to drop nanodevices.

Although the best mode to which the present invention is applied has been described above, the present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the gist of the present invention. Of course.

For example, in the above-described embodiment, other motifs such as DNA junctions, DNA polygons, DNA polyhedra, etc., which are explained using DNA tiles as examples of DNA molecules that are constituent elements of nanostructures, are used. You may use. If the component is not limited to single-stranded DNA or DNA molecules but can be self-assembled, single-stranded RN A or other components such as RNA molecules or proteins may be used.

Claims

The scope of the claims

[1] 1. A nanostructure fabrication apparatus for fabricating a nanostructure in a microchannel, wherein the microchannel is divided into a plurality of regions,

Supply means for supplying a solution containing a self-assembled component of the nanostructure to at least a partial region of the microchannel;

A reaction vessel provided downstream of the microchannel and in which a seed crystal of the nanostructure is fixed;

An apparatus for producing a nanostructure, comprising:

[2] 2. The apparatus for producing a nanostructure according to claim 1, further comprising temperature control means for controlling the temperature of each region of the microchannel and the temperature of the reaction vessel.

[3] 3. The reaction tank has a shape in which a part thereof is opened.

A nanostructure production apparatus according to item 1.

[4] 4. The self-assembling component consists of single-stranded DNA or multiple single-stranded DNAs.

2. The nanostructure production apparatus according to claim 1, which is a DNA molecule.

[5] 5. The apparatus for producing a nanostructure according to claim 4, wherein the DNA molecule is a DNA tile.

[6] 6. A nanostructure fabrication method for fabricating a nanostructure in a microchannel, wherein the microchannel is divided into a plurality of regions,

A solution containing a self-assembled component of the nanostructure is supplied to at least a partial region of the microchannel, provided downstream of the microchannel, and the seed crystal of the nanostructure is fixed. A method for producing a nanostructure, characterized in that the nanostructure is produced in a structured reaction tank.

[7] 7. The method for producing a nanostructure according to claim 6, wherein the temperature of each region of the microchannel and the temperature of the reaction vessel are respectively controlled.

[8] 8. The reaction tank has a shape in which a part thereof is opened.

6. A method for producing a nanostructure according to item 6.

[9] 9. The self-assembling component consists of single-stranded DNA or multiple single-stranded DNAs. 7. The method for producing a nanostructure according to claim 6, wherein the nanostructure is a DNA molecule.

10. The method for producing a nanostructure according to claim 9, wherein the DNA molecule is a DNA tile.