JP2010048714A - Fractionation method of molecule and fractionating chip used for same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a fractionating chip capable of fractionating a molecule to be fractionated by diffusion of a molecule. <P>SOLUTION: The fractionating chip includes a substrate, a channel formed on the substrate, and a plurality of structures disposed in a matrix form in the channel. The respective structures disposed in the channel have asymmetrical shapes to a straight line parallel with the development direction of a lipid film. By performing self development of the lipid film including a molecule to be fractionated in the channel of the fractionating chip, the target molecule to be fractionated can be fractionated according to the diffusion of the molecule. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for fractionating a molecule to be classified contained in a sample and a fractionation chip used therefor.

  Μ-TAS is well known as a technique for manipulating, sorting, and extracting molecules in a minute space. μ-TAS has various advantages such as a short reaction time, high reaction efficiency, and rapid heating and cooling. On the other hand, in μ-TAS, (1) since water is used as a transport medium, the properties of biomolecules (for example, membrane proteins) may be impaired. (2) uncharged molecules are used because electrophoresis is used as a transport driving force. However, there is a possibility that the molecular structure may be changed by an electric field, and (3) the device used is large (centimeter scale).

As a technique for solving the above-described problem, a technique for filtering a molecule by developing a lipid membrane in a flow path in which a plurality of structures are arranged in a matrix is disclosed (for example, see Non-Patent Document 1). In the flow path of the device described in Non-Patent Document 1, structures are arranged so that the interval between the structures is 100 nm or 500 nm. When the lipid membrane is developed in such a channel, the structure of the lipid membrane changes in the gap region between the structures, and the solubility of other molecules in the lipid membrane changes. Therefore, when a lipid membrane containing a specific molecule (a molecule whose solubility decreases in the gap region between structures) is developed in the flow path, this molecule cannot enter the gap region between structures, and the gap between structures Can not pass. Using this phenomenon, molecular filtering can be performed.
H. Nabika, B. Takimoto, N. Iijima and K. Murakoshi, "Observation of self-spreading lipid bilayer on hydrophilic surface with a periodic array of metallic nano-gate", Electrochimica Acta, Volume 53 (2008), Issue 21, Pages 6278-6283.

  In the technique described in Non-Patent Document 1, it is considered that molecules are classified based on factors that determine the chemical potential of the molecules (for example, molecular size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.). ing.

  In the method of separating a molecule by developing a lipid membrane in a channel in which a plurality of structures are arranged in a matrix, the inventor can separate the molecule using a factor other than the above factor as an index. In combination with technology, we thought that the molecules to be separated could be separated with higher accuracy using two factors as indices. And this inventor paid attention to the diffusibility of a molecule | numerator as factors other than the said factor.

  The present invention has been made in view of the above points, and is a method for separating a molecule by developing a lipid membrane in a flow path in which a plurality of structures are arranged in a matrix, wherein the molecule is separated by the diffusibility of the molecule. It is an object of the present invention to provide a sorting method that can be sorted and a sorting chip used therefor.

  The present inventor has found that the above-mentioned problem can be solved by setting the shape of the structure disposed in the flow path to a predetermined shape, and has further studied and completed the present invention.

That is, the first of the present invention relates to the following separation method.
[1] A method for separating molecules to be separated contained in a sample, which includes a substrate, a channel formed on the substrate, and a plurality of structures arranged in a matrix in the channel. Preparing a fractionation chip; a sample containing one or more kinds of molecules to be separated; and a step of preparing a polar lipid; introducing the sample and the polar lipid into a flow path of the fractionation chip; And a step of spontaneously deploying a lipid membrane containing a molecule to be separated and the polar lipid, and each of the structures passes through an apex on the upstream side of the flow path and is parallel to the development direction of the lipid membrane. The distance between the first straight line and the second straight line that passes through the apex on the first side surface side of the flow path and is parallel to the first straight line is such that the first straight line and the second straight line of the flow path Passing through the apex on the side of the first side and parallel to the first straight line Straight and longer than the distance of, and allowed to localize in the region close the first side of the flow path as the diffusion hardly molecules, fractionating the object fractionation molecular fractionation method.
[2] The fractionation method according to [1], wherein the lipid membrane has a lipid bilayer structure.
[3] The separation method according to [2], wherein the step of spontaneously expanding the lipid membrane includes a step of introducing an aqueous electrolyte solution into the flow path of the separation chip.

The second of the present invention relates to the following sorting chip.
[4] A chip for separating molecules to be separated contained in a sample, which is arranged in a matrix in a substrate, a channel formed on the substrate for spontaneous development of a lipid membrane, and the channel. A plurality of structures, and each of the structures passes through an apex on the upstream side of the flow path and is parallel to the development direction of the lipid membrane, and the first of the flow paths The distance between the second straight line passing through the apex on the side surface side and parallel to the first straight line passes through the first straight line and the apex on the second side surface side of the flow path, and the first straight line. Unlike the distance from the third straight line parallel to the straight line, the separation chip has a distance between the one structure and the other structure closest to the structure within a range of 20 to 500 nm.
[5] The sorting chip according to [4], wherein the plurality of structures have substantially the same shape.
[6] The separation chip according to [4] or [5], wherein an interval between the second straight line and the third straight line of the structure is in a range of 100 nm to 3 μm.
[7] Each of the plurality of structures has a substantially rectangular parallelepiped shape having a major axis and a minor axis parallel to the substrate surface, and is arranged so that the directions of the major axes are the same. The sorting chip according to any one of [4] to [6], wherein the direction of the long axis is inclined by 10 to 80 degrees with respect to the development direction of the lipid membrane.
[8] The sorting chip according to [7], wherein the length of the major axis is in the range of 500 nm to 2000 nm, and the length of the minor axis is in the range of 150 nm to 500 nm.

  According to the present invention, a molecule to be separated can be separated based on the diffusibility of the molecule. For example, by combining the present invention with the above-described conventional technique, fractionation by molecular diffusivity and fractionation by a factor that determines the chemical potential of the molecule can be performed simultaneously, and the fractionated molecules can be fractionated with higher accuracy.

  The sorting chip of the present invention is a chip for sorting molecules to be sorted contained in a sample, and includes a substrate, a channel formed on the substrate, and a plurality of structures arranged in a matrix in the channel. Having a body. The sorting chip of the present invention is mainly characterized in that the structure disposed in the flow path has a predetermined shape. As will be described later, by deploying a lipid membrane containing a molecule to be separated in the flow path of the separation chip of the present invention, the target molecule to be separated can be separated according to the diffusivity (diffusion coefficient) of the molecule. it can. In the present specification, the “molecule” means not only one molecule but also a complex composed of two or more molecules. “Separating a molecule” not only means separating the molecule from other molecules, but also increasing the ratio (concentration) of the molecule relative to other molecules.

  The substrate is a member for arranging (forming) the flow path. The magnitude | size of a board | substrate will not be specifically limited if a flow path can be arrange | positioned without difficulty. The material of the substrate is not particularly limited, and can be selected from any material depending on the viewpoint of the method of forming the flow channel and the structure, the properties of the inner surface of the flow channel (for example, whether or not to make it hydrophilic), and the like. . For example, the substrate is a glass plate.

  The channel is a region formed on the substrate for developing the lipid membrane. One channel may be formed on one substrate, or two or more channels may be formed. The structure of the channel is not particularly limited as long as the lipid membrane can be developed. For example, a groove (concave portion) may be formed on the substrate surface to be a flow path, or a plurality of guides (convex portions) may be formed on the substrate and a space between them may be used as the flow path. The width and length of the channel are not particularly limited, and may be set as appropriate according to the type of sample and the number of structures disposed in the sample. The depth of the channel is not particularly limited as long as it is larger than the thickness of the lipid membrane to be developed. The surface (bottom surface) on which the lipid membrane in the flow channel is developed may be hydrophilic or hydrophobic. For example, when a lipid membrane having a lipid bilayer structure is developed (see FIGS. 4C and 9), the surface on which the lipid membrane is developed is preferably hydrophilic.

  The flow path may be connected to an introduction port or an introduction flow path for introducing a sample and polar lipid (lipid constituting the lipid membrane). By providing the introduction port or the like, the user can easily introduce the sample and the polar lipid into the channel. The number of introduction ports (introduction channels) connected to one channel may be one, or two or more. For example, by connecting an inlet (introduction channel) for introducing both the sample and the polar lipid and an inlet (introduction channel) for introducing only the polar lipid to one channel. Thus, it is possible to improve the separation accuracy of the molecules to be separated (see Embodiment 3).

  The structure is a member arranged in a matrix in the flow path. Here, the “matrix shape” means a state in which the two axes do not need to be orthogonal but are arranged in a grid (matrix) orderly. The structure serves as an obstacle to the lipid membrane (and the molecules to be contained therein) that develop in the flow path, and is a member that bears the sorting function of the sorting chip of the present invention. The material of the structure is preferably a material having a high adsorption power to the lipid membrane so that the lipid membrane does not get over the structure. For example, when a lipid membrane having a lipid bilayer structure is developed, the material of the structure may be a metal (for example, gold).

  The shape of the structure is preferably asymmetric with respect to a straight line parallel to the development direction of the lipid membrane when the substrate is viewed in plan. More specifically, as illustrated in FIGS. 1A to 1C, a first straight line that passes through the apex 102 on the upstream side of the flow path of the structure 100 and is parallel to the development direction of the lipid membrane ( And a second straight line (indicated by “2” in the figure) passing through the apex 104 on the first side surface side (right side in the figure) of the flow path and parallel to the first straight line. ) (Indicated by “A” in the figure) through the first straight line (indicated by “1” in the figure) and the apex 106 on the second side surface (left side in the figure) of the flow path, In addition, it is preferably different from a distance (indicated by “B” in the figure) from a third straight line (indicated by “3” in the figure) parallel to the first straight line (in each example of FIG. 1, A is B Longer than). Hereinafter, the distance between the first straight line and the second straight line (indicated by “A” in the figure) is also referred to as “first length”, and the distance between the first straight line and the third straight line (in the figure “ "B") is also referred to as "second length".

  Here, “the apex on the upstream side of the flow path” means the most upstream part of the flow path in one structure. Similarly, the “vertex on the first side surface side of the flow path” means a portion on the first side surface side of the flow path most in one structure, The “vertex” means a portion on the second side surface side of the flow path most in one structure. The “vertices” here do not necessarily have to be strict corners, and may be rounded or planar as shown in FIG.

  The plurality of structures arranged in one flow path do not necessarily have the same shape, but the first length (indicated by “A” in the figure) and the second length (in the figure “ It is preferred that which one of B) is longer is the same. Usually, the shape of the plurality of structures arranged in one flow path is substantially the same shape, and the structures having substantially the same shape are arranged to face the same direction.

  By separating a lipid membrane containing a molecule to be separated in a channel in which the structures having the above shapes are arranged in a matrix, the molecule to be separated can be separated. Here, the principle of separating the molecules to be separated will be described.

  FIG. 2 is a plan view showing an example of a flow path in which a plurality of structures are arranged in a matrix. The broken lines in the figure are lines for indicating the positional relationship between the structures. In this example, a rectangular parallelepiped structure (having an arbitrary height) 100 in which the ratio of the length of the long side to the length of the short side is 4: 1 is inclined so that the long side is 45 degrees with respect to the development direction of the lipid membrane. Is arranged. As a result, in all the structures 100, the first length (“A” in FIG. 1A) is four times longer than the second length (“B” in FIG. 1A). The shortest distance between the structures 100 is the same as the length of the short side. In this figure, it is assumed that a lipid membrane containing a molecule to be separated is spontaneously expanded (described later) from the top to the bottom. The molecules to be separated in the lipid membrane diffuse slowly in the lipid membrane by Brownian motion and slowly downstream (downward in the figure) according to the flow of the lipid membrane (usually about 0.1 to 0.3 μm / s). Move to.

In FIG. 2, it is assumed that one molecule to be separated exists at the position of the point S (the gap region between the two structures). This molecule moves downstream while diffusing in the lipid membrane. In many cases, the molecule is a gap between the points C located almost immediately below the point S or the first side of the flow path from the point S (in the figure). It reaches the gap of the point L located on the left side or the gap of the point R located on the second side surface side (right side in the figure) of the flow path than S (if the diffusion coefficient of the molecule is large, It may reach a far gap). Here, when the probability that the molecule located at the point S reaches the point L is P _L , the P _L is expressed by the following equation (1).
Here, D is the diffusion coefficient of the molecule, v is the development rate of the lipid membrane, and a is the length of the short side of the structure.

Similarly, when the probability of molecules located at the point S reaches the point R and P _R, P _R is represented by the following formula (2).

Here, assuming v = 0.2 μm / s and a = 0.25 μm, va = 0.05. In this case, when the diffusion coefficient of the molecules located at the point S is _{^{2μm 2 / s, P L =}} 0.46, P R = 0.50 , and the diffusion coefficient of molecules located at the point S is 1 [mu] m ² / s Then, P _L = 0.43 and P _R = 0.49. Thus the diffusion coefficient of the molecule is large (e.g., D ≧ 1μm 2 ^{/ s)} when a large difference between the P _L and P _R are not observed. That is, the molecules diffuse in random directions.

On the other hand, if the diffusion coefficient of the molecules located at the point S is _{^{0.1μm 2 / s, P L =}} 0.10, P R = 0.44 next, the diffusion coefficient of the molecules located at the point S is 0.01 [mu] m ² When / s, P _L = 0.05 × 10 ⁻⁴ and P _R = 0.15. Thus the diffusion coefficient of the molecule is small (e.g., D ≦ 0.1μm ² / s) when the value of _{P R} is significantly larger than the _{P L.} That is, the molecules diffuse in a specific direction (right direction in the figure).

Therefore, as shown in FIG. 3, when a lipid membrane containing two kinds of molecules to be separated having different diffusion coefficients is developed, molecules having a large diffusion coefficient diffuse randomly in the channel (“D” in the figure). ≧ 1 μm ² / s ”), molecules having a small diffusion coefficient are unevenly distributed on one side of the channel (indicated by“ D ≦ 0.1 μm ² / s ”in the figure). When the first length is longer than the second length, molecules having a small diffusion coefficient are unevenly distributed on the first side surface side, and when the second length is longer than the first length, the diffusion coefficient is Small molecules are unevenly distributed on the second side. As a result, molecules having a large diffusion coefficient and molecules having a small diffusion coefficient are distinguished. When lipid membranes containing multiple types of molecules to be separated with different diffusion coefficients are deployed, molecules with a smaller diffusion coefficient are more unevenly distributed in regions closer to the side of the channel, and molecules with a larger diffusion coefficient It is unevenly distributed in a region away from the side surface (see FIG. 5C). As a result, a plurality of types of molecules to be sorted with different diffusion coefficients are sorted. As long as the shape of the structure meets the above requirements, even if the deployment speed of the lipid membrane, the arrangement pattern of the structure, the size of the structure, etc. are changed, the molecule can be converted according to its diffusion coefficient according to the same principle. Can be separated.

  Return to the description of the structure. The size of the structure is not particularly limited, but the total of the first length and the second length (the sum of A and B in FIG. 1) is preferably in the range of 100 nm to 3 μm, More preferably, it is in the range of 1 μm. When the total of the first length and the second length exceeds 3 μm, there is a possibility that the molecule to be separated cannot be separated particularly when the lipid membrane is spontaneously developed. For example, in the above formulas (1) and (2), when the lipid membrane development rate (v) is about 0.1 to 0.3 μm / s, the total of the first length and the second length If it exceeds 3 μm, the value of va / D will not fall within the preferred range (about 0.1 to 10), and it may be difficult to separate the molecules to be separated.

  It is preferable that one length is 1.5 times or more with respect to the other length from a viewpoint of improving 1st length and 2nd length with respect to the other length. Moreover, when the diffusion coefficient of the molecule to be separated is large, it is preferable that one length is longer (for example, 3 times or more) than the other length. The preferred ratio of the first length to the second length is determined by the absolute value of the diffusion coefficient of the molecule to be separated, and the larger the diffusion coefficient of the molecule to be separated, the more one length is relative to the other. It is necessary to make it longer. The height of the structure is preferably such a height that the lipid membrane cannot be overcome.

  The distance between structures adjacent to each other in the width direction of the channel (see “c” in FIG. 5B and “d” in FIG. 6B) is such a size that a molecule to be separated and a polar lipid can pass through. If it is, it will not specifically limit, For example, what is necessary is just 20 nm or more. As shown in the second embodiment to be described later, by setting the interval between the structures adjacent in the width direction of the flow channel within the range of 20 to 500 nm, not only the molecular diffusion coefficient but also other factors (size, The molecules can be fractionated using the configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.). When the interval between structures is 500 nm or less, the structure of the lipid membrane changes in the gap region between the structures, and the solubility of molecules in the lipid membrane changes. The change in the solubility of the molecule is determined by the size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc. of the molecule. Therefore, when the interval between the structures is 500 nm or less, it is impossible to pass through the gap between the structures depending on the type of molecule. As a result, in the interstitial region between structures, molecules can be separated by factors other than the diffusion coefficient of the molecules (size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.). On the other hand, the interval between structures adjacent to the flow direction of the flow channel (the direction in which the lipid membrane is developed) is not particularly limited as long as the molecule to be separated and the polar lipid can pass through. Good. An interval between structures adjacent to each other in the flow direction of the flow path is, for example, about 100 nm to 1000 μm. Usually, the interval between structures adjacent to each other in the flow direction of the flow channel (direction of lipid membrane development) is longer than the interval between structures adjacent to each other in the width direction of the flow channel. The “interval between other structures closest to the body” means an interval between adjacent structures in the width direction of the flow path.

  The number of structures (number of rows and columns) arranged in a matrix in the flow path may be set as appropriate according to the type of molecules to be separated, the type of sample, the desired separation accuracy, and the like. For example, when two types of molecules having similar diffusion coefficients are separated, it is preferable to increase the length and width of the flow path and increase the number of structures (number of rows and columns).

  The sorting chip of the present invention can be manufactured by appropriately using a technique known to those skilled in the art such as electron beam lithography.

  Next, a method for fractionating molecules to be separated using the fractionation chip of the present invention (the fractionation method of the present invention) will be described.

  The fractionation method of the present invention comprises (1) a first step of preparing the fractionation chip of the present invention, (2) a second step of preparing a sample containing a molecule to be separated, and a polar lipid, and (3) a first step. And the third step of introducing the sample prepared in the second step and the polar lipid into the flow path of the separation chip prepared in the first step to develop the lipid membrane containing the molecule to be separated.

  In the first step, the above-described sorting chip of the present invention is prepared.

  In the second step, a sample containing one or more kinds of molecules to be separated and a polar lipid are prepared. Here, “polar lipid” refers to an amphiphilic lipid having a hydrophilic group and a hydrophobic group.

  The type of the molecule to be separated is not particularly limited as long as it is a molecule that can diffuse in the lipid membrane or on the surface of the lipid membrane alone or in a state bound to polar lipids. Examples of the classified molecule include proteins such as ion channels, transmembrane proteins, amyloid β protein, cholera toxin, and verotoxin; peptides such as antibacterial peptides and signal transduction peptides; sugar chains; muscle fibers and the like. Examples of the sample include blood, urine, and dilutions thereof.

  The kind of polar lipid is not particularly limited as long as it can form a lipid film in the third step. Two or more types of polar lipids may be prepared. That is, the lipid membrane developed in the third step may be composed of one type of polar lipid, or may be composed of two or more types of polar lipids. Examples of polar lipids include phospholipids (eg dioleoylphosphatidylcholine) and glycolipids (eg ganglioside GM1). For example, when it is desired to separate cytotoxins such as cholera toxin and verotoxin, a lipid (for example, ganglioside) that can bind to the target cytotoxin may be selected (see FIG. 9).

  In the third step, the sample prepared in the second step and the polar lipid are introduced into the flow path (or introduction port) of the separation chip prepared in the first step, and the lipid membrane containing the molecule to be separated is developed. .

As a method for introducing the sample and the polar lipid into the flow channel, a mixture of the sample and the polar lipid prepared in advance may be introduced into the flow channel, or the sample and the polar lipid may be separately introduced into the flow channel. . For example, when a mixture of a sample and a polar lipid is introduced into the channel, a solution containing the sample and the polar lipid is dropped into the channel of the separation chip, and the solvent may be volatilized as necessary. In addition, when introducing the sample and polar lipid separately into the channel, drop the solution containing polar lipid into the channel of the separation chip, volatilize the solvent, and then separate the sample (or solution containing the sample) What is necessary is just to dripping at the flow path of a chip | tip. The latter method is useful when a solvent (for example, ethanol) that dissolves polar lipids denatures a molecule to be separated. The amount ratio (molar ratio) of the sample to the polar lipid is not particularly limited, but is preferably in the range of 10 ⁻⁶ to 30 mol%.

  A method for developing a lipid membrane containing a molecule to be separated is not particularly limited, but it is preferable to develop it spontaneously without applying a driving force from the outside (hereinafter also referred to as “spontaneous deployment”). The lipid membrane is preferably a lipid membrane having a lipid bilayer structure, but may be other lipid membranes.

FIG. 4 is a diagram illustrating an example of a procedure for spontaneously developing a lipid membrane. For convenience of explanation, the structure is not shown in this drawing. First, the polar lipid 110 containing a molecule to be separated is introduced into the channel 112 (or the inlet connected to the channel 112) (FIG. 4A). Next, by introducing the electrolyte aqueous solution 114 into the channel 112, the lipid membrane 116 having a lipid bilayer structure can be spontaneously developed (FIG. 4B). In this example, the sorting chip of the present invention is submerged in the aqueous electrolyte solution 114. Examples of the aqueous electrolyte solution include an aqueous NaCl solution (1 to 100 mM), an aqueous Na ₂ SO ₄ solution (1 to 100 mM), and a phosphate buffer solution (PBS). FIG. 4C is a schematic diagram showing a state in which a lipid membrane 116 having a lipid bilayer structure is spontaneously developed on a substrate 118 having a hydrophilic surface. This lipid membrane 116 contains a molecule 120 to be separated.

  When the lipid membrane containing the molecule to be separated is spontaneously expanded, the lipid membrane expands toward the downstream of the flow path so as to sew between the structures in the flow path (the expansion speed is usually 0.1 to 0.3 μm). / S). The separated molecules in the lipid membrane move downstream in accordance with the flow of the lipid membrane while diffusing in the lipid membrane by Brownian motion. At this time, in the fractionation chip of the present invention, since the shape of the structure is asymmetric with respect to a straight line parallel to the development direction of the lipid membrane, the fractionated molecules in the lipid membrane are flow channels according to the diffusion coefficient of the molecules. It is unevenly distributed at a predetermined position (see FIG. 3). As a result, various molecules contained in the sample are separated in the flow path according to the diffusion coefficient.

  As described above, the fractionation method of the present invention can fractionate a molecule to be fractionated according to its diffusion coefficient using a small number of samples. In addition, the separation method of the present invention is capable of (1) separation without impairing the properties of biomolecules (for example, membrane proteins) because the lipid membrane is used as a transport medium, and (2) non-development because spontaneous development can be used. It can be applied to charged molecules, and (3) has an advantage that the device used is small (micrometer scale).

  For example, after finishing the third step, by performing a process that makes it possible to identify a specific molecule in the flow path of the sorting chip (for example, dropping a fluorescently labeled antibody), molecules contained in a trace amount (Eg, a marker molecule for a particular disease) can be measured. The separation method of the present invention includes, for example, separation (separation) of membrane proteins that are difficult to separate and purify, detection of changes in the behavior of lipid molecules caused by various diseases, screening for specific lipid molecules, lipid or protein that causes disease. The present invention can be applied to analytical applications such as detection, detection of proteins involved in interactions, and detection (testing) of harmful substances.

  Conventionally, a technique has been disclosed in which electrophoresis is performed using a device in which an asymmetric structure is disposed in a flow path to separate charged molecules (for example, Chou C. et al., “Sorting by diffusion: An asymmetric” obstacle course for continuous molecular separation ", PNAS (1999), Vol.96, 13762-13765.). These conventional techniques cannot be applied to uncharged molecules because the molecules are moved by electrophoresis. On the other hand, the present inventor has found that even when a molecule to be separated is dispersed in a lipid membrane, the molecule to be separated can be classified by the diffusion coefficient of the molecule. In this invention, since a molecule | numerator can be moved by spontaneous expansion | deployment, it can apply also to an uncharged molecule | numerator. Moreover, in this invention, since a molecule | numerator is moved by expansion | deployment of a lipid membrane, the point from which the magnitude | size of a structure is small compared with the said prior art differs. In the conventional technique, a molecule having a higher diffusion coefficient (smaller molecule) moves in the channel width direction, whereas in the present invention, a molecule having a lower diffusion coefficient (larger molecule) moves in the channel width direction. The point is also different. This difference is due to the difference in the flow velocity in the direction of movement of the molecules (difference in the medium in which the molecules are diffused).

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

(Embodiment 1)
FIG. 5 is a schematic diagram of the sorting chip according to the first embodiment. FIG. 5A is a perspective view of the entire sorting chip, and FIG. 5B is an enlarged plan view of the flow path portion.

  As shown in FIG. 5A, the separation chip 200 includes a substrate 210, an introduction port 220, a flow path 230, and a plurality of structures 240.

  The substrate 210 is, for example, a glass substrate. The size of the substrate 210 is not particularly limited as long as the channel 230 can be arranged without difficulty.

  The introduction port 220 is a recess formed in the substrate 210 and is connected to the flow path 230. The shape of the inlet 220 is not particularly limited as long as the user can easily drop the sample and the polar lipid.

  The flow path 230 is a recess formed in the substrate 210, and a plurality of structures 240 are arranged in a matrix on the bottom surface thereof. For example, the width of the flow path is in the range of 10 to 10,000 μm, the length of the flow path is in the range of 100 to 10,000 μm, and the depth of the flow path is in the range of 1 to 10,000 μm.

  The structure 240 is a rectangular parallelepiped member arranged in a matrix in the flow channel 230. The structure 240 is arranged such that the long side is inclined by 10 to 80 degrees with respect to the development direction of the lipid membrane. For example, the length of the long side of the structure is in the range of 0.5 to 2 μm, the length of the short piece is in the range of 0.15 to 0.5 μm, and the height is 0.001 to 0.1 μm. Is within the range. In the sorting chip of this embodiment, the shortest distance between structures adjacent in the width direction of the flow path (indicated by “c” in FIG. 5B) is longer than 500 nm.

  In order to separate molecules to be separated using the separation chip 200 having the above-described configuration, for example, (1) a sample and a polar lipid are dropped into the introduction port 220, and (2) an electrolyte solution is introduced into the introduction port 220 and the flow channel 230. It is only necessary to provide and spontaneously develop a lipid membrane containing a molecule to be separated.

FIG. 5C is a schematic diagram showing the distribution of the molecules to be separated in the flow channel after the lipid membrane has been developed. Here, it is assumed that the sample contains three types of molecules (A to C) having different diffusion coefficients. As shown in this figure, by spontaneously expanding the lipid membrane, the molecule A having a large diffusion coefficient (for example, D ≧ 1 μm ² / s) moves to the right side of the flow channel in the expansion direction, and the diffusion coefficient is Medium molecule B (for example, 0.01 μm ² / s <D <1 μm ² / s) moves near the center of the channel, and molecule C having a small diffusion coefficient (for example, D ≦ 0.01 μm ² / s) Moves to the left of the channel in the direction of deployment.

  As described above, the sorting chip according to the present embodiment can sort molecules according to the diffusion coefficient of molecules.

(Embodiment 2)
FIG. 6 is a schematic diagram of a sorting chip according to the second embodiment. FIG. 6A is a perspective view of the entire sorting chip, and FIG. 6B is an enlarged plan view of a flow path portion.

  As illustrated in FIG. 6A, the separation chip 300 includes a substrate 210, an introduction port 220, a flow path 230, and a plurality of structures 240. The sorting chip 300 of the second embodiment is different from the sorting chip of the first embodiment only in the arrangement pattern of the structures 240 in the flow channel 230. The same components as those of the sorting chip according to the first embodiment are denoted by the same reference numerals, and description of overlapping portions is omitted.

  The structures 240 are arranged in a matrix on the bottom surface of the flow channel 230 so that the long side is inclined by 10 to 80 degrees with respect to the development direction of the lipid membrane. In the sorting chip of this embodiment, the shortest distance between structures adjacent in the width direction of the flow path (indicated by “d” in FIG. 6B) is 500 nm or less (for example, 50 nm). .

  In order to separate molecules to be separated using the separation chip 200 having the above-described configuration, for example, (1) a sample and a polar lipid are dropped into the introduction port 220, and (2) an electrolyte solution is introduced into the introduction port 220 and the flow channel 230. It is only necessary to provide and spontaneously develop a lipid membrane containing a molecule to be separated.

  FIG. 6C is a schematic diagram showing the distribution of the classified molecules in the flow channel after the lipid membrane is developed. Here, it is assumed that nine types of molecules (A to I) having different diffusion coefficients or other factors (size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.) are included in the sample.

As shown in this figure, by spontaneously expanding the lipid membrane, molecules A, D and G (for example, D ≧ 1 μm ² / s) having a large diffusion coefficient move to the right side of the flow channel in the expansion direction. , Molecules B, E, and H (for example, 0.01 μm ² / s <D <1 μm ² / s) having a medium diffusion coefficient move to the vicinity of the center of the channel, and molecules C, F, and I having a small diffusion coefficient (For example, D ≦ 0.01 μm ² / s) moves toward the development direction and moves to the left side of the flow path.

  On the other hand, since the shortest distance between the structures 240 is 500 nm or less, each molecule has a gap between the structures 240 due to the other factors (size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.). The ease of passing through is different. As a result, each molecule has a different mobility in the development direction according to the ease of passing through the gaps between the structures 240. Specifically, the molecules G, H, and I that do not easily pass through the gap move to the front portion (portion on the introduction port 220 side) where the structure 240 is disposed, and the molecules D, E, and F moves to the vicinity of the center of the region where the structure 240 is arranged, and the molecules A, B, and C that easily pass through the gap move deeply into the region where the structure 240 is arranged (near the end of the channel 230). .

  As described above, the separation chip according to the present embodiment separates molecules using not only the diffusion coefficient of molecules but also other factors (size, configuration, chirality, hydrophilicity, hydrophobicity, polarity, etc.). can do.

(Embodiment 3)
FIG. 7A is a schematic diagram of a sorting chip according to the third embodiment.

  As shown in FIG. 7A, the separation chip 400 includes a substrate 210, a first introduction port 410, a second introduction port 420, a channel 230, and a plurality of structures 240. The sorting chip 400 of the third embodiment is different from the sorting chip of the first embodiment in that it has a first introduction port 410 and a second introduction port 420. The same components as those of the sorting chip according to the first embodiment are denoted by the same reference numerals, and description of overlapping portions is omitted.

  The first introduction port 410 is a recess formed in the substrate 210 and is connected near the first side surface (the front side surface in FIG. 7A) of the flow path 230. The sample and polar lipid are dropped into the first inlet 410 by the user. The shape of the first inlet 410 is not particularly limited as long as the user can easily drop the sample and the polar lipid.

  The second introduction port 420 is a recess formed in the substrate 210 and is connected near the second side surface of the channel 230 (the back side surface in FIG. 7A). In the second inlet 420, only polar lipids are dropped by the user. The shape of the second inlet 420 is not particularly limited as long as the user can easily drop polar lipids.

  In order to separate molecules to be separated using the separation chip 400 having the above-described configuration, for example, (1) a sample and a polar lipid are dropped into the first introduction port 410, and (2) the polarity is introduced into the second introduction port 420. Only lipid may be dropped, and (3) the electrolyte solution may be provided to the first inlet 410, the second inlet 420, and the flow path 230 to spontaneously develop the lipid membrane containing the molecules to be separated.

FIG. 7B and FIG. 7C are schematic diagrams for explaining the effect of providing the first introduction port 410 and the second introduction port 420. FIG. 7B is a schematic diagram showing a state of movement of molecules to be separated in a separation chip that does not have the first introduction port 410 and the second introduction port 420. FIG. 7C is a schematic diagram showing a state of movement of a molecule to be separated in a separation chip having a first introduction port 410 and a second introduction port 420 (a separation chip in this embodiment). In the figure, solid arrows indicate movement of molecules having a large diffusion coefficient (for example, D ≧ 1 μm ² / s), and broken arrows indicate movement of molecules having a small diffusion coefficient (for example, D ≦ 0.01 μm ² / s). Indicates.

  As shown in FIG. 7B, when a polar lipid 430 containing a molecule to be separated is dropped over the entire width of the channel 230, only the molecule (solid line) having a large diffusion coefficient is in the region “G” in the figure. The molecules with a large diffusion coefficient can be separated with high accuracy, but not only the molecules with a small diffusion coefficient (dashed line) but also the molecules with a large diffusion coefficient (solid line) ) Also move, and molecules with a small diffusion coefficient may not be separated with high accuracy.

  On the other hand, as shown in FIG. 7C, the polar lipid 430 containing the molecule to be separated is dropped into the first inlet 410, and the polarity containing the molecule to be separated in the channel 240 from the first side surface side. When lipid 430 is provided, only molecules having a large diffusion coefficient (solid line) move to the region “G” in the figure, and only molecules (dashed line) having a small diffusion coefficient move to the region “H” in the figure. Therefore, not only molecules having a large diffusion coefficient but also molecules having a small diffusion coefficient can be separated with high accuracy. The second introduction port 420 provides the polar lipid 440 from the second side surface into the flow channel 230 in order to uniformly develop the lipid membrane in the flow channel 240.

  As described above, the classification chip according to the present embodiment can accurately classify not only molecules having a large diffusion coefficient but also molecules having a small diffusion coefficient.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail with reference to an Example, this invention is not limited by these Examples.

  In this example, an example is shown in which cholera toxin B subunit (hereinafter abbreviated as “CTB”) is fractionated by the fractionation method of the present invention.

1. Production of Sorting Chip FIG. 8A is a schematic diagram of the sorting chip produced in this example. As shown in this figure, two guides 510 and a plurality of guides are formed on a cover glass (glass substrate) 500 by an electron beam lithography method using an ultrahigh precision electron beam lithography apparatus (ELS-7700H, Elionix Co., Ltd.). A structure 520 (both made of gold) was formed. A region between the two guides 510 corresponds to a flow path, and a plurality of structures 520 are arranged in a matrix between the two guides 510. In the figure, the length e (width of the flow path) was 200 μm, and the length f (the shortest length of the region where the structure is disposed in the flow path) was also 200 μm. The height of the guide 510 was 30 nm.

  FIG. 8B is an AFM image showing an arrangement pattern of structures in the flow path. As shown in this figure, a rectangular parallelepiped structure (long side: 1 μm, short side: 250 nm, height: 30 nm) was arranged such that the long side was inclined 45 degrees with respect to the lipid membrane development direction (guide direction). . The shortest distance between the structures was set to 250 nm, which is the same as the length of the short side (see FIGS. 2 and 6B).

2. Preparation of Lipid Solution Dioleoylphosphatidylcholine (hereinafter abbreviated as “DOPC”) (Avanti Polar Lipids) was dissolved in chloroform to prepare a 1 mg / mL DOPC solution. Similarly, BODIPY (fluorescent dye) -labeled ganglioside GM1 (hereinafter abbreviated as “GM1”) (Molecular Probes) was dissolved in chloroform to prepare a 0.014 μM GM1 solution. The above-mentioned DOPC solution and GM1 solution were mixed so that the molar ratio of DOPC to GM1 was 99.9: 0.1 to prepare a lipid solution. Since CTB (pentamer) specifically binds to GM1, CTB can be immobilized on the lipid membrane surface by including GM1 in the lipid membrane (see FIG. 9B). In this case as well, CTB bound to GM1 can diffuse in the surface direction on the lipid membrane surface.

3. Preparation of fractionated molecular solution As a fractionated molecular solution, Alexa Fluor 555 (fluorescent dye) -labeled cholera toxin B subunit (Molecular Probes) was dissolved in a phosphate buffer to prepare a 50 μg / mL CTB solution.

4). Separation treatment 2 μL of a lipid solution was dropped into a region where no structure was arranged in the flow path of the separation chip and dried. Next, 400 μL of a phosphate buffer (pH 7.4) was dropped over the entire flow path to spontaneously develop a lipid membrane composed of DOPC and GM1. FIG. 9A is a schematic view showing a state in which a lipid buffer composed of DOPC 540 and GM1 550 is spontaneously developed on the substrate 500 by dropping the phosphate buffer 530.

After developing a lipid membrane composed of DOPC and GM1 for 5 minutes, the phosphate buffer on the flow path was removed. Next, 4 μL of the molecular solution to be separated was dropped into the flow channel, and CTB was bound to GM1 in the lipid membrane. FIG. 9B is a schematic diagram showing a state in which CTB 570 is bound to GM1 550 in the lipid film by dropping the fractionated molecular solution 560. As a preliminary experiment, when the behavior of spontaneous development was examined, even when CTB and GM1 were combined, the behavior of spontaneous development of the lipid membrane was not significantly affected. As a preliminary experiment, the diffusion coefficient of CTB, GM1, and the composite was determined by FRAP and found to be less than 0.1 μm ² / s.

  After the fractionated molecular solution was dropped, the solution was allowed to stand at room temperature to further develop a lipid membrane containing CTB. After the lipid membrane was sufficiently developed to the end of the region where the structures in the channel were arranged (after about 30 minutes), the molecular solution to be separated on the channel was removed to stop the spontaneous development.

  FIG. 10 is a graph showing the CTB distribution in the flow channel after the spontaneous deployment is stopped. The distribution of cholera toxin B subunit was measured by observing the fluorescence of Alexa Fluor 555 labeled with CTB with a fluorescence microscope (BX51, Olympus Corporation). From this graph, it can be seen that CTB (and GM1 complex) is unevenly distributed in the region on the left side of the flow path in the development direction.

  From the above, it is suggested that the cholera toxin B subunit (complex of ganglioside GM1) can be separated by the separation method of the present invention.

  The separation method and the separation chip of the present invention are useful, for example, as a method for separating a target molecule when testing using blood or urine as a specimen and a device used therefor.

Diagram showing examples of structure shapes A plan view showing an example of a channel in which a plurality of structures are arranged in a matrix Schematic diagram showing how the molecules to be separated move in the channel Diagram showing an example of the procedure for spontaneously deploying lipid membranes Schematic diagram showing the sorting chip of the first embodiment Schematic diagram showing the sorting chip of the second embodiment Schematic diagram showing a sorting chip according to the third embodiment Schematic diagram showing the sorting chip produced in the example Schematic diagram showing the procedure for fixing cholera toxin B subunit on the lipid membrane surface The graph which shows the result of an Example

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 1st straight line 2 2nd straight line 3 3rd straight line 100,240 Structure 102 Vertex upstream of flow path 104 Vertex on first side of flow path 106 Vertex on second side of flow path 110,430 Polar lipid containing fractionated molecule 112,230 Channel 114 Electrolyte aqueous solution 116 Lipid membrane 118,210 Substrate 120 Divided molecule 200,300,400 Separation chip 220 Inlet 410 First inlet 420 Second introduction Mouth 440 Polar lipid 500 Glass substrate 510 Guide 520 Structure 530 Phosphate buffer 540 Dioleoylphosphatidylcholine 550 Ganglioside GM1
560 Molecular solution for fractionation 570 Cholera toxin B subunit A 1st length B 2nd length c, d Shortest distance between structures e Channel width f Shortest length of region where structure is arranged

Claims (8)

A method for fractionating molecules to be separated contained in a sample,
Preparing a sorting chip having a substrate, a flow path formed on the substrate, and a plurality of structures arranged in a matrix in the flow path;
Providing a sample containing one or more molecules to be separated, and a polar lipid;
Introducing the sample and the polar lipid into the flow path of the fractionation chip to spontaneously develop the lipid membrane containing the molecule to be fractionated and the polar lipid;
Each of the structures is
A first straight line passing through the apex on the upstream side of the flow path and parallel to the developing direction of the lipid membrane, and a first straight line passing through the apex on the first side face side of the flow path and parallel to the first straight line The distance from the straight line is
Longer than the distance between the first straight line and a third straight line passing through the apex on the second side surface side of the flow path and parallel to the first straight line;
The molecules that are less diffusible are unevenly distributed in a region closer to the first side surface of the flow path to separate the molecules to be separated.
Separation method.   The fractionation method according to claim 1, wherein the lipid membrane has a lipid bilayer structure.   The separation method according to claim 2, wherein the step of spontaneously expanding the lipid membrane includes a step of introducing an electrolyte aqueous solution into a flow path of the separation chip. A chip for separating molecules to be separated contained in a sample,
A substrate,
A channel formed on the substrate for spontaneously developing a lipid membrane;
A plurality of structures arranged in a matrix in the flow path;
Have
Each of the structures is
A first straight line passing through the apex on the upstream side of the flow path and parallel to the developing direction of the lipid membrane, and a first straight line passing through the apex on the first side face side of the flow path and parallel to the first straight line The distance from the straight line is
Unlike the distance between the first straight line and a third straight line that passes through the apex on the second side surface side of the flow path and is parallel to the first straight line,
The distance between the one structure and the other structure closest to the structure is in the range of 20 to 500 nm.
Sorting tip.   The sorting chip according to claim 4, wherein the plurality of structures have substantially the same shape.   The separation chip according to claim 4, wherein an interval between the second straight line and the third straight line of the structure is in a range of 100 nm to 3 μm. Each of the plurality of structures has a substantially rectangular parallelepiped shape having a long axis and a short axis parallel to the substrate surface, and is arranged so that the directions of the long axes are the same.
The direction of the long axis of the structure is tilted by 10 to 80 degrees with respect to the development direction of the lipid membrane,
The sorting chip according to claim 4.   The sorting chip according to claim 7, wherein the length of the major axis is in the range of 500 nm to 2000 nm, and the length of the minor axis is in the range of 150 nm to 500 nm.