JP5230300B2 - Biomolecule function analysis substrate, biomolecule function analysis sample body, and biomolecule function analysis method - Google Patents

Description

  The present invention relates to a biomolecule function analysis substrate, a biomolecule function analysis sample body, and a biomolecule function analysis method for analyzing functions of biomolecules such as membrane proteins.

To understand the functions of biomolecules including proteins, it is essential to know their structures. When a biomolecule functions, the structure of the biomolecule is important because it may change its structure in response to reactions with other molecules or external stimuli.
Conventionally, X-ray diffraction methods, electron beam diffraction methods, and electron microscope methods have been widely applied as structural analysis of biomolecules. However, these techniques require crystallization or freezing of the biomolecule that is the molecule to be analyzed, and the analysis cannot be performed while the biomolecule is functioning. There is a technique that enables analysis in solution, such as nuclear magnetic resonance (NMR), but the scope of application is limited, and it can be applied to large and complex biomolecules such as channel proteins and receptor proteins. It is difficult to apply. Therefore, in order to analyze these proteins, these proteins are divided into functional domains and analyzed.

In addition, scanning probe microscopes that can be analyzed in solution, especially atomic force microscopes (AFMs), are applied to biomolecule analysis as a method that allows biomolecules to be analyzed in a state close to the in vivo conditions. It is coming. In recent years, the technology of AFM has advanced, and a measurement apparatus and measurement method (see Non-Patent Document 1) capable of analyzing resolution at a molecular or atomic level in a solution, and further enabling dynamic analysis at a video rate. A measuring device (see Non-Patent Document 2) has also been developed, and further application to analysis of biomolecules is being studied. Specifically, a method has been studied in which a micropore is formed in a substrate, a biomembrane containing a biomolecule such as a membrane protein is disposed on the micropore, and observation is performed with AFM in that state. According to this observation method, since the biomolecule can be observed without being directly fixed to the substrate, it is expected to be an effective technique for functional analysis of the biomolecule.
Takeshi Fukuma, “Applied PHYSICS LETTERS”, American Physical Society, 87, 2005, 034101-1-3 Ando Toshio et al., “CHMPHYSCHM”, Volume 4, 2003, p. 1196-1202

However, when the biological membrane disposed on the micropore is observed with a scanning probe microscope such as AFM, the probe presses the biological membrane and deflects it by several nm to several tens of nm. Therefore, even in the conventional analysis method, the biomolecule contained in the biomembrane may not express the original function, and it cannot be said that the function of the biomolecule can be analyzed accurately. Further, when the biological membrane is bent, the quantitative analysis of the shape and size is obstructed in the observation of AMF and the like.
The present invention has been made in view of the above circumstances, and does not affect the function expression of the biomolecule, and can accurately analyze the function of the biomolecule, the biomolecule function analysis sample body, and It is an object of the present invention to provide a biomolecule functional analysis method.

The present invention has the following configuration.
[1] A substrate body in which a minute recess composed of a hole having a diameter of 100 nm to 1 μm or a groove having a width of 100 nm to 1 μm is formed, and a nano linear body disposed on the minute recess of the substrate body A biomolecular functional analysis substrate comprising a network structure comprising:
[2] The biomolecule functional analysis substrate according to [1], wherein a fluorescent substance is disposed in a minute recess of the substrate body.
[3] The nano linear body forming the network structure is chemically modified with a component capable of selectively adsorbing a biomolecule to be analyzed, according to [1] or [2] Biomolecular functional analysis substrate.
[4] The biomolecule function analysis substrate according to any one of [1] to [3], and a lipid bilayer film formed on a network structure constituting the biomolecule function analysis substrate. A sample body for biomolecular function analysis characterized by
[5] A lipid bimolecular film is formed on a network structure constituting the biomolecule functional analysis substrate according to any one of [1] to [3], and the biomolecule to be analyzed is formed using the lipid bilayer film And analyzing the function of the biomolecule using a scanning probe microscope apparatus.

  The biomolecule function analysis substrate, biomolecule function analysis sample body, and biomolecule function analysis method of the present invention can accurately analyze the function of a biomolecule without affecting the function expression of the biomolecule.

<Substrates for biomolecular functional analysis>
An embodiment of a biomolecule functional analysis substrate (hereinafter abbreviated as “substrate”) of the present invention will be described.
1 and 2 show the substrate of this embodiment. The substrate 10 according to the present embodiment includes a substrate body 11 in which minute recesses made of holes 11 a are uniformly formed, and a mesh structure 12 disposed on the opening of the hole 11 a of the substrate body 11. Note that the upper right image in FIG. 1 is an enlarged image of one hole 11a.

The material of the substrate body 11 is not particularly limited, and for example, silicon or its oxide can be used. When an electrical or electrochemical method such as a patch clamp method is used to analyze the function of a biomolecule, at least the surface of the substrate body 11 is made of an insulator (for example, silicon oxide). There is a need.
The hole 11a of the substrate body 11 in this embodiment is open in a circular shape. The diameter of the opening part of the hole part 11a is 100 nm-1 micrometer. When the diameter of the hole 11a opening is in the above range, a pseudo cell membrane can be obtained using the substrate 10.

In the hole portion 11a, it is preferable to arrange a fluorescent substance because the function of the biomolecule can be easily observed. For example, when a calcium ion (Ca ²⁺ ) indicator such as fura-2, fluo-3, or fluo-4 is used as the fluorescent substance, the permeation of calcium ions of the ion channel membrane protein can be easily observed.

  As a method for forming the hole 11a, for example, a fine processing technique such as a photolithography method or a dry etching method can be applied.

The network structure 12 is made of a nano linear body. Specifically, the nano linear body has a large number of branches to form a network.
Examples of the nanolinear body include nanotubes, nanowires, and nanofibers, and the material thereof is not particularly limited. However, carbon is preferable in that the nanolinear body can be easily formed.
Here, the nano linear body is a linear body having a diameter of a cross section perpendicular to the length direction of 1 nm to 20 nm. If the diameter of the cross section perpendicular to the length direction of the nanowire is not less than the lower limit, the lipid bilayer membrane can be sufficiently supported. Moreover, if it is below the said upper limit, even if the mesh structure 12 is arrange | positioned on the hole part 11a, it will not interfere with AFM observation of the biological sample for an observation purpose.
In the case of using carbon nanotubes (hereinafter sometimes referred to as CNT) as the nano-linear body, in order to make the diameter within the above range, the type of metal catalyst used in the method of forming the network structure 12 described later, What is necessary is just to select a particle diameter and a density, and the temperature at the time of formation suitably. For example, the larger the catalyst particle diameter, the larger the diameter.

The nanowires forming the network structure 12 are preferably chemically modified with components capable of selectively adsorbing biomolecules. If the nanowire is chemically modified with a component capable of selectively adsorbing biomolecules, it can form a binding point for a biomolecule that is a protein. Immobilized by amide bond. Therefore, the flow of biomolecules in the lipid bilayer membrane can be prevented and observation can be facilitated, and a small amount of biomolecules can be reliably and easily arranged at a predetermined measurement position.
In the case of using CNT as the nano linear body, as a method of chemically modifying CNT, for example, after the nano linear body is oxidized with a mixed solution of concentrated sulfuric acid and concentrated nitric acid to form a carboxyl group (a ) 1-ethyl-3- (3-dimethylamino) propyl) carbodiimide to form a carbodiimide group, (b) N-hydroxysuccinimide to form an N-hydroxysuccinimide ester, etc. Is mentioned.

  In the processing of such a nano linear body, the whole nano linear body is not processed uniformly. As shown in FIG. 2, the nano-linear body has higher chemical reactivity in a portion that is not in contact with the substrate body 11 (that is, a portion above the hole portion 11 a) than in a portion that is in contact with the substrate body 11. In addition, the chemical modification is selectively performed in the portion 12 a not in contact with the substrate body 11.

Further, when the biomolecule A to be analyzed is not electrically neutral such as polarized, the nano-linear body may be capable of applying a voltage. Specifically, as shown in FIG. 3, the electrodes 12d and 12e may be attached to two independent nanowires 12b and 12c that are not connected to each other so that a voltage can be applied. Moreover, an electrode may be attached to the bottom of the hole 11a or the surface of the substrate body 11, and a voltage may be applied between the electrode and the nanowire.
When a voltage is applied to the nanowire, an electric field is generated, and the arrangement of the biomolecule A within the lipid bilayer can be controlled by the electric field. In addition, the expression of the function of the biomolecule A can be controlled by electrical stimulation applied from the outside.

Examples of a method of forming the network structure 12 include a method of forming by a chemical vapor deposition method (CVD method). Examples of the carbon source when the chemical vapor deposition method is applied include methane, ethane, acetylene, methanol, ethanol, and the like.
Further, when the network structure 12 is formed by the chemical vapor deposition method, it is preferable to use a metal catalyst such as iron because a nano linear body can be easily formed. Further, in order to form a nano-linear body on the surface of the substrate body 11, it is preferable to attach a metal catalyst to the surface of the substrate body 11 in advance.

Since the substrate 10 described above has the network structure 12, when the lipid bilayer is formed on the surface of the substrate 10 on which the hole 11a is formed, the lipid bilayer is formed on the hole 11a. It is arranged on the mesh structure 12. Therefore, even when the lipid bilayer is pressed by the probe of the scanning probe microscope apparatus, the lipid bilayer is not easily bent. Therefore, the function of the biomolecule held by the lipid bilayer membrane is hardly affected, and the function of the biomolecule can be accurately analyzed.
Further, since the mesh structure 12 does not close and seal the hole 11a, it is suitable for a substrate for analyzing a biomolecule function.

<Sample for biomolecular function analysis>
Next, a biomolecule function analysis sample body (hereinafter abbreviated as a sample body) using the substrate 10 will be described.
As shown in FIG. 4, the sample body 1 of this embodiment example includes the substrate 10 and a lipid bilayer membrane 20 formed on a network structure 12 constituting the substrate 10.

  The lipid bilayer membrane 20 is a membrane in which lipid molecules such as phospholipids having both polarities form a bilayer structure, and is one of the most basic structures of a biological membrane. In the lipid molecule, hydrophobic end groups are associated with each other inside the membrane, and the hydrophilic end group of the lipid molecule is located on the front side of the membrane, so that the lipid bilayer membrane 20 is formed.

As a method for forming the lipid bilayer membrane 20 on the network structure 12, for example, a substrate having the network structure 12 in a vesicle solution in which the lipid bilayer membrane 20 is diffused into water as spherical particles (vesicles). For example, a method in which 10 is immersed and the vesicles are spread on the surface of the substrate body 11 and the mesh structure 12 in a planar manner.
As lipid molecules forming vesicles, intracellular phospholipids (for example, phosphatidylcholine and the like) or lipid molecules similar thereto are preferably used. As a solvent for the vesicle solution, for example, phosphate buffered saline is used. The solvent of the vesicle solution also becomes a liquid that fills the hole 11a.
The mixing ratio of the solvent and the lipid molecule in the vesicle solution is preferably 0.001 to 1% by mass of the lipid molecule with respect to the solvent.
The particle size of the vesicle in the vesicle solution is preferably larger than the diameter of the opening of the hole 11a.
The vesicle solution may contain a biomolecule to be analyzed in advance.

The periphery of the lipid bilayer membrane 20 is preferably filled with a buffer solution 30 such as phosphate buffered saline as shown in FIG. If the periphery of the lipid bilayer membrane 20 is filled with the buffer solution 30, the network structure 12 corresponds to the cytoskeleton, the lipid bilayer membrane 20 corresponds to the cell membrane, and the buffer solution 30 corresponds to the intracellular solution or the outer solution. Cell membrane can be formed.
The buffer solution 31 above the lipid bilayer membrane 20 can have a composition different from that of the buffer solution 32 below the lipid bilayer membrane 20 by substituting after the lipid bilayer membrane formation. Thereby, the state in which an intracellular fluid and an extracellular fluid differ can be produced.

As shown in FIG. 4, the sample body 1 of the present embodiment example can hold the biomolecule A by the lipid bilayer membrane 20. The state in which the biomolecule A is held by the lipid bilayer membrane 20 is the same as the state in which the biomolecule A exists in the cell membrane of the living body, and thus the function can be sufficiently expressed.
Moreover, in the sample body 1, the lipid bilayer membrane 20 is supported by the network structure 12 on the hole 11a. Therefore, even if the lipid bilayer membrane 20 is pressed by the probe of the scanning probe microscope apparatus, the lipid bilayer membrane 20 containing the biomolecule A is not easily bent and the function of the biomolecule A can be accurately reproduced. Therefore, by using this sample body 1, the function of the biomolecule A can be analyzed accurately.

<Biomolecular function analysis method>
Next, an embodiment of a biomolecule function analysis method using the substrate 10 will be described.
The biomolecule function analysis method of the present embodiment is a method of holding the biomolecule A to be measured on the lipid bilayer membrane 20 of the substrate 10 and analyzing the function of the biomolecule A using a scanning probe microscope apparatus.

Receptor channel proteins and transporters are suitable as biomolecules A to be analyzed by this analysis method.
As a method for retaining the biomolecule A in the lipid bilayer membrane 20, a method of previously containing the biomolecule A in a vesicle solution for forming the lipid bilayer membrane 20, and after forming the lipid bilayer membrane 20, Examples thereof include a method in which a solution containing a vesicle (proteoliposome) reconstituted with biomolecule A is dropped onto the lipid bilayer membrane 20 and fused.

As the scanning probe microscope apparatus, a known scanning probe microscope can be used, but an atomic force microscope (AFM) is preferable because it is suitable for structural analysis of a molecule to be analyzed in a solution.
The scanning probe microscope apparatus includes a cantilever that is a measurement sensor.

In this analysis method, a functional analysis measuring device may be used together with the scanning probe microscope.
Examples of the measurement device for functional analysis include a fluorescence microscope device and a patch clamp device that generates an electric field around a biomolecule to be analyzed by applying a voltage and can measure a variation in current generated by the molecule to be analyzed.

  When a fluorescence microscope is used as the functional analysis measurement device, for example, a fluorescent substance that becomes a calcium indicator is placed together with a buffer solution in the hole 11a in the absence of calcium ions. At this time, no fluorescence is observed in the fluorescent material. Next, the buffer solution on the lipid bilayer membrane 20 is replaced with one containing calcium ions. Even at this stage, there is no tendency since calcium ions and fluorescent substances are not bonded. If the biomolecule A to be analyzed is a protein of a calcium ion channel, calcium ions on the lipid bilayer membrane 20 can enter the hole 11a. For this reason, the calcium ions and the fluorescent material are combined to observe fluorescence. By observing such changes, the activity of the ion channel can be examined.

The patch clamp device includes a pair of electrodes for performing electrical measurement of the biomolecule A and voltage application. At least the tip of the electrode is positioned in the buffer solution. This electrode plays a role of applying a voltage to the biomolecule A to be analyzed and detecting electrical fluctuations of the biomolecule A to be analyzed.
In the present embodiment using the substrate 10, one electrode is preferably provided at the bottom of the hole 11a.
Examples of the material for the electrode include metals such as copper, aluminum, gold, silver, silver chloride, platinum, chromium, and nickel, and conductive substances such as conductive resins.
The thickness of the electrode is preferably 50 to 500 nm.
As a method for forming the electrode, for example, a thin film of the above-mentioned conductive material is deposited on the substrate body by vapor deposition, plating, etc., and a thin film of the conductive material in an unnecessary portion is formed using a fine processing technique such as an etching technique. The method of removing is mentioned.

  According to the patch clamp apparatus, when a voltage is applied to the biomolecule A, the fluctuation of the current generated from the biomolecule A, that is, the fluctuation of the channel current is electrically measured, the fluctuation of the channel current is read, and the function of the biomolecule A is read. The presence or absence and the degree of expression can be examined.

  In the biomolecule function analysis method described above, since the biomolecule A is held by the lipid bilayer film 20 formed on the network structure 12, the lipid bilayer film 20 is pressed by the probe of the scanning probe microscope apparatus. Even when it is done, the function of the biomolecule A is hardly affected. Therefore, the function of the biomolecule A can be analyzed accurately.

  In addition, this invention is not limited to the said embodiment. For example, the substrate main body 11 may be formed with a minute recess made of a groove having a width of 100 nm to 1 μm instead of the hole 11a. In the substrate in which the minute concave portion is the groove portion, as shown in the atomic force microscope image of FIG. 6, the mesh structure 12 is formed on the groove portion 11b. Thus, even in the form in which the mesh structure 12 is formed on the groove 11b, the lipid bimolecular molecule is formed by the probe of the scanning probe microscope apparatus, similarly to the form in which the mesh structure 12 is formed on the hole 11a. Even if the membrane is pressed, it is difficult to bend. Therefore, the function of the biomolecule can be analyzed accurately.

Example 1
A large number of holes having a diameter of 600 nm and a depth of 350 nm are uniformly formed on one surface of the silicon substrate by photolithography, and then a silicon oxide film is deposited on the surface of the silicon substrate on which the holes are formed by depositing about 40 nm. A substrate body was obtained. Next, an iron thin film having a thickness of 0.2 nm was vacuum-deposited on the substrate body, and heat treatment was performed at 900 ° C. to aggregate the iron, thereby forming iron particles. Using the obtained iron particles as a catalyst, a carbon nanotube network structure was formed on the surface of the substrate body by a CVD method using methane as a carbon source under the conditions of a temperature of 900 ° C. and a pressure of 10 kPa to obtain a substrate. . When the surface of the substrate on which the network structure was formed was observed with a scanning electron microscope, this network structure was also formed on the opening of the hole.
The carbon nanotubes constituting the network structure were two- or three-layer multi-walled carbon nanotubes having a diameter of 3 to 10 nm. Therefore, the diameter of the obtained carbon nanotube was equivalent to the cytoskeleton (about 7 to 10 nm) of the biological membrane.
Subsequently, the obtained substrate was attached to 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with 1% by mass of fluorescent modified lipid 4-fluoro-7-nitrobenzofuran with respect to 100% by mass of DPPC. Was immersed in a vesicle solution mixed with phosphatidylethanolamine for 30 minutes. Thereby, the lipid bilayer was developed on the network structure of the substrate.
Next, the proteoliposome reconstituted with the membrane protein was fused with the lipid bilayer membrane. Proteoliposomes were prepared by dialysis. That is, 240 μM lipid vesicle solution (1: 1 mass ratio of phosphocholine derived from egg yolk and phosphatidylserine derived from bovine brain), 4 μL of 800 mM surfactant (n-acetyl-D-glycopyranoside) and 100 ng / ml protein solution 195 μL of (1M Tris-HCl buffer, pH 7.4) was mixed and dialyzed for 5 days using a semipermeable membrane made of cellulose to obtain proteoliposomes.
The state in which the membrane protein was held by the lipid bilayer membrane of the substrate was observed with an AFM (NVB500 manufactured by Olympus). The microscope image is shown in FIG. The white bright spot at the end of the arrow is membrane protein B. Thus, according to this example, an atomic force microscope image of the membrane protein could be obtained accurately.

(Example 2)
A substrate was obtained in the same manner as in Example 1 except that the diameter of the hole was 100 nm and the depth was 100 nm.
Next, the substrate was immersed in a solution in which a fluorescent dye (Alexa 647, 5 mM) was mixed with a phosphate buffer, and the fluorescent dye was placed in the hole.
Moreover, phosphocholine derived from egg yolk and phosphatidylserine derived from bovine brain were mixed at a mass ratio of 1: 1, and 20% by mass of phosphoethanolamine with fluorescein was added thereto to obtain a vesicle solution.
Next, 10 μL of the lipid vesicle solution was dropped onto a substrate on which the fluorescent dye was placed in the hole, and allowed to stand for 30 minutes, and the hole was sealed with a lipid bilayer membrane. Thereafter, the fluorescent dye remaining in the substrate and the solution outside the hole was sufficiently washed with a buffer solution.
When the obtained substrate was observed with a fluorescence microscope, the fluorescent dye in the hole portion looked red and the lipid bilayer membrane looked green. Thereby, it was confirmed that the fluorescent dye was disposed and confined only in the hole.

(Example 3)
Carbon that forms a network structure by immersing the substrate obtained in Example 1 in a mixed solution (volume ratio 3: 1) of concentrated sulfuric acid (concentration 96 mass%) and concentrated nitric acid (concentration 61 mass%) for 10 minutes. A carboxyl group was formed on the surface of the nanotube.
Next, the substrate is immersed in 1-ethyl-3- (3- (dimethylamino) propyl) carbodiimide for 30 minutes and further in N-hydroxysuccinimide for 30 minutes, so that N-hydroxysuccinimide ester is formed on the surface of the carbon nanotube. Formed.
According to the substrate having the network structure thus chemically modified, the membrane protein can be immobilized by the substitution reaction between the N-hydroxysuccinimide ester and the N-terminal or lysine residue of the membrane protein. Can suppress the flow of membrane protein.

It is an atomic force microscope image of one embodiment of the substrate of the present invention. It is sectional drawing which shows one embodiment of the board | substrate of this invention. It is a figure which shows typically the form which enabled it to apply a voltage to a nano linear body. It is sectional drawing which shows one embodiment of the sample body of this invention. It is sectional drawing which shows the usage example of the sample body shown in FIG. It is an atomic force microscope image of other embodiment of the board | substrate of this invention. In Example 2, it is an atomic force microscope image which shows the state by which the biomolecule was hold | maintained at the network structure.

Explanation of symbols

1 Sample body (sample body for biomolecular function analysis)
10 Substrate (Biomolecular Function Analysis Substrate)
DESCRIPTION OF SYMBOLS 11 Substrate body 11a Hole part 11b Groove part 12 Network structure 20 Lipid bilayer membrane A Biomolecule

Claims (5)

  A network comprising a substrate body in which a minute recess composed of a hole having a diameter of 100 nm to 1 μm or a groove having a width of 100 nm to 1 μm is formed, and a nano linear body disposed on the minute recess of the substrate body A biomolecular functional analysis substrate comprising a structure.   The biomolecule functional analysis substrate according to claim 1, wherein a fluorescent substance is disposed in a minute recess of the substrate body.   3. The biomolecule functional analysis according to claim 1, wherein the nano-linear body forming the network structure is chemically modified with a component capable of selectively adsorbing a biomolecule to be analyzed. substrate.   A living body comprising the biomolecule function analysis substrate according to any one of claims 1 to 3 and a lipid bilayer film formed on a network structure constituting the biomolecule function analysis substrate. Sample for molecular function analysis.   A lipid bimolecular film is formed on the network structure constituting the biomolecule functional analysis substrate according to any one of claims 1 to 3, and the biomolecule to be analyzed is held and scanned by the lipid bimolecular film. A biomolecule function analysis method, comprising: analyzing a function of the biomolecule using a probe microscope apparatus.

Images