JP7365022B2 - Supports for biomolecules and their production methods - Google Patents

Description

The present invention relates to a support for biomolecules and a method for producing the same.

Research on sensor devices that examine the behavior of membrane proteins in artificial biological membranes on the surface of minute microchips and biodevices that utilize the functions of membrane proteins has been actively conducted. When creating such a microdevice, an artificial biomembrane such as a lipid membrane, which is efficiently used as a specific reaction field in nature, is placed on the surface of the microchip substrate, and membrane proteins are supported in this artificial biomembrane. It is necessary to do so. At this time, it is required that the function of the membrane protein is not impaired.

The biological membrane placed on the substrate surface can be adsorbed onto the substrate surface, and the most important property is fluidity, so it is possible to provide an optimal environment for membrane proteins to function. can. Non-Patent Document 1 describes a biomolecule detection chip using a biomembrane. This biomolecule detection chip has biological membranes made of lipid molecules and the like separated by partition walls arranged on the surface of a substrate made of a material such as plastic, glass, or silicon. The fluidity of a biomembrane can be confirmed by applying an electric field to the biomembrane parallel to the substrate surface and moving the biomolecules supported on the biomembrane, similar to electrophoresis of proteins supported on a gel. (For example, see Non-Patent Documents 2 and 3).

In normal cases, the biomembrane is provided close to the substrate surface, so that membrane-related biomolecules (transmembrane proteins) supported on the biomembrane easily come into contact with the substrate surface. Contact with the substrate surface becomes noticeable, for example, when membrane-related biomolecules (e.g., cell adhesion receptors, etc.) that have a structure that extends outward from the biomembrane by several tens of nanometers are supported on the biomembrane. . When a functional site of a membrane-related biomolecule comes into contact with a substrate, it may be damaged due to friction between the membrane-related biomolecule and the substrate, adsorption to the substrate surface, etc., or it may become difficult to maintain its function, or the membrane-related biomolecule itself may be damaged. There is a risk that it may degenerate. To solve this problem, conventionally, a layer made of a polymeric material (linker molecule) that can support membrane-related biomolecules and has biocompatibility is formed between the substrate and the biomembrane. A method of separating the membrane from the substrate to avoid contact between the membrane-related biomolecule and the substrate is being considered (see, for example, Non-Patent Document 4).

Groves J T et al., “Micropattern Formation in Supported Lipid Membranes.”, Acc. Chem. Res., Vol. 35, p149-157, 2002. Tanaka M at al., “Frictional Drag and Electrical Manipulation of Recombinant Proteins in Polymer-Supported Membranes.”, Langmuir, Vol. 23, p5638-5644, 2007. Tanaka M at al., “Selective Deposition of Native Cell Membranes on Biocompatible Micropatterns.”, J. Am. Chem. Soc., Vol. 126, p3257-3260, 2004. Tanaka M at al., “Polymer-supported membranes as models of the cell surface.”, Nature, Vol. 437, p656-663, 2005. K. Furukawa, Y. Ueno, M. Takamura, H. Hibino, ACS Sens., 1, pp 710-716, 2016. Y. Ueno, K. Furukawa, K. Matsuo, S. Inoue, K. Hayashi, H. Hibino, Chem. Commun., 49, pp 10346-10348, 2013. S. A. Lange et al., Anal. Chem., 76, pp 1641-1647, 2004. Hillerbrandt H et al., “A Novel Membrane Charge Sensor: Sensitive Detection of Surface Charge at polymer/Lipid Composite Films on Indium Tin Oxide Electrodes”, Journal of Physical Chemistry B, Vol. 106, p477-486, 2002.

When conventionally used polymer chains are immobilized on a substrate as a linker molecule, if the density of the immobilized polymer chains on the substrate is lowered, they become entangled in a ball-like shape (spherical shape), and the linker layer becomes tangled. The thickness decreases, making it easier for membrane-associated biomolecules to contact the substrate surface. On the other hand, if the density of the polymer chains on the substrate is increased, the polymer chains will not become entangled and will have a stable linear structure, making it possible to maintain the thickness of the linker layer. The biomembrane that has been deformed has a reduced degree of freedom of deformation and has low fluidity. When the fluidity of a biomembrane becomes low, membrane-related biomolecules supported on the biomembrane may be denatured or have reduced functionality.

The present invention has been made in view of the above circumstances, and is aimed at suppressing the deterioration of the fluidity of the supporting part and the degeneration and functional deterioration of the biomolecules due to contact with the base material etc. when supporting biomolecules. The purpose of the present invention is to provide a support for biomolecules and a method for producing the same.

The present invention provides the following means to solve the above problems.

(1) A support for biomolecules according to one aspect of the present invention includes a solid substrate, a linker layer formed on one main surface of the solid substrate, and a biological membrane formed on the linker layer. The linker layer has a plurality of paired nucleic acid molecules consisting of a helical first nucleic acid molecule and a second nucleic acid molecule, and one end of the first nucleic acid molecule is on one main surface of the solid substrate. and one end of the second nucleic acid molecule is fixed to the biological membrane.

(2) In the biomolecule support described in (1) above, it is preferable that one end of the second nucleic acid molecule is fixed to a portion of the biomembrane where the biomolecule is not supported.

(3) In the support for biomolecules according to any one of (1) or (2) above, the first nucleic acid molecule and the second nucleic acid molecule each contain 10 to 200 basic elements. It is preferable that it be configured.

(4) In the biomolecule support according to any one of (1) to (3) above, one end of the first nucleic acid molecule is attached to one main surface of the solid substrate via a graphene membrane. It is preferable that it be fixed.

(5) In the support for biomolecules according to any one of (1) to (4) above, the biological membrane is composed of a plurality of overlapping lipid molecule membranes, and the biological membrane is formed of a plurality of overlapping lipid molecule membranes, and Preferably, one end of the second nucleic acid molecule is fixed to the lipid molecule membrane.

(6) A method for producing a support for biomolecules according to one aspect of the present invention includes a graphene film forming step of forming a graphene film on one main surface of a solid substrate, and a step of forming a graphene film on the surface of the graphene film. an adhesion molecule adhesion step of adhering an adhesion molecule for immobilizing molecules; and a first nucleic acid molecule immobilization step of immobilizing one end of the first nucleic acid molecule to a predetermined region on the graphene film via the adhesion molecule. , a plurality of first lipid molecules to which second nucleic acid molecules having a complementary sequence to the first nucleic acid molecule are bound, and a plurality of second lipid molecules to which the second nucleic acid molecules are not bound. a first lipid molecule membrane forming step of forming a lipid molecule membrane; a linker layer forming step of reacting the first nucleic acid molecule and the second nucleic acid molecule to form a linker layer consisting of a pair of both nucleic acid molecules; a second lipid molecule membrane forming step of forming a second lipid molecule membrane consisting only of a plurality of second lipid molecules to which no second nucleic acid molecules are bound on the first lipid molecule membrane.

In the biomolecule support of the present invention, a biomembrane carrying biomolecules is supported by a linker layer using a double helix structure nucleic acid molecule (dsDNA) as a linker molecule. The linker molecules are rigid nucleic acid molecules and do not become entangled in threads even when formed at low density, so the thickness of the linker layer can be kept constant and the fluidity of the biomembrane can be maintained. Can be done. In addition, since there is no entanglement of the linker molecules, the linker layer can have a sufficient thickness and the biofilm carrying the biomolecules can be separated from the solid substrate, which reduces the probability that the biomolecule P will come into contact with the solid substrate. can be reduced. Therefore, when supporting biomolecules, the biomolecule support of the present invention suppresses a decrease in the fluidity of the support membrane (biomembrane), as well as denaturation and functional decline of biomolecules due to contact with a solid substrate. Can be done.

FIG. 1 is a cross-sectional view of a support for biomolecules according to an embodiment of the present invention. 2A to 2C are diagrams showing examples of arrangement of linker molecules constituting the support for the biomolecule in FIG. 1. FIG. (a), (b) are diagrams showing an example of the arrangement of linker molecules constituting the support for the biomolecule in FIG. 1. (a) to (c) are cross-sectional views of an object to be processed during the manufacturing process of a support for biomolecules according to an embodiment of the present invention. FIG. 3 is a cross-sectional view of an object to be processed in the process of manufacturing a support for biomolecules according to an embodiment of the present invention. FIG. 3 is a cross-sectional view of an object to be processed in the process of manufacturing a support for biomolecules according to an embodiment of the present invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS Hereinafter, a support for biomolecules and a method for manufacturing the same according to an embodiment of the present invention will be described in detail with reference to the drawings. Note that the drawings used in the following explanations may show characteristic parts enlarged for convenience in order to make the characteristics easier to understand, and the dimensional ratio of each component may not be the same as the actual one. do not have. Furthermore, the materials, dimensions, etc. exemplified in the following description are merely examples, and the present invention is not limited thereto, and can be practiced with appropriate changes within the scope of the gist thereof.

FIG. 1 is a cross-sectional view schematically showing the configuration of a biomolecule support 100 that supports a biomolecule (membrane-related biomolecule (membrane protein)) P as an embodiment of the present invention. The biomolecule support 100 mainly includes a solid substrate 101 as a base material, a linker layer 102 formed on one main surface 101a of the solid substrate, and a model biological membrane 103 formed on the linker layer 102. and has.

As the solid substrate 101, one made of a material such as plastic, glass, silicon, graphene, etc. can be used. It is preferable that at least one main surface 101a of the solid substrate on which the linker layer 102 is formed has a substantially flat shape.

The linker layer 102 has biocompatibility and consists of a helical first nucleic acid molecule (ssDNA (1)) 104 and a second nucleic acid molecule (ssDNA (2)) 105, which are paired to form a double helical structure. It has a plurality of nucleic acid molecules (dsDNA) 106 as linker molecules. The bases forming the first nucleic acid molecule 104 and the bases forming the second nucleic acid molecule 105 have mutually complementary sequences.

One end 104a of the first nucleic acid molecule is fixed directly or indirectly to one main surface 101a of the solid substrate. When the solid substrate 101 is made of a material other than graphene, a graphene film 107 is formed on one main surface 101a of the solid substrate, as shown in FIG. In this case, one end 103a of the first nucleic acid molecule is indirectly fixed to the solid substrate 101 by being bonded to the graphene film 107. When the solid substrate 101 is made of graphene, one end 104a of the first nucleic acid molecule is directly bonded and fixed to one main surface 101a of the solid substrate (graphene substrate).

In order to achieve a strong adsorption state with the graphene film (graphene substrate), one end 104a of the first nucleic acid molecule is modified with an amino group and is bonded to an adhesive molecule 108 of pyrene alkyl carboxylic acid such as pyrene butanoic acid. It is preferable.

One end 105a of the second nucleic acid molecule, which is located on the opposite side to one end 104a of the first nucleic acid molecule forming a pair, is fixed to a biological membrane (support membrane) 103 that supports the biomolecule P. The model biological membrane 103 is made up of a plurality of overlapping lipid molecule layers, and one end 105a of the second nucleic acid molecule is fixed to the lipid molecule layer closest to the solid substrate 101. FIG. 1 illustrates a case where the model biological membrane 103 is made up of two lipid molecule membranes (a first lipid molecule membrane 109 and a second lipid molecule membrane 110) overlapping each other.

It is preferable that both the first nucleic acid molecule 104 and the second nucleic acid molecule 105 are composed of 10 or more and 200 or less basic elements arranged to form a spiral. In order to take advantage of the strength of the double helix structure, it is preferable that the amount of unpaired surplus base elements, that is, the base elements that do not participate in the formation of the double helix structure, is as small as possible. In other words, the numbers of basic elements constituting the first nucleic acid molecule 104 and the second nucleic acid molecule 105 are approximately the same, and the positions of one end 104a and the other end 104b of the first nucleic acid molecule are the same as those of the second nucleic acid molecule, respectively. It is preferable that the other end 105b and one end 105a are substantially aligned.

Since the nucleic acid molecules 106 are rigid biomolecules, they will not shrink or tangle even in small numbers, and the distance between the model biomembrane 103 and the solid substrate 101 can be kept constant to keep the model biomembrane 103 stable. can be supported. From the viewpoint of maintaining the fluidity of the model biomembrane 103, it is preferable that the model biomembrane 103 has a high degree of freedom in deformation. It is preferable that the number is as small as possible within a range in which the model biological membrane 103 is stably held and the thickness of the linker layer 102 is maintained.

Therefore, for example, the linker layer 102 may be formed so as to support the portion of the model biomembrane 103 where the biomolecule P is not supported. That is, one end 105a of the second nucleic acid molecule is fixed to at least a part of the model biomembrane 103 where the biomolecule P is not supported, and the second end 105a is fixed to the part where the biomolecule P is supported. One end 105a of the nucleic acid molecule does not have to be fixed. At this time, from the viewpoint of stably supporting the model biomembrane 103, it is preferable that the helical structure constituting the nucleic acid molecule 106 is wound around an axis substantially perpendicular to the one surface 101a of the solid substrate. . In this case, one end 104a of the first nucleic acid molecule needs to be fixed to the solid substrate 101 in a region that does not overlap with the portion on which the biomolecule P is supported when viewed in plan from the thickness direction 102d of the linker layer.

In this embodiment, it is assumed that the biological molecule P is supported in the central part of the biological membrane, and the peripheral part where the biomolecule P is not supported is supported by the nucleic acid molecule 106. FIGS. 2(a) to 2(c) illustrate the shape of the region 102A to which the nucleic acid molecule 106 is immobilized. The shape of the fixing region 102A is not limited, and may be a shape surrounding a circular region or a polygonal region as shown in FIG. From the viewpoint of stably supporting P, the higher the symmetry with respect to the central portion 102B, the more preferable. Although FIG. 2 illustrates a case where the fixed area 102A is closed, the fixed area 102A may be open as illustrated in FIGS. 3(a) and 3(b). The shape of the fixed area 102A in this case is not limited either, and may be a plurality of straight lines arranged in parallel as shown in FIG. 3, a wavy line shape, or other shapes. .

4(a) to (c), FIGS. 5(a), (b), and FIG. 6 show the manufacturing process of a biomolecule support 100 to which the method for manufacturing a biomolecule support according to the present embodiment is applied. It is a sectional view of the object to be processed in . Here, a case is illustrated in which the first nucleic acid molecule 104 is fixed to a solid substrate via a graphene film. The method for producing the biomolecule support 100 mainly includes a graphene membrane formation step, an adhesive molecule attachment step, a first nucleic acid molecule fixation step, a first lipid molecule membrane formation step, a linker layer formation step, and a first step. and a step of forming a bilipid molecule membrane.

[Graphene film formation process]
First, a solid substrate 101 made of a material such as plastic, glass, silicon, etc. is prepared, and one main surface 101a of the solid substrate 101 is deposited using a known film forming method such as chemical vapor deposition, as shown in FIG. 4(a). As shown, a graphene film 107 is formed (transferred).

[Adhesive molecule attachment process]
Next, as shown in FIG. 4B, adhesive molecules 108 for fixing the first nucleic acid molecules 104 are attached to the surface of the graphene film 101 using a known film forming method such as sputtering. As the adhesive molecules 108, for example, pyrene alkyl carboxylic acids such as pyrene butanoic acid can be used.

[First nucleic acid molecule fixation step]
Next, a solution containing the first nucleic acid molecule 104 is introduced into a predetermined area on the graphene film 107 (flowing), and as shown in FIG. One end 104a of the first nucleic acid molecule is immobilized. At this time, if one end 104a of the first nucleic acid molecule is modified with an amino group, the first nucleic acid molecule 104 can be more strongly adsorbed to the graphene film 107 through dehydration condensation with the adhesive molecule 108 such as pyrene butadienoic acid. This is preferable because it can (see Non-Patent Document 5).

The nucleic acid molecule 106 formed by combining the first nucleic acid molecule 104 with the second nucleic acid molecule 105 is selected from among the model biological membranes 103 from the viewpoint of suppressing degeneration and functional decline of the biomolecule P due to contact with the nucleic acid molecule 106. It is preferable that the portion on which the biomolecule P is supported and the portion in the vicinity thereof are not supported. Therefore, the region where the first nucleic acid molecule 104 is immobilized (the region where the nucleic acid molecule 106 is formed) is a region that overlaps with the portion where the biomolecule P is not supported when viewed in plan from the thickness direction 102d of the linker layer. It is preferable to have a two-dimensional pattern structure. The procedure for forming the two-dimensional pattern structure will be described later.

[First lipid molecule membrane formation process, linker layer formation process, second lipid molecule membrane formation process]
Next, a plurality of first lipid molecules 109A to which a second nucleic acid molecule 105 having a complementary sequence to the first nucleic acid molecule 104 is bound, and a plurality of second lipid molecules to which a second nucleic acid molecule 105 is not bound. A mixed dispersion of 109B and a dispersion of third lipid molecule 110A are prepared. Examples of processes for forming lipid bilayer membranes using these dispersions include the following two processes (a) and (b).

(A) As shown in FIG. 5, on the substrate, a nucleic acid molecule 106 having at least a part of a double-stranded structure consisting of a first nucleic acid molecule 104 and a second nucleic acid molecule 105, a first lipid molecule 109A, and a second A linker layer 102 made up of a lipid monolayer (first lipid molecule film 109) made of lipid molecules 109B is formed. Subsequently, as shown in FIG. 6, a second lipid molecule membrane 110 made of third lipid molecules 110A is formed on the first lipid molecule membrane 109 to obtain a lipid bilayer membrane.

(a) As shown in FIG. 6, a first lipid molecule membrane 109 and a second lipid molecule membrane 110 consisting of a first lipid molecule 109A and a second lipid molecule 109B are simultaneously formed to obtain a lipid bilayer membrane.

The content of the first lipid molecule 109A in the dispersion can be adjusted as appropriate depending on the density of the first single-stranded nucleic acid molecule (first nucleic acid molecule) 104 immobilized on the substrate, and is 0.05 It is preferably mol% or more and 0.5 mol% or less.
Furthermore, the molar ratio of the first lipid molecule 109A to the second lipid molecule 109B in the dispersion is preferably 1/1000 or more and 1/10 or less. By having the above molar ratio, a more stable lipid bilayer membrane can be formed.
Examples of the solvent for dispersing the first lipid molecule 109A and the second lipid molecule 109B include chloroform and the like.

Further, as a specific method for forming the lipid bilayer membrane (model biological membrane 103) composed of the first lipid molecule membrane 109 and the second lipid molecule membrane 110, for example, the following methods (1) to (3) can be used. Examples include any of the following methods.

(1) Method for transferring a lipid monolayer onto a graphene membrane after preparing it First, a dispersion containing the first lipid molecule 109A and the second lipid molecule 109B is dropped onto the air-liquid interface, and the solvent is evaporated to form a lipid monolayer. A molecular membrane (first lipid molecular membrane 109) is formed. Thereafter, the lipid monolayer is left to stand still at a temperature higher than the phase transition temperature of the lipid molecules, and the lipid monolayer is compressed until the area occupied by the molecules becomes comparable to that of the cell membrane, and then transferred onto a graphene substrate (Figure 5). . A dispersion liquid in which only the third lipid molecules 110A are dispersed in water is added to this surface to form a first lipid molecule membrane 109 consisting of the first lipid molecule 109A and the second lipid molecule 109B, and a third lipid molecule membrane 109 consisting of the third lipid molecule 110A. A stable lipid bilayer membrane composed of a bilipid molecular membrane 110 is prepared (FIG. 6).

(2) Method of producing lipid bilayer vesicles and then transferring them onto a graphene membrane A dispersion containing a first lipid molecule 109A and a second lipid molecule 109B is dropped onto an indium tin oxide (ITO) glass substrate, The solvent is completely removed by evaporation. Thereafter, hydration is performed under an alternating current electric field to create a suspension containing lipid bilayer membrane vesicles. At this time, the first single-stranded nucleic acid molecule 104 immobilized on the adhesive layer and the second single-stranded nucleic acid molecule (second nucleic acid molecule) 105 are at least partially a nucleic acid having a two-stranded structure. At the same time as the molecules 106 are formed, the lipid bilayer membranes fuse and extend laterally with each other, so that a stable lipid bilayer membrane is stacked directly above the linker layer 102 (FIG. 6).

(3) Method of producing a lipid bilayer membrane by dropping a suspension in which lipid molecules are dispersed onto a graphene membrane This method is based on the content disclosed in Non-Patent Document 8. Specifically, first, lipid molecules are dispersed in a 1:1 mixture of water and an organic solvent (for example, ethanol, isopropanol, etc.), and this is dropped onto a graphene substrate. Water is added little by little (for example, 30 μL/min, etc.) for a predetermined period of time to increase the volume fraction of water, thereby spontaneously forming a lipid bilayer membrane. Finally, wash the substrate surface with a sufficient amount of water to remove unnecessary organic solvents and excess lipid molecules, and obtain the desired lipid bilayer membrane (Figure 6).

The first nucleic acid molecules 104 immobilized in the first nucleic acid molecule immobilization step may all have the same structure, or, for example, multiple types of first nucleic acid molecules 104-1, 104 with different chain lengths and sequences. -2, . . . , 104-N (N is a positive integer). When multiple types of first nucleic acid molecules 104-1, 104-2, . 1, 105-2, and 105-N must be combined. In this case, in the first lipid molecule membrane forming step, the first lipid molecule 109A is bound to the same number of second nucleic acid molecules having complementary sequences and corresponding to each of the plurality of types of first nucleic acid molecules. It is sufficient to prepare a solution of In the linker layer forming step, when the object to be treated 100A is made into a mixed solution, the second nucleic acid molecules 105-1, 105-2, and 105-N contained in the mixed solution each spontaneously convert to the corresponding first nucleic acid. It approaches the molecules 104-1, 104-2, . . . , 104-N and forms a pair.

Note that the two-dimensional pattern structure of nucleic acid molecules can be formed by the following procedure, for example. That is, in the adhesive molecule attaching step, the adhesive molecules 108 are uniformly attached to the entire surface of the graphene film 107, and in the next first nucleic acid molecule fixing step, the first nucleic acid molecules are attached using a known patterning method. Molecules 104 are introduced and fixed only at predetermined positions. As a result, the second nucleic acid molecule 105 reacts with the first nucleic acid molecule 104 fixed at a predetermined position to form a pair, so that the desired two-dimensional pattern structure of the nucleic acid molecule is finally formed. can be formed.

Known patterning methods include, for example, a method in which a microchannel structure having the same pattern as the position to be immobilized is used, a solution containing the first nucleic acid molecule is flowed and introduced only to the position to be immobilized, and the solvent is evaporated. (See Non-Patent Document 6). Other known patterning methods include a method of transferring the pattern of the first nucleic acid molecule 104 onto the adhesive molecule 108 using a template having the same pattern as the position to be fixed (microcontact printing method), etc. (see Non-Patent Document 7).

Further, as another example, the two-dimensional pattern structure of a nucleic acid molecule can also be formed by the following procedure. That is, in the adhesive molecule attachment step, the adhesive molecules 108 are attached only to the positions on the surface of the graphene film 107 where the first nucleic acid molecules 104 are desired to be fixed, using a known patterning method such as a microcontact printing method. This limits the immobilization position of the first nucleic acid molecule 104 introduced in the next first nucleic acid molecule immobilization step, so that in this case as well, the desired two-dimensional pattern structure of the nucleic acid molecule is finally formed. can be formed.

As described above, in the biomolecule support 100 according to the present embodiment, the model biomembrane 103 supporting the biomolecule P uses a nucleic acid molecule (dsDNA) 106 having a double helix structure as the linker molecule 106. , supported by a linker layer 102. The linker molecule 106 is a rigid nucleic acid molecule and will not become entangled in a thread even if it is formed at a low density, so the thickness of the linker layer 102 can be kept constant and the fluidity of the model biological membrane 103 can be improved. can be maintained. Furthermore, since the linker molecules 106 are not entangled, the linker layer can have a sufficient thickness and the model biological membrane 103 carrying the biomolecules P can be separated from the solid substrate 101. The probability of coming into contact with 101 can be reduced. Therefore, when the biomolecule support 100 of the present embodiment supports the biomolecule P, the fluidity of the support membrane (model biomembrane 103) is reduced and the biomolecule P is reduced due to contact with the solid substrate 101. Degeneration and functional decline can be suppressed.

In the biomolecule support 100 of this embodiment, the density of the linker molecules 106 can be reduced, so that fixing the linker molecules 106 to the portion on which the biomolecules P are supported can be omitted. As a result, the linker molecule 106 does not exist between the biomolecule P and the solid substrate 101, so not only the probability of contact between the solid substrate 101 and the biomolecule P but also the probability of contact between the linker molecule and the biomolecule P It is possible to further suppress degeneration and functional decline of the biomolecule P. Further, by reducing the number (density) of linker molecules 106 fixed to the model biomembrane 103, the degree of freedom in deforming the model biomembrane 103 increases, and the fluidity of the model biomembrane 103 can be further improved.

The graphene film 107 used in this embodiment not only functions as a scaffold for the nucleic acid molecules 106 but also functions as a sensor material that responds to light and electricity. Therefore, according to the biomolecule support 100 of the present embodiment, it is possible to perform highly sensitive measurements as a microarray sensor, for example, for the purpose of investigating ions and chemical substances released from the biomolecule P.

Note that when detecting the function or biological reaction of the biomolecule P supported on the model biomembrane 103 using a microarray sensor or the like provided on a solid substrate, from the viewpoint of highly sensitive measurement and measurement of a minute area. , it is advantageous for the model biological membrane 103 to be located close to the solid substrate 101. In such a case, by controlling the number of base pairs constituting the nucleic acid molecule 106, the model biomembrane 103 can be brought close to the solid substrate 101 within a range that does not cause the above-mentioned denaturation or functional decline of the biomolecule P. is preferred. Since base pairs are stacked at intervals of 0.34 nm, it is thought that if the number of base pairs is reduced to about 100 or less, highly sensitive measurement and measurement of minute regions will be possible. When the number of base pairs is 100, the thickness of the linker layer 102 is about 30 nm, so the biomolecule P to be detected is a membrane-related biomolecule that has a structure that extends outward from the model biomembrane 103 by about several tens of nanometers. Even if it is, contact with the solid substrate 101 can be avoided.

Hereinafter, the effects of the present invention will be made clearer by way of Examples. It should be noted that the present invention is not limited to the following examples, and can be implemented with appropriate changes without changing the gist thereof.

(Example)
A sample of the biomolecule support according to the above embodiment was produced in the following procedure. First, as a solid substrate, a commercially available silicon wafer was divided into chips of about 1 to several centimeters square. Next, a graphene film grown by chemical vapor deposition on the copper foil was transferred onto one main surface of this solid substrate.

Next, pyrene butanoic acid was introduced onto the surface of the transferred graphene film as an adhesive molecule for fixing single-stranded nucleic acid molecules. Specifically, a 0.5mM dimethylformamide solution of pyrenebutanoic acid succinimide ester, in which the COOH group of pyrenebutanoic acid was activated with succinimide, was dropped onto the graphene surface and allowed to stand for 1 hour, then washed with dimethylformamide, and air-dried. did.

Next, a polymer sheet having a microchannel structure (width and spacing: 10 μm, length of straight portion: 5 mm) was placed on the graphene surface into which pyrene butanoic acid had been introduced. A polymer sheet having a microchannel structure was produced by pouring unpolymerized polydimethylsiloxane (PDMS) into a mold made by photolithography and molding it by polymerization. An aqueous solution (100 μM) of a first nucleic acid molecule with an amino group bound to its 5′ end in advance was introduced into the microchannel. A first nucleic acid molecule (A15) having 15 bases and a first nucleic acid molecule (A37) having 37 bases were patterned onto separate solid substrates and used.

Next, these are allowed to stand at room temperature (approximately 25° C.) for 1 hour, and the COOH group of pyrene butanoic acid adsorbed on graphene is reacted with the amino group bonded to the 5-terminus of the aptamer, resulting in a peptide bond. By washing these with ultrapure water and air drying, the first nucleic acid molecules bound to pyrene butanoic acid are fixed to the graphene membrane so as to form a pattern with a width and spacing of 10 μm and a straight portion length of 5 mm. Ta.

Next, a first lipid molecule (DOPE) is bound to the 5' end of second nucleic acid molecules C-A15 and C-A37, which have complementary sequences to the first nucleic acid molecules A15 and A37, respectively. A second lipid molecule (DOPC) to which no nucleic acid molecules were bound was mixed in chloroform so that the concentration ranged from 0.05 to 0.5 mol%.

Any one of the following (1), (2), and (3) can be used as a method for producing a lipid molecule film lifted from a solid substrate by DNA chains.
(1) After dropping the lipid mixture onto the air-liquid interface and evaporating the solvent, the membrane is compressed until the area occupied by the molecules becomes comparable to that of the cell membrane, and this is transferred to the graphene membrane. A lipid suspension is added to this surface to create a stable membrane.
(2) A suspension of lipid bilayer membrane vesicles prepared by dropping a lipid solution onto an ITO glass substrate, completely removing the solvent, and hydrating it under an alternating current electric field is stabilized by dropping it onto a graphene membrane. Fabricate a membrane.
(3) A suspension of lipid molecules is dropped onto a graphene membrane, and water is gradually added to exchange the solution to create a stable membrane.

In this example, method (3) was used, but a similar first lipid molecule membrane can also be obtained using methods (1) and (2).

To confirm that a lipid molecule membrane is formed and to confirm that the fluidity of the lipid molecule membrane is maintained, the membrane is mixed with approximately 0.5 mol% of a fluorescent label (for example, Texas-Red DHPE). were prepared and performed using confocal fluorescence microscopy images. A15 (or A37) immobilized on the graphene membrane surface forms a double helix structure with C-A15 (or C-A37), and at the same time, the lipid bilayer membranes fuse and extend laterally with each other. , a stable biofilm was deposited on the graphene surface. At this time, fluidity was maintained not only in the patterned part of the first nucleic acid molecule A15 (or A37) immobilized on the graphene membrane surface, but also in the area where the first nucleic acid molecule was not immobilized with an interval of 10 μm therebetween. We were able to create a biological membrane (self-supporting membrane).

Next, membrane proteins (biomolecules) are introduced (supported) into the biological membrane of the sample in (iii'), and the antigen-antibody reaction is confirmed to determine whether there is a structural change (denaturation) or functional decline in the membrane protein. We evaluated the following. The first nucleic acid molecule A37 to be fixed on the surface of the graphene membrane was fluorescently labeled, and the position of the two-dimensional pattern was confirmed using a fluorescence microscope image in parallel.

Because the linker layer is thick enough to avoid contact between membrane proteins and the solid substrate surface, deactivation of membrane protein structure and function occurs in parts of the free-standing membrane that are not supported by nucleic acid molecules. was hardly observed. Even in areas supported by nucleic acid molecules, the density of supporting nucleic acid molecules is relatively low, so it was found that there are membrane proteins whose structure and function have not been deactivated. It was found that the membrane proteins that are more stable are more stable. These results demonstrated the effectiveness of the present invention.

100...Biomolecule supports 100A, 100B...Processed object 101...Solid substrate 101a...One main surface 102 of solid substrate...Linker layer 102A...Fixed region 102B... - Central part 103...Model biological membrane 103A...Self-supporting membrane 103B...Support membrane 104...First nucleic acid molecule 104a...One end of the first nucleic acid molecule 104b...Other than the first nucleic acid molecule End 105...Second nucleic acid molecule 105a...One end 105b of second nucleic acid molecule...Other end 106 of second nucleic acid molecule...Nucleic acid molecule 107...Graphene membrane 108...Adhesion molecule 109. ...Lower lipid molecule membrane (first lipid molecule membrane)
109A...First lipid molecule 109B...Second lipid molecule 110...Upper layer side lipid molecule membrane (second lipid molecule membrane)
110A...Third lipid molecule P...Biomolecule

Claims (5)

a solid substrate;
a linker layer formed on one main surface of the solid substrate;
a biological membrane formed on the linker layer and supporting biomolecules;
The linker layer has a plurality of pairs of nucleic acid molecules forming a double helix structure, each consisting of a helical first nucleic acid molecule and a helical second nucleic acid molecule, and one end of the first nucleic acid molecule is connected to the solid body . fixed to one main surface of the substrate, one end of the second nucleic acid molecule being fixed to the biological membrane,
The biological membrane is made up of a plurality of overlapping lipid molecule membranes, and one end of the second nucleic acid molecule is fixed to the lipid molecule membrane on the side closest to the solid substrate,
The lipid molecule membrane on the side closest to the solid substrate is composed of a plurality of first lipid molecules to which the second nucleic acid molecules are bound and a plurality of second lipid molecules to which the second nucleic acid molecules are not bound. Become,
A support for biomolecules, wherein a molar ratio of the first lipid molecule to the second lipid molecule is 1/1000 or more and 1/10 or less. 2. The support for biomolecules according to claim 1, wherein one end of the second nucleic acid molecule is fixed to a portion of the biomembrane where no biomolecules are supported. The biomolecule support according to claim 1 or 2, wherein both the first nucleic acid molecule and the second nucleic acid molecule are composed of 10 to 200 bases. . The biomolecule according to any one of claims 1 to 3, wherein one end of the first nucleic acid molecule is fixed to one main surface of the solid substrate via a graphene film. support. a graphene film forming step of forming a graphene film on one main surface of the solid substrate;
an adhesive molecule attachment step of attaching an adhesive molecule for fixing the first nucleic acid molecule to the surface of the graphene film;
a first nucleic acid molecule fixing step of fixing one end of the first nucleic acid molecule to a predetermined region on the graphene film via the adhesive molecule;
A first lipid comprising a plurality of first lipid molecules to which second nucleic acid molecules having a complementary sequence to the first nucleic acid molecule are bound, and a plurality of second lipid molecules to which the second nucleic acid molecules are not bound. a first lipid molecule membrane forming step for forming a molecular membrane;
a linker layer forming step of reacting the first nucleic acid molecule and the second nucleic acid molecule to form a linker layer consisting of a pair of both nucleic acid molecules;
A second lipid molecule membrane forming step of forming a second lipid molecule membrane consisting only of a plurality of second lipid molecules to which the second nucleic acid molecules are not bound on the first lipid molecule membrane. The method for producing a biomolecule support according to any one of claims 1 to 4.

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