Lipid membrane giant vesicle, method for preparing lipid membrane giant vesicle, method for detecting target biomolecule contained in lipid membrane giant vesicle, and biosensor

Abstract

To provide a lipid membrane giant vesicle in which a target biomolecule contained in the lipid membrane giant vesicle can be detected with high sensitivity by surface-enhanced Raman scattering, a method for producing the same, a method for detecting a target biomolecule contained therein, and a biosensor including the same.SOLUTION: A lipid membrane giant vesicle contains an aggregate of metal nanocolloids, wherein a metal in the metal nanocolloids is a metal that causes surface-enhanced Raman scattering. A method for producing the lipid membrane giant vesicle has the steps of: (a) preparing a W / O emulsion containing a dispersion of metal nanocolloids; and (b) passing the W / O emulsion through a lipid monolayer formed on a surface of an aqueous solution to form a lipid membrane giant vesicle containing metal nanocolloids in the aqueous solution, wherein the metal in the metal nanocolloids is a metal that causes surface-enhanced Raman scattering.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a giant lipid membrane vesicle, a method for producing a giant lipid membrane vesicle, a method for detecting a target biomolecule contained inside the giant lipid membrane vesicle, and a biosensor. [Background technology]

[0002] Giant unilamellar vesicles (GUVs) have been widely studied and utilized as simplified cell models or as the basis for artificial cells. GUVs are also utilized as micro-volume reaction sites for cell-free protein synthesis and other applications (Non-Patent Document 1). It has also been reported that GUVs can incorporate vesicle contents into GUVs by fusing with vesicles (Non-Patent Document 2). Based on these findings, it is anticipated that GUVs can be utilized as biosensors by analyzing biomolecules produced depending on the GUV's environment, the contents incorporated by fusion, and their reaction products.

[0003] On the other hand, surface-enhanced Raman scattering (SES) is a technique that detects Raman scattering of chemical species adsorbed on metal surfaces in microstructures and nanoparticles of metals such as gold, silver, and copper. 4 ~10 6 Depending on the conditions of surface-enhanced Raman scattering, further signal enhancement has been reported, reaching 10 times the sensitivity of a single molecule. 10 ~10 14 Although it is not easy to actually detect a single molecule, surface-enhanced Raman scattering is attracting attention as a highly sensitive molecular detection method that is expected to significantly enhance sensitivity. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Susanne F. Fenz et al., “Cell-free synthesis of membrane proteins: Tailored cell models out of microsomes”, Biochimica et Biophysica Acta Biomembranes, Vol. 1813,p. 1382-1388, 2014. [Non-patent document 2] Daiya Mombayashi et al., “Factors Facilitating Fusion between Dye-encapsulating Vesicles and Giant Unilamellar Vesicles”, Sensors and Materials, Vol. 33, p. 4361, 2021. [Non-patent document 3] Masayuki Futamata, "Surface-Enhanced Raman Scattering," Surface Science, Vol. 33, No. 4, pp. 216-222, 2012. [Non-patent document 4] EC Le Ru et al., “Principles of Surface-Enhanced Raman Spectroscopy”, Elsevier, 2009. [Non-Patent Document 5] Taro Toyoda, Yiting Zhang, "Effect of oily dispersion medium on giant vesicles prepared by water-in-oil emulsion," BUNSEKI KAGAKU, Vol. 71, No. 1-2, pp. 83-89, 2022. Summary of the Invention [Problem to be solved by the invention]

[0005] GUVs have diameters of several micrometers to several tens of micrometers, which is about the same size as a cell. Therefore, in order to detect trace components encapsulated in GUVs, it is necessary to increase the sensitivity. The present invention has been made in consideration of the above circumstances, and aims to provide a GUV in which target biomolecules contained therein can be detected with high sensitivity using surface-enhanced Raman scattering, a method for producing the GUV, a method for detecting target biomolecules contained therein, and a biosensor including the GUV. [Means for solving the problem]

[0006] That is, the present invention includes the following aspects. [1] A giant lipid membrane vesicle encapsulating an aggregate of metal nanocolloids, wherein the metal in the metal nanocolloids is a metal that causes surface-enhanced Raman scattering. [2] The giant lipid membrane vesicle according to [1], wherein the metal that causes surface-enhanced Raman scattering is at least one metal selected from the group consisting of gold, silver, and copper. [3] The giant lipid membrane vesicle according to [1] or [2], wherein the giant lipid membrane vesicle contains a cation-permeable ionophore or pore-forming protein. [4] A method for producing giant lipid membrane vesicles, comprising: (a) preparing a W / O emulsion containing a metal nanocolloid; and (b) passing the W / O emulsion through a lipid monolayer formed at the oil-water interface of a solution in which an oil phase is formed on top of an aqueous phase, to form giant lipid membrane vesicles encapsulating the metal nanocolloid within the aqueous phase, wherein the metal in the metal nanocolloid is a metal that causes surface-enhanced Raman scattering. [5] The method for producing giant lipid membrane vesicles according to [4], wherein in the step (a), a W / O emulsion containing the metal nanocolloid aggregates is prepared. [6] The method for producing a giant lipid membrane vesicle according to [4] or [5], wherein at least one of the W / O emulsion and the lipid monolayer contains a cation-permeable ionophore or pore-forming protein, and in the step (b), a giant lipid membrane vesicle containing the ionophore or pore-forming protein is formed, and after the step (b), the method further comprises the step (c) of increasing the cation concentration in the external solution of the giant lipid membrane vesicle formed in the step (b) to be higher than the cation concentration in the aqueous solution contained in the giant lipid membrane vesicle, thereby aggregating the metal nanocolloid inside the giant lipid membrane vesicle. [7] A method for detecting a target biomolecule contained inside a giant lipid membrane vesicle, comprising detecting the target biomolecule contained inside the giant lipid membrane vesicle according to any one of [1] to [3] by surface-enhanced Raman scattering. [8] A biosensor comprising the giant lipid membrane vesicle according to any one of [1] to [3]. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a GUV in which target biomolecules contained within the GUV can be detected with high sensitivity using surface-enhanced Raman scattering, a method for producing the GUV, a method for detecting target biomolecules contained within the GUV, and a biosensor including the GUV. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 1 is an explanatory diagram of an example of a metal nanocolloid-encapsulated GUV. [Figure 2] 2(a) to 2(d) are explanatory diagrams of an example of a method for detecting a target biomolecule contained inside a GUV. [Figure 3] Figure 3(a) is an explanatory diagram of an example of a biosensor, and Figure 3(b) is an explanatory diagram of an example of a linker molecule constituting the biosensor. [Figure 4] Figure 4(a) shows the intensity distribution of Raman scattered light (Raman mapping) at the location where the GUV was observed, and Figure 4(b) shows a fluorescence image observed with a fluorescence microscope (FITC filter cube). [Figure 5] Figure 5(a) shows the intensity distribution of Raman scattered light at the location where GUVs were observed when the final NaCl concentration of the GUV external solution was 0 mM. Figure 5(b) shows the intensity distribution of Raman scattered light at the location where GUVs were observed when the final NaCl concentration of the GUV external solution was 50 mM. Figure 5(c) shows the Raman spectra obtained when the intensity distribution of Raman scattered light was measured when the final NaCl concentration of the GUV external solution was 0 mM or 50 mM. DETAILED DESCRIPTION OF THE INVENTION

[0009] [Definition] In this specification, the term "comprise" is a concept that encompasses all of "comprise," "consist essentially of," and "consist only of."

[0010] In this specification, the "particle size of the metal nanocolloid" refers to the average particle size measured on a volume basis using a dynamic light scattering particle size distribution measuring device. The average particle size can be measured, for example, using a dynamic light scattering particle size distribution analyzer "Zetasizer Pro" manufactured by Spectris.

[0011] <Giant lipid membrane vesicles (GUVs)> One aspect of the present invention is a giant lipid membrane vesicle encapsulating aggregates of metal nanocolloids that induce surface-enhanced Raman scattering. The aggregates of the metal nanocolloids can significantly amplify the intensity of Raman scattering obtained by surface-enhanced Raman scattering.

[0012] <Metal nanocolloid> The metal in the metal nanocolloid is preferably one or more metals selected from the group consisting of metals that cause surface-enhanced Raman scattering, more preferably one or more metals selected from the group consisting of gold, silver, and copper, and even more preferably gold.

[0013] The mass concentration or particle number of the metal nanocolloid encapsulated in the GUV is not particularly limited as long as it is a condition that can cause surface-enhanced Raman scattering, and is selected appropriately depending on the size of the GUV, the particle size of the metal nanocolloid, etc. For example, when the diameter of the GUV is 30 μm and the particle size of the metal nanocolloid is 50 nm, the number of metal nanocolloids encapsulated in one GUV is estimated to be about 10,000 on average.

[0014] The metal nanocolloid encapsulated in the GUV of this embodiment preferably has a particle size of 5 to 200 nm, more preferably 10 to 160 nm, and from the viewpoint of the effect of enhancing surface-enhanced Raman scattering light, further preferably 20 to 80 nm.

[0015] It is sufficient that at least a portion of the metal nanocolloids encapsulated in the GUVs form aggregates, and it is preferable that all of them form aggregates. The number of metal nanocolloid aggregates encapsulated in the GUVs is not particularly limited as long as it is a number that can enhance the surface-enhanced Raman scattering light intensity. In this embodiment, the number of metal nanocolloid aggregates encapsulated in the GUVs is preferably several to several tens.

[0016] ≪Lipid molecules≫ GUVs are formed from lipid molecules. Examples of lipid molecules include phospholipids such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylglycerol, as well as sphingolipids. A single lipid molecule or a mixture of multiple lipid molecules may be used. Lipid-soluble components such as cholesterol may also be included as needed.

[0017] <Cation> Increasing the cation concentration inside the GUV can promote the aggregation of metal nanocolloids. Metal nanoparticles generally have an electric charge in solution, and in the case of metal nanoparticles, they are often negatively charged. This surface charge allows metal nanocolloids to be stably dispersed in solution. When the cation concentration in the solution increases, the electrostatic repulsion caused by the negative charge on the surface of the metal nanocolloids is canceled out, promoting aggregation.

[0018] The cations used to increase the cation concentration inside the GUV are not particularly limited as long as they can promote the aggregation of metal nanocolloids, but examples include Na ions, K ions, Ca ions, Mg ions, etc., and one or more cations selected from the group consisting of Na ions and K ions are preferred, with Na ions being more preferred, as they more easily increase the cation concentration inside the GUV.

[0019] <Cation-permeable component> The GUVs of this embodiment may have a cation-permeable component embedded therein. By incorporating a cation-permeable component into the lipid membrane of the GUV, the cation concentration within the GUV can be controlled by changing the cation concentration in the external solution of the GUV. Examples of cation-permeable components include ionophores and pore-forming proteins.

[0020] For example, when the lipid membrane of the GUV contains at least one of an ionophore and a pore-forming protein, the cation concentration inside the GUV can be increased by increasing the cation concentration in the external solution of the GUV, thereby aggregating the metal nanocolloids inside the GUV. It is more preferable to introduce an ionophore as the cation-permeable component contained in the GUV of this embodiment.

[0021] Examples of ionophores to be introduced into GUVs include gramicidin (CAS number: 1405-97-6), valinomycin (CAS number: 2001-95-8), and nystatin (CAS number: 1400-61-9). Gramicidin or nystatin is preferred, and gramicidin is more preferred from the viewpoint of ease of introduction. Examples of the pore-forming protein to be introduced into the GUV include ion channel proteins and α-hemolysin. From the viewpoint of ease of introduction, α-hemolysin derived from Staphylococcus aureus (CAS number: 94716-94-6) is preferred.

[0022] <Method for producing GUV> One aspect of the present invention includes: (a) a step of preparing a W / O emulsion containing metal nanocolloids; and (b) a step of passing the W / O emulsion through a lipid monolayer formed at the oil-water interface of a solution in which an oil phase is formed on the upper surface of an aqueous phase to form a lipid membrane giant vesicle encapsulating the metal nanocolloids in the aqueous phase. This is a method for producing GUV.

[0023] In step (a), the W / O emulsion containing metal nanocolloids can be formed by emulsifying an aqueous dispersion of metal nanocolloids with an oil liquid containing lipid molecules. The metal in the metal nanocolloids is a metal that causes surface-enhanced Raman scattering. As the metal, those mentioned above can be used.

[0024] The aqueous dispersion of metal nanocolloids is prepared by dispersing metal nanocolloids in an aqueous medium. The aqueous medium may be water or an aqueous solution in which an appropriate amount of a non-cationic component such as sucrose is dissolved in water to adjust the osmotic pressure.

[0025] The metal nanocolloids in the aqueous dispersion may be a dispersion of single particles, or a dispersion in which some or all of the metal nanocolloids form aggregates. For example, a dispersion containing aggregates of metal nanocolloids can be prepared by previously dispersing metal nanocolloids in an aqueous medium with a high cation concentration. Also, metal nanocolloids may be dispersed in an aqueous medium with a low cation concentration to form a dispersion of single particles, and then aggregated after being encapsulated in GUV.

[0026] The lipid-containing oil solution is prepared by dissolving the lipid molecules that make up the GUVs in an oil-based medium, such as hexadecane, chloroform, ether, or benzene. The emulsification can be carried out by a conventional method using a homogenizer or the like.

[0027] A W / O emulsion is passed through a lipid monolayer formed at the oil-water interface of a solution in which an oil phase is formed on top of an aqueous phase, forming giant lipid vesicles encapsulating metal nanocolloids within the aqueous phase. This method utilizes the interface passing method (Non-Patent Document 5). Specifically, an oil solution containing lipid molecules that constitute GUVs is slowly layered on top of an aqueous solution. This forms a laminate in which the oil phase is on top of the aqueous phase and a layer of the lipid molecules is formed at the oil-water interface. Next, the W / O emulsion is passed from the oil phase side of the lipid molecular layer to the aqueous phase side by being dropped from above the laminate. This forms a GUV consisting of a lipid bilayer membrane consisting of a lipid molecular layer formed at the oil-water interface in the W / O emulsion and a lipid molecular layer formed at the oil-water interface of the laminate.

[0028] In the method for producing GUVs of this embodiment, pre-aggregated metal nanocolloids can be encapsulated in GUVs in which an ionophore or pore-forming protein is embedded in a lipid membrane, and then the cation concentration of the external solution of the GUV can be controlled to aggregate the metal nanocolloids inside the GUV. Alternatively, pre-aggregated metal nanocolloids can be encapsulated in GUVs, and then the ionophore or pore-forming protein can be embedded in the lipid membrane, and then the cation concentration of the external solution of the GUV can be controlled to aggregate the metal nanocolloids inside the GUV.

[0029] First, at least one of the W / O emulsion and the lipid monolayer at the oil-water interface of the laminate contains a cation-permeable ionophore or a pore-forming protein. Thereby, one or more selected from the group consisting of ionophores and pore-forming proteins can form GUVs embedded in the lipid membrane. Examples of the ionophore or pore-forming protein to be introduced include the same ionophores or pore-forming proteins as those described in the <<Cation-permeable component>>.

[0030] Next, the metal nanocolloids inside the GUV are aggregated (step (c)) by making the cation concentration in the external solution of the formed GUV higher than the cation concentration in the aqueous solution encapsulated by the GUV.

[0031] Since the aggregation of the metal nanocolloids further proceeds, it is preferable to add cations so that the cation concentration in the GUV external solution becomes a final concentration of 5 mM to 100 mM, more preferably to add cations so that the final concentration becomes 10 mM to 80 mM, and even more preferably to add cations so that the final concentration becomes 30 mM to 60 mM.

[0032] An example of the GUV encapsulating metal nanocolloids of the present embodiment is shown in FIG. 1. Note that the present invention is not limited by this example. In FIG. 1, the GUV (reference numeral 1) is formed from lipid molecules (reference numeral 2) and encapsulates metal nanocolloids (reference numeral 3) and target biomolecules (reference numeral 5) inside the GUV. The GUV may contain an ionophore or a pore-forming protein (reference numeral 4).

[0033] <Method for detecting target biomolecules contained inside GUV> By measuring the surface-enhanced Raman scattering of the target biomolecules contained inside the GUV in the present embodiment, the target biomolecules contained inside the GUV can be detected with high sensitivity.

[0034] [[ID=ID=22]]<<Target biomolecule>> The target biomolecules to be detected by surface-enhanced Raman scattering may be biomolecules produced within the GUV or biomolecules incorporated into the GUV. Examples of biomolecules produced within GUVs include, but are not limited to, polypeptide chains. The biological molecules that can be incorporated into GUVs include, but are not limited to, the contents of biological vesicles such as viruses and exosomes, and examples of such contents include DNA, RNA, peptides, proteins, and sugars. In addition, methods for incorporating biomolecules into GUVs include vesicle fusion, such as the fusion of biological vesicles such as liposomes and exosomes.

[0035] It is preferable that the target biomolecules be embedded between the metal nanocolloid aggregates, as this will result in greater sensitivity enhancement due to surface-enhanced Raman scattering. To efficiently incorporate the target biomolecules into the metal nanocolloid aggregates, it is preferable to aggregate the metal nanocolloids in the presence of the target biomolecules. Specifically, the cation concentration in the GUV is increased through an ionophore or a pore-forming protein, causing the metal nanocolloids to aggregate. Examples of the cation concentration to be increased include the same cation concentrations as those described in the "Cations" section above.

[0036] <Method for detecting target biomolecules> To detect target biomolecules contained within GUVs, excitation light is irradiated onto GUVs containing metal nanocolloid aggregates and target biomolecules, and the resulting Raman scattered light is passed through a spectrometer to obtain a Raman spectrum. The obtained Raman spectrum allows for highly sensitive detection of target biomolecules contained within the GUVs. The method for detecting the surface-enhanced Raman scattering is not particularly limited, and any known method can be appropriately selected and used. For example, the surface-enhanced Raman scattering can be detected using an excitation wavelength of 532 nm, 785 nm, or 1064 nm.

[0037] An example of a method for detecting a target biomolecule contained within a GUV according to this embodiment is shown in Figures 2(a) to 2(d). However, the present invention is not limited to this example. As shown in Figures 2(a) and 2(b), a target biomolecule (5) encapsulated in a biological vesicle (6) is incorporated into the GUV (1) through membrane fusion. Next, as shown in Figures 2(c) and 2(d), the concentration of cations (7) inside the GUV is increased, causing the metal nanocolloids to aggregate while incorporating the target biomolecule. Next, as shown in Figure 2(d), excitation light (8) is irradiated onto the metal nanocolloid aggregates incorporating the target biomolecule, and a Raman spectrum can be obtained from the generated Raman scattered light (9). In GUVs that do not encapsulate target biomolecules, the formed metal nanocolloid aggregates do not contain the target biomolecules, so the Raman scattered light at the wavelength corresponding to the characteristic molecular bond of the target molecule is weaker than the Raman scattered light emitted from metal nanocolloid aggregates that have incorporated the target biomolecules.This allows GUVs that encapsulate target biomolecules to be detected and distinguished from GUVs that do not encapsulate target biomolecules.

[0038] <Biosensor> The biosensor of this embodiment includes the GUV described above. For example, the GUV of this embodiment contains a target biomolecule to be measured together with an aggregate of metal nanocolloids. In this case, it is preferable that the target biomolecule is adsorbed onto the surface of the metal nanocolloids. In order to use the GUV as a biosensor, it is necessary to detect trace components contained in the GUV with high sensitivity. The GUV-metal nanocolloid aggregate of this embodiment provides a biosensor with enhanced detection sensitivity for trace amounts of target biomolecules.

[0039] An example of a biosensor according to this embodiment is shown in Figure 3(a). However, the present invention is not limited to this example. In Figure 3(a), the biosensor (reference numeral 10) includes a metal nanocolloid-encapsulated GUV (reference numeral 1), a solid substrate (reference numeral 11), and a surface Raman scattering measurement device (reference numeral 12). The solid substrate is surface-treated with a coating agent (reference numeral 13), and the GUV and the solid substrate are bonded together by linker molecules (reference numeral 14). The linker molecule can be formed, for example, by binding biotin (reference number 14-1) and streptavidin (reference number 14-2), as shown in FIG. 3(b).

[0040] Next, each of the components included in the biosensor of this embodiment will be described in detail below.

[0041] <GUV> As the GUVs containing metal nanocolloid aggregates, <guv>The same can be mentioned.

[0042] ≪Solid substrate≫ The solid substrate is a substrate on which a biosensor is formed. The material of the solid substrate 2 is not particularly limited as long as it can immobilize GUVs, but examples include plastic, glass, and quartz, and glass is preferred from the viewpoint of optical transparency.

[0043] Furthermore, from the viewpoint of ease of immobilization, it is preferable that the solid substrate has a flat substrate surface, and from the viewpoint of preventing GUVs from being destroyed even when they come into contact with the solid substrate, it is preferable that the surface be treated with a coating agent. The coating agent used for surface treatment of the solid substrate is not particularly limited as long as it is not destroyed when the GUVs come into contact with the solid substrate and can bind to the GUVs. Examples of the coating agent include albumin and collagen, and bovine serum albumin (BSA) is more preferred because it is widely used. When the GUV has a positive charge, the coating agent used for surface treatment of the solid substrate is preferably a coating agent having a positive charge, and more preferably poly-L-lysine or poly-L-ornithine.

[0044] <Linker molecule> The linker molecule that bonds the GUV to the solid substrate is not particularly limited as long as it can bond the GUV to the solid substrate, but a biotin-binding protein is preferred, at least one selected from the group consisting of streptavidin, avidin, and neutravidin is more preferred, and streptavidin is even more preferred.

[0045] One method for linking giant lipid membrane vesicles to the solid substrate using linker molecules is to add biotinylated lipids to the GUVs and coating agent, followed by the addition of a biotin-binding protein. The GUVs are immobilized on the solid substrate via the interaction between biotin and the biotin-binding protein.

[0046] <Surface Raman scattering measurement device> The surface Raman scattering measurement device is not particularly limited as long as it can be measured by the same method as the above-described <method for detecting target biomolecules contained inside GUV>.

[0047] <Method for manufacturing a biosensor> The method for manufacturing the biosensor of the present embodiment is not particularly limited as long as it can manufacture a biosensor capable of highly sensitively detecting trace amounts of target biomolecules. For example, a method can be used in which a solid substrate surface-treated with a coating agent and a GUV encapsulating metal nanocolloids are fixed with a linker molecule. As the GUV, a GUV encapsulating metal nanocolloids manufactured by the same method as the above-described <method for manufacturing GUVs> can be used. As a method for fixing the GUV to the solid substrate, it can be fixed to the solid substrate by the same method as the above-described <<linker molecule>>.

[0048] <Method of using a biosensor> The biosensor of the present embodiment is used for detecting target biomolecules. The method of using the biosensor of the present embodiment is not particularly limited as long as it can highly sensitively detect trace amounts of target biomolecules. For example, the following method can be used: Biological vesicles containing target biomolecules are flowed onto a solid substrate on which metal nanocolloid-encapsulated GUVs are fixed, and the target biomolecules are incorporated into the GUVs through membrane fusion between the biological vesicles and the GUVs. The solid substrate on which the GUVs are fixed is preferably placed in a chamber containing the target biomolecules. Furthermore, the GUVs fixed to the solid substrate are preferably modified to selectively react with the biological vesicles. This modification can be achieved by introducing lipid molecules modified with receptor proteins or spike proteins into the lipid membrane of the GUVs. Next, the cation concentration in the external solution of the GUV is increased, thereby increasing the cation concentration within the GUVs and causing the metal nanocolloids to aggregate, forming metal nanocolloid aggregates incorporating the target biomolecules. Subsequently, the target biomolecules contained within the GUVs are detected by irradiating the metal nanocolloid-encapsulated GUVs fixed to the solid substrate with excitation light using a surface Raman scattering measurement device, and detecting Raman scattered light emitted from the metal nanocolloid aggregates incorporating the target biomolecules.

[0049] <Target biomolecule> Examples of target biomolecules to be detected by the biosensor of this embodiment include those similar to those described in the section <<Target Biomolecules>> of the above-mentioned <Giant Lipid Membrane Vesicle>>. [Example]

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0051] [Example 1] We demonstrated surface-enhanced Raman scattering within GUVs when gold nanocolloid aggregates were encapsulated within the GUVs.

[0052] Gold nanocolloid dispersion with a particle size of 50 nm (Sigma Aldrich, product name "50 nm diameter, OD 1, stabilized suspension in 0.1 mM PBS, reactant-free"), mass concentration 4.45 × 10 -2 mg / mL, number of particles 3.5×10 10 900 μL of the solution (particles / mL) was centrifuged at 6,500 G for 10 minutes, and 877.5 μL of the supernatant was removed. The precipitate obtained by centrifugation was redispersed by vortexing to produce a 40x concentrated gold nanocolloid dispersion. To 10 μL of the obtained 40-fold concentrated metal nanocolloid dispersion, 10 μL of a 200 mM sucrose (200 mM) solution containing 10 mM 2,2'-bipyridine was added to obtain a 20-fold concentrated gold nanocolloid dispersion (mass concentration 8.9 × 10) containing 5 mM 2,2'-bipyridine. -2 mg / mL, number of particles 7.0×10 11 particles / mL). Furthermore, 1 μL of NaCl solution (200 mM) was added dropwise to the obtained 20-fold concentrated gold nanocolloid dispersion to a final concentration of 10 mM, causing some of the gold nanocolloids in the dispersion to aggregate, thereby obtaining an aqueous solution containing metal nanocolloid aggregates.

[0053] 16 μL of an aqueous solution containing the metal nanocolloid aggregates was added to 120 μL of a hexadecane solution (0.95 mM) of lipid molecules (a 95:5 mixture (molar ratio) of Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and cholesterol), and a W / O emulsion was prepared by pipetting several times.

[0054] A monolayer of lipid molecules was formed at the oil-water interface by carefully dropping 60 μL of a hexadecane solution (0.95 mM) of lipid molecules (a 95:5 mixture of POPC and cholesterol) onto a microtube containing 100 μL of a 200 mM glucose solution. The W / O emulsion was then dropped onto the monolayer and centrifuged at 300 G for 30 minutes, forcing the W / O emulsion to pass through the interface and form GUVs. For GUV observation, biotin-modified 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was added at 1% (molar ratio) to the lipid molecules to fix the GUV to the cover glass, and NBD-labeled lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl)) was added at 0.5% (molar ratio) for fluorescence observation.

[0055] A cover glass was coated with bovine serum albumin (BSA) (1% biotin-modified BSA). A chamber was created by sandwiching two cover glasses between 2 mm-thick silicone rubber bands (U-shaped), and the chamber was filled with 200 μL of glucose solution (200 mM). Streptavidin (100 μg / mL, 5 μL) was added to the chamber and allowed to stand for 15 minutes. The cover glass surface was then thoroughly rinsed with glucose solution (200 mM) to remove excess streptavidin. Then, 10 μL of the aforementioned gold nanocolloid-encapsulated GUV dispersion was added to the chamber and allowed to stand for 5 minutes. The external solution of the GUV was then replaced with glucose solution (200 mM), and the unfixed GUVs were removed. The GUVs were then used as the observation sample.

[0056] The results of observation using a fluorescence microscope (FITC filter cube) are shown in Figure 4(b). As a result, three GUVs with a diameter of approximately 20 μm were observed.

[0057] The Raman scattering intensity distribution around the GUV was measured and shown in Figure 4(a). Raman scattering was measured using an excitation light of 785 nm, with a measurement time of 0.3 seconds per measurement point, and three measurements were accumulated. The scanning range was 100 × 100 μm. 2 The measurement was performed with a distance of 5 μm between measurement points. In Figure 4(a), Raman mapping was performed at a wavenumber of 1010 cm -1 The Raman scattered light intensity at

[0058] As a result, a significant increase in Raman scattering intensity was observed in the presence of GUVs, and this increase in Raman scattering intensity was observed only in the areas where gold nanocolloid aggregates were present within the GUVs.

[0059] These results demonstrate that surface-enhanced Raman scattering signals can be measured from metal nanocolloid aggregates encapsulated in GUVs.

[0060] [Example 2] We demonstrated the aggregation of gold nanocolloids and the detection of surface-enhanced Raman signals through Na ion permeation in GUVs containing gramicidin, a type of ionophore.

[0061] A 20-fold concentrated gold nanocolloid dispersion (mass concentration 8.9 × 10) was prepared in the same manner as in Example 1. -2 mg / mL, number of particles 7.0×10 11 particles / mL).

[0062] GUVs encapsulating a gold nanocolloid dispersion were prepared in the same manner as in Example 1, except that 0.5% (molar ratio) of gramicidin A (manufactured by Sigma-Aldrich, trade name "Gramicidin A derived from Bacillus brevis") was mixed with lipid molecules (a 95:5 mixture (molar ratio) of POPC and cholesterol).

[0063] In addition, similar to the method described in Example 1, biotin-modified DOPE was mixed at 1% (molar ratio) with the lipid molecules to immobilize them on the substrate, and the fluorescently labeled lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) was mixed at 0.5% (molar ratio) to observe the GUVs.

[0064] The intensity distribution of Raman scattered light in the vicinity of the GUVs was measured using the same method as in Example 1 and is shown in Figure 5(a). The Raman spectrum obtained when the intensity distribution of Raman scattered light was measured is shown in Figure 5(c) (NaCl concentration 0 mM in the figure). As a result, no particularly significant enhancement of Raman scattered light was observed.

[0065] Next, NaCl was added to the external solution of the immobilized GUVs to a final concentration of 50 mM, and the GUVs were observed.

[0066] Figure 5(b) shows the results of measuring the intensity distribution of Raman scattered light at the location where GUVs were observed using the same method as in Example 1. Figure 5(c) shows the Raman spectrum obtained when the intensity distribution of Raman scattered light was measured (NaCl concentration in the figure: 50 mM). As a result, when NaCl was added to the external solution of the GUV to a final concentration of 50 mM, a significant increase in Raman scattering intensity was observed compared to when the NaCl concentration was 0 mM, as shown in Figure 5(c).As shown in Figure 5(b), the increase in Raman scattering intensity was observed only in areas where gold nanocolloid aggregates were present within the GUV.

[0067] The significant enhancement of Raman scattering intensity in the presence of aggregates of gold nanocolloids within the GUVs reflects the fact that Na ions pass through the ion channels formed by gramicidin, increasing the Na ion concentration inside the GUVs and causing the aggregation of gold nanocolloids.

[0068] These results demonstrate that in ionophore-containing GUVs, the surface Raman scattering is enhanced by metal nanocolloids aggregated by cation permeation. [Explanation of symbols]

[0069] 1...Giant lipid vesicle (GUV), 2...Lipid molecule, 3...Metal nanocolloid, 4...Ionophore or pore-forming protein, 5...Target biomolecule, 6...Biological vesicle, 7...Cation, 8...Excitation light, 9...Raman scattered light, 10...Biosensor, 11...Solid substrate, 12...Surface Raman scattering measurement device, 13...Coating agent, 14...Linker molecule, 14-1...Biotin, 14-2...Streptavidin< / guv>

Claims

1. A giant lipid membrane vesicle encapsulating an aggregate of metal nanocolloids, A giant lipid membrane vesicle, wherein the metal in the metal nanocolloid is a metal that causes surface-enhanced Raman scattering.

2. 2. The giant lipid membrane vesicle according to claim 1, wherein the metal that causes surface-enhanced Raman scattering is at least one metal selected from the group consisting of gold, silver, and copper.

3. 2. The giant lipid membrane vesicle according to claim 1, wherein the giant lipid membrane vesicle comprises a cation-permeable ionophore or pore-forming protein.

4. (a) preparing a W / O emulsion containing a metal nanocolloid; (b) passing the W / O emulsion through a lipid monolayer formed at the oil-water interface of a solution in which an oil phase is formed on the upper surface of an aqueous phase, to form giant lipid membrane vesicles encapsulating metal nanocolloids within the aqueous phase; and The method for producing giant lipid membrane vesicles, wherein the metal in the metal nanocolloid is a metal that causes surface-enhanced Raman scattering.

5. In the step (a), a W / O emulsion containing the metal nanocolloid aggregates is prepared. The method for producing the giant lipid membrane vesicle according to claim 4.

6. At least one of the W / O emulsion and the lipid monolayer contains a cation-permeable ionophore or pore-forming protein; In the step (b), a giant lipid membrane vesicle containing the ionophore or pore-forming protein is formed, After the step (b), (c) a step of aggregating the metal nanocolloids inside the giant lipid membrane vesicles by increasing the cation concentration of the external solution of the giant lipid membrane vesicles formed in the step (b) to be higher than the cation concentration of the aqueous solution contained in the giant lipid membrane vesicles; The method for producing a giant lipid membrane vesicle according to claim 4, comprising:

7. A method for detecting a target biomolecule contained inside a giant lipid membrane vesicle, comprising detecting the target biomolecule contained inside the giant lipid membrane vesicle according to any one of claims 1 to 3 by surface-enhanced Raman scattering.

8. A biosensor comprising the giant lipid membrane vesicle according to any one of claims 1 to 3.

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